Design, synthesis and biological evaluation of 1H-pyrrolo[2,3-b]pyridine and 1H-pyrazolo[3,4-b]pyridine derivatives as c-Met inhibitors

Article abstract of DOI:10.1016/j.bioorg.2016.02.009

Five novel 1H-pyrrolo[2,3-b]pyridine or 1H-pyrazolo[3,4-b]pyridine derivatives, with a methylene, sulfur, sulfoxide or cyclopropyl group as a linker, were designed, synthesized and biologically evaluated against c-Met and ALK. The development of these methods of compound synthesis may provide an important reference for the construction of novel 7-azaindole and 7-azaindazole derivatives with a single atom linker. The enzyme assay and cell assay in vitro showed that compound 9 displayed strong c-Met kinase inhibition with IC₅₀ of 22.8 nM, moderate ALK kinase inhibition, and strong cell inhibition with MKN-45 IC₅₀ of 329 nM and EBC-1 IC₅₀ of 479 nM. In order to find the better candidate compounds, compounds 8, 9 and 10 have been selected as tool compounds for further optimization.

Full text of DOI:10.1016/j.bioorg.2016.02.009

Accepted Manuscript
Design, synthesis and biological evaluation of 1H-pyrrolo[2,3-b]pyridine and
1H-pyrazolo[3,4-b]pyridine derivatives as c-Met inhibitors
Na Liu, Yanfen Wang, Gongchao Huang, Conghui Ji, Wei Fan, Haitao Li, Ying
Cheng, Hongqi Tian
PII:
S0045-2068(16)30018-9
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.02.009
YBIOO 1885
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 January 2016
25 February 2016
27 February 2016
Please cite this article as: N. Liu, Y. Wang, G. Huang, C. Ji, W. Fan, H. Li, Y. Cheng, H. Tian, Design, synthesis
and biological evaluation of 1H-pyrrolo[2,3-b]pyridine and 1H-pyrazolo[3,4-b]pyridine derivatives as c-Met
inhibitors, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.02.009
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Design, synthesis and biological evaluation of 1H-pyrrolo[2,3-b]pyridine and
1H-pyrazolo[3,4-b]pyridine derivatives as c-Met inhibitors
Na Liu a,†, Yanfen Wang a,†, Gongchao Huang b,†, Conghui Ji b, Wei Fan a, Haitao Li b, Ying Cheng
b, Hongqi Tian c,*
a
School of Biological Engineering, Tianjin University of Science and Technology, #29, 13th
Avenue, TEDA, Tianjin 300457, China
b Tianjin Binjiang Pharma, Inc., #238 Baidi Road, Nankai District, Tianjin 300192, China
c
Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of
Radiation Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College,
#238 Baidi Road, Nankai District, Tianjin 300192, China
† These authors contributed equally
*
Corresponding author. Tel.: +86 186 0261 3300, Fax.: +86 022 65378920,
tianhongqi@irm-cams.ac.cn
Abstract
Five novel 1H-pyrrolo[2,3-b]pyridine or 1H-pyrazolo[3,4-b]pyridine derivatives, with a
methylene, sulfur, sulfoxide or cyclopropyl group as a linker, were designed, synthesized and
biologically evaluated against c-Met and ALK. The development of these methods of compound
synthesis may provide an important reference for the construction of novel 7-azaindole and
7-azaindazole derivatives with a single atom linker. The enzyme assay and cell assay in vitro
showed that compound 9 displayed strong c-Met kinase inhibition with IC50 of 22.8 nM, moderate
ALK kinase inhibition, and strong cell inhibition with MKN-45 IC50 of 329 nM and EBC-1 IC50
of 479 nM. In order to find the better candidate compounds, compounds 8, 9 and 10 have been
selected as tool compounds for further optimization.
Keywords: c-Met inhibitor, azaindole, azaindazole, synthesis, biological evaluation
1. Introduction

The tyrosine kinase receptor, c-Met, is also known as hepatocyte growth factor receptor
(HGFR), and its ligand, hepatocyte growth factor (HGF), is also known as scatter factor [1]. c-Met
is a heterodimer composed of an extracellular α subunit linked by a disulfide bridge to a
transmembrane catalytic β subunit. Activation of c-Met occurs through autophosphorylation of
Y1234 and Y1235 located in the activation loop [2]. Following activation by its ligand, HGF,
c-Met induces a cell program that facilitates malignant tumor behavior, consisting of cell
proliferation, cell migration, and invasion, increased cell survival, and cell morphogenesis [3].
Aberrant expression of c-Met and HGF is associated with the development of a wide range of
solid tumors, and is regarded as a prognostic marker for malignancy [4]. Because of the known
roles of c-Met and its ligand HGF in the pathogenesis and progression of human malignant tumors,
c-Met inhibitors would be predicted to have a potential role as targets for therapy in oncology.
At present, some small molecule c-Met inhibitors have been reported to have inhibitory
activity against c-Met in human tumors and to inhibit tumor growth [5,6]. Based on the binding
modes with c-Met kinase, these inhibitors have been classified to include Type I inhibitors and
Type II inhibitors [5,7]. Type I inhibitors comprise the majority of the ATP-competitive inhibitors
due to their ability to bind c-Met kinase in the active conformation. Type I inhibitors typically
adopt a ‘U-shaped’ conformation through interactions with both hinge and activation loop residue
Y1230. Type II inhibitors recognize a conformation that is sometimes referred to as ‘DFG-out’,
owing to the rearrangement of this motif. The DFG-out conformation then exposes an additional
hydrophobic binding site that is adjacent to the ATP binding site (Fig. (9). in reference 5).
The clinically established targeted therapeutic compound, Crizotinib, is a Type I c-Met
inhibitor, that displays both anaplastic lymphoma kinase (ALK) and c-Met inhibitory activity [8].
In 2011, Crizotinib has been approved by the US Food and Drug Administration (FDA) for the
treatment of ALK-rearranged non-small cell lung carcinoma (NSCLC) as an ALK and c-Met
inhibitor. The co-crystalline structure of Crizotinib combined with c-Met (Fig. 1) has been
reported by J. Jean Cui and her co-authors [8], in which she revealed that the aminopyridine forms
two hydrogen bonds with the backbone CO of P1158 and NH of M1160 of the kinase hinge. The
2,6-dichloro-3-fluorobenzyloxy ring stacks in a coplanar π-interaction against the phenol side
chain of Y1230 of the activation loop. In the Crizotinib molecule, both the 2-chloro and 3-fluoro
elements on the 3-benzyloxy group point toward the NH of D1222 of the activation loop,

indicating that there may be beneficial electrostatic interactions. The methylene linker allows the
two critical binding elements to wrap around M1211 and form hydrogen bonds with the backbone
of the hinge and π-stacking interactions with Y1230. The R-methyl group is used to rigidify the
benzyl group, and also make favorable hydrophobic interactions with the residues of V1092,
L1157, K1110, and A1108 in the pocket. The 5-pyrazol-4-yl group is bound through the narrow
lipophilic tunnel surrounded by I1084 and Y1159 and provides the inhibition of c-Met. The
terminal piperidine ring that appears to serve as a solubilizing group, attached to the N1 position
of the pyrazol-4-yl, extends out into the solvent surrounding the kinase hinge segment. Bringing
together all of these groups in Crizotinib compound, results in a strong inhibition of c-Met.
Fig. 1. Cocrystalline structure of Crizotinib combined with c-Met
Replacement of the aminopyridine in Crizotinib with 7-azaindole, resulting in the production
of compound 1, has been previously reported. Compound 1 demonstrated c-Met inhibitory activity
[9]. Other 7-azaindole and 7-azaindazole derivatives (compounds 2 and 3) have also been reported
to show c-Met inhibitory activity [10,11]. The structure-activity relationships (SAR) have
suggested that 7-azaindole or 7-azaindazole forms two hydrogen bonds to the backbone of P1158
and M1160 of the kinase hinge. A single atom linker, such as methylene, oxygen, sulfur, sulfone or
cyclopropyl group (Fig. 2) [9-13], is important, and allow two aromatic appendages to wrap
around M1211 and form hydrogen bonds with the backbone P1158 and M1160 of the kinase hinge
and π-stacking interactions with Y1230. Furthermore, small substituent group at the benzylic
position appears to be tolerated (compounds 1, 3 and 5) [9,11,13]. Therefore, exploration of new
7-azaindole or 7-azaindazole derivatives, with a single atom linker, may result in the development

of new c-Met inhibitors.
Fig. 2. The structure of compounds 1, 2, 3, 4, 5 and their c-Met inhibition
2. Results and Discussion
The results of this study included the design, synthesis, and characterization of the five new
compounds, following the replacement of the aminopyridine in Crizotinib with the closely related
1H-pyrrolo[2,3-b]pyridine or 1H-pyrazolo[3,4-b]pyridine. The results also included the design,
characterization and biological evaluation of the five new compounds directed against c-Met,
following the replacement of the linkage between the 7-azaindole or 7-azaindazole and the
2,6-dichloro-3-fluorophenyl with a methylene, sulfoxide or cyclopropyl. The mode of interactions
between Crizotinib and c-Met kinase was reported previously by J. Jean Cui and her co-authors in
the reference 8 (Fig. 1). The new derivatives, taking Crizotinib and compound 1 as a lead scaffold,
were expected to have the similar binding mode. The NH and ring nitrogen of 7-azaindole or
7-azaindazole in new compounds were expected to result in two hydrogen bonds with the
backbone CO of P1158 and NH of M1160 of the kinase hinge region, similar to the
2-aminopyridine of Crizotinib. The halogenated phenyl group was maintained owing to its
potential co-planar π-interaction with the residue Y1230 of the activation loop. Both the 2-chloro
and 3-fluoro elements on the phenyl group were maintained due to the electrostatic interactions
with the backbone NH of D1222. The pyrazole group at the C5 position of the 2-aminopyridine
was maintained due to its compact size and its ability to modulate physicochemical properties.

The terminal piperidine ring, which attached to the N1 position of the pyrazol-4-yl, was
maintained to serve as a solubilizing group.
Compound 2a, with a 3-pyridyl group at the C2 position of 7-azaindole, demonstrated c-Met
inhibition activity [10], indicating that the small lipophilic bulk at the C2 position of 7-azaindole
may be tolerated. In order to check this hypothesis, the aminopyridine in Crizotinib was initially
replaced with 7-azaindole. By the addition of small lipophilic bulk Me or Cl at C2 position of the
7-azaindole, two novel 7-azaindole derivatives 6 and 7 were synthesized and evaluated for c-Met
inhibition. The 7-azaindole was expected to form two hydrogen bonds with the backbone of P1158
and M1160. The Me or Cl substitution at the C2 position of 7-azaindole may be affected the
binding of the substrate with the kinase hinge, and make favorable hydrophobic interactions with
the amino acid residues in the pocket, to improve inhibitory strength and selectivity. But the
enzyme and cell assays of compounds 6 and 7 showed weak kinase and cell inhibition. These
findings suggested that the bulkier substituent at the C2 position of 7-azaindole may be not favor
the formation of hydrogen bonds of 7-azaindole with the backbone of the hinge, and result in
weaker inhibitory effect.
Further optimization resulted from the introduction of 7-azaindazole and replacement of the
linkage between the 7-azaindole and 7-azaindazole and the 2,6-dichloro-3-fluorophenyl.
Following replacement of the aminopyridine in Crizotinib with 7-azaindole and 7-azaindazole, the
methylene linker was replaced with a sulfur and sulfoxide, which was expected to allow the two
aromatic appendages to wrap around M1211 and form hydrogen bonds with the backbone P1158
and M1160 and facilitate π-stacking interactions with Y1230. Then two novel 7-azaindole and
7-azaindazole derivatives, compounds 8 and 9, were synthesized and evaluated against c-Met,
both of which displayed strong c-Met kinase and cell inhibitory activity.
In order to further optimize the linkage, a cyclopropyl group was introduced, which was
expected not only to stabilize the phenyl group, but also to facilitate the hydrophobic interactions,
to allow the two aromatic appendages to wrap around M1211 and form hydrogen bonds to the
backbone P1158 and M1160 and to facilitate π-stacking interactions with Y1230. The other
7-azaindazole derivatives, compound 10, was produced and evaluated against c-Met, which
displayed strong c-Met kinase and cell inhibitory activity.
Compound 11b has been previously synthesized in our laboratory and was submitted for a

patent application (Chinese patent application No. 201210376940.1) on September 29, 2012, but
was authorized to another applicant on October 4, 2012 [14]. Compound 11b has been previously
reported to show strong c-Met inhibitory activity [15]. Meanwhile, the racemic compound of
Crizotinib, compound 11a, was also prepared. Crizotinib, 11a, 11b and Cisplatin were also
evaluated as positive control compounds (Table 1).
Table 1. Structures and biological activities of target compounds 6-10 and positive control
compounds:
Compound
A
kinase IC50 (nM)
ALK
C1156Y L1196M
cell IC50 (nM)
ALK
WT
504
2710
214
135
508
17.6
372
17.3
ND
ALK
No
X
c-Met
MKN-45 EBC-1
C-Me CH-Me
286
982
63
1517
4077
1310
651
1664
3311
725
1979
6519
1494
329
2383
9215
1590
479
6
C-Cl
CH
N
CH-Me
7
S=O
8
9
S
22.8
47.4
14.6
51.2
13.7
ND
578
N
C(CH2)2
2988
133
735
670
1194
129
10
O
S
337
97
11a
2102
7.67
ND
2807
188
1033
ND
1626
ND
11b
Crizotinib
Cisplatin
ND
9736
27467
ND: Not determined.
Finally, five novel 7-azaindole and 7-azaindazole derivatives, with a methylene, sulfur,
sulfoxide or cyclopropyl group as a linker, were synthesized and biologically evaluated against
c-Met and ALK kinase, and MKN-45 and EBC-1 cell line. The results are summarized in Table 1.

Initially, compounds 6 and 7 showed weak c-Met and ALK kinase inhibition and cell inhibition,
indicating that the bulk substituent at the C2 position of 7-azaindole may interrupt the hydrogen
bond formation of 7-azaindole with the backbone of the hinge, resulting in reduced activity.
Following replacement of the methylene linker between 7-azaindole or 7-azaindazole and
2,6-dichloro-3-fluorophenyl with a sulfur, sulfoxide or cyclopropyl group, compounds 8, 9 and 10
were synthesized. The enzyme assay and cell assay in vitro showed that compounds 8, 9 and 10
displayed strong c-Met kinase inhibitory activity, moderate ALK kinase inhibitory activity, and
strong cell inhibitory activity. The kinase selectivity of c-Met against ALK is better than Crizotinib.
Among the test compounds, compound 9 displayed the best c-Met kinase inhibitory activity with
IC50 of 22.8 nM, moderate ALK kinase inhibitory activity, and strong cell inhibitory activity with
MKN-45 IC50 of 329 nM and EBC-1 IC50 of 479 nM. In order to find the better candidate
compounds, compounds 8, 9 and 10 have been selected as tool compounds, with 7-azaindole and
7-azaindazole as core and a single atom as linker (such as sulfur, sulfoxide or cyclopropyl group),
the further structure optimization is ongoing.
3. Chemistry
3.1.
Synthesis
of
target
compound
3-(1-(2,6-Dichloro-3-fluorophenyl)ethyl)-2-methyl-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrr
olo[2,3-b]pyridine (6)
Scheme 1. Reagents and conditions: (i) MsCl, Et3N, DCM; (ii) NaH, DMF, 100 °C, overnight; (iii)

Pd(Ph3P)2Cl2, KOAc, DMSO, 80 °C, 2 h; (iv) PhSO2Cl, NaH, DMF, 0 - 25 °C, 1.5 h; (v) i-Pr2NH,
n-BuLi, MeI, THF, -78 - 25 °C, 3.5 h; (vi) NaOH, MeOH, H2O, 65 °C, 2 h; (vii) TfOH, DCM,
25 °C, 2 h; (viii) Pd(dppf)Cl2, K2CO3, dioxane, H2O, 100 °C, 12 h; (ix) TFA, DCM, 25 °C, 24 h.
Target compound 6 was produced according to the procedures outlined in Scheme 1. The
intermediate 16 was prepared via alkylation of compound 14 with compound 13 followed by
palladium-catalyzed coupling with bis(pinacolato)diboron according to published methods [8].
The intermediate 22 was prepared using the synthetic route shown in Scheme 1. Commercially
available compound 17 was sulfonylated with Benzenesulfonyl chloride under basic conditions
produced compound 18 with a high yield. Methylation of compound 18 was achieved by treatment
of compound 18 with Lithium diisopropylamide (LDA) and Iodomethane (MeI), which generated
compound 19 with a moderate yield. Treatment of compound 19 with Sodium hydroxide produced
compound 20 with a good yield. Fielder-Crafts mutilation of compound 20 with compound 21
generated compound 22 with a good yield. Conventional Suzuki coupling of compound 22 with
compound 16 generated compound 23 with an excellent yield. Subsequent treatment of compound
23 with acid to remove the butoxy carbonyl (Boc) group produced compound 6 with a good yield.
3.2.
Synthesis
of
target
compound
2-Chloro-3-(1-(2,6-dichloro-3-fluorophenyl)ethyl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrr
olo[2,3-b]pyridine (7)
Scheme 2. Reagents and conditions: (i) Br2, t-BuOH, H2O, 25 °C; (ii) Zn, AcOH, 25 °C, 3 h; (iii)
POCl3, 110 °C, 4 h; (iv) TfOH, DCM, 25 °C, 16 h; (v) Pd(dppf)Cl2, K2CO3, dioxane, H2O, 100 °C,
12 h; (vi) TFA, DCM, 25 °C, 24 h.

Target compound 7 was synthesized according to the protocol in Scheme 2. Bromination of
5-bromo-7-azaindole (compound 24) with Bromine produced compound 25 with a high yield.
Reduction-debromination of compound 25 with Zinc powder in Acetic acid generated compound
26 with an excellent yield. Treatment of compound 26 with Phosphorus oxychloride (POCl3)
generated compound 27 with a high yield. Friedel-Crafts alkylation of compound 27 with
compound 21 produced compound 28 with a moderate yield. Suzuki coupling reaction of
compound 28 with compound 16 produced compound 29 with an acceptable yield. Subsequent
treatment of compound 29 with acid produced compound 7 with a moderate yield.
3.3.
Synthesis
of
target
compound
3-((2,6-Dichloro-3-fluorophenyl)sulfide)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b
]pyridine (8)
Scheme 3. Reagents and conditions: (i) S, n-BuLi, THF, -78 °C, 3.5 h; (ii) NIS, acetone, 25 °C, 1
h; (iii) PMBCl, K2CO3, DMF, 25 °C, 4 h; (iv) CuI, K2CO3, ethylene glycol, i-PrOH, 100 °C, 5 h;
(v) Pd(dppf)Cl2, K2CO3, dioxane, H2O, 80 °C, 12 h; (vi) TFA, H2SO4, DCM, 25 °C, 2 h; (vii)

m-CPBA, 0 °C, 2 h.
Target compound 8 was prepared according to the procedure in Scheme 3. Compound 31 was
generated via treatment of 2,4-Dichloro-1-fluorobenzene with n-Butyllithium (n-BuLi) and Sulfur
with a high yield. Iodination of 5-Bromo-7-azaindole (compound 32) with N-Iodosuccinimide
(NIS) produced compound 33 with an excellent yield. Treatment of compound 33 with
1-(chloromethyl)-4-methoxybenzene (PMBCl) under basic conditions produced compound 34
with a high yield. Treatment of compound 34 with compound 31 via a copper-catalyzed reaction
produced compound 35 with a good yield. Suzuki coupling of compound 35 with compound 16
generated compound 36 with a moderate yield. Subsequent treatment of compound 36 with acid to
remove the butoxy carbonyl (Boc) group and the 4-methoxybenzyl (PMB) group produced
compound 37 with a good yield. Oxidation of compound 37 with 3-Chloroperoxybenzoic acid
(m-CPBA) produced compound 8 with a good yield.
3.4.
Synthesis
of
target
compound
3-((2,6-Dichloro-3-fluorophenyl)thio)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]p
yridine (9)
Scheme 4. Reagents and conditions: (i) I2, KOH, DMF, 25 °C, 4 h; (ii) PMBCl, K2CO3, DMF,
25 °C, 4 h; (iii) CuI, K2CO3, ethylene glycol, i-PrOH, 100 °C, 5 h; (iv) Pd(Ph3P)4, Cs2CO3, DME,
H2O, 80 °C, 12 h; (v) TFA, H2SO4, DCM, 25 °C, 2 h.

Target compound 9 was prepared according to the method in Scheme 4. Iodination of
5-Bromo-7-azaindazole (compound 38) with Iodine produced compound 39 with an excellent
yield. Treatment of compound 39 with 1-(Chloromethyl)-4-methoxybenzene produced compound
40 with a high yield. Copper-catalyzed coupling of compound 40 with compound 31 produced
compound 41 with a good yield. Suzuki coupling of compound 41 with compound 16 produced
compound 42 with a moderate yield. Subsequent treatment of compound 42 with acid produced
compound 9 with an excellent yield.
3.5.
Synthesis
of
target
compound
3-(1-(2,6-Dichloro-3-fluorophenyl)cyclopropyl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrazo
lo[3,4-b]pyridine (10)
Scheme 5. Reagents and conditions: (i) n-BuLi, oxirane, THF, -78 °C, 3 h; (ii) NaClO2, TEMPO,
0 - 25 °C, 3 h; (iii) SOCl2, NH3.H2O, DCM, 0 - 25 °C, 4 h; (iv) SOCl2, pyridine, 0 - 25 °C, 2 h; (v)
NaH, BrCH2CH2Br, 0 - 25 °C, 4 h; (vi) DIBAL-H, PhMe, -78 °C, 1 h; (vii) LiHMDS, THF, -78 °C,
1.5 h; (viii) PCC, DCM, 25 °C, 4 h; (ix) NH2NH2, EtOH, 80 °C, 10 h; (x) Pd(Ph3P)4, Cs2CO3,
dioxane, H2O, 80 °C, 12 h; (xi) TFA, DCM, 25 °C, 2 h.

Target compound 10 was synthesized according to the method in Scheme 5. Treatment of
compound 43 with n-BuLi and Oxirane generated compound 44 with a moderate yield. Oxidation
of compound 44 with 2,2,6,6-Tetramethylpiperidine N-oxide (TEMPO) and sodium chlorite
(NaClO2) produced compound 45 with a high yield. Treatment of 45 with Thionyl chloride (SOCl2)
and ammonia water produced compound 46 with a high yield. Dehydration of compound 46 with
Thionyl chloride produced compound 47 with a high yield. Alkylation of compound 47 with
1,2-Dibromoethane produced compound 48 with an excellent yield. Reduction of compound 48
with Di-isobutylaluminum hydride (DIBAL-H) generated compound 49 with a good yield.
Treatment of 5-Bromo-2-fluoropyridine with Lithium bis(trimethylsilyl)amide (LiHMDS) and
compound 49 preduced compound 50 with an excellent yield. Oxidation of compound 50 with
Pyridinium chlorochromate (PCC) produced compound 51 with a high yield. Treatment of 51 with
Hydrazine hydrate produced compound 52 with a moderate yield. Suzuki coupling of compound
52 with compound 16 produced compound 53 with a moderate yield. Subsequent treatment of
compound 53 with acid produced compound 10 with an acceptable yield.
4. Conclusion
In conclusion, five novel 1H-pyrrolo[2,3-b]pyridine or 1H-pyrazolo[3,4-b]pyridine
derivatives with a methylene, sulfur, sulfoxide or cyclopropyl group as a linker, were designed,
synthesized and biologically evaluated as potential c-Met inhibitors. The development of these
methods of compound synthesis may provide an important reference for the construction of novel
7-azaindole and 7-azaindazole derivatives with a single atom linker. The enzyme assay and cell
assay in vitro showed that compound 9 displayed strong c-Met kinase inhibition with IC50 of 22.8
nM, moderate ALK kinase inhibition, and strong cell inhibition with MKN-45 IC50 of 329 nM and
EBC-1 IC50 of 479 nM. In order to find the better candidate compounds, compounds 8, 9 and 10
have been selected as tool compounds, with 7-azaindole and 7-azaindazole as core and with a
single atom as linker (such as sulfur, sulfoxide or cyclopropyl group), the further optimization is
ongoing in our laboratory and the result will be reported in the future.
5. Experimental Methods

5.1. General Methods for Chemistry
All reagents and solvents were used as purchased from commercial sources. Reactions were
carried out under nitrogen atmosphere unless otherwise indicated. Reactions’ time and purity of
the products were monitored by TLC on FLUKA silica gel aluminum cards (0.2 mm thickness)
with fluorescent indicator 254 nm. Column chromatography was run on silica gel (200 - 300 mesh)
1
from Qingdao Ocean Chemicals (Qingdao, Shandong, China). H NMR spectra were recorded on
Bruker ARX-400, 400 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as
an internal standard and Chloroform-D (CDCl3), Dimethylsulfoxid-d6 (DMSO-d6) or
Methanol-d4 (MeOD) as solvents (reported in ppm) with a reference standard of CDCl3 (7.26 ppm)
or DMSO-d6 (2.50 ppm). Multiplicities were given as s (singlet), bs (broad singlet), d (doublet), t
(triplet), dt (double of triplets), and m (multiplet). Mass spectra (MS) were taken in ESI mode on
Agilent 1100 LC-MS (Agilent, Palo Alto, CA, U.S.A.).
5.1.1.
Synthesis
of
target
compound
3-(1-(2,6-Dichloro-3-fluorophenyl)ethyl)-2-methyl-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrr
olo[2,3-b]pyridine (6)
5.1.1.1. 5-Bromo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (18)
To
a
solution of 5-bromo-1H-pyrrolo[2,3-b]pyridine 17 (5 g, 25 mmol) in
N,N-Dimethylformamide (DMF, 40 mL) Sodium hydride (NaH, 0.91 g, 37 mmol) was added at
0 °C and stirred at 25 °C for 20 min. To the mixture Benzenesulfonyl chloride (5.37 g, 30 mmol)
was added dropwise at 0 °C and stirred at 25 °C for 1 h. The mixture was quenched with
Ammonium chloride (NH4Cl) solution, extracted with Bichloromethane (DCM, 50 mL × 2). The
organic phase was washed with water (50 mL × 2), brine (50 mL × 2), dried over Sodium sulfate
(Na2SO4). After filtering, the organic phase was concentrated to give compound 18 as a white
solid (8.5 g, 99.6% yield). 1H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H, Ar-H), 8.18-8.16 (d, J = 7.8
Hz, 2H, Ar-H), 7.96 (s, 1H, Ar-H), 7.74-7.73 (m, J = 4.0 Hz, 1H, Ar-H), 7.59-7.50 (t, J = 7.4 Hz,
1H, Ar-H), 7.47-7.38 (t, 2H, Ar-H), 6.55-6.54 (d, J = 4.0 Hz, 1H, Ar-H).
5.1.1.2. 5-Bromo-2-methyl-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (19)
To a solution of diispropylamine (2.52 g, 24.91 mmol) in anhydrous Tetrahydrofuran (THF,

50 mL) n-BuLi (2 M, 9.25 mL, 23.13 mmol) was added dropwise at -78 °C and stirring at -78 °C
under N2 for 30 min. To the mixture a solution of compound 18 (3 g, 8.90 mmol) in anhydrous
THF (20 mL) was added and stirred at -78 °C for 40 min. To the mixture a solution of compound
MeI (3.78 g, 26.69 mmol) in anhydrous THF (20 mL) was added at -78 °C and stirred 25 °C for 2
h. The mixture was quenched with sat. NH4Cl solution and extracted with Ethyl acetate (EtOAc,
100 mL × 3). The organic phase was washed with brine, dried over Na2SO4, concentrated and
purified by column chromatography on silica (PE:EA = 40:1) to give compound 19 as a white
1
solid (2.0 g, 64.1% yield). H NMR (400 MHz, DMSO-d6) δ 8.36 (s, 1H, Ar-H), 8.17 (s, 1H,
Ar-H), 8.13-8.05 (m, 2H, Ar-H), 7.72-7.70 (t, J = 7.5 Hz, 1H, Ar-H), 7.63-7.59 (t, J = 7.8 Hz, 2H,
Ar-H), 6.56 (s, 1H, Ar-H), 2.71 (s, 3H, CH3). ESI-MS m/z: 351.11, 353.11 (M + H)+.
5.1.1.3. 5-Bromo-2-methyl-1H-pyrrolo[2,3-b]pyridine (20)
To a solution of compound 19 (1.3 g, 3.7 mmol) in Methynol (MeOH, 85 mL) 2 M of
Sodium hydroxide (NaOH) solution (22 mL) was added. After refluxing for 2 h, the mixture was
extracted with EtOAc (100 mL × 3). The organic phase was washed with brine (30 mL × 2), dried
over Na2SO4, concentrated to give compound 20 as a white solid (600 mg, 76.9% yield). 1H NMR
(400 MHz, DMSO-d6) δ 11.69 (s, 1H, NH), 8.13 (s, 1H, Ar-H), 8.01 (s, 1H, Ar-H), 6.14 (s, 1H,
Ar-H), 2.40 (s, 3H, CH3). ESI-MS m/z: 211.16, 213.16 (M + H)+.
5.1.1.4.
(22)
5-Bromo-3-(1-(2,6-dichloro-3-fluorophenyl)ethyl)-2-methyl-1H-pyrrolo[2,3-b]pyridine
To
a
solution of compound 20 (450 mg, 2.13 mmol) in DCM (10 mL)
Trifluoromethanesulfonic
acid
(TfOH,
1.28
g,
8.53
mmol)
and
1-(2,6-Dichloro-3-fluorophenyl)ethanol (1.63 g, 8.53 mmol) was added dropwise. After stirring at
25 °C under N2 for 16 h, the mixture was quenched with sat. Sodium bicarbonate (NaHCO3)
solution, extracted with DCM (100 mL × 3). The organic phase was washed with water (20 mL ×
2), brine (20 mL × 2), dried over Na2SO4. After filtering, the organic phase was concentrated and
purified by column chromatography on silica (DCM:MeOH = 200:1) to give 22 as a white solid
1
(650 mg, 75.8% yield). H NMR (400 MHz, DMSO-d6) δ 11.66 (s, 1H, NH), 8.12 (s, 1H, Ar-H),
7.87 (s, 1H, Ar-H), 7.54-7.50 (dd, J = 8.9, 5.1 Hz, 1H, Ar-H), 7.39-7.34 (t, J = 8.7 Hz, 1H, Ar-H),

5.15-5.12 (q, J = 7.4 Hz, 1H, CH), 2.18 (s, 3H, CH3), 1.86-1.83 (d, J = 7.5 Hz, 3H, CH3).
5.1.1.5.
tert-Butyl
4-(4-(3-(1-(2,6-dichloro-3-fluorophenyl)ethyl)-2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyraz
ol-1-yl)piperidine-1-carboxylate (23)
To a mixture of compound 22 (450 mg, 1.12 mmol), compound 16 (633 mg, 1.68 mmol) and
Potassium carbonate (K2CO3, 464 mg, 3.357 mmol) in Dioxane (16 mL) and H2O (4 mL)
Dichloro(1,1'-bis(diphenylphosphino)ferrocene)palladium (II) (Pd(dppf)Cl2, 91.3 mg, 0.11 mmol)
was added under N2. After stirring at 100 °C under N2 for 12 h, the mixture was diluted with water
(20 mL), extracted with EtOAc (20 mL × 3). The organic phase was washed with brine (20 mL ×
2), dried over Na2SO4, concentrated and purified by column chromatography on silica
(DCM:MeOH = 100:1) to give compound 23 as a white solid (320 mg, 50% yield). 1H NMR (400
MHz, DMSO-d6) δ 11.33 (s, 1H, NH), 8.37-8.29 (m, 1H, Ar-H), 8.22 (s, 1H, Ar-H), 7.90 (s, 1H,
Ar-H), 7.78 (s, 1H, Ar-H), 7.54-7.50 (m, 1H, Ar-H), 7.39-7.32 (t, J = 8.7 Hz, 1H, Ar-H), 5.21-5.15
(q, J = 7.5 Hz, 1H, CH), 4.42-4.31 (m, 1H, CH), 4.20-3.96 (m, 4H, CH2), 2.99-2.78 (m, 4H, CH2),
2.15 (s, 3H, CH3), 1.93-1.92 (d, J =7.5 Hz, 3H, CH3), 1.43 (s, 9H, C(CH3)3).
5.1.1.6.
3-(1-(2,6-Dichloro-3-fluorophenyl)ethyl)-2-methyl-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrr
olo[2,3-b]pyridine (6)
To a solution of compound 23 (250 mg, 0.44 mmol) in DCM (15 mL) Trifluoroacetic acid
(TFA, 8 mL) was added. After stirring at 25 °C for 24 h, the mixture was diluted with NaHCO3
solution, extracted with DCM (20 mL × 3). the organic phase was washed with brine (20 mL × 3),
dried over Na2SO4, concentrated and purified by column chromatography on silica (DCM:MeOH
1
= 6:1) to give compound 6 as a white solid (100 mg, 48.5% yield). H NMR (400 MHz,
DMSO-d6) δ 11.36 (s, 1H, NH), 8.70-8.58 (s, 1H, NH), 8.34 (s, 1H, Ar-H), 8.17 (s, 1H, Ar-H),
7.89 (s, 1H, Ar-H), 7.85 (s, 1H, Ar-H), 7.55-7.49 (m, 1H, Ar-H), 7.39-7.34 (t, J = 8.7 Hz, 1H,
Ar-H), 5.20-5.18 (q, J = 7.6 Hz, 1H, CH), 4.56-4.48 (m, 1H, CH), 3.46-3.39 (m, 2H, CH2),
3.15-3.05 (m, 2H, CH2), 2.28-2.16 (m, 4H, CH2), 2.16 (s, 3H, CH3), 1.92-1.90 (d, J = 7.5 Hz, 3H,
CH3). 13C NMR (400 MHz, DMSO-d6) δ 158.67, 156.01, 146.83, 143.12, 139.16, 136.29, 134.80,

130.54, 129.60, 125.38, 123.04, 121.46, 120.37, 119.25, 115.83, 108.98, 55.63, 42.50, 35.47,
29.12, 17.51, 12.84. ESI-MS m/z: 472.37 (M + H)+.
5.1.2.
Synthesis
of
target
compound
2-Chloro-3-(1-(2,6-dichloro-3-fluorophenyl)ethyl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrr
olo[2,3-b]pyridine (7)
5.1.2.1. 3,3,5-Tribromo-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (25)
To a solution of compound 5-Bromo-1H-pyrrolo[2,3-b]pyridine (3.0 g, 15.2 mmol) in
tert-Butanol (t-BuOH, 15 mL) and H2O (15 mL) Bromine (Br2, 2 mL) was added dropwise. The
mixture was stirred at 25 °C for 1 h at dark place. The reaction mixture was extracted with DCM
(50 mL × 3). The organic phase was washed with water (30 mL × 2), brine (30 mL × 2), dried over
Na2SO4. After filtering, the organic phase was concentrated to give compound 25 as a red solid
(5.64 g, 99.7% yield). ESI-MS m/z: 370.92, 372.91 (M + H)+.
5.1.2.2. 5-Bromo-1H-pyrrolo[2,3-b]pyridin-2(3H)-one (26)
To a solution of compound 25 (5.64 g, 15.2 mmol) in Acetic acid (AcOH, 100 mL) Zn power
(9.88 g, 0.15 mmol) was added. After stirring at 25 °C under N2 for 3 h, the mixture was distilled
to remove excess AcOH and extracted with DCM (100 mL). The organic phase was washed with
water (50 mL × 2), brine (50 mL × 2), dried over Na2SO4. After filtering, the organic phase was
concentrated and purified by column chromatography on silica (DCM:MeOH = 150:1) to give
compound 26 as an off-white solid (2.75 g, 84.8% yield). 1H NMR (400 MHz, DMSO-d6) δ 11.15
(s, 1H, NH), 8.17 (s, 1H, Ar-H), 7.78 (s, 1H, Ar-H), 3.59 (s, 2H, CH2). ESI-MS m/z: 211.01,
213.04 (M - H)-.
5.1.2.3. 5-Bromo-2-chloro-1H-pyrrolo[2,3-b]pyridine (27)
A solution of compound 26 (1.3 g, 6.1 mmol) in POCl3 (9.35 g, 61 mmol, 6 mL) was
refluxed for 4 h. After concentrated, the mixture was diluted with DCM (20 mL) and quenched
with water 20 mL). To the cooled mixture 1M NaOH solution was added to adjust pH to 7 - 8,
extracted with DCM (50 mL × 4), the organic phase was washed with water (30 mL × 2), brine(30
mL × 2), dried over Na2SO4. After filtering, the organic phase was concentrated to give compound

1
27 as a pale yellow solid (1.4 g, 99.1% yield). H NMR (400 MHz, DMSO-d6) δ 12.76 (s, 1H,
NH), 8.27 (s, 1H, Ar-H), 8.14 (s, 1H, Ar-H), 6.49 (s, 1H, Ar-H). ESI-MS m/z: 231.17, 233.17 (M
+ H)+.
5.1.2.4.
(28)
5-Bromo-2-chloro-3-(1-(2,6-dichloro-3-fluorophenyl)ethyl)-1H-pyrrolo[2,3-b]pyridine
To
a
solution of compound 27 (400 mg, 1.73 mmol) in DCM (40 mL)
Trifluoromethanesulfonic acid (1.04 g, 6.92 mmol) and 1-(2,6-Dichloro-3-fluorophenyl)ethanol
(1.45 g, 6.92 mmol) was added dropwise. After stirring at 25 °C under N2 for 16 h, the mixture
was quenched with sat. NaHCO3 solution, extracted with DCM (50 mL × 3). The organic phase
was washed with water (50 mL × 2), brine (50 mL × 2), dried over Na2SO4. After filtering, the
organic phase was concentrated and purified by column chromatography on silica (DCM:MeOH =
1
200:1) to give crude compound 28 as a pale yellow solid (500 mg, 68.5% yield). H NMR (400
MHz, DMSO-d6) δ 11.15 (s, 1H, NH), 8.03 (s, 1H, Ar-H), 7.78 (s, 1H, Ar-H), 7.54-7.50 (dd, J =
8.8, 5.1 Hz, 1H, Ar-H), 7.41-7.37 (t, J = 8.7 Hz, 1H, Ar-H), 5.54-5.49 (q, J = 7.6 Hz, 1H, CH),
1.91-1.85 (d, J = 7.6 Hz, 3H, CH3). ESI-MS m/z: 420.84, 422.93 (M - H)-.
5.1.2.5.
tert-Butyl4-(4-(2-chloro-3-(1-(2,6-dichloro-3-fluorophenyl)ethyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
1H-pyrazol-1-yl)piperidine-1-carboxylate (29)
To a solution of compound 28 (350 m g, 0.83 mmol) in Dioxane (16 mL) and H2O (4 mL)
compound 16 (468.8 mg, 1.24 mmol), K2CO3 (343.4 mg, 2.48 mmol) and then Pd(dppf)Cl2 (67.6
mg, 0.08 mmol) was added under N2. After stirring at 100 °C under N2 for 12 h, the mixture was
diluted with water (150 mL), extracted with EtOAc (50 mL × 3). The organic phase was washed
with brine (50 mL × 2), dried over Na2SO4, concentrated and purified by column chromatography
on silica (DCM:MeOH = 100:1) to give crude compound 29 as a pale yellow solid (119 mg, 24.2%
1
yield). H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H, NH), 8.46 (s, 1H, Ar-H), 8.29 (s, 1H,
Ar-H), 8.01 (s, 1H, Ar-H), 7.85 (s, 1H, Ar-H), 7.55-7.50 (m, 1H, Ar-H), 7.41-7.35 (t, J = 8.7 Hz,
1H, Ar-H), 5.22-5.16 (q, J = 7.6 Hz, 1H, CH), 4.39-4.33 (m, 1H, CH), 4.09-4.01 (m, 2H, CH2),
2.95-2.88 (m, 2H, CH2), 2.05-1.99 (m, 4H, CH2), 1.95 (d, J = 7.6 Hz, 5H, Ch2,CH3), 1.43 (S, 9H,

C(CH3)3). ESI-MS m/z: 591.84 (M - H)-.
5.1.2.6.
2-Chloro-3-(1-(2,6-dichloro-3-fluorophenyl)ethyl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrr
olo[2,3-b]pyridine (7)
To a solution of compound 29 (110 mg, 0.185 mmol) in DCM (15 mL) TFA (4mL) was
added and stirred at 25 °C for 24 h. The mixture was neutralized with NaHCO3 solution, extracted
with DCM (20 mL × 3). The organic phase was washed with brine (30 mL × 2), dried over
Na2SO4. After filtering, the organic phase was concentrated to give a residue which was purified
by column chromatography on silica (DCM:MeOH = 6:1) to give compound 7 as a white solid (46
1
mg, 50.5% yield). H NMR (400 MHz, DMSO-d6) δ 8.47 (s, 1H, Ar-H), 8.38-8.33 (s, 1H, NH),
8.24 (s, 1H, Ar-H), 8.00 (s, 1H, Ar-H), 7.84 (s, 1H, Ar-H), 7.58-7.47 (m, 1H, Ar-H), 7.41-7.36 (t, J
= 8.6 Hz, 1H, Ar-H), 5.22-5.16 (q, J = 7.6 Hz, 1H, CH), 4.35-4.25 (m, 1H, CH), 3.18-3.12 (m, 2H,
CH2), 2.78-2.70 (m, 2H, CH2), 2.08-2.01 (m, 2H, CH2), 1.98-1.90 (m, 5H, CH2, CH3). 13C NMR
(400 MHz, DMSO-d6) δ 158.60, 156.01, 145.85, 142.13, 140.96, 136.51, 132.86, 130.52, 129.72,
128.49, 125.78, 123.58, 121.61, 120.67, 120.29, 116.06, 109.56, 55.64, 42.58, 35.14, 29.13, 17.04.
ESI-MS m/z: 492.38, 494.37 (M + H)+.
5.1.3.
Synthesis
of
target
compound
3-((2,6-Dichloro-3-fluorophenyl)sulfinyl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b
]pyridine (8)
5.1.3.1. 2,6-Dichloro-3-fluorobenzenethiol (31)
To a solution of 2,4-Dichloro-1-fluorobenzene (8.0 g, 48.5 mmol) in THF (63 mL) n-BuLi
(23.27 mL, 2.5 M, 43.6 mmol) was added dropwise at -78 °C and stirred for 1.5 h. To the mixture
Sulfur (3.1 g, 72.7 mmol) was added at -78 °C and stirring for 2 h. To the resulting mixture 10%
Hydrochloric acid (HCl) was added to adjust pH to 5 - 6. The resulting mixture was extracted with
EtOAc (200 mL × 3), the combined organic phase was washed with brine (100 mL × 2), dried
over Na2SO4. After filtered, the organic phase was concentrated to dry to give compound 31 as
1
yellow oil (11.4 g, 99.0% yield). H NMR (400 MHz, CDCl3) δ 7.31-7.19 (m, 1H, Ar-H),
6.95-6.89 (t, 1H, Ar-H), 4.69 (s, 1H, SH).

5.1.3.2. 5-Bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine (33)
To a solution of 5-Bromo-1H-pyrrolo[2,3-b]pyridine (2.0 g, 10.2 mmol) in Acetone (20 mL)
NIS (2.7 g, 12.0 mmol) was added at 25 °C and stirred for 1 h. The mixture was concentrated and
then extracted with EtOAc (30 mL × 2), the organic phase was washed with sat. Sodium
thiosulfate (Na2S2O3) solution (30 mL × 2), brine (30 mL × 2), dried over Na2SO4. After filtering,
the organic phase was concentrated to give compound 33 as an off-white solid (2.9 g, 88.0% yield).
1H NMR (400 MHz, CDCl3) δ 9.25 (s, 1H, NH), 8.35 (s, 1H, Ar-H), 7.90 (s, 1H, Ar-H), 7.43 (s,
1H, Ar-H).
5.1.3.3. 5-Bromo-3-iodo-1-(4-methoxybenzyl)-1H-pyrrolo[2,3-b]pyridine (34)
To a solution of compound 33 (2.9 g, 9.0 mmol) in DMF (20 mL) K2CO3 (3.7 g, 26.8 mmol)
and 1-(Chloromethyl)-4-methoxybenzene (PMBCl, 1.7 g, 10.8 mmol) was added. After stirring at
25 °C for 4 h, the mixture was diluted with water (50 mL), extracted with EtOAc (20 mL × 3), the
organic phase was washed with brine (50 mL × 2), dried over Na2SO4. After filtering, the organic
phase was concentrated and purified by column chromatography on silica (PE:EA = 30:1) to give
1
compound 34 as a white solid (3.6 g, 90.3% yield). H NMR (400 MHz, CDCl3) δ 8.37 (s, 1H,
Ar-H), 7.85 (s, 1H, Ar-H), 7.25 (s, 1H, Ar-H), 7.21-7.17 (d, J = 8.6 Hz, 2H, Ar-H), 6.88-6.84 (t, J
= 8.0 Hz, 2H, Ar-H), 5.37 (s, 2H, CH2), 3.78 (s, 3H, CH3).
5.1.3.4.
5-Bromo-3-((2,6-dichloro-3-fluorophenyl)thio)-1-(4-methoxybenzyl)-1H-pyrrolo[2,3-b]Pyridine
(35)
To a solution of compound 34 (3.6 g, 8.1 mmol) in Isopropanol (i-PrOH, 50 mL) compound
31 (1.9 g, 9.7 mmol), K2CO3 (2.2 g, 16.2 mmol), Cuprous iodide (CuI, 1.5 g, 8.1 mmol) and
ethylene glycol (1.0 g, 16.2 mmol) was added. After stirring at 100 °C under N2 for 5 h, the
mixture was filtered and washed with EtOAc (100 mL). The organic phase was washed with
ammonia water (50 mL × 2), brine (50 mL × 2). The organic phase was dried over Na2SO4,
concentrated and purified by column chromatography on silica (PE:EA = 100:1) to give
1
compound 35 as a white solid (2.5 g, 70.0% yield). H NMR (400 MHz, CDCl3) δ 8.35 (s, 1H,

Ar-H), 8.19 (s, 1H, Ar-H), 7.55 (s, 1H, Ar-H), 7.35-7.30 (m, 1H, Ar-H), 7.19-7.13 (m, 2H, Ar-H),
7.06-7.00 (m, 1H, Ar-H), 6.87-6.80 (m, 2H, Ar-H), 5.36 (s, 2H, CH2), 3.79 (s, 3H, CH3).
5.1.3.5.
tert-Butyl
4-(4-(3-((2,6-dichloro-3-fluorophenyl)thio)-1-(4-methoxybenzyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-1
H-pyrazol-1-yl)piperidine-1-carboxylate (36)
To a solution of compound 35 (2.5 g, 4.9 mmol), Cesium carbonate (Cs2CO3, 4.8 g, 14.7
mmol), compound 16 (2.6 g, 6.8 mmol) in Dioxane (16 mL) and H2O (4 mL)
Tetrakis(triphenylphosphine)platinum (Pd(Ph3P)4, 170 mg, 0.15 mmol) was added under N2. After
stirring at 80 °C under N2 for 12 h, the mixture was diluted with water (30 mL), extracted with
EtOAc (20 mL × 3). The organic phase was washed with brine (30 mL × 2), dried over Na2SO4.
After filtering, the organic phase was concentrated and purified by column chromatography on
1
silica (PE:EA = 10:1) to give compound 36 as an off-white solid (1.4 g, 41.7% yield). H NMR
(400 MHz, CDCl3) δ 8.43 (s, 1H, Ar-H), 8.12-8.08 (m, 1H, Ar-H), 7.72 (s, 1H, Ar-H), 7.65 (s, 1H,
Ar-H), 7.50 (s, 1H, Ar-H), 7.36-7.30 (m, 1H, Ar-H), 7.21-7.16 (m, 2H, Ar-H), 7.07-7.01 (m, 1H,
Ar-H), 6.88-6.80 (m, 2H, Ar-H), 5.36 (s, 2H, CH2), 4.33-4.26 (m, 3H, CH, CH2), 3.79 (s, 3H,
CH3), 2.89-2.81 (m, 2H, CH2), 2.15-2.09 (m, 2H, CH2), 1.95-1.89 (m, 2H, CH2), 1.49 (s, 9H,
C(CH3)3).
5.1.3.6.
3-((2,6-Dichloro-3-fluorophenyl)thio)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]py
ridine (37)
To a solution of compound 36 (1.4 g, 2.1 mmol) in DCM (10 mL) TFA (5 mL) and
concentrated Sulfuric acid (con. H2SO4, 1 mL) was added. After stirring at 25 °C for 2 h, to the
mixture ammonia water was added to adjust pH to 7 - 8, extracted with DCM (20 mL × 2). The
organic phase was washed with sat. NaHCO3 solution (30 mL × 2), brine (30 mL × 2), dried over
Na2SO4. After filtering, the organic phase was concentrated to give compound 37 as a tan solid
1
(761.5 mg, 78.4% yield). H NMR (400 MHz, CDCl3) δ 9.19 (s, 1H, Ar-H), 8.43-8.39 (m, 1H,
Ar-H), 8.15-8.11 (m, 1H, Ar-H), 7.76 (s, 1H, Ar-H), 7.66 (s, 1H, Ar-H), 7.53 (s, 1H, Ar-H),
7.37-7.31 (m, 1H, Ar-H), 7.09-7.05 (m, 1H, Ar-H), 4.50-4.45 (m, 1H, CH), 3.73-3.67 (m, 2H,

CH2), 3.22-3.17 (m, 2H, CH2), 2.49-2.41 (m, 4H, CH2).
5.1.3.7.
3-((2,6-Dichloro-3-fluorophenyl)sulfinyl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b
]pyridine (8)
To a solution of compound 37 (761.5 mg, 1.6 mmol) in DCM (10 mL) m-CPBA (334.0 mg,
1.8 mmol) was added and stirred at 0 °C for 2 h. The mixture was quenched with sat. Na2S2O3
solution, extracted with DCM (10 mL × 2). The organic phase was washed with sat. NaHCO3
solution (30 mL × 2), brine (30 mL × 2), dried over Na2SO4. After filtering, the organic phase was
concentrated and purified by column chromatography on silica (DCM:MeOH = 10:1) to give
compound 8 as an off-white solid (608.0 mg, 77.2% yield). 1H NMR (400 MHz, CDCl3) δ 8.67 (s,
1H, Ar-H), 8.32 (s, 1H, Ar-H), 8.21 (s, 1H, Ar-H), 7.99-7.97 (d, 2H, Ar-H), 7.67-7-65 (d, 2H,
Ar-H), 4.56-4.51 (m, 1H, CH), 3.43-3.35 (m, 2H, CH2), 3.13-3.07 (m, 2H, CH2), 2.27-2.14 (m,
4H, CH2). 13C NMR (400 MHz, DMSO-d6) δ 158.74, 156.25, 147.74, 142.84, 139.34, 136.57,
132.02, 131.95, 130.38, 129.44, 126.04, 122.88, 120.73, 119.69, 117.35, 112.64, 55.76, 42.59,
29.18. ESI-MS m/z: 478.23, 480.22 (M + H)+.
5.1.4.
Synthesis
of
target
compound
3-(1-(2,6-Dichloro-3-fluorophenyl)ethyl)-2-methyl-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrr
olo[2,3-b]pyridine (9)
5.1.4.1. 5-Bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine (39)
To a solution of 5-Bromo-1H-pyrazolo[3,4-b]pyridine (2.0 g, 10.1 mmol) in DMF (20 mL)
Potassium hydroxide (KOH, 1.2 g, 21.4 mmol) was added at 25 °C. After stirring for 10 min,
Iodine (2.8 g, 11.1 mmol) was added and stirred at 25 °C for 4 h. The resulting mixture was
diluted with water (30 mL), extracted with EtOAc (20 mL × 3). The organic phase was washed
with sat. Na2S2O3 solution (30 mL × 2), brine (30 mL × 2), dried over Na2SO4, and concentrated to
give compound 39 as a brown solid (2.8 g, 85.6% yield). 1H NMR (400 MHz, DMSO-d6) δ 14.30
(s, 1H, NH), 8.65 (s, 1H, Ar-H), 8.21 (s, 1H, Ar-H).
5.1.4.2. 5-Bromo-3-iodo-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-b]pyridine (40)

To a solution of compound 39 (2.8 g, 8.6 mmol) in DMF (20 mL) K2CO3 (3.6 g, 26.0 mmol)
and 1-(Chloromethyl)-4-methoxybenzene (1.7 g, 10.9 mmol) was added and stirred at 25 °C for 4
h. The mixture was diluted with water (50 mL), extracted with EtOAc (20 mL × 3). The organic
phase was washed with brine (50 mL × 3), dried over Na2SO4. After filtering, the organic layer
was concentrated and purified by column chromatography on silica (PE:EA = 100:1) to give
1
compound 40 as a white solid (3.6 g, 90.3% yield). H NMR (400MHz, CDCl3) δ 8.58 (s, 1H,
Ar-H), 7.92 (s, 1H, Ar-H), 7.35-7.31 (d, J = 8.4 Hz, 2H, Ar-H), 6.84-6.80 (d, J = 8.8 Hz, 2H, Ar-H),
5.60 (s, 2H, CH2), 3.76 (s, 3H, CH3).
5.1.4.3.
5-Bromo-3-((2,6-dichloro-3-fluorophenyl)thio)-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-b]pyridine
(41)
To a solution of compound 40 (3.6 g, 8.1 mmol) in i-PrOH (50 mL) compound 31 (1.9 g, 9.7
mmol), K2CO3 (2.2 g, 16.2 mmol), CuI (1.5 g, 8.1 mmol) and ethylene glycol (1.0 g, 16.2 mmol)
was added. The mixture was stirred at 100 °C under N2 for 5 h. The resulting mixture was filtered
and washed with EtOAc (50 mL), the combined organic phase was washed with ammonia water
(50 mL × 2), brine (50 mL × 2). The organic phase was dried over Na2SO4, concentrated and
purified by column chromatography on silica (PE:EA = 100:1) to give compound 41 as a white
1
solid (2.5 g, 70.0% yield). H NMR (400 MHz, CDCl3) δ 8.52 (s, 1H, Ar-H), 7.92 (s, 1H, Ar-H),
7.40-7.36 (d, J = 4.8 Hz, 1H, Ar-H), 7.28-7.23 (d, J = 8.4 Hz, 2H, Ar-H), 7.16-7.12 (d, J = 7.6 Hz,
1H, Ar-H), 6.81-6.77 (d, J = 8.4 Hz, 2H, Ar-H), 5.57 (s, 2H, CH2), 3.76 (s, 3H, CH3).
5.1.4.4.
tert-Butyl
4-(4-(3-((2,6-dichloro-3-fluorophenyl)thio)-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)-
1H-pyrazol-1-yl)piperidine-1-carboxylate (42)
To a solution of compound 41 (2.5 g, 4.9 mmol) in Glycol dimethyl ether (DME, 16 mL) and
H2O (4 mL) compound 16 (2.6 g, 6.8 mmol), Cs2CO3 (4.8 g, 14.7 mmol) and then Pd(Ph3P)4 (170
mg, 0.15 mmol) was added under N2. After stirring at 80 °C under N2 for 12 h, the mixture was
diluted with water (30 mL), extracted with EtOAc (20 mL × 3). The organic phase was washed
with brine (20 mL × 3), dried over Na2SO4, concentrated and purified by column chromatography

on silica (PE:EA = 25:1) to give compound 42 as an off-white solid (1.6 g, 47.8% yield). 1H NMR
(400MHz, CDCl3) δ 8.65 (s, 1H, Ar-H), 7.82 (s, 1H, Ar-H), 7.71 (s, 1H, Ar-H), 7.64 (s, 1H, Ar-H),
7.39-7.35 (dd, J= 4.8 Hz, 1H, Ar-H), 7.31-7.26 (d, J= 8.8 Hz, 2H, Ar-H), 7.15-7.11 (t, 1H, Ar-H),
6.82-6.76 (m, J= 8.8 Hz, 2H, Ar-H), 5.61 (s, 2H, CH2), 4.39-4.26 (m, 3H, CH, CH2), 3.76 (s, 3H,
CH3), 2.95-2.88 (m, 2H, CH2), 2.19-2.16 (m, 2H, CH2), 2.01-1.92 (m, 2H, CH2), 1.49 (s, 9H,
C(CH3)3).
5.1.4.5.
3-((2,6-Dichloro-3-fluorophenyl)thio)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrazolo[3,4-b]p
yridine (9)
To a solution of compound 42 (1.6 g, 2.3 mmol) in DCM (20 mL) TFA (5 mL) and con.
H2SO4 (1 mL) was added. After stirring at 25 °C for 2 h, to the mixture ammonia water was added
to adjust pH to 7 - 8, extracted with DCM (20 mL × 2). The organic phase was washed with sat.
NaHCO3 solution (20 mL × 2), brine (20 mL × 2), dried over Na2SO4. After filtering, the organic
1
phase was concentrated to give compound 9 as an off-white solid (870.5 mg, 80.3% yield). H
NMR (400 MHz, DMSO-d6) δ 8.89 (s, 1H, Ar-H), 8.38 (s, 1H, Ar-H), 8.15 (s, 1H, Ar-H), 8.02 (s,
1H), 7.75-7.68 (m, 1H, Ar-H), 7.65-7.58 (m, 1H, Ar-H), 4.57-4.47 (m, 1H, CH), 3.38-3.32 (m, 2H,
CH2), 3.10-3.03 (m, 2H, CH2), 2.30-2.17 (m, 4H, CH2). 13C NMR (400 MHz, DMSO-d6) δ
158.53, 156.05, 151.55, 148.55, 136.76, 135.50, 131.97, 130.25, 127.41, 126.25, 123.72, 122.98,
119.47, 119.16, 115.24, 55.97, 42.65, 29.34. ESI-MS m/z: 463.21, 465.21 (M + H)+.
5.1.5.
Synthesis
of
target
compound
3-(1-(2,6-Dichloro-3-fluorophenyl)cyclopropyl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrazo
lo[3,4-b]pyridine (10)
5.1.5.1. 2-(2,6-Dichloro-3-fluorophenyl)ethanol (44)
To a solution of 2,4-Dichloro-1-fluorobenzene (165.0 g, 1.0 mol) in THF (500 mL) n-BuLi
(420 mL, 2.5 M, 1.05 mol) was added at -78 °C. After stirring at -78 °C for 1 h, Oxirane (44.0 g,
1.0 mmol) was added and stirred at -78 °C for 2 h. The mixture was quenched with sat. NH4Cl
solution, extracted with EtOAc (200 mL × 3). The organic phase was washed with water (100 mL
× 2), brine (100 mL × 2), dried over Na2SO4. After filtering, the organic phase was concentrated to

1
give compound 44 as a colorless liquid (156.7 g, 75.0% yield). H NMR (400 MHz, CDCl3) δ
7.29-7.25 (m, 1H, Ar-H), 7.02-6.97 (m, 1H, Ar-H), 3.89-3.84 (m, 2H, CH2), 3.29-3.24 (m, 2H,
CH2).
5.1.5.2. 2-(2,6-Dichloro-3-fluorophenyl)acetic acid (45)
To a solution of compound 44 (156.7 g, 750.0 mmol) and 2,2,6,6-Tetramethylpiperidine
N-oxide (TEMPO, 129.0 g, 825.0 mmol) in phosphate buffer (PH = 6.5, 500 mL) Sodium chlorite
solution was added dropwise at 0 °C while stirring. After stirring at 25 °C for 3 h, to the mixture
HCl was added to adjust pH to 2-3, extracted with EtOAc (200 mL × 3). The organic phase was
washed with water (100 mL × 2), brine (100 mL × 2), dried over Na2SO4. After filtering, the
1
organic phase was concentrated to give compound 45 as a white solid (150.5 g, 90.0% yield). H
NMR (400 MHz, CDCl3) δ 7.39-7.36 (m, 1H, Ar-H), 7.19-7.14 (m, 1H, Ar-H), 4.02 (s, 2H, CH2).
5.1.5.3. 2-(2,6-Dichloro-3-fluorophenyl)acetamide (46)
A solution of compound 45 (150.5 g, 674.9 mmol) in SOCl2 (100 mL) was refluxed for 1 h.
The mixture was distilled to remove excessive SOCl2, the residue was redissolved in DCM and
added dropwise to ammonia water (300 mL) at 0 °C. After stirring at 25 °C for 3 h, the mixture
was filtered and washed with water (100 mL × 2), dried to give compound 46 as a white solid
1
(142.3 g, 95.0% yield). H NMR (400 MHz, DMSO-d6) δ 7.58 (s, 1H, NH), 7.54-7.50 (m, 1H,
Ar-H), 7.42-7.37 (m, 1H, Ar-H), 7.09 (s, 1H, NH), 3.80 (s, 2H, CH2).
5.1.5.4. 2-(2,6-Dichloro-3-fluorophenyl)acetonitrile (47)
To a solution of compound 46 (142.3 g, 641.0 mmol) in Pyridine (1000 mL) SOCl2 (380 g,
3.2 mmol) was added dropwise at 0 °C. After stirring at 0 °C for 2 h, the mixture was quenched
with cooled water, extracted with petroleum ether (200 mL × 3). The organic phase was washed
with sat. Copper(II) sulfate (CuSO4) solution (100 mL × 2), brine (100 mL × 2), dried over
Na2SO4. After filtering, the organic phase was concentrated to give compound 47 as an off-white
solid (85.9 g, 65.7% yield). 1H NMR (400 MHz, CDCl3) δ 7.34-7.30 (m, 1H, Ar-H), 7.11-7.06 (m,
1H, Ar-H), 4.08 (s, 2H, CH2).

5.1.5.5. 1-(2,6-Dichloro-3-fluorophenyl)cyclopropanecarbonitrile (48)
To a solution of compound 47 (85.9 g, 421.1 mmol) in DMF (500 mL) NaH (60% purity,
42.1 g, 1.05 mol) was added at 0 °C. After stirring at 25 °C for 1 h, 1,2-Dibromoethane (95 g, 505
mmol) was added and stirred at 25 °C for 3 h. The mixture was quenched with sat. NH4Cl solution,
extracted with EtOAc (200 mL × 3). The organic phase was washed with water (100 mL × 2),
brine (100 mL × 2), dried over Na2SO4. After filtering, the organic phase was concentrated and
purified by column chromatography on silica (PE:EA = 100:1) to give compound 48 as an
1
off-white solid (83.1 g, 85.8% yield). H NMR (400 MHz, CDCl3) δ 7.35-7.32 (m, 1H, Ar-H),
7.17-7.13 (m, 1H, Ar-H), 1.98-1.94 (m, 2H, CH2), 1.50-1.46 (m, 2H, CH2).
5.1.5.6. 1-(2,6-Dichloro-3-fluorophenyl)cyclopropanecarbaldehyde (49)
To a solution of compound 48 (10.0 g, 43.47 mmol) in anhydrous Toluene (100 mL)
Di-isobutylaluminum hydride (DIBAL-H, 1M, 65.2 mL, 65.2 mmol) was added dropwise and
stirred at -78 °C under N2 for 1 h. The mixture was quenched with 1N of HCl at 0 °C and stirred
for 30 min, extracted with DCM. The organic phase was washed with brine, dried over Na2SO4,
concentrated to give compound 49 as an off-white solid (7.5 g, 74.0% yield). 1H NMR (400 MHz,
CDCl3) δ 8.88 (s, 1H, CHO), 7.33-7.30 (m, 1H, Ar-H), 7.13-7.09 (m, 1H, Ar-H), 1.92-1.89 (m, 2H,
CH2), 1.61-1.57 (m, 2H, CH2).
5.1.5.7. (5-Bromo-2-fluoropyridin-3-yl)(1-(2,6-dichloro-3-fluorophenyl)cyclopropyl)methanol (50)
To a solution of 5-Bromo-2-fluoropyridine (6.2 g, 35.4 mmol) in anhydrous THF (50 mL)
Lithium bis(trimethylsilyl)amide (LiHMDS, 1M, 48.3 mL, 48.3 mmol) was added dropwise and
stirred at -78 °C under N2 for 30 min. To the mixture a solution of 49 (7.5 g, 32.2 mmol) in
anhydrous THF (20 mL) was added and stirred at -78 °C for 1 h. The mixture was quenched with
sat. NH4Cl solution at 0 °C, extracted with EtOAc (100 mL × 2). The organic phase was washed
with brine, dried over Na2SO4, concentrated and purified by column chromatography on silica
1
(PE:EA = 50:1) to give compound 50 as an off-white solid (11.0 g, 83.6% yield). H NMR (400
MHz, CDCl3) δ 8.17-8.15 (m, 1H, Ar-H), 7.67-7.65 (m, 1H, Ar-H), 7.30-7.18 (m, 1H, Ar-H),
7.08-7.02 (m, 1H, Ar-H), 4.95-4.92 (m, 1H, OH), 2.73-2.66 (m, 1H, CH), 1.53-1.01 (m, 4H,
C(CH2)2).

5.1.5.8.
(5-Bromo-2-fluoropyridin-3-yl)(1-(2,6-dichloro-3-fluorophenyl)cyclopropyl)methanone
(51)
To a solution of compound 50 (11.0 g, 26.9 mmol) in DCM (50 mL) Pyridinium
chlorochromate (PCC, 11.6 g, 53.8 mmol) was added and stirred at 25 °C for 4 h. The mixture was
concentrated and purified by column chromatography on silica (PE:EA = 50:1) to give compound
1
51 as an off-white solid (10.6 g, 96.8% yield). H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H, Ar-H),
7.76-7.73 (m, 1H, Ar-H), 7.31-7.25 (m, 1H, Ar-H), 7.11-7.04 (m, 1H, Ar-H), 2.18-7.15 (m, 2H,
CH2), 1.69-1.65 (m, 2H, CH2).
5.1.5.9. 5-Bromo-3-(1-(2,6-dichloro-3-fluorophenyl)cyclopropyl)-1H-pyrazolo[3,4-b]pyridine (52)
To a solution of compound 51 (10.6 g, 26.0 mmol) in Ethanol (EtOH, 100 mL) 50%
Hydrazine hydrate (15.6 g, 156 mmol) was added and refluxed for 10 h. The mixture was distilled
to remove excess EtOH and diluted with water. The formed solid was filtered and washed with
water. The resulting solid was purified by column chromatography on silica (PE:EA = 15:1) to
give compound 52 as an off-white solid (5.8 g, 55.3% yield). 1H NMR (400 MHz, CDCl3) δ 10.37
(s, 1H, NH), 8.49 (s, 1H, Ar-H), 7.53 (s, 1H, Ar-H), 7.42-7.37 (m, 1H, Ar-H), 7.20-7.16 (m, 1H,
Ar-H), 2.10-2.04 (m, 2H, CH2), 1.59 (m, 2H, CH2).
5.1.5.10.
tert-butyl
4-(4-(3-(1-(2,6-Dichloro-3-fluorophenyl)cyclopropyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)-1H-pyrazo
l-1-yl)piperidine-1-carboxylate (53)
To a solution of compound 52 (5.8 g, 14.5 mmol), Cs2CO3 (14.2 g, 43.5 mmol), compound
16 (8.24 g, 21.8 mmol) in dioxane (75 mL) and H2O (25 mL) Pd(Ph3P)4 (508 mg, 0.44 mmol) was
added. After stirring at 80 °C under N2 for 12 h, the mixture was diluted with water (50 mL),
extracted with EtOAc (50 mL × 3). The organic phase was washed with brine (50 mL × 2), dried
over Na2SO4. After filtering, the organic phase was concentrated and purified by column
chromatography on silica (DCM:MeOH = 25:1) to give compound 53 as a pale yellow solid (5.4 g,
1
65.7% yield). H NMR (400 MHz, CDCl3) δ 10.63 (s, 1H, NH), 8.61 (s, 1H, Ar-H), 7.63 (s, 1H,
Ar-H), 7.60 (s, 1H, Ar-H), 7.42-7.38 (m, 2H, Ar-H), 7.20-7.16 (m, 1H, Ar-H), 4.31-4.25 (m, 4H,

CH2), 2.93-2.89 (m, 3H, CH, CH2), 2.17-1.99 (m, 2H, CH2), 1.98-1.93 (m, 2H, CH2), 1.61-1.57
(m, 2H, CH2), 1.49 (s, 9H, C(CH3)3).
5.1.5.11.
3-(1-(2,6-Dichloro-3-fluorophenyl)cyclopropyl)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-1H-pyrazo
lo[3,4-b]pyridine (10)
To a solution of compound 53 (2.7 g, 4.8 mmol) in DCM (20 mL) TFA (5 mL) was added.
After stirring at 25 °C for 2 h, the mixture was added ammonia water to adjust pH to 7 - 8,
extracted with DCM (20 mL × 2). The organic phase was washed with sat. NaHCO3 solution (30
mL × 2), brine (30 mL × 2), dried over Na2SO4. After filtering, the organic phase was concentrated
1
to give compound 10 as an off-white solid (1.3 g, 27.5% yield). H NMR (400 MHz, MeOD) δ
8.66 (s, 1H, Ar-H), 7.99 (m, 1H, Ar-H), 7.64 (s, 1H, Ar-H), 7.56-7.53 (m, 1H, Ar-H), 7.40-7.34 (m,
2H, Ar-H), 4.37-4.29 (m, 1H, CH), 3.24-3.20 (m, 2H, CH2), 2.82-2.75 (m, 2H, CH2), 2.15-2.10
(m, 4H, CH2), 2.02-1.92 (m, 2H, CH2), 1.62-1.57 (m, 2H, CH2). 13C NMR (400 MHz, DMSO-d6)
δ 158.44, 155.99, 151.90, 147.43, 145.09, 140.24, 136.45, 133.17, 129.99, 125.34, 124.59, 121.70,
119.49, 117.33, 111.55, 56.00, 42.65, 29.40, 24.85, 20.75. ESI-MS m/z: 472.36 (M + H)+.
5.2. Kinase Assays
5.2.1. C-Met Z-LYTE Assay
The c-Met kinase activity of five target compounds and three positive compounds were
evaluated using standard Z-LYTE Assays (fluorescence resonance energy transfer, FRET).
The test compounds or reference controls were dissolved in dimethyl sulfoxide (DMSO) as
stock solution. The DMSO solution was serial diluted (duplicated with 3-fold dilution) to make
that the concentrations were from 10 μM to 0.17 nM in 11 doses. The enzyme reaction mixture of
c-Met Z-LYTE assay contained 2.5 nM c-Met, 1 μM Peptide substrate (Tyr 6) and 17 μM ATP in a
buffer containing 50 mM HEPES (pH 7.0), 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA at a final
volume of 10 μL. The enzyme reaction were carried out at room temperature in black Proxiplate
384-Plus plate (PerkinElmer) for 90 min. 5 μL of the detection reagents A (Invitrogen) was added
at final concentrations of 64-fold dilution. The plates were incubated at room temperature for 60
min and then read in the Envision plate reader (PerkinElmer).

Graphs corresponding to the % of inhibition of c-Met activity by the different compound
concentrations were presented in Graph 1. Using the curve and the GraphPad Prism software, the
50% inhibition concentration (IC50) of each compound against c-Met kinase activity was
calculated.
Graph 1. The curves corresponding to the percentage of c-Met kinase inhibition according to the
concentrations of the different compounds.
5.2.2. ALK HTRFAssays
The ALK kinase activity of five target compounds and three positive compounds were
evaluated using standard homogeneous time-resolved fluorescence (HTRF) assay (High
Fluorescence Resonance Energy Transfer).
The test compounds or reference controls were dissolved in DMSO as stock solution. The
DMSO solution was serial diluted (duplicated with 3-fold dilution) to make that the concentrations
were from 10 μM to 0.17 nM in 11 doses. The enzyme reaction mixture of ALK WT/ ALK
C1156Y/ ALK L1196M for HTRF assay contained 0.5 nM ALK WT/ 0.15 nM ALK C1156Y/
0.15 nM ALK L1196M, 1 μM biotin-TK peptide, and 30 μM ATP in a kinase reaction buffer
containing 50 mM HEPES (pH 7.5), 5 mM MgCl2, 0.01% BSA, 0.1 mM Orthovanadate at a final
volume of 10 μL. The enzyme reaction were carried out at room temperature in white Proxiplate
384-Plus plate (PerkinElmer) for 90 min. 10 μL of the detection reagents contained 6.7 nM
TK-Antibody and 16.67 nM XL665 in a detection buffer containing 50 mM HEPES (pH 7.5), 0.8
M KF, 20 mM EDTA, 0.01% BSA were added. The plates were incubated at room temperature for
60 min and then read using the Envision plate reader.

Graphs corresponding to the % of inhibition of ALK WT, ALK C1156Y and ALK L1196M
activity by the different compound concentrations were presented in Graphs 2-4. Using the curve
and the GraphPad Prism software, the IC50 of each compound against ALK kinase activity was
calculated.
Graph 2. The curves corresponding to the percentage of ALK WT kinase inhibition according to
the concentrations of the different compounds.
Graph 3. The curves corresponding to the percentage of ALK C1156Y kinase inhibition
according to the concentrations of the different compounds.