Indazoles as potential c-met inhibitors: Design, synthesis and molecular docking studies

Article abstract of DOI:10.1016/j.ejmech.2013.04.004

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is considered as an attractive target for small molecule drug discovery. In this study, a series of indazoles were designed, synthesized and evaluated as novel c-Met inhibitors. The results showed that the majority of the compounds exhibited significant inhibition on c-Met and compound 4d showed highest activity against c-Met with IC₅₀ value of 0.17 μM in TR-FRET-based assay and IC₅₀ value of 5.45 μM in cell-based assay as compared to other tested compounds. Molecular docking experiments verified the results and explained the molecular mechanism of pretty activities to c-Met.

Full text of DOI:10.1016/j.ejmech.2013.04.004

European Journal of Medicinal Chemistry 65 (2013) 112e118
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Indazoles as potential c-met inhibitors: Design, synthesis
and molecular docking studies
,
,
Lianbao Ye a 1, Xiaomin Ou a, Yuanxin Tian b 1, Bangwei Yu a, Yan Luo a, Binghong Feng a,
Hansen Lin a, Jiajie Zhang b , Shuguang Wu
,
b,
*
*
a Medicinal Chemistry Department, Guangdong Pharmaceutical University, Guangzhou 510006, Guangdong, China
b School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, Guangdong, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is
considered as an attractive target for small molecule drug discovery. In this study, a series of indazoles
were designed, synthesized and evaluated as novel c-Met inhibitors. The results showed that the ma-
jority of the compounds exhibited significant inhibition on c-Met and compound 4d showed highest
Received 31 December 2012
Received in revised form
31 March 2013
Accepted 2 April 2013
Available online 29 April 2013
activity against c-Met with IC50 value of 0.17 mM in TR-FRET-based assay and IC50 value of 5.45 mM in cell-
based assay as compared to other tested compounds. Molecular docking experiments verified the results
and explained the molecular mechanism of pretty activities to c-Met.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
c-Met inhibitor
Indazole
Bioisosterism principles
Molecular docking
Suzuki coupling reaction
Isomer
1. Introduction
implications for specificity and activity, but the body of data
available remains incomplete [8,9].
c-Met has been shown to be deregulated and associated with
high tumor grade and poor prognosis in a number of human can-
cers. c-Met can become activated by a variety of mechanisms,
including gene amplification and mutation inducing motility,
invasiveness and tumor genicity into the transformed cells. Acti-
vation leads to various signaling cascades that induce survival, as
well as mitogenic and motogenic responses [1]. Targeting the ATP
binding site of c-Met is a popular strategy for inhibition of the ki-
nase, many small molecules selectively targeting the ATP binding
site of c-Met kinase have been identified and exerted significant
therapeutic effects in treating human cancers clinically [2].
Impressive numbers of c-Met kinase inhibitors have been devel-
oped over the past 10 years and a number of novel therapeutic
agents that target the c-Met/HGF pathway have been tested in
early-phase clinical studies with promising results [3e7]. Currently,
the different binding modes of the available c-Met inhibitors have
Several pharmaceutical companies including Pfizer, SGX, Sugen,
Bayer and formerly Pharmacia, have or have had also ongoing
programs in this area. SGX’s SGX-523 (2) is a potent c-Met inhibitor
which was withdrawn from Phase I clinical trial due to a reversible
acute renal failure in one dose group caused by a metabolite
compound crystallizing in the kidney [10], and Pfizer’s PF-
04217903 (1) and Janssen’s JNJ-33377605 (3) are currently in
Phase I clinical trials [11,12]. In our previous study, we designed and
synthesized spiro[indoline-3,40-piperidine]-2-ones and 2,3,4,5-
tetrahydro-1H-pyrido[4,3-b]indoles as potent c-Met inhibitors us-
ing compounds 1 and 3 as lead compounds [13,14], but the syn-
thetic construction of these compounds requires a number of
synthetically challenging manipulations and the raw material is
relatively expensive. In an ongoing effort to discover novel c-Met
inhibitors and build diversification of chemical library, we
continued to develop ten structurally relevant novel compounds
4ae4e and 5ae5e by replacing 3H-[1,2,3]triazolo[4,5-b]pyrazine of
1, 3H-[1,2,4]triazolo [4,3-b]pyridazine of 2 and 3 with cheap and
readily available indazole moiety (Figs. 1 and 2), and investigated
their inhibitions on c-Met, verified the results and explained the
molecular mechanism of pretty activities to c-Met through mo-
lecular docking experiments. The synthetic route for indazoles
* Corresponding authors. Tel.: þ86 20 62789415; fax: þ86 20 61648548.
E-mail addresses: yelb7909@163.com (L. Ye), tyx523@163.com (Y. Tian),
zhangjj@smu.edu.cn (J. Zhang), shuguang@smu.edu.cn (S. Wu).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.004

L. Ye et al. / European Journal of Medicinal Chemistry 65 (2013) 112e118
113
HO
N
N
R
N
N
N
N
N
N
N
N
N
N
N
1
4a-4e
F
N
+
R
F
N
N
N
N
N
N
N
S
N
N
N
2
N
N
N
N
N
5a-5e
N
N
N
3
Fig. 1. Design of compounds.
was convenient for raw materials availability and industrial
manufacture.
gave information about carbon atoms and all the Mþ ion peaks
corresponding to molecular weight of confirmed novel compounds.
2.2. Evaluation of biological activity
2. Results and discussions
The results of anti-c-Met enzyme activities in vitro showed that
all the title compounds were active against c-Met enzyme to some
extent. Among the compounds tested, most of them showed higher
activity. Noticeably, the IC50 values of 4ae4e were, lower than those
of their isomers 5ae5e against the c-Met enzyme in vitro, in which
2.1. Chemistry
The target compounds 4ae4e and 5ae5e were prepared as
shown in Scheme 1. The 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)pyrazole groups (7e9) reacted with 6-bromo-1H-indazole (6)
to obtain 6-(1-methyl-pyrazol-3-yl)-1H-indazole (10), 6-[1-(2-
tetrahydro-2H-pyran-2-yloxyethyl)-1H-pyrazol-4-yl]-1H-indazole
(11), and 6-{1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclohexyl]-
1H-pyrazol-4-yl}-1H-indazole (12) with yields of 68%, 55%, and 65%
respectively via Suzuki coupling reaction. This coupling reaction
with pinacol borane esters gave good yields under basic conditions
using DMF/H2O as a solvent and Pd (PPh3)4 as a catalyst and pro-
vided a range of applicability [15].
4d had the best inhibitory effect with IC50 of 0.17
mM. The IC50
values derived from in vitro screening studies revealed that all the
compounds posed moderate cytotoxicity against MKN-45 cell lines
(Table 1). In accordance with the results of c-Met enzyme, Com-
pounds 4ae4e, 5b and 5e showed higher inhibition against MKN-
45 cell lines with IC50 values of 12.86, 14, 6.72, 11, 5.45,12 and
13.42
mM respectively. Compound 4e was found to show highest
activity against MKN-45 cell line with an IC50 value of 5.45
mM.
These results indicated that indazoles could afford good potency
against activity, so we could elementary make sure that designed
compounds should be well worth studying based on molecular
docking theory and preliminary biological tests. Further research
works on activities are currently under investigation and will be
reported in due course.
The indazole compounds 10, 11, and 12 reacted with commer-
cially available 6-chloromethylquinoline to give three pairs of iso-
mers 4a and 5a, 4b and 5b, 4d and 5d as a result of two isomeric
forms of indazole. Then 4b and 5b, 4d and 5d transformed to 4c and
5c, 4e and 5e by hydrolysis. Due to the distinct difference in polarity
between isomers allowed them to be separated easily by silica gel
column chromatography. Using this producer, two series of com-
pounds (4ae4e and 5ae5e) were synthesized in overall yield of
17.2e26%.
2.3. Docking studies
A systematic study of the indazole scaffold revealed that the 6-
position would be suitable for further potency optimization studies.
It was found that a range of substituents could be accommodated at
this position.
Docking experiments indicated that our synthesized com-
pounds adopted a U-shape binding mode with the active site of c-
Met (shown in Fig. 3), which was the typical conformation of type-I
c-Met inhibitors [2]. A direct hydrogen bond was formed between
the backbone NH of Met1160 and the quinoline-N, other notable
The derivatives were characterized by 1H NMR, 13C NMR,
elemental analyses and ESI-MS. The analytical data for target com-
pounds can be seen from Experimental and showed that the char-
acteristic signals of 1H NMR for indazole were 7.20e8.10 ppm, 7.60e
8.90 ppm for quinoline, and w5.70 ppm for methane between
indazole and quinoline, and substituents data of 6-position in
indazole was consistent with the reference. 13C NMR and ESI-MS
interaction was
pep stacking interaction between the indazole
group core with Tyr1230 and Met1211. Comparing 5ae5e, the
methylene of 4ae4e could better match the sub hydrophobic
pocket composed of Val1109, Lys1110, Ala1226 and Val1092 and
enhanced the
pep interaction. Therefore compounds 4ae4e
showed more potent activities than 5ae5e. The binding modes of

114
L. Ye et al. / European Journal of Medicinal Chemistry 65 (2013) 112e118
H3C
H3C
N
N
N
N
N
N
N
N
N
N
5a
4a
O
O
N
O
N
O
N
N
N
N
N
N
N
N
N
5b
4b
N
N
N
N
HO
Si
N
N
N
N
HO
N
5c
4c
Si
O
O
N
N
N
N
N
N
N
N
N
N
4d
5d
N
HO
HO
N
N
N
N
N
N
N
N
N
4e
5e
Fig. 2. Novel indazoles.
4b and 5b were shown in Fig. 4. Both of them formed two hydrogen
bonds with Asn1167 and Arg1166 except the hydrogen bonds
composed of Met1160 and quinoline group. The interaction
extruded 5a to make methylene match the sub hydrophobic pocket,
4. Experimental part
All chemicals were obtained from Aladdin or J&K. Solvents were
purified and dried by standard procedures, and stored over 3-A
ꢀ
and then promoted the
p
e
p
interaction from indazole group and
molecular sieves. Reactions were followed by TLC using SILG/UV
254 silica-gel plates. Flash chromatography (FC): silica gel (SiO2;
Tyr1230, so the 5b also had potent activity. The same phenomena
appeared in compounds 4e and 5e. The activities of 4a and 5a were
the lowest in their own series. The docking studies suggested that
their short adjacent hydrophilic ring pyrazole could not match the
active sites exactly to stabilize the conformation. So we deduced
that the 1-position substituent of indazole was better for the U-
40
Digital NMR Spectrometer, rep.
m
m, 230e400 mesh). 1H NMR and 13C NMR spectra: Bruker
d
in parts per million (ppm), J in
hertz (Hz). EI-MS: Waters ZQ4000.
4.1. Synthesis of compounds
shape conformation and would enhance activity through the pep
interaction. Moreover the hydrophilic group interacted with res-
idue of adjacent solvent area outside pocket could increase the
activity, such as 4d and 4b.
4.1.1. Synthesis of 10
In a three-necked round bottom flask (50 ml) equipped with a
condenser and magnetic stirring bar, Pd(PPh3)4 (11.6 mg,
0.01 mmol) was added portion wise to a solution of the 6-bromo-
1H-indazole (6; 1.97 g, 10 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)pyrazole (7; 2.50 g, 12 mmol), K2CO3
(4.145 mg, 30 mmol) in DMF/H2O (4/1, 15 ml), and nitrogen was
bubbled through the mixture for 5 min. Then, the mixture was
stirred for 18 h at 80 ꢀC (LCeMS control), then cooled to r.t., H2O
(10 ml) was added, and the mixture was extracted with CH2Cl2
(3 ꢁ 30 ml). The org. layer was dried (1 g of Na2SO4), concentrated
3. Conclusions
In conclusion, our strategy to replace 3H-[1,2,3]triazolo[4,5-b]
pyrazine of 1, 3H-[1,2,4]triazolo[4,3-b]pyridazine of 2 and 3 with
indazole moiety was successful in leading to compounds, which
demonstrated inhibition of c-Met kinase activity. These compounds
would be useful as lead compounds of developing c-Met inhibitors.

L. Ye et al. / European Journal of Medicinal Chemistry 65 (2013) 112e118
115
R
H
N
N
H
N
R
Br
O
a
N
N
b
N
N
B
+
N
O
10 R=CH3
6
7
8
R=CH3
O
11
O
R=
R=
O
R=
O
12
9
Si
O
Si
O
R=
R
N
N
N
R
N
N
N
N
+
N
N
N
4a R=CH3
5a R=CH3
4b
c
c
O
5b
O
c
O
4c
4e
R=
5c
5e
R=
O
5d
4d
c
Si
O
R=
Si
O
R=
Scheme 1. Synthesis of 4ae4e and 5ae5e. Reagents and conditions: (a) Pd(PPh3)4, DMF/H2O, N2,K2CO3, 80 ꢀC, 18 h; (b) 6-chloromethylquinoline, KOH, THF, r.t., 2 h; (c) 4 M HCl, 1,4-
dioxane, THF, r.t., 1 h.
to obtain crude product, which was purified by FC with MeOH/
4.1.6. Synthesis of 4c
CH2Cl2.
In a three-necked round bottom flask (50 ml) equipped with a
condenser and magnetic stirring bar, a solution of 4 M HCl in 1,4-
dioxane was added portion wise to a solution of 4b (45.4 mg,
0.1 mmol) in THF (3 ml), the mixture was stirred for 1 h at r.t., and
the solvent was removed under reduced pressure, 10% NaOH (5 ml)
was added, the mixture was extracted with CH2Cl2 (3 ꢁ 5 ml). The
organic layer was dried (1 g of Na2SO4) and concentrated to afford
the crude product, which was purified by FC with MeOH/CH2Cl2.
4.1.2. Synthesis of 11 and 12
Compounds 11 and 12 were prepared in analogy to 10.
4.1.3. Synthesis of 4a and 5a
In a double-necked round bottom flask (100 ml) equipped with
magnetic stirring bar, a mixture of 10 (39.6 mg, 0.2 mmol), 6-
chloromethylquinoline (53.3 mg, 0.3 mmol), and THF (5 ml) was
stirred for 2 h in nitrogen at r.t. (TLC control). The solvent was
removed under reduced pressure, and the crude product was puri-
fied by FC with MeOH/CH2Cl2 to obtain the target products 4a and 5a.
4.1.7. Compounds 5c, 4e and 5e were prepared in analogy to 4c.
4.1.7.1. 6-(1-Methyl-pyrazol-3-yl)-1H-indazole (10). Yield: 68%:
mp: 213.1e214.7 ꢀC; 1H NMR (400 MHz, CDCl3):
d
¼ 3.97 (s, 3H),
4.1.4. Synthesis of 4b and 5b
Compounds 4b and 5b were prepared in analogy to 4a and 5a.
4.1.5. Synthesis of 4d and 5d
Compounds 4d and 5d were prepared in analogy to 4a
and 5a.
Table 1
Inhibition of 4ae4e and 5ae5e on c-Met and MKN-45.
Compound
c-Met IC50
(
mM)
MKN-45 IC50 (mM)
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
0.42
0.29
0.38
0.17
0.64
37.00
0.45
0.83
19.00
0.58
12.86
14.00
6.72
11.00
5.45
105.60
12.00
110.00
1300.00
13.42
Fig. 3. Compact U-shaped binding modes of all compounds (PDB code: 3dkf).

116
L. Ye et al. / European Journal of Medicinal Chemistry 65 (2013) 112e118
4.1.7.5. 1-(Quinolin-6-ylmethyl)-6-[1-(2-tetrahydro-2H-pyran-2-
yloxyethyl)-1H-pyrazol-4-yl]-1H-indazole (4b). Yield: 38%: mp:
284.3e285.8 ꢀC; 1H NMR (400 MHz, CDCl3):
d
¼ 1.45e1.79 (m, 6H),
3.45 (m, 1H), 3.67 (m, 1H), 3.81 (m, 1H), 4.11 (m, 1H), 4.36e4.39 (m,
2H), 4.56 (t, J ¼ 3.6 Hz, 1H), 5.78 (s, 2H), 7.27 (dd, J ¼ 1.6, 8.8 Hz, 1H),
7.45 (dd, J ¼ 4.0, 8.0 Hz, 1H), 7.64 (dd, J ¼ 1.2, 8.8 Hz, 1H), 7.66 (dd,
J ¼ 2.0, 8.8 Hz,1H), 7.71 (s,1H), 7.80 (dd, J ¼ 0.8, 2.0 Hz,1H), 7.83 (dd,
J ¼ 0.8, 11.6 Hz, 1H), 7.95 (d, J ¼ 0.8 Hz, 1H), 8.16e8.18 (m, 2H),
8.92 ppm (dd, J ¼ 2.0, 4.4 Hz, 1H); 13C NMR (75 MHz, (D6) DMSO):
d
¼ 21.1, 26.2, 30.7, 56.4, 61.9, 63.4, 64.1, 106.5, 108.7 (C-7), 119.1,
121.2,121.7,123.9 (C-3a),126.1,127.2,128.3,128.5,129.1,130.9,131.8,
132.9 (C-3),134.1,135.4,136.1,141.1 (C-7a),145.2,149.3 ppm; EI-MS:
454.04 [M þ Hþ]. Anal. Calcd for C27H27N5O2 (453.22): C 71.50, H
6.00, N 15.44. Found: C 71.52, H 5.97, N 15.44.
4.1.7.6. 1-(Quinolin-6-ylmethyl)-6-[1-(2-hydroxyethyl)-1H-pyrazol-
4-yl]-1H-indazole (4c). Yield: 36.0 mg (97%): mp: 301.3e303.1 ꢀC.
1H NMR (400 MHz, CDCl3):
d
¼ 4.03 (t, J ¼ 4.8 Hz, 2H), 4.28 (t,
J ¼ 4.4 Hz, 2H), 5.82 (s, 2H), 7.32 (dd, J ¼ 1.2, 8.0 Hz, 1H), 7.40 (dd,
J ¼ 1.2, 2.0 Hz, 1H), 7.52 (m, 1H), 7.61 (s, 1H), 7.70e7.72 (m, 2H), 7.76
(dd, J ¼ 1.2, 8.8 Hz, 1H), 7.81 (d, J ¼ 0.8 Hz, 1H), 8.08 (d, J ¼ 0.8 Hz,
Fig. 4. Conformations of 4b (gray) and 5b (magenta) (For interpretation of the refer-
ences to color in this figure legend, the reader is referred to the web version of this
article.).
1H), 8.26 (m,1H), 8.33 (m,1H), 8.91 ppm (dd, J ¼ 1.6, 4.4 Hz,1H); 13
C
NMR (75 MHz, (D6) DMSO):
d
¼ 58.3, 59.4, 63.1, 108.6 (C-7), 118.7,
7.31 (dd, J ¼ 1.2, 8.4, 1H), 7.56 (t, J ¼ 1.2, 1H), 7.67 (s, 1H), 7.73 (d,
121.2, 121.5, 123.9 (C-3a), 126.5, 127.2, 128.6, 128.8, 129.1, 130.9,
132.4, 133.1 (C-3), 134.1, 135.4, 136.1, 140.5 (C-7a), 145.3, 149.2 ppm;
EI-MS: 370.08 [M þ Hþ]. Anal. Calcd for C22H19N5O (369.16): C
71.53, H 5.18, N 18.96. Found: C 71.53, H 5.21, N 18.94.
J ¼ 8.0, 1H), 7.83 (d, J ¼ 0.4, 1H), 8.03 (s, 1H), 10.27ppm (br s, 1H); 13
C
NMR (75 MHz, (D6) DMSO):
d
¼ 44.3, 109.1, 119.3, 121.4, 124.7, 126.3,
127.5, 128.5, 132.1, 134.3, 142.8 ppm; EI-MS: 199.00 [M þ Hþ]. Anal.
Calcd for C11H10N4 (198.09): C 66.65, H 5.08, N 28.27. Found: C
66.71, H 5.03, N 28.26.
4.1.7.7. 1-(Quinolin-6-ylmethyl)-6-{1-[(1R,4R)-4-(tert-butyldimethylsi-
lyloxy)cyclohexyl]-1H-pyrazol-4-yl}-1H-indazole (4d). Yield: 40%: mp:
4.1.7.2. 6-[1-(2-Tetrahydro-2H-pyran-2-yloxyethyl)-1H-pyrazol-4-
313.2e314.3 ꢀC. 1H NMR (400 MHz, CDCl3):
d
¼ 0.07 (s, 6H), 0.91 (s,
yl]-1H-indazole (11). Yield: 55%: mp: 244.3e246.1 ꢀC; 1H NMR
9H), 1.47e1.56 (m, 2H), 1.81e1.92 (m, 2H), 1.98e2.04 (m, 2H), 2.16e
2.21 (m, 2H), 3.70 (m, 1H), 4.12 (m, 1H), 5.30 (s, 2H), 7.30 (dd, J ¼ 1.2,
8.4 Hz,1H), 7.39 (dd, J ¼ 4.4, 8.0 Hz,1H), 7.41 (d, J ¼ 1.2 Hz, 1H), 7.56 (d,
J ¼ 1.2 Hz, 1H), 7.61 (dd, J ¼ 2.0, 8.8 Hz, 1H), 7.66 (d, J ¼ 0.8 Hz, 1H),
7.74 (dd, J ¼ 0.8, 8.0 Hz, 1H), 7.77 (d, J ¼ 1.2 Hz,1H), 8.05 (d, J ¼ 1.2 Hz,
1H), 8.08e8.12 (m, 2H), 8.88 ppm (dd, J ¼ 1.6, 4.4 Hz, 1H); 13C NMR
(400 MHz, CDCl3):
d
¼ 1.47e1.59 (m, 6H), 3.47e1.59 (m, 1H), 3.68
(m, 1H), 3.83 (m, 1H), 4.11 (m, 1H), 4.36e4.39 (m, 2H), 4.57 (t,
J ¼ 4.0, 1H), 7.31 (dd, J ¼ 1.2, 8.4, 1H), 7.56 (s, 1H), 7.73 (d, J ¼ 8.4,
1H), 7.81 (d, J ¼ 0.8, 1H), 7.85 (d, J ¼ 0.8, 1H), 8.03 (s, 1H), 10.27 ppm
(br s, 1H); 13C NMR (75 MHz, (D6) DMSO):
d
¼ 19.1, 25.3, 30.2, 57.1,
62.8, 64.1, 106.6, 108.3, 119.0, 121.2, 123.7, 125.1, 128.3, 128.7, 131.4,
(75 MHz, (D6) DMSO):
d
¼ ꢂ5.2, 18.7, 25.8, 26.1, 33.1, 62.3, 65.1, 73.9,
133.6, 142.7 ppm; EI-MS: 313.06 [M þ Hþ]. Anal. Calcd for
108.5 (C-7), 118.6, 121.3, 121.7, 124.1 (C-3a), 126.7, 127.5, 129.1, 129.5,
129.7, 131.2, 131.9, 132.8 (C-3), 133.9, 135.4, 136.2, 141.0 (C-7a), 144.9,
148.8 ppm; EI-MS: 538.16 [M þ Hþ]. Anal. Calcd for C32H39N5OSi
(537.29): C 71.47, H 7.31, N 13.02. Found: C 71.49, H 7.30, N 13.03.
C
17H20N4O2 (312.16): C 65.37, H 6.45, N 17.94. Found: C 65.35, H
6.47, N 17.94.
4.1.7.3. 6-{1-[(1R,4R)-4-(tert-Butyldimethylsilyloxy)cyclohexyl]-1H-
pyrazol-4-yl}-1H-indazole (12). Yield: 65%: mp: 237.1e238.5 ꢀC; 1H
4.1.7.8. 1-(Quinolin-6-ylmethyl)-6-[1-((1R,4R)-4-hydroxy-cyclo-
NMR (400 MHz, CDCl3):
d
¼ 0.08 (s, 6H), 0.91 (s, 9H), 1.48e1.58 (m,
hexyl)-1H-pyrazol-4-yl]-1H-indazole (4e). Yield: 99%: mp: 325.4e
2H), 1.84e1.94 (m, 2H), 2.00e2.04 (m, 2H), 2.21 (m, 2H), 3.72 (m,
1H), 4.14 (m, 1H), 7.29 (d, J ¼ 8.4, 1H), 7.55 (s, 1H), 7.70 (s, 1H), 7.71
(d, J ¼ 8.4, 1H), 7.82 (s, 1H), 8.04 ppm (s, 1H); 13C NMR (75 MHz, (D6)
327.1 ꢀC; 1H NMR (400 MHz, CDCl3):
d
¼ 1.57e1.66 (m, 2H), 1.88e
1.94 (m, 2H), 2.14e2.17 (m, 2H), 2.23e2.26 (m, 2H), 3.77 (m, 1H),
4.23 (m, 1H), 5.89 (s, 2H), 7.34 (dd, J ¼ 1.6, 8.4 Hz, 1H), 7.42 (s, 1H),
7.70e7.74 (m, 2H), 7.79 (d, J ¼ 8.4 Hz,1H), 7.85e7.88 (m, 2H), 7.99 (d,
J ¼ 8.8 Hz,1H), 8.11 (s,1H), 8.71 (d, J ¼ 8.0 Hz,1H), 8.93 (d, J ¼ 8.4 Hz,
1H), 8.98 ppm (d, J ¼ 5.6 Hz, 1H); 13C NMR (75 MHz, (D6) DMSO):
DMSO):
d
¼ ꢂ4.6, 19.1, 25.3, 25.4, 32.8, 43.7, 65.1, 107.9, 117.8, 119.6,
123.5, 126.1, 128.2, 128.4, 132.81, 133.2, 143.3 ppm; EI-MS: 397.09
[M þ Hþ]. Anal. Calcd for C22H32N4OSi (396.23): C 66.62, H 8.13, N
14.13. Found: C 66.57, H 8.12, N 14.18.
d
¼ 25.3, 31.7, 62.3, 64.7, 74.1, 108.7 (C-7),119.1, 121.3, 121.6,124.1 (C-
3a), 126.9, 127.4, 128.7, 128.8, 129.3, 131.1, 132.5, 133.4 (C-3), 133.9,
135.7, 135.9, 139.8 (C-7a), 145.3, 149.0 ppm; EI-MS: 424.05
[M þ Hþ]. Anal. Calcd for C26H25N5O (423.21): C 73.74, H 5.95, N
16.54. Found: C 73.75, H 5.94, N 16.53.
4.1.7.4. 1-(Quinolin-6-ylmethyl)-6-(1-methyl-pyrazol-3-yl)-1H-
indazole (4a). Yield: 23.7 mg (35%): mp: 276.5e277.7 ꢀC; 1H NMR
(400 MHz, CDCl3):
d
¼ 3.93 (s, 3H), 5.79 (s, 2H), 7.29 (dd, J ¼ 0.8,
8.0 Hz,1H), 7.38 (dd, J ¼ 4.4, 8.4 Hz,1H), 7.41 (dd, J ¼ 0.8, 2.0 Hz,1H),
7.57 (d, J ¼ 0.8 Hz, 1H), 7.60 (dd, J ¼ 2.0, 8.8 Hz, 1H), 7.61 (s, 1H), 7.74
(dd, J ¼ 1.2, 8.8 Hz, 1H), 7.76 (d, J ¼ 0.8 Hz, 1H), 8.05 (d, J ¼ 0.8 Hz,
1H), 8.07e8.10 (m, 2H), 8.88 ppm (dd, J ¼ 2.0, 4.4 Hz, 1H); 13C NMR
4.1.7.9. 2-(Quinolin-6-ylmethyl)-6-(1-methyl-pyrazol-3-yl)-1H-
indazole (5a). Yield: 30%: mp: 278.5e280.1 ꢀC; 1H NMR (400 MHz,
CDCl3):
d
¼ 3.96 (s, 3H), 5.78 (s, 2H), 7.26 (dd, J ¼ 1.6, 8.8 Hz, 1H),
(75 MHz, (D6) DMSO):
d
¼ 43.1, 61.9 (eCH2e), 108.7 (C-7), 118.6,
7.43 (dd, J ¼ 4.4, 8.4 Hz, 1H), 7.63e7.65 (m, 2H), 7.66 (s, 1H), 7.71 (d,
J ¼ 1.2 Hz, 1H), 7.80 (dd, J ¼ 1.6, 2.0 Hz, 1H), 7.82 (d, J ¼ 0.8 Hz, 1H),
7.95 (d, J ¼ 0.8 Hz, 1H), 8.13e8.15 (m, 2H), 8.92 ppm (dd, J ¼ 1.6,
121.3,121.7,125.4 (C-3a),126.1,126.9,127.6,128.2,128.9,131.3,132.1,
132.9, 133.8 (C-3), 135.6, 136.1, 140.4 (C-7a), 145.3, 148.7 ppm; EI-
MS: 340.07 [M þ Hþ]. Anal. Calcd for C21H17N5 (339.15): C 74.32,
H 5.05, N 20.63. Found: C 74.26, H 5.10, N 20.64.
4.4 Hz, 1H); 13C NMR (75 MHz, (D6) DMSO):
d
¼ 42.7, 60.9, 108.5 (C-
7), 118.6, 121.1 (C-3a), 121.7, 122.8 (C-3), 124.6, 126.1, 126.9, 127.6,

L. Ye et al. / European Journal of Medicinal Chemistry 65 (2013) 112e118
117
128.2, 128.9, 131.5, 132.1, 133.8, 135.5, 136.0, 145.3, 148.3 (C-7a),
149.1 ppm; EI-MS: 340.07 [M þ Hþ]. Anal. Calcd for C21H17N5
(339.15): C 74.32, H 5.05, N 20.63. Found: C 74.31, H 5.07, N 20.62.
determined using an IMAP Time Resolved Fluorescence Resonance
Energy Transfer (TR-FRET) assay. 50 nM 6 His-tagged recombinant
human c-Met residues 974-end (Millipore) was incubated in 20 mM
Tris, 10 mM MgCl2, 2.5 mM MnCl2, 0.01% Tween 20 and 2 mM DTT
4.1.7.10. 2-(Quinolin-6-ylmethyl)-6-[1-(2-tetrahydro-2H-pyran-2-
yloxyethyl)-1H-pyrazol-4-yl]-1H-indazole (5b). Yield: 32%: mp:
with 5
volume of 25
tested at 10 concentrations starting from 20
tration of 1% DMSO. The reaction was stopped by addition of 50
m
M ATP and 200 nM 5FAM-KKKSPGEYVNIGFG-NH2 in a total
l for 60 min at ambient temperature. Inhibitors were
M at a final concen-
l of
m
287.1e288.6 ꢀC; 1H NMR (400 MHz, CDCl3):
d
¼ 1.42e1.72 (m, 6H),
m
3.42 (m,1H), 3.63 (m,1H), 3.79 (m,1H), 4.08 (m,1H), 4.35 (dt, J ¼ 1.2,
4.8 Hz, 2H), 4.54 (t, J ¼ 3.6 Hz, 1H), 5.80 (s, 2H), 7.31 (dd, J ¼ 1.6,
8.4 Hz, 1H), 7.41 (dd, J ¼ 4.0, 8.4 Hz, 1H), 7.42 (d, J ¼ 1.2 Hz, 1H), 7.61
(d, J ¼ 1.6 Hz, 1H), 7.64 (dd, J ¼ 2.0, 6.0 Hz, 1H), 7.75 (dd, J ¼ 0.8,
8.4 Hz,1H), 7.78 (dd, J ¼ 0.8, 5.2 Hz,1H), 8.06 (d, J ¼ 0.8 Hz,1H), 8.11e
8.14 (m, 2H), 8.89 ppm (dd, J ¼ 1.6, 4.4 Hz, 1H); 13C NMR (75 MHz,
m
IMAP stop solution containing 60% buffer A:40% buffer B and a 1 in
400 dilution of beads and Terbium reagent. Plates were read after an
overnight incubation at 4 ꢀC on an Analyst HT reader. Reported IC50
s
are from a minimum of 2 experiments (n ¼ 2). Data analysis was
carried out using a four parameter curve fit. The standard errors of
the mean were calculated and expressed as a percentage of the
mean IC50. The average for this value was 12%.
(D6) DMSO):
d
¼ 19.8, 25.3, 30.4, 56.6, 61.9, 62.8, 64.1,107.3,107.9 (C-
7), 119.1, 120.9, 121.3, 122.9 (C-3), 124.5 (C-3a), 126.1, 126.8, 128.3,
128.7, 129.1, 131.7, 132.2, 133.8, 134.9, 136.1, 145.1, 148.4 (C-7a),
149.1 ppm; EI-MS: 454.04 [M þ Hþ]. Anal. Calcd for C27H27N5O2
(453.22): C 71.50, H 6.00, N 15.44. Found: C 71.52, H 5.97, N 15.44.
4.3. Cell assay
MKN-45 cells (purchased from Riken Cell Bank of Japan) were
seeded in RPMI 1640 medium containing 10% FCS and pretreated
for 1 h at 37 ꢀC with increasing concentrations of compound or
DMSO. Cells were then lysed in RIPA buffer containing appropriate
inhibitors. Western blotting was carried out following standard
procedures and probed with the following antibodies: anti-
phosphor c-Met (pYpYpY1230/1234/1235), anti-c-Met (Cell
Signaling Technologies Inc.), following the manufacturer’s protocol.
Total cellular protein loadings were semi-quantified by probing
with anti-GAPDH.
4.1.7.11. 2-(Quinolin-6-ylmethyl)-6-[1-(2-hydroxyethyl)-1H-pyrazol-
4-yl]-1H-indazole (5c). Yield: 98%: mp: 304.7e306.3 ꢀC; 1H NMR
(400 MHz, CDCl3):
d
¼ 4.06 (t, J ¼ 4.8 Hz, 2H), 4.30 (t, J ¼ 4.8 Hz, 2H),
5.80 (s, 2H), 7.26 (dd, J ¼ 1.6, 8.0 Hz, 1H), 7.50 (m, 1H), 7.66 (dd,
J ¼ 1.2, 8.8 Hz, 1H), 7.69 (d, J ¼ 8.4 Hz, 1H), 7.73e7.74 (m, 2H), 7.81
(dd, J ¼ 1.2, 2.8 Hz, 1H), 7.87 (d, J ¼ 0.8 Hz, 1H), 7.97 (d, J ¼ 0.8 Hz,
1H), 8.23 (m, 2H), 8.94 ppm (dd, J ¼ 1.6, 4.4 Hz, 1H); 13C NMR
(75 MHz, (D6) DMSO):
d
¼ 58.1, 59.3, 61.2, 109.1 (C-7), 119.2, 121.1,
121.5, 123.1 (C-3), 124.5 (C-3a), 126.2, 126.9, 128.4, 128.9, 129.2,
131.7, 132.2, 134.1, 135.0, 135.9, 145.3, 148.5 (C-7a), 149.3 ppm; EI-
MS: 370.01 [M þ Hþ]. Anal. Calcd for C22H19N5O (369.16): C 71.53,
H 5.18, N 18.96. Found: C 71.49, H 5.19, N 18.97.
4.4. Molecular docking
Molecular docking was performed with Surflex-Dock program
that is interfaced with Sybyl 7.3. The programs adapted an empir-
ical scoring function and a patented searching engine [16,17].
Ligand was docked into the corresponding protein’s binding site
guided by protomol, which is an idealized representation of a
ligand that makes every potential interaction with binding site. In
this work, the protomol was established by ligand from the crystal
structure of c-Met (PDB entry: 3dkf). Beginning of docking, all the
water and ligands were removed and the random hydrogen atoms
were added. Then the receptor structure was minimized in 10,000
cycles with Powell method in Sybyl 7.3. All the compounds were
constructed using Sketch Molecular module. Hydrogen and Gas-
teigereHückel charges were added to every molecule. Then their
geometries are optimized by conjugate gradient method in TRIPOS
force field. The energy convergence criterion is 0.001 kcal/mol.
Default values are chosen to finish this work except that the
threshold was 1 when the protomol was generated.
4.1.7.12. 2-(Quinolin-6-ylmethyl)-6-{1-[(1R,4R)-4-(tert-butyldime-
thylsilyloxy)cyclohexyl]-1H-pyrazol-4-yl}-1H-indazole
(5d).
¼ 0.08
Yield: 31%: mp: 311.2e312.5 ꢀC; 1H NMR (400 MHz, CDCl3):
d
(s, 6H), 0.90 (s, 9H), 1.45e1.54 (m, 2H), 1.79e1.91 (m, 2H), 1.97e2.03
(m, 2H), 2.14e2.20 (m, 2H), 3.68 (m, 1H), 4.03 (m, 1H), 5.28 (s, 2H),
7.24 (dd, J ¼ 1.6, 8.8 Hz, 1H), 7.41 (dd, J ¼ 4.4, 8.4 Hz, 1H), 7.62e7.65
(m, 2H), 7.63 (s, 1H), 7.70 (d, J ¼ 1.2 Hz, 1H), 7.78 (dd, J ¼ 1.6, 2.0 Hz,
1H), 7.80 (d, J ¼ 0.8 Hz, 1H), 7.91 (d, J ¼ 0.8 Hz, 1H), 8.11e8.14 (m,
2H), 8.91 ppm (dd, J ¼ 1.6, 4.4 Hz, 1H); 13C NMR (75 MHz, (D6)
DMSO):
d
¼ ꢂ4.9, 17.8, 24.8, 25.7, 32.5, 60.1, 63.9, 73.9, 108.1 (C-7),
118.2, 120.9, 121.3, 123.0 (C-3), 124.2 (C-3a), 126.1, 126.7, 128.5,
128.9, 129.2, 131.6, 132.1, 133.8, 135.2, 135.9, 144.7, 148.1 (C-7a),
149.2 ppm; EI-MS: 538.09 [M þ Hþ]. Anal. Calcd for C32H39N5OSi
(537.29): C 71.47, H 7.31, N 13.02. Found: C 71.46, H 7.30, N 13.04.
4.1.7.13. 2-(Quinolin-6-ylmethyl)-6-[1-((1R,4R)-4-hydroxy-cyclo-
hexyl)-1H-pyrazol-4-yl]-1H-indazole (5e). Yield: 99%: mp: 327.7e
329.3 ꢀC; 1H NMR (400 MHz, CDCl3):
d
¼ 1.59e1.67 (m, 2H), 1.90e
Acknowledgments
1.95 (m, 2H), 2.16e2.19 (m, 2H), 2.25e2.28 (m, 2H), 4.12 (m, 1H),
4.27 (m, 1H), 6.01 (s, 2H), 7.28 (dd, J ¼ 1.6, 8.8 Hz, 1H), 7.47 (dd,
J ¼ 4.4, 8.4 Hz,1H), 7.64e7.69 (m, 2H), 7.67 (s,1H), 7.78 (d, J ¼ 1.2 Hz,
1H), 7.81 (dd, J ¼ 1.6, 2.0 Hz, 1H), 7.87 (d, J ¼ 0.8 Hz, 1H), 8.03 (d,
J ¼ 0.8 Hz, 1H), 8.13e8.16 (m, 2H), 8.94 ppm (dd, J ¼ 1.6, 4.4 Hz, 1H);
This study was supported by grants from Guangdong Strategic
Emerging Project of Guangdong Provincial Department of Science
and Technology (no. 2012A080800012), International Science and
Technology Cooperation Base of Guangdong Provincial Department
of Science and Technology (no. 2009B050900006) and Science and
Technology Bureau of Guangzhou (no. 2009A1-E011-8 and no.
2010V1-E00531-3).
13C NMR (75 MHz, (D6) DMSO):
d
¼ 25.3, 31.3, 61.1, 63.7, 72.1, 108.2
(C-7), 118.9, 120.9, 121.4, 123.3 (C-3), 124.1 (C-3a), 125.9, 126.8,
128.4, 128.9, 129.2, 131.6, 132.4, 134.3, 135.1, 136.2, 145.4, 148.6 (C-
7a), 149.4 ppm; EI-MS: 424.05 [M þ Hþ]. Anal. Calcd for C26H25N5O
(423.21): C 73.74, H 5.95, N 16.54. Found: C 73.74, H 5.93, N 16.55.
Appendix A. Supplementary data
4.2. c-Met kinase assay
Supplementary data associated with this article can be found
in the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.
04.004. These data include MOL files and InChiKeys of the most
important compounds described in this article.
The c-Met kinase activities were evaluated according to a pub-
lished literature [3]. IC50 values for inhibitors of c-Met were

118
L. Ye et al. / European Journal of Medicinal Chemistry 65 (2013) 112e118
[9] S. Eathiraj, R. Palma, E. Volckova, M. Hirschi, Discovery of a novel mode of
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