Synthesis, and docking studies of phenylpyrimidine-carboxamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety as c-Met inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.02.046

Four series of phenylpyrimidine-carboxamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety (14a-e, 15a-g, 16a-e and 17a-g) were designed, synthesized and evaluated for the IC₅₀ values against three cancer cell lines (A549, PC-3 and MCF-7). Four selected compounds (15e, 16a-b and 17a) were further evaluated for the activity against c-Met kinase, HepG2 and Hela cell lines. Most of the compounds showed excellent cytotoxicity activity and selectivity with the IC₅₀ valuables in single-digit μM to nanomole range. Eleven of them are equal to more active than positive control Foretinib against one or more cell lines. The most promising compound 15e showed superior activity to Foretinib against A549, PC-3 and MCF-7 cell lines, with the IC₅₀ values of 0.14 ± 0.08 μM, 0.24 ± 0.07 μM and 0.02 ± 0.01 μM, which were 4.6, 1.6 and 473.5 times more active than Foretinib (0.64 ± 0.26 μM, 0.39 ± 0.11 μM, 9.47 ± 0.22 μM), respectively. Structure-activity relationships (SARs) and docking studies indicated that the replacement of phenylpicolinamide scaffold with phenylpyrimidine fragment of the target compounds was benefit for the activity. What's more, the introduction of fluoro atom to the aminophenoxy part played no significant impact on the activity and any substituent group on aryl group is unfavourable for the activity.

Full text of DOI:10.1016/j.bmc.2016.02.046

Accepted Manuscript
Synthesis, and docking studies of phenylpyrimidine-carboxamide derivatives
bearing 1H-pyrrolo[2,3-b]pyridine moiety as c-Met inhibitors
Wufu Zhu, Wenhui Wang, Shan Xu, Jianqiang Wang, Qidong Tang, Chunjiang
Wu, Yanfang Zhao, Pengwu Zheng
PII:
S0968-0896(16)30136-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.02.046
BMC 12848
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
10 February 2016
27 February 2016
29 February 2016
Please cite this article as: Zhu, W., Wang, W., Xu, S., Wang, J., Tang, Q., Wu, C., Zhao, Y., Zheng, P., Synthesis,
and docking studies of phenylpyrimidine-carboxamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety as c-
Met inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.02.046
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Synthesis, and docking studies of phenylpyrimidine-carboxamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine
moiety as c-Met inhibitors
Wufu Zhu1,† ,*, Wenhui Wang1,†, Shan Xu1,†, Jianqiang Wang2, Qidong Tang1, Chunjiang Wu1, Yanfang Zhao3, Pengwu
Zheng1
1 School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, P. R. China.
2Fushun Center for Drug Control, Fushun, 113000, China;
3Key Laboratory of Original New Drugs Design and Discovery of Ministry of Education, Shenyang Pharmaceutical
University, 103 Wenhua Road, Shenhe District, Shenyang 110016, P. R. China.
*Correspondence author.
Tel/Fax: +86 791 8380-2393;
E-mail address: zhuwf@jxstnu.edu.cn, zhuwufu-1122@163.com (W. Zhu), zhengpw@126.com (P. Zheng).
Four series of phenylpyrimidine-carboxamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety (14a–e, 15a–g,
16a–e and 17a–g) were designed, synthesized and evaluated for the IC50 values against three cancer cell lines (A549,
PC-3 and MCF-7). Four selected compounds (15e, 16a–b and 17a) were further evaluated for the activity against c-Met
kinase, HepG2 and Hela cell lines. Most of the compounds showed excellent cytotoxicity activity and selectivity with
the IC50 valuables in single-digit μM to nanomole range. Eleven of them are equal to or more active than positive
control Foretinib against one or more cell lines. The most promising compound 15e showed superior activity to
Foretinib against A549, PC-3 and MCF-7 cell lines, with the IC50 values of 0.14±0.08 μM, 0.24±0.07 μM and
0.02±0.01 μM, which were 4.6, 1.6 and 473.5 times more active than Foretinib (0.64 ± 0.26 μM, 0.39 ± 0.11 μM, 9.47
± 0.22 μM ), respectively. Structure–activity relationships (SARs) and docking studies indicated that the replacement of
phenylpicolinamide scaffold with phenylpyrimidine fragment of the target compounds was benefit for the activity.
What’s more, the introduction of fluoro atom to the aminophenoxy part played no significant impact on the activity and
any substituent group on aryl group is unfavourable for the activity.
Key words: Phenylpyrimidine; 1H-Pyrrolo[2,3-b]pyridine; Synthesis; Docking; c-Met inhibitors; Antitumor activity
† These authors contribute equally to this work
Corresponding author. Tel./fax: +86 791 83802393.
E-mail address:zhuwf@jxstnu.edu.cn, zhuwufu-1122@163.com (W. Zhu), zhengpw@126.com (P. Zheng).
1

1 Introduction
The c-Met receptor tyrosine kinase is aberrantly activated in many solid tumors. c-Met inhibitors may have
therapeutic application in the treatment of various types of cancers. Cabozantinib[1], a small molecule c-Met kinase
inhibitor, was approved by FDA for the treatment of patients with progressive metastatic medullary thyroid cancer on
November 29, 2012[2]. In recent years, many derivatives of Cabozantinib were reported, such as Foretinib[3], compounds
I, II [4-5]. The studies revealed that these compounds exhibited strong antitumor activity.
In our previous study, we reported a series of quinoline derivatives bearing phenylpicolinamide scaffold as potential
c-Met inhibitors [6]. In order to investigate the influence of the quinoline nucleus to the activity of target compounds, we
kept the phenylpicolinamide scaffold unchanged and replaced the quinoline nucleus with 1H-pyrrolo[2,3-b]pyridine
moiety to yield several series of new compounds[7]. These 1H-pyrrolo[2,3-b]pyridine derivatives exhibited excellent
antitumor activity. Structure–activity relationships (SARs) shown that the 1H-pyrrolo[2,3-b]pyridine scaffold benefit to
the antitumor activity.
In this study, further modifications were carried out on these 1H-pyrrolo[2,3-b]pyridine derivatives. According to
the theory of bioisosterism, we replaced the phenylpicolinamide scaffold with phenylpyrimidine moiety to get
compounds 14a–e and 15a–g. What’s more, fluoro atom was introduced to the aminophenoxy part of compounds 14a–e
and 15a–g to study the effect to the activity and yielded 16a–e and 17a–g.
Herein we disclosed the synthesis and antitumor activity against A549, PC-3, MCF-7, Hela and HepG2 cancer cell
lines, and c-Met kinase of phenylpyrimidine-carboxamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety.
Moreover, docking studies were presented in this paper as well.
Fig. 1 Structures of small-molecule c-Met inhibitors and target compounds.
2. Chemistry
2

The preparation of target compounds 14a–e, 15a–g 16a–e and 17a–g was described in Scheme 1.
The key intermediates 4-(4-aminophenoxy)-N-methylpicolinamide 13a,b were
achieved
from
4-chloro-1H-pyrrolo[2,3-b]pyridine 10 via substitution reaction with 4-nitrophenol or 2-fluoro-4-nitrophenol 11a,b and
reduction with hydrazine hydrate in sequence as shown in Scheme 1.
Substituted 1-phenyl ethanone 1a-i was reacted with N,N-Dimethylformamide dimethyl acetal (DMF-DMA) yielding
compounds 2a–i, which were then condensed with formamidine acetate to obtain compounds 3a–i. Subsequently,
compounds 3a–i reacted with sulfuric acid and formamide with the absence of different catalyst to get 4a–e and 7a–g,
respectively. Compounds 4a–e and 7a–g were hydrolyzed to corresponding carboxylic acid 5a–e and 8a–g, which were
then chlorination with oxalyl chloride to obtain 6a–e and 9a–g, respectively. Finally, reaction of amides 13a,b with
phenylpyrimidine-carbonyl chloride 6a–e or 9a–g promoted by DIPEA in dichloromethane at room temperature yielded
target compounds 14a–e, 15a–g 16a–e and 17a–g, respectively.
Scheme 1 Synthetic route of target compounds. Reagents and conditions: (a) DMF-DMA, 80℃, 8-12 h; (b)
formamidine acetate, EtONa, EtOH, 70℃, 1h, reflux, 15-20 h; (c) acetamide, 98%H2SO4, FeSO4·7H2O, 30%H2O2,
0-10℃, 1 h, 10-15℃, 30 min; (d) 20%H2SO4, 100℃, 5-10 h; (e) SOCl2, reflux, 1h, CH2Cl2, r.t., 0.5h; (f) acetamide,
98%H2SO4, FeSO4·7H2O, tert-Butyl hydroperoxide, 0-10℃, 1 h, 10-15℃, 30 min; (g) diphenyl ether, 190℃, 1 h; (h)
.
N2H4 H2O, FeCl3, activated carbon, EtOH, 10 min; (i) DIPEA, CH2Cl2, r.t., 0.5h.
3

3. Results and discussion
3.1 Biological evaluation
Taking c-Met inhibitor Foretinib as reference compound, the target compounds (14a–e, 15a–g, 16a–e and 17a–g)
were evaluated for the cytotoxicity against three cancer cell lines A549 (human lung cancer), PC-3 (human prostatic
cancer) and MCF-7(human breast cancer) by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) cell
proliferation assay. In addition, four selected compounds (15e, 16a–b and 17a) were evaluated for the IC50 values
against HepG2, Hela cell lines and c-Met kinase in vitro by MTT assay or the Mobility shift assay with ATP
concentration at Km, together with reference compound Foretinib. The results expressed as IC50 values were
summarized in Tables 1–5 and the values are the average of at least two independent experiments.
Table 1 Structures and cytotoxicity of compounds 14a-e
IC50 (μM) a
Compd. No.
R
A549
PC3
6.14±1.01
7.80±1.08
17.32±1.41
NAc
MCF-7
6.41±1.00
10.20±1.06
12.44±1.39
NA
14a
14b
4-CH3
3-Cl
4-OCH3
4-Cl
4-F
2.38±1.10
4.12±1.00
13.34±1.31
22.38±1.15
NA
14c
14d
14e
NA
NA
Foretinibb
-
0.64 ± 0.26
0.39 ± 0.11
9.47 ± 0.22
a The values are an average of two separate determinations.
b Used as a positive control
c No Activity
Table 2 Structures and cytotoxicity of compounds 15a-g
4

IC50 (μM) a
PC3
Compd. No.
R
A549
MCF-7
4.45±0.94
15.86±0.97
7.77±0.95
6.24±1.12
0.02±0.01
3.84±0.96
1.62±0.78
9.47 ± 0.22
15a
15b
4-OCH3
4-F
4.41±1.11
12.99±0.92
6.53±1.06
9.38±1.02
0.14±0.08
9.87±0.94
5.41±1.00
0.64 ± 0.26
9.83±1.25
NAc
15c
4-Cl
4-CF3
H
15.62±0.95
2.16±1.64
0.24±0.07
2.61±0.89
9.88±1.19
0.39 ± 0.11
15d
15e
15f
4-Br
4-CH3
-
15g
Foretinibb
a The values are an average of two separate determinations.
b Used as a positive control
c No Activity
Table 3 Structures and cytotoxicity of compounds 16a-e
IC50 (μM) a
PC3
Compd. No.
R
A549
MCF-7
7.17±1.02
1.58±0.84
NAc
16a
16b
4-CH3
3-Cl
1.15±0.14
0.45±0.20
4.47±1.21
15.43±1.23
8.94±1.07
0.64 ± 0.26
0.52±0.12
1.38±0.11
12.14±1.08
14.08±1.44
14.59±1.43
0.39 ± 0.11
16c
4-NO2
4-OCH3
4-Cl
16d
8.96±1.51
9.66±1.31
9.47 ± 0.22
16e
Foretinibb
-
a The values are an average of two separate determinations.
b Used as a positive control
c No Activity
Table 4 Structures and cytotoxicity of compounds 17a-g
5

Compd. No.
R
IC50 (μM) a
PC3
A549
MCF-7
4.64±1.18
10.41±0.96
NA
17a
17b
H
4-OCH3
4-F
0.18±0.04
4.21±1.57
14.24±0.66
3.36±0.14
2.22±0.82
6.98±1.05
8.60±0.67
0.64 ± 0.26
0.04±0.01
2.57±0.84
NAc
17c
17d
4-Cl
4-CF3
4-Br
4-CH3
-
3.20±1.33
4.12±0.98
11.34±1.01
24.92±0.68
0.39 ± 0.11
26.94±1.62
2.84±0.70
10.33±1.40
6.73±0.83
9.47 ± 0.22
17e
17f
17g
Foretinibb
a The values are an average of two separate determinations.
b Used as a positive control
c No Activity
Table 5 Cytotoxicity and c-Met kinase inhibitory activity of selected compounds 15e, 16a, 16b, 17a
Compd. No.
IC50 (µM)a
Hela
c-Met
0.12
HepG2
3.11±0.75
16.41±0.89
9.34±0.64
9.61±0.77
-
15e
16a
0.40±0.15
5.51±0.52
3.35±0.49
2.86±0.54
-
3.40
16b
2.20
17a
0.98
Foretinibb
0.014
a The values are an average of two separate determinations.
b Used as a positive control
As shown in Tables 1–4, most of the four series of compounds showed excellent cytotoxicity activity against
different cancer cells with potency from the single-digit μM to single-digit nanomole range. And the activity of the
second, the third and the fourth series (15a–g, 16a–e and 17a–g) is much better than that of the first series (14a–e).
Eleven of the target compounds showed better activity than positive control Foretinib against one or more cancer lines.
6

Among them, compounds 15e, 16a–b and 17a showed superior activity to positive control Foretinib against all the three
cancer cell lines. The most promising compound 15e exhibit best activity against A549, PC-3 and MCF-7 cell lines with
IC50 values of 0.14±0.08 μM, 0.24±0.07 μM and 0.02±0.01 μM, which were 4.6, 1.6 and 473.5 times more active than
Foretinib (0.64 ± 0.26 μM, 0.39 ± 0.11 μM, 9.47 ± 0.22 μM ), respectively. The results suggested that the replacement
of phenylpicolinamide scaffold with phenylpyrimidine fragment of the target compounds was benefit for the activity.
What’s more, the introduction of fluoro atoms to the aminophenoxy part played no significant impact on the activity.
Furthermore, different substitutions of aryl group affected the cytotoxicity of target compounds. It’s seem to be that
the presence of electron withdrawing group (-F, -Cl, -Br, -NO2) at C-4 position decreased the activity of the target
compounds, such as compounds 14d, 14e, 15b-d, 15f, 16c, 16e, 17c-d and 17f. However, the electron withdrawing
group Cl atom at C-3 position is better than that at C-4 group. For example, the activity of compounds14b, 16b is better
than compounds 14d, 16e. In addition, compounds without substituent group on aryl group exhibit the best activity,
such as compounds 15e, 16a–b and 17a. The results suggest that any substituent group on aryl group is unfavourable
for the activity.
Activity against c-Met kinase, Hela and HepG2 cell lines of compounds 15e, 16a–b and 17a was further carried out
in this paper to investigate the target and selectivity of these compounds. According to the results of Table 5, we can
easily find that the four selected compounds showed moderate to excellent inhibitory on c-Met kinase, Hela and HepG2
cell lines. Compounds 15e and 17a are more active than that of compounds 16a–b, especially compounds 15e with IC50
value of 0.12 μM on c-Met kinase. The results prompt us that the compounds may be a series of c-Met kinase inhibitors.
3.2 Molecular docking study
To explore the binding modes of target compounds with the active site of c-Met, molecular docking simulation
studies were carried out by using SURFLEX-DOCK module of SYBYL package version. Based on the in vitro
inhibition results, we selected compound 15e, our best c-Met inhibitor in this study, as the ligand example, and the
structure of c-Met was selected as the docking model (PDB ID code : 3LQ8 [8]).
7

Fig. 2 Binding poses of compound 15e with c-Met. The proteins were denoted by green ribbon. Compound 15e and
lead compound were denoted by orange and cyan sticks, respectively. H-bonding interactions between the 15e, lead
compound and c-Met were denoted by dashed lines in yellow.
The binding modes of compound 15e and lead compound Foretinib were shown in Fig. 2. As depicted in Fig. 2,
compound 15e and lead compound can mostly overlap in the binding model and pyrrolo[2,3-b]pyridine nucleus,
phenylpyrimidine-carboxamide moiety formed three hydrogen bonds with residues MET1160 and LYS1110,
respectively. Analysis of compound 15e’s binding mode in the active binding site demonstrated that the docking mode
of the 15e is similar to the lead compound with the same H-bond between pyrrolo[2,3-b]pyridine nucleus,
phenylpyrimidine-carboxamide moiety and MET1160, LYS1110. The three hydrogen bonds really play an important
role in increasing the inhibitory potency of pyrrolo[2,3-b]pyridine derivatives against c-Met kinase according to the
docking results. Furthermore, the docking results also give us a new direction to design new c-Met inhibitors. The
above-mentioned results of SARs analysis and molecular docking study may allow the rational design of more potent
c-Met inhibitors.
4. Conclusions
In summary, we designed and synthesized four series of phenylpyrimidine-carboxamide derivatives bearing
1H-pyrrolo[2,3-b]pyridine nucleus and evaluated for the IC50 values against three cancer cell lines and c-Met kinase.
Eleven of them are equal to more active than positive control Foretinib against one or more cell lines. The most
promising compound 15e showed superior activity to Foretinib, with the IC50 values of 0.14±0.08 μM, 0.24±0.07 μM
and 0.02±0.01 μM, which were 4.6, 1.6 and 473.5 times more active than Foretinib (0.64 ± 0.26 μM, 0.39 ± 0.11 μM,
8

9.47 ± 0.22 μM ), respectively. Structure–activity relationships (SARs) and docking studies indicated that the
replacement of phenylpicolinamide scaffold with phenylpyrimidine fragment of the target compounds was benefit for
the activity. What’s more, the introduction of fluoro atom to the aminophenoxy part played no significant impact on the
activity and any substituent group on aryl group is unfavourable for the activity. Further study will be carried out to
identify the exact action mechanism in near future.
5. Experimental
5.1. Chemistry
All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil,
Switzerland) and were 4 uncorrected. NMR spectra were performed using Bruker 400 MHz spectrometers (Bruker
Bioscience, Billerica, MA, USA) with TMS as an internal standard. Mass spectra (MS) were taken in ESI mode
on Agilent 1100 LCMS (Agilent, Palo Alto, CA, USA). All the materials were obtained from commercial
suppliers and used without purification, unless otherwise specified. Yields were not optimized. TLC analysis was
carried out on silica gel plates GF254 (Qindao Haiyang Chemical, China). All the materials were obtained from
commercial suppliers and used without purification, unless otherwise specied. Yields were not optimized.
5.2 General procedure for the preparation of compounds 2a-i.
The mixture of 1-phenyl ethanone (0.1mol) and DMF-DMA (39.8mL, 0.3mol) were heated to 80 ℃, and
monitored by TLC. Then the mixture was cooled to room temperature, poured into the mixture solution of
petroleum ether and ether(120mL/40mL), and stirred 0.5 h. The precipitated solid was filtered off and dried to
obtain the title compound 2a-i.
5.3 Preparation of compounds 3a-i.
Formamidine acetate(9.9 g, 0.096 mol) and sodium ethoxide(10.9 g, 0.16 mol) were dissolved in ethanol at
70 ℃ for 1 h. Then 2a-i was added in the reaction solution slowly, refluxed for 15~20 h and monitored by TLC.
The solution was concentrated in vacuum, the residue was resolved with dichloromethane (300 mL), washed with
brine (60 mL), dried over anhydrous Na2SO4, and concentrated in vacuum to give the light yellow solids 3a-i.
5.4 General procedure for the key intermediates 4a-e.
The preparation of the key intermediates 4a-e has been illustrated in detail in our previous work [6], and so
the synthesis method would not be listed here.
5.5 General procedure for the key intermediates 7a-g.
The preparation of the key intermediates 7a-g was illustrated in Sakamoto’s work[9].
Concentrated sulfuric acid(20 mL) was added drop-wise to the mixture of 3a-i (0.06 mol) and
formamide(100mL) at 0-10℃. At the same time, 65% tert-butyl hydroperoxide(50 mL)and saturated FeSO4·7H2O
(aq.100mL) were added drop-wise. The reaction solution was stirred at 10-15 ℃ for 0.5 h and then poured into
water(2000 mL), basified with potassium hydroxide to pH 9, and extracted with dichloromethane (200 mL×3).
The organic layer was dried over anhydrous Na2SO4, and concentrated in vacuum to obtain the title compound
9

7a-g.
5.6 General procedure for the preparation of 5a-e and 8a-g
The mixture of an appropriate 4a-e or 7a-g (0.025 mol) and 20% H2SO4 (100 mL) was reflxed at 100℃ for
5-10 h and monitored by TLC. The reaction mixture was poured into ice/water with stirring and filtered off and
dried to obtain the desired target compounds 5a-e or 8a-g.
5.7 General procedure for the key intermediates 6a-e and 9a-g
The compounds 5a-e or 8a-g (0.02 mmol) were dissolved in thionyl chloride (8mL) and refluxed for 1 h.
Then the reaction mixture was evaporated to yield the corresponding Chloride which were dissolved in
dichloromethane (10mL). The solution was used for the next step without further purification.
5.8 . General procedure for the key intermediates 13a,b
The mixture of 4-chloro-1H-pyrrolo[2,3-b]pyridine (10 g, 66 mmol) and 4-nitrophenol 11a (13.62 g, 98
mmol) or 2-fluoro-4-nitrophenol 11b ( 15.56 g, 99 mmol) were refluxed in diphenyl ether at 190℃ for 1 h and
monitored by TLC. The mixture was cooled to 70 ℃ and poured into 400 mL ethyl acetate in an ice bath for 30
min, light yellow solid was precipitated, filtered off and dried to obtain the title compound 12a or 12b.
Then 12a or 12b (20 mmol) was refluxed with hydrazine hydrate (200 mmol), Ferric chlorid（10 mmol) and an
appropriate amount of activated carbon in ethanol (100 mL)for 10min and monitored by TLC. The reaction
solution was cooled. Then the insoluble was filtered, the filtrate was evaporated, after that water was added and
stirred at room temperature for 30 min. Afterwords, the yellow solid was filtered off and dried to obtain the
desired target compounds 13a or 13b.
5.9 General procedure for the preparation of target compounds 14a-e, 15a-g, 16a-e, 17a-g
A solution of phenylpyrimidine-carbonyl chloride 6a-e or 9a-g (0.82 mmol) in dichloromethane (10 mL) was
added drop-wise to a solution of aniline 13a,b (0.41 mmol) and diisopropylethylamine (0.49 mmol) in
dichloromethane (10 mL) in an ice bath. Upon completion of the addition, the reaction mixture was removed from
the ice bath and placed in room temperature for 30 min and monitored by TLC. The mixture was washed with
10% K2CO3 (50 mL ×3) followed by brine (50 mL ×1), and the organic phase was separated, dried, and
evaporated to yield 14a-e, 15a-g, 16a-e and 17a-g which were recrystallized by isopropanol.
5.9.1 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-(p-tolyl)pyrimidine-2-carboxamide (14a)
1
Characters: light brown powder; Yield: 67.52%; ESI-MS m/z: [M+H]+422.5; m.p:234.9 ~ 239.6; H NMR (400
MHz, DMSO) δ 11.73 (s, 1H), 10.98 (s, 1H), 9.43 (s, 1H), 8.58 (s, 1H), 8.24 (d, J = 7.9 Hz, 2H), 8.10 (d, J = 5.5
Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 7.7 Hz, 2H), 7.38 – 7.30 (m, 1H), 7.25 (d, J = 8.4 Hz, 2H), 6.46 (d,
J = 5.3 Hz, 1H), 6.23 (s, 1H), 2.42 (s, 3H).
5.9.2 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-(3-chlorophenyl)pyrimidine-2-carboxamide (14b)
1
Characters: light brown powder; Yield: 70.21%; ESI-MS m/z: [M+H]+442.9; m.p:243.1 ~ 247.8; H NMR (400
MHz, DMSO) δ 11.64 (s, 1H), 10.90 (s, 1H), 9.39 (s, 1H), 8.57 (s, 1H), 8.26 (s, 1H), 8.19 (d, J = 7.4 Hz, 1H),
10

8.01 (d, J = 4.9 Hz, 1H), 7.94 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.27 (s, 1H),
7.15 (d, J = 8.4 Hz, 2H), 6.36 (d, J = 4.9 Hz, 1H), 6.13 (s, 1H).
5.9.3 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-(4-methoxyphenyl)pyrimidine-2-carboxamide (14c)
+
1
Characters: light yellow powder; Yield: 67.14%; ESI-MS m/z: [M+H] 438.2; m.p:201.3~204.5; H NMR (400
MHz, DMSO) δ 11.78 (s, 1H), 11.03 (s, 1H), 9.40 (s, 1H), 8.55 (s, 1H), 8.33 (d, J = 8.9 Hz, 2H), 8.10 (d, J = 5.4
Hz, 1H), 8.05 (d, J = 8.9 Hz, 2H), 7.39 – 7.36 (m, 1H), 7.25 (d, J = 8.9 Hz, 2H), 7.15 (d, J = 8.9 Hz, 2H), 6.44 (d,
J = 5.4 Hz, 1H), 6.23 (s, 1H), 3.88 (s, 3H).
5.9.4 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-(4-chlorophenyl)pyrimidine-2-carboxamide (14d)
1
Characters: light yellow powder; Yield: 69.08%; ESI-MS m/z: [M+H]+442.9; m.p:244.1~255.4; H NMR (400
MHz, DMSO) δ 11.78 (s, 1H), 11.08 (s, 1H), 9.48 (s, 1H), 8.64 (d, J = 6.6 Hz, 1H), 8.38 (d, J = 8.4 Hz, 2H), 8.09
(d, J = 5.4 Hz, 1H), 8.05 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H), 7.37 (s, 1H), 7.26 (d, J = 8.7 Hz, 2H),6.44
(d, J = 5.3 Hz, 1H), 6.23 (s, 1H).
5.9.5 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-4-(4-fluorophenyl)pyrimidine-2-carboxamide (14e)
+
1
Characters: light brown powder; Yield: 66.98%; ESI-MS m/z: [M+H] 426.1; m.p:184.2~186.8; H NMR (400
MHz, DMSO) δ 11.74 (s, 1H), 11.02 (s, 1H), 9.46 (s, 1H), 8.62 (s, 1H), 8.44 – 8.40 (m, 2H), 8.09 (d, J = 5.4 Hz, 1H),
8.04 (d, J = 8.7 Hz, 2H), 7.44 (t, J = 8.8 Hz, 2H), 7.36 (s, 1H), 7.24 (d, J = 8.7 Hz, 2H), 6.44 (d, J = 5.3 Hz, 1H), 6.22 (s,
1H).
5.9.6 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-6-(4-methoxyphenyl)pyrimidine-4-carboxamide (15a)
1
Characters: light yellow powder; Yield: 68.93%; ESI-MS m/z: [M+H]+438.5; m.p:226.7 ~ 228.6; H NMR (400
MHz, DMSO) δ 11.73 (s, 1H), 10.97 (s, 1H), 9.40 (s, 1H), 8.58 (s, 1H), 8.36 (d, J = 8.3 Hz, 2H), 8.10 (d, J = 4.6
Hz, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.37 (s, 1H), 7.25 (d, J = 8.2 Hz, 2H), 7.15 (d, J = 8.1 Hz, 2H), 6.46 (d, J = 4.5
Hz, 1H), 6.23 (s, 1H), 3.88 (s, 3H).
5.9.7 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-6-(4-fluorophenyl)pyrimidine-4-carboxamide (15b)
1
Characters: light yellow powder; Yield: 67.42%; ESI-MS m/z: [M+H]+426.4; m.p: 235.1 ~ 241.3; H NMR (400
MHz, DMSO) δ 11.74 (s, 1H), 11.00 (s, 1H), 9.46 (d, J = 1.3 Hz, 1H), 8.61 (dd, J = 7.5, 1.3 Hz, 1H), 8.45 – 8.40
(m, 2H), 8.09 (d, J = 5.4 Hz, 1H), 8.06 – 8.02 (m, 2H), 7.47 – 7.41 (m, 2H), 7.37 – 7.35 (m, 1H), 7.27 – 7.22 (m,
2H), 6.45 (d, J = 5.4 Hz, 1H), 6.22 (dd, J = 3.4, 2.0 Hz, 1H).
5.9.8 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-6-(4-chlorophenyl)pyrimidine-4-carboxamide (15c)
1
Characters: light yellow powder; Yield: 67.42%; ESI-MS m/z: [M+H]+442.9; m.p: 271.1 ~ 277.4; H NMR (400
MHz, DMSO) δ 11.74 (s, 1H), 11.02 (s, 1H), 9.48 (d, J = 1.3 Hz, 1H), 8.63 (t, J = 4.7 Hz, 1H), 8.36 (dd, J = 8.1,
11

6.2 Hz, 2H), 8.09 (d, J = 5.4 Hz, 1H), 8.06 – 8.02 (m, 2H), 7.67 (dd, J = 9.1, 2.3 Hz, 2H), 7.38 – 7.34 (m, 1H),
7.27 – 7.22 (m, 2H), 6.45 (d, J = 5.4 Hz, 1H), 6.22 (dd, J = 3.4, 2.0 Hz, 1H).
5.9.9 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-6-(4-(trifluoromethyl)phenyl)pyrimidine-4-carboxamide
(15d)
Characters: yellow powder; Yield: 69.02%; ESI-MS m/z: [M+H]+476.4; m.p: 262.9 ~ 264.9; 1H NMR (400 MHz,
DMSO) δ 11.74 (s, 1H), 11.06 (s, 1H), 9.55 (d, J = 1.3 Hz, 1H), 8.72 (d, J = 1.3 Hz, 1H), 8.55 (d, J = 8.2 Hz, 2H),
8.10 (d, J = 5.4 Hz, 1H), 8.07 – 8.02 (m, 2H), 7.97 (d, J = 8.4 Hz, 2H), 7.38 – 7.35 (m, 1H), 7.27 – 7.23 (m, 2H),
6.45 (d, J = 5.4 Hz, 1H), 6.22 (dd, J = 3.4, 2.0 Hz, 1H).
5.9.10 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-6-phenylpyrimidine-4-carboxamide (15e)
Characters: yellow powder; Yield: 67.77%; ESI-MS m/z: [M+H]+408.4; m.p:189.4~190.2; 1H NMR (400 MHz,
DMSO) δ 11.74 (s, 1H), 11.02 (s, 1H), 9.48 (d, J = 1.2 Hz, 1H), 8.62 (d, J = 1.2 Hz, 1H), 8.34 (dd, J = 7.5, 2.1 Hz,
2H), 8.10 (d, J = 5.4 Hz, 1H), 8.07 – 8.02 (m, 2H), 7.65 – 7.59 (m, 3H), 7.38 – 7.34 (m, 1H), 7.25 (d, J = 9.0 Hz,
2H), 6.45 (d, J = 5.4 Hz, 1H), 6.22 (dd, J = 3.4, 2.0 Hz, 1H).
5.9.11 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-6-(4-bromophenyl)pyrimidine-4-carboxamide (15f)
Characters: yellow powder; Yield: 68.46%; ESI-MS m/z: [M+H]+487.3; m.p:263.1~266.3; 1H NMR (400 MHz,
DMSO) δ 11.74 (s, 1H), 11.03 (s, 1H), 9.48 (s, 1H), 8.64 (s, 1H), 8.29 (d, J = 8.4 Hz, 2H), 8.09 (d, J = 5.4 Hz,
1H), 8.04 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.36 (s, 1H), 7.25 (d, J = 8.8 Hz, 2H), 6.44 (d, J = 5.4 Hz,
1H), 6.22 (s, 1H).
5.9.12 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)phenyl)-6-(p-tolyl)pyrimidine-4-carboxamide (15g)
Characters: yellow powder; Yield: 66.72%; ESI-MS m/z: [M+H]+422.5; m.p:239.6~244.2; 1H NMR (400 MHz,
DMSO) δ 11.75 (s, 1H), 11.00 (s, 1H), 9.43 (s, 1H), 8.57 (s, 1H), 8.23 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 5.4 Hz,
1H), 8.04 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 7.36 (s, 1H), 7.24 (d, J = 8.8 Hz, 2H), 6.44 (d, J = 5.4 Hz,
1H), 6.23 (s, 1H), 2.40 (s, 3H).
5.9.13 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-4-(p-tolyl)pyrimidine-2-carboxamide (16a)
1
Characters: khaki powder; Yield: 67.89%; ESI-MS m/z: [M+H]+440.4; m.p: 228.0 ~ 233.9; H NMR (400 MHz,
DMSO) δ 11.79 (s, 1H), 11.20 (s, 1H), 9.45 (s, 1H), 8.59 (s, 1H), 8.24 (d, J = 7.8 Hz, 2H), 8.18 – 8.11 (m, 1H),
8.10 (d, J = 5.3 Hz, 1H), 7.91 (d, J = 8.6 Hz, 1H), 7.50 – 7.43 (m, 2H), 7.41 – 7.37 (m, 2H), 6.42 (t, J = 8.1 Hz,
1H), 6.28 (s, 1H), 2.42 (s, 3H).
5.9.14 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-4-(3-chlorophenyl)pyrimidine-2-carboxamide
(16b)
12

1
Characters:light green powder; Yield: 64.59%; ESI-MS m/z: [M+H]+460.9; m.p:254.4 ~ 257.6; H NMR (400
MHz, DMSO) δ 11.78 (s, 1H), 11.23 (s, 1H), 9.51 (s, 1H), 8.69 (s, 1H), 8.38 (s, 1H), 8.32 (d, J = 7.5 Hz, 1H), 8.15
(s, 1H), 8.11 (d, J = 7.0 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.69 (s, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.45 (t, J = 8.6
Hz, 1H), 7.39 (s, 1H), 6.42 (s, 1H), 6.27 (s, 1H).
5.9.15
N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-4-(4-nitrophenyl)pyrimidine-2-carboxamide
(16c)
1
Characters:khaki powder; Yield: 63.47%; ESI-MS m/z: [M+H]+471.4; m.p:288.5~290.4; H NMR (400 MHz,
DMSO) δ 11.82 (s, 1H), 11.32 (s, 1H), 9.60 (s, 1H), 8.78 (s, 1H), 8.62 (d, J = 7.6 Hz, 3H), 8.44 (s, 3H), 8.10 (s,
1H), 7.47 (s, 1H), 7.40 (s, 1H), 6.41 (s, 1H), 6.28 (s, 1H).
5.9.16 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-4-(4-methoxyphenyl)pyrimidine-2-carbox-
amide (16d)
1
Characters: light yellow powder; Yield: 66.54%; ESI-MS m/z: [M+H]+456.1; m.p:212.0~214.6; H NMR (400
MHz, DMSO) δ 11.84 (s, 1H), 11.23 (s, 1H), 9.40 (s, 1H), 8.55 (s, 1H), 8.33 (d, J = 8.7 Hz, 2H), 8.12 - 8.17 (m,
1H), 8.09 (d, J = 5.4 Hz, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.46 (t, J = 9.1 Hz, 1H), 7.40 (s, 1H), 7.14 (d, J = 8.8 Hz,
2H), 6.40 (d, J = 5.4 Hz, 1H), 6.28 (s, 1H), 3.86 (d, J = 5.0 Hz, 3H)
5.9.17 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-4-(4-chlorophenyl)pyrimidine-2-carboxamide
(16e)
+
1
Characters: light yellow powder; Yield: 68.77%; ESI-MS m/z: [M+H] 460.1; m.p: 245.0~247.3; H NMR (400
MHz, DMSO) δ 11.83 (s, 1H), 11.29 (s, 1H), 9.50 (s, 1H), 8.66 (s, 1H), 8.38 (d, J = 8.6 Hz, 2H), 8.09 (d, J = 5.4
Hz, 1H), 7.92 (d, J = 9.3 Hz, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.47 (s, 1H), 7.41 – 7.39 (m, 1H), 6.40 (d, J = 5.5 Hz,
1H), 6.28 (d, J = 2.0 Hz, 1H), 5.77 (s, 1H).
5.9.18 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-6-phenylpyrimidine-4-carboxamide (17a)
1
Characters: light yellow powder; Yield: 68.61%; ESI-MS m/z: [M+H]+426.4; m.p:226.8 ~ 228.9; H NMR (400
MHz, DMSO) δ 11.69 (s, 1H), 11.13 (s, 1H), 9.40 (s, 1H), 8.54 (s, 1H), 8.24 (s, 2H), 8.05 (d, J = 5.2 Hz, 1H), 8.00
(s, 1H), 7.82 (d, J = 9.6 Hz, 1H), 7.54 (s, 3H), 7.35 (t, J =8.9 Hz, 1H), 7.30 (s, 1H), 6.32 (d, J = 5.3 Hz, 1H), 6.18
(s, 1H).
5.9.19 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-6-(4-methoxyphenyl)pyrimidine-4-carbox-
amide (17b)
1
Characters: light brown powder; Yield: 72.57%; ESI-MS m/z: [M+H]+456.4; m.p:249.0 ~ 251.4; H NMR (400
MHz, DMSO) δ 11.78 (s, 1H), 11.17 (s, 1H), 9.41 (d, J = 9.4 Hz, 1H), 8.54 (s, 1H), 8.32 (d, J = 8.2 Hz, 2H), 8.12
- 8.16 (m, 2H), 7.90 (d, J = 8.5 Hz, 1H), 7.43 (t, J = 9.1, 1H), 7.37 (s, 1H), 7.15 (d, J = 8.2 Hz, 2H), 6.41 (d, J =
13

5.0 Hz, 1H), 6.28 (s, 1H), 3.91 (s, 3H).
5.9.20 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-6-(4-fluorophenyl)pyrimidine-4-carboxamide
(17c)
1
Characters: white powder; Yield: 70.36%; ESI-MS m/z: [M+H]+444.4; m.p:269.8 ~ 274.1; H NMR (400 MHz,
DMSO) δ 11.79 (s, 1H), 11.23 (s, 1H), 9.48 (d, J = 1.3 Hz, 1H), 8.64 (d, J = 1.3 Hz, 1H), 8.45 – 8.40 (m, 2H),
8.12 – 8.16 (m, 1H), 8.09 (d, J = 5.4 Hz, 1H), 7.92 – 7.88 (m, 1H), 7.47 (t, J = 3.8 Hz, 1H), 7.45 (d, J = 3.4 Hz,
1H), 7.43 (d, J = 3.2 Hz, 1H), 7.40 – 7.37 (m, 1H), 6.41 (d, J = 5.4 Hz, 1H), 6.27 (dd, J = 3.4, 2.0 Hz, 1H).
5.9.21 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-6-(4-chlorophenyl)pyrimidine-4-carboxamide
(17d)
1
Characters: light brown powder; Yield: 71.11%; ESI-MS m/z: [M+H]+460.9; m.p:256.4 ~ 265.9; H NMR (400
MHz, DMSO) δ 11.79 (s, 1H), 11.23 (s, 1H), 9.49 (d, J = 1.3 Hz, 1H), 8.65 (d, J = 1.3 Hz, 1H), 8.40 – 8.35 (m,
2H), 8.12 – 8.16 (m, 1H), 8.09 (d, J = 5.4 Hz, 1H), 7.93 – 7.88 (m, 1H), 7.70 – 7.65 (m, 2H), 7.45 (t, J = 9.1 Hz,
1H), 7.40 – 7.37 (m, 1H), 6.41 (d, J = 5.4 Hz, 1H), 6.27 (dd, J = 3.4, 2.0 Hz, 1H).
5.9.22 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-6-(4-(trifluoromethyl)phenyl)pyrimidine-4-
carboxamide (17e)
1
Characters: light brown crystals; Yield: 72.45%; ESI-MS m/z: [M+H]+494.4; m.p:264.5 ~ 278.3; H NMR (400
MHz, DMSO) δ 11.79 (s, 1H), 11.27 (s, 1H), 9.56 (d, J = 1.3 Hz, 1H), 8.74 (d, J = 1.3 Hz, 1H), 8.56 (d, J = 8.2 Hz, 2H),
8.12 – 8.16 (m, 1H), 8.09 (d, J = 5.4 Hz, 1H), 7.98 (d, J = 8.3 Hz, 2H), 7.93 – 7.89 (m, 1H), 7.46 (t, J = 9.1 Hz, 1H),
7.41 – 7.37 (m, 1H), 6.41 (d, J = 5.4 Hz, 1H), 6.27 (dd, J = 3.4, 2.0 Hz, 1H).
5.9.23 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-6-(4-bromophenyl)pyrimidine-4-carboxamide (17f)
Characters: yellow powder; Yield: 71.70%; ESI-MS m/z: [M+H]+505.3; m.p:257.9~260.5; 1H NMR (400 MHz, DMSO)
δ 11.81 (s, 1H), 11.24 (s, 1H), 9.49 (s, 1H), 8.65 (s, 1H), 8.29 (d, J = 8.5 Hz, 2H), 8.12 – 8.16 (m, 1H), 8.09 (d, J = 5.5
Hz, 1H), 7.91 (d, J = 9.3 Hz, 1H), 7.81 (d, J = 8.5 Hz, 2H), 7.45 (t, J = 9.1 Hz, 1H), 7.40 (d, J = 2.3 Hz, 1H), 6.41 (d, J
= 5.4 Hz, 1H), 6.28 (s, 1H).
5.9.24 N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-6-(p-tolyl)pyrimidine-4-carboxamide (17g)
Characters: white powder; Yield: 71.67%; ESI-MS m/z: [M+H]+440.44; m.p: 250.4~251.9; 1H NMR (400 MHz, DMSO)
δ 11.79 (s, 1H), 11.21 (s, 1H), 9.44 (s, 1H), 8.58 (s, 1H), 8.24 (d, J = 8.1 Hz, 2H), 8.12 – 8.16 (m, 1H), 8.09 (d, J = 5.4
Hz, 1H), 7.90 (d, J = 8.9 Hz, 1H), 7.46 (d, J = 9.1 Hz, 1H), 7.43 (s, 1H), 7.41 (s, 1H), 7.39 (s, 1H), 6.40 (d, J = 5.4 Hz,
1H), 6.27 (s, 1H), 2.42 (s, 3H).
5. 10 Cytotoxicity assay in vitro
14

The cytotoxic activities of target compounds (14a–e, 15a–g, 16a–e and 17a–g) were evaluated with A549, PC-3,
MCF-7,HepG2 and Hela cell lines by the standard MTT assay in vitro, with compounds c-Met inhibitors Foretinib as
positive control. The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10% fetal
bovine serum (FBS). Approximately 4×103 cells, suspended in MEM medium, were plated onto each well of a 96-well
plate and incubated in 5% CO2 at 37°C for 24 h. The test compounds at indicated final concentrations were added to the
culture medium and the cell cultures were continued for 72 h. Fresh MTT was added to each well at a terminal
concentration of 5μg/mL and incubated with cells at 37°C for 4 h. The formazan crystals were dissolved in 100 μL
DMSO each well, and the absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the reference
wavelength) was measured with the ELISA reader. All of the compounds were tested three times in each of the cell
lines. The results expressed as inhibition rates or IC50 (half-maximal inhibitory concentration) were the averages of two
determinations and calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
5. 11 c-Met kinase assay in vitro
The selected compounds (15e, 16a–b and 17a) are tested for their activity against c-Met kinase through the mobility
shift assay [8-9]. All kinase assays were performed in 96-well plates in a 50 μL reaction volume. The kinase buffer
contains 50 mM HEPES, pH 7.5, 10 mM MgCl2, 0.0015% Brij-35 and 2 mM DTT. The stop buffer contains 100 mM
HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3 and 50 mM EDTA. Dilute the compounds to 500 μM by
100% DMSO, then transfer 10 μL of compound to a new 96-well plate as the intermediate plate, add 90 μL kinase
buffer to each well. Transfer 5 μL of each well of the intermediate plate to 384-well plates. The following amounts of
enzyme and substrate were used per well: kinase base buffer, FAM-labeled peptide, ATP and enzyme solution. Wells
containing the substrate, enzyme, DMSO without compound were used as DMSO control. Wells containing just the
substrate without enzyme were used as low control. Incubate at room temperature for 10 min. Add 10 μL peptide
solution to each well. Incubate at 28 °C for specified period of time and stop reaction by 25 μL stop buffer. At last
collect data on Caliper program and convert conversion values to inhibition values. Percent inhibition = (max −
conversion)/(max − min) × 100. “max” stands for DMSO control; “min” stands for low control.
5. 12 Docking studies
For docking purposes, the three-dimensional structure of the c-Met (PDB code: 3LQ8) were obtained from RCSB
Protein Data Bank [4]. Hydrogen atoms were added to the structure allowing for appropriate ionization at physiological
pH. The protonated state of several important residues, such as CYS919, and ASP1046 were adjusted by using
SYBYL6.9.1 (Tripos, St. Louis, USA) in favor of forming reasonable hydrogen bond with the ligand. Molecular
docking analysis was carried out by the SURFLEX-DOCK module of SYBYL 6.9.1 package to explore the binding
model for the active site of c-Met with its ligand. All atoms located within the range of 5.0 Å from any atom of the
cofactor were selected into the active site, and the corresponding amino acid residue was, therefore, involved into the
active site if only one of its atoms was selected. Other default parameters were adopted in the SURFLEX-DOCK
calculations. All calculations were performed on Silicon Graphics workstation.
15

Acknowledgments
We gratefully acknowledge the generous support provided by The National Natural Science Funds (81460527),
Science and Technology Project Founded by the Education Department of Jiangxi Province (GJJ150796), Top-notch
talent project of Jiangxi Science & Technology Normal University (2015QNBJRC001), Program of Key
Laboratory of Drug Design and Optimization, Jiangxi Science & Technology Normal University (300098010306) and
College Students' Science and Technology Innovation Project of Jiangxi Province (YC2015-X28).
References
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Administration Updated: 11/30/2012, last accessed: 11/6/2015
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57, 162-175.
16

Legends
Fig. 1 Structures of small-molecule c-Met inhibitors and target compounds.
Fig. 2 Binding poses of compound 15e with c-Met. The proteins were denoted by green ribbon. Compound 15e
and lead compound were denoted by orange and cyan sticks, respectively. H-bonding interactions between the 15e,
lead compound and c-Met were denoted by dashed lines in yellow.
Scheme 1 Synthetic route of target compounds.
Reagents and conditions: (a) DMF-DMA, 80℃, 8-12 h; (b) formamidine acetate, EtONa, EtOH, 70℃, 1h, reflux,
15-20 h; (c) acetamide, 98%H2SO4, FeSO4·7H2O, 30%H2O2, 0-10℃, 1 h, 10-15℃, 30 min; (d) 20%H2SO4, 100℃,
5-10 h; (e) SOCl2, reflux, 1h, CH2Cl2, r.t., 0.5h; (f) acetamide, 98%H2SO4, FeSO4·7H2O, tert-Butyl hydroperoxide,
.
0-10℃, 1 h, 10-15℃, 30 min; (g) diphenyl ether, 190℃, 1 h; (h) N2H4 H2O, FeCl3, activated carbon, EtOH, 10 min;
(i) DIPEA, CH2Cl2, r.t., 0.5h.
Table 1 Structures and cytotoxicity of compounds 14a-e
a The values are an average of two separate determinations.
b Used as a positive control
c No Activity
Table 2 Structures and cytotoxicity of compounds 15a-g
a The values are an average of two separate determinations.
b Used as a positive control
c No Activity
Table 3 Structures and cytotoxicity of compounds 16a-e
a The values are an average of two separate determinations.
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b Used as a positive control
c No Activity
Table 4 Structures and cytotoxicity of compounds 17a-g
a The values are an average of two separate determinations.
b Used as a positive control
Table 5 Cytotoxicity and c-Met kinase inhibitory activity of selected compounds 15e, 16a, 16b, 17a
a The values are an average of two separate determinations.
b Used as a positive control
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Graphical Abstract
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