Discovery of novel pyrrolopyrimidine/pyrazolopyrimidine derivatives bearing 1,2,3-triazole moiety as c-Met kinase inhibitors

Article abstract of DOI:10.1111/cbdd.13192

Six series of pyrrolo[2,3-d]pyrimidine and pyrazolo[3,4-d]pyrimidine derivatives bearing 1,2,3-triazole moiety were designed and synthesized, and some bio-evaluation was also carried out. As a result, four points can be summarized: Firstly, some of compounds exhibited excellent cytotoxicity activity and selectivity with the IC₅₀ values in single-digit μm level. In particular, the most promising compound 16d showed equal activity to lead compound foretinib against A549, HepG2, and MCF-7 cell lines, with the IC₅₀ values of 4.79?±?0.82, 2.03?±?0.39, and 2.90?±?0.43?μm, respectively. Secondly, the SARs and docking studies indicated that the in vitro antitumor activity of pyrrolo[2,3-d]pyrimidine derivatives bearing 1,2,3-triazole moiety was superior to the pyrazolo[3,4-d]pyrimidine derivatives bearing 1,2,3-triazole moiety. Thirdly, three selected compounds (16d, 18d, and 20d) were further evaluated for inhibitory activity against the c-Met kinase, and the 16d could inhibit the c-Met kinase selectively by experiments of enzyme-based selectivity. What is more, 16d could induce apoptosis of HepG2 cells and inhibitor the cell cycle of HepG2 on G2/M phase by acridine orange staining and cell cycle experiments, respectively.

Full text of DOI:10.1111/cbdd.13192

DR WUFU ZHU (Orcid ID : 0000-0001-6075-0450)
Article type
: Research Article
Discovery of novel pyrrolopyrimidine/pyrazolopyrimidine derivatives bearing 1,2,3-triazole moiety
as c-Met kinase inhibitors
Linxiao Wang1,†, Xiaobo Liu1,†, Yongli Duan1, Xiaojing Li2,Bingbing Zhao1, Caolin Wang1, Zhen Xiao1,
Pengwu Zheng, Qidong Tang1,*,Wufu Zhu1,*
1Jiangxi Provincial Key Laboratory of Drug Design and Evaluation,School of Pharmacy,Jiangxi Science
& Technology Normal University,Nanchang 330013,P.R. China
2College of Service,Naval University of Academy of PLA,Tianjin 300450,PR China
*Correspondence author.
Tel/Fax: +86 791 8380-2393;
E-mail address:zhuwf@jxstnu.edu.cn, zhuwufu-1122@163.com (W. Zhu).tangqidongcn@126.com
Abstract: Six series of pyrrolo[2,3-d]pyrimidine and pyrazolo[3,4-d]pyrimidine derivatives bearing
1,2,3-triazole moiety were designed, synthesized, and some bio-evaluation were also carried out. As a
result, four points can be summarized: Firslty, some of compounds exhibited excellent cytotoxicity activity
and selectivity with the IC50 values in single-digit µM level. Especially, the most promising compound 16d
showed equal activity to lead compound Foretinib against A549, HepG2 and MCF-7 cell lines, with the
IC50 values of 4.79 ± 0.82 µM, 2.03 ± 0.39 µM and 2.90 ± 0.43 µM, respectively. Secondly, The SARs and
docking studies indicated that the in vitro antitumor activity of pyrrolo[2,3-d]pyrimidine derivatives
bearing 1,2,3-triazole moiety was superior to the pyrazolo[3,4-d]pyrimidine derivatives bearing
1,2,3-triazole moiety. Thirdly, Three selected compounds (16d, 18d and 20d) were further evaluated for
inhibitory activity against the c-Met kinase, and the 16d could inhibit the c-Met kinase selectively by
experiments of enzyme-based selectivity. What's more, 16d could induce apoptosis of HepG2 cells and
inhibitor the cell cycle of HepG2 on G2/M phase by acridine orange (AO) staining and cell cycle
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13192
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experiments, respectively.
Keywords: Pyrrolopyrimidine/pyrazolopyrimidine, 1,2,3-triazole, Antitumor activity, Apoptosis and Cell
cycle, c-Met, Docking Study1
1. Introduction
c-Met, the receptor tyrosine kinase encoded by MET proto oncogene located on chromosome 7b and
7q21–q31 that encoded a 1408 amino acid transmembrane glycoprotein[1] . c-Met is expressed in both
normal and malignant cells which is an epithelial/endothelial cell surface transmembrane receptor with
specificity for hepatocyte growth factor (HGF) or also known as scatter factor (SF) [1-3]
.
c-Met inhibitors, a class of small molecules which can inhibit the enzymatic activity of the c-Met
kinase and have therapeutic application in the treatment of various types of cancers[4]. Cabozantinib, the first
small-molecule c-Met inhibitor, was approved by FDA on November 29, 2012. In recent years, many
Cabozantinib derivatives were reported, such as Foretinib, compounds 3 and 4 (The structures were shown
in Fig.1[5-7] ).
Many researches showed that nearly all the Cabozantinib derivatives show excellent activity and
contain two obvious structural characteristics ( Foretinib was used as a template): moiety A and C were
belonged to the HD/HR (Hydrogen bond donor or Hydrogen bond receptor) structure, while the moiety B
and D were belong to HI (Hydrophobic interaction) structure (Fig.1, Fig.2). And many bioactivity structure
have been reported that introduced into the moiety A, such as substituted quinoline, substituted pyridine
and pyrrolo[2,3-b]pyridine series (1-2, 3 and 4, respectively). But, nearly all the Cabozantinib derivatives
reserve the moiety C (a 5-atom linker) which has two obvious structural characteristics. One is the ‘5
atoms regulation’, which means six chemical bonds distance existing between moiety B and moiety D; the
other one is that the 5-atom linker containing HR/HD structure which could form hydrogen-bond with the
[8-9]
c-Met.
Furthermore, the moiety B and moiety D may form hydrophobic bond bound to c-Met
preferably.
In our previous research, several series of pyrrolo[2,3-b]pyridine derivatives (compounds 5 and 6,
Fig.1) were designed and synthesized as potent c-Met inhibitors according to the ‘5 atom regulation’.[10-11]
† 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), tangqidong@126.com
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Most of these compounds exhibited potent activity, especially the most promising compound 6 with the
IC50 values in the nanomole level. What's more, the triazole also contain other boiactivity, such as
anti-vaccinia, adenovirus and HIV-1 inhibitors and can be prepared in a high yield and without azide
isolation or purification.[12-17] These results show that 1,2,3-triazole moiety may be beneficial to the in vitro
activity.
In continue to our previous work and inspired by compound 6, further modification was concentrated
on pyrrolo[2,3-b]pyridine moiety. Firstly, reserving the 1,2,3-triazole scaffold of compound 6 and replacing
the pyrrolo[2,3-b]pyridine moiety with pyrrolo[2,3-d]pyrimidine moiety to study the effects of increasing
the hydrogen receptor to the compounds activity and the first two serials compounds (15a-f and 16a-f )
were prepared. And then the methyl group was introduced to N-7 position of pyrrolo[2,3-d]pyrimidine
moiety to investigate the effects of electron donor effect to the target compounds and the second two series
of compounds were obtained. Thus, four series of target compounds 15a-f, 16a-f, 17a-f and 18a-f were
achieved.
Furthermore,
pyrrolo[2,3-d]pyrimidine
moiety
was
further
modified
with
pyrazolo[3,4-d]pyrimidine to study the effects of pyrrolopyrimidine /pyrazolopyrimidine scaffold to
activity. As a result, the latter two series of compounds 19a-f and 20a-f were prepared. And the design
strategy for all target compounds was described in Fig.2.
Herein we disclosed the synthesis and antitumor activity of pyrrolo[2,3-d]pyrimidine or
pyrazolo[3,4-d]pyrimi -dine bearing 1,2,3-triazole moiety against A549 (human lung cancer), HepG2
(human liver cancer), MCF-7 (human breast cancer) cancer cell lines, and c-Met kinase. Moreover,
experiments of enzyme-based selectivity, acridine orange single staining, cell cycle and docking studies
were presented within this paper as well.
(Fig.1 should be here)
(Fig. 2 should be here)
2. Materials and Methods
2.1 chemistry
2.1.1 Reagents and materials
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
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were obtained from commercial suppliers and used without purification, unless otherwise specified.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 specified.
2.1.2 Synthesis
General procedure for the key intermediate 9a-f
4-Aminophenol or 2-fluoro-4-aminophenol 8a-b (0.1mol) which was dissolved in tetrahydrofuran
(50ml), was added to a 1,4-dioxane/H2O (50ml, 5:1) solution of compounds 7a-c (0.1mol), sodium
carbonate and hydrogen sodium at 80℃ for 2 hours. Then the solution was concentrated in vacuum and
washed with water, filtered to give a solid .
4-((1H-pyrrolo[3,4-d]pyrimidine-4-yl)oxy)aniline (9a)
Light yellow solid; Yield: 61%; M.P.: 186-188℃; 1H NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 8.26 (s,
1H), 7.39 – 7.33 (m, 1H), 6.87 (d, J = 8.7 Hz, 2H), 6.59 (d, J = 8.7 Hz, 2H), 6.23 (dd, J = 3.3, 1.7 Hz, 1H),
5.04 (s, 2H) ; MS (ESI) m/z(%):227.09 [M+H] +.
3-Fluoro-4-((1H-pyrrolo[3,4-d]pyrimidine-4-yl)oxy)aniline (9b)
Light yellow solid; Yield: 56%; M.P.: 189-191℃; 1H NMR (400 MHz, DMSO-d6) δ 12.17 (s, 1H), 8.26 (s,
1H), 7.43 (s, 1H), 6.99 (t, J = 8.9 Hz, 1H), 6.44 (dd, J = 8.7, 5.4 Hz, 3H), 5.34 (s, 2H) ; MS (ESI)
m/z(%):245.08[M+H] +.
4-((1-Methyl-1H-pyrrolo[3,4-d]pyrimidine-4-yl)oxy)aniline (9c)
Light yellow solid; Yield: 58%; M.P.: 193-195℃; 1H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 1H), 7.53 (d,
J = 3.5 Hz, 1H), 6.97 (d, J = 8.7 Hz, 2H), 6.69 (d, J = 8.7 Hz,2H), 6.34 (d, J = 3.5 Hz, 1H), 5.17 (s, 2H),
3.89 (s,3H) ; MS (ESI) m/z(%):241.10 [M+H] +.
3-Fluoro-4-((1-methyl-1H-pyrrolo[3,4-d]pyrimidine-4-yl)oxy)aniline (9d)
1
Light yellow solid; Yield: 59%; M.P.: 194-197℃; H NMR (400 MHz, DMSO-d6) δ 8.50 (t, J = 3.2 Hz,
1H), 8.47 (s, 1H), 8.32 (d, J = 9.0 Hz, 1H), 7.89 (t, J = 8.4 Hz, 1H), 7.73 (d, J = 3.5 Hz,1H), 6.82 (d, J =
3.5 Hz,1H), 5.27 (s, 2H), 3.96 (s,3H) ; MS (ESI) m/z(%):259.10 [M+H] +.
4-((1-Methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)aniline(9e)
Light yellow solid; Yield: 55%; M.P.: 201-202℃;1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H), 7.67 (s,
1H), 6.96 (d, J = 8.7 Hz,2H), 6.64 (d, J = 8.7 Hz, 2H), 5.20 (s, 2H), 4.02 (s, 3H) ;MS (ESI) m/z(%):242.10
[M+H] +.
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3-Fluoro-4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)aniline(9f)
Light yellow solid; Yield: 58%; M.P.: 202-205℃; 1H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 7.99 (s,
1H), 6.98 (t, J = 8.9 Hz, 1H), 6.42 (d, J = 10.8 Hz, 1H), 6.35 (d, J = 8.7 Hz, 1H), 5.36 (s, 2H), 3.95 (s,
3H) ;MS (ESI) m/z(%):259.11 [M+H] +.
General procedure for preparation of compounds 12a-f.
The intermediates 1-aryl-5-methyl (or trifluoromethyl)-1,4,5-trisubstituted-1,2,3-triazoles 12a-f were
synthesized in high yield by a one-pot three-component reaction of arylboronic acids 10a-g, sodium
azide,and active methylene ketones, such as ethyl acetoacetate (or ethyl 4,4,4-trifluoroacetoacetate) 11a-b
in the presence of Cu(OAc)2 and piperidine using a DMSO/H2O (10:1) mixture as solvent. [14-15] The
reaction was monitored by TLC until completed. Finally the solution was filtered and washed with a plenty
of water to give a white solid.
General procedure for preparation of 13a-f
Appropriate intermediate 12a-f (0.11 mol), sodium carbonate (0.2 mol) were dissolved in
Ethanol/H2O (50ml, 5:1) ,and heated to 80℃ for 5-6 hour. Subsequently, the mixture solution acidified to
pH 2-3 to yield the compounds 13a-f, respectively.
General procedure for the key intermediate 14a-f
The compounds 13a-f （ 0.001 mol ） and appropriate DMF (0.0001 mol) were dissolved in
dichloromethane, then appropriate oxalyl chloride was added slowly and monitored by TLC. The solution
was used for next step without further purification.
General procedure for the preparation of target compounds 15a-f, 16a-f, 17a-f, 18a-f, 19a-f and
20a-f.
A solution of phenylpyrdazinone carbonyl chloride 14a-f (0.82 mmol) in dichloromethane (10 mL)
was added drop-wise to a solution of aniline 9a-f (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 concentrated in vacuum to yield 15a-f, 16a-f, 17a-f, 18a-f, 19a-f and 20a-f which were
recrystallized by isopropanol.
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N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(3-chloro-4-fluorophenyl)-5-(trifluoromethyl)-
1H- 1,2,3-triazole-4-carboxamide (15a)
Yield: 62%; M.P.: 206–207℃; ESI-MS m/z:518.06 [M+H] +; 1H NMR (400 MHz, DMSO-d6) δ 12.11（s，
1H）,11.13 (s, 1H), 8.33 (s, 1H), 8.21 (dd, J = 6.4, 2.4 Hz, 1H), 7.89 (d, J = 9.0 Hz, 2H), 7.86 – 7.83 (m,
1H), 7.76 (t, J = 8.9 Hz, 1H), 7.51 (d, J = 3.4 Hz, 1H), 7.26 (d, J = 8.9 Hz, 2H), 6.51 (d, J = 3.4 Hz, 1H);
13C NMR (101 MHz, DMSO-d6) δ 161.66, 160.00, 159.97, 157.46, 156.39, 152.45, 150.05, 148.96, 146.53,
142.09, 135.27, 132.12, 129.20, 127.95, 122.18(2C), 121.81(2C), 118.02, 117.79, 104.71, 97.37.
N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(3,4-difluorophenyl)-5-(trifluoromethyl)-1H-
1,2,3-triazole-4-carboxamide (15b)
Yield: 64%; M.P.: 206–208℃; ESI-MS m/z:501.10 [M+H] +; 1H NMR (400 MHz, DMSO-d6) δ 12.11（s，
1H）,11.13 (s, 1H), 8.33 (s, 1H), 8.10 (t, J = 7.7 Hz, 2H), 7.80 (dd, J = 18.5, 9.1 Hz, 2H), 7.72 (s, 1H), 7.51
(d, J = 3.4 Hz, 1H), 7.26 (d, J = 8.8 Hz, 2H), 6.51 (d, J = 3.4 Hz, 1H).
N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-chlorophenyl)-5-(trifluoromethyl)-1H-
1,2,3-triazole-4-carboxamide (15c)
Yield: 62%; M.P.: 203–205℃; ESI-MS m/z:500.07 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 12.14（s，
1H）,11.11 (s, 1H), 8.34 (s, 1H), 7.88 (d, J = 8.8 Hz, 2H), 7.82 – 7.74 (m, 4H), 7.51 (d, J = 3.3 Hz, 1H),
7.26 (d, J = 8.7 Hz, 2H), 6.50 (d, J = 3.3 Hz, 1H) .
N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4
-carboxamide (15d)
Yield: 65%; M.P.: 207–208℃; ESI-MS m/z:429.14 [M+H] + ; 1H NMR (400 MHz, DMSO-d6) δ 12.21（s，
1H）,10.78 (s, 1H), 8.45 (s, 1H), 8.03 (d, J = 8.9 Hz, 2H), 7.86 (dd, J = 8.8, 4.9 Hz, 2H), 7.62 (dd, J = 10.6,
6.8 Hz, 3H), 7.34 (d, J = 8.9 Hz, 2H), 6.59 (d, J = 3.4 Hz, 1H), 3.93 (s, 3H), 2.60 (s, 3H).
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N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-phenyl-5-(trifluoromethyl)-1H-1,2,3-triazole-4
-carboxamide (15e)
Yield: 63%; M.P.: 210–211℃; ESI-MS m/z:411.15 [M+H] + ; 1H NMR (400 MHz, DMSO-d6) δ 12.13（s，
1H）,10.78 (s, 1H), 8.45 (s, 1H), 8.03 (d, J = 8.9 Hz, 2H), 7.76 (d, J = 6.9 Hz, 5H), 7.62 (d, J = 3.4 Hz, 1H),
7.34 (d, J = 8.9 Hz, 2H), 6.59 (d, J = 3.5 Hz, 1H), 2.69 (s,3H).
N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-5-(trifluoromethyl)-1-(2-(trifluoromethyl)pheny
l)-1H- 1,2,3-triazole-4-carboxamide (15f)
Yield: 67%; M.P.: 213–215℃; ESI-MS m/z:533.11 [M+H] + ; 1H NMR (400 MHz, DMSO-d6) δ 12.12（s，
1H）, δ 11.30 (s, 1H), 8.45 (s, 1H), 8.25 (d, J = 7.2 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.17 – 8.08 (m, 2H),
8.01 (d, J = 8.9 Hz, 2H), 7.63 (d, J = 3.4 Hz, 1H), 7.38 (d, J = 8.9 Hz, 2H), 6.61 (d, J = 3.5 Hz, 1H).
N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-fluorophenyl)-1-(3-chloro-4-fluorophenyl)-5-(trifluor
o- methyl)-1H-1,2,3-triazole-4-carboxamide (16a)
Yield: 71%; M.P.: 209–211℃; ESI-MS m/z: 536.06 [M+H] +; 1H NMR (400 MHz, DMSO-d6) δ 12.14（s，
1H）,11.26 (s, 1H), 8.27 (s, 1H), 8.06 (d, J = 10.2 Hz, 1H), 7.88 (d, J = 12.8 Hz, 1H), 7.79 – 7.72 (m, 1H),
7.67 (d, J = 6.2 Hz, 2H), 7.51 (d, J = 3.5 Hz, 1H), 7.45 – 7.40 (m, 1H), 6.58 (d, J = 3.5 Hz, 1H)
N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-fluorophenyl)-1-(3,4-difluorophenyl)-5-(trifluoro-
methyl)-1H-1,2,3-triazole-4-carboxamide (16b)
+ 1
Yield: 67%; M.P.: 213–214℃; ES I-MS m/z: 519.09 [M+H] ; H NMR (400 MHz, DMSO-d6) δ12.28 (s,
1H),11.26 (s, 1H), 8.27 (s, 1H), 8.05 (t, J = 8.9 Hz, 1H), 7.88 (d, J = 12.6 Hz, 1H), 7.76 – 7.70 (m, 1H),
7.69 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 3.5 Hz, 1H), 7.44 – 7.40 (m, 1H), 6.58 (d, J = 3.4 Hz, 1H).
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N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-fluorophenyl)-1-(4-chlorophenyl)-5-(trifluoromethyl)-
1H-1,2,3-triazole-4-carboxamide (16c)
Yield: 72%; M.P.: 211–212℃; ES I-MS m/z:518.06 [M+H] +;1H NMR (400 MHz, DMSO-d6) δ 12.29 (s,
1H), 11.27 (s, 1H), 8.29 (s, 1H), 7.93 (d, J = 12.9 Hz, 1H), 7.82 – 7.75 (m, 4H), 7.72 (d, J = 8.7 Hz, 1H),
7.51 (t, J = 2.9 Hz, 1H), 7.45 (t, J = 8.9 Hz, 1H), 6.61 (s, 1H)
N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl)-5-methyl-1H-1,2,3-
triazole-4-carboxamide (16d)
+ 1
1
Yield: 58%; M.P.: 218–219℃; ESI-MS m/z: 447.13 [M+H] ; H NMR (400 MHz, DMSO-d6) H NMR
(400 MHz, ) δ 12.27 (s, 1H), 10.83 (s, 1H), 8.29 (s, 1H), 7.97 (d, J = 12.8 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H),
7.66 (m, 4H), 7.50 (s, 1H), 7.40 (t, J = 8.9 Hz, 1H), 6.59 (s, 1H), 2.58 (s, 3H).; 13C NMR (101 MHz,
DMSO-d6) δ 160.43, 159.18, 154.22, 152.02, 151.79, 149.45, 137.47, 136.75, 134.73, 129.58, 129.20,
129.09(2C), 124.95(2C), 123.62, 116.20, 108.15, 103.69, 101.01, 96.70, 8.97.
N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-fluorophenyl)-5-methyl-1-phenyl-1H-1,2,3-triazole-4-
carboxamide (16e)
Yield: 69%; M.P.: 214–216℃; ES I-MS m/z: 429.14 [M+H] +;1H NMR (400 MHz, DMSO-d6) δ 12.28 (s,
1H), 10.84 (s, 1H), 8.29 (s, 1H), 7.98 (d, J = 13.3 Hz, 1H), 7.76 (d, J = 9.2 Hz, 1H), 7.70 – 7.57 (m, 5H),
7.50 (s, 1H), 7.40 (t, J = 9.0 Hz, 1H), 6.60 (s, 1H), 2.58 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 161.76,
160.81, 158.59, 153.54, 152.40, 149.94, 142.85, 141.29, 136.96, 129.68, 127.91(2C), 125.57, 124.46,
116.47, 115.52(2C), 108.34, 104.02, 101.50, 97.77, 9.46.
N-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-3-fluorophenyl)-5-(trifluoromethyl)-1-(2-(trifluorometh
yl)phenyl)-1H-1,2,3-triazole-4-carboxamide (16f)
+ 1
Yield:65%; M.P.: 217–219℃; ES I-MS m/z: 551.10 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 12.40 (s,
1H), 11.44 (s, 1H), 8.41 (s, 1H), 8.25 (d, J = 7.6 Hz, 1H), 8.21 (d, J = 7.5 Hz, 1H), 8.17 – 8.08 (m, 2H),
8.05 (d, J = 12.9 Hz, 1H), 7.85 (d, J = 9.0 Hz, 1H), 7.63 (s, 1H), 7.57 (t, J = 8.7 Hz, 1H), 6.72 (s, 1H).
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1-(3-Chloro-4-fluorophenyl)-N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-5-(trifluo
ro-methyl)-1H-1,2,3-triazole-4-carboxamide (17a)
+ 1
Yield: 59%; M.P.: 213–215℃; ES I-MS m/z: 532.08 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 11.13 (s,
1H), 8.33 (s, 1H), 8.21 (dd, J = 6.4, 2.4 Hz, 1H), 7.89 (d, J = 9.0 Hz, 2H), 7.86 – 7.83 (m, 1H), 7.76 (t, J =
8.9 Hz, 1H), 7.51 (d, J = 3.4 Hz, 1H), 7.26 (d, J = 8.9 Hz, 2H), 6.51 (d, J = 3.4 Hz, 1H), 3.82 (s, 3H).
1-(3,4-Difluorophenyl)-N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-5-(trifluoromet
hyl)-1H-1,2,3-triazole-4-carboxamide (17b)
+ 1
Yield: 63%; M.P.: 217–219℃; ES I-MS m/z: 515.12 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 11.13 (s,
1H), 8.33 (s, 1H), 8.10 (t, J = 7.7 Hz, 2H), 7.80 (dd, J = 18.5, 9.1 Hz, 1H), 7.72 (s, 1H), 7.51 (d, J = 3.4 Hz,
1H), 7.26 (d, J = 8.8 Hz, 2H), 6.51 (d, J = 3.4 Hz, 1H), 3.82 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ
161.66, 160.10, 156.40, 152.45, 150.05, 148.95, 142.11, 135.27, 131.60, 129.27, 124.45, 122.20(2C),
121.78(2C), 118.63, 118.44, 117.06, 116.85, 104.69, 101.49, 97.37, 39.90, 31.18.
1-(4-Chlorophenyl)-N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-5-(trifluoromethyl
)-1H-1,2,3-triazole-4-carboxamide (17c)
+ 1
Yield: 65%; M.P.: 220–221℃; ES I-MS m/z: 514.09 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 11.11 (s,
1H), 8.34 (s, 1H), 7.88 (d, J = 8.8 Hz, 2H), 7.82 – 7.74 (m, 4H), 7.51 (d, J = 3.3 Hz, 1H), 7.26 (d, J = 8.7
Hz, 2H), 6.50 (d, J = 3.3 Hz, 1H), 3.82 (s, 3H).
1-(4-Fluorophenyl)-5-methyl-N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1H-1,2,3
-triazole-4-carboxamide (17d)
+
1
Yield: 71%; M.P.: 218–219℃; ES I-MS m/z: 443.15 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 10.78
(s, 1H), 8.45 (s, 1H), 8.03 (d, J = 8.9 Hz, 2H), 7.86 (dd, J = 8.8, 4.9 Hz, 2H), 7.62 (dd, J = 10.6, 6.8 Hz,
3H), 7.34 (d, J = 8.9 Hz, 2H), 6.59 (d, J = 3.4 Hz, 1H), 3.93 (s, 3H), 2.60 (s, 3H); 13C NMR (101 MHz,
DMSO-d6) δ 161.26, 160.82, 158.92, 152.01, 149.59, 148.00, 137.43, 135.32, 131.18, 128.69, 127.53(2C),
121.41(2C), 121.16(2C), 116.27(2C), 104.24, 101.00, 96.92, 30.67, 8.86.
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5-Methyl-N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-phenyl-1H-1,2,3-triazole-4
-carboxamide (17e)
Yield: 69%; M.P.: 215–218℃; ES I-MS m/z: 425.16 [M+H] +;1H NMR (400 MHz, DMSO-d6) δ 10.78 (s,
1H), 8.45 (s, 1H), 8.03 (d, J = 8.9 Hz, 2H), 7.76 (d, J = 6.9 Hz, 5H), 7.62 (d, J = 3.4 Hz, 1H), 7.34 (d, J =
8.9 Hz, 2H), 6.59 (d, J = 3.5 Hz, 1H), 3.93 (s, 3H), 2.69 (s,3H); 13C NMR (101 MHz, DMSO-d6) δ 161.75,
159.45, 152.43, 150.08, 148.38, 138.17, 137.66, 135.86, 135.25, 129.69(2C), 129.19, 125.43(2C), 122.55,
121.97(2C), 121.63(2C), 104.68, 97.40, 39.49, 31.18, 9.46.
N-(4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-5-(trifluoromethyl)-1-(2-(trifluoromet
hyl)
phenyl)-1H-1,2,3-triazole-4-carboxamide (17f)
+ 1
Yield: 72%; M.P.: 224–225℃; ES I-MS m/z: 547.12 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 11.29 (s,
1H), 8.45 (s, 1H), 8.25 (d, J = 7.2 Hz, 1H), 8.20 (d, J = 7.6 Hz, 1H), 8.15 – 8.08 (m, 2H), 8.01 (d, J = 8.9
Hz, 2H), 7.63 (d, J = 3.4 Hz, 1H), 7.38 (d, J = 8.9 Hz, 2H), 6.61 (d, J = 3.5 Hz, 1H), 3.93 (s, 3H); 13C
NMR (101 MHz, DMSO-d6) δ 161.16, 155.61, 152.00, 149.58, 148.60, 141.49, 134.67, 133.80, 132.38,
131.53, 129.52, 128.77, 127.20, 124.76, 123.38, 121.65(2C), 121.44(2C), 119.53, 104.24, 101.00, 96.89,
30.68.
1-(3-Chloro-4-fluorophenyl)-N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-
5-(trifl--uoromethyl)-1H-1,2,3-triazole-4-carboxamide (18a)
+ 1
Yield: 65%; M.P.: 213–214℃; ES I-MS m/z: 550.07 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 11.26 (s,
1H), 8.27 (s, 1H), 8.16 (dd, J = 6.5, 2.5 Hz, 1H), 7.88 (dd, J = 12.7, 2.2 Hz, 1H), 7.83 – 7.77 (m, 1H), 7.70
(dd, J = 16.2, 7.7 Hz, 2H), 7.51 (d, J = 3.5 Hz, 1H), 7.39 (t, J = 8.8 Hz, 1H), 6.58 (d, J = 3.5 Hz, 1H), 3.77
(s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 160.34, 159.50, 157.00, 156.11, 154.27, 152.03, 149.41, 141.25,
136.08, 135.17, 131.60, 129.16, 128.70, 127.36, 123.88, 117.51, 117.28, 116.43, 108.55, 108.32, 103.68,
96.68, 30.74.
1-(3,4-Difluorophenyl)-N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-5-
(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamide (18b)
+ 1
Yield: 63%; M.P.: 214–216℃; ES I-MS m/z: 533.11 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 11.26 (s,
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1H), 8.27 (s, 1H), 8.05 (t, J = 8.9 Hz, 1H), 7.88 (d, J = 12.6 Hz, 1H), 7.76 – 7.70 (m, 1H), 7.67 (d, J = 8.0
Hz, 2H), 7.51 (d, J = 3.5 Hz, 1H), 7.39 (t, J = 8.9 Hz, 1H), 6.58 (d, J = 3.4 Hz, 1H), 3.74 (s, 3H).
1-(4-Chlorophenyl)-N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-5-(trifluo
ro-
methyl)-1H-1,2,3-triazole-4-carboxamide (18c)
Yield: 71%; M.P.: 217–218℃; ES I-MS m/z: 532.08 [M+H] +;1H NMR (400 MHz, DMSO-d6) δ 11.24 (s,
1H), 8.27 (s, 1H), 7.88 (d, J = 12.4 Hz, 1H), 7.73 (m, 3H), 7.70 – 7.64 (m, 2H), 7.51 (d, J = 3.5 Hz, 1H),
7.39 (t, J = 8.9 Hz, 1H), 6.58 (d, J = 3.4 Hz, 1H), 3.77 (s, 3H).
N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-fluorophenyl)-5-methyl-
1H-1,2,3-triazole-4-carboxamide (18d)
Yield: 66%; M.P.: 219–220℃; ES I-MS m/z: 461.14 [M+H] +;1H NMR (400 MHz, DMSO-d6) δ 10.88 (s,
1H), 8.34 (s, 1H), 7.98 (dd, J = 13.0, 2.3 Hz, 1H), 7.79 – 7.72 (m, 2H), 7.56 (d, J = 3.5 Hz, 1H), 7.52 (dd, J
= 11.9, 5.5 Hz, 3H), 7.40 (t, J = 8.9 Hz, 1H), 6.63 (d, J = 3.5 Hz, 1H), 3.83 (s, 3H), 2.56 (s, 3H).
N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-5-methyl-1-phenyl-1H-1,2,3
-triazole-4-carboxamide (18e)
Yield: 67%; M.P.: 218–221℃; ES I-MS m/z: 443.15 [M+H] +;1H NMR (400 MHz, DMSO-d6) δ 10.87 (s,
1H), 8.34 (s, 1H), 7.98 (dd, J = 13.0, 2.2 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.73 – 7.61 (m, 5H), 7.56 (d, J
= 3.5 Hz, 1H), 7.41 (t, J = 9.0 Hz, 1H), 6.63 (d, J = 3.5 Hz, 1H), 3.83 (s, 3H), 2.58 (s, 3H); 13C NMR (101
MHz, DMSO-d6) δ 160.43, 159.18, 154.22, 152.02, 151.79, 149.45, 137.47, 136.75, 134.73, 129.58,
129.20(2C), 129.09, 124.95(2C), 123.62, 116.18, 108.07, 103.69, 101.00, 96.70, 30.74, 8.97.
N-(3-fluoro-4-((7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)phenyl)-5-(trifluoromethyl)-1-(2-
( trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-carboxamide (18f)
+ 1
Yield: 69%; M.P.: 221–224℃; ES I-MS m/z: 565.11 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 11.36 (s,
1H), 8.34 (s, 1H), 8.12 (dd, J = 14.0, 7.4 Hz, 2H), 8.07 – 7.90 (m, 3H), 7.74 (d, J = 8.1 Hz, 1H), 7.56 (s,
1H), 7.46 (t, J = 8.8 Hz, 1H), 6.64 (d, J = 3.4 Hz, 1H), 3.83 (s, 3H); 13C NMR (101 MHz, DMSO) δ 160.34,
155.82, 154.25, 152.04, 151.82, 149.43, 141.12, 136.07, 135.36, 135.24, 133.80, 132.41, 131.82, 131.49,
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129.51, 129.18, 127.21, 123.88, 116.56, 108.69, 108.46, 103.69, 96.68, 30.75.
1-(3-Chloro-4-fluorophenyl)-N-(4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-5-
(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamide (19a).
Yield: 57%; M.P.: 221–225℃; ES I-MS m/z: 533.08 [M+H] +; 1H NMR (400 MHz, DMSO-d6) δ 11.17 (s,
1H), 8.54 (s, 1H), 8.25 – 8.19 (m, 1H), 8.17 (s, 1H), 7.93 (d, J = 8.9 Hz, 2H), 7.89 – 7.83 (m, 1H), 7.76 (t,
J = 8.9 Hz, 1H), 7.34 (d, J = 8.9 Hz,2H), 4.04 (s, 3H).
1-(3,4-Difluorophenyl)-N-(4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-5-
(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamide (19b)
Yield: 64%; M.P.: 223–224℃; ES I-MS m/z: 516.11 [M+H] +;1H NMR (400 MHz, DMSO-d6) δ 11.10 (s,
1H), 8.47 (s, 1H), 8.10 (s, 1H), 8.04 (dd, J = 10.0, 7.0 Hz, 1H), 7.86 (d, J = 8.7 Hz, 2H), 7.73 (q, J = 9.1
Hz, 1H), 7.65 (s, 1H), 7.27 (d, J = 8.7 Hz, 2H), 3.97 (s, 3H).
1-(4-Chlorophenyl)-N-(4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-5-(trifluorometh
yl)-1H-1,2,3-triazole-4-carboxamide (19c)
+ 1
Yield: 65%; M.P.: 225–226℃; ES I-MS m/z: 515.09 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 11.08 (s,
1H), 8.47 (s, 1H), 8.10 (s, 1H), 7.85 (d, J = 8.9 Hz, 2H), 7.72 (dd, J = 8.2, 2.8 Hz, 4H), 7.27 (d, J = 8.7 Hz,
2H), 3.97 (s, 3H).
1-(4-Fluorophenyl)-5-methyl-N-(4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-1H-1,2,
3-triazole-4-carboxamide (19d)
Yield: 69%; M.P.: 224–226℃; ES I-MS m/z: 444.15 [M+H] +;1H NMR (400 MHz, DMSO-d6) δ 10.63 (s,
1H), 8.47 (s, 1H), 8.05 (s, 1H), 7.89 (d, J = 8.9 Hz, 2H), 7.68 (dd, J = 8.8, 5.0 Hz, 2H), 7.44 (t, J = 8.7 Hz,
2H), 7.23 (d, J = 8.9 Hz, 2H), 3.97 (s, 3H).
5-Methyl-N-(4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-1-phenyl-1H-1,2,3-triazole
-4-carboxamide (19e).
Yield: 65%; M.P.: 226–228℃; ES I-MS m/z: 426.16 [M+H] +;1H NMR (400 MHz, DMSO-d6) δ 10.62 (s,
1H), 8.48 (s, 1H), 8.04 (s, 1H), 7.89 (d, J = 9.0 Hz, 2H), 7.62 – 7.51 (m, 5H), 7.23 (d, J = 8.9 Hz, 2H), 4.01
(s, 3H).
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N-(4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-5-(trifluoromethyl)-1-(2-
(trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-carboxamide (19f).
+ 1
Yield: 66%; M.P.: 227–229℃; ES I-MS m/z: 548.12 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 11.14 (s,
1H), 8.48 (s, 1H), 8.10 – 8.00 (m, 2H), 7.94 (p, J = 7.5 Hz, 3H), 7.88 – 7.84 (m, 2H), 7.28 (d, J = 9.0 Hz,
2H), 3.97 (s, 3H).
1-(3-Chloro-4-fluorophenyl)-N-(3-fluoro-4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)
-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamide (20a).
+ 1
Yield: 67%; M.P.: 227–228℃; ES I-MS m/z: 551.07 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 11.38 (s,
1H), 8.58 (s, 1H), 8.42 (s, 1H), 8.24 (dd, J = 6.6, 2.6 Hz, 1H), 8.01 (dd, J = 12.7, 2.4 Hz, 1H), 7.89 (q, J =
4.2 Hz, 1H), 7.82 – 7.74 (m, 2H), 7.54 (t, J = 8.8 Hz, 1H), 4.09 (s, 3H).
1-(3,4-Difluorophenyl)-N-(3-fluoro-4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-5-
(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamide (20b)
+ 1
Yield: 65%; M.P.: 225–227℃; ES I-MS m/z: 534.10 [M+H] ; H NMR (400 MHz, DMSO-d6) δ 11.37 (s,
1H), 8.57 (s, 1H), 8.41 (s, 1H), 8.13 (s, 1H), 7.99 (d, J = 12.3 Hz, 1H), 7.79 (dt, J = 17.7, 9.4 Hz, 3H), 7.53
(t, J = 8.9 Hz, 1H), 4.08 (s, 3H).
1-(4-Chlorophenyl)-N-(3-fluoro-4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-5-
(trifluoromethyl)-1H-1,2,3-triazole-4-carboxamide (20c)
+
Yield:68%; M.P.: 230–231℃; ES I-MS m/z: 533.08 [M+H] ; 1H NMR (400 MHz, DMSO-d6) δ 11.35 (s,
1H), 8.58 (s, 1H), 8.41 (s, 1H), 8.11 (s, 1H), 7.99 (d, J = 12.8 Hz, 1H), 7.84 – 7.69 (m, 4H), 7.53 (t, J = 8.9
Hz, 1H), 4.08 (s, 3H).
N-(3-fluoro-4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-1-(4-fluorophenyl)-5-methyl
-1H-1,2,3-triazole-4-carboxamide (20d).
Yield: 67%; M.P.: 223–225℃; ES I-MS m/z: 462.14 [M+H] +;1H NMR (400 MHz, DMSO-d6) δ 10.94 (s,
1H), 8.58 (s, 1H), 8.39 (s, 1H), 8.04 (d, J = 12.8 Hz, 1H), 7.81 (d, J = 8.7 Hz, 2H), 7.79 – 7.73 (m, 1H),
7.51 (dt, J = 17.6, 8.8 Hz, 3H), 4.07 (s, 3H), 2.58 (s, 3H).
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N-(3-fluoro-4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-5-methyl-1-phenyl-1H-1,2,3-
triazole-4-carboxamide (20e).
Yield: 72%; M.P.: 226–228℃; ES I-MS m/z: 444.15 [M+H] +;1H NMR (400 MHz, DMSO-d6) δ 10.94 (s,
1H), 8.58 (s, 1H), 8.40 (s, 1H), 8.04 (d, J = 13.0 Hz, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.68 (s, 5H), 7.49 (t, J =
8.6 Hz, 1H), 4.08 (s, 3H), 2.60 (s, 3H).
N-(3-fluoro-4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-5-(trifluoromethyl)-1-(2-
(trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-carboxamide (20f)
Yield: 67%; M.P.: 228–231℃; ES I-MS m/z: 566.11 [M+H] +;1H NMR (400 MHz, DMSO-d6) δ 11.42 (s,
1H), 8.58 (s, 1H), 8.41 (s, 1H), 8.14 (dd, J = 13.9, 7.5 Hz, 2H), 8.07 – 7.97 (m, 3H), 7.78 (d, J = 9.0 Hz,
1H), 7.54 (t, J = 8.9 Hz, 1H), 4.08 (s, 3H).
2.2 Biology
2.2.1 Cytotoxicity assay in vitro
The cytotoxic activities of target compounds (15a-f, 16a-f, 17a-f, 18a-f, 19a-f and 20a-f) were
evaluated with A549, HepG2 and MCF-7cell 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 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.
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2.2.2 Tyrosine kinases assay in vitro
The selected compounds ( 16d, 18d and 20d ) are tested for their activity against one or several
tyrosine kinases (c-Met, Flt-3, VEGFR-2, c-Kit and EGFR kinases) through the mobility shift assay.[18,19]
(The rationale of measuring the activity of different types of tyrosine kinases : the tyrosine kinase can
catalyze the phosphorylation of the substrate, and this process requires the involvement of ATP. Then the
activity of the sample enzyme was estimated by making a standard curve after measuring the radioactivity
of the reaction product.) 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 MgCl 2, 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 500µM by 100% DMSO, then transfer 10 µL of compound to a new
96-well plate as the intermediate plate, add 90µL 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 10µL peptide
solution to each well. Incubate at 28℃ 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.
2. 3 Acridine orange (AO) single staining
The cancer cell apoptotic of target compounds 16d were evaluated with HepG2 cancer cell lines by
acridine orange (AO) single staining. The cancer cell lines were cultured in minimum essential medium
(MEM) supplement with 10% fetal bovine serum (FBS). Approximately 2×103 cells, suspended in MEM
medium, were plated onto each well of a 24-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 12 h. Fresh acridine orange was added to each well at a terminal concentration of 10μg/mL
and keep in dark place to stained for 15 min. Then, the staining cells washed three times with PBS and
placed under fluorescence microscopy to observe cell morphology.
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2. 4 Analyzing of cell cycle
HepG2 cells were seeded in 16-well plates at a density of 1 × 106 cells/well in DMEM, then treated
with 2.03 µM concentration of 16d and treated with 2.42 µM concentration of Foretinib for 12 h,
respectively. Cultured cells were stained with propidium iodide (PI) in the dark at 4 ℃ for 30 min and
analyzed by ACEA NovoCyteTM [20]
.
2. 5 Docking studies
For docking purposes, the three-dimensional structure of the c-Met (PDB code: 3LQ8) was obtained
from RCSB Protein Data Bank.[7]Hydrogen atoms were added to the structure allowing for appropriate
ionization at physiological pH. The protonated state of several important residue were adjusted by using
AutoDock vina 1.1.2 in favor of forming reasonable hydrogen bond with the ligand. And using AutoDock
vina 1.1.2 to produce twenty ligand conformation, the best molecular conformation was used as a ligand.
Molecular docking analysis was carried out by the Discover Studio 2.5 Visualization 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 AutoDock calculations. All calculations were performed on Silicon Graphics workstation.
3. Results and discussion
3.1 Chemistry
The key intermediates 9a-f were synthesized from 4-cholor-1-substituted-pyrrolo[2,3-d]pyrimdine
7a-b or 4-cholor-1-methyl-pyrrolo[2,3-d]pyrimidine 7c via substitution reaction with 4-aminophenol or
2-fluoro-4-aminophenol 8a-b as show in Scheme 1.
The intermediates 1-aryl-5-methyl (or trifluoromethyl)-1,4,5-trisubstituted-1,2,3-triazoles 12a-f were
synthesized in high yield by a one-pot three-component reaction of arylboronic acids 10a-g, sodium
azide,and active methylene ketones, such as ethyl acetoacetate (or ethyl 4,4,4-trifluoroacetoacetate) 11a-b
[12]
in the presence of Cu(OAc)2 and piperidine using a DMSO/H2O (10:1) mixture as solvent.
Following
by hydrolysis reaction and chlorination reaction, 13a-f and 14a-f were obtained. Finally, substitution
reaction of amides 9a-d and 9e-f with carbonyl-chloride 14a-f promoted by DIPEA in dichloromethane at
room temperature to obtain the target compounds 15a-f, 16a-f, 17a-f, 18a-f, 19a-f and 20a-f, respectively.
(Scheme 1 should be here)
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3.2 Biology
Taking Foretinib as reference compound, the target compounds (15a-f, 16a-f, 17a-f, 18a-f, 19a-f and
20a-f) were evaluated for the cytotoxicityagainst three cancer cell lines A549, HepG2 and MCF-7 by
3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. The selected
compounds ( 16d, 18d and 20d ) are tested for their activity against one or several tyrosine kinases (c-Met,
Flt-3, VEGFR-2, c-Kit and EGFR kinases) through the mobility shift assay. The results expressed as IC50
values were summarized in Tables 1-3 and the values are the average of at least two independent
experiments.
Table 1. Activity of target compounds 15a-f, 16a-f, 17a-f, 18a-f, 19a-f and 20a-f.
IC50a (μM)
Compounds
R1
Z
R2
No.
A549
HepG2
MCF-7
NA
15a
15b
15c
15d
15e
15f
H
H
H
H
H
H
F
CF3
CF3
CF3
CH3
CH3
CF3
CF3
CF3
CF3
CH3
CH3
CF3
CF3
CF3
3-F-4-Cl
3-F-4-F
4-Cl
27.54 ± 2.68
13.36 ± 2.34
19.76 ± 1.98
12.53 ± 1.22
32.21 ± 2.28
NA
56.84 ± 4.71
14.13 ± 3.26
23.63 ± 3.14
6.20 ± 0.98
37.91 ± 2.92
12.54 ± 2.41
27.44 ± 3.83
6.82 ± 0.97
11.41 ± 2.32
2.03 ± 0.39
18.30 ± 2.76
8.05 ± 1.07
NA
NA
32.06 ± 2.47
4.71 ± 0.67
NA
4-F
H
2-CF3
3-F-4-Cl
3-F-4-F
4-Cl
39.34 ± 3.27
NA
16a
16b
16c
16d
16e
16f
10.53 ± 2.12
5.11 ± 0.43
7.56 ± 1.17
4.79 ± 0.82
12.32 ± 1.22
31.92 ± 3.56
42.35 ± 2.26
21.74 ± 2.21
F
49.28 ± 4.67
22.5 ± 2.79
2.90 ± 0.43
NA
F
F
4-F
F
H
F
2-CF3
3-F-4-Cl
3-F-4-F
27.61 ± 3.81
NA
17a
17b
H
H
30.32 ± 3.28
NA
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17c
17d
17e
17f
H
H
H
H
F
CF3
CH3
CH3
CF3
CF3
CF3
CF3
CH3
CH3
CF3
CF3
CF3
CF3
CH3
CH3
CF3
CF3
CF3
CF3
CH3
CH3
CF3
4-Cl
4-F
25.44 ± 1.74
20.37 ± 1.36
46.37 ± 2.64
NA
46.68 ± 3.32
7.83 ± 0.87
45.64 ± 2.84
35.62 ± 3.54
31.21 ± 2.56
9.84 ± 1.17
16.47 ± 2.18
5.37 ± 1.68
20.18 ± 2.26
14.61 ± 1.37
NA
NA
9.48±1.52
NA
H
2-CF3
3-F-4-Cl
3-F-4-F
4-Cl
NA
18a
18b
18c
18d
18e
18f
15.74 ± 1.83
11.93 ± 1.36
8.92 ± 1.52
7.54 ± 1.27
23.25 ± 1.58
51.32 ± 3.85
NA
NA
F
NA
F
34.51 ± 3.86
4.73 ± 1.02
NA
F
4-F
F
H
F
2-CF3
3-F-4-Cl
3-F-4-F
4-Cl
38.27 ± 3.68
NA
19a
19b
19c
19d
19e
19f
H
H
H
H
H
H
F
33.56 ± 2.75
42.36 ± 2.93
25.22 ± 2.68
NA
NA
NA
NA
NA
4-F
11.25 ± 1.27
NA
14.83 ± 1.54
NA
H
2-CF3
3-F-4-Cl
3-F-4-F
4-Cl
NA
NA
NA
20a
20b
20c
20d
20e
22.68 ± 1.52
19.62 ± 1.83
15.52 ± 2.08
10.34 ± 2.18
42.26 ± 2.79
NA
NA
F
15.83 ± 2.61
24.27 ± 2.63
9.72 ± 1.25
30.22 ± 2. 31
NA
F
49.34 ± 3.92
6.43 ± 1.76
NA
F
4-F
F
H
20f
F
2-CF3
-
NA
30.47 ± 2.66
2.42 ± 0.25
NA
Foretinibb
3.71 ± 0.41
4.89 ± 0.62
aThe values are an average of two separate determinations.
bUsed as a positive control.
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c NA: Not active (IC50> 60 μM).
Table 2. c-Met kinase inhibitory activity of selected compounds16d, 18d and 20d.
IC50a (μM)
c-Met
Compounds No.
16d
18d
0.79 ± 0.11
1.23 ± 0.13
1.67 ± 0.19
0.019 ± 0.003
20d
Foretinibb
aThe values are an average of two separate determinations.
bUsed as a positive control.
Table 3 Inhibition of tyrosine kinases by compound 16d
Kinase
Flt-3
Enzyme IC 50 (μM)
5.23 ± 0.35
27.6 ± 1.25
20.2 ± 1.42
>100
VEGFR-2
c-Kit
EGFR
As showed in Tables 1-3, some of compounds displayed excellent anticancer activity against three
cancer cells lines (A549, HepG2 and MCF-7) with single-digit µM. According to the Table 1-3, we could
know that the first four series of compounds (15a-f, 16a-f, 17a-f and 18a-f) showed more activity than the
other two series of compounds (19a-f and 20a-f). And The most promising compound 16d exhibited the
best activity against A549, HepG2 and MCF-7 cell lines with the IC50 values of 4.79 ± 0.82 µM, 2.03 ±
0.39 µM and 2.90 ± 0.43 µM, which were equal to the lead drug Foretinib, respectively. The results
suggested that the pyrrolo[2,3-b]pyrimidine moiety was a privileged scaffold for anticancer activity.
What’s more, those compounds without substituents (Y = H) at N-7 position of pyrrolopyrimidines/
pyrazolopyrimidines moiety show more activity than that substituted with -CH3 group. That’s why the first
and second series compounds (15a-f and 16a-f) were more activity than that of the third and fourth series
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(17a-f and 18a-f).
Then, R1 group (R1= H/F) were introduced to C-2 position of aminophenoxy moiety to investigate the
effect of electron withdrawing effect to the target compounds activity. The result revealed that the
aminophenoxy moiety substituting with substituent (R1= F) was benefit to the activity of the target
compounds (16a-f) compared with H-atom substituent (15a-f). What's more,, the same trend also was
observed in compounds 18a-f/17a-f and 20a-f/19a-f.
Furthermore, different substituents at aryl group also affected the cytotoxicity of target compounds. In
general, it’s seem to be that target compounds with electron drawing groups (EWGs) show better in vitro
activity, such as compounds 15b, 15d, 16b-d, 18b-d and so on. Among these compounds, the EWGs on
the 4-C position of aryl was more preferred, such as compounds 15d, 16c-d, 18c-d and so on. Especially,
the compound 16d substituted with F atom on the 4-C position of aryl showed the best activity.
Activity against c-Met kinase of compounds 16d (IC50 = 0.79 ± 0.11 μM), 18d (IC50 = 1.23 ± 0.13 μM)
and 20d (IC50 = 1.67 ± 0.19 μM) was further carried out in this paper to investigate the in vitro activity of
these compounds. According to the results of Table 2, we could easily find that the three selected
compounds showed moderate to excellent inhibitory on c-Met kinase. Compounds 16d showed more
active than the compounds 18d and 20d, with IC50 value of 0.79 μM against c-Met kinase.
In order to examine whether compound 16d was a selectivity c-Met inhibitor or not, experiments of
enzyme-based selectivity was carried out to meansure the another four tyrosine kinases (Flt-3, VEGFR-2,
c-Kit and EGFR ). As shown in Table 3, compound 16d exhibited medium inhibitory effects against Flt-3
(IC50 = 5.23 ± 0.35 μM),VEGFR-2 (IC 50 = 27.6 ± 1.25 μM), c-Kit (IC 50 = 20.2 ± 1.42 μM) and EGFR
(IC 50 >100 μM), which was 5.9-, 31.0-, 22.7- and 112.3- fold lower than that of c-Met. These data
indicated that compound 16d could inhibit the c-Met kinase selectively.
3.3 Acridine orange (AO) single staining
As showed in the Figure.3, the control group cell (Fig. 3a) was stained with acridine orange (AO) and
shape of the cell was full and the edge was clear, which treated with nothing. But in the Fig. 3b, the cell
shape was abnormal with cell shrinkage, chromatin condensation and the DNA fragments were stained
with Orange after 2.03 ± 0.39 μM concentration of compound 16d acted on the HepG2 cells. It claimed
that the compounds 16d could induce apoptosis of HepG2 cells.
(Fig. 3 should be here)
3.4 Analyzing of cell cycle
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The cell growth inhibitory potency of the tested compounds prompted us to evaluate their effects on
the cell mitosis. For cell cycle progression analysis, the effect of the 16d at IC50 concentrations on cell
cycle progression was investigated in HepG2 cells via flow cytometry. As shown in Fig.4, 16d arrested
efficiently the cell cycle progression in G2/ M phase, with increasing the percentage of cells in G2/M
phase in a IC50 concentration (2.03 μM ) from 26.85 to 49.09 compared with Foretinib (2.42 μM). And the
index of Coefficient of Variation (CV) G1 and G2 are below the 7% which show that the data is reliable.
(Fig. 4 should be here)
3.5 Docking studies
3.5.1 Binding modes of target compounds with c-Met
To explore the binding modes of target compounds with the active site of c-Met, molecular docking
simulation studies were carried out by using AutoDock vina 1.1.2 and analyzed by Discover Studio 2.5
Visualization. The three selected compounds (16d, 18d and 20d) occupy the ATP site of c-Met kinase and
all can form hydrogen bond with c-Met in the pyrrolo[2,3-d]pyrimidines moiety and 1,2,3-triazole moiety
(Fig. 5a). And basing on the in vitro inhibition results, we selected compound 16d, our best c-Met inhibitor
in this study, as the ligand example, and the c-Met protein was selected as the docking model (PDB ID
code: 3LQ8 ).The binding modes of compound 16d and c-Met were depicted in Fig.5b, Fig.5c. Visual
inspection of the pose of compound 16d into c-Met binding site revealed that compound 16d was tightly
embedded into the active binding pocket . In the binding mode, compound 16d is potently bound to the
active binding site of c-Met via four hydrogen bond and one pi–pi interactions. The nitrogen atom and
hydrogen atom of pyrrolo[2,3-d]pyrimidines moiety formed two hydrogen bonds with MET A：1160; the
aminophenoxy moiety can form a pi-pi interaction with PHE A: 1223; the two nitrogen atom of
1,2,3-triazole moiety via two hydrogen bond interaction with ASP A: 1222; the aryl group is firmly
embedded in the hydrophobic pocket of the c-Met kinase so that show better activity. Contributing to the
hydrogen bond, pi-pi and hydrophobic interaction, it may be a probable explanation for its nice activity. In
general, these results of the molecular docking study showed that 1,2,3-triazole derivatives bearing
pyrrolo[2,3-d]pyrimidine moiety could act synergistically to interact with the active binding site of c-Met,
suggested that compound 16d may be a potential inhibitor of c-Met.
(Fig. 5 should be here)
3.5.2 Analysis of the interaction between c-Met and selected compounds 16d, 18d and 20d
To explore the strong and weak of activity of target compounds with c-Met, the binding modes of
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three selected compounds (16d, 18d and 20d) were labeled with distances between the c-Met and target
compounds, and the results were analyzed by Discover Studio Visualization 2.5 which described in Fig.6a,
Fig.6b and Fig.6c. What's more, all the information was summarized in the Fig.6d. In the bonding modes,
the distance between the 16d and c-Met is shorter than 18d and 20d, which claimed that the interaction
between 16d and c-Met is stronger than 18d and 20d. And it may be a reasonable explanation why the 16d
show the best activity among all the target compounds.
(Fig. 6 should be here)
4. Conclusion
In summary, we designed and synthesized six series of pyrrolopyrimidine and pyrazolopyrimidine
derivatives bearing 1,2,3-triazole moiety and evaluated for the IC50 values against three cancer cell lines
(A549, HepG2 and MCF-7). Some selected compounds (16d, 18d and 20d) were further evaluated for the
activity against c-Met, Flt-3,VEGFR-2, c-Kit and EGFR kinases. The most promising compound 16d
showed equal activity to lead compound Foretinib against A549, HepG2, MCF-7 cell lines, with the IC50
values of 4.79 ± 0.82µM, 2.03 ± 0.39 µM and 2.90 ± 0.43 µM, respectively. SARs and docking studies
indicated that pyrrolo[2,3-d]pyrimidine moiety may be a privileged structure compared with
pyrazolo[3,4-d]pyrimidine .And EWGs on the aryl group could increase the activity of compounds,
especially compound 16d which the 4-position of the aryl group substituted with F atom show the best
activity. What's more, 16d could induce apoptosis of HepG2 cells and inhibitor the cell cycle of HepG2 on
G2/M phase by acridine orange (AO) staining and Cell cycle experiments, respectively. And further study
found that 16d could inhibitor the c-Met kinase selectively by the experiment of enzyme-based selectivity.
According to these results, it's claimed that the pyrrolo[2,3-d]pyrimidine bearing 1,2,3-triazole moiety may
be a privileged scaffold and compound 16d may be a potential inhibitor of c-Met.
5. Acknowledgments
We gratefully acknowledge the generous support provided by The National Natural Science Funds of
China (No. 21662014,81460527), Outstanding Youth Foundation of Jiangxi, Natural Science Foundation
of Jiangxi, China (20171BCB23078), Natural Science Foundation of Jiangxi, China (20171ACB21052 &
20171BAB215073 & 20161BAB215216), Science and Technology Project Founded by the Education
Department of Jiangxi Province (GJJ160787), Graduate Students' Science and Technology Innovation
Project of Jiangxi Science & Technology Normal University (YC2016-X16).
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Conflict of interest
The authors declare that there are no conflicts of interests.
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Legends to the figures and schemes
Fig. 1. Structures of small-molecule c-Met inhibitors
Fig. 2. Structures and design strategy for target compounds 15a-f, 16a-f, 17a-f, 18a-f, 19a-f and 20a-f
Scheme 1. Synthetic route of target compounds
Reagents and conditions: (a) sodium carbonate, sodium hydroxide, tetrahydrofuran , 1,4-dioxane/H2O
(5:1), 80℃, 1 h; (b) Cu(OAc) 2 , DMSO/H 2 O (10:1),Piperidine, rt to 80 ℃, 24 h, 88–92%; (c) sodium
carbonate , ethanol/H2O(5:1), 80℃，3-5 h; (d) oxalyl chloride , DMF, CH2Cl2 , rt, 5 minute; (e) DIPEA,
CH2Cl2 , rt,0.5 h.
Fig. 3. Morphologic changes of HepG2 cells under inverted microscopy and fluorescence
microscopy
Fig. 4 . Identification of apoptotic stage of HepG2 by cell cycle. Cell cycle experiments were carried out to
verify the duration of apoptosis of cancer cells under the action of IC50 concentration of Foretinib (Fig.4a)
and 16d (Fig.4b).
Fig. 5 The protonated state of several important residue were adjusted by using AutoDock vina v1.02 in
favor of forming reasonable hydrogen bond with the ligand and Molecular docking analysis was carried
out by the Discover Studio Visualization to explore the binding model for the active site of c-Met with its
ligand. Docking simulations show that the three selected compounds (16d, 18d and 20d) occupy the ATP
site of c-Met kinase (Fig.5a). Then We employed 3D interaction map (Figure.5b) , 2D diagram (Figure.5c :
Foretinib was used as a template) to display the interaction between 16d and the targeted protein(3LQ8). In
the binding model, compound 16d is nicely bound to c-Met via four hydrogen bond and one pi–pi
interaction.
Fig. 6 The binding modes of three selected compounds (16d, 18d and 20d) were labeled with distances
between the c-Met and target compounds and analysed by Discover Studio 2.5, which described in Fig.6a,
Fig.6b and Fig.6c and all the information were summarized in the Fig.6d.
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