Discovery of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives as novel, potent and selective c-Met kinase inhibitors: Synthesis, SAR study, and biological activity

Article abstract of DOI:10.1016/j.ejmech.2018.03.049

A series of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives were designed, synthesized and evaluated for their biological activity. Most of these compounds showed potent activities against c-Met kinase and cell growth inhibition. The most promising compound, 7d, has the IC₅₀ values of 2.02 and 88 nM to inhibit c-Met kinase activity and cell growth in the MKN45 cell line, respectively. In addition, 7d is highly selective to c-Met and exhibits over 2500-fold selective inhibition to 16 tyrosine kinases evaluated.

Full text of DOI:10.1016/j.ejmech.2018.03.049

Accepted Manuscript
Discovery of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives as novel, potent and
selective c-Met kinase inhibitors: Synthesis, SAR study, and biological activity
Li Zhang, Jingyun Zhao, Beichen Zhang, Tao Lu, Yadong Chen
PII:
S0223-5234(18)30292-7
10.1016/j.ejmech.2018.03.049
EJMECH 10314
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 January 2018
Revised Date: 17 March 2018
Accepted Date: 17 March 2018
Please cite this article as: L. Zhang, J. Zhao, B. Zhang, T. Lu, Y. Chen, Discovery of [1,2,4]triazolo[3,4-
b][1,3,4]thiadiazole derivatives as novel, potent and selective c-Met kinase inhibitors: Synthesis,
SAR study, and biological activity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.03.049.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives as
novel, potent and selective c-Met kinase inhibitors: Synthesis, SAR
study, and biological activity
Li Zhanga, Jingyun Zhaoa, Beichen Zhanga, Tao Lub,c,* and Yadong Chenb,*
a
Department of Organic Chemistry, School of Science, China Pharmaceutical
University, 639 Longmian Avenue, Nanjing 211198, China
b
Laboratory of Molecular Design and Drug Discovery, School of Science, China
Pharmaceutical University, 639 Longmian Avenue, Nanjing 211198, China
c
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, China
Abstract: A series of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives were
designed, synthesized and evaluated for their biological activity. Most of these
compounds showed potent activities against c-Met kinase and cell growth inhibition.
The most promising compound, 7d, has the IC50 values of 2.02 and 88 nM to inhibit
c-Met kinase activity and cell growth in the MKN45 cell line, respectively. In
addition, 7d is highly selective to c-Met and exhibits over 2500-fold selective
inhibition to 16 tyrosine kinases evaluated.
Keywords: [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole, synthesis, c-Met, inhibitors, SAR
1. Introduction
Mesenchymal-epithelial transition factor (c-Met), the receptor of hepatocyte
growth factor (HGF), is a receptor tyrosine kinase (RTK) which plays an important
role in cell growth during embryo development and postnatal organ regeneration [1-5].
Overexpression or aberrant activation of c-Met has been reported to be associated
with the formation and development of many types of cancers in lung, breast and
gastric cancers [6-8]. Consequently, c-Met kinase has been considered as an attractive

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target for molecular targeted therapy in cancers. A number of small molecule
inhibitors binding to the ATP binding pocket of c-Met protein have been developed to
inhibit the activity of c-Met kinase (Fig. 1) [9-14]. Some of them have advanced to
the clinical trials currently or have been approved by U.S. Food and Drug
Administration [15]. Because of the high homology of RTKs, it remains a challenge
to develop a highly selective c-Met inhibitor which is expected to produce less side
effects than other RTKs in cancer treatment [16-18].
Fig. 1. Structures of reported c-Met kinase inhibitors
In our previous high throughput screening campaign study [19-20], we have
discovered
a
novel
c-Met
inhibitor,
HL-11,
containing
a
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole scaffold that inhibited c-Met kinase at an IC50
value of 0.19 µM. In the crystal structure of compound HL-11 with c-Met kinase
domain (Fig. 2), the [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole group of HL-11 interacts
with Tyr1230 via a π-π staking and forms a hydrogen bond with Asp1222. An
important hydrogen bond formed between the oxygen atom of methoxybenzene group
in HL-11 and Met1160 in the hinge region of c-Met protein, was considered to be
critical for the inhibition activity of HL-11 to c-Met. Another hydrogen bond
between the N-H on the indole ring of HL-11 residue Arg1208 is also identified in the
crystal structure (Fig. 2).

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Fig. 2. The crystal structure of HL-11 in c-Met kinase domain (PDB ID: 5YA5)
Herein,
we
designed
and
synthesized
a
series
of
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives based on the analysis of the crystal
structure between HL-11 and c-Met. These new compounds were evaluated for their
inhibition activities to c-Met kinase and cell growth in the PC-3, MKN45 and EBC-1
cancer cell lines. In addition, selectivity profile and inhibition of phosphorylation of
c-Met kinase in MKN45 cell line were determined in this work.
2. Chemistry
The synthetic routes of 2a, 2b and 4a-4c were shown in Scheme 1.
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives 2a and 2b were readily prepared
from
a
two-step cyclisation of 2-(4-hydroxyphenyl)acetic acid with
thiocarbohydrazide and carboxylic acid. Hydrolysis of 2b with NaOH afforded phenol
3. Subsequent, treatment of 3 with appropriate chlorides afforded 4a-4c in the
presence of Cs2CO3.
As
illustrated
in
Scheme
2,
nucleophilic
substitution
of
4-Chloro-7-methoxyquinoline and 2-hydroxyacetic acid afforded intermediate 5.
7a-7m were prepared from acid 5 using the similar method as those for the
preparation of 2a. Finally, treating 7c with appropriate bromides in the present of
KOH obtained compounds 8a-8c.

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Scheme 1. Reagents and conditions: a) thiocarbohydrazide, CH3SO3H, sulfolane, H2O,
90oC; b) different carboxylic acid, POCl3, sulfolane, 85oC; c) NaOH, THF, rt; d)
appropriate chlorides, Cs2CO3, DMF, 80oC.
Scheme 2. Reagents and conditions: a) 2-hydroxyacetic acid, KOH; b) 6M HCl, rt; c)
thiocarbohydrazide, CH3SO3H, sulfolane, H2O, 90oC; d) appropriate carboxylic acids,
POCl3, sulfolane, 85oC; e) appropriate bromides, KOH, DMF, rt.
3. Results and discussion
3.1 In vitro enzymatic assays and structure-activity relationship

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Table 1. Chemical structures of compounds 2a-2b, 4a-4c and their c-Met kinase
inhibition activities
Compd.
2a
R1
R2
c-Met kinase IC50 (µM)
>10
2b
4a
>10
>10
4b
4c
>10
>10
HL-11a
Me
0.19
a Used as a positive control.

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Table 2. Chemical structures of compounds 7a-7m, 8a-8c and their c-Met kinase
inhibition activities
c-Met kinase
c-Met kinase
Compd.
7a
R1
Compd.
R1
IC50 (µM)
IC50 (µM)
0.0156
7j
0.0286
7b
7c
7d
7e
0.110
0.0167
0.00202
0.0173
7k
7l
0.0817
0.0145
0.440
7m
8a
0.0314

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7f
0.150
8b
8c
0.0273
0.201
N
7g
0.0649
7h
7i
0.0263
0.0145
HL-11f
0.0793
crizotiniba
0.00829
a Used as a positive control.
The synthesized compounds were evaluated for their c-Met kinase inhibition
activity using hot-SpotSM kinase assay method using crizotinib, HL-11 and HL-11f
as positive controls. To study the SAR, we investigated several alternative choices of
the hinge-binder groups (see the R2 substitutents in Table 1) and a series of
hydrophilic groups and aromatic heterocycles installed in the position of methoxy
group in HL-11 to occupy the solvent accessible region. Among compounds in Table
1, 2a, 2b and 4a-4c showed very weak c-Met kinase inhibition activity, indicating that
such modification in HL-11 was not favorable. 7-Methoxyquinolin-4-oxy, a
commonly used hinge region group of c-Met inhibitor, was introduced instead of the
methoxyphenyl in HL-11 and several substituted phenyl rings or aromatic
heterocyclic rings were assessed to replace the indole ring of HL-11 (Table 2). The
c-Met kinase inhibition activities of 7a and 11f showed that the hydrogen bond

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between N-H in HL-11 and Arg1208 was not critical. Compound 7f with a
hydrophobic substituent at the 5-position of indole showed less inhibition activity
than 7g with a hydrophilic or no substituent (HL-11f). Compounds 7b-7e, 7h-7j and
7l demonstrated good c-Met kinase inhibition activity, suggesting that the electron
density of the aromatic groups in the hydrophilic region have little influence on their
kinase inhibition activites. The inhibition to c-Met kinase did not improved when
either methoxyethyl (8a), cyclopropylmethyl (8b) or cyclopentyl (8c) were introduced
in the pyrrolyl fragment of 7c. Compound 7d with N-methyl pyrazole group displayed
the highest inhibitory potency giving an IC50 value of 2.02 nM. 7d was docked to
c-Met kinase and the overlayed results revealed that 7d had a similar binding mode as
the
lead
compound
HL-11
(Fig.
3).
Specifically,
the
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole groups of 7d and HL-11 overlapped almost
completely. The quinoline N and the methoxy group of 7d also formed two hydrogen
bonds with residues Met1160 and Tyr1159 respectively.
Fig. 3. Binding mode of compound 7d (green) overlayed with HL-11 (light magenta)
at the c-Met kinase domain. The formed hydrogen bonds were marked with green and
light magenta respectively.
3.2 In vitro cell growth inhibition assays
Table 3. Cytotoxic activities of compounds 7a-7m and 8a-8c against SNU-5, MKN45,
EBC-1 cell lines in vitroa

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Antiproliferative activity
Antiproliferative activity
IC50
at 5 µM
at 1 µM
（nM）
Compd.
SNU-5 MKN45 EBC-1
MKN45
60.8%
EBC-1
91.6%
EBC-1
312.6
7a
7b
67.7% 74.1% 87.0%
14.5% 28.1% 86.3%
71.5% 70.5% 85.9%
68.7% 71.9% 84.4%
72.4% 76.8% 83.7%
54.4% 47.7% 42.3%
60.0% 61.2% 84.0%
65.7% 73.0% 83.7%
74.5% 72.0% 85.2%
65.7% 66.6% 85.4%
65.8% 71.0% 83.1%
66.3% 71.3% 81.4%
44.7% 29.8% 84.5%
46.1% 73.0% 78.6%
52.2% 66.6% 75.9%
ND
7.4%
90.6%
90.9%
80.5%
ND
ND
353.2
88
7c
71.1%
73.6%
79.3%
ND
7d
7e
149
7f
ND
7g
75.3%
73.8%
76.4%
37.8%
68.5%
13.2%
ND
82.2%
91.6%
89.1%
91.5%
89.4%
86.7%
89.3%
89.0%
87.2%
41.3%
86.5%
93.1%
85.6
289.2
161
7h
7i
7j
472.1
186.7
504.3
403.3
327.4
350.8
ND
7k
7l
7m
8a
71.1%
41.1%
ND
8b
8c
42%
47.6% 72.1%
HL-11fb
70.2% 76.3% 87.3%
78.1%
82.6%
106.6
39
Crizotinibb 80.0% 77.7% 94.4%
a ND: not determined. b Used as a positive control.

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The inhibitory effects of compounds 7a-7m and 8a-8c were evaluated in several
c-Met overexpressed human cancer cell lines, including SNU-5 (human gastric
cancer), MKN45 (human gastric cancer) and EBC-1 (human lung cancer) cells
[21-23]. The results are summarized in Table 3. Most of the compounds showed
potent antiproliferative activity against these cell lines, especially EBC-1 cells.
Compounds with indole substituent (7a, 7g and HL-11f) showed better cellular
potency than compounds with single ring in hydrophilic region (7b, 7c, 7e, 7h-7m
and 8a-8c) except 7d.
3.3 Enzymatic selectivity assays
Table 4. Kinase selectivity profile of compound 8c
Kinase
FLT3
Inhibition at 5µM
69.68% (1.37µM)
14.91%
Kinase
c-Kit
Inhibition at 5µM
10.93%
ABL1
PDGFRα
EGFR
-3.29%
AXL
11.11%
-10.68%
-14.57%
-7.71%
RON/MST1R
RET
-15.45%
FGFR1
TIE2/TEK
TRKA
TRKB
-0.73%
ALK
-1.59%
-11.80%
0.04%
FLT1/VEGFR1
KDR/VEGFR2
0.28%
-3.93%
LCK
-0.81%
To further examine the selectivity profile of 7d, 7d was evaluated against a panel
of 16 tyrosine kinases, including FLT3, ABL1, AXL, RON, RET, ALK, FLT1, KDR,
c-Kit, PDGFRα, EGFR, FGFR1, TIE2, TRKA, TRKB and LCK. As shown in Table 4,
compared to its inhibition activity against c-Met kinase (IC50: 2.02 nM), 7d
demonstrated more than 2500-fold less potency against these selected kinases except
FLT3 (IC50: 1.37 µM，over 650-fold of its c-Met IC50), indicating that 7d is a highly

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selective c-Met inhibitor.
3.4 Western blot analysis
Fig. 4. Compound 7d inhibited c-Met phosphorylation in MKN45 cells.
We further investigated if c-Met kinase inhibition of 7d could be recapitulated in
vitro in western blotting assay using the MKN45 cell line. MKN45 cells were treated
with different doses of 7d for 1h and the cell lysates were prepared for western
blotting analysis [21]. As shown in Fig. 4, compound 7d inhibited HGF-induced
c-Met phosphorylation in a dose-dependent manner in MKN45 cells and c-Met
phosphorylation was completely inhibited at a concentration of 0.4 µM.
4. Conclusion
In summary, a series of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives were
synthesized and evaluated as novel c-Met inhibitors. SAR studies was carried out and
compound 7d was identified as the most potent and selective c-Met inhibitor with an
IC50 value of 2.02 nM against c-Met kinase activity and gave over 2500-fold
selectivity against other 16 tyrosine kinases evaluated. Moreover, 7d exhibited high
antiproliferative activities against several cancer cell lines (SNU-5, MKN45 and
EBC-1) and displayed high inhibitory effect on c-Met phosphorylation in the MKN45
cancer cell line. Further optimization and in vivo efficacy experiment of 7d are
currently ongoing in our lab and the results will be reported in due course.
5. Materials and methods
5.1. Instrumentation and chemicals

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All reactions were performed under N2 unless otherwise indicated. Reagents and
solvents were purchased as reagent grade and were used without further purification.
Flash column chromatography was performed over silica gel (200 - 300 m) using a
mixture of ethyl acetate (EA), petroleum ether (PE), dichloromethane (DCM) and
1
13
MeOH. H NMR and C NMR spectra were obtained at room temperature using a
Bruker Avance 300 spectrometer. The abbreviations s, d, t, q and m signify singlet,
doublet, triplet, quartet and multiplet, respectively. Because of the poor solubility, 13C
NMR analysis of compounds 4a, 7b, 7e-7h, 7l-7m, 8a-8c were not applicable. High
resolution mass spectrometry (HRMS) was obtained on a Q-TOF micro spectrometer.
Melting points were determined with a Micro melting point apparatus. TLC plates
were visualized by exposure to ultraviolet light.
5.2. Experimental
5.2.1. General procedure for preparation of 2a-2b, 4a-4c, 7a-7m
The synthesis methods of 2a-2b, 4a-4c and 7a-7m would not be shown here because
their preparation has been presented in detail in our previous study [20].
5.2.1.1.
4-((6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)
phenyl 1-methyl-1H-pyrazole-4-carboxylate (2a)
White solid; Yield: 27%; m.p.: 167-168 oC; 1H NMR (300 MHz, DMSO-d6) δ 8.61 (s,
1H), 8.53 (s, 1H), 8.10 (s, 1H), 8.01 (s, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.18 (d, J = 8.0
13
Hz, 2H), 4.45 (s, 2H), 3.93 (s, 3H), 3.92 (s, 3H); C NMR (75 MHz, DMSO-d6) δ
161.23, 159.65, 152.83, 149.60, 146.62, 141.33, 138.17, 135.59, 133.63, 132.23,
130.30(2C), 122.59(2C), 113.15, 112.42, 39.49 (2C), 30.21; HRMS (ESI) m/z calcd
for C19H17N8O2S+ [M+H]+: 421.11897; found: 421.11983.
5.2.1.2.
4-((6-(1H-indol-2-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)methyl)phenyl
1H-indole-2-carboxylate (2b)
White solid; Yield: 27%; m.p.: 280-281oC; 1H NMR (300 MHz, DMSO-d6) δ 9.35 (s,
1H), 8.60 (s, 1H), 8.09 (s, 1H), 7.14 (d, J = 7.9 Hz, 2H), 6.75 – 6.66 (m, 2H), 4.26 (s,
13
2H), 3.93 (s, 3H); C NMR (75 MHz, DMSO-d6) δ 160.33, 159.44, 152.95, 149.68,

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146.79, 138.62, 138.31, 133.95, 130.41(2C), 127.69, 127.18, 126.82, 126.57, 125.70,
125.55, 122.77, 122.64(2C), 122.06, 121.11, 120.91, 113.16, 112.88, 109.97, 107.94,
30.26; HRMS (ESI) m/z calcd for C27H18N6O2SNa+ [M+Na]+: 513.11148; found:
513.11159.
5.2.1.3.
4-(2-(4-((6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)m
ethyl)phenoxy)ethyl)morpholine (4a)
White solid; Yield: 43%; m.p.: 155-157oC; 1H NMR (300 MHz, DMSO-d6) δ: 8.59 (s,
1H), 8.08 (d, J = 0.8 Hz, 1H), 7.31 – 7.20 (m, 2H), 6.95 – 6.84 (m, 2H), 4.32 (s, 2H),
4.05 (t, J = 5.7 Hz, 2H), 3.93 (s, 3H), 3.61 – 3.51 (m, 4H), 2.68 (t, J = 5.4 Hz, 2H),
2.46 (s, 4H); HRMS (ESI) m/z calcd for C20H24N7O2S+ [M+H]+: 426.17067; found:
426.17247.
5.2.1.4.
N-methyl-2-(4-((6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol
-3-yl)methyl)phenoxy)acetamide (4b)
White solid; Yield: 37%; m.p.: 243-245oC; 1H NMR (300 MHz, DMSO-d6) δ 8.59 (s,
1H), 8.08 (d, J = 0.8 Hz, 1H), 7.99 (s, 1H), 7.33 – 7.23 (m, 2H), 6.97 – 6.87 (m, 2H),
4.42 (s, 2H), 4.33 (s, 2H), 3.93 (s, 3H), 2.63 (d, J = 4.7 Hz, 3H); 13C NMR (75 MHz,
DMSO-d6) δ 168.42, 159.51, 157.13, 152.69, 146.99, 138.17, 132.25, 130.26(2C),
128.74, 115.30(2C), 112.41, 67.53, 39.51, 30.00, 25.80; HRMS (ESI) m/z calcd for
C17H17N7O2SNa+ [M+Na]+: 406.10566; found: 406.10698.
5.2.1.5.
N,N-dimethyl-2-(4-((6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadi
azol-3-yl)methyl)phenoxy)acetamide (4c)
White solid; Yield: 54%; m.p.: 170-172oC; 1H NMR (300 MHz, DMSO-d6) δ: 8.59 (s,
1H), 8.08 (s, 1H), 7.25 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 4.76 (s, 2H), 4.33
13
(s, 2H), 3.32 (s, 3H), 2.98 (s, 3H), 2.83 (s, 3H); C NMR (75 MHz, DMSO-d6) δ
167.55, 159.51, 157.53, 152.68, 147.06, 138.18, 132.23, 130.08, 128.27, 115.15,
112.42, 66.19, 39.50, 36.03, 35.39, 30.00; HRMS (ESI) m/z calcd for C18H20N7O2S+
[M+H]+: 398.13937; found: 398.14084.

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5.2.1.6.
3-(((7-methoxyquinolin-4-yl)oxy)methyl)-6-(1H-pyrrol-2-yl)-[1,2,4]triazolo[3,4-b][1,
3,4]thiadiazole (7a)
1
Gray solid; Yield:18%; m.p.: 205-206oC; H NMR (300 MHz, DMSO-d6) δ 12.38 (s,
1H), 8.73 (d, J = 5.3 Hz, 1H), 7.99 (d, J = 9.2 Hz, 1H), 7.35 (d, J = 2.6 Hz, 1H), 7.23
(d, J = 5.3 Hz, 1H), 7.16 (dt, J = 6.8, 2.5 Hz, 2H), 6.99 (s, 1H), 6.30 (dt, J = 4.4, 2.3
Hz, 1H), 5.77 (s, 2H), 3.90 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 160.97, 160.36,
159.86, 154.07, 152.35, 151.15, 143.30, 125.89, 123.33, 120.85, 118.72, 115.44,
111.25, 107.75, 101.21, 60.17, 55.89; HRMS (ESI) m/z calcd for C18H15N6O2S+
[M+H]+: 379.09717; found: 379.09795.
5.2.1.7.
3-(((7-methoxyquinolin-4-yl)oxy)methyl)-6-(1-methyl-1H-pyrrol-2-yl)-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazole (7b)
White solid; Yield: 22%; m.p.: 214-215oC; 1H NMR (300 MHz, DMSO-d6) δ: 8.71 (d,
J = 5.2 Hz, 1H), 8.04 (d, J = 9.2 Hz, 1H), 7.34 (d, J = 2.6 Hz, 1H), 7.29 – 7.12 (m,
3H), 6.95 (dd, J = 4.1, 1.7 Hz, 1H), 6.24 (dd, J = 4.0, 2.5 Hz, 1H), 5.85 (s, 2H), 3.90
(s, 3H), 3.81 (s, 3H); HRMS (ESI) m/z calcd for C19H17N6O2S+ [M+H]+: 393.11282;
found: 393.11352.
5.2.1.8.
3-(((7-methoxyquinolin-4-yl)oxy)methyl)-6-(1H-pyrrol-3-yl)-[1,2,4]triazolo[3,4-b][1,
3,4]thiadiazole (7c)
1
Brown solid; Yield: 14%; m.p.: 209-211oC; H NMR (300 MHz, DMSO-d6) δ: 11.76
(s, 1H), 8.73 (d, J = 5.3 Hz, 1H), 8.02 (d, J = 9.2 Hz, 1H), 7.71 (dt, J = 3.3, 1.8 Hz,
1H), 7.35 (d, J = 2.6 Hz, 1H), 7.27 – 7.11 (m, 2H), 6.99 (q, J = 2.4 Hz, 1H), 6.57 (q, J
13
= 2.3 Hz, 1H), 5.81 (s, 2H), 3.90 (s, 3H); C NMR (75 MHz, DMSO-d6) δ 163.77,
160.94, 160.37, 154.13, 152.30, 151.18, 143.26, 123.29, 122.17, 121.68, 118.68,
115.54, 113.70, 107.76, 107.11, 101.36, 60.39, 55.88; HRMS (ESI) m/z calcd for
C18H15N6O2S+ [M+H]+: 379.09717; found: 379.09827.
5.2.1.9.
3-(((7-methoxyquinolin-4-yl)oxy)methyl)-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazol

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o[3,4-b][1,3,4]thiadiazole (7d)
Brown solid; Yield: 27%; m.p.: 203-205oC; 1H NMR (300 MHz, DMSO-d6) δ: 8.73 (d,
J = 5.2 Hz, 1H), 8.62 (s, 1H), 8.09 (s, 1H), 8.01 (d, J = 9.2 Hz, 1H), 7.35 (d, J = 2.5
Hz, 1H), 7.23 (d, J = 5.3 Hz, 1H), 7.17 (dd, J = 9.2, 2.5 Hz, 1H), 5.81 (s, 2H), 3.92 (s,
13
3H), 3.90 (s, 3H); C NMR (75 MHz, DMSO-d6) δ 170.09, 160.96, 160.38, 154.26,
152.23, 151.05, 143.39, 138.24, 132.43, 123.27, 118.72, 115.49, 112.25, 107.61,
101.30, 60.31, 55.89, 39.52; HRMS (ESI) m/z calcd for C18H16N7O2S+ [M+H]+:
394.10807; found: 394.11007.
5.2.1.10.
3-(((7-methoxyquinolin-4-yl)oxy)methyl)-6-(1-methyl-1H-indol-2-yl)-[1,2,4]triazolo[
3,4-b][1,3,4]thiadiazole (7e)
Brown solid; Yield: 11%; m.p.: 232-234oC; 1H NMR (300 MHz, DMSO-d6) δ: 8.08 (d,
J = 9.1 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.62 (d, J = 8.7 Hz, 1H), 7.49 – 7.33 (m,
3H), 7.25 (d, J = 5.4 Hz, 1H), 7.23 – 7.13 (m, 2H), 5.92 (s, 2H), 3.90 (s, 3H), 3.09 (s,
3H); HRMS (ESI) m/z calcd for C23H19N6O2S+ [M+H]+: 443.12847; found:
443.12930.
5.2.1.11.
6-(5-fluoro-1H-indol-2-yl)-3-(((7-methoxyquinolin-4-yl)oxy)methyl)-[1,2,4]triazolo[3
,4-b][1,3,4]thiadiazole (7f)
1
White solid; Yield: 15%; m.p.: 236-238oC; H NMR (300 MHz, DMSO-d6) δ: 12.47
(s, 1H), 8.76 (dd, J = 5.3, 1.6 Hz, 1H), 8.07 – 7.97 (m, 1H), 7.52 – 7.31 (m, 4H), 7.31
– 7.23 (m, 1H), 7.17 (d, J = 9.1 Hz, 2H), 5.84 (s, 2H), 3.89 (d, J = 1.6 Hz, 3H);
HRMS (ESI) m/z calcd for C22H16FN6O2S+ [M+H]+: 5447.10340; found: 447.10379.
5.2.1.12.
6-(5-methoxy-1H-indol-2-yl)-3-(((7-methoxyquinolin-4-yl)oxy)methyl)-[1,2,4]triazol
o[3,4-b][1,3,4]thiadiazole (7g)
1
White solid; Yield: 11%; m.p.: 252-253oC; H NMR (300 MHz, DMSO-d6) δ: 12.22
(s, 1H), 8.75 (d, J = 5.2 Hz, 1H), 8.01 (d, J = 9.1 Hz, 1H), 7.40 – 7.31 (m, 2H), 7.31 –
7.22 (m, 2H), 7.20 – 7.13 (m, 1H), 7.11 (d, J = 2.6 Hz, 1H), 6.94 (dd, J = 8.7, 2.5 Hz,
1H), 5.82 (s, 2H), 3.89 (s, 3H), 3.77 (s, 3H); HRMS (ESI) m/z calcd for C23H19N6O3S+

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[M+H]+: 459.12339; found: 459.12486.
5.2.1.13.
3-(((7-methoxyquinolin-4-yl)oxy)methyl)-6-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4
]thiadiazole (7h)
1
Light brown solid; Yield: 28%; m.p.: 228-229oC; H NMR (300 MHz, DMSO-d6) δ:
8.88 – 8.80 (m, 2H), 8.74 (d, J = 5.3 Hz, 1H), 8.05 (d, J = 9.1 Hz, 1H), 7.95 – 7.88 (m,
2H), 7.35 (d, J = 2.6 Hz, 1H), 7.25 (d, J = 5.3 Hz, 1H), 7.18 (dd, J = 9.2, 2.6 Hz, 1H),
5.91 (s, 2H), 3.90 (s, 3H); HRMS (ESI) m/z calcd for C19H15N6O2S+ [M+H]+:
391.09717; found: 391.09884.
5.2.1.14.
6-(4-methoxyphenyl)-3-(((7-methoxyquinolin-4-yl)oxy)methyl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole (7i)
1
White solid; Yield: 24%; m.p.: 184-186oC; H NMR (300 MHz, Chloroform-d) δ:
8.66 (d, J = 5.5 Hz, 1H), 8.07 (d, J = 9.2 Hz, 1H), 7.73 (d, J = 8.9 Hz, 2H), 7.40 (d, J
= 2.2 Hz, 1H), 7.08 (dd, J = 9.2, 2.5 Hz, 1H), 7.02 (d, J = 5.5 Hz, 1H), 6.94 (d, J = 8.9
Hz, 2H), 5.72 (s, 2H), 3.88 (s, 3H), 3.82 (s, 3H); 13C NMR (75 MHz, Chloroform-d) δ
167.42, 163.34, 161.14, 160.65, 154.75, 151.51, 151.09, 143.07, 128.97(2C), 123.16,
121.45, 118.67, 115.82, 114.88(2C), 106.96, 100.03, 59.77, 55.64, 55.48; HRMS (ESI)
m/z calcd for C21H18N5O3S+ [M+H]+: 420.1125; found: 420.1143.
5.2.1.15.
6-(3-methoxyphenyl)-3-(((7-methoxyquinolin-4-yl)oxy)methyl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole (7j)
1
Yellow solid; Yield: 25%; m.p.: 201-203oC; H NMR (300 MHz, DMSO-d6) δ: 8.65
(d, J = 5.3 Hz, 1H), 8.05 (d, J = 9.2 Hz, 1H), 7.56-7.51 (m, 3H),7.38-7.30 (m, 4H),
7.06-7.02 (m, 2H), 6.95 (d, J = 5.3 Hz, 1H), 5.70 (s, 2H), 3.85 (s, 3H), 3.78 (s, 3H);
13C NMR (75 MHz, Chloroform-d) δ 167.68, 161.12, 160.55, 160.22, 154.75, 151.60,
151.22, 143.24, 130.63, 130.20, 123.11, 119.77, 119.02, 118.68, 115.81, 112.02,
107.08, 100.01, 59.75, 55.62, 55.48; HRMS (ESI) m/z calcd for C21H18N5O3S+
[M+H]+: 420.11249; found: 420.11374.
5.2.1.16.

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6-(4-fluorophenyl)-3-(((7-methoxyquinolin-4-yl)oxy)methyl)-[1,2,4]triazolo[3,4-b][1,
3,4]thiadiazole (7k)
1
White solid; Yield: 24%; m.p.: 195-196oC; H NMR (300 MHz, Chloroform-d) δ:
8.66 (d, J = 5.4 Hz, 1H), 8.06 (d, J = 9.2 Hz, 1H), 7.81 (dd, J = 8.5, 5.1 Hz, 2H), 7.36
(d, J = 2.4 Hz, 1H), 7.15 (d, J = 8.3 Hz, 2H), 7.10 – 7.03 (m, 1H), 6.98 (d, J = 5.4 Hz,
1H), 5.72 (s, 2H), 3.88 (s, 3H); 13C NMR (75 MHz, Chloroform-d) δ 167.13, 166.51,
163.74, 161.14, 161.14, 160.51, 154.73, 151.65, 151.28, 143.32, 129.59, 129.47,
125.38, 123.07, 118.69, 117.13, 116.83, 115.80, 107.12, 100.00, 59.74, 55.49; HRMS
(ESI) m/z calcd for C20H15FN5O2S+ [M+H]+: 408.09250; found: 408.09302.
5.2.1.17.
6-(3-fluorophenyl)-3-(((7-methoxyquinolin-4-yl)oxy)methyl)-[1,2,4]triazolo[3,4-b][1,
3,4]thiadiazole (7l)
1
Yellow solid; Yield: 17%; m.p.: 209-210oC; H NMR (300 MHz, Chloroform-d) δ:
8.73 (d, J = 5.5 Hz, 1H), 8.14 (d, J = 9.2 Hz, 1H), 7.68 – 7.58 (m, 2H), 7.53 (td, J =
8.0, 5.3 Hz, 1H), 7.46 (d, J = 2.6 Hz, 1H), 7.37 – 7.30 (m, 1H), 7.16 (dd, J = 9.2, 2.5
Hz, 1H), 7.08 (d, J = 5.5 Hz, 1H), 5.82 (s, 2H), 3.95 (s, 3H); HRMS (ESI) m/z calcd
for C20H15FN5O2S+ [M+H]+: 408.09250; found: 408.09461.
5.2.1.18.
3-(((7-methoxyquinolin-4-yl)oxy)methyl)-6-(3,4,5-trifluorophenyl)-[1,2,4]triazolo[3,4
-b][1,3,4]thiadiazole (7m)
1
Yellow solid; Yield: 13%; m.p.: 233-235oC; H NMR (300 MHz, DMSO-d6) δ: 8.74
(d, J = 5.6 Hz, 1H), 8.13 (d, J = 9.2 Hz, 1H), 7.56-7.51 (m, 3H), 7.19 (dd, J = 9.2, 2.4
Hz, 1H), 7.12 (d, J = 5.6 Hz, 1H), 5.84 (s, 2H), 3.97 (s, 3H); HRMS (ESI) m/z calcd
for C20H13F3N5O2S+ [M+H]+: 444.07366; found: 444.07575.
5.2.2 General procedure for preparation of 8a-8c
7c (200 mg, 0.53 mmol) and brominated alkane (1.06 mmol) were dissolved in 15 mL
DMF, and then KOH (84 mg, 1.5 mmol) was added. The mixture was stirred at room
temperature for 12 hours. 45 mL water was added to the reaction mixture and the
mixture was extracted with EA (30mL×3). The organic layer was washed with brine
(45mL×3), dried over Na2SO4 and concentrated. The residue was purified by silica gel

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chromatography to afford 8a-8c.
5.2.2.1.
6-(1-(2-methoxyethyl)-1H-pyrrol-3-yl)-3-(((7-methoxyquinolin-4-yl)oxy)methyl)-[1,2
,4]triazolo[3,4-b][1,3,4]thiadiazole (8a)
White solid; Yield: 36%; m.p.: 141-142oC; 1H NMR (300 MHz, DMSO-d6) δ: 8.72 (d,
J = 5.1 Hz, 1H), 8.02 (d, J = 9.2 Hz, 1H), 7.71 (t, J = 2.0 Hz, 1H), 7.35 (d, J = 2.6 Hz,
1H), 7.26 – 7.12 (m, 2H), 7.04 – 6.96 (m, 1H), 6.54 (dd, J = 3.0, 1.8 Hz, 1H), 5.80 (s,
1H), 4.12 (t, J = 5.2 Hz, 2H), 3.90 (s, 2H), 3.62 (t, J = 5.2 Hz, 2H), 3.24 (s, 2H);
HRMS (ESI) m/z calcd for C21H21N6O3S+ [M+H]+: 437.13904; found: 437.13963.
5.2.2.2.
6-(1-(cyclopropylmethyl)-1H-pyrrol-3-yl)-3-(((7-methoxyquinolin-4-yl)oxy)methyl)-[
1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (8b)
White solid; Yield: 28%; m.p.: 131-133oC; 1H NMR (300 MHz, DMSO-d6) δ: 8.72 (d,
J = 5.2 Hz, 1H), 8.02 (d, J = 9.2 Hz, 1H), 7.76 (s, 1H), 7.35 (d, J = 2.5 Hz, 1H), 7.26
– 7.11 (m, 2H), 7.06 (d, J = 2.6 Hz, 1H), 6.59 – 6.51 (m, 1H), 5.80 (s, 2H), 3.90 (s,
3H), 3.82 (d, J = 7.1 Hz, 2H), 1.25 – 1.16 (m, 1H), 0.57 – 0.48 (m, 2H), 0.37 (dd, J =
9.9, 4.8 Hz, 2H); HRMS (ESI) m/z calcd for C22H21N6O2S+ [M+H]+: 433.14412;
found: 433.14559.
5.2.2.3.
6-(1-cyclopentyl-1H-pyrrol-3-yl)-3-(((7-methoxyquinolin-4-yl)oxy)methyl)-[1,2,4]tri
azolo[3,4-b][1,3,4]thiadiazole (8c)
White solid; Yield: 28%; m.p.: 81-82oC; 1H NMR (300 MHz, DMSO-d6) δ: 8.72 (d, J
= 5.3 Hz, 1H), 8.02 (d, J = 9.2 Hz, 1H), 7.78 (t, J = 1.9 Hz, 1H), 7.35 (d, J = 2.6 Hz,
1H), 7.22 (d, J = 5.3 Hz, 1H), 7.17 (dd, J = 9.1, 2.5 Hz, 1H), 7.08 (t, J = 2.6 Hz, 1H),
6.55 (dd, J = 3.0, 1.8 Hz, 1H), 5.80 (s, 2H), 4.57 – 4.45 (m, 1H), 3.90 (s, 3H), 2.19 –
2.05 (m, 2H), 1.79 (s, 4H), 1.63 (s, 2H); HRMS (ESI) m/z calcd for C23H23N6O2S+
[M+H]+: 447.15977; found: 447.16079.
5.3 In vitro assays
5.3.1 Biochemical assay
Kinase inhibitory activities were determined using Hot-SpotSM kinase assay

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performed by Reaction Biology Corp. (Malvern PA, USA). After the substrate was
prepared in freshly prepared reaction buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 1
mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1%
DMSO), 5 nM of human GST-tagged target kinase was delivered into the substrate
solution and mixed gently. The testing compounds were dissolved in 100% DMSO to
specific concentration and added into the kinase reaction mixture by Acoustic
technology (Echo550; nanoliter range). The reaction mixture was incubated for 20
33
min at room temperature. P-ATP (Specific activity 10 µCi/µL) was delivered into
the reaction mixture to initiate the reaction and incubated at room temperature for 2
hours. The kinase activities were detected by filter-binding method. IC50 values were
obtained using Prism2 software (GraphPad).
5.3.2 In vitro cell activity assay
In vitro cell activity was determined using Cell Titer-Glo (CTG) assay [24-25].
SNU-5, EBC-1, MKN45 cells in exponential growth were plated onto each well of
96-well plate at a density of 4000 cells per well and maintained in RPMI-1640 media
o
supplemented with 10% FPS. The cells were incubated in 5% CO2 at 37 C for 12 h.
The tested compounds at the indicated final concentrations were added to the culture
medium. The cells were then incubated at 37 oC under 5% CO2 for 72 h. 50 µL CTG
solution was added to each well and incubated at room temperature for 10 min. The
fluorescence signal values were determined with Envision2104 plate reader. The %
inhibitory was calculated as follows: cell inhibitory = (1- Vsample / Vvehicle control) x
100%.
5.3.3 Western blot analysis
MKN45 cells in exponential growth were placed onto each well of 6-well plate
at a density of 2x104 cells per well and maintained in RPMI-1640 media
supplemented with 10% FPS. Cells were then treated with ice-cold RIPA lysis buffer
supplemented with a protease inhibitor cocktail to get whole-cell extracts. Protein
concentration was determined by BCA assay. Cell lysate containing 30µg protein was
separated by SDS-PAGE electrophoresis and transferred onto PVDF membranes. The
membranes were blocked with 5% skim milk and incubated with primary antibodies

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o
at 4 C overnight. Membranes were then washed with TBST and incubated with
horseradish peroxidase conjugated secondary antibody in 5% BSA in TBST for 1 h at
room temperature. The membranes were visualized by ECL chemiluminescence
system (Chemidoc XRS+, Bio-Rad, CA, USA).
5.4. Docking studies
Crystal structure of c-Met (PDB ID: 5YA5) was downloaded from the Protein
Data Bank (www.rcsb.org). After the waters were deleted and the hydrogen atoms
were added to the protein, the protein was prepared using Protein Preparation Wizard
in the Schrodinger suite [26]. 3D structures of compounds 7d was built and
minimized by the Ligand Preparation module in Schrodinger. The Glide module with
extra precision (XP) was selected for molecular docking. 10 best conformations were
minimized by a post docking program. The image files were generated using pymol
system.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of
China (Grant No. 81473078).
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ACCEPTED MANUSCRIPT
Highlights:
1. A series of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives were designed and
synthesized.
2. The target compounds showed potent antitumor activity.
3. Compound 7d showed nanomolar c-Met kinase and subnanomolar cell growth
inhibitory activity.
4. Compound 7d inhibiting the phosphorylation of c-Met kinase in MKN45 cell line.
5. Compound 7d was highly selective to c-Met with over 2500-fold selectivity.