Synthesis, biological evaluation, and molecular docking of ((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl) piperazin-1-yl)methyl) benzohydrazide derivatives

Article abstract of DOI:10.1177/1747519820911278

A series of ((4-([1,2,4]triazolo[4,3-b][1,2,4,5] methyl) benzo-hydrazide derivatives was designed, synthesized, and evaluated for their inhibition activities against five tumor cells and c-Met kinase in vitro. These compounds were fully characterized by <

Full text of DOI:10.1177/1747519820911278

911278CHL0010.1177/1747519820911278Journal of Chemical ResearchWu et al.
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Research Paper
Journal of Chemical Research
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Synthesis, biological evaluation, and molecular
docking of ((4-([1,2,4]triazolo[4,3-b]
[1,2,4,5]tetrazin-6-yl) piperazin-1-yl)methyl)
benzohydrazide derivatives
https://doi.org/10.1177/1747519820911278
DOI: 10.1177/1747519820911278
journals.sagepub.com/home/chl
Xiaoyu Wu1, Feng Xu2 , Zhenzhen Yang2, Zhonglu Ke2, Lei Shi2,
Can Ye2, Qidong Yan2 and Shijie Zhang3
Abstract
A series of ((4-([1,2,4]triazolo[4,3-b][1,2,4,5] methyl) benzo-hydrazide derivatives was designed, synthesized, and
evaluated for their inhibition activities against five tumor cells and c-Met kinase in vitro. These compounds were fully
characterized by 1H NMR, 13C NMR, MS, and elemental analysis. Antitumor experiments indicated that some compounds
exhibited significant inhibition activities against A549 and Bewo. Especially, the IC50 values of 5f (12μM), 5h (7.1μM),
6a (8.4μM), and 6d (9.2μM) demonstrated better antitumor activities against A549 than the positive agent cisplatin
(13.3μM), and the IC50 value of 6a (5.2μM) exhibited better antitumor activity against Bewo than cisplatin (7.7μM).
Keywords
anticancer, c-Met, molecular docking, synthesis, tetrazine, triazole
Date received: 21 December 2019; accepted: 14 February 2020
A series of ((4-([1,2,4]triazolo[4,3-b][1,2,4,5] methyl) benzohydrazide derivatives were designed, synthesized, and
evaluated for their inhibition activities against five tumor cells and c-Met kinase in vitro.
Introduction
1Laboratory Department, Taizhou Central Hospital (Taizhou university
Hospital), Taizhou, P.R. China
Cancer is a class of diseases in which a group of cells dis-
play uncontrolled growth, invasion, and sometimes metas-
tasis.1,2 According to the World Health Organization
(WHO), cancer is the second leading cause of death after
cardiovascular disease all around the globe and accounted
for 8.8million deaths in 2015 worldwide.3,4
2Biopharmaceutical Research and Development Centre, Taizhou
Vocational & Technical College, Taizhou, P.R. China
3Academy of Chinese Medical Sciences, Zhejiang Chinese Medical
University, Hangzhou, P.R. China
Corresponding author:
Feng Xu, Biopharmaceutical Research and Development Centre,
Taizhou Vocational & Technical College, Taizhou 318000, P.R. China.
Email: xufeng901@126.com
c-Met is a unique member of the receptor tyrosine
kinases (RTKs) expressed in both normal and malignant

2
Journal of Chemical Research 00(0)
Figure 1. Examples of c-Met inhibitors: benzoyl hydrazide and triazolotetrazine derivatives.
cells. It is a cell-surface receptor for hepatocyte growth fac- [1,2,4,5] tetrazine derivatives exhibit significant anticancer
tor (HGF), a pleiotropic cytokine that conveys a unique activities against A549, Bewo, and MCF-7 (e.g.
combination of pro-migratory, anti-apoptotic, and mito- TZXU-4: IC50 =21.68, 20.72, and 2.81μM, respectively);19
genic signals.5,6 Under normal physiological conditions, 3-alkyl(aryl)-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazine deriv-
the pleiotropic effects of the HGF/c-Met axis are essential atives also show significant antiproliferative activities
for embryogenesis and tissue homeostasis.7,8 Aberrant against MCF-7, Bewo, and HL-60 cells (e.g. TZXU-5:
HGF/c-Met signaling has been identified in a wide range of IC50 =17.63, 0.73, and 0.73μM, respectively) (Figure 1).20
human malignancies, including brain, colorectal, gastric,
Based on the above information, we designed a series of
lung, stomach, breast, and head and neck cancers.9,10 new triazolotetrazine derivatives with benzohydrazide
Moreover, both c-Met overexpression and MET amplifica- groups at the 6-position to investigate further how these
tion have been associated with poor clinical outcomes for substituents located at the 6-position of the [1,2,4]
cancer patients. Of particular note, amplification of the triazolo[4,3-b][1,2,4,5]tetrazine ring influence antitumor
MET oncogene is observed in epidermal growth factor activity.
receptor (EGFR)-mutant non-small-cell lung carcinomas
after tyrosine kinase inhibitor failure.11–14 Therefore, c-Met
Results and discussion
has become one of the most promising therapeutic targets
in anticancer drug discovery.15
The synthetic routes to the 6-substituted-[1,2,4]triazolo[4,3-
There have been a number of studies on developing
small molecule c-Met inhibitors, and various c-Met inhibi-
tors have been reported.16 Among these, benzoyl hydrazide
derivatives I–III have been synthesized and found to dis-
play c-Met inhibitory activity with IC50 values of 69, 1.3,
and 8.1μM, respectively (Figure 1), which has led to them
emerging as promising and attractive scaffolds in the anti-
cancer field.17
Recently, a series of triazolotetrazine derivatives has
been synthesized and found to exhibit significant antican-
cer activities. 6-Alkylamino-[1,2,4]triazolo[4,3-b][1,2,4,5]
tetrazine derivatives (TZXU-1–3) show strong antiprolif-
erative activities against MCF-7, Bewo, and HL-60 cells (
IC50 =1.09–2.24μM) and c-Met kinase inhibitory activities
(IC50 =7.9–23.70μM);18 6-alkoxyl-[1,2,4]triazolo[4,3-b]
b][1,2,4,5] tetrazines are summarized in Scheme 1. Methyl
4-(piperazin-1-ylmethyl) benzoate 1 was prepared from
methyl 4-(bromomethyl)benzoate and piperazine using
dichloromethane as the solvent,21 which was then reacted
with 6-(3,5-dimethyl-1H-pyrazol-1-yl)-[1,2,4]triazolo[4,3-
b][1,2,4,5]tetrazine 2 in ethyl acetate at 50°C to yield com-
pound 3.18 Compound 3 was then heated with 80%
hydrazine hydrate to obtain compound 4. Finally, the title
compounds 5 and 6 were prepared from compound 4 by
treatment with aromatic aldehydes and acyl chlorides,
respectively. The reactions were monitored by thin layer
chromatography (TLC) plates.
The chemical structures of the compounds synthe-
sized were elucidated on the basis of 1H NMR, 13C NMR,
and elemental analysis. In the 1H NMR spectra of

Wu et al.
3
Scheme 1. Synthetic routes to the target compounds 5 and 6.
Table 1. Antitumor activities against A549, Bewo, Hela,
HepG2, and HT29 cell lines in vitro (IC50 in μM)a.
compounds 5 and 6, the characteristic signal due to the
CH proton on the triazole ring appeared at 9.46–9.48 ppm
and 9.54–9.61 ppm, respectively, and the signal due to
the CH2 protons on the benzyl group appeared at 3.61–
3.66 ppm and 4.11–4.66 ppm, respectively. In the 13C
NMR spectra for compounds 5 and 6, the signals
observed in the region of 163.0–163.7 ppm and 165.6–
175.1 ppm accounted for the carbons at the 6-position of
the triazolotetrazine ring, respectively, and the signals
observed in the region of 61.8–62.5 ppm and 58.7–
62.2 ppm accounted for the carbons (CH2) on the benzyl
group, respectively.
To test the antitumor activities of the compounds synthe-
sized, we evaluated antiproliferative activities of com-
pounds 3–6 against A549, Bewo, Hela, HepG2, and HT29
cells by the MTT assay. The results are summarized in
Table 1. As illustrated in Table 1, the active analogues
showed remarkable cytotoxic activity. Particularly, it
should be noticed that compound 6a (8.4μM for A549,
5.2μM for Bewo, 19.1μM for Hela, 2.4μM for HepG2,
and 31.1μM for HT29, respectively) showed the strongest
biological activity, comparable to the positive control cispl-
atin (13.3μM for A549, 7.7μM for Bewo, 6.7μM for Hela,
2.3μM for HepG2, and 14.0μM for HT29, respectively).
Overall, the inhibitory activities of compound 6 are better
than those of compound 5. This is because the effect of the
benzoyl group attached to the benzoyl hydrazide binding to
the c-Met receptor is better than that of benzylidene group.
For compound 5, comparing 5a-5e and 5f-5h, shows that
Compounds
IC50 (μM)
A549
Bewo
Hela
HepG2
HT29
3
4
5a
5b
5c
5d
5e
5f
5g
5h
5i
6a
6b
6c
6d
6e
6f
21.2
13.3
106.2
102.1
239.3
150.6
61.7
12.0
65.8
7.1
25.7
8.4
19.7
49.2
9.2
18.5
30.8
134.8
92.7
162.8
98.2
39.6
48.8
85.9
19.3
21.6
5.2
15.5
68.5
38.2
37.9
40.1
37.9
39.7
7.7
42.2
32.9
215.2
186.7
242.2
92.7
60.2
88.2
133.0
16.0
24.3
19.1
20.2
51.4
18.8
28.4
26.6
11.3
13.4
6.7
2.1
6.7
45.4
48.0
204.2
85.6
213.8
174.9
64.9
69.4
178.5
28.7
28.7
31.1
105.4
78.9
34.2
31.3
33.3
39.1
33.8
14.0
128.9
47.8
129.4
23.8
31.2
53.0
123.8
11.9
8.6
2.4
18.2
28.5
8.9
6.5
7.6
7.9
6.5
2.3
38.5
24.7
27.8
38.9
13.3
6g
6h
Cisplatinb
aAverage of three independent experiments.
bCisplatin was used as positive control.
electron-withdrawing groups such as F, Cl on the phenyl (7.1μM), 6a (8.4μM), and 6d (9.2μM) show better antitu-
ring enhance the inhibitory activities and electron-donating
groups such as N(CH3)2, OCH3, OH decrease the inhibitory
activities. For A549 cell line, compounds 5f (12μM), 5h
mor activity than the positive cisplatin (13.3μM). For com-
pounds 6a-6f, the inhibitory activities of compounds with
an ortho-substituent on the phenyl ring are superior to those

4
Journal of Chemical Research 00(0)
Figure 2. (a) Compound 6a (colored by atom: carbons: gray, nitrogen: blue, oxygen: red) is bond into c-Met (entry 4DEH in the
Protein Data Bank). The dotted lines show the hydrogen bonds, C–H—π and π—π interactions. (b) The surface model structure of
compound 6a binding model with c-Met complex.
with para- or meta-substitution. In addition, comparing 6a- molecule 6a was well embedded in the active pocket of
6f and 6g-6h, the inhibitory activities of the latter with c-Met kinase. This molecular docking result, along with the
alkyl R2 substitution are almost the same order of magni- biological assay data, suggests that compound 6a is a
tude as those of former with phenyl substitution.
potential inhibitor of c-Met kinase.
To examine whether the compounds inhibit c-Met
kinase, we screened compounds 5h, 6a, and 6d against
c-Met kinase via homogeneous time-resolved fluorescence
immunoassay (HTRF). Compounds 5h, 6a, and 6d dis-
played strong c-Met kinase inhibitory activity with IC50
values of 19.00, 8.26, and 22.4μM, respectively (the posi-
tive control staurosporine has an IC50 of 0.042μM for
c-Met kinase), which demonstrated that the antitumor
activities of the synthetic compounds probably correlate to
their c-Met kinase inhibitory activities.
To gain a better understanding of the potency of the
compounds studied and to guide further SAR studies, we
proceeded to examine the interaction of compound 6a with
the c-Met crystal structure (4DEH.pdb). The molecular
docking was performed by inserting compound 6a into the
binding site of c-Met kinase. All docking runs were applied
using the Surflex-Dock of Sybyl-X 2.0.
Conclusion
In summary, a series of ((4-([1,2,4]triazolo[4,3-b][1,2,4,5]
tetrazin-6-yl)piperazin-1-yl) methyl) benzohydrazide deriv-
atives has been prepared and evaluated for anticancer activ-
ity against five cancer cell lines (A549, Bewo, Hela, HepG2,
and HT29). Compounds 5f (12μM), 5h (7.1μM), 6a
(8.4μM), and 6d (9.2μM) demonstrated better antitumor
activities against A549 than the positive agent cisplatin
(13.3μM); 6a (5.2μM) exhibited better antitumor activity
against Bewo than cisplatin (7.7μM). The molecular dock-
ing of 6a and c-Met kinase (4DEH) suggested that com-
pound 6a was a potential inhibitor of c-Met kinase.
Experiment
The binding modes of compound 6a and c-Met kinase
are depicted in Figure 2(a). As illustrated in this figure,
compound 6a is potently bound to the active site of c-Met
kinase via hydrophobic interactions and the binding is sta-
bilized by three hydrogen bonds, one C–H—π and one
π—π interaction.
The two nitrogen atoms of the triazolotetrazine ring
formed two hydrogen bonds with the same amido hydrogen
of ARG1086 (bond length: 2.23 and 2.60Å, respectively),
and the oxygen atom of benzoyl group formed one hydro-
gen bond with the amido hydrogen of ASP1222 (bond
length: 2.12Å). A C–H—π interaction was predicted to
form between the methyl hydrogen of LEU1157 and the
o-chlorophenyl of 6a (Cg1, Cg1 is centroid of o-chlorophe-
nyl ring of 6a) (C–H—Cg1=2.72 Å). A π—π interaction
was predicted to form between the two phenyl rings in 6a
(Cg2, Cg2 is centroid of phenyl ring in 6a) and TYR1236
(Cg3, Cg3 is centroid of phenyl ring in TYR1236) (Cg2—
Cg3=4.06Å) (Figure 2(a)). The enzyme surface model is
shown in Figure 2(b), which reveals that the target
Materials and methods
Melting points were carried out on a XRC-1 apparatus and
are uncorrected (Beijing Technical Instrument Co., Beijing,
China). Infrared spectra were recorded using KBr disks for
solid materials on a Nicolex FI-IR-170 instrument. The 1H
NMR and 13C NMR spectra were run on a Bruker AC400
(400MHz). Compounds were dissolved in DMSO-d6, and
chemical shifts were referenced to TMS (tetramethylsi-
lane). Mass spectra were obtained on an Agilent 1260 Ion
Trap LC/MS 500 analysis system. Elemental analyses were
performed on a Thermo-Finnigan Flash EA 1112 instru-
ment. TLC was carried out on silica gel UV-254 plates.
General procedure for the synthesis of Methyl 4-((4-([1,2,4]
triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin-1-yl)methyl)
benzoate (3)
Step 1. Methyl 4-(bromomethyl)benzoate (7.3g,
30mmol) in dichloromethane (50mL) was added drop-
wise to a stirred solution of anhydrous piperazine (12.9g,

Wu et al.
0.15mol) and dichloromethane (150mL) at 0°C. The
5
377 [(M+Na)+, 100]. Anal. calcd (%) for C16H18N8O2: C,
54.23; H, 5.12; N, 31.62; found: C, 54.10; H, 5.11; N,
31.70.
mixture was stirred at room temperature overnight. Af-
ter the reaction was over (the reaction course was moni-
tored by TLC (AcOEt)), the mixture was washed by a
saturated aqueous NaHCO3 solution (2×100mL) and
dried over Na2SO4. Finally, the solvent was evaporated
under reduced pressure and the resulting product was
used directly in the next step (yield: 5.6g, 75.2%).
((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin-1-yl)
methyl)benzohydrazide (4). Orange solid, m.p. 176–178°C;
1H NMR (400MHz, DMSO-d6): δ 9.77 (s, 1H, NH), 9.48
(s, 1H, CH), 7.82 (d, 2H, J=7.8Hz, Ar), 7.43 (d, 2H, J=7.8
Hz, Ar), 3.84 (t, 4H, J=6.2Hz, 2CH2), 3.61(s, 2H, CH2),
2.57(t, 4H, J=6.2 Hz, 2CH2), 2.38(brs, 2H, NH2). 13C NMR
(100MHz, DMSO-d6): δ 166.2, 156.6, 136.9, 132.6, 129.6,
129.4, 129.2 (2C), 127.5 (2C), 61.9, 53.1 (2C), 52.3 (2C).
ESI-MS: m/z (%): 377 [(M+Na)+, 100]. Anal. calcd (%)
for C15H18N10O: C, 50.84; H, 5.12; N, 39.53; found: C,
50.95; H, 5.10; N, 39.44.
Step 2. 6-(3,5-dimethyl-1H-pyrazol-1-yl)-[1,2,4]
triazolo[4,3-b][1,2,4,5]tetrazine 2 (13.5g, 62.0mmol)
was added to a mixture of methyl 4-(piperazin-1-yl-
methyl) benzoate 1 (14.6g, 62.0mmol) and ethyl acetate
(300mL). The reaction was carried out for 1h at 50°C.
After the reaction was over (the reaction course was
monitored by TLC (AcOEt/PE=1/1)), the solvent was
evaporated under reduced pressure, and 95% ethanol
(50mL) was added. The yellow solid sediment was fil-
tered and recrystallized in 95% ethanol to yield orange
solid (14.2g, 62.0%).
4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin-
1-yl)methyl)-N′-(4-(dimethylamino)benzylidene) benzohydra-
zide (5a). Yield: 78%. Orange solid, m.p. 149–151°C. H
1
NMR (400MHz, DMSO-d6): δ 11.57 (s, 1H, NH), 9.46 (s,
1H, CH), 8.30 (s, 1H, N=CH), 7.88 (d, 2H, J=8.0 Hz, Ar),
7.51 (m, 4H, Ar), 6.75 (d, 2H, J=7.8 Hz, Ar), 3.86 (t, 4H,
J=5.8 Hz, 2CH2), 3.63 (s, 2H, CH2), 2.97 (s, 6H, 2CH3),
2.58 (t, 4H, J=5.8Hz, 2CH2). 13C NMR (100MHz, DMSO-
d6): δ 163.0, 155.6, 151.9, 150.1, 149.0, 142.0, 136.9,
133.1, 129.3(2C), 128.8(2C), 128.0(2C), 122.1, 112.3(2C),
61.9, 52.3(2C), 44.7(2C), 40.2(2C). ESI-MS: m/z (%): 486
[(M+H)+, 40], 508 [(M+Na)+, 100]. Anal. calcd (%) for
C24H27N11O: C, 59.37; H, 5.60; N, 31.73; found: C, 59.51;
H, 5.57; N, 31.63.
General procedure for the synthesis of ((4-([1,2,4]triazolo [4,3-
b][1,2,4,5]tetrazin-6-yl)piperazin-1-yl) methyl) benzohy-drazide
(4). Methyl 4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5] piperazin-
1-yl)methyl)benzoate 3 (2.0g, 5.4mmol) and 80% hydra-
zine hydrate (30mL) in a 50-mL three-necked bottle and
stirred at room temperature for 1h. After the reaction was
over (the reaction course was monitored by TLC (AcOEt)),
the mixture was filtered, washed twice with water and
recrystallized in 95% ethanol to yield an orange solid
(1.25g, 65.2%). CAUTION: Safety precautions must be
taken when the reaction solution is filtered, as 80% hydra-
zine hydrate may release a lot of heat during filtration.
4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin-
1-yl)methyl)-N′-(3,4-dihydroxybenzylidene)benzohydrazide
(5b). Yield: 85%. Orange solid, m.p. 268–270°C. 1H NMR
(400MHz, DMSO-d6):δ 11.61 (s, 1H, NH), 9.47 (brs, 3H,
CH+2OH), 8.27 (s, 1H, N=CH), 7.88 (d, 2H, J=7.1Hz,
Ar), 7.49 (d, 2H, J=7.1Hz, Ar), 7.25 (s, 1H, Ar), 6.92(d,
1H, J=7.3Hz, Ar), 6.79 (d, 1H, J=7.3Hz, Ar), 3.87 (t, 4H,
J=5.9Hz, 2CH2), 3.64 (s, 2H, CH2), 2.59 (t, 4H, J=5.9Hz,
2CH2). 13C NMR (100MHz, DMSO-d6): δ 163.1, 155.5,
150.1, 148.7, 148.4, 146.2, 142.1, 136.8, 132.9, 129.3 (2C),
128.0 (2C), 126.2, 121.1, 116.0. 113.1, 61.8, 52.2 (2C),
44.7 (2C). ESI-MS: m/z (%): 497 [(M+Na)+, 100]. Anal.
calcd (%) for C22H22N10O3: C, 55.69; H, 4.67; N, 29.52;
found: C, 55.81; H, 4.63; N, 29.44.
General procedure for the synthesis of compounds 5a-5i. Inter-
mediate 4 (1.0g, 2.7mmol), the aromatic aldehyde
(2.7mmol), and 95% ethanol (20 mL) were added to a
50-mL single-necked bottle and stirred at room temperature
for 2–5h. After the reaction was over (the reaction course
was monitored by TLC (AcOEt/EtOH=10/1)), the mixture
was filtered and recrystallized in 95% ethanol to obtain an
orange solid.
General procedure for the synthesis of compounds 6a-6h. Inter-
mediate 4 (1.0g, 2.7mmol), the acyl chloride (2.7mmol),
and chloroform (20mL) were added to a 50-mL single-
necked bottle and stirred at room temperature for 5–15h.
After the reaction was over (the reaction course was moni-
tored by TLC (AcOEt/EtOH=10/1)), the mixture was fil-
tered and recrystallized in 95% ethanol to obtain a yellow
solid.
4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin-
1-yl)methyl)-N′-(3-hydroxy-4-methoxybenzylidene) benzohy-
drazide (5c). Yield: 82%. Orange solid, m.p. 210–212°C.
1H NMR (400MHz, DMSO-d6): δ 11.68 (s, 1H, NH), 9.48
(s, 1H, CH), 9.37 (s, 1H, OH), 8.30 (s, 1H, N=CH), 7.89
(d, 2H, J=8.1 Hz, Ar), 7.51 (d, 2H, J=8.1 Hz, Ar), 7.28 (s,
1H, Ar), 7.06 (d, 1H, J=8.4Hz, Ar), 6.98 (d, 1H, J=8.4Hz,
Ar), 3.87 (t, 4H, J=6.1 Hz, 2CH2), 3.81 (s, 3H, OCH3),
3.65 (s, 2H, CH2), 2.59 (t, 4H, J=6.1Hz, 2CH2). 13C NMR
(100MHz, DMSO-d6): δ 163.2, 155.6, 150.2, 150.1, 148.3,
147.3, 142.2, 136.9, 132.9, 129.3 (2C), 128.1 (2C), 127.6,
120.8, 112.7. 112.3, 61.9, 56.0, 52.3 (2C), 44.8 (2C). ESI-
Methyl4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piper-
azin-1-yl)methyl)benzoate (3). Orange solid, m.p. 141–
1
143°C; H NMR (400MHz, DMSO-d6): δ 9.48 (s, 1H,
CH), 7.95 (d, 2H, J=8.0Hz, Ar), 7.52 (d, 2H, J=8.0Hz,
Ar), 3.85 (brs, 7H, 2CH2+CH3), 3.65 (s, 2H, CH2), 2.58 (t,
4H, J=6.0 Hz, 2CH2). 13C NMR (100MHz, DMSO-d6): δ
166.6, 155.6, 150.1, 144.2, 136.9, 129.7(2C), 129.5(2C),
128.9, 61.8, 52.6(2C), 52.3(2C), 44.7. ESI-MS m/z (%):
+
MS: m/z (%): 511 [(M+Na) , 100]. Anal. calcd (%) for

6
Journal of Chemical Research 00(0)
C23H24N10O3: C, 56.55; H, 4.95; N, 28.67; found: C, 56.40; (4C), 61.9, 52.3 (2C), 44.7 (2C). ESI-MS: m/z (%): 488
H, 4.91; N, 28.78.
[(M+H)+, 100]. Anal. calcd (%) for C22H21N11O3: C,
54.21; H, 4.34; N, 31.61; found: C, 54.03; H, 4.36; N,
4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin- 31.69.
1-yl)methyl)-N′-(3,4-dimethoxybenzylidene)benzohydrazide
(5d). Yield: 88%. Orange solid, m.p. 185–187°C. 1H NMR 4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin-
(400MHz, DMSO-d6): δ 11.75 (s, 1H, NH), 9.48 (s, 1H, 1-yl)methyl)-N′-(4-fluorobenzylidene)benzohydrazide
CH), 8.39 (s, 1H, N=CH), 7.90 (d, 2H, J=8.0Hz, Ar), 7.51 (5h). Yield: 91%. Orange solid, m.p. 182–184°C. 1H NMR
(d, 2H, J=8.0Hz, Ar), 7.35 (s, 1H, Ar), 7.20 (d, 1H, J=8.3 (400MHz, DMSO-d6): δ 11.89 (s, 1H, NH), 9.48 (s, 1H,
Hz, Ar), 7.03 (d, 1H, J=8.3Hz, Ar), 3.87 (t, 4H, J=6.0Hz, CH), 8.46 (s, 1H, N=CH), 7.90 (d, 2H, J=8.0Hz, Ar), 7.80
2CH2), 3.83 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.66 (s, (dd, 2H, J1 =8.4Hz, J2 =5.6Hz, Ar), 7.52 (d, 2H, J=8.0Hz,
2H, CH2), 2.60 (t, 4H, J=6.0Hz, 2CH2). 13C NMR Ar), 7.32 (t, 2H, J=8.8Hz, Ar), 3.88 (t, 4H, J=5.8Hz,
(100MHz, DMSO-d6): δ 163.3, 155.6, 151.2, 150.2, 149.5, 2CH2), 3.65 (s, 2H, CH2), 2.59 (t, 4H, J=5.8Hz, 2CH2). 13C
148.4, 142.2, 136.9, 133.4, 129.4 (2C), 128.1 (2C), 127.5, NMR (100MHz, DMSO-d6): δ 163.6 (J=241Hz), 163.4,
122.4, 111.9. 108.5, 61.8, 56.0, 55.9, 52.3 (2C), 44.7 (2C). 155.6, 150.2, 147.0, 142.4, 136.9, 132.7, 131.4, 129.7 (2C)
ESI-MS: m/z (%): 503 [(M+H)+, 100], 525 [(M+Na)+, (J=15Hz), 129.4(2C), 128.2 (2C), 116.4 (2C) (J=22Hz),
80]. Anal. calcd (%) for C24H26N10O3: C, 57.36; H, 5.21; N, 61.9, 52.3 (2C), 44.8 (2C). ESI-MS: m/z (%): 461 [(M+H)+,
27.87; found: C, 57.47; H, 5.24; N, 27.80.
5], 483 [(M+Na)+, 100]. Anal. calcd (%) for C22H21FN10O:
C, 57.38; H, 4.60; N, 30.42; found: C, 57.52; H, 4.62; N,
5-4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin- 30.32.
1-yl)methyl)-N′-(4-hydroxybenzylidene)benzohydrazide
1
(5e). Yield: 75%. Orange solid, m.p. 148°C (dec.). H 4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin-
NMR (400MHz, DMSO-d6):δ 11.66 (s, 1H, NH), 9.99 1-yl)methyl)-N′-benzylidenebenzohydrazide (5i). Yield: 87%.
(brs, 1H, OH), 9.48 (s, 1H, CH), 8.35 (s, 1H, N=CH), 7.89 Orange solid, m.p. 213–215°C. 1H NMR (400MHz,
(d, 2H, J=8.1Hz, Ar), 7.56 (d, 2H, J=8.5Hz, Ar), 7.50 (d, DMSO-d6): δ 11.86 (s, 1H, NH), 9.47 (s, 1H, CH), 8.47 (s,
2H, J=8.1Hz, Ar), 6.84 (d, 2H, J=8.5Hz, Ar), 3.87 (t, 4H, 1H, N=CH), 7.92 (d, 2H, J=7.0Hz, Ar), 7.73 (d, 2H,
J=5.9Hz, 2CH2), 3.65 (s, 2H, CH2), 2.59 (t, 4H, J=5.9Hz, J=7.0Hz, Ar), 7.46–7.53 (m, 5H, Ar), 3.88 (t, 4H,
2CH2). 13C NMR (100MHz, DMSO-d6): δ 163.1, 159.9, J=5.9Hz, 2CH2), 3.66 (s, 2H, CH2), 2.60 (t, 4H, J=5.9Hz,
155.6, 150.2, 148.5, 142.2, 136.9, 133.0, 129.3 (2C), 128.0 2CH2). 13C NMR (100MHz, DMSO-d6): δ 163.4, 155.6,
(2C), 125.8 (2C), 119.6, 116.2 (2C), 62.1, 52.2 (2C), 44.8 150.2, 148.1, 142.4, 136.9, 134.8, 132.7, 130.6, 129.3(4C),
(2C). ESI-MS: m/z (%): 481 [(M+Na)+, 100]. Anal. calcd 128.2(2C), 127.6(2C), 61.9, 52.3(2C), 44.8(2C). ESI-MS:
(%) for C22H22N10O2: C, 57.63; H, 4.84; N, 30.55; found: C, m/z (%): 465 [(M+Na)+, 100]. Anal. calcd (%) for
57.75; H, 4.80; N, 30.48.
C22H22N10O: C, 59.72; H, 5.01; N, 31.66; found: C, 59.85;
H, 4.98; N, 31.57.
4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin-
1-yl)methyl)-N′-(2,4-dichlorobenzylidene)benzohydrazide N′-(4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6yl)pipera-
(5f). Yield: 94%. Orange solid, m.p. 147–149°C. 1H NMR zin-1-yl)methyl)benzoyl)-2-fluorobenzohydrazide (6a). Yield:
(400MHz, DMSO-d6): δ 11.75 (s, 1H, NH), 9.48 (s, 1H, 75%. Yellow solid, m.p. 184–186°C. 1H NMR (400MHz,
CH), 8.39 (s, 1H, N=CH), 7.90 (d, 2H, J=8.0Hz, Ar), 7.51 DMSO-d6): δ 10.67 (s, 1H, NH), 10.42 (s, 1H, NH), 9.54(s,
(d, 2H, J=8.0Hz, Ar), 7.35 (s, 1H, Ar), 7.20 (d, 1H, 1H, CH), 7.36–8.31 (m, 8H, Ar), 4.11(s, 2H, CH2), 3.86 (t,
J=8.3Hz, Ar), 7.03 (d, 1H, J=8.3Hz, Ar), 3.87 (t, 4H, 4H, J=6.2Hz, 2CH2), 2.98 (t, 4H, J=6.2Hz, 2CH2). 13C
J=5.7Hz, 2CH2), 3.66 (s, 2H, CH2), 2.60 (t, 4H, J=5.7Hz, NMR (100MHz, DMSO-d6): δ 165.6, 164.0, 162.1
2CH2). 13C NMR (100MHz, DMSO-d6): δ 163.6, 155.5, (J=253Hz), 155.4, 150.2, 148.3, 137.0, 133.6 (J=6.5Hz),
150.3, 149.9, 143.1, 136.9, 135.6, 134.4, 129.9 (2C), 128.6 130.6(J=2.4Hz), 128.5(2C), 128.2(2C), 125.1(J=2.8Hz),
(2C), 128.3, 126.0, 120.9, 111.7. 100.0, 62.5, 52.2 (2C), 124.6, 122.7 (J=15.3Hz), 116.8 (J=32.3Hz), 58.7,
+
44.8 (2C). ESI-MS: m/z (%): 511 [(M+H) , 100]. Anal. 50.1(2C), 41.9(2C). ESI-MS: m/z (%): 477 [(M+H)+,
calcd (%) for C22H20Cl2N10O: C, 51.67; H, 3.94; N, 27.39; 100]. Anal. calcd (%) for C22H21FN10O2: C, 55.46; H, 4.44;
found: C, 51.79; H, 3.93; N, 27.32.
N, 29.40; found: C, 55.35; H, 4.46; N, 29.45.
4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin- 4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin-
1-yl)methyl)-N′-(4-nitrobenzylidene)benzohydrazide 1-yl)methyl)-N′-(4-chlorobenzoyl)benzohydrazide (6b). Yield:
(5g). Yield: 90%. Orange solid, m.p. 153–155°C. 1H NMR 70%. Yellow solid, m.p. 178–180°C. 1H NMR (400MHz,
(400MHz, DMSO-d6): δ 12.19 (s, 1H, NH), 9.48 (s, 1H, DMSO-d6): δ 10.69 (s, 1H, NH), 10.66 (s, 1H, NH), 9.60 (s,
CH), 8.56 (s, 1H, N=CH), 8.32 (d, 2H, J=8.5Hz, Ar), 8.01 1H, CH), 8.02 (d, 2H, J=7.0Hz, Ar), 7.95 (d, 2H, J=7.9Hz,
(d, 2H, J=8.5Hz, Ar), 7.93 (d, 2H, J=8.0Hz, Ar), 7.54 (d, Ar), 7.79 (d, 2H, J=7.0Hz, Ar), 7.62 (d, 2H, J=7.9Hz,
2H, J=8.0Hz, Ar), 3.88 (t, 4H, J=6.1Hz, 2CH2), 3.66 (s, Ar), 4.66 (s, 2H, CH2), 3.86 (t, 4H, J=6.1Hz, 2CH2), 3.25
2H, CH2), 2.60 (t, 4H, J=6.1Hz, 2CH2). 13C NMR (t, 4H, J=6.1Hz, 2CH2). 13C NMR (100MHz, DMSO-d6):
(100MHz, DMSO-d6): δ 163.7, 155.6, 150.2, 148.3, 145.6, δ 165.7, 165.3, 155.3, 150.2, 137.3, 137.1, 132.0, 131.7,
141.1, 136.9, 132.3, 129.4 (2C), 128.5 (2C), 128.3, 124.6 129.9 (3C), 129.2 (4C), 128.3 (2C), 58.9, 50.3 (2C), 42.0

Wu et al.
7
(%): 473 [(M+H)+, 7], 495 [(M+Na)+, 6]. Anal. calcd (%)
for C23H24N10O2: C, 58.46; H, 5.12; N, 29.64; found: C,
58.61; H, 5.10; N, 29.43.
(2C). ESI-MS: m/z (%): 515 [(M+Na)+, 100]. Anal. calcd
(%) for C22H21ClN10O2: C, 53.61; H, 4.29; N, 28.42; found:
C, 53.70; H, 4.26; N, 28.35.
4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin-
1-yl)methyl)-N′-(cyclohexanecarbonyl)benzohydrazide
(6g). Yield: 54%. Yellow solid, m.p. 185–187°C. 1H NMR
(400MHz, DMSO-d6): δ 10.36 (s, 1H, NH), 9.85 (s, 1H,
NH), 9.56 (s, 1H, CH), 7.92 (d, 2H, J=7.6Hz, Ar), 7.69 (d,
2H, J=7.6Hz, Ar), 4.20 (s, 2H, CH2), 3.85 (t, 4H, J=5.8Hz,
2CH2), 3.12 (t, 4H, J=5.8Hz, 2CH2), 2.24–2.29 (m, 1H,
CH), 1.63–1.74 (m, 4H, 2CH2), 1.15–1.40 (m, 6H, 3CH2).
13C NMR (100MHz, DMSO-d6): δ 175.1, 165.4, 155.4,
150.1, 137.0, 133.3, 131.2, 129.7 (2C), 128.1 (2C), 59.6,
52.7, 50.8 (2C), 42.5 (2C), 29.5 (2C), 25.6 (3C). ESI-MS:
m/z (%): 465 [(M+H)+, 60]. Anal. calcd (%) for
C22H28N10O2: C, 56.88; H, 6.08; N, 30.15; found: C, 57.98;
H, 6.06; N, 30.11.
4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin-
1-yl)methyl)-N′-(4-Nitrobenzoyl)benzohydrazide (6c). Yield:
70%. Yellow solid, m.p. 178–180°C. 1H NMR (400MHz,
DMSO-d6): δ 10.98 (s, 1H, NH), 10.76 (s, 1H, NH), 9.57 (s,
1H, CH), 8.33 (d, 2H, J=8.6Hz, Ar), 8.17 (d, 2H, J=8.4Hz,
Ar), 8.00 (d, 2H, J=7.4Hz, Ar), 7.74 (d, 2H, J=7.4Hz,
Ar), 4.11(s, 2H, CH2), 3.86 (t, 4H, J=6.3Hz, 2CH2), 3.14
(t, 4H, J=6.3Hz, 2CH2). 13C NMR (100MHz, DMSO-d6):
δ 166.3, 165.7, 155.4, 150.5, 150.2, 137.0, 136.8, 131.2
(2C), 129.8, 129.5 (2C), 128.2 (2C), 128.0, 124.2 (2C),
62.2, 50.7 (2C), 42.8 (2C). ESI-MS: m/z (%): 504 [(M+H)+,
100]. Anal. calcd (%) for C22H21N11O4: C, 52.48; H, 4.20;
N, 30.60; found: C, 52.61; H, 4.18; N, 30.52.
N′-(4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl) pipera-
zin-1-yl)methyl)benzoyl)-3-chlorobenzohydrazide (6d). Yield:
83%. Yellow solid, m.p. 181–183°C. 1H NMR (400MHz,
DMSO-d6): δ 10.74 (s, 1H, NH), 10.69 (s, 1H, NH), 9.58(s,
1H, CH), 8.01 (s, 1H, Ar), 7.98 (d, 2H, J=7.5Hz, Ar), 7.90
(d, 1H, J=7.8Hz, Ar), 7.75 (d, 2H, J=7.5Hz, Ar), 7.70 (d,
1H, J=7.8Hz, Ar), 7.59 (t, 1H, J=7.8Hz, Ar), 4.27 (s, 2H,
CH2), 3.86 (t, 4H, J=6.2Hz, 2CH2), 3.19 (t, 4H, J=6.2Hz,
2CH2). 13C NMR (100MHz, DMSO-d6): δ 165.7, 165.0,
155.4, 150.2, 137.0, 134.9, 133.9, 133.3, 132.3, 131.5,
131.2, 129.8 (2C), 128.2 (2C), 127.7, 126.7, 59.4, 50.6(2C),
42.5(2C). ESI-MS: m/z (%): 493 [(M+H) +, 20], 515
[(M+Na)+, 30]. Anal. calcd (%) for C22H21ClN10O2: C,
53.61; H, 4.29; N, 28.42; found: C, 53.72; H, 4.25; N,
28.36.
4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin-
1-yl)methyl)-N′-hexanoylbenzohydrazide (6h). Yield: 61%.
Yellow solid, m.p. 185–187°C. 1H NMR (400MHz,
DMSO-d6): δ 10.42 (s, 1H, NH), 9.94 (s, 1H, NH), 9.58 (s,
1H, CH), 7.96 (d, 2H, J=7.7Hz, Ar), 7.78 (d, 2H, J=7.7Hz,
Ar), 4.41 (s, 2H, CH2), 3.86 (t, 4H, J=5.6Hz, 2CH2),3.56
(t, 4H, J=5.6Hz, 2CH2), 2.19 (t, 2H, J=7.3Hz, CH2),
1.49–1.56 (m, 2H, CH2), 1.25–1.32 (m, 4H, 2CH2), 0.84–
0.90 (m, 3H, CH3). 13C NMR (DMSO-d6, ppm): δ 175.0,
172.1, 165.3, 155.3, 150.1, 137.0, 133.7, 131.9 (2C), 128.3
(2C), 58.9, 50.2 (2C), 42.0 (2C), 34.2, 31.2, 25.2, 22.4,
14.4. ESI-MS: m/z (%): 453 [(M+H)+, 85], 475 [(M+Na)+,
27]. Anal. calcd (%) for C21H28N10O2: C, 55.74; H, 6.24; N,
30.95; found: C, 55.57; H, 6.26; N, 31.07.
In vitro cancer cell growth inhibition assay. The antiproliferative
activities of the compounds 3–6 against several human cancer
cell lines were assayed by standard MTT assay procedures.
Cells were cultured in DMEM (Dulbecco’s Modified Eagle
Medium) medium at 37°C with 5% CO2 and 95% air, supple-
mented with 10% (v/v) bovine calf serum. Cells were plated in
96-well plates at a density of 10,000 cells per well. After 24h,
the cells were treated with various concentrations of com-
pounds from 0.4 to 500μM. Wells containing culture medium
without cells were used as blanks and cisplatin was assayed
over the same time as a positive control. The cells were further
incubated for 72h. The cytotoxicity was measured by adding
5mg/mL of MTT to each well with incubation for another 4h.
The formazan crystals were dissolved by adding 150μL of
DMSO to each well. The optical density of each well was then
measured on a microplate spectrophotometer at a wavelength
of 570nm. The IC50 value was determined from plots of %
viability against the dose of each compound added. Each assay
was performed in triplicate. Original data can be obtained
from Supplemental material.
N′-(4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)piperazin
-1-yl)methyl)benzoyl)-3-fluorobenzohydrazide (6e). Yield:
85%. Yellow solid, m.p. 207–209°C. H NMR (400MHz,
1
DMSO-d6): δ 10.72 (s, 1H, NH), 10.70 (s, 1H, NH), 9.60(s,
1H, CH), 8.02 (d, 2H, J=8.2Hz, Ar), 7.81 (d, 3H, J=8.2Hz,
Ar), 7.72 (d, 1H, J=8.3Hz, Ar), 7.46–7.67 (m, 2H, Ar), 4.47
(s, 2H, CH2), 3.87 (t, 4H, J=6.0Hz, 2CH2), 3.51 (t, 4H,
J=6.0Hz, 2CH2). 13C NMR (100MHz, DMSO-d6): δ 165.6,
165.0, 162.4 (J=242Hz), 155.3, 150.2, 137.1, 135.1
(J=6.9Hz), 133.8, 132.2, 131.4 (J=7.0Hz), 129.9(2C),
128.3(2C), 124.1 (J=2.7Hz), 116.2 (J=21.8Hz), 114.7
(J=22.7Hz), 58.7, 50.2(2C), 41.9(2C). ESI-MS: m/z (%):
477 [(M+H)+, 20]. Anal. calcd (%) for C22H21FN10O2: C,
55.46; H, 4.44; N, 29.40; found: C, 55.59; H, 4.40; N, 29.29.
N′-(4-((4-([1,2,4]triazolo[4,3-b][1,2,4,5]tetrazin-6-yl)pipera-
zin -1-yl)methyl)benzoyl)-3-methylbenzohydrazide (6f). Yield:
65%. Yellow solid, m.p. 174–176°C. 1H NMR (400MHz,
DMSO-d6): δ 10.63 (s, 1H, NH), 10.54 (s, 1H, NH), 9.61 (s,
1H, CH), 8.02 (d, 2H, J=8.2Hz, Ar), 7.81 (d, 1H, J=8.2Hz,
Ar), 7.73–7.75 (m, 3H, Ar), 7.38–7.42 (m, 2H, Ar), 4.47 (s,
2H, CH2), 3.87 (t, 4H, J=5.9Hz, 2CH2), 3.52 (t, 4H,
J=5.9Hz, 2CH2), 2.37 (s, 3H, CH3). 13C NMR (100MHz,
DMSO-d6): δ 166.4, 165.6, 155.3, 150.2, 138.3, 137.1,
134.0, 132.9, 132.2, 130.2, 128.9 (2C), 128.5, 128.3 (2C),
126.9, 125.0, 58.7, 50.1 (2C), 41.9 (2C), 21.4. ESI-MS: m/z
Molecular docking. Molecular docking was performed with the
Surflex-Dock program interfaced with SybylX-2.0. The pro-
grams adapted an empirical scoring function and a patented
searching engine.22 The ligand was docked into the corre-
sponding protein-binding site guided by protomol, which is an

8
Journal of Chemical Research 00(0)
idealized representation of a ligand that makes every potential Funding
interaction with a binding site. In this work, The crystal struc-
ture of c-Met complexed with 5-phenyl-3-(quinolin-
6-ylmethyl)-3,5,6,7-tetrahydro-4H-[1,2,3]triazolo[4,5-c]
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: This
study was supported by Zhejiang Public Welfare Technology
pyridin-4-one (PDB entry: 4DEH) was extracted from the Research Project (LGF19B020001), a scientific research project
of the Education Department of Zhejiang Province (Y201840472),
Taizhou Science and Technology Planning Project (1902gy27),
and a research project of Taizhou Vocational and Technical
College (2019YB04).
Brookhaven Protein Database (PDB; http://www.rcsb.org/
pdb). At the beginning of docking, all the water and ligands
were removed and random hydrogen atoms were added. Next,
the receptor structure was minimized over 10,000 cycles using
the Powell method in SybylX-2.0. All the compounds were
constructed using a sketch molecular module. Hydrogen and
Gasteiger-Hückel charges were added to every molecule.
Then their geometries were optimized by the conjugate gradi-
ent method in the TRIPOS force field. The energy conver-
gence criterion is 0.001kcal/mol. Finally, ligand-based mode
was adopted to generate the “protomol,” leaving the threshold
at their default value of 1.
ORCID iD
Feng Xu
https://orcid.org/0000-0002-1696-2242
Supplemental material
Supplemental material for this article is available online.
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c-Met kinase assay in vitro. HTRF uses two fluorescence labels:
europium cryptate (fluorescence donor, EuK) and crosslinked
allophycocyanin (fluorescence acceptor, XL665). When both
fluorescence molecules are in proximity (<10nm), the energy
of EuK excited by nitrogen laser (λ=340nm) is transferred
nonradiatively to XL665, resulting in long-lived emission at
λ=665 nm. The nonspecific fluorescence from unbound
XL665 and from some other components in media or plastic
have short decay times and can exclude their interference of
the detection signal by delaying detected time. On the other
hand, the free EuK excited by nitrogen laser (λ=340nm),
resulting in long-lived emission at λ=620nm, which is used as
a background signal. These two specific signals at 665 and
620nm were measured by multifunctional microplate reader,
and the strength of detection signal from the reaction system
can reflect the activity of tested compounds against c-Met
kinase. The experimental method of HTRF was described as
follows: (1) tested compounds (4μL, diluted with buffer solu-
tion to nine different concentrations: 100,000, 20,000, 4000,
800, 160, 32, 6.4, 1.28, and 0.256nM) and c-Met kinase (2μL)
were added to each well, to which the mixture (4μL, V/V=1/1)
of TK Substrate-biotin (1µΜ) and ATP (3μM) was added.
Then each well was kept at room temperature for 40min. (2)
The mixture (10µL, V/V=1/1) of SA-XL665 (0.125µΜ) and
TK antibody-cryptate (diluted to 100 times) was added to the
above wells, after keeping the mixture at room temperature for
1h, the fluorescence signal was measured by multifunctional
microplate reader. Original data can be obtained from Supple-
mental material.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.