Synthesis and biological evaluation of novel 6-alkoxy-3-aryl-[1,2,4]triazolo[4,3-b][1,2,4,5] tetrazines

Article abstract of DOI:10.1177/1747519819861865

A series of 6-alkoxy-3-aryl-[1,2,4]triazolo[4,3-b][1,2,4,5] tetrazine derivatives is synthesized and evaluated for their antitumor activities. These compounds exhibit potent antiproliferative activities against A549, Bewo, and MCF-7 cells. Molecular docking is performed to study the inhibitor–c-Met kinase interactions, and the results show that 6-ethoxyl-3-phenylethyl-[1,2,4]triazolo[4,3-b][1,2,4,5] tetrazine is potently bound to c-Met kinase with two hydrogen bonds and one π–π interaction. Based on these preliminary results, it is thought that compound 6-ethoxyl-3-phenylethyl-[1,2,4]triazolo[4,3-b][1,2,4,5] tetrazine with potent inhibitory activity may be a potential anticancer agent.

Full text of DOI:10.1177/1747519819861865

8
research-arti
6cle2019 1865CHL0010.1177/1747519819861865Journal of Chemical ResearchShi et al.
Research Paper
Journal of Chemical Research
–6
1
Synthesis and biological evaluation of novel
-alkoxy-3-aryl-[1,2,4]triazolo[4,3-b][1,2,4,5]
tetrazines
© The Author(s) 2019
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6
1
1
1
1
Run-Jie Shi , Zhen-Zhen Yang , Ye-Tao Gao , Wen-Jin Cai ,
1
1
2
Can Ye , Feng Xu
and John Wang
Abstract
A series of 6-alkoxy-3-aryl-[1,2,4]triazolo[4,3-b][1,2,4,5] tetrazine derivatives is synthesized and evaluated for their
antitumor activities. These compounds exhibit potent antiproliferative activities against A549, Bewo, and MCF-7 cells.
Molecular docking is performed to study the inhibitor–c-Met kinase interactions, and the results show that 6-ethoxyl-
3-phenylethyl-[1,2,4]triazolo[4,3-b][1,2,4,5] tetrazine is potently bound to c-Met kinase with two hydrogen bonds and
one π–π interaction. Based on these preliminary results, it is thought that compound 6-ethoxyl-3-phenylethyl-[1,2,4]
triazolo[4,3-b][1,2,4,5] tetrazine with potent inhibitory activity may be a potential anticancer agent.
Keywords
anticancer, c-Met, molecular docking, synthesis, tetrazines, triazoles
Date received: 3 May 2019; accepted: 15 June 2019
A series of novel 6-alkoxy-3-aryl-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazines is synthesized and their anticancer activities
against A549, Bewo, and MCF-7 are tested. The results suggest that these compounds display potent antiproliferative
activities.
Introduction
(m-methylpheny)-3,6-dimethyl-1,4-dihydro-1,2,4,
-tetrazine-1,4-dicarboamide (ZGDHu-1), has been found
with strong inhibition on the proliferation of PANC-1 pancre-
5
Cancer is one of the most important problems in public
health and is the second leading cause of death in the world.
1
12
atic cancer cells via apoptosis and G2/M cell cycle arrest;
-alkylamino-1,2,4,5-tetrazine derivative I exhibits potent
Currently, cancer chemotherapy is one of the most effective
methods to treat cancer, which represents a constant, global,
and interdisciplinary research effort and can heavily promote
and extend the quality of life. New antitumor therapies point
to novel compounds with potently selective anticancer
effects and must therefore exhibit a cytotoxic effect on
3
13
antiproliferativeactivityagainstH1975cells; and3-(4-meth-
ylimidazole-1-yl)imidazo[1,2-b][1,2,4,5]tetrazine (II), an
imidazotetrazine derivative, is being used for therapy of
malignant tumors in human and animals (Figure 1).1
4
2–4
malignant cells, without damaging normal cells.
Most of the known synthetic anticancer drugs are hetero-
cyclic compounds, and several of them correspond to nitro-
1Pharmaceutical Research Institute, Taizhou Vocational & Technical
College, Taizhou, P.R. China
ImmuOn Therapeutics, Nantong, P.R. China
5–7
2
gen heterocycles. In the last decades, a large number of
,2,4,5-tetrazine derivatives have been synthesized and their
antitumor activity has been reported, which has led to them
1
Corresponding author:
Feng Xu, Pharmaceutical Research Institute, Taizhou Vocational &
Technical College, Taizhou 318000, P.R. China.
8–11
emerging as a promising and attractive scaffolds. Among
them, a 1,4-dihydro-1,2,4,5-tetrazine derivative, N,N′-di Email: xufeng901@126.com

2
Journal of Chemical Research 00(0)
Figure 1. Structures of 1,2,4,5-tetrazine derivatives with anticancer activities.
Our research group has synthesized numerous [1,2,4]
triazolo[4,3-b][1,2,4,5]tetrazine derivatives, and some
of them (TZXU-1-3) showed potent antiproliferative
activities against MCF-7, Bewo, and HL-60 cells and
c-Met kinase inhibitory activities (Figure 1).15–17 All
thesesyntheticcompoundsbelongto6-alkylamino-[1,2,4]
triazolo[4,3-b][1,2,4,5]tetrazine derivatives; however,
literature focused on 6-alkoxy-[1,2,4]triazolo[4,3-b]
[
1,2,4,5] tetrazine derivatives are rare. On the basis of
the above research, we have prepared some new [1,2,4]
triazolo[4,3-b][1,2,4,5]tetrazines and have attempted to
investigate how the alkoxy groups located at the 6-posi-
tion of the triazolotetrazine ring influence antitumor
activity. This letter reports the synthesis of 10 [1,2,4]
triazolo[4,3-b][1,2,4,5]tetrazines. Subsequently, in vitro
bioassays were performed to evaluate the anticancer
Scheme 1. Synthesis of the target compounds 5.
activities against human lung adenocarcinoma cells with different aromatic aldehydes in ethanol to give com-
2
0
(
A549), human placenta choriocarcinoma cells (Bewo), pounds to 3 via the Schiff reaction. Compounds 3 were
and human breast cancer cells (MCF-7). Furthermore, then subjected to intramolecular cyclization with lead
2
1
molecular docking was performed to study the inhibitor tetraacetate as the oxidizing agent. Finally, the title com-
c-Met kinase interactions.
pounds 5 were prepared from the compound 4 by treat-
ment with alkyl alcohols.
The structures of the synthesized compounds were
1
13
Results and discussion
confirmed from their H NMR, C NMR, infrared (IR),
and elemental analysis data. All of these compounds pro-
vided satisfactory spectroscopic data, which were consist-
Chemical synthesis
Scheme 1 details the synthesis and structures of the title ent with the assigned structures. It should be noted in this
compounds. The starting material 1 was prepared using context that to the best of our knowledge, all of the title
1
8
the published method. Compound 1 was then reacted products are reported for the first time. The 1H NMR and
1
3
with 80% hydrazine hydrate in acetonitrile at room tem-
C NMR spectra of the title compounds are given in the
,
19
perature to yield compound 2 which was then heated supplementary data.

Shi et al.
3
Anticancer activity in vitro
the R1 group was critical for enhancing the anticancer
potency of the target compounds, and compounds with an
electron-donating group (e.g. Me) on the phenyl ring were
more effective than those with an electron-withdrawing
group (e.g. Cl). This trend is the reverse of our previously
reported work regarding the substituted group on the
phenyl ring (connected to the piperazine ring) located at
the 6-position of the triazolotetrazine ring. That is elec-
To test the antitumor activities of the synthesized com-
pounds, we evaluated the antiproliferative activities of
compounds 5 against A549, Bewo, and MCF-7 cells by the
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
,
16
mide (MTT) assay. The results are summarized in Table 1.
The active analogs were found to display cytotoxic activ-
ity. In terms of the bioactivities against A549, all the com-
pounds were no more effective than the positive control
cisplatin (IC =33.23μM), except for compounds 5b
tron-withdrawing groups (e.g. NO , Cl) on the phenyl ring
2
were more effective than an electron-donating group (e.g.
Me). This result is of interest when taking into considera-
tion structure optimization.
5
0
(
IC =8.08μM) and 5j (IC =21.68μM). Particularly, all
5
0
50
of the tested compounds presented higher anticancer activ-
ities against MCF-7 than the positive control cisplatin
(
IC =50.09μM), and compound 5j (IC =2.81μM)
5
0
50
Binding mode analysis
showed the most potent biological activity against MCF-7,
and its inhibitory effect was close to that of TZXU-1 To gain a better understanding of the potency of the target
IC =2.24μM). Notably, when the R group was the compounds and to guide further structure–activity relation-
(
5
0
2
same (e.g. ethyl), the anticancer activity results against ship (SAR) studies, we continued to investigate the interac-
MCF-7 followed the order: 5h>5f>5c>5g. This tion effect between compound 5j and the c-Met crystal
revealed that the substituted group on the phenyl ring of structure (3EFJ.pdb). The molecular docking was per-
formed by inserting compound 5j into c-Met kinase at the
active site. All docking runs were applied in Surflex-Dock
of Sybyl-X 2.0.
Table 1. Antitumor activities against MCF-7, Bewo, and HL-60
cell lines in vitro (IC50 in μM) .
a
The binding modes of compound 5j and c-Met kinase
Compound
IC50 (μM)
are depicted in Figure 2(a). As illustrated in Figure 2, com-
pound 5j is potently bound to the active site of c-Met kinase
via hydrophobic interactions and binding is stabilized by
two hydrogen bonds and one π–π interaction.
The two nitrogen atoms of the triazole ring formed
two hydrogen bonds with the same amido hydrogen of
LYS1110 (bond lengths: 1.94 and 2.79 Å, respectively),
and the π–π interaction was predicted to form between
the two phenyl rings in 5j (Cg1, Cg1 is centroid of the
phenyl ring in 5j) and PHE1223 (Cg2, Cg2 is centroid of
the phenyl ring in PHE1223) (Cg1–Cg2 = 3.83 Å; Figure
A549
Bewo
MCF-7
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
49.50
8.08
86.72
106.96
90.89
70.03
69.22
83.13
147.91
56.97
87.76
20.72
34.86
1.30
14.40
12.39
23.50
6.89
23.47
9.76
26.67
4.72
9.68
88.42
45.89
54.78
43.37
85.66
64.00
79.49
21.68
33.23
–
2
(a)). The enzyme surface model is shown in Figure 2(b),
j
2.81
50.09
2.24
which revealed that the target molecule 5j was well-
embedded in the active pocket of c-Met kinase. This
molecular docking result, along with the biological assay
data, suggested that compound 5j is a potential inhibitor
of c-Met kinase.
Cisplatin
TZXU-1
b
a
Values presented are the mean of three experiments.
Data taken from the literature.
b
15
Figure 2. (a) Compound 5j (colored by atom: carbons: gray; nitrogen: blue; oxygen: red) is bound into c-Met (entry 3EFJ in the
Protein Data Bank). The dotted lines show the hydrogen bonds and π–π interaction. (b) The surface model structure of binding
compound 5j with the c-Met complex.

4
Journal of Chemical Research 00(0)
Experimental
3-(4-Fluorophenyl)-6-isobutoxy-[1,2,4]triazolo[4,3-b][1,2,4, 5]
tetrazine (5a). Yellow solid, m.p. 128–130°C; IR (KBr,
Materials and methods
−1
cm ): 3074, 2974, 1606, 1547, 1057, and 861. 1H NMR
) δ: 8.45 (t, J=8.8Hz, 2H), 7.52 (t,
tus and are uncorrected (Beijing Technical Instrument Co.). J=8.8Hz, 2H), 4.29 (d, J=6.3Hz, 2H), 2.23 (m, 1H), 1.06
IR spectra were recorded as KBr disks of solid materials on (d, J=6.3Hz, 2H); 13C NMR (100MHz, DMSO-d
Melting points (m.p.s) were obtained on an XRC-1 appara- (400MHz, DMSO-d
6
) δ:
6
a Nicolex FI-IR-170 instrument. The 1H NMR spectra 165.2, 159.4, 152.1, 144.3, 130.2, 130.1, 121.6, 117.2, 117.0,
+
were run on a Bruker AC400 (400 MHz) spectrometer. 75.9, 27.6, 19.3 (2C); MS (ES): m/z (%) 289.2 ((M+H) ,
Compounds were dissolved in DMSO-d or CDCl , and 100). Anal. calcd for C13H13FN O: C, 54.16; H, 4.55; N,
6
3
6
chemical shifts were referenced to tetramethylsilane 29.15; O, 5.55; found: C, 54.25; H, 4.54; N, 29.12; O, 5.57.
TMS). Mass spectra were obtained on an Agilent 1260 Ion
Trap LC-MS 500 analysis system. Elemental analyses were 3-(4-Fluorophenyl)-6-methoxy-[1,2,4]triazolo[4,3-b][1,2,4, 5]tet-
(
−1
performed on a Thermo-Finnigan Flash EA 1112 instru- razine (5b). Yellow solid, m.p. 178–180°C; IR (KBr, cm ):
ment. Thin-layer chromatography (TLC) was carried out 3099, 2977, 1605, 1559, 1052, and 852; 1H NMR (400MHz,
on silica gel UV-254 plates.
DMSO-d
6
) δ: 8.47–8.50 (m, 2H), 7.51 (dd, J =10.0,
1
J2 =8.0Hz, 2H), 4.19 (s, 3H); 13C NMR (100MHz, DMSO-
d ) δ: 165.3, 159.9, 152.1, 144.3, 130.2, 130.3, 121.5, 117.2,
6
General procedure for the synthesis of
compounds 4a–f
+
117.0, 57.2. MS (ES): m/z (%) 247.2 ((M+H) ,100). Anal.
calcd for C H FN O: C, 48.78; H, 2.87; N, 34.13; O, 6.50;
10
7
6
Lead tetraacetate (5.0g, 11.4mmol) in chloroform (10mL) found: C, 48.69; H, 2.86; N, 34.20; O, 6.48.
was added to a refluxing mixture of compound 3 (10mmol)
and chloroform (20mL). The color of the solution changed 3-(2,4-Dichlorophenyl)-6-ethoxy-[1,2,4]triazolo[4,3-b][1,
from wine-red to orange. After substrate 3 was completely 2,4,5]tetrazine (5c). Yellow solid, m.p. 146–148°C; IR
−1
consumed (the reaction course was monitored by TLC (KBr, cm ): 3098, 2979, 1596, 1549, 1052, and 826; 1H
AcOEt)), evaporation of the chloroform gave crude yellow NMR (400MHz, DMSO-d ) δ: 7.97 (s, 1H), 7.80 (d,
compound 4, which was recrystallized from absolute J=8.8Hz, 1H), 7.71 (d, J=8.8Hz, 1H), 4.40 (q, J=7.0Hz,
(
6
ethanol.
2H), 1.06 (t, J=7.0Hz, 3H); 13C NMR (100MHz, DMSO-
Compounds 4a, 4b, 4d, and 4e have been described in
our previous report.
d ) δ: 159.5, 151.5, 143.7, 137.6, 134.8, 134.1, 130.7,
6
1
7
128.6, 122.7, 66.5, 14.2; MS (ES): m/z (%) 311.1 ((M+H)
+
,
+
1
00), 333.1 ((M+Na) , 50); Anal. calcd for C H Cl N O:
1
1
8
2
6
3
-(4-Chlorophenyl)-6-(3,5-dimethyl-1H-pyrazol-1-yl)-[1,2,4] C, 42.46; H, 2.59; N, 27.01; O, 5.14; found: C, 42.57; H,
triazolo[4,3-b][1,2,4,5]tetrazine (4c). Yellow solid, m.p. 2.59; N, 26.93; O, 5.13.
−
1
1
1
(
1
98–200°C; IR (KBr, cm ): 2976, 1529, 1396, 1452,
+
3-(2,4-Dichlorophenyl)-6-isopropoxy-[1,2,4]triazolo[4,3-b][1,
036, and 830. MS (ES): m/z 327 (M+H) ; 1H NMR
400MHz, DMSO-d ) δ: 8.43 (d, 2H), 7.75 (d, 2H), 6.25 (s, 2,4,5]tetrazine (5d). Yellow solid, m.p. 128–130°C; IR
6
−1
H), 2.80 (s, 3H), 2.41 (s, 3H); Anal. calcd (%) for (KBr, cm ): 3094, 2988, 1547, 1399, 1053, and 824; 1H
) δ: 7.98 (s, 1H), 7.79 (d,
C H ClN : C, 51.46; H, 3.39; N, 34.29; found: C, 51.52; NMR (400MHz, DMSO-d
1
4
11
8
6
H, 3.37; N, 34.22.
J=8.4Hz, 1H), 7.72 (d, J=8.4Hz, 1H), 5.06 (sext, J=6.1
Hz, 1H), 1.42(d, J=4.1 Hz, 6H); 13C NMR (100MHz,
6
-(3,5-Dimethyl-1H-pyrazol-1-yl)-3-phenethyl-[1,2,4]tri- DMSO-d
azolo[4,3-b][1,2,4,5]tetrazine (4f). Red solid, m.p. 143– 130.7, 128.5, 122.7, 74.8, 21.5 (2C); MS (ES): m/z (%)
325.1 ((M+H) , 100), 347.1 ((M+Na) , 23); Anal. calcd
for C H Cl N O: C, 44.33; H, 3.10; N, 25.85; O, 4.92;
6
) δ: 158.9, 151.4, 143.6, 137.5, 134.8, 134.1,
−
1
+
+
1
45°C; IR (KBr, cm ): 3260, 3071, 1589, 1488, and 1418;
+
MS (ES): m/z 321 (M+H) ; 1H NMR (400 MHz, DMSO-
12 10
2
6
d6) δ: 7.16–7.27 (m, 5H, ArH), 6.35 (s, 1H), 3.45(t, found: C, 44.43; H, 3.11; N, 25.82; O, 4.91.
J=7.4Hz, 2H), 3.17(d, J=7.4Hz, 2H), 2.58 (s, 3H), 2.27 (s,
3
3
H); Anal. calcd (%) for C H N : C, 59.99; H, 5.03; N, 3-(2,4-Dichlorophenyl)-6-isobutoxy-[1,2,4]triazolo[4,3-b][1,2,
16 16 8
4.98; found: C, 59.90; H, 5.02; N, 35.03.
4,5]tetrazine (5e). Yellow solid, m.p. 168–170°C; IR
−1
(KBr, cm ): 3058, 2977, 1583, 1538, 1038, and 821; 1H
NMR (400MHz, DMSO-d ) δ: 7.97 (s, 1Hz), 7.80 (d,
6
General procedure for the synthesis of
J=8.4Hz, 1H), 7.22 (d, J=8.4Hz, 1H), 4.13(d, J=6.4Hz,
compounds 5a–j
2
H), 2.14 (m, 1H), 1.00 (d, J=6.7Hz, 6H); 13C NMR
) δ: 159.7, 151.5, 143.6, 137.6, 134.8,
mixed and heated to reflux for 5–10 h. After 4 was com- 134.0, 130.7, 128.5, 122.7, 75.9, 27.6, 19.2 (2C); MS (ES):
Compound 4 (3mmol) and the alkyl alcohol (20mL) were (100MHz, DMSO-d
6
+
+
pletely consumed (the reaction course was monitored by m/z (%) 340.2 ((M+2) , 100), 361.2 ((M+Na) , 25);
TLC (PE/AcOEt=1:1)), the mixture was cooled to room Anal. calcd for C13 O: C, 46.03; H, 3.57; N, 24.78;
H
12Cl N
2 6
temperature and the excess alcohol evaporated. The residue O, 4.72; found: C, 46.12; H, 3.57; N, 24.70; O, 4.70.
was added cooled ethanol (3mL), resulting in a significant
amount of a yellow solid, which was purified by prepara- 3-(4-Chlorophenyl)-6-ethoxy-[1,2,4]triazolo[4,3-b][1,2,4,5] tet-
−1
tive thin-layer chromatography over silica gel PF 254 (2 razine (5f). Yellow solid, m.p. 220–222°C; IR (KBr, cm ):
mm, PE/AcOEt=2:1).
3090, 2994, 1539, 1054, and 851; 1H NMR (400MHz,

Shi et al.
5
Modified Eagle Medium (DMEM) medium at 37°C with
DMSO-d ) δ: 8.41 (d, J=8.6Hz, 2H), 7.73 (d, J=8.6 Hz,
6
5
% CO and 95% air, supplemented with 10% (v/v) bovine
2
H), 4.56 (q, J=7.0Hz, 2H), 1.50 (t, J=7.0Hz, 3H); 13C
2
calf serum. Cells were plated in 96-well plates at a density
of 10,000 cells per well. After 24 h, the cells were treated
with various concentrations of compounds from 0.5 to
NMR (100MHz, DMSO-d ) δ: 159.2, 152.2, 144.1, 136.5,
6
1
2
4
30.0 (2C), 129.3 (2C), 123.9, 66.5, 14.3; MS (ES): m/z (%)
+
77.0 ((M+H) , 100). Anal. calcd for C H ClN O: C,
11
9
6
−1
5
0μgmL . Wells containing culture medium without cells
7.75; H,3.28; N, 30.37; O, 5.78; found: C, 47.90; H, 3.27;
were used as blanks and cisplatin was assayed over the
same time as a positive control. The cells were further incu-
bated for 72h. The cytotoxicity was measured by adding
N, 30.27; O, 5.77.
6
-Ethoxy-3-phenyl-[1,2,4]triazolo[4,3-b][1,2,4,5]tetrazine
−1
−1
5mgmL of MTT to each well with incubation for another
h. The formazan crystals were dissolved by adding 150μL
(5g). Yellow solid, m.p. 120–122°C; IR (KBr, cm ):
4
3
068, 2986, 1573, 1541, 1054, 747, and 705; 1H NMR
of DMSO to each well. The optical density of each well
was then measured on a microplate spectrophotometer at a
(
400MHz, DMSO-d ) δ: 8.43–8.45 (m, 2H), 7.65–7.71 (m,
6
3
1
1
H, ArH), 4.58 (q, J=7.0Hz, 2H), 1.53(t, J=7.0Hz, 3H);
wavelength of 570nm. The IC value was determined from
3C NMR (100MHz, DMSO-d ) δ: 159.2, 152.2, 144.1,
50
6
plots of % viability against the dose of each compound
added. Each assay was performed in triplicate.
31.8, 130.7 (2C), 128.8 (2C), 125.9, 66.4, 14.3; MS (ES):
+
m/z (%) 243.1 ((M+H) , 100); Anal. calcd for C H N O:
C, 54.54; H, 4.16; N, 34.69; O, 6.60; found: C, 54.61; H,
1
1
10
6
4
.14; N,34.75; O, 6.58.
Molecular docking
Molecular docking was performed with the Surflex-Dock
program interfaced with Sybyl-X 2.0. The programs adapted
an empirical scoring function and a patented searching
engine. The ligand was docked into the corresponding pro-
tein binding site guided by protomol, which is an idealized
representation of a ligand that makes every potential inter-
action with a binding site. In this work, the crystal structure
of c-Met complexed with 2-benzyl-5-{4-[(6,7-dimethox-
yquinolin-4-yl)oxy]-3-fluorophenyl}-3-methylpyrimidin-
3
-(4-Methoxyphenyl)-6-ethoxy-[1,2,4]triazolo[4,3-b][1,2, 4,5]
tetrazine (5h). Yellow solid, m.p. 143–145°C; IR (KBr,
cm ): 3086, 2991, 1529, 1044, and 850; 1H NMR
−
1
22
(
400MHz, DMSO-d ) δ: 8.38 (d, J=8.6Hz, 2H), 7.19 (d,
6
J=8.6Hz, 2H), 4.56 (q, J=7.0Hz, 2H), 3.83 (s, 3H), 1.50
(
1
6
t, J=7.0Hz, 3H); 13C NMR (100 MHz, DMSO-d ) δ:
6
59.2, 152.2, 144.3, 136.2, 129.5 (2C), 125.7 (2C), 123.6,
+
6.4, 55.3, 14.2; MS (ES): m/z (%) 273.1 ((M+H) , 100);
Anal. calcd for C H N O: C, 52.94; H, 4.44; N, 30.87; O,
1
2
12
6
4(3H)-one (PDB entry: 3EFJ) was extracted from the
1
1.75; found: C, 52.82; H, 4.46; N, 30.95; O, 11.78.
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 Sybyl-X 2.0. All the com-
pounds 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 gradient method in the TRIPOS force field.
3
-Phenylethyl-6-methoxy-[1,2,4]triazolo[4,3-b][1,2,4,5] tetra-
−1
zine (5i). Yellow solid, m.p. 98–100°C; IR (KBr, cm ):
098, 2945, 1579, 1551, 1041, 745, and 707; 1H NMR
400MHz, DMSO-d ) δ: 7.16–7.27 (m, 5H, ArH), 4.09 (s,
3
(
6
3
H), 3.37 (t, J=7.7Hz, 2H), 3.15 (t, J=7.7Hz, 2H); 13C
NMR (100MHz, DMSO-d ) δ: 159.8, 151.5, 147.8, 140.5,
6
1
28.9 (2C), 128.8 (2C), 126.8, 57.0, 31.6, 25.4; MS (ES):
+
m/z (%) 257.3 ((M+H) , 100); Anal. calcd for C H N O:
C, 56.24; H, 4.72; N, 32.79; O, 6.24; found: C, 56.35; H,
1
2
12
6
−1
The energy convergence criterion is 0.001kcalmol .
Finally, ligand-based mode was adopted to generate the
4
.71; N, 32.75; O, 6.22.
“protomol,” leaving the threshold at their default value of 1.
3
-Phenylethyl-6-ethoxyl-[1,2,4]triazolo[4,3-b][1,2,4,5] tetra-
−1
zine (5j). Yellow solid, m.p. 83–85°C; IR (KBr, cm ): Conclusion
024, 2999, 1601, 1541, 1037, 748, and 698; 1H NMR
400MHz, DMSO-d ) δ: 7.14–7.26 (m, 5H, ArH), 4.47 (q,
3
(
In summary, several of 6-alkoxy-3-aryl-[1,2,4]triazolo[4,3-
b][1,2,4,5]tetrazine derivatives have been prepared and
evaluated for their anticancer activities against three cancer
cell lines (MCF-7, Bewo, and HL-60). Compound 5b
showed substantially strong anticancer activity against
6
J=7.0Hz, 2H), 3.36 (t, J=7.7Hz, 2H), 3.13 (t, J=7.7Hz,
H), 1.46 (t, J=7.0Hz, 3H); 13C NMR (100MHz, DMSO-
d6) δ: 159.1, 151.5, 147.7, 140.5, 128.9 (2C), 128.8 (2C),
26.8, 66.2, 31.6, 25.4, 14.2; MS (ES): m/z (%) 271.5
2
1
A549 in vitro with an IC value of 8.08μM. Compound 5j
+
+
50
(
(M+H) , 100), 293.3 ((M+Na) , 83); Anal. calcd for
showed the most potent biological activity against MCF-7
and Bewo with IC50 values of 2.81 and 20.72μM, respec-
tively, and the molecular docking result between 5j and
c-Met kinase (3EFJ) suggested that compound 5j was a
potential inhibitor of c-Met kinase.
C H N O: C, 57.77; H, 5.22; N, 31.09; O,5.92; found: C,
1
3
14
6
5
7.70; H, 5.20; N, 31.15; O, 5.93.
Biological evaluation
In vitro cancer cell growth inhibition assay
Declaration of conflicting interests
The antiproliferative activities of the compounds 5 against The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
several human cancer cell lines were assayed by standard
MTT assay procedures. Cells were cultured in Dulbecco’s

6
Journal of Chemical Research 00(0)
Funding
8. Rao GW, Guo YM and Hu WX. Chem Med Chem 2012; 7:
9
73.
. Rao GW, Wang C and Wang J. Bioorg Med Chem Lett 2013;
3: 6474.
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 the Science and Technology Innovation project of
college students in Zhejiang (2018R477002), the Zhejiang Public
Welfare Technology Research Project (LGF19B020001), and the
Research Project of Taizhou Vocational and Technical College
9
2
1
1
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13. Canete-Molina A, Espinosa-Bustos
C and Gonzalez-
Castro M. Arab J Chem 2017; https://doi.org/10.1016/j.ara-
bjc.2017.04.002.
ORCID iD
Feng Xu
https://orcid.org/0000-0002-1696-2242
14. Danilenko VN, Charushin VN and Rusinov GL. RU Patent
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Supplemental material
2
Supplemental material for this article is available online.
4
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