Discovery of highly potent tubulin polymerization inhibitors: Design, synthesis, and structure-activity relationships of novel 2,7-diaryl-[1,2,4]triazolo[1,5-a]pyrimidines

Article abstract of DOI:10.1016/j.ejmech.2021.113449

By removing 5-methyl and 6-acetyl groups in our previously reported compound 3, we designed a series of novel 2,7-diaryl-[1,2,4]triazolo[1,5-a]pyrimidine derivatives as potential tubulin polymerization inhibitors. Among them, compound 5e displayed low nanomolar antiproliferative efficacy on HeLa cells which was 166-fold higher than the lead analogue 3. Interestingly, 5e displayed significant selectivity in inhibiting cancer cells over HEK-293 (normal human embryonic kidney cells). In addition, 5e dose-dependently arrested HeLa in G2/M phase through the alterations of the expression levels of p-cdc2 and cyclin B1, and caused HeLa cells apoptosis by regulation of expressions of cleaved PARP. Further evidence demonstrated that 5e effectively inhibited tubulin polymerization and was 3-fold more powerful than positive control CA-4. Moreover, molecular docking analysis indicated that 5e overlapped well with CA-4 in the colchicine-binding site. These studies demonstrated that 2,7-diaryl-[1,2,4]triazolo[1,5-a]pyrimidine skeleton might be used as the leading unit to develop novel tubulin polymerization inhibitors as potential anticancer agents.

Full text of DOI:10.1016/j.ejmech.2021.113449

European Journal of Medicinal Chemistry 220 (2021) 113449
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of highly potent tubulin polymerization inhibitors: Design,
synthesis, and structure-activity relationships of novel 2,7-diaryl-
[1,2,4]triazolo[1,5-a]pyrimidines
Xian-Sen Huo 1, Xie-Er Jian 1, Jie Ou-Yang 1, Lin Chen, Fang Yang, Dong-Xin Lv,
Wen-Wei You, Jin-Jun Rao**, Pei-Liang Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Science, Southern Medical University, Guangzhou, 510515, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
By removing 5-methyl and 6-acetyl groups in our previously reported compound 3, we designed a series
of novel 2,7-diaryl-[1,2,4]triazolo[1,5-a]pyrimidine derivatives as potential tubulin polymerization in-
hibitors. Among them, compound 5e displayed low nanomolar antiproliferative efficacy on HeLa cells
which was 166-fold higher than the lead analogue 3. Interestingly, 5e displayed significant selectivity in
inhibiting cancer cells over HEK-293 (normal human embryonic kidney cells). In addition, 5e dose-
dependently arrested HeLa in G2/M phase through the alterations of the expression levels of p-cdc2
and cyclin B1, and caused HeLa cells apoptosis by regulation of expressions of cleaved PARP. Further
evidence demonstrated that 5e effectively inhibited tubulin polymerization and was 3-fold more
powerful than positive control CA-4. Moreover, molecular docking analysis indicated that 5e overlapped
well with CA-4 in the colchicine-binding site. These studies demonstrated that 2,7-diaryl-[1,2,4]triazolo
[1,5-a]pyrimidine skeleton might be used as the leading unit to develop novel tubulin polymerization
inhibitors as potential anticancer agents.
Received 16 December 2020
Received in revised form
3 April 2021
Accepted 4 April 2021
Available online 16 April 2021
Keywords:
2,7-Diaryl-[1,2,4]triazolo[1,5-a]pyrimidine
Anticancer agents
Tubulin inhibitors
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
bioavailability, and its phosphate disodium CA-4P is under evalu-
ation in phase III trials for the treatment of anaplastic thyroid
Microtubules are highly dynamic frameworks composed by
a
-
cancer and other advanced solid tumors [12,13]. Hence, CA-4 has
always been considered as a promising lead compound as a tubulin
polymerization inhibitor since its discovery, and extensive in-
vestigations strongly demonstrated that the cis-configuration of
the olefinic double bond is essential for antiproliferative activity,
and fixing this bond through incorporation into heterocycle rings
has recently become a well-verified strategy [14e18]. In addition
[1,2,4]triazolo[1,5-a]pyrimidine scaffold is highly privileged in
medicinal chemistry due to its versatile pharmacological activity
profile, such as anticancer [19,20], antimicrobial [21], antiviral [22],
herbicidal [23e26], and fungicidal activities [27]. Our research
group has explored a great number of fused bicyclic systems as cis-
restricted CA-4 analogues [28e33], and more recently we also re-
ported a series of 6-acetyl-5-methyl- [1,2,4]triazolo[1,5-a]pyrimi-
dine derivatives as new tubulin inhibitors, exampled by analogue 3
which showed potent antiproliferative properties in inducing G2/M
arrest and remarkable antitubulin activity [28]. The potent anti-
tumor activity made the class of compounds worth further
evaluation.
and -tubulin dimmers, which are involved in abundant funda-
b
mental cellular processes, including maintenance of cytoskeleton,
cell division, spindle formation and intracellular trafficking [1].
These aforementioned vital roles make tubulin an extremely
attractive target for clinically effective anticancer drugs [2e5].
In last decades, great deals of chemically diverse natural and
synthetic compounds targeting tubulin have been explored [6e10],
among which, a natural cis-stilbene derivative CA-4 (Fig. 2) pre-
sents strong antiproliferative activities toward multiple cancer cell
lines, including cells bearing a multidrug resistance phenotype, and
inhibits tubulin polymerization through interacting with tubulin at
the colchicine binding site [11]. However, CA-4 possessed poor
activity in vivo because of poor water solubility and low
* Corresponding author.
** Corresponding author.
E-mail addresses: raojj@smu.edu.cn (J.-J. Rao), plzhao@smu.edu.cn (P.-L. Zhao).
Authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113449
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

X.-S. Huo, X.-E. Jian, J. Ou-Yang et al.
European Journal of Medicinal Chemistry 220 (2021) 113449
Fig. 1. Structures of several reported tubulin inhibitors 1e3, and newly designed compounds 4aeu and 5aef.
Fig. 2. Predicted binding mode of (A) 3 (amber stick) and (B) corresponding 4 (amber stick) with tubulin (PDB code: 5lyj) and overlapping with CA-4 (yellow stick).
Our previous docking studies indicated that 5-methyl and 6-
acetyl groups of the triazolopyrimidine core would be more steri-
cally restrictive of the molecule in the binding pocket. The
modeling studies further revealed that both compound 3 and cor-
responding 4 without two substituents could dock within the
tubulinꢀcolchicine binding site. But 3,4,5-trimethoxyphenyl of
compound 3 can’t overlap with that of CA-4, while analogue 4
superimposed well with CA-4 (Fig. 2). Hence, in our current study, a
series of new [1,2,4]triazolo[1,5-a]pyrimidine derivatives 4aeu
were designed by removing 5-methyl and 6-acetyl groups in
compound 3. Meanwhile, we shifted 3,4,5-trimethoxyphenyl from
C-2 to C-7 position to attain the derivatives with typical structure
5aef (Fig. 1). To the best of our knowledge, these target compounds
were unexplored so far as tubulin polymerization inhibitors.
AcOH at reflux to generate the required products 4aeu and 5aef in
yields ranging from 52 to 84%. All final analogues were fully char-
acterized through various spectroscopic techniques including 1H
NMR, 13C NMR, and HRMS, which were provided in the Experi-
mental Section.
3. Results and discussion
3.1. In vitro cell growth inhibitory activity
The in vitro cell growth inhibitory activity of 27 newly synthe-
sized derivatives 4aeu and 5aef were first screened toward HeLa
(human cervical cancer cell line) by the conventional MTT assay
with our previously reported compound 3 and CA-4 as positive
controls. The results were illustrated in Table 1.
2. Chemistry
In general, almost all investigated compounds 4aeu (except for
4g, 4m, 4p and 4r) having 3,4,5-trimethoxyphenyl group in posi-
tion 2 of triazolopyrimidine ring, didn’t display potent anti-
proliferative efficacy toward HeLa possessing IC50 values higher
Synthesis of newly designed 2,7-diaryl- [1,2,4]triazolo[1,5-a]
pyrimidine analogues 4aeu and 5aef was outlined in Scheme 1.
The key intermediates 3-substituted 5-amino-1,2,4-triazoles 9 and
16 for the final cyclization reaction were obtained in four steps
starting with appropriate benzoic acids according to reported
procedure [34]. Meanwhile, commercially available ketones 10 and
17 were reacted with an excess of N,N-dimethylformamide
dimethyl acetal (DMFDMA) under solvent-free conditions at 90 ꢁC
to give arylpropenones 12 with good yields [33,34], which were
then condensed with 5-amino-1,2,4-triazoles 9 and 16 in EtOH or
than 30
amino-4-methoxyphenyl substituent showed the highest cytotox-
icity with an IC50 value of 0.40 M. Interestingly, most corre-
mM. Notably, among these compounds, 4m with the 3-
m
sponding compounds 5aef bearing 3,4,5-trimethoxyphenyl in
position 7 of the fused core displayed excellent antitumor activities
with IC50 values of nanomolar range, which revealed that the po-
sition of 3,4,5-trimethoxyphenyl group played a crucial role in
antiproliferative activity. Within series of 5aef, compounds with
2

X.-S. Huo, X.-E. Jian, J. Ou-Yang et al.
European Journal of Medicinal Chemistry 220 (2021) 113449
Scheme 1. Synthesis of 2,7-diaryl- [1,2,4]triazolo[1,5-a]pyrimidines 4aeu and 5aef. Reagents and conditions: (a) conc. H2SO4, ethanol, reflux; (b) NH2NH2$H2O, ethanol, reflux; (c)
S-methylisothiourea sulfate; (d) 5% NaOH, 36 h, reflux; (e) 90 ꢁC.; (f) AcOH, 80 ꢁC; or EtOH, HCl, 80 ꢁC.
Table 1
Growth inhibitory effects of compounds 4aeu and 5aef toward HeLa cell line.
Compd.
R
IC50 mean SDa
(m
M)
Compd.
R
IC50 mean SDa
(mM)
4a
4b
3,4-(CH3O)2Ph
4-CH3Ph
>30
>30
4p
4q
4-NH2Ph
29.94 2.80
>30
4c
3,4,5-(CH3O)3Ph
>30
4r
28.56 3.36
4d
4e
4-CF3Ph
>30
>30
4s
4t
>30
>30
3-F-4-CH3OPh
4f
2-CH3Ph
>30
4u
>30
4g
4h
4i
4j
4k
4l
4m
4n
4o
4-CH3CONHPh
4-CO2CH3Ph
3-NO2-4-CH3OPh
4-CH3OPh
22.16 3.28
>30
>30
>30
>30
>30
0.40 0.03
>30
5a
5b
5c
5d
5e
5f
3,4-Cl2Ph
4-CH3OPh
4-CH3Ph
3-NH2-4-CH3OPh
3-HO-4-CH3OPh
3-CH3CONH-4-CH3OPh
\
\
>30
0.90 0.02
>30
0.11 0.01
0.06 0.01
0.28 0.04
9.96 0.05
0.005 0.0002
3,4-Cl2Ph
3-Cl-4-CH3OPh
3-NH2-4-CH3OPh
3,5-Cl2-4- CH3CONHPh
4-NO2Ph
3
CA-4
>30
a
SD: standard deviation of three independent experiments.
3

X.-S. Huo, X.-E. Jian, J. Ou-Yang et al.
European Journal of Medicinal Chemistry 220 (2021) 113449
electron-donating groups displayed much higher cytotoxicity than
those having electron-withdrawing groups (5b vs. 5a, 5d vs. 5a, 5e
vs. 5a, 5f vs. 5a), which suggested that the introduction of electron-
donating groups at phenyl ring of position 2 in triazolopyrimidine
scaffold is more favorable for the antiproliferative potency. Partic-
ularly, in agreement with CA-4, analogue 5e featuring 3-hydroxy-4-
methoxyphenyl moiety, was found to be the most potent having
the IC50 value of 60 nM and was about 166-fold more than that of
3.3. HeLa cell apoptosis and caspase-3 activation
Subsequently, an Annexin V-FITC/PI assay was used to identify
the cell apoptosis analysis of HeLa cells. As demonstrated in Fig. 5,
after treatment with 5e at the concentrations of 30, 60, and 120 nM,
percentages of total apoptotic cells from 3.6% (control) increased to
14.1% (30 nM), 19.4% (60 nM), and 33.6% (120 nM), respectively.
Hence, the results revealed that analogue 5e possibly possessed the
anti-proliferative effect through dose-dependently inducing of
cellular apoptosis.
To explore the correlation between antiproliferative efficacy and
cellular apoptosis, we investigated the cleavage of poly(ADP-ribose)
polymerase (PARP) which is one of the important caspase-3-
mediated cleavage targets in apoptosis induction. Additionally,
Bax which is a key pro-apoptotic family protein was also observed.
As shown in Fig. 6 in HeLa cells, 5e induced a concentration-
dependent increase in the expression of cleaved PARP after 24 h
of treatment (30, 60 and 120 nM), which revealed that 5e caused
cancer cells apoptosis through activation of caspase-3. Whereas,
there is no obvious change in the expression level of the Bax after
treatment for 24 h. These findings indicated that analogue 5e
caused cell apoptosis through regulating expression of cleaved
PARP.
the lead analogue 3 (IC50 ¼ 9.96
mM). These results indicated that
2,7-diaryl- [1,2,4]triazolo[1,5-a]pyrimidine scaffold could be used
as the leading unit to develop anticancer agents.
Next, some highly active analogues possessing excellent activity
toward HeLa were chosen to further explore their antiproliferative
activity profile on three another cancer types, such as human colon
cancer cell line HCT116, human lung adenocarcinoma cell line
A549, and human hepatoma cells HepG2, and the normal human
embryonic kidney cell line (HEK-293). CA-4 was also utilized as a
positive control. As depicted in Table 2, the results suggested that
these derivatives revealed meaningful growth inhibitory activity on
diverse cancer types. Particularly, 5e displayed potent activities
against A549, HepG2, and HCT116 with IC50 values of 6.05, 14.69,
and 18.43
cytotoxic activity (IC50 > 100
demonstrated noticeable cytotoxity activity with the IC50 value of
45.69 M. These findings manifested that 5e could have significant
mM, respectively. Interestingly, 5e exhibited no obvious
mM) toward HEK-293, whereas CA-4
m
3.4. Effect of compounds on tubulin polymerization
selectivity on normal human cells.
Next, four highly active compounds 4m, 5d, 5e, and 5f were
evaluated for their ability to inhibit tubulin polymerization in vitro
using the typical tubulin inhibitor CA-4 as the positive control. The
assay presented in Table 3 suggested that these analogues notably
blocked tubulin polymerization, and inhibition percentages were
49, 94, and 79 for compounds 5d, 5e, and CA-4, respectively, at the
3.2. HeLa cell cycle arrest and G2/M-related proteins regulation
To explore potential antitumor mechanism of this series of an-
alogues, the most promising compound 5e was selected to inves-
tigate for its effect on cell cycle progression of HeLa cells using flow
cytometry analysis. In this work, Hela cells were treated with 30, 60
and 120 nM concentration of 5e for 24 h, respectively. As depicted
in Fig. 3, treatment with indicated concentrations of 5e, percent-
ages of cells at the G2/M phase of the cell cycle were increased from
16.18 to 46.82%, with respect to the control (12.49%). The results
indicated that analogue 5e concentration-dependently caused a
significant G2/M arrest, which was a representative characteristic
for tubulin polymerization inhibitors.
It is well known that mitosis-promoting factor generated be-
tween cell cycle regulatory proteins cyclin B1 and cdc2 acts as a
crucial role in G2eM phase transition. Hence, it was considered of
interest to study alterations of these proteins expression in order to
gain insight in the mechanism involved in 5e-induced G2/M arrest.
The results shown in Fig. 4 indicated that 5e concentration-
dependently upregulated cyclin B1 protein level and significantly
decreased expression of cdc2 protein, which suggested that G2/M-
phase arrest induced by 5e might be associated with regulation of
expressions of p-cdc2 and cyclin B1.
concentration of 10
indicated that 5e, the greatest compound for antiproliferative ac-
tivity, also displayed the best IC50 value (1.3 M), in contrast to CA-4
M). The results revealed that effect on the tubulin
mM. As shown in Fig. 7, additional screening
m
(IC50 ¼ 4.22
m
polymerization positively correlated well with antiproliferative
activity, indicating that these [1,2,4]triazolo[1,5-a]pyrimidines
were potent tubulin polymerization inhibitors.
To verify whether analogues 5e and colchicine occupy the same
binding site in tubulin, a fluorescence based assay was carried out,
according to our previously reported method [28]. And the results
(Fig. 8) indicated that 5e competitively inhibited colchicine binding
to tubulin in a concentration-dependent manner. Therefore, that 5e
binds at the colchicine binding site of tubulin.
3.5. Molecular studies
To gain the potential binding features for the greatest derivative
5e in the tubulin, a series of molecular docking simulations were
carried out using the co-crystallized structure of tubulin with CA-4
(PDB: 5lyj). Overview of the binding site possessing the best
Table 2
Growth inhibitory effects of several compounds toward A549, HCT-116, HepG2, and the normal human embryonic kidney cell line HEK293.
comp.
IC50 mean SDa
T47D
(mM)
HT29
A549
HCT-116
HepG2
HEK293
4m
5d
5e
5f
CA-4
>100
11.47 1.64
83.05 7.01
0.24 0.07
1.66 0.35
1.96 0.61
>100
91.06 4.33
>100
18.43 1.34
22.44 0.62
6.10 0.14
41.19 0.66
21.06 0.52
14.69 0.51
>100
>100
>100
>100
82.14 1.69
45.69 5.15
6.51 0.54
3.49 0.97
5.67 0.29
0.027 0.01
18.91 1.16
6.05 4.67
34.65 1.14
0.021 0.005
5.29 0.05
4

X.-S. Huo, X.-E. Jian, J. Ou-Yang et al.
European Journal of Medicinal Chemistry 220 (2021) 113449
Fig. 3. Effects of 5e on HeLa cell cycle progress. Flow cytometry analysis of HeLa stained by propidium iodide and treated using 5e for 24 h. (A) Control; (B) 5e, 30 nM; (C) 5e, 60 nM;
(D) 5e, 120 nM.
Fig. 4. Effects of 5e on some G2/M regulatory proteins. HeLa cells were harvested and lysed for the detection of cyclin B1 and p-cdc2 expressions by western blot analysis. Data are
presented as the mean SD of three independent experiments. *P < 0.05, **P < 0.01 vs the control.
docking score was shown in Fig. 9. Of interest is that 5e adopts
pretty similar location with CA-4 in the colchicine-binding site
with the Asn258 of
3-hydroxyl-4-methyloxyphenyl ring established a hydrogen bond
with -Val181. Notably, a critical hydrogen bond was observed
between one of the oxygen atoms in 3,4,5-trimethoxylphenyl
group and thiol hydrogen of -Cys241 which is a typical interac-
tion frequently reported for tubulin polymerization inhibitors
a chain. Furthermore, the 3- hydroxyl group of
which is located on the interface between
a/
b-subunits and
a
extended slightly out toward -subunit. The triazolopyrimidine
b
ring of the 5e went deep into the pocket, and the nitrogen atom at
N-4 position provided two potential hydrogen bond interactions
b
5

X.-S. Huo, X.-E. Jian, J. Ou-Yang et al.
European Journal of Medicinal Chemistry 220 (2021) 113449
Fig. 5. Analyses of apoptosis induction in Hela cells. Detection of apoptotic cells after Annexin-V/PI staining by flow cytometry analysis. Cells were harvested and stained with
Annexin-V/PI for analysis after treatment with different concentrations of compound 5e (30, 60 and 120 nM) and control (untreated cells) for 24 h. The diverse cell stages were
given as live (Q4), early apoptotic (Q3), late apoptotic (Q2), and necrotic cells (Q1).
binding in colchicine-site [37,38]. All these observations indicated
that 5e was a novel potential tubulin inhibitor targeting at the
colchicine-binding site.
polymerization and was 3-fold more potent than CA-4 (4.22
mM)
with the IC50 value of 1.3 M. The observations revealed that 2,7-
m
diaryl- [1,2,4]triazolo[1,5-a]pyrimidine core might be used as the
leading unit to develop novel tubulin polymerization inhibitors as
potential anticancer agents.
4. Conclusion
We have synthesized a series of new 2,7-diaryl- [1,2,4]triazolo
[1,5-a]pyrimidines as potential tubulin polymerization inhibitors,
through removing 5-methyl and 6-acetyl groups in our previously
reported compound 3. Some analogus exhibited increased anti-
proliferative activities in the nanomolar range toward HeLa cells,
and the most potent analogue 5e possessed 166-fold improvement
than the parent compound 3 for antiproliferative efficacies in HeLa
cells. Interestingly, 5e demonstrated significant selectivity in
inhibiting cancer cells over HEK-293, a normal human embryonic
kidney cell line. Further mechanism analysis suggested that 5e
dose-dependently caused G2/M arrest in HeLa cells through the
alterations in the expression of p-cdc2 and cyclin B1, and induced
cells apoptosis by up-regulating cleaved PARP expressions. Further
evidence demonstrated that 5e effectively inhibited tubulin
5. Experimental section
5.1. General chemistry
1H and 13C NMR spectra were measured by a Mercury-Plus 400
spectrometer using DMSO‑d6 or CDCl3 as solvents. Chemical shift
(d) is reported in ppm with tetramethylsilane (TMS) as internal
reference. HRMS analyses were carried out through an Agilent
QTOF 6540 mass spectrometer. Flash column chromatography was
performed with silica gel (mesh 200e300), and reactions were
monitored by TLC on 0.25 mm silicagel GF254 plates. Melting points
(mp) were determined with a Buchi B-545 apparatus. Yields were
of purified product and were not optimized. All starting materials
were commercially available unless otherwise indicated.
6

X.-S. Huo, X.-E. Jian, J. Ou-Yang et al.
European Journal of Medicinal Chemistry 220 (2021) 113449
Fig. 6. Effects of 5e on some apoptosis-related protein expression (cleaved PARP and Bax). Data are presented as the mean SD of three independent experiments. *P < 0.05,
**P < 0.01 vs the control.
Table 3
Inhibitory activities of tubulin polymerization by four target compounds.
Comp.
R
Tubulin polymerization
%inhibitiona
IC50
NDc
12.2 0.6
1.3 0.1
ND
(m
M)b
4m
5d
5e
3-NH2-4-CH3OPh
3-NH2-4-CH3OPh
3-OH-4-CH3OPh
12
49
94
16
79
5f
3-CH3CONH-4-CH3OPh
CA-4/
4.22 0.15
a
Samples were determined at 10 mM concentration.
IC50 values were shown as the mean SD of three independent experiments.
ND: Not determined.
b
c
Fig. 7. Effect of compound 5e on tubulin polymerization. Purified tubulin protein at
2 mg/mL in a reaction buffer incubated at 37 ꢁC in the presence of 5e (0.8, 1.5, 3, 6
M),
m
CA-4 (6
mM) or vehicle DMSO. Tubulin polymerization reaction was monitored at
OD340 nm every minute at 37 ꢁC over a 20 min period. Data were expressed as the
mean SD from at least three independent experiments. ***P < 0.001 vs the control.
5.2. Procedure for synthesis of intermediates 9, 16aeu, and 12aeu
Fig. 8. Plot of fluorescence intensity of formation of the tubulin-colchicine complex by
various concentrations of compound 5e. Tubulin-compound 5e complex was formed
The intermediates 3-substituted 5-amino-1,2,4-triazoles 9 and
16aeu were prepared according to reported procedures [34].
Arylpropenones 12aeu were synthesized through the following
procedure: A solution of 1-arylethanones 10 or 17 (2 mmol) and
DMF-DMA (8 mL) was stirred at 90 ꢁC for 4e6 h, then was poured
into water, extracted with EtOAc, and dried. The EtOAc was then
distilled off and the product was washed with cold petroleum ether
to afford the compounds 12aeu, respectively.
by incubating 4
m
M tubulin with compound 5e (20, 40, 80
mM) and CA-4 (20 mM) for
45 min at 37 ꢁC. 4
mM colchicine was then added to the solution of this complex to get
tubulin-colchicine complex. Fluorescence spectra were recorded (excitation at 340 nm,
emission at 435 nm) after incubating for 45 min at 37 ꢁC by using a Tecan Spark
multimode reader. Spectra comprised of multiple scans from which blank values
(buffer alone) were subtracted. ***P < 0.001, ****P < 0.0001 vs the control.
7

X.-S. Huo, X.-E. Jian, J. Ou-Yang et al.
European Journal of Medicinal Chemistry 220 (2021) 113449
(dd, J1 ¼ 2.0 Hz, J2 ¼ 8.4 Hz,1H, ArH), 7.79 (d, J ¼ 12.0 Hz, 1H, CH),
7.90 (d, J ¼ 2.0 Hz,1H, ArH).
5.2.9. (E)-3-(dimethylamino)-1-(p-tolyl)prop-2-en-1-one (12b)
Yield, 72%; mp: 89.0e90.9 ꢁC, Lit. 94e96 ꢁC [35]; 1H NMR
(400 MHz, CDCl3) d: 2.49 (s, 3H, CH3), 2.89 (s, 3H, CH3), 3.07 (s, 3H,
CH3), 5.71 (d, J ¼ 12.0 Hz, 1H, CH), 7.22 (d, J ¼ 8.0 Hz, 2H, ArH),7.81
(d, J ¼ 12.0 Hz, 1H, CH),8.01 (d, J ¼ 8.0 Hz, 2H, ArH).
5.2.10. (E)-3-(dimethylamino)-1-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one (12c)
Yield, 53%; mp: 125.1e127.0 ꢁC, Lit. 123e125 ꢁC [36]; 1H NMR
(400 MHz, DMSO‑d6) d: 2.94 (s, 3H, CH3), 3.15 (s, 3H, CH3), 3.71 (s,
3H, CH3O), 3.85 (s, 6H, 2 ꢂ CH3O), 5.83 (d, J ¼ 12.4 Hz, 1H, CH), 7.18
(s, 2H, ArH), 7.70 (d, J ¼ 12.0 Hz, 1H, CH).
Fig. 9. Docking of 5e (amber stick) into the colchicine-site of tubulin (PDB code: 5lyj),
and overlapping with CA-4 (yellow stick).
5.2.11. (E)-3-(dimethylamino)-1-(4-(trifluoromethyl)phenyl)prop-
2-en-1-one (12d)
Yield, 65%; mp: 88.1e89.9 ꢁC, Lit. 88e90 ꢁC [34]; 1H NMR
5.2.1. 3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-5-amine (9)
(400 MHz, CDCl3) d: 2.90 (s, 3H, CH3), 3.12 (s, 3H, CH3), 5.71 (d,
Yield, 71%; mp: 189.1e191.9 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d:
J ¼ 12.0 Hz, 1H, CH), 7.70 (d, J ¼ 8.0 Hz, 2H, ArH), 7.78 (d, J ¼ 12.0 Hz,
3.69 (s, 3H, CH3O), 3.82 (s, 6H, 2 ꢂ CH3O), 6.03 (s, 2H, NH2), 7.19
1H, CH),7.98 (d, J ¼ 8.0 Hz, 2H, ArH).
(s,2H, ArH), 12.04 (s, 1H, NH).
5.2.12. (E)-3-(dimethylamino)-1-(3-fluoro-4-methoxyphenyl)prop-
2-en-1-one (12e)
5.2.2. 3-(3,4-dichlorophenyl)-1H-1,2,4-triazol-5-amine (16a)
Yield, 60%; mp: >300 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d: 6.17 (s,
Yield, 74%; mp: 100.2e102.1 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
2H, NH2), 7.67 (d, J ¼ 8.4 Hz, 1H, ArH), 7.83 (dd, J1 ¼ 1.6 Hz,
2.91 (s, 3H, CH3), 3.12 (s, 3H, CH3), 3.92 (s, 3H, CH3O), 5.75 (d,
J ¼ 12.0 Hz, 1H, CH), 7.03 (d, J ¼ 8.8 Hz, 1H, ArH), 7.76 (d, J ¼ 12.0 Hz,
1H, CH), 7.89 (d,J ¼ 8.4 Hz, 1H, ArH), 8.01 (d, J ¼ 12.0 Hz, 1H, ArH).
J2 ¼ 8.4 Hz 1H, ArH), 8.00 (d, J ¼ 1.6 Hz, 1H, ArH), 12.43 (s, 1H, NH).
5.2.3. 3-(4-methoxyphenyl)-1H-1,2,4-triazol-5-amine (16b)
Yield, 69%; mp: 223.1e225.0 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d:
3.79 (s, 3H, CH3O), 6.19 (s, 2H, NH2), 6.98 (d, J ¼ 8.8 Hz, 2H, ArH),
5.2.13. (E)-3-(dimethylamino)-1-(o-tolyl)prop-2-en-1-one (12f)
Yield, 78%; mp: 95.1e97.0 ꢁC; 1H NMR (400 MHz, CDCl3)
d: 2.26
7.82 (d, J ¼ 8.8 Hz, 2H, ArH), 12.42 (s, 1H, NH).
(s, 3H, CH3), 2.92 (s, 3H, CH3), 3.12 (s, 3H, CH3), 5.78 (d, J ¼ 12.0 Hz,
1H, CH), 7.24 (t, J ¼ 8.0 Hz, 2H, ArH), 7.32 (d, J ¼ 7.6 Hz,1H, ArH), 7.39
(d, J ¼ 7.2 Hz, 1H, ArH), 7.73 (d, J ¼ 12.0 Hz, 1H, CH).
5.2.4. 3-(p-tolyl)-1H-1,2,4-triazol-5-amine (16c)
Yield, 58%; mp: 209.5e211.2 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d:
2.37 (s, 3H, CH3), 6.17 (s, 2H, NH2), 7.34 (d, J ¼ 8.0 Hz, 2H, ArH), 7.69
(d, J ¼ 8.4 Hz, 2H, ArH), 12.21 (s, 1H, NH).
5.2.14. (E)-N-(4-(3-(dimethylamino)acryloyl)phenyl)acetamide
(12g)
Yield, 56%; mp: 151.9e153.2 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
5.2.5. 3-(3-amino-4-methoxyphenyl)-1H-1,2,4-triazol-5-amine
(16d)
2.17 (s, 3H, CH3), 2.93 (s, 3H, CH3), 3.08 (s, 3H, CH3), 5.71 (d,
J ¼ 12.0 Hz, 1H, CH), 7.62 (d, J ¼ 7.6 Hz, 2H, ArH), 7.76 (d, J ¼ 12.0 Hz,
1H, CH), 7.85 (d, J ¼ 8.0 Hz, 2H, ArH), 8.74 (s, 1H, NH).
Yield, 75%; mp: 184.3e185.9 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d:
3.82 (s, 3H, CH3O), 4.98 (s, 2H, NH2), 6.11 (s, 2H, NH2), 6.94 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.28 (d, J ¼ 1.6 Hz, 1H, ArH), 7.44 (d, J ¼ 8.4 Hz,
1H, ArH), 12.14 (s, 1H, NH).
5.2.15. (E)-methyl 4-(3-(dimethylamino)acryloyl)benzoate (12h)
Yield, 62%; mp: 110.5e111.9 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
5.2.6. 5-(5-amino-1H-1,2,4-triazol-3-yl)-2-methoxyphenol (16e)
2.97 (s, 3H, CH3), 3.19 (s, 3H, CH3), 3.95 (s, 3H, CH3O), 5.72 (d,
J ¼ 12.4 Hz, 1H, CH), 7.84 (d, J ¼ 12.4 Hz, 1H, CH), 7.94 (d, J ¼ 7.6 Hz,
2H, ArH), 8.09 (d, J ¼ 7.6 Hz, 2H, ArH).
Yield, 67%; mp: 193.1e194.5 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d:
3.83 (s, 3H, CH3O), 6.15 (s, 2H, NH2), 6.98 (d, J ¼ 8.4 Hz, 1H, ArH),
7.30 (s, 1H, ArH), 7.54 (d, J ¼ 8.4 Hz, 1H, ArH), 9.21 (s, 1H, OH), 12.31
(s, 1H, NH).
5.2.16. (E)-3-(dimethylamino)-1-(4-methoxy-3-nitrophenyl)prop-
2-en-1-one (12i)
5.2.7. N-(5-(5-amino-1H-1,2,4-triazol-3-yl)-2-methoxyphenyl)
Yield, 76%; mp: 163.1e165.0 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
acetamide (16f)
Yield, 62%; mp: 194.3e195.5 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d:
2.98 (s, 3H, CH3), 3.19 (s, 3H, CH3), 4.02 (s, 3H, CH3O), 5.67 (d,
J ¼ 12.0 Hz, 1H, CH), 7.13 (d, J ¼ 8.4 Hz, 1H, ArH), 7.86 (d, J ¼ 12.0 Hz,
1H, CH), 8.19 (d, J ¼ 8.8 Hz, 1H, ArH), 8.40 (s, 1H, ArH).
2.13 (s, 3H, CH3), 3.82 (s, 3H, CH3O), 6.13 (s, 2H, NH2), 7.19 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.67 (s, 1H, ArH), 7.88 (d, J ¼ 8.4 Hz, 1H, ArH),
9.12 (s, 1H, NH), 12.22 (s, 1H, NH).
5.2.17. (E)-3-(dimethylamino)-1-(4-methoxyphenyl)prop-2-en-1-
one (12j)
5.2.8. (E)-1-(3,4-dimethoxyphenyl)-3-(dimethylamino)prop-2-en-
1-one (12a)
Yield, 81%; mp: 109.4e111.3 ꢁC, Lit. 113e115 ꢁC [36]; 1H NMR
Yield, 78%; mp: 112.1e113.8 ꢁC; 1H NMR (400 MHz, CDCl3)
d
:
(400 MHz, CDCl3) d: 2.91 (s, 3H, CH3), 3.13 (s, 3H, CH3), 3.81 (s, 3H,
2.91 (s, 3H, CH3), 3.09 (s, 3H, CH3), 3.83 (s, 3H, CH3O), 3.90 (s, 3H,
CH3O),5.73 (d, J ¼ 12.0 Hz, 1H, CH), 7.01 (d, J ¼ 8.4 Hz, 1H, ArH), 7.65
CH3O), 5.81 (d, J ¼ 12.0 Hz,1H, CH), 6.97 (d, J ¼ 8.4 Hz, 2H, ArH), 7.67
(d, J ¼ 12.4 Hz, 1H, CH), 7.89 (d, J ¼ 8.4 Hz, 2H, ArH).
8

X.-S. Huo, X.-E. Jian, J. Ou-Yang et al.
European Journal of Medicinal Chemistry 220 (2021) 113449
5.2.18. (E)-1-(3,4-dichlorophenyl)-3-(dimethylamino)prop-2-en-1-
5.3. Procedure for synthesis of compounds 4aeu and 5aef
one (12k)
Yield, 73%; mp: 94.1e95.7 ꢁC; 1H NMR (400 MHz, CDCl3)
d
: 2.95
To a solution of 3-substituted 5-amino-1,2,4-triazoles 9 or 16
(1 mmol) in 6 mL of glacial acetic acid (or 8 mL of ethanol with
catalytic amount of HCl for preparation of 4 m, 4p, 5d, 5e) was
added to appropriate 3-(dimethylamino)prop-2-en-1-one 12
(1 mmol), and the resulting mixtures were stirred for 2e5 h at
80 ꢁC, and detected by TLC. After cooling, the reaction mixtures
were poured into ice/water, extracted with EtOAc, washed with
H2O, then dried in vacuum to provide a precipitate, which was
crystallized by ethanol to afford the desired compounds 4aeu and
5aef.
(s, 3H, CH3), 3.17 (s, 3H, CH3), 5.91 (d, J ¼ 12.0 Hz, 1H, CH), 7.76 (d,
J ¼ 8.8 Hz, 1H, ArH), 7.93 (d, J ¼ 12.0 Hz, 1H, CH), 8.21 (dd,
J1 ¼ 2.0 Hz, J2 ¼ 8.4 Hz, 1H, ArH), 8.33 (d, J ¼ 2.0 Hz, 1H, ArH).
5.2.19. (E)-1-(3-chloro-4-methoxyphenyl)-3-(dimethylamino)prop-
2-en-1-one (12l)
Yield, 77%; mp: 124.6e126.1 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
2.94 (s, 3H, CH3), 3.13 (s, 3H, CH3), 3.74 (s, 3H, CH3O), 5.81 (d,
J ¼ 12.0 Hz, 1H, CH), 7.23 (d, J ¼ 8.8 Hz, 1H, ArH),7.80 (d, J ¼ 12.0 Hz,
1H, CH), 8.18 (dd, J1 ¼ 2.0 Hz, J2 ¼ 8.8 Hz, 1H, ArH), 8.39 (d,
J ¼ 2.0 Hz, 1H, ArH).
5.3.1. 7-(3,4-Dimethoxy-phenyl)-2-(3,4,5-trimethoxy-phenyl)-
[1,2,4]triazolo[1,5-a]pyrimidine (4a)
5.2.20. (E)-N-(2,6-dichloro-4-(3-(dimethylamino)acryloyl)phenyl)
Yield, 68%; mp: 228.9e229.8 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
acetamide (12n)
3.95 (s, 3H, CH3O), 3.99 (s, 6H, 2 ꢂ CH3O), 4.04 (s, 3H, CH3O),4.06 (s,
3H, CH3O), 7.11 (d, J ¼ 8.4 Hz, 1H, ArH), 7.22 (d, J ¼ 4.8 Hz, 2H, ArH),
7.67 (s, 2H, ArH), 7.80 (dd, J1 ¼ 2.0 Hz, J2 ¼ 8.4 Hz,1H, ArH), 8.12 (d,
J ¼ 2.0 Hz,1H, ArH), 8.79 (d, J ¼ 4.8 Hz, 1H, ArH). 13C NMR (100 MHz,
Yield, 62%; mp: 196.5e197.9 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
2.13 (s, 3H, CH3), 2.99 (s, 3H, CH3), 3.15 (s, 3H, CH3), 5.90 (d,
J ¼ 12.0 Hz, 1H, CH), 7.97 (d, J ¼ 12.0 Hz, 1H, CH), 8.25 (s,2H, ArH),
10.05 (s, 1H, NH).
CDCl3)
d: 165.22, 153.72, 153.37, 152.06, 148.71, 147.18, 140.11,
125.81, 123.08, 122.07, 112.52, 111.05, 107.77, 104.48, 60.92, 56.15,
56.09, 55.96. HRMS (ESI) m/z: calcd for C22H22N4O5 (M þ Hþ)
423.1663 found 423.1667.
5.2.21. (E)-3-(dimethylamino)-1-(4-nitrophenyl)prop-2-en-1-one
(12o)
Yield, 59%; mp: 151.2e153.5 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
2.95 (s, 3H, CH3), 3.13 (s, 3H, CH3), 5.94 (d, J ¼ 12.0 Hz, 1H, CH), 7.90
(d, J ¼ 12.0 Hz, 1H, CH), 8.20 (d, J ¼ 8.8 Hz, 2H, ArH), 8.46 (d,
J ¼ 8.4 Hz, 2H, ArH).
5.3.2. 7-p-Tolyl-2-(3,4,5-trimethoxy-phenyl)-[1,2,4]triazolo[1,5-a]
pyrimidine (4b)
Yield, 65%; mp: 187.0e187.4 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
2.53 (s, 3H, CH3), 3.94 (s, 3H, CH3O), 4.00 (s, 6H, 2 ꢂ CH3O), 7.19 (d,
J ¼ 4.4 Hz, 1H, ArH), 7.46 (d, J ¼ 8.0 Hz, 2H, ArH), 7.66 (s, 2H, ArH),
8.15 (d, J ¼ 8.0 Hz, 2H, ArH), 8.80 (d, J ¼ 4.4 Hz, 1H, ArH). 13C NMR
5.2.22. (E)-3-(dimethylamino)-1-(furan-2-yl)prop-2-en-1-one
(12q)
Yield, 76%; mp: 92.6e93.7 ꢁC; 1H NMR (400 MHz, CDCl3)
d: 2.92
(100 MHz, CDCl3) d: 165.45, 157.23, 153.82, 153.39, 147.75, 142.56,
(s, 3H, CH3), 3.07 (s, 3H, CH3), 5.62 (d, J ¼ 12.0 Hz, 1H, CH), 6.78 (d,
J ¼ 3.6 Hz, 1H, ArH), 7.53 (d, J ¼ 12.0 Hz, 1H, CH), 7.68 (s, 1H, ArH),
8.29 (d, J ¼ 3.2 Hz, 1H, ArH).
140.24, 129.58, 129.34, 126.97, 125.86, 108.40, 104.82, 60.90, 56.30,
21.60. HRMS (ESI) m/z: calcd for C21H20N4O3(M þ Hþ) 377.1608
found 377.1609.
5.2.23. (E)-3-(dimethylamino)-1-(naphthalen-1-yl)prop-2-en-1-
5.3.3. 2,7-Bis-(3,4,5-trimethoxy-phenyl)-[1,2,4]triazolo[1,5-a]
one (12r)
pyrimidine (4c)
Yield, 82%; mp: 91.1e93.0 ꢁC; 1H NMR (400 MHz, CDCl3)
d: 2.91
Yield, 59%; mp: 268.1e268.9 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
(s, 3H, CH3), 3.09 (s, 3H, CH3), 5.71 (d, J ¼ 12.0 Hz, 1H, CH),7.54 (d,
J ¼ 8.8 Hz, 2H, ArH), 7.58 (d, J ¼ 6.8 Hz,1H, ArH), 7.67 (t, J ¼ 7.6 Hz,
1H, ArH), 7.72 (d, J ¼ 12.0 Hz, 1H, CH), 7.81 (d, J ¼ 6.4 Hz, 1H,
ArH),7.89 (d, J ¼ 8.0 Hz, 1H, ArH), 8.14 (d, J ¼ 8.4 Hz, 1H, ArH).
3.95 (s, 3H, CH3O), 3.99 (s, 6H, 2 ꢂ CH3O), 4.02 (s, 9H, 3 ꢂ CH3O),
7.24 (d, J ¼ 4.4 Hz, 1H, ArH), 7.60 (s, 2H, ArH), 7.67 (s, 2H, ArH), 8.82
(d, J ¼ 4.0 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3)
d: 165.40, 157.35,
153.80, 153.41, 153.28, 147.14, 141.34, 140.25, 125.69, 124.56, 108.19,
107.15, 104.52, 61.05, 60.92, 56.31, 56.15. HRMS (ESI) m/z: calcd for
5.2.24. (E)-3-(dimethylamino)-1-(thiophen-2-yl)prop-2-en-1-one
(12s)
C
23H24N4O6 (M þ Hþ) 453.1769 found 453.1768.
Yield, 74%; mp: 107.5e109.4 ꢁC, Lit. 106e108 ꢁC [35]; 1H NMR
5.3.4. 7-(4-Trifluoromethyl-phenyl)-2-(3,4,5-trimethoxy-phenyl)-
[1,2,4]triazolo[1,5-a]pyrimidine (4d)
(400 MHz, CDCl3) d: 2.90 (s, 3H, CH3), 3.11 (s, 3H, CH3), 5.72 (d,
J ¼ 12.0 Hz, 1H, CH), 7.21 (t, J ¼ 4.4 Hz, 1H, ArH), 7.69 (d, J ¼ 12.0 Hz,
Yield, 61%; mp: 198.1e198.7 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
1H, CH), 7.81 (d, J ¼ 5.2 Hz, 1H, ArH), 8.16 (d, J ¼ 3.6 Hz, 1H, ArH).
3.94 (s, 3H, CH3O), 3.99 (s, 6H, 2 ꢂ CH3O), 7.23 (d, J ¼ 4.0 Hz, 1H,
ArH), 7.63 (s, 2H, ArH), 7.92 (d, J ¼ 8.0 Hz, 2H, ArH), 8.34 (d,
J ¼ 8.0 Hz, 2H, ArH), 8.88 (d, J ¼ 4.0 Hz, 1H, ArH).13C NMR (100 MHz,
5.2.25. (E)-3-(dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one
(12t)
CDCl3)
d: 165.88, 157.06, 153.99, 153.45, 145.98, 140.47, 133.25,
Yield, 81%; mp: 79.8e81.4 ꢁC, Lit. 72e74 ꢁC [35]; 1H NMR
129.81, 125.86, 125.83, 125.43, 122.15, 109.13, 104.83, 60.91, 56.31.
HRMS (ESI) m/z: calcd for C21H17F3N4O3 (M þ Hþ) 431.1326 found
431.1325.
(400 MHz, CDCl3) d: 2.95 (s, 3H, CH3), 3.13 (s, 3H, CH3), 5.75 (d,
J ¼ 12.0 Hz, 1H, CH), 7.21 (d, J ¼ 3.6 Hz, 1H, ArH), 7.73 (d, J ¼ 12.0 Hz,
1H, CH), 8.31 (d, J ¼ 7.6 Hz, 1H, ArH), 8.67 (s, 1H, ArH), 9.12 (s, 1H,
ArH).
5.3.5. 7-(3-Fluoro-4-methoxy-phenyl)-2-(3,4,5-trimethoxy-
phenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (4e)
5.2.26. (E)-3-(3-(dimethylamino)acryloyl)-2H-chromen-2-one
Yield, 70%; mp: 174.6e175.6 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
(12u)
3.95 (s, 3H, CH3O), 4.00 (s, 6H, 2 ꢂ CH3O), 4.04 (s, 3H, CH3O), 7.18 (d,
J ¼ 4.0 Hz, 1H, ArH), 7.21 (d, J ¼ 8.8 Hz, 1H, ArH), 7.65 (s, 2H, ArH),
8.05 (d, J ¼ 8.4 Hz, 1H, ArH), 8.18 (d, J ¼ 12.0 Hz, 1H, ArH),8.79 (d,
Yield, 76%; mp: 161.1e163.2 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
2.91 (s, 3H, CH3), 3.12 (s, 3H, CH3), 5.78 (d, J ¼ 12.0 Hz, 1H, CH), 7.40
(t, J ¼ 8.0 Hz, 2H, ArH), 7.67 (d, J ¼ 12.0 Hz, 1H, CH), 7.72 (d,
J ¼ 8.0 Hz, 2H, ArH), 9.21 (s, 1H, ArH).
J ¼ 4.4 Hz, 1H, ArH).13C NMR (100 MHz, CDCl3)
d: 165.52, 157.24,
153.74, 153.42, 150.66, 146.02, 140.33, 126.28, 125.66, 122.18, 117.32,
9

X.-S. Huo, X.-E. Jian, J. Ou-Yang et al.
European Journal of Medicinal Chemistry 220 (2021) 113449
113.17, 113.15, 107.92, 104.79, 60.91, 56.34, 56.29. HRMS (ESI) m/z:
calcd for C21H19FN4O4 (M þ Hþ) 411.1463 found 411.1463.
128.34, 125.35, 108.57, 104.73, 60.91, 56.24. HRMS (ESI) m/z: calcd
for C20H16Cl2N4O3 (M þ Hþ) 431.0672 found 431.06720.
5.3.6. 7-o-Tolyl-2-(3,4,5-trimethoxy-phenyl)-[1,2,4]triazolo[1,5-a]
5.3.12. 7-(3-Chloro-4-methoxy-phenyl)-2-(3,4,5-trimethoxy-
phenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (4l)
pyrimidine (4f)
Yield, 67%; mp: 160.9e161.5 ꢁC; 1H NMR (400 MHz, CDCl3)
d
:
Yield, 68%; mp: 206.3e206.7 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
2.27 (s, 3H, CH3), 3.92 (s, 3H, CH3O), 3.96 (s, 6H, 2 ꢂ CH3O), 7.03 (s,
d
: 3.76 (s, 3H, CH3O), 3.90 (s, 6H, 2 ꢂ CH3O), 4.01 (s, 3H, CH3O), 7.43
1H, ArH), 7.44 (t, J ¼ 8.0 Hz, 2H, ArH), 7.50 (d, J ¼ 7.6 Hz, 1H, ArH),
(d, J ¼ 8.8 Hz,1H, ArH), 7.50 (s, 2H, ArH), 7.65 (d, J ¼ 4.8 Hz, 1H, ArH),
8.38 (dd, J1 ¼ 2.0 Hz, J2 ¼ 8.8 Hz, 1H, ArH), 8.55 (d, J ¼ 2.0 Hz, 1H,
7.54 (d, J ¼ 7.2 Hz, 1H, ArH), 7.59 (s, 2H, ArH), 8.83 (s, 1H, ArH).13
C
NMR (100 MHz, CDCl3)
d
: 165.71, 156.61, 153.64, 153.36, 148.99,
ArH),8.85 (d, J ¼ 4.8 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO‑d6)
d:
140.24, 137.50, 130.87, 130.78, 130.17, 129.63, 126.07, 125.76, 110.83,
104.82, 60.88, 56.31, 20.06. HRMS (ESI) m/z: calcd for C21H20N4O3
(M þ Hþ) 377.1608 found 377.1610.
164.30, 157.50, 157.09, 154.96, 153.63, 145.47, 140.13, 131.52, 130.72,
126.01, 122.76, 121.68, 113.22, 109.19, 104.58, 60.57, 57.03, 56.34.
HRMS (ESI) m/z: calcd for C21H19ClN4O4 (M þ Hþ) 427.1168 found
427.1163.
5.3.7. N-(4-(2-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[1,5-a]
pyrimidin-7-yl)phenyl)acetamide (4g)
5.3.13. 2-Methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-[1,2,4]triazolo
Yield, 63%; mp: 236.8e237.2 ꢁC; 1H NMR (400 MHz, CDCl3)
d
:
[1,5-a]pyrimidin-7-yl]- phenylamine (4 m)
2.28 (s, 3H, CH3), 3.93 (s, 3H, CH3O), 3.95 (s,6H,2 ꢂ CH3O), 7.16 (d,
J ¼ 4.4 Hz, 1H, ArH), 7.59 (s, 2H, ArH), 7.86 (d, J ¼ 8.0 Hz, 2H, ArH),
8.14 (s, 1H, NH), 8.24 (d, J ¼ 8.4 Hz, 2H, ArH), 8.76 (d, J ¼ 4.4 Hz, 1H,
Yield, 79%; mp: 109.2e111.1 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d:
3.76 (s, 3H, CH3O), 3.91 (s, 3H, CH3O), 3.92 (s, 6H, 2 ꢂ CH3O), 5.15 (s,
2H, NH2), 7.07 (d, J ¼ 8.4 Hz, 1H, ArH), 7.46 (d, J ¼ 4.0 Hz, 1H, ArH),
7.56 (s, 2H, ArH), 7.60 (d, J ¼ 8.4 Hz, 1H, ArH),7.75 (s, 1H, ArH),8.82
ArH). 13C NMR (100 MHz, CDCl3)
d: 168.90, 165.30, 157.16, 153.82,
153.35, 147.02, 141.41, 140.15, 130.49, 125.71, 124.90, 119.39, 108.18,
104.74, 60.89, 56.25, 24.66. HRMS (ESI) m/z: calcd for C22H21N5O4
(M þ Hþ) 420.1667 found 420.1666.
(d, J ¼ 4.4 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO‑d6)
d: 164.31,
157.26, 154.84, 153.64, 149.65, 147.91, 140.05, 138.28, 126.24, 122.20,
119.06, 114.41, 110.75, 108.77, 104.76, 60.58, 56.43, 55.98. HRMS
(ESI) m/z: calcd for C21H21N5O4 (M þ Hþ) 408.1667 found 408.1667.
5.3.8. 4-[2-(3,4,5-Trimethoxy-phenyl)-[1,2,4]triazolo[1,5-a]
pyrimidin-7-yl]-benzoic acid methyl ester (4h)
5.3.14. N-(2,6-dichloro-4-(2-(3,4,5-trimethoxyphenyl)-[1,2,4]
triazolo[1,5-a]pyrimidin-7-yl)phenyl)
Yield, 71%; mp: 202.4e202.7 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
3.94 (s, 3H, CH3O), 3.99 (s, 6H, 2 ꢂ CH3O), 4.02 (s, 3H, CH3O), 7.24 (s,
1H, ArH), 7.63 (s, 2H, ArH), 8.29 (s, 4H, ArH), 8.86 (s, 1H, ArH). 13
NMR (100 MHz, CDCl3) : 166.05, 165.75, 157.09, 153.94, 153.42,
acetamide (4n). Yield, 52%; mp: 273.1e273.5 ꢁC; 1H NMR
C
(400 MHz, DMSO‑d6) d: 2.14 (s, 3H, CH3), 3.76 (s, 3H, CH3O), 3.89 (s,
d
6H, 2 ꢂ CH3O), 7.49 (s, 2H, ArH), 7.76 (d, J ¼ 4.8 Hz, 1H, ArH), 8.49
146.44, 140.37, 133.84, 132.84, 129.91, 129.41, 125.51, 109.10, 104.76,
60.90, 56.28, 52.52. HRMS (ESI) m/z: calcd for C22H20N4O5 (M þ Hþ)
421.1507 found 421.1505.
(s,2H, ArH), 8.95 (d, J ¼ 4.4 Hz, 1H, ArH), 10.13 (s, 1H, NH). 13C NMR
(100 MHz, DMSO‑d6)
d: 168.72, 164.43, 156.91, 155.31, 153.65,
144.07, 140.17, 136.40, 134.07, 130.33, 129.87, 125.86, 110.51, 104.52,
60.59, 56.29, 22.88. HRMS (ESI) m/z: calcd for C22H19Cl2N5O4
(M þ Hþ) 488.0887 found 488.0881.
5.3.9. 7-(4-Methoxy-3-nitro-phenyl)-2-(3,4,5-trimethoxy-phenyl)-
[1,2,4]triazolo[1,5-a]pyrimidine (4i)
Yield, 73%; mp: 222.9e223.7 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
5.3.15. 7-(4-Nitro-phenyl)-2-(3,4,5-trimethoxy-phenyl)-[1,2,4]
d
: 3.76 (s, 3H, CH3O), 3.91 (s, 6H, 2 ꢂ CH3O), 4.09 (s, 3H, CH3O), 7.54
triazolo[1,5-a]pyrimidine(4o)
(s, 2H, ArH), 7.65 (d, J ¼ 8.8 Hz,1H, ArH), 7.77 (d, J ¼ 4.0 Hz,1H, ArH),
8.63 (d,J ¼ 8.8 Hz, 1H, ArH), 8.91 (d,J ¼ 3.6 Hz, 1H, ArH), 9.26 (s, 1H,
Yield, 62%; mp: 246.8e247.2 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d:
3.76 (s, 3H, CH3O), 3.90 (s, 6H, 2 ꢂ CH3O), 7.51 (s, 2H, ArH), 7.72 (d,
J ¼ 4.4 Hz, 1H, ArH), 8.50 (d, J ¼ 8.8 Hz, 2H, ArH), 8.56 (d, J ¼ 8.4 Hz,
2H, ArH), 8.99 (d, J ¼ 4.4 Hz,1H, ArH). 13C NMR (100 MHz, DMSO‑d6)
ArH). 13C NMR (100 MHz, DMSO‑d6)
d: 164.29,157.08,155.10,154.87,
153.65, 144.47, 140.12, 139.07, 136.01, 127.61, 125.86, 121.66, 115.23,
109.28, 104.48, 60.59, 57.73, 56.29. HRMS (ESI) m/z: calcd for
C
d: 164.61, 156.94, 155.46, 153.68, 149.34, 145.17, 140.27, 136.02,
21H19N5O6 (M þ Hþ) 438.1408 found 438.1408.
131.57, 125.89, 124.13, 110.97, 104.78, 60.59, 56.48. HRMS (ESI) m/z:
calcd for C20H17N5O5 (M þ Naþ) 430.1122 found 430.1117.
5.3.10. 7-(4-Methoxy-phenyl)-2-(3,4,5-trimethoxy-phenyl)-[1,2,4]
triazolo[1,5-a]pyrimidine (4j)
5.3.16. 4-(2-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[1,5-a]
Yield, 60%; mp: 174.2e174.6 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d
:
pyrimidin-7-yl)aniline (4p)
3.76 (s, 3H, CH3O), 3.91 (s, 9H, 3 ꢂ CH3O), 7.23 (d,J ¼ 8.8 Hz, 2H,
ArH), 7.52 (s, 2H, ArH), 7.57 (d, J ¼ 4.8 Hz, 1H, ArH), 8.37 (d,
J ¼ 8.8 Hz, 2H, ArH), 8.85 (d,J ¼ 4.4 Hz, 1H, ArH). 13C NMR (100 MHz,
Yield, 84%; mp: 239.1e240.1 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d:
3.76 (s, 3H, CH3O), 3.92 (s, 6H, 2 ꢂ CH3O), 6.14 (s, 2H, NH2), 6.79 (d,
J ¼ 8.8 Hz, 2H, ArH), 7.47 (d, J ¼ 5.2 Hz,1H, ArH), 7.54 (s, 2H, ArH),
8.25 (d, J ¼ 8.8 Hz, 2H, ArH), 8.72 (d, J ¼ 4.8 Hz, 1H, ArH). 13C NMR
DMSO‑d6) d: 164.35, 162.43, 157.17, 154.95, 153.65, 147.01, 140.13,
131.95, 126.16, 122.00, 114.69, 108.97, 104.72, 60.58, 56.44, 56.00.
HRMS (ESI) m/z: calcd for C21H20N4O4 (M þ Hþ) 393.1558 found
393.1557.
(100 MHz, DMSO‑d6) d: 164.07, 157.44, 154.32, 153.64, 153.14,
147.75, 140.01, 131.87, 126.36, 115.59, 113.60, 106.95, 104.65, 60.58,
56.39. HRMS (ESI) m/z: calcd for C20H19N5O3 (M þ Hþ) 378.1561
found 378.1563.
5.3.11. 7-(3,4-Dichloro-phenyl)-2-(3,4,5-trimethoxy-phenyl)-[1,2,4]
triazolo[1,5-a]pyrimidine (4k)
5.3.17. 7-(furan-2-yl)-2-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo
Yield, 63%; mp: 213.2e213.5 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d
:
[1,5-a]pyrimidine (4q)
3.76 (s, 3H, CH3O), 3.90 (s, 6H, 2 ꢂ CH3O), 7.49 (s, 2H, ArH), 7.71 (d,
J ¼ 4.8 Hz, 1H, ArH), 7.96 (d, J ¼ 8.8 Hz, 1H, ArH), 8.31 (dd,
J1 ¼ 2.0 Hz, J2 ¼ 8.4 Hz,1H, ArH), 8.61 (d, J ¼ 2.0 Hz,1H, ArH), 8.93 (d,
Yield: 57%; mp: 206.8e207.7 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
3.97 (s,3H, CH3O), 4.05 (s, 6H, 2 ꢂ CH3O), 6.83 (dd, J1 ¼ 1.6 Hz,
J2 ¼ 3.6 Hz, 1H, ArH), 7.54 (d, J ¼ 4.8 Hz,1H, ArH), 7.72 (s, 2H, ArH),
7.82 (s, 1H, ArH), 8.33 (d, J ¼ 3.2 Hz, 1H, ArH), 8.82 (d, J ¼ 4.8 Hz, 1H,
J ¼ 4.4 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO‑d6)
d: 165.77, 157.08,
153.91, 153.44, 144.97, 140.42, 136.34, 133.45, 131.35, 130.92, 129.43,
ArH).13C NMR (100 MHz, CDCl3)
d:165.67, 156.70, 153.43, 153.36,
10

X.-S. Huo, X.-E. Jian, J. Ou-Yang et al.
European Journal of Medicinal Chemistry 220 (2021) 113449
146.61, 143.20, 140.32, 136.73, 125.76, 120.69, 113.46, 104.77, 104.03,
60.95, 56.30. HRMS (ESI) m/z: calcd for C18H16N4O4 (M þ Hþ)
353.1245 found 353.1244.
(100 MHz, DMSO‑d6) d: 164.53, 161.69, 157.22, 154.79, 153.14,
146.75, 140.78, 128.92, 125.01, 123.14, 114.87, 109.53, 107.91, 60.66,
56.64, 55.74. HRMS (ESI) m/z: calcd for C21H20N4O4 (M þ Hþ)
393.1558 found 393.1557.
5.3.18. 7-(naphthalen-1-yl)-2-(3,4,5-trimethoxyphenyl)-[1,2,4]
triazolo[1,5-a]pyrimidine (4r)
5.3.24. 2-(p-tolyl)-7-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[1,5-
Yield, 69%; mp: 199.1e200.5 ꢁC; 1H NMR (400 MHz, CDCl3)
d
:
a]pyrimidine (5c)
3.90 (s, 9H, 3 ꢂ CH3O), 7.22 (d, J ¼ 4.4 Hz, 1H, ArH), 7.50 (s, 2H, ArH),
7.58 (d, J ¼ 8.8 Hz, 2H, ArH), 7.62 (d, J ¼ 6.8 Hz,1H, ArH), 7.71 (t,
J ¼ 7.6 Hz, 1H, ArH), 7.87 (d, J ¼ 6.4 Hz, 1H, ArH), 8.03 (d, J ¼ 8.0 Hz,
1H, ArH), 8.16 (d, J ¼ 8.4 Hz, 1H, ArH), 8.91 (d, J ¼ 4.4 Hz, 1H, ArH).
Yield, 58%; mp: 221.0e221.2 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
2.45 (s, 3H, CH3), 4.01 (s, 9H, 3 ꢂ CH3O), 7.21 (d, J ¼ 4.8 Hz, 1H, ArH),
7.33 (d, J ¼ 8.0 Hz, 2H, ArH), 7.57 (s, 2H, ArH), 8.28 (d, J ¼ 8.0 Hz, 2H,
ArH), 8.80 (d, J ¼ 4.4 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3)
d:
13C NMR (100 MHz, CDCl3)
d: 165.65, 156.92, 153.67, 153.29, 147.66,
165.78, 157.32, 153.65, 153.27, 147.20, 141.25, 140.89, 129.40, 127.63,
127.39, 124.70, 108.14, 107.14, 61.01, 56.41, 21.50. HRMS (ESI) m/z:
calcd for C21H20N4O3 (M þ Hþ) 377.1607 found 377.1609.
140.13, 133.63, 131.67, 130.40, 128.74, 128.54, 127.71, 127.24, 126.66,
125.69, 125.07, 124.86, 111.72, 104.78, 60.86, 56.23. HRMS (ESI) m/z:
calcd for C24H20N4O3 (M þ Hþ) 413.1608 found 413.1608.
5.3.25. 2-methoxy-5-(7-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo
5.3.19. 7-(thiophen-2-yl)-2-(3,4,5-trimethoxyphenyl)-[1,2,4]
[1,5-a]pyrimidin-2-yl)aniline(5d)
triazolo[1,5-a]pyrimidine (4s)
Yield, 60%; mp: 227.2e228.0 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d:
Yield, 63%; mp: 196.4e197.2 ꢁC; 1H NMR (400 MHz, CDCl3)
d
:
3.82 (s, 3H, CH3O), 3.85 (s, 3H, CH3O), 3.94 (s, 6H, 2 ꢂ CH3O), 5.00 (s,
2H, NH2), 6.96 (d, J ¼ 8.4 Hz, 1H, ArH), 7.50 (d, J ¼ 8.4 Hz, 1H, ArH),
7.58 (d, J ¼ 1.6 Hz, 1H, ArH), 7.65 (d, J ¼ 4.4 Hz, 1H, ArH), 7.71 (s, 2H,
3.96 (s, 3H, CH3O), 4.04 (s, 6H, 2 ꢂ CH3O), 7.37 (t, J ¼ 4.4 Hz, 1H,
ArH), 7.43 (d, J ¼ 4.8 Hz, 1H, ArH), 7.73 (s, 2H, ArH), 7.86 (d,
J ¼ 5.2 Hz, 1H, ArH), 8.46 (d, J ¼ 3.6 Hz, 1H, ArH), 8.76 (d, J ¼ 4.8 Hz,
ArH), 8.85 (d, J ¼ 4.8 Hz, 1H, ArH). 13C NMR (100 MHz, DMSO‑d6)
d:
1H, ArH). 13C NMR (100 MHz, CDCl3)
d: 165.38, 156.89, 153.42,
165.31, 157.15, 154.53, 153.13, 148.88, 146.65, 140.67, 138.35, 125.14,
123.28, 116.08, 112.32, 110.88, 109.35, 107.84, 60.65, 56.64, 55.80.
HRMS (ESI) m/z: calcd for C21H21N5O4 (M þ Hþ) 408.1667 found
408.1667.
153.15, 141.01, 140.36, 133.71, 132.39, 130.72, 128.23, 125.66, 105.17,
104.84, 60.93, 56.28. HRMS (ESI) m/z: calcd for C18H16N4O3S
(M þ Hþ) 369.1016 found 369.1017.
5.3.20. 7-Pyridin-3-yl-2-(3,4,5-trimethoxy-phenyl)-[1,2,4]triazolo
5.3.26. 2-methoxy-5-(7-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo
[1,5-a]pyrimidine (4t)
[1,5-a]pyrimidin-2-yl)phenol(5e)
Yield, 55%; mp: 235.8e236.2 ꢁC; 1H NMR (400 MHz, CDCl3)
d
:
Yield, 66%; mp: 177.0e177.6 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d:
3.93 (s, 3H, CH3O), 3.98 (s, 6H, 2 ꢂ CH3O), 7.25 (d, J ¼ 3.6 Hz,1H,
3.82 (s, 3H, CH3O), 3.85 (s, 3H, CH3O), 3.91 (s, 6H, 2 ꢂ CH3O), 7.09 (d,
ArH), 7.62 (s, 3H, ArH), 8.60 (d, J ¼ 7.6 Hz,1H, ArH), 8.87 (s, 2H, ArH),
J ¼ 8.4 Hz, 1H, ArH), 7.68 (d, J ¼ 4.8 Hz, 1H, ArH), 7.70 (s, 1H, ArH),
9.42 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3)
d: 165.86, 157.01,
7.72 (s, 3H, ArH), 8.87 (d, J ¼ 4.8 Hz, 1H, ArH), 9.40 (s, 1H, OH). 13
C
153.99, 153.44, 152.34, 149.81, 144.70, 140.45, 136.72, 126.23,
125.39, 123.48, 108.75, 104.80, 60.90, 56.30. HRMS (ESI) m/z: calcd
for C19H17N5O3 (M þ Hþ) 364.1404 found 364.1404.
NMR (100 MHz, DMSO‑d6) d: 164.71, 157.18, 154.70, 153.13, 150.41,
147.14, 146.70, 140.74, 125.06, 123.30, 119.05, 114.13, 112.70, 109.45,
107.90, 60.66, 56.64, 56.00. HRMS (ESI) m/z: calcd for C21H20N4O5
(M þ Hþ) 409.1507 found 409.1504.
5.3.21. 3-[2-(3,4,5-Trimethoxy-phenyl)-[1,2,4]triazolo[1,5-a]
pyrimidin-7-yl]-chromen-2-one (4u)
5.3.27. N-(2-methoxy-5-(7-(3,4,5-trimethoxyphenyl)-[1,2,4]
triazolo[1,5-a]pyrimidin-2-yl)phenyl)
Yield, 63%; mp: 219.1e219.5 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
3.94 (s, 3H, CH3O), 3.98 (s, 6H, 2 ꢂ CH3O), 7.48 (t, J ¼ 8.0 Hz, 2H,
ArH), 7.61 (s, 2H, ArH), 7.74 (d, J ¼ 8.0 Hz, 2H, ArH), 7.86 (d,
J ¼ 4.0 Hz, 1H, ArH), 8.87 (d, J ¼ 4.0 Hz, 1H, ArH), 9.39 (s, 1H, ArH).
acetamide (5f). Yield, 63%; mp: 236.8e237.2 ꢁC; 1H NMR
(400 MHz, DMSO‑d6) d: 2.15 (s, 3H, CH3), 3.82 (s, 3H, CH3O), 3.93 (s,
3H, CH3O), 3.96 (s, 6H, 2 ꢂ CH3O), 7.22 (d, J ¼ 8.4 Hz, 1H, ArH), 7.73
(d, J ¼ 4.8 Hz, 1H, ArH), 7.79 (s, 2H, ArH), 7.98 (dd, J1 ¼ 1.6 Hz,
J2 ¼ 8.4 Hz, 1H, ArH), 8.88 (d, J ¼ 4.8 Hz, 1H, ArH), 9.02 (s, 1H, ArH),
13C NMR (100 MHz, CDCl3)
d: 165.26, 157.96, 156.90, 154.20, 153.92,
153.43, 147.74, 140.49, 140.44, 134.36, 129.49, 125.42, 125.35, 118.22,
116.92, 116.42,111.07, 104.79, 60.91, 56.28. HRMS (ESI) m/z: calcd for
9.25 (s, 1H, NH). 13C NMR (100 MHz, DMSO‑d6)
d: 169.12, 164.62,
C
23H18N4O5 (M þ Hþ) 431.1350 found 431.1347.
157.27, 154.75, 153.14, 151.55, 146.57, 140.80, 128.30, 124.89, 123.55,
122.78, 120.30, 111.70, 109.34, 107.88, 60.66, 56.64, 56.33, 24.41.
HRMS (ESI) m/z: calcd for C23H23N5O5 (M þ Hþ) 450.1772 found
450.1772.
5.3.22. 2-(3,4-dichlorophenyl)-7-(3,4,5-trimethoxyphenyl)-[1,2,4]
triazolo[1,5-a]pyrimidine (5a)
Yield, 59%; mp: 204.2e205.0 ꢁC; 1H NMR (400 MHz, CDCl3)
d:
3.82 (s, 3H, CH3O), 3.93 (s, 6H, 2 ꢂ CH3O), 7.68 (s, 2H, ArH), 7.74 (d,
J ¼ 4.8 Hz, 1H, ArH), 7.83 (d, J ¼ 8.4 Hz,1H, ArH), 8.16 (dd, J1 ¼1.6 Hz,
J2 ¼ 8.4 Hz, 1H, ArH), 8.32 (d, J ¼ 1.6 Hz, 1H, ArH), 8.93 (d, J ¼ 4.4 Hz,
5.4. Biological evaluation
5.4.1. Antiproliferative activity
1H, ArH). 13C NMR (100 MHz, CDCl3)
d
: 162.32, 157.15, 155.61,153.17,
HeLa, A549, HCT-116, and HepG2 cell lines were grown at 37 ꢁC
in a CO2 incubator, in RPMI-1640 or DMEM medium supplemented
with 10% fetal bovine serum. Exponentially growing cells were
plated in 96-well plates with the density of 2 £ 103 cells per well
and were incubated for attachment at 37 ꢁC for 24 h. Subsequently,
culture medium was changed, and cells were treated with tested
compounds, DMSO (0.1%) and CA-4, and incubated at 37 ꢁC for 48 h.
147.20, 133.74, 132.30, 131.97, 131.31, 128.79, 127.23, 124.74, 110.26,
107.95, 99.93, 60.67, 56.64. HRMS (ESI) m/z: calcd for C20H16Cl2N4O3
(M þ Hþ) 431.0672 found 431.0670.
5.3.23. 2-(4-methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)-[1,2,4]
triazolo[1,5-a]pyrimidine (5b)
Yield, 56%; mp: 194.0e194.6 ꢁC; 1H NMR (400 MHz, DMSO‑d6)
d
:
20 mL of MTT solution (5 mg/mL) was then added into per well, and
3.80 (s, 3H, CH3O),3.85 (s, 3H, CH3O), 3.94 (s, 6H, 2 ꢂ CH3O), 7.13 (d,
J ¼ 8.8 Hz,2H, ArH), 7.70 (d, J ¼ 4.4 Hz, 1H, ArH), 7.73 (s, 2H, ArH),
8.20 (d, J ¼ 9.2 Hz, 2H, ArH), 8.88 (d, J ¼ 4.8 Hz, 1H, ArH). 13C NMR
plates were incubated at 37 ꢁC for a further 4 h. Then reduced MTT
crystals were dissolved, and absorbance was determined by a
microplate reader at 570 nm. Finally, the growth inhibitory effects
11

X.-S. Huo, X.-E. Jian, J. Ou-Yang et al.
European Journal of Medicinal Chemistry 220 (2021) 113449
were represented as IC50 values which were calculated with the
prism statistical package.
field by a distanceedependent dielectric and powell gradient al-
gorithm using a convergence criterion of 0.005 kcal/mol/Å). The
surflex module was employed in the docking studies, and all other
related parameters were kept at default.
5.4.2. Flow cytometric analysis for cell cycle distribution
2 £ 105 Human cervical cancer cells (HeLa) were treated with
indicated concentrations (30, 60, 120 nM) of 5e and the control for
24 h. Subsequently, treated cells were collected, washed with PBS,
and fixed by ice-cold ethanol (75%) at 4 ꢁC overnight. Cells were
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
then washed, treated using RNase (50 m
g/mL) for 30 min at 37 ꢁC,
and stained with propidium iodide. Cell cycle distribution was
finally analyzed with flow cytometry (Beckman Coulter).
Acknowledgments
5.4.3. Apoptosis assay
After treatment with different concentrations of 5e and vehicle
This work was supported by grants from the Natural Science
Foundation of Guangdong Province, China (Grant No.
2018B030311067), and the Science and Technology Program of
Guangzhou City, China (Grant No. 201707010198).
for 24 h, cells were collected and incubated using 5
FITC in binding buffer (containing 140 mM NaCl, 10 mM HEPES,
2.5 mM CaCl2, pH 7.4) and 10 L PI solution for 15 min. Thereafter,
mL Annexin-V/
m
the solution was put in the medium and incubated for extra 10 min.
Apoptosis was analyzed by FlowJo 7.6 software.
Appendix A. Supplementary data
5.4.4. Western blot assays
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113449.
HeLa cells were homogenized using lysis buffer, after being
treated by the process of cell cycle analysis. The protein concen-
trations were determined using a BSA-100 protein quantitative
analysis kit (Biocolor Bioscience & technology, Shanghai, China).
Cell lyses were then boiled at 100 ꢁC for 5 min for SDS-
polyacrylamide gel electrophoresis (PAGE), after being added the
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