Synthesis, and biological evaluation of 3,6-diaryl-[1,2,4]triazolo[4,3-a]pyridine analogues as new potent tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112625

On the basis of our previous work, twenty-nine novel [1,2,4]triazolo[4,3-a]pyridines possessing 3,4,5-trimethoxylphenyl groups were designed, synthesized, and evaluated as tubulin polymerization inhibitors. The bioassay results revealed that some of the compounds displayed excellent antiproliferative efficacies in the nanomolar range against HeLa cells, and the most promising derivative 7i demonstrated almost comparable activity to that of the reference CA-4 and sixty-two fold more potent than the parent compound 6 with an IC₅₀ value of 12 nM. Importantly, 7i exhibited high selectivity over the normal human embryonic kidney HEK-293 cells (IC₅₀ > 100 μM). Further mechanism studies revealed that 7i significantly arrested cell cycle at G2/M phase, induced apoptosis with a dose-dependent manner, and disrupted microtubule networks. Additionally, the most active compound 7i effectively inhibited tubulin polymerization with a value similar to that of CA-4 (3.4 and 4.2 μM, respectively). Furthermore, molecular docking analysis suggested that 7i well occupied the colchicine binding pocket of tubulin. The present study highlights that compound 7i is a novel potential tubulin polymerization inhibitor and deserves further investigation for the treatment of cancers.

Full text of DOI:10.1016/j.ejmech.2020.112625

Journal Pre-proof
Synthesis, and biological evaluation of 3,6-diaryl-[1,2,4]triazolo[4,3-a]pyridine
analogues as new potent tubulin polymerization inhibitors
Fang Yang, Xie-Er Jian, Peng-Cheng Diao, Xian-Sen Huo, Wen-Wei You, Pei-Liang
Zhao
PII:
S0223-5234(20)30597-3
DOI:
https://doi.org/10.1016/j.ejmech.2020.112625
EJMECH 112625
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 March 2020
Revised Date: 8 June 2020
Accepted Date: 25 June 2020
Please cite this article as: F. Yang, X.-E. Jian, P.-C. Diao, X.-S. Huo, W.-W. You, P.-L. Zhao,
Synthesis, and biological evaluation of 3,6-diaryl-[1,2,4]triazolo[4,3-a]pyridine analogues as new potent
tubulin polymerization inhibitors, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
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Synthesis,
and
biological
evaluation
of
3,6-diaryl-[1,2,4]triazolo[4,3-a]pyridine analogues as new potent
tubulin polymerization inhibitors
Fang Yang 1, Xie-Er Jian 1, Peng-Cheng Diao, Xian-Sen Huo, Wen-Wei You, Pei-Liang Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical
Science, Southern Medical University, Guangzhou 510515, P.R.China

Synthesis,
and
biological
evaluation
of
3,6-diaryl-[1,2,4]triazolo[4,3-a]pyridine analogues as new potent
tubulin polymerization inhibitors
5
10
15
20
Fang Yang 1, Xie-Er Jian 1, Peng-Cheng Diao, Xian-Sen Huo, Wen-Wei You, Pei-Liang Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical
Science, Southern Medical University, Guangzhou 510515, P.R.China
ABSTRACT: On the basis of our previous work, twenty-nine novel
[1,2,4]triazolo[4,3-a]pyridines possessing 3,4,5-trimethoxylphenyl groups were designed,
synthesized, and evaluated as tubulin polymerization inhibitors. The bioassay results revealed that
some of the compounds displayed excellent antiproliferative efficacies in the nanomolar range
against HeLa cells, and the most promising derivative 7i demonstrated almost comparable activity
to that of the reference CA-4 and sixty-two fold more potent than the parent compound 6 with an
IC50 value of 12 nM. Importantly, 7i exhibited high selectivity over the normal human embryonic
kidney HEK-293 cells (IC50 > 100 μM). Further mechanism studies revealed that 7i significantly
arrested cell cycle at G2/M phase, induced apoptosis with a dose-dependent manner, and disrupted
microtubule networks. Additionally, the most active compound 7i effectively inhibited tubulin
polymerization with a value similar to that of CA-4 (3.4 and 4.2 μM, respectively). Furthermore,
molecular docking analysis suggested that 7i well occupied the colchicine binding pocket of tubulin.
The present study highlights that compound 7i is a novel potential tubulin polymerization inhibitor
and deserves further investigation for the treatment of cancers.
Keywords: [1,2,4]Triazolo[4,3-a]pyridine; Synthesis;
Antiproliferative activity; Tubulin
25
polymerization.
——————————
Corresponding author. E-mail address: plzhao@smu.edu.cn (P.-L. Zhao)
1 These authors contributed equally to this work.
1

30
35
40
45
50
55
1. Introduction
Microtubules are one of the most successful therapeutic targets in tumour cells, and composed
of α/β-tubulin dimmers, which provide a highly dynamic framework for maintaining cell structure,
protein trafficking, and supporting the process of mitosis [1-3]. Microtubule-targeting agents were
found to bind to be at least four different sites, among which inhibitors binding to the
colchicine-site have been always attracting considerable attention in anti-cancer therapy due to their
advantages such as simple structure, broad therapeutic index, and significant ability to overcome
clinically relevant multidrug resistance [4-7].
In recent decades, a great deal of tubulin polymerization inhibitors with diverse backbones
targeting the colchicines-site have been investigated [8-12]. Among them, 1,2,4-triazole fused
heterocycles have recently drawn increasing interest as a privileged skeleton for the discovery of
antitumor agents [13-16]. As shown in Fig. 1, [1,2,4]triazole fused thiadiazole 1 displayed excellent
antiproliferative activity on three cancer cell lines with IC50 values of 22–29 nM in the three cell
lines [17]. Most interestingly, 1,2,4-triazole fused six-membered aromatic rings, presented as
compounds 2 and 3, exerted outstanding antitumor potencies in the low nanomolar range [18-19].
Recently, triazoloquinazolinone 4 has been developed as a significant tubulin polymerization
inhibitor and vascular disrupting agent (Fig. 1) [20].
Previously, we also reported a series of novel tubulin polymerization inhibitors, exampled by
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine 5 and [1,2,4]triazolo[1,5-a]pyrimidine derivative 6 both
bearing a 3,4,5-trimethoxyphenyl moiety [21-22], which is crucial for maintaining antiproliferative
activities and is a common group shared by many colchicine-binding site inhibitors. On the other
hand, pyridine ring is regarded as a privileged scaffold in medicinal chemistry and is frequently
observed within a number of anticancer agents, particularly in the small-molecule tubulin inhibitors
[23-26]. Inspired by the above considerations and in continuation of our efforts on optimizing the
structure of the analogue 6, we fused 1,2,4-triazole moiety with pyridine ring instead of pyrimidine
core to generate compounds 7a–u and 8a–h which are also bioisosteres based on the
[1,2,4]triazolo[4,3-b]pyridazine framework 2. Herein, we described the detailed synthetic routes
and their biological efficiency including antiproliferative activities, cytotoxicities toward a
representative normal human cell line HEK-293, as well as their tubulin polymerization inhibitory
2

effects.
60
Fig. 1. Chemical structures of several representative tubulin inhibitors containing the 1,2,4-triazole fused
heterocycle scaffold, and the general structure of target compounds 7a–u and 8a–h.
2. Chemistry
65
70
75
Synthesis of target compounds 7a–u and 8a–h was accomplished using a three-step procedure,
respectively, illustrated in Scheme 1. In brief, the condensation of commercially available
5-bromo-2-hydrazinylpyridine 9 with 3,4,5-trimethoxybenzaldehyde led to the formation of
3,4,5-trimethoxybenzylidenehydrazinyl substituted pyridine derivative 10 [27], which was cyclized
into the corresponding key intermediate 6-bromo-3-phenyl-[1,2,4]triazolo[4,3-a]pyridine 11.
Subsequently, intermediate 11 was efficiently coupled with the appropriate arylboronic acids using a
catalyst formed in situ from tris(dibenzylideneacetone)dipalladium and xantphos in the presence of
K3PO4 in
a
mixed solution of DMSO/H2O under N2 atmosphere, and provided
3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine derivatives 7a–u in yields ranging from
60 to 84%. Meanwhile, the designed target compounds 8a–h bearing 3,4,5-trimethoxyphenyl
moiety at C-6 position were also obtained from a three-step synthesis in a similar manner, starting
from 5-bromo-2-hydrazinylpyridine 9 and various benzaldehydes. All target derivatives were fully
13
characterized by 1H NMR, C NMR, and HRMS spectroscopic techniques, and purity of the most
potent compound 7i was 97.05% based on HPLC analysis. The spectral data were in full agreement
with the expected structures (see Experimental Section and Supporting Information).
3

80
85
Scheme 1. Synthesis of the target compounds 7a–u and 8a–h. Reagents and conditions: (a)
3,4,5-trimethoxybenzaldehyde, ethanol, rt, 4h; (b) CH2Cl2, iodobenzene diacetate, N2, rt, 10h; (c) DMF, H2O,
o
arylboronic acids, Pd2(dba)3, xantphos, K3PO4, N2, 80 C, 8-12h.; (d) substituted benzaldehyde, ethanol, rt, 3-5h;
(e) CH2Cl2, iodobenzene diacetate, N2, rt, 8-10h; (f) DMF, H2O, (3,4,5-trimethoxyphenyl)boronic acid, Pd2(dba)3,
xantphos, K3PO4, N2, 80 oC, 8-12h
.
3. Results and Discussion
3.1 In vitro antiproliferative activity
90
The in vitro antiproliferative efficacy of synthesized novel 1,2,4-triazolo[4,3-a]pyridine
derivatives were first evaluated against human cervical cancer cells (HeLa), through
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results expressed
as IC50 (μM) were summarized in Table 1, compared to our previously reported compound 6 and the
reference Combretastatin A-4 (CA-4) which is a well-known tubulin inhibitor binding to the
colchicine-site. Moreover, some highly active compounds were selected to futher explore their
cytotoxic activity against a representative normal cell lines HEK-293 (human embryonic kidney
cells). Here, the IC50 value represents the concentration of one compound resulting in a 50%
inhibition in cell growth after 48 h incubation, and is the average of at least three independent
95
4

experiments.
As shown in Table 1, generally, except for compounds 7k–m and 7t, nearly all of the tested
100
105
110
115
120
derivatives 7a–u with 3,4,5-trimethoxyphenyl at C-3 position of [1,2,4]triazolo[4,3-a]pyridine core
demonstrated remarkable antiproliferative activities in a nanomolar range. In this series, fourteen
analogues 7a–j and 7n–q displayed much more potent activities with IC50 values from 0.012 to
0.651 μM than our parent compound 6 (IC50 = 0.75 μM), but were still less active than that of CA-4
(IC50 = 4.7 nM). In particular, derivative 7i featuring 3-amino-4-methoxyphenyl moiety, was found
to be the most active compound with an IC50 value of 12 nM, which was approximately comparable
with that of CA-4 and was almost sixty-two fold more potent than the lead analogue 6, suggesting
that the 3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine provides a useful skeleton for the
development of anticancer agents. Interestingly, replacement of the 2-methoxy phenyl substituent
(7p) by a 2-methoxypyridine group (7u) significantly decreased the antiproliferative activity. While
substituting phenyl moiety with bulky naphthalenyl group, compound 7m completely lost its
inhibitory activity when compared to the 7a–l and 7n–s. However, all of the derivatives 8a–h,
which have 3,4,5-trimethoxyphenyl on the C-6 position of the core, always showed much lower
activities than the corresponding analogus 7a–u (for example, 8a vs 7a, 8b vs 7e, 8c vs 7h, 8d vs 7c,
8e vs 7d, 8f vs 7j, 8g vs 7f, 8h vs 7i), indicating that 3,4,5-trimethoxyphenyl on the C-3 position of
fused-ring was critical for antiproliferative activity.
Most interestingly, all of the eight representative analogues 7a, 7c, 7f–j and 7p with potent
antiproliferative efficacy did not exhibite inhibitory effect (IC50 > 100 μM) on the normal human
embryonic kidney cells, HEK-293, while both the parent compound 6 and reference CA-4 displayed
significant cytotoxicity with an IC50 value of 29.94, and 45.69 μM, respectively. The results
manifested that these designed compounds might possess excellent selectivity over normal human
cells, indicating a high safety index.
5

Table 1 Antiproliferative activities of derivatives 7a–u and 8a–h against HeLa cells, and
125
cytotoxicity of some representative compounds.
IC50 (μM)a
IC50 (μM)a
Comp.
R
Comp
R
HeLa
HEK-293b
HeLa
HEK-293b
NT
NT
NT
NT
7a
7b
7c
7d
4-CH3C6H4
3,4-(OCH3)2C6H3
4-ClC6H4
0.034±0.003
0.119±0.015
0.065±0.003
0.347±0.044
>100
NTc
7q
7r
7s
7t
4-F,2-OCH3C6H3
2-OCF3C6H4
0.153±0.027
22.19±1.32
7.90±0.37
>25
>100
NT
2,4-(OCH3)2C6H3
2-OH,4-OCH3C6H3
3,4-Cl2C6H3
7e
4-OCH3C6H4
0.194±0.019
NT
7u
20.63±2.57
NT
>100
>100
>100
>100
>100
NT
NT
NT
NT
NT
NT
NT
NT
7f
7g
7h
7i
4-FC6H4
0.025±0.003
0.023±0.002
8a
8b
8c
8d
8e
8f
4-CH3C6H4
4-OCH3C6H4
4-OCH3,3-NO2C6H3
4-ClC6H4
>25
>25
4-NO2C6H4
4-OCH3,3-NO2C6H3 0.062±0.007
3-NH2,4-OCH3C6H3 0.012±0.001
>25
>25
7j
3-OH,4-OCH3C6H3
4-CF3C6H4
0.045±0.007
>25
3.4-Cl2C6H3
>25
7k
7l
3-OH,4-OCH3C6H3
4-FC6H4
22.09±1.30
>25
NT
4-OHC6H4
>25
8g
7m
>25
NT
8h
3-NH2,4-OCH3C6H3
3.97±0.14
NT
NT
NT
29.94±0.69
7n
7o
7p
3-OCH3C6H4
3-F,6-OCH3C6H3
2-OCH3C6H4
0.446±0.075
0.651±0.082
0.084±0.015
6
0.75±0.02
45.69 ± 5.15
CA-4
0.0047±0.0002
>100
a 50% inhibitory concentration and mean ± SD of at least three independent experiments performed in duplicate.
b Normal human embryonic kidney (HEK-293) cell lines.
c NT: not tested.
130
Moreover, some potent analogues with excellent antiproliferative activity on HeLa were
further tested against three additional different cancer cell lines, including human lung
adenocarcinoma cell line (A549), and human breast cell line (MCF-7 and T47D), and were also
compared with the reference compound CA-4. The results are presented in Table 2, which revealed
some representative compounds effectively inhibited the proliferation of a variety of cancer types.
135
6

Among them, 7i was found to be the most active analogue, and exhibited potent activities against
A549, MCF-7, and T47D, suggesting that 7i could be a potential anticancer agent worthy of
further investigation.
Table 2 Antiproliferative activities of representative compounds against three cancer cell lines.
IC50 (μM)
Compd.
R
A549
MCF-7
>100
T47D
>100
7a
7b
4-CH3C6H4
3,4-(OCH3)2C6H3
4-FC6H4
6.26±0.91
87.08±0.94
78.62±4.10
58.67±1.08
10.40±2.17
17.76±3.04
9.28±0.81
>100
>100
>100
75.97±0.96
>100
7f
7h
4-OCH3,3-NO2C6H3
3-NH2,4-OCH3C6H3
3-OH,4-OCH3C6H3
2-OCH3C6H4
75.71±0.83
40.40±6.17
>100
7i
27.91±3.22
>100
7j
13.69±2.779
0.17±0.07
31.54±2.05
7p
CA-4
\
0.021±0.04
0.0019±0.0003
140
aIC50 is the concentration of compound required to decrease cell growth by 50% and values reported
represent the mean ± SD (standard deviation) of three independent experiments.
3.2. Cell cycle analysis
Encouraged by the preliminary antiproliferative screening results, the anticancer mechanism of
these compounds was further explored, and the most promising analogue 7i was selected to
examine the influence on the cell cycle. In this study, HeLa cells were incubated with increased
concentrations of 7i (12, 24, and 48 nM) for 24 h. As illustrated in Fig. 2, the percentage of cells at
the G2/M phase in presence of the compound was 13.90%, 26.00%, and 92.65%, respectively,
while 11.14% of G2/M phase was detected for the control group. These findings demonstrated that
compound 7i caused a clear G2/M arrest pattern in a concentration-dependent manner.
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7

150
155
Fig. 2. Effect of compound 7i on cell cycle in HeLa cells. Flow cytometry of HeLa cells treated with 7i for
24h. (A) Control; (B) 7i, 12 nM; (C) 7i, 24 nM; (D) 7i, 48 nM.
It has been known mitosis promoting factor formed between cdc2 and cyclin B1, plays a key
role during the transition from interphase to mitotic phase and controls cell cycle progression
through increasing distribution in the G2/M phase [28-29]. Hence, we studied alterations in
expression of cell cycle regulatory proteins to achieve insight into the mechanism of 7i-induced
G2/M arrest in HeLa. As presented in Figure 3, 7i concentration-dependently decreased cyclin B1
and cdc2 protein levels, which indicated that 7i-caused G2/M arrest might be correlated with the
alterations of expression of cyclin B1 and cdc2.
160
Figure 3. Effects of 7i on G2/M regulatory proteins. HeLa cells were treated for 24 h with the indicated
concentration of 7i (12, 24 and 48 nM). The cells were harvested and lysed for the detection of cyclin B1 and
8

p-cdc2.
165
3.3. Cell apoptosis study
In order to investigate the mode of cell death produced by 7i, cell apoptosis analysis of HeLa
cells incubated with the increasing concentrations of the compound was performed using an
Annexin V-FITC/PI assay. As shown in Fig. 4, when the cells were incubated with 7i at 12, 24, and
48 nM for 24 h, the total numbers of early and late apoptotic cells were 8.44%, 26.87% and 53.3%,
respectively, whereas that of control group was only 6.31%. Therefore, these results indicated that
compound 7i effectively induced cell apoptosis in HeLa cells via a dose-dependent manner.
170
Fig. 4. Flow cytometric analysis of apoptotic cells after treatment of HeLa cells with derivative 7i at different
concentrations (12, 24, 48 nM) and no treatment (control) as a reference control for 24 h. The lower left quadrant
represents live cells, the lower right stands for early apoptotic cells, the upper right represents late apoptotic cells
and the upper left is for cells necrosis.
175
Activation of caspase-3 plays a vital role for the initiation and execution of the apoptotic
process. Among the caspase-3, PARP cleavage, an essential indicator of apoptosis induction, is a
9

180
key indicator of caspase-3 activation. Hence, in order to understand the correlation of
antiproliferative activity with cellular death by apoptosis by these analogues, we examined the
activation of caspase-3 in HeLa cells exposed to compound 7i (12, 24 and 48 nM). Results indicated
that a significant increase in the levels of cleaved PARP was observed by treatment with 7i for 24 h
via a concentration-dependent manner (Fig. 5), suggesting that 7i induced apoptosis in cancer cells
by activation of caspase-3. Additionally, in this study, it was clear that the important anti-apoptotic
protein Bcl-2 was down regulated after 24 h of treatment compare to the control, which revealed
that the induction of apoptosis by 7i is associated with Bcl-2 down-regulation.
185
Fig. 5. Western blotting analysis of the expression of cleaved PARP and Bcl-2. HeLa cells were treated for 24 h
with the indicated concentration of 7i (12, 24 and 48 nM). Cell lysates were collected, and the expression levels of
PARP and Bcl-2 were determined by western blot analysis. β-actin was used as a loading control.
190
3.4. In vitro tubulin polymerization assay
To examine whether microtubule system could be a potential target of these compounds, the
above seven highly active derivatives 7a, 7b, 7f, 7h, 7i, 7j, and 7p were initially chose to evaluate
their in vitro tubulin polymerization inhibition at the concentration of 10 μM, comparing to the
reference tubulin polymerization inhibitor CA-4. As shown in Table 3, most of the tested
compounds significantly inhibited tubulin polymerization. Particularly, compounds 7a, 7b, and 7i
inhibited tubulin polymerization by 77, 68 and 81 %, respectively, while 79 % inhibition was
195
10

200
observed with CA-4. Further screening revealed that 7i (Fig. 6), the most potent analogue on HeLa
cells, also exhibited much higher anti-tubulin activity than CA-4 (IC50 = 4.2 μM) with an IC50 value
of 3.4 μM. These results suggested that influence on the tubulin polymerization inhibition
correlated well with their antiproliferative efficacy, and tubulin was the most likely the target of
these compounds.
205
Table 3 Tubulin polymerization inhibitory activities of the most potent compounds
Tubulin polymerization
Comp.
R
%inhibitiona
IC50(μM)b
4.1±0.1
11.3±1.0
NTc
4-CH3C6H4
3.4-(OCH3)2C6H3
4-FC6H4
77
68
18
36
81
14
16
79
7a
7b
7f
4-OCH3,3-NO2C6H3
3-NH2,4-OCH3C6H3
NT
7h
7i
3.4±0.1
NT
3-OH,4-OCH3C6H3
2-OCH3C6H4
7j
NT
7p
4.2±0.1
CA-4
a Compounds were tested at a final concentration of 10μM.
b IC50 values are presented as mean values of three independent experiments done in quadruplicates.
c NT: Not test
.
210
Fig. 6. Effects of 7i on tubulin polymerization in vitro. Tubulin in reaction buffer was incubated at 37°C in the
presence of the control (1% DMSO), compound 7i (0.6, 1.5, 2.5, 5 μM) or the reference CA-4 (1.5 μM).
Absorbance at 340 nm was monitored at 37 °C every minute, and polymerizations were followed by an increase in
11

215
220
fluorescence emission at 340 nm over a 20 min period. Data are presented as the mean ± SD from three
independent experiments. **P < 0.01 vs the vehicle control.
3.5. Immunocytochemistry
To substantiate the direct effects of these analogus on tubulin, immunohistochemistry was
carried out to investigate alterations in the microtubule network caused by compound 7i in cultured
HeLa cells, compared with that of CA-4 at the same level of concentration. As depicted in Fig. 7,
the microtubule network exhibited normal arrangement and organization in the untreated HeLa cells.
Whereas, exposure to 12 nM of 7i for 6h led to significantly disrupt microtubule organization,
which was similar to those of CA-4. These results indicated that compound 7i showed similar
effects to CA-4 on the microtubule network and thus revealed that 7i inhibited the tubulin
polymerization.
225
Fig. 7. Compound 7i and CA-4 induced microtubules depolymerization in HeLa cells. HeLa cells were processed
with 7i and CA-4 at 12 nM concentration for 6h, followed by immunofluorescent staining with anti-α-tubulin
antibody (green), anti-β-tubulin antibody (red) and DIPA (blue, for nuclear staining). Images were captured with
laser scanning confocal microscope.
230
3.6. Molecular studies
In order to illustrate the possible binding mode, the most active compound 7i was chose to
perform the molecular docking simulations on the colchicine-site of tubulin. As given in Fig. 8, 7i
and CA-4 adopted quite similar location at the colchicine binding pocket in the co-crystallized
tubulin structure (PDB: 5lyj). The triazolopyridine core of the 7i was extended deeply in the
β-subunit of the tubulin and the N-1 of the triazole ring formed a hydrogen-bonding interaction with
235
12

residue β-Asp251. Meanwhile, the much stronger hydrogen bond existed between the oxygen atom
of 3,4,5-trimethoxylphenyl moiety and the residue β-Cys241 which was consistent with that of the
4-methoxyl group of CA-4. Furthermore, the 3-amino moiety of phenyl ring established two
additional hydrogen bonds with α-Val181 and β-Asn350. The docking studies further indicated that
compound 7i may be a potential tubulin inhibitor binding to the colchicine site.
240
Fig. 8. Proposed binding model for 7i (green stick) overlaid with CA-4 (yellow stick) in colchicine-site of
tubulin (PDB code: 5lyj). The main interacting residues are shown and labeled. The black dashed lines were the
potential H-bond between Cys241 (2.3Å), Val181 (3.0Å), Asp251 (3.6Å), Asn350 (3.9Å). Final figure for
docking pose was generated by PyMOL.
245
4. Conclusion
250
On the basis of our previous work, a series of novel 3,4,5-trimethoxyphenyl substituted
[1,2,4]triazolo[4,3-a]pyridines were designed and synthesized with a triazolopyrimidine 6 as the
parent compound. Some target compounds exhibited improved antiproliferative efficacy in a
nanomolar range against HeLa cells, and the most promising derivative 7i displayed excellent
antiproliferative efficacy with an IC50 value of 12 nM, which was approximately comparable to that
of the reference CA-4, and was almost sixty-two fold more potent than the analogue 6. The results
indicated that the bioisosteric replacement of the pyrimidine moiety of triazolo[4,3-a]pyrimidine
with a pyridine ring successfully enhanced potent activity. Most interestingly, 7i exhibited
outstanding selectivity over the normal human embryonic kidney HEK-293 cells (IC50 > 100 μM),
which manifested that the compound might have much lower toxicity. Further mechanistic studies
demonstrated that 7i significantly arrested cell cycle at G2/M phase, induced apoptosis with a
dose-dependent manner, and also disrupted microtubule networks. In addition, tubulin
255
260
13

polymerization assay suggested that analogue 7i manifested much higher anti-tubulin activity than
CA-4. Furthermore, molecular docking studies revealed that 7i well occupied the colchicine-site in
a similar way with CA-4. The preliminary observations highlight that compound 7i is a novel
potential tubulin polymerization inhibitor and deserves further investigation for the treatment of
cancers.
265
5. Experimental protocols
5.1 Chemistry
1
Melting points were measured on a Buchi B-545 melting point apparatus and uncorrected. H
270
and 13C NMR spectra were determined on a Mercury-Plus 400 spectrometer in CDCl3 or DMSO-d6
solution at room temperature, and chemical shift (δ) was expressed in parts per million (ppm) using
TMS as an internal standard. High-resolution mass spectra (HRMS) performed on an Agilent
QTOF 6540 mass spectrometer. Unless otherwise noted, all of chemicals were purchased from
commercial suppliers and used without further purification.
275
5.2 General procedure for the preparation of derivatives 7a–u and 8a–h.
5.2.1. General procedure for the preparation of the 7a–u.
(E)-5-bromo-2-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)pyridine 10.
A solution of 5-bromo-2-hydrazinylpyridine 8 (2.2 g, 10mmol) in ethanol (80 mL) was added to
3,4,5-trimethoxybenzaldehyde (2.0 g, 10 mmol) and the resulting mixture was stirred for 4 h at
room temperature. The crude product was then filtered, washed with ethanol (10 mL× 3) and dried
to give compound 10 as a solid in the yield of 91%, which was used for next step reaction without
purification.
280
6-bromo-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine 11.
To the solution of 10 (5 mmol) in DCM (80 mL) was added iodobenzene diacetate (5 mmol) at
285
room temperature under nitrogen atmosphere. The resulting mixture was stirred for 10 h, poured
into water, and extracted with ethyl acetate (80 mL×3). The solvents were then removed in vacuo,
and the solid was filtered off, washed with methanol (3 mL ×2) and dried to afford 11.
6-Bromo-3-(3,4,5-trimethoxy-phenyl)-[1,2,4]triazolo[4,3-a]pyridine. Yield, 83%; mp:
223.4–225.1°C; 1H NMR (400 MHz, CDCl3) δ: 3.95 (s, 6H, 2×CH3O), 3.96 (s, 3H, CH3O), 7.07 (s,
14

290
2H, Ph-H), 7.59 (d, J = 9.2 Hz, 1H, Pyridine-H), 7.91 (d, J = 9.2 Hz, 1H, Pyridine-H), 8.44 (s, 1H,
Pyridine-H). 13C NMR (100 MHz, CDCl3) δ: 153.74, 150.46, 147.58, 137.94, 133.45, 129.32,
128.06, 126.81, 123.86, 105.25, 60.62, 56.88.
General procedure for the preparation of the 7a–u.
A mixture of 11 (0.40 mmol), Pd2(dba)3 (0.04 mmol), xantphos (0.05 mmol), K3PO4 (0.48 mmol)
and substituted arylboronic acids (0.41 mmol) in DMF/H2O (20 mL, 5:1) was degassed and purged
with N2 for about three times. After stirred at 80°C for about 8-12 h (indicated by TLC) under N2
atmosphere, the reaction mixture was poured into H2O (50 mL) and extracted with ethyl acetate (80
mL×3). The combined organics were washed with brine (10 mL×3), dried over anhydrous Na2SO4,
filtered and concentrated under vacuum to give a residue, which was purified by column
chromatography using a mixture of petroleum ether and acetone (3:1) as an eluent to provide the
target compounds 7a–u in yields of 60–84%.
295
300
5.2.1.1. 6-p-Tolyl-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (7a). Yield, 80%; mp:
1
184.7–185.9°C; H NMR (400 MHz, CDCl3) δ: 2.43 (s, 3H, CH3), 3.95 (s, 6H, 2×CH3O), 3.96
(s,3H, CH3O), 7.07 (s, 2H, Ph-H), 7.31 (d, J = 8 Hz, 2H, Ph-H), 7.44 (d, J = 8 Hz, 2H, Ph-H), 7.57
(d, J = 9.2 Hz, 1H, Pyridine-H), 7.88 (d, J = 9.2 Hz, 1H, Pyridine-H), 8.39 (s, 1H, Pyridine-H). 13C
NMR (100 MHz, CDCl3) δ: 153.96, 149.85, 146.93, 139.81, 138.68, 133.25, 130.01, 128.78,
128.39, 126.73, 121.85, 118.88, 116.52, 105.81, 60.97, 56.46, 21.10. HRMS (ESI) m/z: calcd for
C22H21N3O3 (M+H+) 376.1656 found 376.1645.
305
5.2.1.2. 6-(3,4-Dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (7b).
1
310
315
Yield, 81%; mp: 101.9–102.2°C; H NMR (400 MHz, CDCl3) δ: 3.78 (s, 3H, CH3O), 3.81 (s, 3H,
CH3O), 3.85 (s,3H, CH3O), 3.90 (s,6H, 2×CH3O), 7.07 (d, J = 8.4 Hz, 1H, Ph-H), 7.28 (s, 2H,
Ph-H), 7.30 (s, 1H, Ph-H), 7.36 (s, 1H, Ph-H), 7.80 (d, J = 9.6 Hz, 1H, pyridine), 7.93 (d, J = 9.6 Hz,
13
1H, pyridine), 8.66 (s, 1H, pyridine). C NMR (100 MHz, DMSO-d6) δ: 153.89, 149.67, 149.55,
149.50, 146.82, 139.23, 129.00, 128.82, 127.55, 122.34, 120.28, 119.81, 115.80, 112.62, 111.21,
106.22, 60.55, 56.61, 56.01, 55.97. HRMS (ESI) m/z: calcd for C23H23N3O5 (M+H+) 422.1710
found 422.1701.
5.2.1.3. 6-(4-Chlorophenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (7c). Yield,
1
78%; mp: 166.7–168.5; H NMR (400 MHz, DMSO-d6) δ: 3.78 (s, 3H, CH3O), 3.88 (s, 6H,
2×CH3O), 7.24 (s, 2H, Ph-H), 7.56 (d, J = 8.4 Hz, 2H, Ph-H), 7.80 (m, 3H, Ph-H, Pyridine-H), 7.96
(d, J = 9.2 Hz, 1H, Pyridine-H), 8.69 (s, 1H, Pyridine-H). 13C NMR (100 MHz, CDCl3) δ: 154.02,
149.75, 147.08, 139.96, 134.89, 134.66, 129.54, 128.17, 127.91, 127.74, 121.64, 119.34, 116.89,
320
15

105.87, 60.98, 56.49. HRMS (ESI) m/z: calcd for C21H18ClN3O3 (M+H+) 396.1109 found 396.1103.
5.2.1.4. 6-(3,4-Dichlorophenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (7d).
Yield, 79%; mp: 194.4–195.0°C; 1H NMR (400 MHz, CDCl3) δ: 3.95 (s, 6H, 2×CH3O), 3.97 (s, 3H,
CH3O), 7.04 (s, 2H, Ph-H), 7.39 (dd, J1 = 1.6 Hz, J2 = 8 Hz, 1H, Ph-H), 7.49 (d, J = 9.6 Hz, 1H,
Pyridine-H), 7.58 (d, J = 8.4 Hz, 1H, Ph-H), 7.64 (s, 1H, Ph-H), 7.91 (d, J = 9.6 Hz, 1H,
Pyridine-H), 8.39 (s, 1H, Pyridine-H). 13C NMR (100 MHz, CDCl3) δ: 154.05, 149.67, 147.19,
140.05, 136.19, 133.59, 133.08, 131.29, 128.84, 127.49, 126.65, 126.11, 121.45, 119.72, 117.13,
105.88, 60.98, 56.49. HRMS (ESI) m/z: calcd for C21H17Cl2N3O3 (M+H+) 430.0720 found 430.
0719.
325
330
5.2.1.5. 6-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (7e). Yield,
1
81%; mp: 184.3–186.0°C; H NMR (400 MHz, CDCl3) δ: 3.88 (s, 3H, CH3O), 3.96 (s, 6H,
2×CH3O), 3.97 (s,3H, CH3O), 7.04 (d, J = 8.4 Hz, 2H, Ph-H), 7.07 (s, 2H, Ph-H), 7.48 (d, J = 8.4
Hz, 2H, Ph-H), 7.55 (d, J = 9.6 Hz, 1H, Pyridine-H), 7.89 (d, J = 9.6 Hz, 2H, Pyridine-H), 8.36 (s,
13
335
1H, Pyridine-H). C NMR (100 MHz, CDCl3) δ: 160.07, 153.97, 149.79, 146.90, 139.83, 128.52,
128.50, 128.38, 128.05, 121.89, 118.44, 116.51, 114.76, 105.85, 60.98, 56.48, 55.38. HRMS (ESI)
m/z: calcd for C22H21N3O4 (M+H+) 392.1605 found 392.1604.
5.2.1.6. 6-(4-Fluorophenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (7f). Yield,
1
75%; mp: 186.6–186.8°C; H NMR (400 MHz, DMSO-d6) δ: 3.78 (s, 3H, CH3O), 3.89 (s, 3H,
340
345
2×CH3O), 7.24 (s, 2H, Ph-H), 7.34 (t, J1 = 8.8 Hz, J2 = 17.6 Hz, 2H, Ph-H), 7.77 (d, J = 9.6 Hz, 1H,
Pyridine-H), 7.83 (dd, J1 = 5.6 Hz, J2 = 8.4 Hz, 2H, Ph-H), 7.95 (d, J = 9.6 Hz, 1H, Pyridine-H),
8.65 (s, 1H, Pyridine-H). 13C NMR (100 MHz, DMSO-d6) δ: 162.68 (d, J = 243.8 Hz, 1C), 153.87,
149.59, 147.05, 139.29, 132.76 (d, J = 2.9 Hz, 1C), 129.70 (d, J = 8.2 Hz, 2C), 128.77, 126.54,
122.21, 121.11, 116.34 (d, J = 21.5 Hz, 2C), 116.00, 106.47, 60.53, 56.66. HRMS (ESI)
m/z: calcd for C21H18FN3O3 (M+H+) 380.1405 found 380.1399.
5.2.1.7. 6-(4-Nitrophenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (7g). Yield,
1
84%; mp: 172.5–173.3°C; H NMR (400 MHz, CDCl3) δ: 3.95 (s, 6H, 2×CH3O), 3.97 (s, 3H,
CH3O), 7.05 (s, 2H, Ph-H), 7.58 (d, J = 9.6 Hz, 1H, Pyridine-H), 7.74 (d, J = 8.4 Hz, 2H, Ph-H),
7.97 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.37 (d, J = 8.8 Hz, 2H, Ph-H), 8.49 (s, 1H, Pyridine-H). 13C
NMR (100 MHz, CDCl3) δ: 154.10, 149.67, 147.85, 147.37, 142.55, 140.17, 127.77, 127.26,
126.67, 124.59, 121.31, 120.54, 117.43, 105.96, 61.00, 56.54. HRMS (ESI) m/z: calcd for
C21H18N4O5 (M+H+) 407.1350 found 407.1345.
350
355
5.2.1.8.
6-(4-Methoxy-3-nitrophenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine
1
(7h). Yield, 75%; mp: 181.7–182.4°C; H NMR (400 MHz, CDCl3) δ: 3.96 (s, 6H, 2×CH3O), 3.98
(s, 3H, CH3O), 4.05 (s,3H, CH3O), 7.06 (s, 2H, Ph-H), 7.26 (d, J = 8.8 Hz, 1H, Ph-H), 7.53 (d, J =
16

9.6 Hz, 1H, Pyridine-H), 7.75 (t, J1 = 2 Hz, J2 = 8.4 Hz, 1H, Ph-H), 7.94 (d, J = 9.6 Hz, 1H,
Pyridine-H), 8.07 (d, J = 2 Hz, 1H, Ph-H), 8.42 (s, 1H, Pyridine-H). 13C NMR (100 MHz, CDCl3) δ:
154.06, 153.17, 149.61, 147.16, 140.02, 139.89, 132.34, 128.63, 127.45, 126.36, 124.18, 121.43,
119.46, 117.17, 114.55, 105.82, 60.98, 56.77, 56.47. HRMS (ESI) m/z: calcd for C22H20N4O6
(M+H+) 437.1456 found 437.1464.
360
365
5.2.1.9. 2-Methoxy-5-(3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)aniline (7i).
1
Yield, 62%; mp: 195.1–195.5°C; H NMR (400 MHz, CDCl3) δ: 3.92 (s, 3H, CH3O), 3.95 (s, 6H,
2×CH3O), 3.97 (s, 3H, CH3O), 6.89 (s, 3H, Ph-H), 7.06 (s, 2H, Ph-H), 7.53 (d, J = 9.2 Hz, 1H,
13
Pyridine-H), 7.85 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.33 (s, 1H, Pyridine-H). C NMR (150 MHz,
CDCl3) δ: 153.99, 149.90, 147.77, 146.89, 139.79, 137.04, 129.06, 129.05, 128.71, 121.99, 118.43,
116.91, 116.27, 113.00, 110.82, 105.89, 61.05, 56.53, 55.67. HRMS (ESI) m/z: calcd for
C22H22N4O4 (M+H+) 407.1714 found 407.1717.
5.2.1.10. 2-Methoxy-5-(3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)phenol (7j).
1
Yield, 78%; mp: 183.2–183.7°C; H NMR (400 MHz, CDCl3) δ: 2.20 (s, 1H, OH), 3.96 (s, 6H,
370
375
2×CH3O), 3.97 (s, 6H, 2×CH3O), 6.97 (d, J = 8.4 Hz, 1H, Ph-H), 7.04 (s, 1H, Ph-H), 7.07 (s, 2H,
Ph-H), 7.15 (s, 1H, Ph-H), 7.55 (d, J = 9.2 Hz, 1H, Pyridine-H), 7.87 (d, J = 9.6 Hz, 1H,
Pyridine-H), 8.36 (s, 1H, Pyridine-H). 13C NMR (100 MHz, CDCl3) δ: 153.96, 149.80, 147.16,
146.92, 146.36, 139.78, 129.41, 128.54, 128.43, 121.81, 118.63, 118.61, 116.40, 113.11, 111.23,
105.78, 60.99, 56.45, 56.06. HRMS (ESI) m/z: calcd for C22H21N3O5 (M+H+) 408.1554 found
408.1559.
5.2.1.11.
6-(4-Trifluoromethylphenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine
1
(7k). Yield, 75%; mp: 199.3–199.5°C; H NMR (400 MHz, CDCl3) δ: 3.91 (s, 3H, CH3O), 3.94
(s,6H, 2×CH3O), 6.71 (s, 2H, Ph-H), 7.57 (d, J = 9.6 Hz, 1H, Pyridine-H), 7.88 (d, J = 8 Hz, 2H,
Ph-H), 7.93 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.04 (d, J = 8 Hz, 2H, Ph-H), 8.34 (s, 1H, Pyridine-H).
13C NMR (100 MHz, CDCl3) δ: 153.88, 150.19, 145.67, 138.85, 131.85 (d, J = 1.8 Hz, 2C), 130.24
(d, J = 1.1 Hz, 2C), 129.84, 129.00, 128.41, 126.37 (q, J1 = 3.8 Hz, J2 = 7.5 Hz, Hz, 1C), 123.31 (d,
J = 204.8 Hz, 1C), 118.84, 116.69, 104.79, 60.94, 56.41. HRMS (ESI) m/z: calcd for C22H28F3N3O3
(M+H+) 430.1373 found 430.1365.
380
5.2.1.12. 4-(3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)phenol (7l). Yield, 62%;
mp: 212.5–213.1°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.70 (s, 3H, CH3O), 3.86 (s, 6H, 2×CH3O),
6.99 (s, 2H, Ph-H), 7.01 (s, 2H, Ph-H), 7.76 (d, J = 9.6 Hz, 1H, Pyridine-H), 7.80 (d, J = 8.4 Hz, 2H,
Ph-H), 7.91 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.52 (s, 1H, Pyridine-H), 10.04 (s, 1H, OH). 13C NMR
(100 MHz, DMSO-d6) δ: 159.40, 153.71, 149.45, 147.10, 138.06, 132.27, 130.18, 129.08, 127.91,
120.65, 117.59, 116.48, 115.85, 105.32, 60.48, 56.56. HRMS (ESI) m/z: calcd for C21H19N3O4
(M+H+) 378.1449 found 378.1450.
385
390
17

5.2.1.13. 6-Naphthalen-2-yl-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (7m). Yield,
1
60%; mp: 185.6–185.9°C; H NMR (400 MHz, CDCl3) δ: 3.92 (s, 3H, CH3O), 3.93 (s,6H,
2×CH3O), 6.72 (s, 2H, Ph-H), 7.56 (dd, J1 = 1.6 Hz, J2 = 9.2 Hz, 1H, Pyridine-H), 7.61-7.64 (m, 2H,
Naphthalene-H), 7.94 (dd, J1 = 0.8 Hz, J2 = 9.2 Hz, 1H, Pyridine-H), 7.97-7.99 (m, 3H,
Naphthalene-H), 8.10 (d, J = 8.4 Hz, 1H, Naphthalene-H), 8.38 (s, 1H, Naphthalene-H), 8.45 (s, 1H,
Pyridine-H). 13C NMR (100 MHz, CDCl3) δ: 153.84, 149.98, 147.11, 138.68, 133.81, 133.18,
132.14, 129.34, 129.28, 128.64, 128.41, 128.00, 127.93, 127.51, 127.10, 125.08, 123.95, 119.22,
116.63, 104.69, 60.95, 56.36. HRMS (ESI) m/z: calcd for C25H21N3O3 (M+H+) 412.1656 found
412.1656.
395
400
405
5.2.1.14. 6-(3-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (7n).
1
Yield, 71%; mp: 127.2–127.6°C; H NMR (400 MHz, CDCl3) δ: 3.89 (s, 3H, CH3O), 3.95 (s,6H,
2×CH3O), 3.97 (s, 3H, CH3O), 7.00 (d, J = 8.4 Hz, 1H, Ph-H), 7.06 (s, 3H, Ph-H), 7.12 (d, J = 7.2
Hz, 1H, Ph-H), 7.43 (t, J1 = 7.6 Hz, J2 = 15.6 Hz, 1H, Pyridine-H), 7.60 (d, J = 9.6 Hz, 1H,
13
Pyridine-H), 7.93 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.41 (s, 1H, Pyridine-H). C NMR (100 MHz,
CDCl3) δ: 160.24, 154.01, 149.46, 146.90, 139.94, 137.44, 130.47, 129.01, 128.87, 121.41, 119.42,
119.26, 116.48, 113.55, 113.19, 105.84, 61.00, 56.47, 55.37. HRMS (ESI) m/z: calcd for
C22H21N3O4 (M+H+) 392.1606 found 392.1604
5.2.1.15. 6-(5-Fluoro-2-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine
1
(7o). Yield, 79%; mp: 193.2–194.°C; H NMR (400 MHz, DMSO-d6) δ: 3.77 (s, 3H, CH3O), 3.80
410
415
(s,3H, CH3O), 3.88 (s,6H, 2×CH3O), 7.17 (dd, J1 = 4.8 Hz, J2 = 9.2 Hz, 1H, Ph-H), 7.20 (s, 2H,
Ph-H), 7.23-7.28 (m, 1H, Ph-H), 7.42 (dd, J1 = 3.2 Hz, J2 = 9.2 Hz, 1H, Ph-H), 7.61 (dd, J1 = 1.6
Hz, J2 = 9.6 Hz, 1H, Pyridine-H), 7.88 (dd, J1 = 0.8 Hz, J2 = 9.6 Hz, 1H, Pyridine-H), 8.61 (m, J1 =
1.2 Hz, J2 = 2.4 Hz, 1H, Pyridine-H). 13C NMR (100 MHz, DMSO-d6) δ: 156.77 (d, J = 235.1 Hz,
1C), 153.85, 153.33 (d, J = 1.8 Hz, 1C), 149.41, 146.89, 139.32, 130.75, 126.63 (d, J = 8.1 Hz, 1C),
124.16 (d, J = 1.6 Hz, 1C), 123.27, 122.20, 117.57 (d, J = 24.1 Hz, 1C), 116.09 (d, J = 22.4 Hz, 1C),
114.82, 113.64 (d, J = 8.3 Hz, 1C), 106.41, 60.55, 56.73, 56.63. HRMS (ESI) m/z: calcd for
C22H20FN3O4 (M+H+) 410.1511 found 410.1505.
5.2.1.16. 6-(2-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]Pyridine-H (7p).
Yield, 77%; mp: 180.5–180.9°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.76 (s, 3H, CH3O), 3.81 (s,3H,
CH3O), 3.87 (s,6H, 2×CH3O), 7.06 (t, J1 = 7.6 Hz, J2 = 15.2 Hz, 1H, Ph-H), 7.17 (d, J = 8.4 Hz, 1H,
Ph-H), 7.20 (s, 2H, Ph-H), 7.43 (t, J1 = 7.6 Hz, J2 = 15.2 Hz, 1H, Ph-H), 7.49 (d, J= 7.2 Hz, 1H,
Ph-H), 7.60 (d, J= 9.6 Hz, 1H, Pyridine-H), 7.87 (d, J= 9.6 Hz, 1H, Pyridine-H), 8.53 (s, 1H,
420
425
13
Pyridine-H). C NMR (100 MHz, DMSO-d6) δ: 156.87, 153.84, 149.42, 146.75, 139.21, 131.10,
130.88, 130.38, 125.25, 125.23, 122.58, 122.29, 121.41, 114.77, 112.29, 106.31, 60.55, 56.60,
56.10. HRMS (ESI) m/z: calcd for C22H21N3O4 (M+H+) 392.1605 found 392.1602.
18

5.2.1.17. 6-(4-Fluoro-2-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine
(7q). Yield, 75%; mp: 203.1–204.8°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.76 (s, 3H, CH3O), 3.83
(s, 3H, CH3O), 3.87 (s, 6H, 2×CH3O), 6.90 (t, J1 = 8.4 Hz, J2 = 16.8 Hz, 1H, Ph-H), 7.09 (d, J =
11.6 Hz, 1H, Pyridine-H), 7.19 (s, 2H, Ph-H), 7.51 (d, J = 8 Hz, 1H, Ph-H), 7.56 (d, J = 10.4 Hz,
13
430
1H, Pyridine-H), 7.87 (d, J = 9.6 Hz, 1H, Ph-H), 8.51 (s, 1H, Pyridine-H). C NMR (100 MHz,
DMSO-d6) δ: 163.56 (d, J = 243.4 Hz, 1C), 158.32 (d, J = 10.2 Hz, 1C), 153.83, 149.40, 146.76,
139.21, 132.11(d, J = 10.1 Hz, 1C), 131.04, 124.48, 122.70, 122.25, 121.71 (d, J = 3.2 Hz, 1C),
114.80, 107.63 (d, J = 20.9 Hz, 1C), 106.30, 100.60 (d, J = 25.8 Hz, 1C), 60.54, 56.60. HRMS (ESI)
m/z: calcd for C22H20FN3O4 (M+H+) 410.1511 found 410.1506.
435
440
5.2.1.18. 6-(2-Trifluoromethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine
(7r). Yield, 82 %; mp: 187.2–187.9°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.76 (s, 3H, CH3O), 3.87
(s, 6H, 2×CH3O), 7.19 (s, 2H, Ph-H), 7.55-7.62 (m, 4H, Ph-H), 7.75 (d, J= 6.4 Hz, 1H, Pyridine-H),
7.97 (d, J= 9.6 Hz, 1H, Pyridine-H), 8.65 (s, 1H, Pyridine-H). 13C NMR (150 MHz, CDCl3) δ:
154.02, 149.68, 147.11, 146.30 (d, J = 1.31 Hz, 1C), 139.88, 131.16, 130.25, 130.16, 130.16,
129.43, 127.75, 123.91, 122.08, 121.84, 121.62, 120.32 (q, J1 = 257.34 Hz, J2 = 514.68 Hz, 1C),
116.43, 105.73, 61.00, 56.38. HRMS (ESI) m/z: calcd for C22H18F3N3O4 (M+H+) 446.1322 found
446.1316.
5.2.1.19. 6-(2,4-Dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (7s).
1
Yield, 79%; mp: 105.9–106.5°C; H NMR (400 MHz, DMSO-d6) δ: 3.70 (s, 3H, CH3O), 3.87 (s,
445
450
6H, 2×CH3O), 3.89 (s, 3H, CH3O), 3.91 (s, 3H, CH3O), 6.78 (d, J= 8.4 Hz, 1H, Ph-H), 6.84 (s, 1H,
Ph-H), 6.93 (s, 2H, Ph-H), 7.56 (d, J = 8.4 Hz, 1H, Ph-H), 7.79 (d, J = 9.2 Hz, 1H, Pyridine-H),
7.91 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.16 (s, 1H, Pyridine-H). 13C NMR (100 MHz, DMSO-d6) δ:
163.08, 158.65, 153.67, 149.31, 145.69, 137.99, 133.23, 132.03, 128.85, 127.18, 121.95, 115.59,
107.96, 106.50, 105.03, 99.45, 60.46, 56.40, 55.99, 55.96. HRMS (ESI) m/z: calcd for C23H23N3O5
(M+H+) 422.1711 found 422.1707.
5.2.1.20. 5-Methoxy-2-[3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl]-phenol (7t).
1
Yield, 70%; mp: 195.2–195.8°C; H NMR (400 MHz, CDCl3) δ: 3.51 (s, 1H, OH), 3.91 (s, 3H,
CH3O), 3.93 (s, 6H, 2×CH3O), 4.01 (s, 3H, CH3O), 6.71 (s, 2H, Ph-H), 7.06 (d, J = 8.4 Hz, 1H,
Ph-H), 7.37 (dd, J1 = 2 Hz, J2 = 8.4 Hz, 1H, Ph-H), 7.51 (dd, J1 = 1.6 Hz, J2 = 9.6 Hz, 1H,
Pyridine-H), 7.69 (d, J = 2 Hz, 1H, Ph-H), 7.86 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.37 (s, 1H,
Pyridine-H). 13C NMR (100 MHz, CDCl3) δ: 162.21, 158.61, 153.88, 148.79, 145.31, 138.79,
132.03, 129.57, 129.14, 125.78, 120.08, 116.61, 107.01, 104.80, 104.33, 102.49, 60.96, 56.41,
55.41. HRMS (ESI) m/z: calcd for C22H21N3O5 (M+H+) 408.1554 found 408.1556.
455
460
5.2.1.22. 6-(2-Methoxypyridin-3-yl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (7u).
1
Yield, 68 %; mp: 203.5–204.0°C; H NMR (400 MHz, DMSO-d6) δ: 3.76 (s, 3H, CH3O), 3.88
19

(s,6H, 2×CH3O), 3.92 (s, 3H, CH3O), 7.13 (dd, J1 = 4.8 Hz, J2 = 6.4 Hz, 1H, Pyridine-H), 7.21 (s,
2H, Ph-H), 7.67 (d, J= 9.6 Hz, 1H, Pyridine-H), 7.90 (d, J= 9.6 Hz, 1H, Pyridine-H) 7.96 ((d, J=
13
6.8 Hz, 1H, Pyridine-H), 8.24 (d, J= 4.8 Hz, 1H, Pyridine-H), 8.69 (s, 1H, Pyridine-H). C NMR
(100 MHz, DMSO-d6) δ: 160.75, 153.84, 149.44, 147.10, 146.89, 139.64, 139.25, 130.28, 123.52,
123.27, 122.17, 119.65, 118.03, 115.06, 106.32, 60.54, 56.61, 53.95. HRMS (ESI) m/z: calcd for
C21H20N4O4 (M+H+) 393.1558 found 393.1557.
465
5.2.2 General procedure for the preparation of derivatives 8a–h.
(E)-5-bromo-2-(2-(substituted benzylidene)hydrazinyl)pyridine 12a–h.
A solution of 5-bromo-2-hydrazinylpyridine 8 (2.2 g, 10mmol) in ethanol (80 mL) was added to
appropriate benzaldehydes (10 mmol) and the resulting mixture was stirred for about 3-5 h at room
temperature. The crude product was filtered, washed with ethanol (10 mL× 3) and dried to give
compounds 12a–h in the yield of 73-89%, which was used for next step reaction without
purification.
470
6-Bromo-3-substituted -[1,2,4]triazolo[4,3-a]pyridine 13a–h.
475
To the solution of 12a–h (0.25mmol) in CH2Cl2 (100 mL) was added iodobenzene diacetate
(1.0mmol) at room temperature under nitrogen atmosphere. The resulting mixture was stirred for
8-10 h. After completion of the reaction, the mixture was poured into water and extracted with
dichloromethane. The organic extract was washed with brine, dried, and evaporated to provide a
crude solid, which was washed with methanol (3 mL ×2) and dried to afford 13a–h in yields of
70-83%.
480
485
1
6-Bromo-3-p-tolyl-[1,2,4]triazolo[4,3-a]pyridine (13a). Yield, 81%; mp: 185.3–187.2°C; H
NMR (400 MHz, DMSO-d6) δ: 2.44 (s, 3H, CH3), 7.35 (d, J = 7.6 Hz, 2H, Ph-H), 7.83 (d, J = 9.6
Hz, 1H, Pyridine-H), 7.86 (d, J = 7.6 Hz, 2H, Ph-H), 7.97 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.59 (s,
13
1H, Pyridine-H). C NMR (150 MHz, CDCl3) δ: 148.98, 146.84, 140.92, 130.66, 130.18, 128.18,
123.12, 122.65, 117.44, 109.33, 21.55.
6-Bromo-3-(4-methoxy-phenyl)-[1,2,4]triazolo[4,3-a]pyridine (13b). Yield, 79%; mp:
204.9.3–206.8°C; 1H NMR (400 MHz, CDCl3) δ: 3.91 (s, 3H, CH3O), 3.92 (s, 3H, CH3O), 7.11 (d,
J = 8.4 Hz, 2H, Ph-H), 7.49 (d, J = 9.2 Hz, 1H, Pyridine-H), 7.80 (d, J = 8.4 Hz, 2H, Ph-H), 7.88 (d,
J = 9.6 Hz, 1H, Pyridine-H), 8.32 (s, 1H, Pyridine-H). 13C NMR (150 MHz, CDCl3) δ: 161.43,
148.65, 146.62, 131.00, 129.85, 122.62, 117.97, 117.32, 114.99, 109.52, 55.52.
490
6-Bromo-3-(4-methoxy-3-nitro-phenyl)-[1,2,4]triazolo[4,3-a]pyridine (13c). Yield, 76%; mp:
20

1
215.5–217.4°C; H NMR (400 MHz, DMSO-d6) δ: 4.05 (s, 3H, CH3O), 7.60 (d, J = 8.8 Hz, 1H,
Ph-H), 7.90 (d, J = 9.6 Hz, 1H, Pyridine-H), 7.99 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.31 (dd, J1 = 2.4
Hz, J2 = 8.8 Hz, 1H, Ph-H), 8.55 (d, J = 2 Hz, 1H, Ph-H), 8.69 (s, 1H, Pyridine-H). 13C NMR (150
MHz, CDCl3) δ: 154.39, 149.25, 144.42, 139.94, 134.17, 131.22, 125.13, 122.04, 118.50, 117.65,
114.81, 110.29, 56.95.
495
500
505
510
515
520
525
6-Bromo-3-(4-chloro-phenyl)-[1,2,4]triazolo[4,3-a]pyridine
(13d).
Yield,
83%;
mp:
170.1–172.3°C; 1H NMR (400 MHz, CDCl3) δ: 7.55 (d, J = 9.2 Hz, 1H, Pyridine-H), 7.58 (d, J = 8
Hz, 2H, Ph-H), 7.84 (d, J = 8 Hz, 2H, Ph-H), 7.93 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.34 (s, 1H,
13
Pyridine-H). C NMR (150 MHz, CDCl3) δ: 150.72, 148.14, 132.61, 132.44, 131.21, 128.64,
128.17, 123.87, 107.83, 105.15.
6-Bromo-3-(3,4-dichloro-phenyl)-[1,2,4]triazolo[4,3-a]pyridine (13e). Yield, 80%; mp:
182.6–184.4°C; 1H NMR (400 MHz, DMSO-d6) δ: 7.91 (d, J = 9.6 Hz, 1H, Pyridine-H), 7.84 (d, J
= 8.4 Hz, 1H, Ph-H), 7.99 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.22 (d, J = 8.8 Hz, 1H, Ph-H), 8.38 (s,
1H, Ph-H), 8.69 (s, 1H, Pyridine-H). 13C NMR (150 MHz, CDCl3) δ: 149.31, 144.59, 135.10,
134.09, 131.58, 131.23, 130.00, 127.08, 125.96, 122.22, 117.60, 110.23.
5-(6-Bromo-[1,2,4]triazolo[4,3-a]pyridin-3-yl)-2-methoxy-phenol (13f). Yield, 77%; mp:
1
235.5–237.4°C; H NMR (400 MHz, DMSO-d6) δ: 3.71 (s, 3H, CH3O), 7.17 (d, J = 8.4 Hz, 1H,
Ph-H), 7.39 (s, 1H, Ph-H), 7.40 (d, J = 8.4 Hz, 1H, Ph-H), 7.86 (d, J = 9.6 Hz, 1H, Pyridine-H),
13
7.93 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.59 (s, 1H, Pyridine-H), 9.50 (s, 1H, OH). C NMR (150
MHz, CDCl3) δ: 148.84, 148.68, 146.56, 130.81, 122.75, 120.66, 118.82, 117.30, 114.50, 111.42,
109.37, 56.13.
6-Bromo-3-(4-fluoro-phenyl)-[1,2,4]triazolo[4,3-a]pyridine
(13g).
Yield,
79%;
mp:
1
161.5–163.2°C; H NMR (400 MHz, DMSO-d6) δ: 7.46 (t, J = 8.8 Hz, 2H, Ph-H), 7.83 (d, J = 9.6
Hz, 1H, Pyridine-H), 7.96 (d, J = 9.6 Hz, 1H, Pyridine-H), 7.92 (dd, J1 = 5.6 Hz, J2 = 8.4 Hz, 2H,
Ph-H), 8.66 (s, 1H, Pyridine-H). 13C NMR (150 MHz, CDCl3) δ: 160.53 (d, J = 284.5 Hz), 155.43,
149.95, 133.51, 132.49 (d, J = 10.5 Hz), 129.05 (d, J = 2.7 Hz), 125.95, 117.43 (d, J = 24.3 Hz),
109.65, 103.26.
5-(6-Bromo-[1,2,4]triazolo[4,3-a]pyridin-3-yl)-2-methoxy-phenylamine (13h). Yield, 70%; mp:
202.7–204.9°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.96 (s, 3H, CH3O), 6.22 (s, 2H, NH2), 6.98 (d,
J = 8.4 Hz, 1H, Ph-H), 7.28 (d, J = 8.4 Hz, 1H, Ph-H), 7.22 (s, 1H, Ph-H), 7.51 (d, J = 9.6 Hz, 1H,
13
Pyridine-H), 7.89 (d, J = 9.2 Hz, 1H, Pyridine-H), 8.46 (s, 1H, Pyridine-H). C NMR (150 MHz,
CDCl3) δ: 150.97, 149.88, 145.46, 138.24, 137.53, 134.57, 123.95, 119.51, 115.44, 110.01, 108.65,
100.24, 55.86.
21

General procedure for the preparation of the 8a–h.
To the solution of 13 (0.40 mmol), Pd2(dba)3 (0.04 mmol), xantphos (0.05 mmol), K3PO4 (0.48
mmol) and (3,4,5-trimethoxyphenyl)boronic acid (0.41 mmol) in DMF/H2O (20 mL, 5:1) was
degassed and purged with N2 for about three times. After stirred at 80°C for about 8-12 h
(indicated by TLC) under N2 atmosphere, the reaction mixture was poured into H2O (50 mL) and
extracted with ethyl acetate (80 mL×3). The combined organics were washed with brine (10 mL×3),
dried over anhydrous Na2SO4, filtered and concentrated under vacuum to give a residue, which was
purified by column chromatography using a mixture of petroleum ether and acetone (3:1) as an
eluent to give the target compounds 8a–h in yields of 59–84%.
530
535
540
5.2.2.1. 3-p-Tolyl-6-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (8a). Yield, 84%; mp:
1
182.1–182.9°C; H NMR (400 MHz, DMSO-d6) δ: 2.44 (s, 3H, CH3), 3.71 (s, 3H, CH3O), 3.87
(s,6H, 2×CH3O), 7.00 (s, 2H, Ph-H), 7.45 (d, J = 7.6 Hz, 2H, Ph-H), 7.79 (d, J = 9.6 Hz, 1H,
Pyridine-H), 7.89 (d, J = 7.6 Hz, 2H, Ph-H), 7.94 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.58 (s, 1H,
Pyridine-H). 13C NMR (100 MHz, CDCl3) δ: 153.80, 149.76, 148.00, 140.55, 138.65, 132.18,
130.05, 129.03, 128.50, 128.13, 123.66, 119.26, 116.47, 104.70, 60.94, 56.39, 21.46. HRMS (ESI)
m/z: calcd for C22H21N3O3 (M+H+) 376.1656 found 376.1683.
5.2.2.2. 3-(4-Methoxyphenyl)-6-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (8b). Yield,
80%; mp: 205.7–206.1°C; 1H NMR (400 MHz, CDCl3) δ: 3.91 (s, 3H, CH3O), 3.92 (s, 3H, CH3O),
3.93 (s,6H, 2×CH3O), 6.70 (s, 2H, Ph-H), 7.13 (d, J = 8.4 Hz, 2H, Ph-H), 7.50 (d, J = 9.2 Hz, 1H,
Pyridine-H), 7.80 (d, J = 8.4 Hz, 2H, Ph-H), 7.87 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.29 (s, 1H,
Pyridine-H). 13C NMR (100 MHz, CDCl3) δ: 161.07, 153.80, 149.69, 146.93, 138.65, 132.23,
129.74, 128.94, 128.35, 119.18, 118.84, 116.49, 114.81, 104.71, 60.94, 56.39, 55.42. HRMS (ESI)
m/z: calcd forC22H21N3O4 (M+H+) 392.1610 found 392.1586.
545
5.2.2.3. 3-(4-Methoxy-3-nitrophenyl)-6-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (8c).
1
550
555
Yield, 78%; mp: 202.8–203.0°C; H NMR (400 MHz, DMSO-d6) δ: 3.71 (s, 3H, CH3O), 3.87 (s,
6H, 2×CH3O), 4.05 (s,3H, CH3O), 7.02 (s, 2H, Ph-H), 7.61 (d, J = 8.8 Hz, 1H, Ph-H), 7.87 (d, J =
9.6 Hz, 1H, Pyridine-H), 7.96 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.28 (dd, J1 = 2.4 Hz, J2 = 8.8 Hz,
1H, Ph-H), 8.52 (d, J = 2 Hz, 1H, Ph-H), 8.66 (s, 1H, Pyridine-H). 13C NMR (100 MHz, CDCl3) δ:
154.23, 153.92, 149.99, 144.63, 138.85, 134.46, 131.58, 129.91, 128.87, 128.34, 124.78, 118.97,
118.63, 116.67, 114.87, 104.55, 60.96, 56.87, 56.35. HRMS (ESI) m/z: calcd for C22H20N4O6
(M+H+) 437.1455 found 437.1445.
5.2.2.4. 3-(4-Chlorophenyl)-6-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (8d). Yield,
1
77%; mp: 230.1–232.5°C; H NMR (400 MHz, CDCl3) δ: 3.91 (s, 3H, CH3O), 3.94 (s, 6H,
22

2×CH3O), 6.70 (s, 2H, Ph-H), 7.53 (d, J = 9.2 Hz, 1H, Pyridine-H), 7.59 (d, J = 8 Hz, 2H, Ph-H),
13
560
7.82 (d, J = 8 Hz, 2H, Ph-H), 7.89 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.28 (s, 1H, Pyridine-H). C
NMR (100 MHz, CDCl3) δ: 153.85, 149.98, 146.00, 138.79, 136.40, 131.96, 129.70, 129.45,
128.71, 125.13, 118.92, 116.60, 104.75, 60.94, 56.41. HRMS (ESI) m/z: calcd for C21H18ClN3O3
(M+H+) 396.1115 found 396.1097.
5.2.2.5. 3-(3,4-Dichlorophenyl)-6-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (8e).
Yield, 81%; mp:140.2–140.5°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.71 (s, 3H, CH3O), 3.87 (s, 6H,
2×CH3O), 7.03 (s, 2H, Ph-H), 7.85 (d, J = 9.6 Hz, 1H, Pyridine-H), 7.89 (d, J = 8.4 Hz, 1H, Ph-H),
7.98 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.02 (d, J = 8.8 Hz, 1H, Ph-H), 8.28 (s, 1H, Ph-H), 8.67 (s, 1H,
Pyridine-H). 13C NMR (100 MHz, CDCl3) δ: 153.88, 150.10, 144.87, 138.84, 134.66, 133.88,
131.76, 131.45, 129.93, 129.85, 128.95, 127.07, 126.53, 118.75, 116.66, 104.71, 60.96, 56.38.
HRMS (ESI) m/z: calcd for C21H17Cl2N3O3 (M+H+) 430.0725 found 430.0710.
565
570
5.2.2.6. 2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)phenol (8f).
1
Yield, 59%; mp: 253.0–253.7°C; H NMR (400 MHz, DMSO-d6) δ: 3.71 (s, 3H, CH3O), 3.87 (s,
6H, 2×CH3O), 6.99 (s, 2H, Ph-H), 7.15 (d, J = 8.4 Hz, 1H, Ph-H), 7.37 (s, 1H, Ph-H), 7.42 (d, J =
8.4 Hz, 1H, Ph-H), 7.76 (d, J = 9.6 Hz, 1H, Pyridine-H), 7.91 (d, J = 9.6 Hz, 1H, Pyridine-H), 8.55
13
575
(s, 1H, Pyridine-H), 9.50 (s, 1H, OH). C NMR (100 MHz, DMSO-d6) δ: 153.76, 149.64, 149.53,
147.49, 146.84, 138.16, 132.28, 129.13, 127.97, 120.68, 119.69, 119.42, 115.88, 115.49, 113.04,
105.37, 60.49, 56.61, 56.08. HRMS (ESI) m/z: calcd for C22H21N3O5 (M+H+) 409.1559 found
409.1588.
5.2.2.7. 3-(4-Fluorophenyl)-6-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridine (8g). Yield,
1
580
585
75%; mp: 175.5–177.4°C; H NMR (400 MHz, DMSO-d6) δ: 3.71 (s, 3H, CH3O), 3.87 (s, 3H,
2×CH3O), 7.01 (s, 2H, Ph-H), 7.47 (t, J1 = 8.8 Hz, J2 = 17.6 Hz, 2H, Ph-H), 7.81 (d, J = 9.6 Hz, 1H,
Pyridine-H), 7.95 (d, J = 9.6 Hz, 1H, Pyridine-H), 7.89 (dd, J1 = 5.6 Hz, J2 = 8.4 Hz, 2H, Ph-H),
8.59 (s, 1H, Pyridine-H). 13C NMR (100 MHz, DMSO-d6) δ: 163.38 (d, J = 246.2 Hz, 1C), 153.74,
149.77, 146.13, 138.23, 132.11, 131.14, 131.09 (d, J = 8.7 Hz, 2C), 129.44, 128.18, 123.61 (d, J =
3.1 Hz, 1C), 120.78, 116.77 (d, J = 21.7 Hz, 2C), 115.81, 105.48, 60.49, 56.62. HRMS (ESI)
m/z: calcd for C21H18FN3O3 (M+H+) 380.1410 found 380.1397.
5.2.2.8. 2-Methoxy-5-[6-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl]-phenylamine
(8h). Yield, 70%; mp: 188.2–190.9°C; 1H NMR (400 MHz, CDCl3) δ: 3.91 (s, 3H, CH3O), 3.94 (s,
6H, 2×CH3O), 3.96 (s, 3H, CH3O), 4.06 (s,2H, NH2), 6.70 (s, 2H, Ph-H), 6.97 (d, J = 8.4 Hz, 1H,
Ph-H), 7.18 (d, J = 8.4 Hz, 1H, Ph-H), 7.22 (s, 1H, Ph-H), 7.48 (d, J = 9.6 Hz, 1H, Pyridine-H),
7.85 (d, J = 9.2 Hz, 1H, Pyridine-H), 8.34 (s, 1H, Pyridine-H). 13C NMR (100 MHz, CDCl3) δ:
153.77, 149.63, 148.66, 147.32, 138.56, 137.24, 132.35, 128.71, 128.27, 119.53, 119.16, 118.11,
116.38, 114.45, 110.49, 104.71, 60.94, 56.39, 55.59. HRMS (ESI) m/z: calcd for C22H22N4O4
590
23

(M+H+) 407.1719 found 407.1704.
595
5.3 Biological evaluation
5.3.1 Antiproliferative activity
The antitumor activities of tested samples were measured against human epithelial cervical
cancer (HeLa) cell lines by the typical MTT assay in vitro. The cancer cell lines were cultured in
RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in a humidified 5% CO2
atmosphere at 37 °C. All of tested samples were prepared by dissolving in dimethyl sulfoxide
(DMSO) at 50 mM concentration and diluted with the medium into a series of concentrations.
Exponentially growing cells were plated in 96-well plates (2×103 cells/well) and incubated for
attachment at 37 °C for 24 h. The culture medium was then changed, and cells grew in medium
with the tested compounds. DMSO (0.1%) and CA-4 were used as negative and positive control,
respectively. Cells were incubated at 37 °C for 48 h. After the treatment period, 20 μL of MTT
solution (5 mg/mL) was added to each well, and the plates were incubated for 4 h at 37°C. The
medium was then aspirated and formazan crystals were dissolved in DMSO (200μL) for about 10
mins. The absorbance at 570 nm (Abs) of the suspension was measured by a microplate reader
(Bio-Rad laboratories, USA). Percentage of relative viability was calculated using the following
equation: % inhibition = (Abscontrol－Abscompound)/Abscontrol×100%. The IC50 values of the tested
samples were measured by treating cells with drugs of various concentrations and analyzed by use
of the prism statistical package.
600
605
610
5.3.2 Cell cycle analysis
Flow cytometric analysis was performed to investigate the effect of 7i on cell cycle phase
distribution of human cervical cancer cell lines (HeLa). The cells were plated in 6-well plate and
incubated overnight at 37 °C. After grew to about 70% confluence in 60 mm dish, cells were
incubated with different concentrations (12, 24, and 48 nM) or vehicle for 24 h. Control and treated
cells were harvested, washed with PBS, and fixed in 75% ice-cold ethanol at 4 °C overnight.
Subsequently, the cells were washed with PBS, incubated with 50 μg/mL of RNase at 37 °C for 30
min, and stained with 50 μg/mL of propidium iodide, and finally subjected to flow cytometry
(Beckman Coulter).
615
620
5.3.3 Apoptosis analysis
24

HeLa cells were seed into 6-well plates (2×105 cells/well), incubated with compound 7i at
indicated concentrations or vehicle for 24h. Cells were harvested and incubated with 5 μL of
Annexin-V/FITC in binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 at pH 7.4)
and PI solution (10 μL) for 15 min. PI solution (10 μL) was then added to the medium for another
10 min incubation. Almost 10000 events were collected for each sample and analyzed with flow
cytometry. Finally, the percentag of apoptotic cells was examined using FlowJo 7.6 software.
5.3.4 Immunocytochemistry
625
630
635
640
Confocal microscopy was performed to explore the effects of the most potent compound 7i on
HeLa cytoskeleton. HeLa cells (9 × 103 cells/well) were plated on glass coverslips in 24-well plates
and allowed to attach overnight. After treatment with 1-fold IC50 concentration of 7i, CA-4 or 0.1%
DMSO (control) for 6 h, cells were rinsed twice with PBS, fixed with 4% paraformaldehyde, and
then permeabilized with 0.1% Triton X-100. After blocking with 5% BSA for 60 min, the
coverslips were incubated at 4°C overnight with primary antibodies (α-tubulin mouse monoclonal
antibody and β-tubulin rabbit monoclonal antibody). After washing, the cells were incubated with
the following secondary antibodies: Alexa Fluor® 555 conjugate anti-rabbit IgG and FITC
AffiniPure goat anti-mouse IgG. Nuclei were visualized with DAPI, and then, images were obtained
by a laser scanning confocal microscope (Carl Zeiss LSM 880).
5.3.5 In vitro tubulin polymerization assay
The tubulin polymerization activity assay in vitro was carried out according to our previously
reported method [16].
5.3.6 Molecular modeling
Molecular modelling studies were carried out with the SURFLEX module of SYBYL 7.3
package [30], and the crystal strucure of tubulin complexed with Combretastatin A-4 (PDB: 5lyj)
was down loaded from the PDB data bank (http://www.rcsb.org/pdb) [31]. Structural energy
minimization process was performed using the Tripos force field with a distance–dependent
dielectric and powell gradient algorithm with a convergence criterion of 0.005 kcal/mol Å.
Additionally, partial atomic charges were calculated using Gasteiger–Hückel method and all the
other parameters were assigned default values in the docking process. Finally, the best 20 ranked
conformations with different scores were obtained and the best ranking pose was visualized with
PyMOL [32].
645
650
25

Acknowledgments
This present work was financially supported by the Natural Science Foundation of Guangdong
655
Province, China (Grant No. 2018B030311067), and the Science and Technology Program of
Guangzhou City, China (Grant No. 201707010198).
Appendix A. Supplementary data
Supplementary
data
related
to
this
article
can
be
found
at
http://dx.doi.org/10.1016/j.ejmech.2020.xx.xxx.
660
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