Design, synthesis and bioevaluation of antitubulin agents carrying diaryl-5,5-fused-heterocycle scaffold

Article abstract of DOI:10.1016/j.ejmech.2017.05.065

A series of 3,6-diaryl-1H-pyrazolo[5,1-c][1,2,4]triazoles (I) and 3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles (II) as antitubulin agents were designed, synthesized and bioevaluated. Compounds (II) 4a, 4d, 4f, 4j, 4l and 4n showed potent antiproli

Full text of DOI:10.1016/j.ejmech.2017.05.065

Accepted Manuscript
Design, synthesis and bioevaluation of antitubulin agents carrying diaryl-5,5-fused-
heterocycle scaffold
Qile Xu, Maolin Sun, Zhaoshi Bai, Yueting Wang, Yue Wu, Haiqiu Tian, Daiying Zuo,
Qi Guan, Kai Bao, Yingliang Wu, Weige Zhang
PII:
S0223-5234(17)30436-1
10.1016/j.ejmech.2017.05.065
EJMECH 9495
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 April 2017
Revised Date: 16 May 2017
Accepted Date: 31 May 2017
Please cite this article as: Q. Xu, M. Sun, Z. Bai, Y. Wang, Y. Wu, H. Tian, D. Zuo, Q. Guan,
K. Bao, Y. Wu, W. Zhang, Design, synthesis and bioevaluation of antitubulin agents carrying
diaryl-5,5-fused-heterocycle scaffold, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.05.065.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and bioevaluation of antitubulin agents carrying
diaryl-5,5-fused-heterocycle scaffold
Qile Xu,a Maolin Sun,a Zhaoshi Bai,b Yueting Wang,a Yue Wu,a Haiqiu Tian,a Daiying Zuo,b Qi
Guan,a Kai Bao,c* Yingliang Wub* and Weige Zhanga*
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education,
Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016,
China.
b
Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe
District, Shenyang 110016, China.
c
Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe
District, Shenyang 110016, China
* Corresponding authors:
E-mail addresses: zhangweige2000@sina.com (W. Zhang), yingliang_1016@163.com (Y. Wu),
baokai@syphu.edu.cn (K. Bao).

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Abstract
A
series
of
3,6-diaryl-1H-pyrazolo[5,1-c][1,2,4]triazoles
(I)
and
3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles (II) as antitubulin agents were
designed, synthesized and bioevaluated. Compounds (II) 4a, 4d, 4f, 4j, 4l and 4n
showed potent antiproliferative activity at sub-micromolar or nanomolar
concentrations against SGC-7901, A549 and HT-1080 cell lines, indicating that the
bioisosteric replacement of the carbonyl group and B-ring of SMART and ABI with a
5,5-fused-heterocycle scaffold successfully maintained potent antiproliferative activity.
Compound 4f exhibited the most excellent antiproliferative activity against the three
cancer cell lines (IC50 = 0.022-0.029 µM). Consistent with its potent antiproliferative
activity, 4f also displayed excellent antitubulin activity (IC50 = 0.77 µM). Furthermore,
compound 4f could dramatically affect cell morphology and microtubule networking,
while cell cycle studies demonstrated that 4f significantly induced SGC-7901cells
arrest in G2/M phase. In addition, molecular docking studies supported the biological
assay data and suggested that 4f may be a potential antitubulin agent.
Keywords: Antiproliferative activity; Tubulin; Immunofluorescence; Cell cycle arrest;
Molecular modeling

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1. Introduction
Microtubule is one of the most attractive targets for anticancer drugs discovery
[1]. Paclitaxel, vinblastine and colchicine represent the well-known microtubule
targeting agents by binding three different binding sites (taxane, vinca and colchicine
sites) of tubulin [1-3]. These agents interfere with the dynamic equilibrium of
microtubules by either inhibition of tubulin polymerization or promotion the
polymerization of tubulin, and both effects lead to the cell cycle arrest and limit cell
proliferation thus resulting in cell death [1,4]. Given the tremendously successful
clinical use of taxanes and vinca alkaloids, the colchicine binding site inhibitors
(CBSIs) have aroused great interest among medicinal chemists [5-10].
Several outstanding CBSIs, such as 4-substituted methoxybenzoyl-aryl-thiazole
(SMART 1, Figure 1) and 2-aryl-4-benzoyl-imidazole (ABI 2, Figure 1), exhibit
potent antiproliferative activity, which contain three aromatic rings (A, B and C) and a
carbonyl group between ring-A and ring-B [11,12]. Ring-closing is one strategy of
bioisosterism in medicinal chemistry and represents a widely used method for rational
design of new drugs [13,14]. Indeed, by applying this strategy, medicinal chemists
successfully discovered some bioactive molecules including CBSIs [15,16]. Thus, we
attempt to use the ring-closing strategy to design a series of new CBSIs carrying fused
heterocyclic scaffold, the bioisosteres of the carbonyl group and B-ring (Figure 1).
The target compounds were classified into the following two groups according to the
5,5-fused-heterocycle scaffold: (I) pyrazolo[5,1-c][1,2,4]triazole (3a-e), (II)
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (4a-p).

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Figure 1. Design strategy and structures of new tubulin inhibitors
In view of the structural characteristics of SMART and ABI, we suspected that a
certain angle∠ABC and distance between A and C (A, B and C represent the
geometric center of ring-A, ring-B and ring-C, respectively) may necessary for the
potent activity. In order to verify our speculation, calculation of the angle ABC and
the distance between A and C of the skeletal structure of the target compounds,
SMART and ABI, were performed with the Gaussian 09 software package. The angle
and distance of compounds I were slightly larger than those of SMART and ABI,
while the angle and distance of compounds II were pretty close to that of ABI (Figure
2). Herein, we describe the synthesis, bioevaluation and the preliminary
structure-activity
relationships
of
the
3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles
and

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3,6-diaryl-1H-pyrazolo[5,1-c][1,2,4]triazoles as new antitubulin agents.
Figure 2. Caculation of∠ABC and distance between A and C of the SMART, ABI and the target
compounds..
2. Results and discussion
2.1. Synthesis
Figure 3. Reagents and conditions: (a) CS2, KOH, MeOH, 25 °C; (b) N2H4·H2O, H2O, MW,
100 °C; (c) EtOH, MW, 80 °C; (d) (i) Ac2O, reflux, (ii) HCl, MeOH, reflux; (e) POCl3, reflux.

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The target compounds 3a-e and 4a-p were synthesized according to the synthetic
route outlined in Figure 3. The hydrazides 5 were reacted with carbon disulphide and
potassium hydroxide in methanol to afford corresponding dithiocarbazinates 6 and
then underwent ring closure with an excess of 80% hydrazine monohydrate to give the
key intermediate aryl triazoles 7 [17]. Then the aryl triazoles 7 were treated with
α-bromoacetophenones to give the compounds 8 in ethanol under microwave
irradiation (150 W, 80 °C). Compounds 8 were converted into the corresponding
target compounds 3a-e via desulfurization ring contraction [18]. On the other hand, a
mixture of compounds 7 and substituted benzoic acids in phosphorus oxychloride was
heated to reflux to get target compounds 4a-p.
2.2. In vitro antiproliferative activity
The antiproliferative activities of target compounds 3a-e and 4a-p were evaluated
against three human cancer cell lines (gastric adenocarcinoma SGC-7901 cells, lung
adenocarcinoma A549 cells and fibrosarcoma HT-1080 cells) using MTT assay with
SMART (1) and ABI (2) as reference. SMART (1) and ABI (2) gave IC50 values of
0.019-0.029, 0.15-0.98 µM, respectively. As depicted in Table 1, six target compounds
(4a, 4d, 4f, 4j, 4l and 4n) showed potent antiproliferative activity at sub-micromolar
or nanomolar concentrations against the three different cancer cells. The angle and
distance of 3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles (118.4º, 779 pm) were
more
close
to
that
of
ABI
(117.4º,
766
pm)
than
3,6-diaryl-1H-pyrazolo[5,1-c][1,2,4]triazoles (123.5º, 787 pm) and the results of in
vitro antiproliferative activity showed that compounds II were more active than
compounds I. Compounds 3a-e with a pyrazolo[5,1-c][1,2,4]triazole moiety displayed
only
modest
antiproliferative
activity.
Replacement
of
the
pyrazolo[5,1-c][1,2,4]triazole scaffold with the [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
tended to greatly enhance the activity (3d vs 4e, 3e vs 4f). When ring-A was a
2,3,4-trimethoxyphenyl moiety, compound 4d with a 3-amino-4-methoxyphenyl
group as ring-C exhibited the most active antiproliferative activity (0.062-0.082 µM).
However, the 3-nitro-4-methoxy-substituted 4c displayed a dramatic decrease in its
antiproliferative activity. Compounds 4e, 4i, 4k, 4m and 4o with a nitro group at

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ring-C displayed similar antiproliferative activity with 4c, suggesting that the
electron-withdrawing nitro group is disfavored. Using 3-amino-4-methoxy
substituents at ring-C, we examined the cytotoxic effect of compounds 4f, 4j, 4l, 4n
and 4p by replacement of 2,3,4-trimethoxy substituents with other substituents
(3,4,5-trimethoxy, 3,4-methylenedioxy, 3,4-dimethoxy, 3-methoxy, 4-methoxy) at
ring-A. These compounds (4f, 4j, 4l and 4n) also showed potent antiproliferative
activity
and
broaden
the
SAR
by
modifications
on
ring-C.
3,4,5-Trimethoxy-substituted compound 4f exhibited the most excellent
antiproliferative activity against the three cancer cell lines (IC50 = 0.022-0.029 µM),
which was comparable to SMART (IC50 = 0.019-0.029 µM) but more effective than
ABI (IC50 = 0.15-0.98 µM).
Table 1. Antiproliferative activity of 3a-e, 4a-p, SMART and ABI.
Compd
Antiproliferative activity (IC50± SD, µM) a
A549
SGC-7901
33.5 ± 1.9
HT-1080
51.3 ± 2.6
29.3 ± 1.2
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
4f
26.8 ± 1.1
26.4 ± 2.0
26.5 ± 2.2
6.12 ± 0.13
48.3 ± 1.5
1.21 ± 0.09
6.81 ± 0.21
35.6 ± 3.8
58.2 ± 2.9
13.8 ± 1.6
17.4 ± 2.0
17.8 ± 1.5
0.186 ± 0.012
3.75 ± 0.02
32.5 ± 1.2
0.803 ± 0.021
2.29 ± 0.09
0.089 ± 0.008
1.88 ± 0.05
35.1 ± 1.5
33.8 ± 1.4
0.062 ± 0.007
23.2 ± 1.3
0.082 ± 0.008
26.5 ± 2.1
0.073 ± 0.016
30.2 ± 1.2
0.022 ± 0.006
24.0 ± 1.4
0.029 ± 0.011
27.7 ± 0.9
0.027 ± 0.013
26.8 ± 1.3
4g
4h
4i
25.6 ± 2.1
29.7 ± 1.7
24.5 ± 0.8
37.8 ± 3.1
32.8 ± 2.5
30.3 ± 3.0
4j
0.057 ± 0.011
37.7 ± 1.6
0.272 ± 0.007
30.4 ± 1.7
0.327 ± 0.018
32.8 ± 2.1
4k
4l
0.083 ± 0.009
34.7 ± 2.4
0.522 ± 0.021
29.5 ± 0.5
0.339 ± 0.032
33.0 ± 0.8
4m

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4n
4o
0.059 ± 0.004
0.12 ± 0.06
26.9 ± 1.9
0.088 ± 0.017
27.3 ± 1.4
22.1 ± 0.7
14.2 ± 1.1
14.9 ± 0.9
16.4 ± 1.5
4p
SMART (1) b
ABI (2) b
0.019 ± 0.008
0.81 ± 0.08
0.029 ± 0.009
0.98 ± 0.11
0.028 ± 0.011
0.15 ± 0.05
a IC50: the half maximal inhibitory concentration. b Used as positive controls.
2.3. Tubulin polymerization
To explore the relationship between the antiproliferative activities of these compounds
and tubulin, compound 4f was evaluated for its effects on tubulin polymerization in
comparison to the positive control SMART (1) and the negative control paclitaxel. As
shown in Figure 4, 4f significantly inhibited tubulin polymerization (IC50: 0.77 µM)
which was different from the negative control paclitaxel. Furthermore, compound 4f
inhibited tubulin polymerization in a dose-dependent manner. These results suggested
that the molecular target of this series of compounds was most likely tubulin.
Figure 4. Effects of 4f on tubulin polymerization. Tubulin had been pre-incubated for 1 min with
4f (0.33 µM, 1.1 µM, 3.3 µM and 10.0 µM), SMART (1.0 µM), paclitaxel (5.0 µM) or vehicle
DMSO (control) before GTP was added to start the tubulin polymerization reactions.
Polymerization of microtubule was monitored kinetically by using a spectrophotometer.
2.4. Analysis of immunofluorescence staining
To confirm the direct effects of compound 4f on tubulin, we further investigated
alterations in the microtubule network induced by 4f and SMART (1) in cultured

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SGC-7901 cells using immunofluorescence techniques. As given in Figure 5, the
microtubule network without drug treatment displays normal arrangement and
organization in control cells. Whereas SGC-7901 cells were treated with compound 4f
or SMART (at their 2-fold IC50 concentrations, respectively) and the results
demonstrated that microtubules became short and wrapped around the nucleus in
comparison with the control. These results indicated that 4f similar to SMART could
destabilize microtubules.
Figure 5. Effects of 4f on tubulin assembly in SGC-7901 cells by immunofluorescence assay.
SGC-7901 cells were treated with DMSO (control), 4f (0.044 µM) or SMART (0.038 µM) for 48
h.
2.5. Cell cycle study
Because most tubulin inhibitors induce cell cycle arrest in the G2/M phase [19], we
next investigated the time-effects on cell cycle distribution of SGC-7901cells treated
with 4f (2-fold IC50) by flow cytometry. As given in Figure 6, as time went on,
compound 4f was found to significantly increase the percentage of cells in the sub-G1
and G2/M phases, with an accompanying decrease in cells in the G0/G1 and S phases.
The biggest percentage of G2/M phase appeared after the cells were treated with
compound 4f for 24 h. At the same time, polyploidy was induced increasingly by 4f.
Thus, cell cycle studies demonstrated that 4f significantly induced SGC-7901cells

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arrest in G2/M phase in a time-dependent manner (Figure S1).
Figure 6. The statistical graph of cell cycle distribution. Cell cycle distribution of SGC-7901 cells
treated for 0, 6, 12, 24, 48, 72 h with 4f (0.044 µM).
2.6. Molecular modeling study
To further understand the binding interactions, molecular docking studies of 4f was
carried out with tubulin crystal structure (PDB: 1SA0) using the CDOCKER program
of Discovery Studio 3.0 software. As given in Figure 7, the 3,4,5-trimethoxybenzoyl
ring-A of 4f was located deeply into the β-subunit of tubulin. The ring-B and ring-C
of 4f extends toward the α/β-tubulin interface and several important amino acids of
tubulin formed hydrogen bond interactions with 4f. The residue of β-Cys241 forms a
hydrogen bond with the oxygen of the methoxy group (ring-A). Furthermore, sulfur
atom of ring-B forms a hydrogen bond with the residue β-Asn101. These molecular
docking results further supported the above biological assay data and suggested that
4f may be a potential tubulin inhibitor.

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Figure 7. The binding mode of compound 4f in the colchicine binding site of tubulin (left).
Overlay of 4f in the binding site (right).
3. Conclusions
A series of new tubulin inhibitors with a 5,5-fused-heterocycle scaffold were designed
and synthesized as the analogs of SMART and ABI. We proposed the two parameters
of angle∠ABC and distance between A and C to examine the relationship between
the designed compounds and the lead compounds. The angle and distance of
3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles were more close to that of ABI
(117.4º, 766 pm) than 3,6-diaryl-1H-pyrazolo[5,1-c][1,2,4]triazoles, and SAR studies
indicated that compounds with a [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole scaffold were
more potent than those with the pyrazolo[5,1-c][1,2,4]triazole scaffold. Six target
compounds (4a, 4d, 4f, 4j, 4l and 4n) showed potent antiproliferative activity at
sub-micromolar or nanomolar concentrations against the three different cancer cells,
indicating that the bioisosteric replacement of the carbonyl group and ring-B of
SMART and ABI with a 5,5-fused-heterocycle scaffold successfully maintained
potent antiproliferative activity. Compound 4f exhibited the most excellent
antiproliferative activity against the three cancer cell lines, which was comparable to
SMART but more effective than ABI. Consistent with its potent antiproliferative
activity, 4f also displayed excellent antitubulin activity with an IC50 value of 0.77 µM.
Furthermore, compound 4f could significantly affect cell morphology and
microtubule networking and cell cycle studies demonstrated that 4f significantly
induced SGC-7901cells arrest in G2/M phase. Molecular docking studies suggested
that 4f may be a potential tubulin inhibitor.
4. Experimental Section
4.1. Chemistry
All reagents and solvents were obtained from commercially available sources and
were used without purification. The microwave reactions were performed on a

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discover-sp single mode microwave reactor from CEM Corporation. Reactions were
monitored by TLC with silica gel plates under UV light (λ = 254 nm). The melting
points (uncorrected) were measured on a hot-stage microscope (X-4, Beijing Taike
Ltd.). 1H NMR and 13C NMR spectra were obtained on a Bruker AVANCE 300 or 600
in CDCl3 or DMSO-d6 (TMS as internal standard). Mass spectra (MS) were measured
on an Agilent 1100-sl mass spectrometer with an electrospray ionization source from
Agilent Co. Ltd.
4.1.2. General synthetic procedure for the key intermediates 7.
The substituted hydrazides 5 (10 mmol) were stirred with potassium hydroxide (15
mmol) in absolute methanol. Then CS2 (12 mmol) was slowly added and the mixture
was stirred at 25 °C for 1-6 h. After the reaction completed, the solid was precipitated
and dried to afford compounds 6. The compounds 6 was refluxed with excess 80%
hydrazine hydrate in water under microwave irradiation at 250 W, 100 °C, for 60 min.
After completion of the reaction, the mixture was acidified with concentrated HCl and
the precipitate solid was filtered, washed with water, dried and recrystallized from
aqueous methanol to obtain the intermediates 7.
4.1.3. General synthetic procedures for 3a-e.
To a mixture of each of intermediates 7 (10 mmol) in absolute ethylalcohol (15 mL),
was added the appropriate α-haloketones (10 mmol). The mixture was heated at 80 °C
under microwave irradiation until the reaction was complete. Water was added and the
precipitate 8 formed was collected. Then compounds 8 were added slowly to acetic
anhydride and the mixture was refluxed for 4 h. Then the mixture was poured into
cold water. Precipitated solid was filtered, washed with water and dried. Finally, the
precipitated solid was added to a mixture of methanol (30 mL) and HCl (4 mL, 10 M)
and the mixture was refluxed for 6–24 h. Finally, the solvent was removed in vacuo
and the remaining materials were washed with sodium bicarbonate solution and the
remaining materials were recrystallized from the proper solvent.
4.1.4. General synthetic procedures for 4a-p.
A mixture of intermediates 7 (4 mmol) and substituted benzoic acid (4 mmol) in
phosphorus oxychloride (10 mL) was heated under reflux for 2 h [20]. After the

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reaction completed, the solution was poured into 200g of ice. Precipitate was filtered,
washed with water and recrystallized from the proper solvent.
4.1.5.
3-(3,4,5-Trimethoxyphenyl)-6-(4-methylphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazole
(3a).
o
1
Brown solid; yield: 81%; M.p.: 119-122 C; H-NMR (600 MHz, CDCl3): δ 9.73 (s,
1H), 7.88 (s, 2H), 7.82 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 6.02 (s, 1H), 4.01
(s, 6H), 3.94 (s, 3H), 2.40 (s, 3H); 13C-NMR (150 MHz, CDCl3): δ 160.1, 153.4 (2C),
148.4, 139.5, 139.4, 138.4, 130.9, 129.3 (2C), 126.0 (2C), 121.1, 103.8 (2C), 75.0,
60.9, 56.2 (2C), 21.3; ESI-MS: m/z = 365.3 [M+H]+, 387.3 [M+Na]+.
4.1.6.
3-(3,4,5-Trimethoxyphenyl)-6-(4-methoxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]triazole
(3b).
Red solid; yield: 84%; M.p.: 146-147 oC; 1H-NMR (600 MHz, CDCl3): δ 9.89 (s, 1H),
7.87 (s, 2H), 7.86 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 5.99 (s, 1H), 4.01 (s,
13
6H), 3.93 (s, 3H), 3.86 (s, 3H); C-NMR (150 MHz, CDCl3): δ 159.9, 159.8, 153.4
(2C), 148.4, 139.5, 139.3, 127.3 (2C), 126.5, 121.2, 114.0 (2C), 103.8 (2C), 74.6, 60.9,
56.2 (2C), 55.3; ESI-MS: m/z = 381.3 [M+H]+.
4.1.7.
3-(3,4,5-Trimethoxyphenyl)-6-(4-chlorophenyl)-1H-pyrazolo[5,1-c][1,2,4]triazole
(3c).
Red solid; yield: 82%; M.p.: 150-151 oC; 1H-NMR (600 MHz, CDCl3): δ 9.65 (s, 1H),
7.86 (s, 2H), 7.85 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 6.05 (s, 1H), 4.02 (s,
13
6H), 3.94 (s, 3H); C-NMR (150 MHz, CDCl3): δ 158.7, 153.4 (2C), 148.4, 139.7,
139.4, 134.3, 132.3, 128.8 (2C), 127.3 (2C), 120.9, 103.8 (2C), 75.3, 60.9, 56.2 (2C);
ESI-MS: m/z = 385.0 [M+H]+, 407.1 [M+Na]+.
4.1.8.
3-(3,4,5-Trimethoxyphenyl)-6-(3-nitro-4-methoxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]tr
iazole (3d).
Yellow solid; yield: 76%; M.p.: 149-151 oC; 1H-NMR (400 MHz, DMSO-d6): δ 13.24

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(s, 1H), 8.41 (d, J = 2.0 Hz, 1H), 8.25 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H), 7.80 (s, 2H),
7.44 (d, J = 8.8 Hz, 1H), 6.50 (s, 1H), 3.97 (s, 3H), 3.93 (s, 6H), 3.76 (s, 3H);
13C-NMR (100 MHz, DMSO-d6): δ 156.7, 153.7 (2C), 152.3, 149.2, 139.9, 139.4,
137.8, 131.7, 126.9, 122.3, 121.5, 115.2, 103.6 (2C), 75.7, 60.7, 57.3, 56.4 (2C);
ESI-MS: m/z = 426.1 [M+H]+, 448.1 [M+Na]+.
4.1.9.
3-(3,4,5-Trimethoxyphenyl)-6-(3-amino-4-methoxyphenyl)-1H-pyrazolo[5,1-c][1,2,4]t
riazole (3e).
o
1
Brown solid; yield: 69%; M.p.: 135-137 C; H-NMR (600 MHz, CDCl3): δ 9.86 (s,
1H), 7.85 (s, 2H), 7.31 (s, 2H), 6.84 (d, J = 8.8 Hz, 1H), 5.95 (s, 1H), 4.00 (s, 6H),
13
3.92 (s, 3H), 3.89 (s, 3H); C-NMR (150 MHz, CDCl3): δ 160.2, 153.4 (2C), 148.3,
147.9, 139.5, 139.4, 135.9, 126.8, 121.2, 116.8, 112.9, 110.3, 103.8 (2C), 74.8, 60.9,
56.2 (2C), 55.5; ESI-MS: m/z = 396.4 [M+H]+.
4.1.10.
3-(2,3,4-Trimethoxyphenyl)-6-(4-methylphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazo
le (4a).
White solid; yield: 72%; M.p.: 100-103 oC; 1H-NMR (600 MHz, CDCl3): δ 7.75 (d, J
= 8.2 Hz, 2H), 7.52 (d, J = 8.6 Hz, 1H), 7.30 (d, J = 8.2 Hz, 2H), 6.82 (d, J = 8.6 Hz,
1H), 4.01 (s, 3H), 3.94 (s, 3H), 3.91 (s, 3H), 2.42 (s, 3H); 13C-NMR (150 MHz,
CDCl3): δ 166.0, 156.0, 153.4, 152.5, 145.3, 143.4, 142.3, 130.0 (2C), 126.9 (2C),
126.8, 125.6, 112.5, 107.2, 61.6, 60.8, 56.1, 21.5; ESI-MS: m/z = 383.1 [M+H]+.
4.1.11.
3-(2,3,4-Trimethoxyphenyl)-6-(4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadia
zole (4b).
Brown solid; yield: 69%; M.p.: 112-114 oC; 1H-NMR (600 MHz, CDCl3): δ 7.69 (d, J
= 8.7 Hz, 2H), 7.44 (d, J = 8.6 Hz, 1H), 6.88 (d, J = 8.7 Hz, 2H), 6.74 (d, J = 8.6 Hz,
1H), 3.93 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H), 3.76 (s, 3H); 13C-NMR (150 MHz,
CDCl3): δ 165.4, 162.9, 155.8, 153.3, 152.4, 145.1, 142.2, 128.6 (2C), 125.4, 121.7,
114.6 (2C), 112.5, 107.2, 61.5, 60.7, 56.0, 55.4; ESI-MS: m/z = 399.1 [M+H]+.
4.1.12.

ACCEPTED MANUSCRIPT
3-(2,3,4-Trimethoxyphenyl)-6-(3-nitro-4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazole (4c).
White solid; yield: 75%; M.p.: 195-197 oC; 1H-NMR (600 MHz, DMSO-d6): δ 8.44 (d,
J = 2.4 Hz, 1H), 8.22 (dd, J = 8.9 Hz, J = 2.4 Hz, 1H), 7.57 (d, J = 8.9 Hz, 1H), 7.45
(d, J = 8.6 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H ), 4.03 (s, 3H), 3.90 (s, 6H), 3.82 (s, 3H);
13C-NMR (150 MHz, DMSO-d6): δ 164.4, 156.3, 155.1, 153.6, 152.2, 144.7, 142.2,
140.0, 133.1, 125.9, 124.0, 121.6, 115.9, 112.1, 108.5, 61.9, 60.9, 57.8, 56.5; ESI-MS:
m/z = 444.1 [M+H]+, 466.1 [M+Na]+.
4.1.13.
3-(2,3,4-Trimethoxyphenyl)-6-(3-amino-4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,
4]thiadiazole (4d).
o
1
Yellow solid; yield: 66%; M.p.: 139-142 C; H-NMR (600 MHz, DMSO-d6): δ 7.40
(d, J = 8.6 Hz, 1H), 7.23 (d, J = 2.3 Hz, 1H), 7.11 (dd, J = 8.4 Hz, J = 2.3 Hz, 1H),
7.01 (d, J = 8.6 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H ), 5.20 (s, 2H), 3.90 (s, 3H), 3.90 (s,
3H), 3.85 (s, 3H), 3.83 (s, 3H); 13C-NMR (150 MHz, DMSO-d6): δ 166.8, 156.1,
153.1, 152.2, 150.3, 144.7, 142.2, 139.2, 125.9, 121.9, 116.3, 112.7, 111.0, 110.7,
108.4, 61.9, 60.9, 56.5, 56.0; ESI-MS: m/z = 414.3 [M+H]+, 436.3 [M+Na]+.
4.1.14.
3-(3,4,5-Trimethoxyphenyl)-6-(3-nitro-4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazole (4e).
o
1
Yellow solid; yield: 65%; M.p.: 246-248 C; H-NMR (600 MHz, DMSO-d6): δ 8.49
(s, 1H), 8.26 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.55 (s, 2H), 4.04 (s, 3H),
3.90 (s, 6H), 3.76 (s, 3H); 13C-NMR (150 MHz, DMSO-d6): δ 165.0, 155.2, 153.7,
153.7 (2C), 139.9, 139.6, 133.2, 124.3, 121.5, 121.0, 116.1, 103.7 (2C), 103.0, 60.6,
57.8, 56.3 (2C); ESI-MS: m/z = 444.1 [M+H]+, 466.1 [M+Na]+.
4.1.15.
3-(3,4,5-Trimethoxyphenyl)-6-(3-amino-4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,
4]thiadiazole (4f).
White solid; yield: 61%; M.p.: 153-155 oC; 1H-NMR (600 MHz, DMSO-d6): δ 7.56 (s,
2H), 7.24 (s, 1H), 7.17 (d, J = 7.8 Hz, 1H), 6.97 (d, J = 8.2 Hz, 1H), 5.25 (s, 2H), 3.90

ACCEPTED MANUSCRIPT
13
(s, 6H), 3.86 (s, 3H), 3.76 (s, 3H); C-NMR (150 MHz, DMSO-d6): δ 167.6, 153.9,
153.6 (2C), 150.4, 145.5, 139.4, 139.2, 121.8, 121.2, 116.3, 111.0, 110.8, 103.6 (2C),
60.6, 56.4 (2C), 56.0 ; ESI-MS: m/z = 414.1 [M+H]+, 436.1 [M+Na]+.
4.1.16.
3-(3,4-Methylenedioxyphenyl)-6-(4-methylphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadi
azole (4g).
o
1
White solid; yield: 69%; M.p.: 162-165 C; H-NMR (600 MHz, CDCl3): δ 7.96 (dd,
J = 8.2 Hz, J = 1.4 Hz, 1H), 7.86 (d, J = 1.4 Hz, 1H), 7.80 (d, J = 8.1 Hz, 2H), 7.33 (d,
13
J = 8.1 Hz, 2H), 6.97 (d, J = 8.2 Hz, 1H), 6.06 (s, 2H), 2.45 (s, 3H); C-NMR (150
MHz, CDCl3): δ 166.7, 153.8, 149.3, 148.0, 146.4, 143.6, 130.1 (2C), 127.0 (2C),
126.6, 121.0, 119.6, 108.7, 106.7, 101.5, 21.6; ESI-MS: m/z = 337.1 [M+H]+, 359.0
[M+Na]+.
4.1.17.
3-(3,4-Methylenedioxyphenyl)-6-(4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thia
diazole (4h).
o
1
White solid; yield: 74%; M.p.: 145-147 C; H-NMR (600 MHz, CDCl3): δ 7.92 (dd,
J = 8.1 Hz, J = 1.3 Hz, 1H), 7.82-7.83 (m, 3H), 6.99 (d, J = 8.8 Hz, 2H), 6.94 (d, J =
8.1 Hz, 1H), 6.03 (s, 2H), 3.87 (s, 3H); 13C-NMR (150 MHz, CDCl3): δ 166.3, 163.1,
154.0, 149.3, 148.0, 146.3, 128.8 (2C), 121.7, 121.0, 119.6, 114.8 (2C), 108.6, 106.7,
101.5, 55.6; ESI-MS: m/z = 353.1 [M+H]+, 375.0 [M+Na]+.
4.1.18.
3-(3,4-Methylenedioxyphenyl)-6-(3-nitro-4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3
,4]thiadiazole (4i).
White solid; yield: 65%; M.p.: 135-137 oC; 1H-NMR (600 MHz, DMSO-d6): δ 8.56 (s,
1H), 8.29 (d, J = 8.7 Hz, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.74 (s, 1H), 7.59 (d, J = 8.9
13
Hz, 1H ), 7.16 (d, J = 8.1 Hz, 1H), 6.16 (s, 2H), 4.05 (s, 3H); C-NMR (150 MHz,
DMSO-d6): δ 164.9, 155.0, 150.8, 149.4, 148.3, 145.2, 140.1, 133.3, 124.0, 121.5,
121.1, 119.5, 115.8, 109.3, 106.3, 102.2, 56.4; ESI-MS: m/z = 398.3 [M+H]+, 420.3
[M+Na]+.
4.1.19.

ACCEPTED MANUSCRIPT
3-(3,4-Methylenedioxyphenyl)-6-(3-amino-4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,
3,4]thiadiazole (4j).
o
1
Yellow solid; yield: 56%; M.p.: 182-185 C; H-NMR (600 MHz, DMSO-d6): δ 7.85
(dd, J = 8.2 Hz, J = 1.7 Hz, 1H), 7.74 (d, J = 1.7 Hz, 1H), 7.31 (d, J = 2.3 Hz, 1H),
7.19 (dd, J = 8.3 Hz, J = 2.3 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H ), 6.99 (d, J = 8.3 Hz,
1H), 6.16 (s, 2H), 3.87 (s, 3H); 13C-NMR (150 MHz, DMSO-d6): δ 167.6, 153.8,
150.5, 149.3, 148.2, 145.6, 139.2, 121.8, 120.9, 119.8, 116.6, 111.0, 110.8, 109.3,
106.3, 102.1, 56.0; ESI-MS: m/z = 368.1 [M+H]+.
4.1.20.
3-(3,4-Dimethoxyphenyl)-6-(3-nitro-4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]th
iadiazole (4k).
o
1
Yellow solid; yield: 67%; M.p.: 136-139 C; H-NMR (600 MHz, DMSO-d6): δ 8.53
(d, J = 2.2 Hz, 1H), 8.28 (dd, J = 8.8 Hz, J = 2.2 Hz, 1H), 7.92 (dd, J = 8.5 Hz, J = 1.9
Hz, 1H), 7.78 (d, J = 1.9 Hz, 1H), 7.59 (d, J = 9.0 Hz, 1H ), 7.18 (d, J = 8.5 Hz, 1H),
4.05 (s, 3H), 3.89 (s, 3H), 3.85 (s, 3H); 13C-NMR (150 MHz, DMSO-d6): δ 164.7,
155.1, 154.0, 150.9, 149.3, 145.9, 140.0, 133.2, 124.0, 121.5, 119.4, 118.2, 115.9,
112.3, 109.2, 57.8, 56.0, 55.9; ESI-MS: m/z = 414.1 [M+H]+, 436.3 [M+Na]+.
4.1.21.
3-(3,4-Dimethoxyphenyl)-6-(3-amino-4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]t
hiadiazole (4l).
o
1
Pale yellow solid; yield: 59%; M.p.: 160-162 C; H-NMR (600 MHz, DMSO-d6): δ
7.88 (dd, J = 8.4 Hz, J = 1.9 Hz, 1H), 7.79 (d, J = 1.9 Hz, 1H), 7.28 (d, J = 2.3 Hz,
1H), 7.19 (dd, J = 8.2 Hz, J = 2.3 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H ), 6.98 (d, J = 8.2
13
Hz, 1H), 5.24 (s, 2H), 3.89 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H); C-NMR (150 MHz,
DMSO-d6): δ 167.4, 153.7, 150.8, 150.4, 149.3, 145.7, 139.2, 121.9, 119.3, 118.5,
116.4, 112.2, 111.0, 110.9, 109.3, 56.0, 56.0, 55.9; ESI-MS: m/z = 384.4 [M+H]+,
406.3 [M+Na]+.
4.1.22.
3-(3-Methoxyphenyl)-6-(3-nitro-4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadi
azole (4m).

ACCEPTED MANUSCRIPT
o
1
Pale yellow solid; yield: 76%; M.p.: 236-238 C; H-NMR (600 MHz, DMSO-d6): δ
8.50 (d, J = 2.2 Hz, 1H), 8.25 (dd, J = 8.8 Hz, J = 2.2 Hz, 1H), 7.88 (d, J = 7.7 Hz,
1H ), 7.77 (s, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.50-7.53 (m, 1H), 7.11 (dd, J = 8.2 Hz, J
13
= 2.2 Hz, 1H ), 4.04 (s, 3H), 3.85 (s, 3H); C-NMR (150 MHz, DMSO-d6): δ 165.1,
159.9, 155.1, 154.6, 145.7, 140.0, 133.3, 130.8, 126.9, 124.1, 121.5, 118.6, 116.5,
115.9, 111.4, 57.8, 56.4; ESI-MS: m/z = 384.1 [M+H]+, 406.3 [M+Na]+.
4.1.23.
3-(3-Methoxyphenyl)-6-(3-amino-4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thia
diazole (4n).
o
1
Brown solid; yield: 69%; M.p.: 169-170 C; H-NMR (600 MHz, DMSO-d6): δ 7.87
(d, J = 7.8 Hz, 1H), 7.80-7.81 (m, 1H), 7.51-7.54 (m, 1H ), 7.29 (d, J = 2.3 Hz, 1H),
7.17 (dd, J = 8.3 Hz, J = 2.3 Hz, 1H ), 7.13 (dd, J = 8.2 Hz, J = 2.5 Hz, 1H ), 6.97 (d,
J = 8.4 Hz, 1H), 5.25 (s, 2H), 3.87 (s, 3H), 3.86 (s, 3H); 13C-NMR (150 MHz,
DMSO-d6): δ 167.8, 159.9, 150.5, 145.6, 139.2, 130.7, 130.6, 127.2, 121.8, 118.5,
116.5, 116.2, 111.4, 111.0, 110.8, 56.0, 55.7; ESI-MS: m/z = 354.4 [M+H]+, 376.3
[M+Na]+.
4.1.24.
3-(4-Methoxyphenyl)-6-(3-nitro-4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadi
azole (4o).
Yellow solid; yield: 73%; M.p.: 226-229 oC; 1H-NMR (600 MHz, CDCl3): δ 8.44 (d, J
= 2.4 Hz, 1H), 8.30 (d, J = 8.9 Hz, 2H), 8.07 (dd, J = 8.9 Hz, J = 2.4 Hz, 1H), 7.27 (d,
13
J = 8.9 Hz, 1H ), 7.08 (d, J = 8.9 Hz, 2H), 4.08 (s, 3H), 3.90 (s, 3H); C-NMR (150
MHz, CDCl3): δ 163.6, 161.3, 155.7, 153.2, 146.7, 140.0, 132.4, 128.0 (2C), 124.3,
121.8, 117.9, 114.4 (2C), 114.3, 57.0, 53.3; ESI-MS: m/z = 384.1 [M+H]+, 406.1
[M+Na]+.
4.1.25.
3-(4-Methoxyphenyl)-6-(3-amino-4-methoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thia
diazole (4p).
o
1
Pale yellow solid; yield: 67%; M.p.: 169-171 C; H-NMR (600 MHz, DMSO-d6): δ
8.21 (d, J = 8.6 Hz, 2H), 7.30 (d, J = 1.9 Hz, 1H), 7.17 (dd, J = 8.4 Hz, J = 1.9 Hz,

ACCEPTED MANUSCRIPT
1H), 7.16 (d, J = 8.6 Hz, 2H ), 6.97 (d, J = 8.4 Hz, 1H), 5.23 (s, 2H), 3.87 (s, 3H),
13
3.85 (s, 3H); C-NMR (150 MHz, DMSO-d6): δ 167.4, 161.1, 153.6, 150.4, 145.7,
139.2, 127.9 (2C), 121.9, 118.5, 116.5, 114.9 (2C), 111.0, 110.9, 56.0, 55.7; ESI-MS:
m/z = 354.3 [M+H]+, 376.3 [M+Na]+.
4.2. Biology
4.2.1. MTT assay
The antiproliferative activities of target compounds 3a-e, 4a-p, ABI and SMART
were determined by a standard MTT assay, as previously described [21].
4.2.2. Tubulin polymerization assay
Tubulin polymerization experiment was followed a previously reported method [22].
4.2.3. Immunofluorenscence assay
Immunofluorenscence assay studies were performed following a previously reported
method [23].
4.2.4. Cell cycle analysis
Cell cycle analysis studies were performed according to procedures described
previously [23-25].
4.2.5. Molecular docking
Molecular docking studies were performed following a previously documented
method [24].
Acknowledgements
We gratefully acknowledge the National Natural Science Foundation of China
(81673293, 81502932, 81602969), the Natural Science Foundation of Liaoning
Province (201602687, 201610163L10) and State Key Laboratory of Natural
Medicines and Active Substance (No. GTZK201603) for their generous financial
support.
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A series of antitubulin agents with a 5,5-fused-heterocycle scaffold were designed.
Compound 4f exhibited potent antiproliferative and antitubulin activities.
Compound 4f induced cell cycle arrest at G2/M phase.
The binding mode of 4f was determined by docking studies.