Synthesis and Bioevaluation of 3,6-Diaryl-[1,2,4]triazolo[4,3-b] Pyridazines as Antitubulin Agents

Article abstract of DOI:10.1021/acsmedchemlett.6b00252

A series of 3,6-diaryl-[1,2,4]triazolo[4,3-b]pyridazines were designed as a class of vinylogous CA-4 analogues. The easily isomerized (Z,E)-butadiene linker of vinylogous CA-4 was replaced by a rigid [1,2,4]triazolo[4,3-b]pyridazine scaffold. Twenty-one target compounds were synthesized and exhibited moderate to potent antiproliferative activity. The compound 4q with a 3-amino-4-methoxyphenyl moiety as the B-ring, comparable to CA-4 (IC₅₀ = 0.009-0.012 μM), displayed the highly active antiproliferative activity against SGC-7901, A549, and HT-1080 cell lines with IC₅₀ values of 0.014, 0.008, and 0.012 μM, respectively. Tubulin polymerization experiments indicated that 4q effectively inhibited tubulin polymerization, and immunostaining assay revealed that 4q significantly disrupted tubulin microtubule dynamics. Moreover, cell cycle studies revealed that compound 4q dramatically arrested cell cycle progression at G2/M phase in A549 cells. Molecular modeling studies showed that 4q could bind to the colchicine binding site on microtubules.

Full text of DOI:10.1021/acsmedchemlett.6b00252

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Letter
Synthesis and Bioevaluation of 3,6-Diaryl-
[1,2,4]Triazolo[4,3-b]Pyridazines as Antitubulin Agents
Qile Xu, Yueting Wang, Jingwen Xu, Maolin Sun, Haiqiu Tian,
Daiying Zuo, Qi Guan, Kai Bao, Yingliang Wu, and Weige Zhang
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00252 • Publication Date (Web): 17 Oct 2016
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Synthesis and Bioevaluation of 3,6-Diaryl-[1,2,4]Triazolo[4,3-
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Qile Xu,a Yueting Wang,a Jingwen Xu,b Maolin Sun,a Haiqiu Tian,a Daiying Zuo,b Qi Guan,a Kai Bao,a,c
Yingliang Wu,b,* Weige Zhanga,*
a Key Laboratory of StructureꢀBased Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical Universiꢀ
ty, 103 Wenhua Road, Shenhe District, Shenyang 110016, China.
b Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016,
China.
c Gordon Center for Medical Imaging, Division of Nuclear Medicine and Molecular Imaging, Department of Radiology,
Massachusetts General Hospital and Harvard Medical School, Boston, MA 02214, USA.
KEYWORDS: [1,2,4]Triazolo[4,3-b]pyridazine; Combretastatin A-4; Tubulin; Colchicine binding site; Molecular modelling
ABSTRACT: A series of 3,6ꢀdiarylꢀ[1,2,4]triazolo[4,3ꢀb]pyridazines were designed as a class of vinylogous CAꢀ4 analogues. The
easily isomerized (Z,E)ꢀbutadiene linker of vinylogous CAꢀ4 was replaced by a rigid [1,2,4]triazolo[4,3ꢀb]pyridazine scaffold.
Twenty one target compounds were synthesized and exhibited moderate to potent antiproliferative activity. The compound 4q with
a 3ꢀaminoꢀ4ꢀmethoxyphenyl moiety as the Bꢀring, comparable to CAꢀ4 (IC50 = 0.009ꢀ0.012 ꢁM), displayed the highly active antiꢀ
proliferative activity against SGCꢀ7901, A549 and HTꢀ1080 cell lines with IC50 values of 0.014 ꢁM, 0.008 ꢁM, and 0.012 ꢁM, reꢀ
spectively. Tubulin polymerization experiments indicated that 4q effectively inhibited tubulin polymerization and immunostaining
assay revealed that 4q significantly disrupted tubulin microtubule dynamics. Moreover, cell cycle studies revealed that compound
4q dramatically arrested cell cycle progression at G2/M phase in A549 cells. Molecular modeling studies showed that 4q could
bind to the colchicine binding site on microtubules.
Tubulin which plays a key role in cell mitosis is one of the
most effective molecular targets for anticancer drugs discovꢀ
ery.1,2 Microtubule targeting drugs disrupt tubulin/microtubule
dynamics by binding to distinct sites such as taxol, vinca alkaꢀ
loid and colchicine binding sites and arrests cells during mitoꢀ
sis, leading to cell death.3,4 Combretastatin Aꢀ4 (CAꢀ4, 1, Figꢀ
ure 1) is a well known antitubulin agent by binding to the colꢀ
chicine site.5 In recent years, a wide variety of CAꢀ4 analogues
have been developed for the structureꢀactivity relationships
study.6,7 In brief, the existence of the 3,4,5ꢀtrimethoxy group
on the Aꢀring and the Zꢀrestricted configuration have been
reported as preconditions for effective antiproliferative activiꢀ
ty.8 However, the cisꢀolefin bond of CAꢀ4 easily isomerizes to
its inactive transform (E geometry) under light, heat and protic
media resulting in a sharp reduction in both antiproliferative
and antitubulin activities.9 Therefore, to restrict the comꢀ
Figure 1. Design strategy and structures for the target compounds.
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Scheme 1.
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Reagents and conditions: (a) (1) glyoxylic acid, AcOH, reflux, (2) NH4OH, (3) N2H4·H2O, reflux; (b) POCl3, 100 °C, 2 h; (c)
MeOH, H2SO4, reflux; (d) N2H4·H2O, MeOH, reflux; (e) nꢀbutyl alcohol, MW, 120 °C.
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pounds in the desired Zꢀrestricted configuration, a wide variety
of strategies have been designed, particularly by rigidifying
the olefin bond with heterocyclic moieties (fiveꢀmembered
and sixꢀmembered rings, Figure 1).10ꢀ13 Interestingly, when the
cisꢀolefin bridge of CAꢀ4 was replaced by a four carbons
(Z,E)ꢀbutadiene linker, the resulting vinylogous CAꢀ4 (2, Figꢀ
ure 1) actually retained potent bioactivity.14 Furthermore, viꢀ
nylogous CAꢀ4 analogue 3 whose Bꢀring was a phenyl group
was discovered to more active than CAꢀ4 in antitubulin activiꢀ
ty.14 These dienic analogues of CAꢀ4 also tend to isomerizaꢀ
tion to the more stable but inactive (E,E)ꢀisomeric derivaꢀ
tives.14,15
and 5c were reduced by hydrazine hydrate to get correspondꢀ
ing aminoꢀcompounds 4h, 4n, 4q and 5d.20
To explore the ability of various 3,6ꢀdiarylꢀ
[1,2,4]triazolo[4,3ꢀb]pyridazines to inhibit cancer cells, the
target compounds and reference compounds CAꢀ4 (1) were
screened for antiꢀproliferative activity against gastric adenoꢀ
carcinoma SGCꢀ7901 cells, lung adenocarcinoma A549 cells
and fibrosarcoma HTꢀ1080 cells. As illustrated in Table 1, the
majority of these target compounds exhibited moderate to
potent antiproliferative activity. When ringꢀA was a 3,4,5ꢀ
trimethoxyphenyl, a comparison of compounds 4a-j revealed
an electronic properties substitution effect at paraꢀsubstitution
of ringꢀB. The compounds with electronꢀdonating groups such
as CH3 (4a), OCH3 (4c), NH2 (4h), SCH3 (4i) on paraꢀ
substitution of ringꢀB gave higher activities than those with
electronꢀwithdrawing groups such as CF3 (4b), NO2 (4g), CN
(4j) with the exception of compounds 4d-f with paraꢀhalogen
substituted electronꢀwithdrawing groups (e.g., F, Cl, Br). For
example, compound 4e with a chlorine substituent on paraꢀ
substitution of ringꢀB displayed potent antiproliferative activiꢀ
ty which may be due to chlorine being a relative small and
weak electronꢀwithdrawing group in contrast with other elecꢀ
tronꢀwithdrawing groups. However, for the electronic effects
to metaꢀsubstitution of ringꢀB, only compound 4n with an
electronꢀdonating group NH2 exhibited moderate antiproliferaꢀ
tive activity (4k-m vs 4n). Futhermore, to explore the effect of
disubstitution on the Bꢀring, methoxyl, fluorine, nitro and
amino groups were introduced in the mꢀ and pꢀpositions reꢀ
spectively (4o-q). Interestingly, the compound 4q with a 3ꢀ
aminoꢀ4ꢀmethoxyphenyl moiety as the Bꢀring, comparable to
CAꢀ4 (IC50 = 0.009ꢀ0.012 ꢁM), displayed the highly active
antiproliferative activity against SGCꢀ7901, A549 and HTꢀ
1080 cell lines with IC50 values of 0.014 ꢁM, 0.008 ꢁM, and
0.012 ꢁM, respectively. Moreover, replacement of the 3,4,5ꢀ
Over the years, researchers have been highly interested in
compounds containing the [1,2,4]triazolo[4,3ꢀb]pyridazine
scaffold, on account of their characteristic chemical structure
and various biological properties including anticancer activiꢀ
ty.16ꢀ19 As part of our search for new antitubulin agents,20ꢀ24 we
hypothesized that the replacement of the easily isomerized
(Z,E)ꢀbutadiene linker of vinylogous CAꢀ4 analogues with a
rigid [1,2,4]triazolo[4,3ꢀb]pyridazine scaffold could be a sucꢀ
cessful strategy to unravel a class of antitubulin agents (Figure
1). Thus,
a
series of 3,6ꢀdiarylꢀ[1,2,4]triazolo[4,3ꢀ
b]pyridazines were synthesized and distinguished into two
types. Type I target compounds (4a-q) include a 3,4,5ꢀ
trimethoxyphenyl unit as the Aꢀring which is an essential
component to induce cytotoxicity in the compounds based on
CAꢀ4 pharmacophores, while type II target compounds (5a-d)
include a 2,3,4ꢀtrimethoxy substitutions on the Aꢀring for
comparison (Figure 1).25 To explore the SAR of target comꢀ
pounds, various substitutions with electronꢀwithdrawing (F, Cl,
Br, CF3, CN and NO2) and electronꢀdonating (CH3, OCH3,
SCH3 and NH2) groups were introduced at different positions
of the Bꢀring. In addition, the most potent compound 4q was
selected to investigate its mechanism of activity and the possiꢀ
ble binding mode of 4q on tubulin.
trimethoxyꢀsubstituted
Aꢀring
with
2,3,4ꢀtrimethoxyꢀ
substituted Aꢀring (5a-d) induced a reduction in antiproliferaꢀ
tive activity of resulting compound (4a vs 5a, 4c vs 5b, 4p vs
5c, 4q vs 5d). Compound 5b showed moderate antiproliferaꢀ
tive activity and this result was consistent with the data in the
reported literature.19
Target compounds 4a-q and 5a-d were prepared as outlined
in Scheme 1. In brief, substituted acetophenones 6 were reactꢀ
ed with glyoxylic acid in acetic acid and then treated with
hydrazine to afford the desired pyridazinones 7 in 80ꢀ95%
yield. Subsequently, pyridazinones 7 were treated with phosꢀ
phorus oxychloride to afford the key intermediates 8. On the
other hand, the substituted benzoic acids 9 were reacted with
excess methanol by using concentrated sulfuric acid as catalyst
to give the corresponding esters 10 which were further reacted
with 80% hydrazine monohydrate in methanol to get hydraꢀ
zides 11 in 60ꢀ90% yield. Finally, intermediates 8 were reactꢀ
ed with hydrazides 11 to afford the desired compounds in nꢀ
butyl alcohol under microwave irradiation (150 W, 120 ºC) in
the absence of catalysts.17 The nitroꢀcompounds 4g, 4m, 4p
Table 1. Antiproliferative activity of target compounds 4a-q,
5a-d and CAꢀ4.
IC50 (ꢁM) a
Comp.
4a
SGCꢀ7901
A549
HTꢀ1080
0.016 ± 0.009
2.21 ± 0.11
0.26 ± 0.21
2.02 ± 0.07
0.025 ± 0.006
0.023 ± 0.012
10.4 ± 1.3
0.14 ± 0.07
1.86 ± 0.22
4b
4c
0.051 ± 0.009
2.18 ± 0.06
0.017 ± 0.011
0.021 ± 0.010
0.45 ± 0.03
4d
4e
0.051 ± 0.009
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4f
4g
1.28 ± 0.03
2.38 ± 0.05
1.23 ± 0.09
0.098 ± 0.012
30.9 ± 0.5
0.78 ± 0.05
3.85 ± 0.07
2.35 ± 0.08
0.28 ± 0.03
51.6 ± 0.9
0.15 ± 0.07
4.94 ± 0.09
1.62 ± 0.06
0.073 ± 0.013
33.5 ± 2.1
polymerization experiment strongly implicated a direct interꢀ
action of 4q with tubulin.
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4h
4i
To further confim the influence of inhibition of tubulin
polymerization in cells, we explored microtubules structure
and distribution in cultured A549 cells by using the indirect
immunofluorescence assay. A549 cells treated with DMSO
showed a normal arrangement and organization throughout the
cells (Figure 3). Instead, A549 cells treated with compound 4q
and CAꢀ4 (at their respective 2ꢀfold IC50 concentrations)
demonstrated a destruction of the tubulin network (Figure 3).
In short, the results further confirmed that tubulin was the
molecular target for compound 4q.
4j
4k
4l
63.1 ± 0.7
47.9 ± 1.3
60.5 ± 1.4
59.0 ± 0.9
52.2 ± 1.7
54.5 ± 1.6
4m
4n
4o
86.4 ± 2.3
90.5 ± 3.4
50.0 ± 1.5
0.98 ± 0.06
4.17 ± 0.07
2.28 ± 0.01
0.014 ± 0.002
4.53 ± 0.06
0.42 ± 0.06
9.14 ± 0.07
0.91 ± 0.09
0.012 ± 0.003
2.98 ± 0.05
4.23 ± 0.08
4.57 ± 0.03
0.008 ± 0.006
10.4 ± 0.07
6.98 ± 0.06
11.4 ± 1.1
0.39 ± 0.07
1.69 ± 0.03
6.85 ± 0.05
0.012 ± 0.004
0.17 ± 0.09
5.57 ± 0.09
18.2 ± 0.7
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4p
4q
5a
5b
5c
5d
CAꢀ4 b
2.62 ± 0.05
0.009 ± 0.002
0.92 ± 0.04
0.009 ± 0.006
a IC50: 50% inhibitory concentration (detemined by standard
MTT assay). Each experiment was carried out in triplicate.
b Used as a positive control.
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To investigate whether the antiproliferative activity of these
new compounds was due to interaction with tubulin, the highly
active compound 4q was evaluated for its inhibition of tubulin
polymerization. CAꢀ4 (1) and paclitaxel were employed as
positive and negative controls, respectively. As shown in Figꢀ
Figure 3. Effects of 4q (0.016 ꢁM) and CAꢀ4 (0.018 ꢁM) on
inhibition of tubulin polymerization in A549 cells by immunoꢀ
fluorescence. The left and middle panels represent the tubulin
assembly stained with FITC and DAPI and the right panel
represents a merge of the corresponding left and middle panels.
To investigate whether the potent compound 4q could arrest
cell cycle distribution, the effect of compound 4q on the cell
cycle were analyzed by flow cytometry. First, A549 cells were
treated with 4q of different concentrations (0.5ꢀ, 1ꢀ, 2ꢀfold IC50)
for 12 h (Figure 4A). On the other hand, A549 cells were
treated with 4q or CAꢀ4 (at their 2ꢀfold IC50 concentrations,
respectively) for 0, 12, 24 and 48 h respectively (Figure 4B).
Cell cycle analysis revealed that 4q caused a significant cell
cycle arrest at the G2/M phase in both concentration and time
dependent manners.
To further understand the possible binding mode of these
new compounds with the colchicine binding site of tubulin,
molecular modeling study of the most potent compound 4q,
CAꢀ4 and vinylogous CAꢀ4 analogue 3 were performed by
using CDOCKER protocol in Discovery Studio 3.0 software
package (PDB: 1SA0). The docking study (Figure 5A) showed
that compound 4q superimposed well with CAꢀ4 and 3 in colꢀ
chicine binding site, 4q displays a distorted conformation simꢀ
ilar to CAꢀ4 and 3, which may be responsible for its bioactiviꢀ
ty. For compound 4q, the oxygen atom of the para methoxy
group on Aꢀring formed a hydrogen bond with the thiol group
of Cys β241 and the amino nitrogen atom on Bꢀring formed
another hydrogen bond with the sulphur atom of Met β259
(Figure 5B). Additionally, the Ala β250 residue formed a diꢀ
rect hydrogen bond with the [1,2,4]triazolo[4,3ꢀb]pyridazine
linker. The docking study and tubulin polymerization assay
Figure 2. Effects of 4q and CAꢀ4 on tubulin polymerizaꢀ
tion. Tubulin had been incubated with 4q (0.33, 1.1, 3.3
and 10 ꢁM), paclitaxel (5.0 ꢁM), CAꢀ4 (0.5, 1.0, 2.0 and
4.0 ꢁM) or DMSO (vehicle control) at room temperature.
Values are the mean ± SD of three different experiments
performed in triplicates.
ure 2, 4q displayed antitubulin activity with an IC50 value of
1.80 ꢁM, which is less active than that of CAꢀ4 (IC50 = 0.64
ꢁM). Moreover, 4q and CAꢀ4 inhibited tubulin polymerization
in a concentrationꢀdependent manner (Figure 2). The tubulin
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suggested that 4q could bind at the colchicine binding site of
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Figure 4. (A) 4q caused G2/M phase arrest in a concentraꢀ
tionꢀdependent manner. A549 cells were treated with difꢀ
ferent concentrations (0.004 ꢁM to 0.016 ꢁM ) of 4q for 12
h, then stained with PI and subjected to flow cytometric
analysis. (B) 4q and CAꢀ4 induced G2/M phase arrest in a
timeꢀdependent manner. A549 cells were treated with 4q or
CAꢀ4 for time 12, 24 and 48 h, then stained with PI and
subjected to flow cytometric analysis.
In conclusion, we designed and synthesized a set of 3,6ꢀdiarylꢀ
[1,2,4]triazolo[4,3ꢀb]pyridazines as a new class of vinylogous
CAꢀ4 analogues. The structure of these compounds are unique
as they involved a rigid [1,2,4]triazolo[4,3ꢀb]pyridazine scafꢀ
fold as the linker to fix the Z,Eꢀdiene configuration of Aꢀring
and Bꢀring. These target compounds exhibited moderate to
potent antiproliferative activity with IC50 values from 0.008 to
90.5 ꢁM. Interestingly, the compound 4q with a 3ꢀaminoꢀ4ꢀ
methoxyphenyl moiety as the Bꢀring, comparable to CAꢀ4
(IC50 = 0.009ꢀ0.012 ꢁM), and displayed the highly active antiꢀ
proliferative activity against SGCꢀ7901, A549 and HTꢀ1080
cell lines with IC50 values of 0.014 ꢁM, 0.008 ꢁM, and 0.012
ꢁM, respectively. The tubulin polymerization assay suggested
that 4q effectively inhibited tubulin polymerization and imꢀ
munofluorescence staining experiment revealed that 4q signifꢀ
icantly disrupted tubulin microtubule dynamics. Furthermore,
cell cycle analysis studies revealed that compound 4q signifiꢀ
cantly arrested cell cycle progression at G2/M phase in A549
cells. Additionally, the results of docking study together with
other two in vitro tubulin experiments showed that 4q may
bind to colchicine binding site of tubulin. Our work not only
expands the exploration of the linker modification of tubulin
inhibitor CAꢀ4 but also provides a set of rigid analogues of
vinylogous CAꢀ4 with potent antiproliferative activity.
Figure 5. (A) Possible binding mode of compound 4q (vioꢀ
let), CAꢀ4 (green) and vinylogous CAꢀ4 analogue 3 (yellow)
in the colchicine binding site. (B) Overlay of 4q in the bindꢀ
ing site. Hydrogen bonds are displayed by green dashed lines
(hydrogen bond distance < 3Å).
AUTHOR INFORMATION
Corresponding Author
Tel: +86 24 23986422, Fax: +86 24 23986393 (W. Zhang);
Tel: +86 24 23986278, Fax: +86 24 23986278 (Y. Wu).
Eꢀmail addresses: zhangweige2000@sina.com (W. Zhang), yingꢀ
liang_1016@163.com (Y. Wu).
Author Contributions
Q.X., Y.W., J.X., M.S., H.T. and D.Z. performed the experiments.
Q.X., Q.G., K.B., Y.W. and W.Z. analyzed and interpreted the
data. Q.X., K.B. and W.Z. wrote the paper.
Funding Sources
This work was funded by the National Natural Science Foundaꢀ
tion of China (81502932) and State Key Laboratory of Natural
Medicines and Active Substance (No. GTZK201603)
ASSOCIATED CONTENT
ACKNOWLEDGMENT
Supporting Information
We gratefully acknowledge Program for Innovative Research
Team of the Ministry of Education and Program for Liaoning
Innovative Research Team in University.
The Supporting Information is available free of charge on the
ACS Publications website. The supporting information includes
experimental procedures, data for compounds
REFERENCES
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Table of Contents
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Figure 1. Design strategy and structures for the target compounds.
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Figure 2. Effects of 4q and CA-4 on tubulin polymerization. Tubulin had been incubated with 4q (0.33, 1.1,
3.3 and 10 µM), paclitaxel (5.0 µM), CA-4 (0.5, 1.0, 2.0 and 4.0 µM) or DMSO (vehicle control) at room
temperature. Values are the mean ± SD of three different experiments performed in triplicates.
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Figure 3. Effects of 4q (0.016 µM) and CA-4 (0.018 µM) on inhibition of tubulin polymerization in A549 cells
by immunofluorescence. The left and middle panels represent the tubulin assembly stained with FITC and
DAPI and the right panel represents a merge of the corresponding left and middle panels.
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Figure 4. (A) 4q caused G2/M phase arrest in a concentration-dependent manner. A549 cells were treated
with different concentrations (0.004 µM to 0.016 µM ) of 4q for 12 h, then stained with PI and subjected to
flow cytometric analysis. (B) 4q and CA-4 induced G2/M phase arrest in a time-dependent manner. A549
cells were treated with 4q or CA-4 for time 12, 24 and 48 h, then stained with PI and subjected to flow
cytometric analysis.
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Figure 5. (A) Possible binding mode of compound 4q (violet), CA-4 (green) and vinylogous CA-4 analogue 3
(yellow) in the colchicine binding site.
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(B) Overlay of 4q in the binding site. Hydrogen bonds are displayed by green dashed lines (hydrogen bond
distance < 3Å).
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Reagents and conditions: (a) (1) glyoxylic acid, AcOH, reflux, (2) NH4OH, (3) N2H4·H2O, reflux; (b) POCl3,
100 °C, 2 h; (c) MeOH, H2SO4, reflux; (d) N2H4·H2O, MeOH, reflux; (e) nꢀbutyl alcohol, MW, 120 °C.
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