Microwave-assisted synthesis, molecular docking and antiproliferative activity of (3/5-aryl-1,2,4-oxadiazole-5/3-yl)(3,4,5-trimethoxyphenyl)methanone oxime derivatives

Article abstract of DOI:10.1039/c5md00150a

A series of (3/5-aryl-1,2,4-oxadiazole-5/3-yl)(3,4,5-trimethoxyphenyl)methanone oxime derivatives were synthesized via a rapid and facile microwave-assisted synthesis method of building a 1,2,4-oxadiazole skeleton using mandelic acid as the starting material. Twenty-four target compounds were evaluated for their in vitro antiproliferative activities against three human cancer cell lines (SGC-7901, A549 and HT-1080). Among them, 16b exhibited the highest potency against different tumour cell lines, especially the A549 cell line (IC₅₀ = 87 nM). Structure-activity relationship (SAR) studies revealed that the aryl substituent at the C-5 position on the 1,2,4-oxadiazole ring is superior to that at the C-3 position. An oxime as a connector can obviously increase the potency, contrary to that in SMART derivatives. Moreover, 16b significantly induced a cell cycle arrest in the G2/M phase and caused microtubule destabilization. Molecular docking studies provided a theoretical binding mode of 16b at the colchicine site in the tubulin dimer. Our work laid the foundation for further structure-guided design of novel tubulin polymerization inhibitors.

Full text of DOI:10.1039/c5md00150a

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Microwave-assisted synthesis, molecular docking and antiproliferative activity of
(3/5-aryl-1,2,4-oxadiazole-5/3-yl)(3,4,5-trimethoxyphenyl)methanone oxime
derivatives
Qi Guan,a,1 Dongjie Feng,a,1 Zhaoshi Bai,b Yuanhang Cui,a Daiying Zuo,b Min’an Zhai,a Xuewei Jiang,b
Wenbo Zhou,a Kai Bao, a,c Yingliang Wu,*b and Weige Zhang*a
a Key Laboratory of StructureꢀBased 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 Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center
and Harvard Medical School, Boston, MA 02215, USA.
* Corresponding authors:
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), yingliang_1016@163.com (Y. Wu).
1 These two authors contributed equally to this work
Graphical Abstract

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Abstract
A series of (3/5ꢀarylꢀ1,2,4ꢀoxadiazoleꢀ5/3ꢀyl)(3,4,5ꢀtrimethoxyphenyl)methanone oxime derivatives
were synthesized via a rapid and facile microwaveꢀassisted synthesis method of building
1,2,4ꢀoxadiazole skeleton using mandelic acid as the starting material. Twenty four target compounds
were evaluated for their in vitro antiproliferative activities against three human cancer cell lines
(SGCꢀ7901, A549 and HTꢀ1080). Among them, 16b exhibited the most potency against different
tumour cell lines, especially A549 cell lines (IC50 = 87 nM). Structureꢀactivity relationship (SAR)
studies revealed the aryl substituent at Cꢀ5 position on 1,2,4ꢀoxadiazole ring is superior to that at Cꢀ3
position. Oxime as a connector can obviously increase the potency, contrary to that in SMART
derivatives. Moreover, 16b significantly induced cell cycle arrest in the G2/M phase and caused
microtubule destabilization. Molecular docking studies provide a theoretical binding mode of 16b at
the colchicine site in the tubulin dimer. Our work laid the foundation for further structureꢀguided
design of novel tubulin polymerization inhibitors.
Keywords: Antiproliferative activity, Tubulin, Microwave, 1,2,4ꢀoxadiazole, Molecular docking

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Introduction
Microtubules, composed of α,βꢀtubulin heterodimers, play an important role in a wide range of cellular
functions including shape maintenance, vesicle transport, cell motility and division. Formation of
microtubules from tubulin, which is crucial for mitosis, is a dynamic process that involves
polymerization and depolymerization.1 Therefore, anticancer therapy based on microtubule targeting
agents (MTAs) is receiving growing attention in drug discovery, with several clinical drugs developed
(e.g., paclitaxel and vinblastine). MTAs are known to interact with tubulin via at least four binding sites:
the laulimalide, taxane (stabilisers of the microtubules), vinca alkaloid and colchicine (1, Fig. 1) sites
(destabilisers of the microtubules).2,3 Compared to other categories of binding site agents, colchicine
binding site inhibitors usually possess relatively simple and diverse structures (e.g., CAꢀ4 2, TN16 3
and Nocodazoles 4, Fig. 1), which provides many possibilities for optimization and new scaffold
design.
4ꢀSubstituted methoxybenzoylꢀarylꢀthiazole (SMART 5, Fig. 1) and its analogues (ABI 6 and
RABI 7, Fig. 1), as potential anticancer agents targeting tubulin by binding to the colchicine binding
site, show low nanomolar inhibition on various cancer cell lines and exhibit excellent potency in
vivo.4ꢀ6 Compared with their structures, these agents have some similar templates containing three
conjugated aromatic rings (Aꢀ, Bꢀ, and Cꢀrings, respectively) with a ketone linkage between Aꢀ and
Bꢀrings. And the 3,4,5ꢀtrimethoxyphenyl moiety as Aꢀring plays an important role in these templates to
enhance antiproliferative activity.
Fig. 1. The structures of some tubulin inhibitors and anticancer agents bearing 1,2,4ꢀoxadiazole
fragments.

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Ring bioisosterism is widely used as a rational approach for the discovery of new anticancer
agents.7 Recently, there has been great interest in compounds containing the 1,2,4ꢀoxadiazole scaffold
because of their unique chemical structure and broad spectrum of biological properties including
anticancer activity.8,9 For example, the 3,5ꢀdisubstitutedꢀ1,2,4ꢀoxadiazoles (8–10, Fig. 1) are reported in
literatures for their anticancer potential, especially 10 as a tubulin inhibitor.10ꢀ12
In view of these observations, herein we describe using the oxadiazole moiety as a biomimetic
replacement of the central thiazole ring of SMART (5) to afford
a new series of
(3/5ꢀarylꢀ1,2,4ꢀoxadiazoleꢀ5/3ꢀyl)(3,4,5ꢀtrimethoxyphenyl)methanone oxime derivatives 11–16 (Fig. 2).
Moreover, we developed a microwaveꢀassisted synthesis method of building 1,2,4ꢀoxadiazole skeleton
in good yields. The newly synthesized derivatives were evaluated for their biological activity to explore
the structureꢀactivity relationships (SARs) studies.
O
O
X
X
O
O
O
N
O
O
N
N
O
N
Ar
Ar'
O
O
15a, Ar'=phenyl, X=O
11a, Ar=phenyl, X=O
O
15b, Ar'=4-methyphenyl, X=O
15c, Ar'=4-fluorophenyl, X=O
16a, Ar'=phenyl, X=NOH
16b, Ar'=4-methyphenyl, X=NOH
16c, Ar'=4-fluorophenyl, X=NOH
11b, Ar=4-methyphenyl, X=O
11c, Ar=4-methoxyphenyl, X=O
11d, Ar=4-bromophenyl, X=O
11e, Ar=2-fluorophenyl, X=O
11f, Ar=2-chloropyridin-4-yl, X=O
11g, Ar=thiophen-2-yl, X=O
O
A
HN
N
O
O
B
C
S
N
N
F
11h, Ar=thiophen-3-yl, X=O
12a, Ar=phenyl, X=NOH
12b, Ar=4-methyphenyl, X=NOH
12c, Ar=4-methoxyphenyl, X=NOH
12d, Ar=4-bromophenyl, X=NOH
12e, Ar=2-fluorophenyl, X=NOH
12f, Ar=2-chloropyridin-4-yl, X=NOH
12g, Ar=thiophen-2-yl, X=NOH
12h, Ar=thiophen-3-y, X=NOH
13b, Ar=4-methyphenyl, X=NOCH3
14b, Ar=4-methyphenyl, X=NOCH2CH3
SMART (5)
10
Fig. 2. Structures and design strategy for the target compounds.
Results and Discussion
Chemistry
The synthetic routes for (3ꢀarylꢀ1,2,4ꢀoxadiazolꢀ5ꢀyl)(3,4,5ꢀtrimethoxyphenyl)methanone oxime
derivatives (11–14) are outlined in Scheme 1. The benzonitriles (17a–h) as starting material were
converted into corresponding amidoximes (18a–h) via an addition reaction with hydroxylamine.13 The
compound 20a was first prepared by heating as previously described.14 The yield of the desired product
20a increased steadily from 14% to 50% when we extended the reaction time from 30 min to 600 min,

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but prolonged heating did not increase the yield (entry 1–5, Table 1). Recently, microwave irradiation
has become a very useful tool in synthetic organic chemistry because it could increase both reaction
rates and yields via more efficient heating compared to standard thermal conduction.15,16 When
microwave irradiation was applied to synthesis of 20a, we noted that the reaction rate was significantly
faster (entry 1 vs entry 7). As can be seen from entries 6–8 in Table 1, when we elevated the reaction
temperature from 110°C to 120°C, the yield of 20a increased from 20% to 69%. However, it was
observed that the reaction temperature over 120°C resulted in a decrease in the yield of 20a (entries 6–
8). No matter whether to extend or shorten the time at 120°C the yield was reduced (entries 7, 9 and
10). Hence, the best result was obtained when 18a was treated with 19 under microwave irradiation at
120°C for 30 min (entry 7). Electronic effects on the overall reaction are not significant since similar
yields of 20b–h (48–67%) were obtained from substituted amidoximes (18b–h) under the optimized
condition (Scheme 1). Then 20a–h underwent oxidation using pyridinium chlorochromate (PCC) to
yield the corresponding ketones (11a–h),17 and the following transformations into oxime isomers (12a–
h) were performed in an excess of hydroxylamine hydrochloride and sodium acetate in refluxing
ethanol for 5 h.18 12b was alkylated respectively with dimethyl sulfate and diethyl sulfate in DMF to
provide corresponding 13b and 14b.
Scheme 1. Synthesis of compounds 11–14; reagents and conditions: (i) NH2OH·HCl, NaOH aq. (1 M),
H2O, RT, 0.5 h; 100°C, 3 h; (ii) 3,4,5ꢀtrimethoxymandelic acid (19), HOBt, EDC·HCl, CH3CN, RT, 0.5
h; toluene, MW 120°C, 0.5 h; (iii) PCC, CH2Cl2, 35°C, 3 h; (iv) NH2OH·HCl, CH3COONa,
CH3CH2OH, reflux, 10 h; (v) (CH3)2SO4 or (CH2CH3)2SO4, Na2CO3, DMF, 50°C, 3 h.
Table 1. Temperature, time and yield data of newly synthesized compound 20a using conventional and
microwave irradiation techniques.
Entry
1
Heating
Temperature (°C)
Time (min)
Yield (%)c
Ca
reflux
30
14

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Ca
Ca
reflux
reflux
reflux
reflux
110
60
120
240
600
30
25
40
2
3
4
5
6
7
8
9
Ca
49
Ca
50
Mb
Mb
Mb
Mb
Mb
20
120
30
69
130
30
56
120
15
trace
44
10
120
45
a C — Conventional.
b M — Microwave irradiation.
c Isolated yields.
The synthetic routes for (5ꢀarylꢀ1,2,4ꢀoxadiazolꢀ3ꢀyl)(3,4,5ꢀtrimethoxyphenyl)methanone oxime
derivatives (15–16) are outlined in Scheme 2. In brief, 3,4,5ꢀtrimethoxybenzaldehyde 21 reacted with
sodium cyanide giving cyanohydrin 22.19 Then protection of the hydroxyl by using
3,4ꢀdihydroꢀ2Hꢀpyran (THP) provided 23,20 which was treated with hydroxylamine hydrochloride and
fresh sodium methoxide in refluxing methanol to provide to amidoxime 24. Subsequently, 24
underwent cyclization with esters 25a–c followed by acidification to afford corresponding
intermediates 26a–c.21 The desired compounds 15a–c and 16a–c were obtained by the same methods
which were described in Scheme 1.

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Scheme 2. Synthesis of compounds 15–16; reagents and conditions: (i) NaCN, Na2S2O5, H2O; (ii)
TsOH cat., THP, anhydrous toluene, under N2 atmosphere, 50°C; (iii) NH2OH.HCl, CH3ONa, RT, 5 h;
(iv) (a) CH3CH2OH, Na, reflux, 2 h; (b) HCl aq. (6 M), RT, 1h; (v) PCC, CH2Cl2, 35°C, 3 h; (vi)
NH2OH.HCl, CH3COONa, CH3CH2OH, reflux, 5 h.
The oxime derivatives were mainly obtained as mixtures of Z and E isomers (see experimental
section). Most of them could not be separated chromatographically except 12a, 12c, 12f and 12g. The
stereochemistry of oxime was established by 1H NMR spectrum. For the representative compound 12c,
1
Z and E oximes were separated. The major component (71%) of the mixture exhibited the H NMR at
δ=10.63 (NOH) and 6.91 (ArꢀH), whereas the minor isomer showed peaks at δ=10.05 (NOH) and 6.95
(ArꢀH) (Fig. 3). By analogy to literature examples,22ꢀ24 we assigned the Z and E configurations,
respectively, to the major and minor components of the 12c isomer mixture. When a single product
isomer was placed in the solvent and exposed to air under ambient atmosphere for some time
1
(overnight to several days), then we found the Z/E tautomerism, which was clearly indicated by H
NMR, for example (Z)ꢀ12c (Fig. 4). Therefore, the isomer mixtures were selected for evaluation
without separation.

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Fig. 3. 1H NMR (300 MHz) for compound (Z)ꢀ12c and (E)ꢀ12c in CDCl3.
Fig. 4. Tautomerism from (Z)-12c to (E)-12c. Overnight, NMR sample contained (Z)-12c and (E)-12c
mixtures in CDCl3; ratio is about 6:1.
Biological Studies
In vitro antiproliferative activity
Using cancer cell lines, we investigated the antiproliferative activities of target compounds. CAꢀ4 (2)
and nocodazole (4) were used as a positive control. MTT assays were performed on three human
carcinoma cell lines (gastric adenocarcinoma SGCꢀ7901 cells, lung adenocarcinoma A549 cells and
fibrosarcoma HTꢀ1080 cells). As shown in Table 2, some target compounds showed moderate
antiproliferative activity against different cancer cells with potencies in ꢁM range. The IC50 values of
the most promising compound, 16b, were 0.092 ± 0.011, 0.087 ± 0.009 and 0.68 ± 0.10 ꢁM against the

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SGCꢀ7901, A549 and HTꢀ1080 cell lines, respectively. With oxime as the linker, the aryl group at the
1,2,4ꢀoxadiazole Cꢀ3 position slightly reduced the potency compared with the Cꢀ5 position (12b vs
16b). However, the methylated and ethylated products of the oxime were considerably less active (12b
vs 13b and 14b). Replacement of the oxime linkage with a carbonyl linkage caused a deleterious effect
on the activity of resulting compound (11b vs 12b, 15b vs 16b). Interestingly, this is an opposite result
of modification of SMART analogues. Moreover, the influence of electronic effects against activity on
the Cꢀring is not significant. A variety of groups on the Cꢀring all caused obviously losses of activity
except of 4ꢀmethylphenyl group. In general, an oxime as the linker and a 4ꢀmethylphenyl group on the
Cꢀring are necessary for antiproliferative activity.
Table 2. In vitro antiproliferative activities of the target compounds on three human carcinoma cell
lines.
Compound
IC50 (ꢁM) ± SDa
A549
SGCꢀ7901
HTꢀ1080
>30
11a
11b
11c
11d
11e
11f
>30
25.3 ± 3.2
>30
>30
27.6 ± 2.1
20.3 ± 1.0
>30
13.9 ± 1.5
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
11g
11h
12a
12b
12c
12d
12e
12f
>30
>30
>30
>30
>30
>30
>30
0.77 ± 0.08
>30
9.8 ± 1.1
>30
2.3 ± 0.3
21.3 ± 2.6
10.5 ± 1.1
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
12g
12h
13b
14b
15a
15b
15c
16a
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
21.5 ± 2.7
>30
28.8 ± 3.2
13.4 ± 1.9
>30
>30
>30
>30
>30
19.1 ± 2.2
26.4 ± 3.1

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16b
0.092 ± 0.011
0.087 ± 0.009
22.1 ± 2.5
0.68 ± 0.10
14.4 ± 1.7
23.7 ± 3.3
0.016 ± 0.003
0.15 ± 0.002
16c
CA-4 (2)b
0.024 ± 0.003
0.085 ± 0.009
0.012 ± 0.002
0.13 ± 0.003
Nocodazole (4)b
a
IC50: Concentration of the compound (ꢁM) producing 50% cell growth inhibition after 72 h of drug
exposure, as determined by the MTT assay. Each experiment was carried out in triplicate.
b Used as a positive control.
Analysis of immunofluorescence staining
In an attempt to further identify whether the antiproliferative activities of 16b was derived from an
interaction with tubulin, we examined the effect of 16b on the cellular microtubule network via tubulin
immunostaining. As shown in Fig. 5, the fibrous microtubules (green) and the cell nucleus (blue) were
visualized upon immunofluorescence staining of A549 cells. CAꢀ4 (2) at 24 nM concentration could
depolymerize cellular microtubules. 16b displayed at 160 nM concentration a similar effect in
microtubule depolymerization.
Fig. 5. CAꢀ4 (2) and 16b depolymerise the microtubule network of A549 cancer cells. Untreated cells
(Control) and cells treated with CAꢀ4 or 16b for 48 h, were fixed and stained with antiꢀαꢀtubulinꢀFITC
specific antibodies followed by DAPI. Microtubules and unassembled tubulin are shown in green, and
nuclei, which were stained with DAPI, are shown in blue.
Cell cycle analysis
Because the inhibition of tubulin polymerization is often implicated in G2/M phase arrest in various
cancer cell lines, the effect of 16b at various concentrations on cell cycle progression was investigated
in A549 cells via flow cytometry (Fig. 6). The effect of CAꢀ4 (2) on the cell cycle of A549 cells was
also comparatively examined as a positive control. Cell cycle analysis revealed that the cells
accumulated in G2/M phase of the cell cycle in a concentrationꢀdependent manner, which was

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consistent with the behavior of CAꢀ4 (2), the tubulinꢀbinding agent.
Fig. 6. (A) Cell cycle distribution of A549 cancer cells treated for 12 h with different concentrations of
16b (B) The statistical graph of cell cycle distribution.
Molecular modelling
Due to the high structural similarity with SMART which inhibits tubulin polymerization by binding to
the colchicine site in tubulin, based on the tubulin immunostaining studies described above, we believe
that the target compounds also bind to the colchicine binding site in tubulin. Therefore, the theoretical
binding mode of 16b, a highly potent compound, at the colchicine site in the tubulin dimer was
investigated by using the CDOCKER program in the Discovery Studio 3.0 software. We tried to dock
E and Z isomeric forms of 16b into two different tubulin crystal structures (PDB ID code: 1SA0 or
3HKD).16,20,25 The Z isomer is more stable than the E isomer by forming an intramolecular hydrogen
bond between protonated oxime and 2 position nitrogen atom on 1,2,4ꢀoxadiazole (Fig. 7B). Hence, the
docking result only showed the poses of (Z)-16b. In addition, (Z)-16b demonstrated best CDOCKER

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docking score of ꢀ55.57 in 3HKD compared with a score of ꢀ44.27 in 1SA0. This result is consistent
with the previous literature that TN16ꢀtubulin complex (3HKD) fits the scaffolds of RABI better than
the colchicineꢀtubulin complex (1SA0).6
The overview of the binding site of (Z)-16b and TNꢀ16 (the native ligand) in relation to the α,βꢀ
tubulin dimer is shown in Fig. 7A, and Fig. 6B illustrated the close view of the potential binding pose.
As shown in Fig. 7B, the binding orientations of (Z)-16b (yellow) superimposed well with TNꢀ16
(green) in the binding pocket. Furthermore, the key residue βꢀVAL238 forms the hydrogen bonds
with oxime OH of (Z)-16b and the pyrrolidinedione NH of the native ligand, respectively. The
3,4,5ꢀtrimethoxybenzene group (A ring) of (Z)-16b extends toward the α,βꢀtubulin interface, and the
oxygen of one methoxyl group forms a hydrogen bond with the residue αꢀTHR179. The results of the
computational modelling analysis further support the above results of the immunofluorescence studies.
Fig. 7. (A) The overview of the binding modes of 16b and the native ligand TNꢀ16 in tubulin crystal
structure 3HKD. (B) The close view of the potential binding pose of 16b and TNꢀ16 in 3HKD. The
dashed red lines represent hydrogen bonds, and the distance between ligands and protein is less than
2.7 Å.
Conclusions
In summary, we have described herein a series of novel inhibitors of tubulin polymerization, based on
the 1,2,4ꢀoxadiazole molecular skeletons, which were derived from thiazole ring modification of
SMART structures. Among them, 16b exhibited excellent antiproliferative activity with IC50 values at
the submicromolar level against three human cancer cell lines. The results show that oxime as a
connector between “B” ring and “C” ring played an important role for the high potency. The in vitro
immunofluorescence staining assay demonstrated that 16b, similar to CAꢀ4 (2), caused microtubule
destabilization. Besides, 16b could also arrest cancer cell cycle in the G2/M phase in A549 cells and

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subsequently induced cell apoptosis. A docking study was then performed and offered some insights
into the further modification of the target compounds. Our work laid the foundation for further
development of novel tubulin polymerization inhibitors.

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Experimental Section
Chemistry
General considerations: Unless otherwise noted, all materials were obtained from commercially
available sources and were used without purification. The reaction process was monitored by TLC with
silica gel plates (thickness 250ꢁm, Indicator Fꢀ254) under UV light. The products were purified using
column chromatography (60 Å, 200ꢀ300 mesh, Qingdao Ocean Chemicals) or silica gel plates (0.25
mm layer, Qingdao Ocean Chemicals) with the designated solvents. The melting points were measured
on a hotꢀstage microscope (Xꢀ4, Beijing Taike Ltd.) and are uncorrected. Mass spectra (MS) were
measured on an Agilent 1100ꢀsl mass spectrometer with an electrospray ionization source from Agilent
Co. Ltd. High resolution accurate mass determinations (HRMS) for some target compounds were
obtained on a Bruker Micromass Time of Flight mass spectrometer equipped with electrospray
ionization (ESI). NMR spectra were obtained on a Bruker AVANCE 300 (1H, 300 MHz; 13C, 75 MHz)
and Bruker AVANCE 600 (1H, 600 MHz; 13C, 150 MHz) in CDCl3 or DMSOꢀd6 (internal standard
tetramethylsilane).
General synthetic procedures for amidoxime (18a–h)
The stirring solution of appropriate benzonitrile (3.6 mmol) and hydroxylamine hydrochloride (25.2
mmol) in water was basified with a 1 M NaOH solution to pH 10. The mixture was stirred at 30°C for
1 hour, then under reflux for another 2 hours to full conversion and checked by TLC. After the reaction
was completed, the mixture was cooled to RT. The precipitate was filtered and washed with water,
which was used for the next step without further purification.
General synthetic procedures for (3-aryl-1,2,4-oxadiazole-5-yl)(3,4,5-trimethoxyphenyl)methanol
(20a–h)
The 3,4,5ꢀtrimethoxymandelic acid 19 (0.94 mmol), appropriate amidoxime (0.94 mmol), HOBt (1.04
mmol) and EDC.HCl (0.94 mmol) in dry acetonitrile (0.5 mL) were stirred together for 30 minutes in a
microwave cavity (closed vessel mode). After adding the dry toluene (3 mL), microwave irradiation at
150 W was used and the mixture was held at 120°C for 30 minutes. After the reaction was completed,
the mixture was poured into water (20 mL) and extracted with CH2Cl2 (20 mL × 3). The combined
organic layers were washed with water, dried over Na2SO4, and the solvent was then evaporated under

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reduced pressure. The residue was purified by column chromatography (nꢀhexane:EtOAc = 2:1 as
eluent) on silica gel to afford pure product.
General synthetic procedures for 3-aryl-5-benzoyl-1,2,4-oxadiazole (11a–h)
To a solution of compound 20a–h (1 mmol) in CH2Cl2 (5 mL) was added PCC (2 mmol), then the
mixture was stirred for 2ꢀ3 hours. When the reaction was completed (TLC control), the mixture was
poured into water (20 mL) and extracted with CH2Cl2 (20 mL × 3). The combined organic layers were
washed with water, dried over Na2SO4, and the solvent was then evaporated under reduced pressure.
The residue was purified by column chromatography (CH2Cl2 as eluent) on silica gel to afford pure
product.
General synthetic procedures for compounds (12a–h)
An excess of hydroxylamine hydrochloride was added to a solution of compound 11a–h (1 mmol) and
sodium acetate (10 mmol) in absolute ethanol (5 mL). The mixture was heated at 80°C for 5 hours.
When the reaction was completed (TLC control), the mixture was poured into water (20 mL) and
extracted with EtOAc (20 mL × 3). The combined organic layers were washed with water, dried over
Na2SO4, and the solvent was then evaporated under reduced pressure. The residue was purified by
column chromatography (methanol: CH2Cl2 = 1:50 as eluent) on silica gel to afford pure product.
(Z+E)-[3-(4-Methyphenyl)-1,2,4-oxadiazole-5-yl](3,4,5-trimethoxyphenyl)methanone methoxime
(13b)
To a solution of anhydrous K2CO3 (2 mmol) and dimethyl sulfate (1 mmol) in DMF (5 mL) was added
dropwise the solution of compound 12b (1 mmol) in DMF (0.5 mL) and the mixture was heated under
stirring at 50°C for 3h. The reaction was diluted with H2O (100 mL) then extracted with EtOAc (3×100
mL) and the combined organic phases were washed with H2O (3×100 mL) and brine (50 mL), dried
over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography
(nꢀhexane:EtOAc = 2:1 as eluent) on silica gel to afford pure product.
2-Hydroxy-2-(3,4,5-trimethoxyphenyl)acetonitrile (22)
Sodium cyanide (2.5 g, 51 mmol) was dissolved in water (100 mL). 3,4,5ꢀTrimethoxylbenzaldehyde
(10 g, 51 mmol) was added, and the mixture was stirred for 5 minutes at 0°C. Then, a solution of

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sodium disulfite (9.69 g, 51 mmol) in water (150 mL) was added, and the mixture stirred for 1 hour at
RT. The white solid product was filtered off and used in the next step without purification.
2-[(Tetrahydro-2H-pyran-2-yl)oxy]-2-(3,4,5-trimethoxyphenyl)acetonitrile (23)
In a roundꢀbottomed flask maintained under N2, appropriate amounts of compound 22 (4 mmol) and
3,4ꢀdihydroꢀ2Hꢀpyran (10 mmol) as well as 1.5 mol% of pꢀTsOH·H2O were dissolved in dry toluene
(30 mL). The resulting mixture was heated to 55°C for 2 hours. The solvent was removed under
reduced pressure and the residue was extracted with EtOAc (20 mL × 3) and washed with water, dried
over anhydrous sodium sulfate and concentrated under vacuum to afford crude product which was used
for the next step without further purification.
N-hydroxy-2-[(tetrahydro-2H-pyran-2-yl)oxy]-2-(3,4,5-trimethoxyphenyl)acetimidamide (24)
To a solution of sodium (9.8 mmol) in absolute methanol (10 mL) was added hydroxylamine
hydrochloride (9.8 mmmol) followed by compound 23 (6.5 mmol). After stirring for 5 hours, the
mixture was poured into water (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic
layers were washed with water, dried over Na2SO4, and the solvent was then evaporated under reduced
pressure. The residue was purified by column chromatography (nꢀhexane:EtOAc = 1:1 as eluent) on
silica gel to afford pure product. Brown oil (3.64 g, 79%).
General synthetic procedure for compounds (26a–c)
Sodium metal (1.0 mmol) was added into absolute ethanol (6 mL) and stirred under N2. After 10
minutes at RT, corresponding methyl benzoate 23a–c (1.0 mmol) and compound 24 (1.0 mmol) were
added to the mixture, which was then heated under reflux for 3 hours. When the reaction was
completed (TLC control), the mixture was cooled to RT. The residue was dissolved in a mixture of 10
mL of water and HCl aq. (3 mL, 12 M) and stirred at RT for 1 h. When the reaction was completed
(TLC control), the mixture was poured into water (20 mL) and extracted with EtOAc (20 mL × 3). The
combined organic layers were washed with water, dried over Na2SO4, and the solvent was then
evaporated under reduced pressure. The residue was purified by column chromatography
(nꢀhexane:EtOAc = 1:1 as eluent) on silica gel to afford pure product.
General synthetic procedures for 3-aryl-5-benzoyl-1,2,4-oxadiazole (15a–c)

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To a solution of compound 24a–c (1 mmol) in CH2Cl2 (5 mL) was added PCC (2 mmol), then the
mixture was stirred for 2ꢀ3 hours. When the reaction was completed (TLC control), the mixture was
poured into water (20 mL) and extracted with CH2Cl2 (20 mL × 3). The combined organic layers were
washed with water, dried over Na2SO4, and the solvent was then evaporated under reduced pressure.
The residue was purified by column chromatography (CH2Cl2 as eluent) on silica gel to afford pure
product.
Biology
Cell culture: The human gastric adenocarcinoma cell line SGCꢀ7901, the human fibrosarcoma cell line
HTꢀ1080 and the human pulmonary carcinoma cell line Aꢀ549 were cultured in RPMIꢀ1640 medium
containing 10% FBS, 100 U/mL streptomycin and 100 U/mL penicillin at 37°C in a humidified
atmosphere containing 5% CO2. All cell lines were purchased from the American Type Culture
Collection (ATCC, Manassas, VA).
MTT assay: The in vitro antiproliferative activities of CAꢀ4 (2), Nocodazole (4) and all of the target
compounds were determined with the MTT (Sigma) assay. Briefly, cells were seeded into 96ꢀwell
plates at a density of 1ꢀ3 × 104/well (depending on the cell growth rate). Twentyꢀfour hours later,
triplicate wells were treated with media and the agents. After 72 h of incubation at 37°C in 5% CO2, the
drugꢀcontaining medium was removed and replaced with 100 mL of fresh medium containing 5 mg/mL
MTT solution. After 4 h of incubation, the medium with MTT was removed, and 100 mL of dimethyl
sulphoxide (DMSO) was added to each well. The plates were gently agitated until the purple formazan
crystals were dissolved, and the OD490 was determined using a microplate reader (MK3, Thermo,
Germany). The data were calculated and plotted as the per cent viability compared to the control. The
50% inhibitory concentration (IC50) was defined as the concentration that reduced the absorbance of the
untreated wells by 50% of the vehicle in the MTT assay.
Immunofluorescence assay: Immunostaining was carried out to detect the microtubuleꢀassociated
tubulin protein after exposure to CAꢀ4 (2) and the investigated compound 16b. A549 cells were seeded
at 1 × 104 cells per well on a 24ꢀwell plate and grown for 24 h. The cells were treated with the vehicle
or twice the IC50 concentration of CAꢀ4 (2) or 16b for 12 h. The control and treated cells were fixed
with 4% formaldehyde in PBS for 30 min at ꢀ20 °C, washed twice with PBS and permeabilized with

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0.1% (v/v) Triton Xꢀ100 in PBS for 5 min. The cells were then blocked with 5% bovine serum albumin
(BSA) in PBS for 10 min. The primary αꢀtubulin antibody (Santa Cruz, CA) was diluted (1:100) with 2%
BSA in PBS and incubated overnight at 4°C. The cells were washed with PBS to remove unbound
primary antibody, and the cells were then incubated with FITCꢀconjugated antimouse secondary
antibody and diluted (1:1000) with 2% BSA in PBS for 3 h at 37°C. The cells were washed with PBS
to remove unbound secondary antibody, the nuclei were stained with 4,6ꢀdiaminoꢀ2ꢀphenolindol
dihydrochloride (DAPI) and immunofluorescence was then detected using a fluorescence microscope
(Olympus, Tokyo, Japan).
Cell cycle analysis: A549 cells (8 × 104 cells) were incubated with various concentrations of CAꢀ4 (2),
16b or 0.05% DMSO for the indicated times. The cells were collected by centrifugation, washed with
PBS and fixed in iceꢀcold 70% ethanol. The fixed cells were harvested by centrifugation and
resuspended in 500 ꢁl of PBS containing 1 mg/mL RNase. After 30 min of incubation at 37°C, the cells
were stained with 50 mg/mL propidium iodide (PI) at 4°C in the dark for 30 min. The samples were
then analyzed by FACScan flow cytometry (BectonꢀDickinson, Franklin Lakes, NJ, USA). The
experiments were repeated at least three times.
Molecular modelling
The molecular modelling studies were performed with Accelrys Discovery Studio 3.0. The crystal
structures of tubulin complexed with DAMAꢀcolchicine (PDB: 1SA0) and TN16 (PDB: 3HKD) were
retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb). In the docking process, the
protein protocol was prepared via several operations, including the standardization of atom names,
insertion of missing atoms in residues and removal of alternate conformations, insertion of missing
loop regions based on SEQRES data, optimization of short and medium sized loop regions with the
Looper Algorithm, minimization of remaining loop regions, calculation of pK, and protonation of the
structure. The receptor model was then typed with the CHARMm force field, and a binding sphere with
radius of 9.0 Å was defined with the original ligand (DAMAꢀcolchicine or TN16) as the binding site.
The 16b was drawn with Chemdraw and fully minimized using the CHARMm force field. Finally, 16b
was docked into the binding site using the CDOCKER protocol with the default settings.

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Author information
Corresponding Authors*
For Weige Zhang: Tel, +86 (024)23986422; Fax, +86 (024)23986422; Email:
zhangweige2000@sina.com
For Yingliang Wu: Tel, +86 (024)23986443; Fax, +86 (024)23986278; Email:
yingliang_1016@163.com
Acknowledgements
We gratefully acknowledge the National S&T Major Project (2012ZX09103101ꢀ060), the Liaoning
S&T Project (2013010434ꢀ401), and the Excellent Talent Supporting Project of Liaoning Province
(LR20013046) for their generous financial support. This work was also supported by the Program for
Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research
Team in University.

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