Synthesis and biological evaluation of novel 3,4-diaryl-1,2,5-selenadiazol analogues of combretastatin A-4

Article abstract of DOI:10.1016/j.ejmech.2014.09.046

A set of novel selenium-containing heterocyclic analogues of combretastatin A-4 (CA-4) have been designed and synthesised using a rigid 1,2,5-selenadiazole as a linker to fix the cis-orientation of ring-A and ring-B. All of the target compounds were evalu

Full text of DOI:10.1016/j.ejmech.2014.09.046

European Journal of Medicinal Chemistry 87 (2014) 1e9
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel 3,4-diaryl-1,2,5-
selenadiazol analogues of combretastatin A-4
Qi Guan a, Fushan Yang a, Dandan Guo b, Jingwen Xu b, Mingyang Jiang a, Chunjiang Liu b,
,
,
, *
Kai Bao a c, Yingliang Wu b **, Weige Zhang a
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 Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of novel selenium-containing heterocyclic analogues of combretastatin A-4 (CA-4) have been
designed and synthesised using a rigid 1,2,5-selenadiazole as a linker to fix the cis-orientation of ring-A
and ring-B. All of the target compounds were evaluated for their in vitro anti-proliferative activities.
Among these compounds, compounds 3a, 3i, 3n and 3q exhibited superior potency against different
tumour cell lines with IC50 values at the nanomolar level. Moreover, compound 3n significantly induced
cell cycle arrest in the G2/M phase, inhibited tubulin polymerisation into microtubules and caused
microtubule destabilisation. A molecular modelling study of compound 3n was performed to elucidate
its binding mode at the colchicine site in the tubulin dimer and to provide a basis for the further
structure-guided design of novel CA-4 analogues.
Received 5 August 2014
Received in revised form
30 August 2014
Accepted 12 September 2014
Available online
Keywords:
Synthesis
Anti-proliferative activity
Tubulin
© 2014 Elsevier Masson SAS. All rights reserved.
Combretastatin A-4
3,4-Diaryl-1,2,5-selenadiazol
Molecular modelling
1. Introduction
Combretastatin A-4 (1a, Fig. 1) is a natural product that was first
extracted from the bark of the South African willow tree Combretum
Microtubules, the cytoskeletal polymers of
a
- and
b
-tubulin
caffrum in 1989 and inhibits tubulin polymerisation by interacting
with the colchicine binding site on tubulin [7e9]. This cis-stilbene
shows potent cytotoxicity against a broad spectrum of human
cancer cell lines, including multidrug resistant cancer cell lines [10].
Its soluble prodrug CA-4P (1b, Fig. 1) is currently under Phase II/III
clinical evaluation in the USA as an agent used in combination
treatment for various multidrug resistant solid tumours. Because of
the structural simplicity of CA-4 (1a), large structure-activity re-
lationships (SARs) studies of this compound and its analogues have
been carried out by numerous academic and industrial groups
[10,11]. SAR studies have demonstrated that the cis-orientation of
the double bond and the presence of a 3,4,5-trimethoxyphenyl as
ring-A are fundamental requirements for potent cytotoxicity [10].
Unfortunately, CA-4 (1a) and other olefinic analogues are prone to
isomerisation into their inactive trans-forms during storage and
administration. To avoid the stability problems of CA-4 (1a), suit-
able conformationally restricted rings, such as heterocyclic rings,
have been used in place of the olefin group between ring-A and
ring-B [12e14]. In 2005, G. Sorba and co-workers reported that the
heterodimers, have an essential role in vital cellular functions, such
as motility, division, shape maintenance and intracellular transport
[1,2]. Interfering with either the assembly or disassembly of mi-
crotubules and spindle poisons arrests dividing cells in the G2/M
phase of the cell cycle to result mitotic catastrophe and finally
apoptotic cell death [1,3]. Therefore, anticancer therapy based on
microtubule targeting agents (MTAs) is receiving growing attention
in drug discovery [4,5]. MTAs are known to interact with tubulin via
at least four binding sites: the laulimalide, taxane (stabilisers of the
microtubules), vinca alkaloid and colchicine sites (destabilisers of
the microtubules). Given the success of taxanes and vinca alkaloids,
which have established tubulin as a valid target in cancer therapy,
research efforts have been focused on developing colchicine bind-
ing site agents for cancer treatment [6].
* Corresponding author.
** Corresponding author.
E-mail addresses: yingliang_1016@163.com (Y. Wu), zhangweige2000@sina.com
(W. Zhang).
http://dx.doi.org/10.1016/j.ejmech.2014.09.046
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

2
Q. Guan et al. / European Journal of Medicinal Chemistry 87 (2014) 1e9
Fig. 1. Combretastatin A-4 (CA-4, 1a), its water-soluble derivate (CA-4P, 1b), furazan analogue (2) and general structure of the target compounds.
cis-locked analogue (2, Fig. 1) of CA-4 embodying a furazan ring
exhibited very potent antitumour activity [15].
As illustrated in Table 1, most of the target compounds showed
moderate-to-excellent anti-proliferative activity against different
Recently, the application of organoselenium compounds in
cancer prevention and treatment has become a fascinating field
[16e19]. A large body of evidence shows that selenium effectively
inhibits tumourigenesis in both animal models and epidemiological
studies [19]. As shown in Fig. 1, we hope that the isosteric substi-
tution of the oxygen atom in the furazan ring by selenium retains
the structural features required for its potent activity. In this study,
we report the synthesis, biological evaluation and the preliminary
SARs of 3,4-diaryl-1,2,5-selenadiazole analogues of CA-4.
cancer cells, with potencies in the single-digit mM range, indicating
that the introduction of the 1,2,5-selenadiazole moiety as a suitable
mimic for the cis-olefin present in CA-4 (1a) maintained potent
anti-proliferative activity. A comparison of compounds 3a, 3i, 3l
and 3o revealed a substitution effect at C4 of ring-B. Compound 3a,
which contains a methoxy group, showed the most potent activity
against SGC-7901, HT-1080 and KB cells, with IC50 values of 0.063,
0.084 and 0.031 mM, respectively. The conversion of the 4-OMe to
4-OEt (3i) resulted in a 5 ~ 12-fold decrease in potency. When the 4-
Br and 4-OH were incorporated, the anti-proliferative activities of
compounds 3l and 3o were almost lost. In the ring-B modification,
additional eNH2 and eOH groups accompanied by a 4-OMe were
found to improve the biological activity. However, compound 3c,
which contains a nitro group accompanied by a 4-OMe group,
displayed a dramatic decrease in its biological activity, indicating
that the electron-withdrawing NO2 group is disfavoured. A similar
phenomenon was observed for compound 3j, which contains a
bromine atom at the C3 position. The addition of an extra benzyl
group or methyl group, as in compound 3b or 3s, led to decrease in
biological activity. Moreover, switching ring-B from 3-amino-4-
2. Results and discussion
2.1. Chemistry
The synthetic methods for the preparation of target compounds
are summarised as follows (Scheme 1). In this synthetic work, we
opted for a synthetic route that involves an initial umpolung of the
substituted benzaldehydes 5 or 6 via transformation to their cor-
responding
a-hydroxyphosphonates 7 or 8. Next, the hydroxyl
group was protected by 3,4-dihydro-2H-pyran (THP), followed by a
Horner-Wadsworth-Emmons (HWE)-type reaction with a suitable
second benzaldehyde, yielding deoxybenzoins 11aeg and 12aed
[14,20]. Compounds 13aeg and 14aed were synthesised in a mi-
crowave (MW) reactor by treating the appropriate deoxybenzoin
with selenium(IV) oxide in dimethylsulphoxide (DMSO) at 150 ꢀC
(150 W) for 2 min, and the following transformations into dioxime
15aeg and 16aed were performed in an excess of hydroxylamine
hydrochloride and pyridine in refluxing ethanol for 3 days [15,21].
These intermediates were then directly used for the next step to
furnish the desired 1,2,5-selenadiazoles 3ael and 4aed upon
dehydrating with selenium(IV) oxide in refluxing N,N-dime-
thylformamide (DMF) [22]. Subsequently, the deprotection of 3b,
3eeg and 4b with AlCl3 afforded 3mep and 4n at yields of 75e89%
[23], and the amino-substituted 3q, 3r and 4q were obtained by
reducing the nitro group of 3c, 3h and 4c in a refluxing Fe(OH)2
solution [24]. Finally, 3q was alkylated with methyl iodide in DMF
to provide 3s.
methoxyphenyl (3n) to b-naphthalene (3d) resulted in a signifi-
cant loss of activity. When ring-A was replaced with a 3,4-
methylenedioxyphenyl, compounds 4a, 4c, 4d, 4n and 4q showed
much lower activities. Consequently, this finding indicates that the
combination of the 3,4,5-trimethoxyphenyl with the 3-hydroxy-4-
methoxyphenyl (or 3-amino-4-methoxyphenyl) is the most
favourable modification for the anti-proliferative activity of 3,4-
diaryl-1,2,5-selenadiazole analogues.
2.2.2. Inhibition of tubulin polymerisation
To confirm that the anti-proliferative activities of these com-
pounds were related to the microtubule system, compound 3n, one
of the most active compounds in this series of CA-4 analogues, was
evaluated for its inhibition of tubulin polymerisation (Fig. 2).
Compound 3n exhibited antitubulin activity, with an IC50 value of
2.89
(IC50 ¼ 0.93
merisation of tubulin in
(Fig. 2). Treatment with 1, 2, 4 and 8
m
M, which is slightly less potent than that of CA-4
M). Furthermore, compound 3n inhibited the poly-
concentration-dependent manner
M of compound 3n inhibited
m
a
m
tubulin polymerisation by 20.4%, 48.83%, 65.0% and 97.6%,
respectively.
2.2. Biological evaluation
2.2.1. In vitro anti-proliferative activity
In an attempt to evaluate the ability of various 3,4-diaryl-1,2,5-
selenadiazole derivatives to inhibit cancer cells, the target com-
pounds 3aee, 3ies, 4a, 4c, 4d, 4n, 4q and reference compound CA-4
(1a) were screened for anti-proliferative activity against three hu-
man cancer cell lines (gastric adenocarcinoma SGC-7901 cells,
fibrosarcoma HT-1080 cells, and mouth epidermal carcinoma KB
cells) using a standard MTT assay (Table 1). The reported IC50 values
are the average of at least three independent experiments.
2.2.3. Analysis of immunofluorescence staining
In an attempt to further identify the cellular changes that may
be relevant to the anti-proliferative activity of compound 3n, we
evaluated its effect on the microtubule network via tubulin im-
munostaining. A microscopic analysis of HT-1080 cells stained with
a-tubulin antibodies demonstrated a well organised microtubular
network in control cells (Fig. 3). As visualised in Fig. 3 and similar to
CA-4 (1a), compound 3n at a concentration that was twice the IC50

Q. Guan et al. / European Journal of Medicinal Chemistry 87 (2014) 1e9
3
Scheme 1. Reagents and conditions: (i) Dimethyl phosphite, NaOMe, MeOH, 50 ꢀC; (ii) TsOH cat., 3,4-dihydro-2H-pyran (THP), anhydrous toluene, under N2 atmosphere, 50 ꢀC; (iii)
(a) n-BuLi, anhydrous THF, ꢁ55 ꢀC; (b) Substituted benzaldehydes, anhydrous THF, ꢁ55e0 ꢀC; (c) HCl aq. (12 M), MeOH, rt.; (iv) Selenium(IV) oxide, DMSO, microwave, 150 ꢀC,
150 W, 2 min; (v) NH2OH$HCl in Py/EtOH, 90 ꢀC; (vi) Selenium(IV) oxide, DMF, reflux; (vii) AlCl3, CH2Cl2, 0 ꢀC; (viii) Fe(OH)2, reflux; (ix) MeI, K2CO3, DMF, 60 ꢀC.
significantly impacted the microtubule network in HT-1080 cells
and induced cell rounding, detachment, and a loss of the radial
distribution of structured microtubules. These results indicate that
compound 3n can destabilise microtubules.
computational modelling analysis further support the results of the
inhibition of tubulin polymerisation: compound 3n does bind to
the colchicine-binding site of microtubules.
3. Conclusion
2.2.4. Cell cycle analysis
Because the inhibition of tubulin polymerisation is often
implicated in G2/M phase arrest in various cancer cell lines, the
effect of compound 3n at various concentrations on cell cycle
progression was investigated in HT-1080 cells via flow cytometry
(Fig. 4). The effect of CA-4 (1a) on the cell cycle of HT-1080 cells was
also comparatively examined as a positive control. After 24 h of
exposure, compound 3n was found to obviously increase the per-
centage of cells in the G2/M and sub-G1 phases, with a concomitant
decrease in cells in the G0/G1 and S phases. Compound 3n was
demonstrated to clearly cause significant G2/M phase arrest in a
concentration-dependent manner, which was consistent with the
behaviour of CA-4 (1a), the tubulin-binding agent.
To the best of our knowledge, we designed and synthesised new
selenium-containing heterocyclic analogues of CA-4 for the first
time; this process involved a rigid 1,2,5-selenadiazole as linker to
fix the cis-orientations of ring-A and ring-B. Of these compounds,
compounds 3a, 3i, 3n and 3q exhibited excellent anti-proliferative
activities with IC50 values at the nanomolar level, especially com-
pounds 3n and 3q, which were more potent than the standard CA-4
(1a). The in vitro immunofluorescence staining and tubulin poly-
merisation assay demonstrated that compound 3n, similar to CA-4
(1a), inhibited tubulin polymerisation into microtubules and
caused microtubule destabilisation, which suggests that compound
3n acts as a microtubule destabiliser by disrupting tubulin-
microtubule dynamics. Furthermore, a flow cytometric study
showed that compound 3n significantly induced cell cycle arrest in
the G2/M phase in HT-1080 cells. A docking study was then per-
formed and offered some insights into the further modification of
the target compounds. In summary, this work not only widens the
exploration of olefin modification but also provides a series of
useful analogues of CA-4 (1a) that are potent anti-proliferative
agents.
2.3. Molecular modelling
To better understand the interactions between the newly syn-
thesised compounds and tubulin, the potential binding mode of
compound 3n, 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 with the tubulin crystal
structure (PDB: 1SA0) [25,26], as in our previous study. To compare
the binding features of compound 3n with CA-4 (1a), both com-
pounds were docked into tubulin. In the binding models shown in
Fig. 5A, the binding orientations of compound 3n (purple) and CA-4
(orange) superimposed well with each other and with DAMA-
colchicine (cyan), the ligand used in the tubulin crystal structure.
4. Experimental
4.1. Chemistry
4.1.1. General
As evident in Fig. 5A, a hydrogen bond is present between Val
in the -T5 loop of tubulin and the hydroxyl group on ring-B of
compound 3n and CA-4 (1a). This hydrogen bond is also observed
between Val 181 and the carbonyl group on ring-C of DAMA-
a
181
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
a
a
(thickness 250 mm, Indicator F-254) under UV light. The products
colchicine. In addition, the trimethoxybenzene rings of these
compounds are well overlapped with each other, and hydrogen
bonds formed between the oxygen of two methoxyl groups and the
were purified using column chromatography (60 Å, 200e300
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 hot-stage microscope (X-4,
key residue Cysb241 in the active site. The results of the

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Q. Guan et al. / European Journal of Medicinal Chemistry 87 (2014) 1e9
Table 1
with ethyl acetate (100 mL ꢂ 3). The combined organic layer was
washed with water and brine and then dried over Na2SO4, filtered
and concentrated to yield the crude product, which was used
without further purification.
In vitro anti-proliferative activities of the target compounds as measured with MTT
test in three human tumour cell lines.
Compound
IC50 (m
M) SDa
SGC-7901
HT-1080
KB
4.1.3. General synthetic procedures for deoxybenzoins 11aeg and
12aed
In a round-bottomed flask maintained under a nitrogen atmo-
sphere, appropriate amounts of a-hydroxyphosphonate (10 mmol)
3a
3b
3c
3d
3e
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
0.063 0.007
>30
>30
2.35 0.44
>30
0.23 0.04
>30
12.79 0.51
10.87 0.32
>30
0.019 0.002
>30
0.084 0.002
>30
24.78 0.04
1.11 0.25
>30
0.16 0.01
2.59 0.18
3.46 0.32
7.40 0.27
5.98 0.33
0.021 0.006
14.98 0.13
16.40 0.83
0.026 0.008
10.09 0.22
0.98 0.01
>30
0.031 0.005
>30
10.10 0.03
0.77 0.16
10.73 0.17
0.42 0.03
3.06 0.24
0.93 0.02
7.86 0.14
>30
0.0039 0.0003
15.81 0.05
4.80 0.47
0.011 0.002
0.099 0.013
1.52 0.02
5.21 0.48
>30
and 3,4-dihydro-2H-pyran (17 mmol) as well as 1.5 mol% of p-
TsOH$H2O were dissolved in dry toluene (30 mL). The resulting
mixture was heated to 65 ꢀC for 2 h, and dry THF (5 mL) was then
added to the mixture. Upon cooling to ꢁ55 ꢀC, 10.5 mmol of n-
butyllithium (15% in hexane, 1.6 mol/L) was added dropwise to the
mixture, and the contents of the flask were stirred for 1 h under a
nitrogen atmosphere, after which the solvent for the appropriate
benzaldehyde (10 mmol) in anhydrous THF was added dropwise
and stirred for another 4 h. Upon solvent removal via rotary
evaporation, the residue was dissolved in a mixture of 20 mL of
MeOH and HCl aq. (3 mL, 12 M) and stirred at room temperature for
1 h. Next, the flask was stored at ꢁ10 ꢀC to allow the deoxybenzoin
to precipitate as a solid. Pure product as a white solid could be
obtained from this crude mixture via recrystallisation from meth-
anol (10 mL). For example:
>30
0.021 0.005
0.14 0.04
>30
8.28 0.46
>30
17.33 0.16
3.68 0.24
>30
3s
4a
4c
4d
4n
4q
1ab
>30
23.31 0.39
0.73 0.05
13.54 0.14
0.0076 0.0010
10.37 0.21
2.38 0.11
1.87 0.07
0.013 0.002
0.028 0.003
Bold values show the IC50 values of target compounds that are lower than those of
the positive control CA-4 (1a).
4.1.3.1. 1-(3,4,5-trimethoxyphenyl)-2-(naphthalen-2-yl)ethanone
a
IC50: Concentration of the compound (mM) producing 50% cell growth inhibition
(11d). White solid; yield: 73%; M.p.: 68e70 ꢀC; 1H NMR (300 MHz,
after 72 h of drug exposure, as determined by the MTT assay. Each experiment was
carried out in triplicate.
CDCl3):
d 7.80 (m, 3H, Ph-H), 7.72 (s, 1H, Ph-H), 7.45 (m, 2H, Ph-H),
b
Used as a positive control.
7.39 (d, J ¼ 8.3 Hz, 1H, Ph-H), 7.30 (s, 2H, Ph-H), 4.40 (s, 2H, COCH2),
3.89 (3H, s, OCH3), 3.86 (6H, s, OCH3). MS (ESI) m/z 337.2 [M þ H]þ,
359.1 [M þ Na]þ.
4.1.3.2. 1-(3,4-methylenedioxyphenyl)-2-[3-(benzyloxy)-4-
methoxyphenyl]ethanone (12b). White solid; yield: 79%; M.p.:
98e101 ꢀC; 1H NMR (300 MHz, CDCl3):
J2 ¼ 1.5 Hz, 1H, Ph-H), 7.46 (s, 2H, Ph-H), 7.42 (s, 1H, Ph-H), 7.35 (t,
d
7.56 (dd, J1 ¼ 8.2 Hz,
Fig. 2. Effects of 3n on tubulin polymerisation. Tubulin had been pre-incubated for
1 min with 3n at 1 mM, 2 mM, 4 mM and 8 mM, CA-4 at 2 mM, taxol at 5 mM or vehicle
DMSO at room temperature before GTP was added to start the tubulin polymerisation
reactions. The reaction was monitored at 37 ꢀC. Taxol and CA-4 were included as
positive controls.
Beijing Taike Ltd.) and are uncorrected. Mass spectra (MS) were
measured on an Agilent 1100-sl mass spectrometer with an elec-
trospray ionisation source from Agilent Co. Ltd. High resolution
accurate mass determinations (HRMS) for all final target com-
pounds were obtained on a Bruker Micromass Time of Flight mass
spectrometer equipped with electrospray ionisation (ESI). NMR
spectra were obtained on a Bruker AVANCE 400 (1H, 300 MHz) and
Bruker AVANCE 600 (13C, 150 MHz) in CDCl3 or DMSO-d6 (internal
standard tetramethylsilane).
Fig. 3. CA-4 (1a) and compound 3n depolymerise the microtubule network of HT-1080
cancer cells. Untreated cells (Control) and cells treated with CA-4 or compound 3n at
twice the IC50 concentration for 12 h, were fixed and stained with anti-a-tubulin-FITC
specific antibodies followed by DAPI. Microtubules and unassembled tubulin are
shown in green, and nuclei, which were stained with DAPI, are shown in blue. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
4.1.2. Synthesis of a-hydroxyphosphonates 7 and 8
A solution of an appropriate benzaldehyde (0.1 mol), dimethyl
phosphite (0.12 mol), and sodium methoxide (0.015 mol) in anhy-
drous methanol (25 mL) was vigorously stirred for 3 h at 50 ꢀC. The
solvent was then removed under reduced pressure and extracted

Q. Guan et al. / European Journal of Medicinal Chemistry 87 (2014) 1e9
5
Fig. 4. Compound 3n affects the cell cycle distribution in HT-1080 cancer cells. (A) Untreated cells (Control) and cells treated with compound 3n at different concentrations and CA-
4 at 25 nM concentration for 24 h; (B) The statistical graph of cell cycle distribution.
J ¼ 7.8 Hz, 2H, Ph-H), 7.29 (m, 1H, Ph-H), 6.83 (m, 4H, Ph-H), 6.01 (s,
2H, OCH2O), 5.11 (s, 2H, OCH2), 4.09 (s, 2H, COCH2), 3.85 (s, 3H,
OCH3). MS (ESI) m/z 377.3 [M þ H]þ, 399.3 [M þ Na]þ.
J2 ¼ 1.8 Hz, 1H, Ph-H), 7.94 (m, 3H, Ph-H), 7.65 (m, 1H, Ph-H), 7.57
(m, 1H, Ph-H), 7.28 (s, 2H, OCH3), 3.96 (s, 3H, OCH3), 3.89 (s, 6H,
OCH3). MS (ESI) m/z 351.2 [M þ H]þ, 373.3 [M þ Na]þ.
4.1.3.3. 1-(3,4-methylenedioxyphenyl)-2-(4-methoxy-3-nitrophenyl)
4.1.4.2. 1-(4-Methoxy-3-nitrophenyl)-2-(3,4-methylenedioxyphenyl)
ethanone (12c). Pale yellow solid; yield: 75%; M.p.: 143e147 ꢀC; 1H
ethane-1,2-dione (14c). Orange oil; yield: 68%; 1H NMR (300 MHz,
NMR (300 MHz, CDCl3):
d
7.76 (s, 1H, Ph-H), 7.62 (d, J ¼ 8.0 Hz, 1H,
CDCl3):
d
8.44 (d, J ¼ 8.3 Hz, 1H, Ph-H), 8.17 (dd, J1 ¼ 8.9 Hz,
Ph-H), 7.45 (d, J ¼ 12.1 Hz, 2H, Ph-H), 7.07 (d, J ¼ 9.0 Hz, 1H, Ph-H),
6.88 (d, J ¼ 8.1 Hz, 1H, Ph-H), 6.07 (s, 2H, OCH2O), 4.22 (s, 2H,
COCH2), 3.96 (s, 3H, OCH3). MS (ESI) m/z 316.2 [M þ H]þ, 338.1
[M þ Na]þ.
J2 ¼ 2.3 Hz, 1H, Ph-H), 7.50 (m, 2H, Ph-H), 7.20 (d, J ¼ 9.0 Hz, 1H, Ph-
H), 6.89 (d, J ¼ 8.0 Hz, 1H, Ph-H), 6.10 (s, 2H, OCH2O), 4.06 (s, 3H,
OCH3). MS (ESI) m/z 330.4 [M þ H]þ, 352.2 [M þ Na]þ.
4.1.5. General synthetic procedures for dioxime 15aeg and 16aed
An excess of hydroxylamine hydrochloride (10 mmol) was
added to a solution of an appropriate amount of benzil (1 mmol) in
dry pyridine (3 mL) and ethanol (7 mL). The resulting solution was
heated at 90 ꢀC for 3 days, worked up by dilution of EtOAc and
washed with HCl aq. (15 mL, 0.5 M), water (30 mL) and brine
(30 mL). After drying over Na2SO4 and the evaporation of the sol-
vent, the crude product was used without future purification.
4.1.4. General synthetic procedures for benzils 13aeg and 14aed
A mixture of an appropriate amount of deoxybenzoin (1 mmol)
and selenium dioxide (2 mmol) in DMSO (2 mL) was placed into the
MW cavity (closed vessel mode, Pmax ¼ 250 PSI). MW irradiation of
150 W was used, and the temperature was ramped from 25 to
150 ꢀC. Once 150 ꢀC was reached after approximately 1 min, the
reaction mixture was held at this temperature for 2 min while
stirring, then cooled, diluted with water, and extracted with ethyl
acetate; the organic layer was washed with brine, dried over
Na2SO4, and filtered. The removal of the solvent yielded a residue
that was purified by silica gel column chromatography (n-
hexane:dichloromethane ¼ 1:3 as eluent) to furnish 13aeg and
14aed. For example:
4.1.6. General synthetic procedures for 3,4-diaryl-1,2,5-
selenadiazoles 3ael and 4aed
A mixture of an appropriate amount of dioxime (1 mmol), se-
lenium dioxide (1.5 mmol), and N,N-dimethylformamide (10 mL)
was refluxed for 1 h with stirring. The reaction mixture was poured
into water and extracted with EtOAc (20 mL ꢂ 3). The combined
organic layer was washed with brine and dried over Na2SO4. After
filtration and the evaporation of the solvent, the crude product was
4.1.4.1. 1-(Naphthalen-2-yl)-2-(3,4,5-trimethoxyphenyl)ethane-1,2-
dione (13d). Pale yellow solid; yield: 71%; M.p.: 129e131 ꢀC; 1H
NMR (300 MHz, CDCl3):
d
8.41 (s, 1H, Ph-H), 8.10 (dd, J1 ¼ 8.8 Hz,

6
Q. Guan et al. / European Journal of Medicinal Chemistry 87 (2014) 1e9
Fig. 5. (A) Predicted modes for compound 3n (purple) and CA-4 (orange) binding in the colchicine-binding site of tubulin (PDB code: 1SA0), and overlapping with DAMA-colchicine
(cyan, the native ligand of 1SA0); (B) Docking conformation of compound 3n (purple) in the colchicine-binding site of tubulin. The dashed green lines represent hydrogen bonds,
and the distance between ligands and protein is less than 2.8 Å. Surrounding amino acid side chains are shown in grey stick format and labelled. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
purified by column chromatography (n-hexane: EtOAc ¼ 10:1 as
H), 5.01 (s, 2H, PhCH2), 3.87 (s, 3H, OCH3), 3.65 (s, 6H, OCH3). 13
NMR (150 MHz, CDCl3):
C
eluent) on silica gel to afford pure products.
d
164.6, 164.2, 152.9 (ꢂ2), 136.6, 128.6 (ꢂ2),
128.1, 127.3 (ꢂ2), 121.9, 116.2, 115.5, 106.6 (ꢂ2), 70.1, 60.9, 56.0 (ꢂ2).
MS (ESI): m/z 483.2 [M þ H]þ, 505.2 [M þ Na]þ.
4.1.6.1. 3-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-
selenadiazole (3a). White solid; yield: 44%; M.p.: 94e100 ꢀC; 1H
NMR (300 MHz, CDCl3):
J ¼ 8.7 Hz, 2H, Ph-H), 6.67 (s, 2H, Ph-H), 3.89 (s, 3H, OCH3), 3.83 (s,
3H, OCH3), 3.69 (s, 6H, OCH3). 13C NMR (150 MHz, CDCl3):
164.6,
d
8.85 (d, J ¼ 8.7 Hz, 2H, Ph-H), 6.86 (d,
4.1.6.6. 3-(4-ethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-
selenadiazole (3i). White solid; yield: 44%; M.p.: 139e143 ꢀC; 1H
d
NMR (300 MHz, CDCl3):
d
7.39 (d, J ¼ 8.7 Hz, 2H, Ph-H), 6.88 (d,
164.3, 160.3, 152.8 (ꢂ2), 138.8, 131,2, 130.6 (ꢂ2), 128.6, 113.6 (ꢂ2),
J ¼ 8.7 Hz, 2H, Ph-H), 6.67 (s, 2H, Ph-H), 4.05 (q, J ¼ 7.0 Hz, 2H,
OCH2CH3), 3.89 (s, 3H, OCH3), 3.69 (s, 6H, OCH3), 1.43 (t, J ¼ 7.0 Hz,
106.5 (ꢂ2), 60.9, 55.9 (ꢂ2), 55.3; HRMS (ESI): calcd for
18H18N2O4Se [M þ H]þ: 407.0510, found: 407.0517.
3H, OCH2CH3). 13C NMR (150 MHz, CDCl3):
d 164.6, 164.3, 159.6,
C
152.8 (ꢂ2), 138.8, 131.3, 130.6 (ꢂ2), 128.5, 114.1 (ꢂ2), 106.5 (ꢂ2),
63.5, 60.8, 55.9 (ꢂ2), 14.6; HRMS (ESI): calcd for C19H21N2O4Se
[M þ H]þ, C19H20N2NaO4Se [M þ Na]þ: 421.0667, 443.0486, found:
421.0687, 443.0482.
4.1.6.2. 3-[3-(benzyloxy)-4-methoxyphenyl]-4-(3,4,5-
trimethoxyphenyl)-1,2,5-selenadiazole (3b). White solid; yield: 47%;
M.p.: 132e138 ꢀC; 1H NMR (300 MHz, CDCl3):
d 7.31 (m, 5H, Ph-H),
7.04 (m, 2H, Ph-H), 6.86 (d, J ¼ 18.4 Hz,1H, Ph-H), 6.64 (s, 2H, Ph-H),
5.01 (s, 2H, OCH2), 3.91 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 3.69 (s, 6H,
4.1.6.7. 3-(3-bromo-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-
OCH3). 13C NMR (150 MHz, CDCl3):
d
164.4, 164.4, 152.9 (ꢂ2), 150.6,
1,2,5-selenadiazole (3j). White solid; yield: 39%; M.p.: 103e108 ꢀC;
1H NMR (300 MHz, CDCl3):
d
7.78 (d, J ¼ 1.9 Hz, 1H, Ph-H), 7.31 (dd,
147.8, 138.9, 136.6, 131.4, 128.6, 128.5 (ꢂ2), 127.9, 127.1 (ꢂ2), 122.8,
106.5 (ꢂ2), 71.0, 60.9, 56.0, 56.0 (ꢂ2); HRMS (ESI): calcd for
J1 ¼ 8.7 Hz, J2 ¼ 1.9 Hz, 1H, Ph-H), 6.85 (d, J ¼ 8.7 Hz, 1H, Ph-H), 6.67
C
25H24N2O5Se [M þ H]þ: 513.0929, found: 513.0906.
(s, 2H, Ph-H), 3.92 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.72 (s, 6H,
OCH3). 13C NMR (150 MHz, CDCl3):
d164.2, 162.9, 156.6, 153.0 (ꢂ2),
139.1, 134.2, 131.0, 129.7, 129.5, 111.4, 111.1, 106.6 (ꢂ2), 60.9, 56.3,
4.1.6.3. 3-(4-Methoxy-3-nitrophenyl)-4-(3,4,5-trimethoxyphenyl)-
1,2,5-selenadiazole (3c). Pale yellow solid; yield: 41%; M.p.:
56.0 (ꢂ2); HRMS (ESI): calcd for C18H18BrN2O4Se [M þ H]þ,
C
18H17BrN2NaO4Se [M
þ
Na]þ: 484.9615, 506.9435, found:
110e115 ꢀC; 1H NMR (300 MHz, CDCl3):
d
8.09 (d, J ¼ 2.2 Hz, 1H, Ph-
484.9617, 506.9429.
H), 7.60 (dd, J1 ¼8.8 Hz, J2 ¼ 2.3 Hz,1H, Ph-H), 7.06 (d, J ¼ 8.8 Hz,1H,
Ph-H), 6.65 (s, 2H, Ph-H), 3.99 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.74
(s, 6H, OCH3). 13C NMR (150 MHz, CDCl3):
d
164.1, 161.7, 153.2 (ꢂ2),
4.1.6.8. 3-(3-bromo-4-ethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-
1,2,5-selenadiazole (3k). White solid; yield: 42%; M.p.: 113e115 ꢀC;
139.4, 139.3, 134.5, 130.7, 128.4, 126.6, 113.0, 106.5 (ꢂ2), 61.0, 56.7,
56.1 (ꢂ2); HRMS (ESI): calcd for C18H18N3O6Se [M þ H]þ: 452.0361,
found: 452.0317.
1H NMR (300 MHz, CDCl3):
d
7.77 (d, J ¼ 1.9 Hz, 1H, Ph-H), 7.27 (dd,
J1 ¼ 8.4 Hz, J2 ¼ 1.9 Hz, 1H, Ph-H), 6.82 (d, J ¼ 8.4 Hz, 1H, Ph-H), 6.66
(s, 2H, Ph-H), 4.11 (q, J ¼ 6.9 Hz, 2H, OCH2CH3), 3.87 (s, 3H, OCH3),
3.72 (s, 6H, OCH3), 1.48 (t, J ¼ 6.8 Hz, 3H, OCH2CH3). 13C NMR
4.1.6.4. 3-(naphthalen-2-yl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-
selenadiazole (3d). White solid; yield: 48%; M.p.: 113e117 ꢀC; 1H
(150 MHz, CDCl3):
d
164.1, 162.9, 156.0, 152.9 (ꢂ2), 139.1, 134.2,
131.0,129.5,129.4,112.1,111.8,106.6 (ꢂ2), 64.8, 60.9, 56.0 (ꢂ2),14.5;
HRMS (ESI): calcd for C19H20BrN2O4Se [M þ H]þ, C19H19BrN2NaO4Se
[M þ Na]þ: 498.9772, 520.9591, found: 498.9746, 520.9572.
NMR (300 MHz, CDCl3):
(m, 3H, Ph-H), 6.69 (s, 2H, Ph-H), 3.86 (s, 3H, OCH3), 3.54 (s, 6H,
OCH3). 13C NMR (150 MHz, CDCl3):
d 8.03 (s, 1H, Ph-H), 7.82 (m, 3H, Ph-H), 7.51
d
164.8, 164.5, 152.9 (ꢂ2), 139.0,
133.6, 133.2, 132.8, 130.9, 129.1, 128.3, 127.8, 127.6, 127.0, 126.6,
126.2, 106.6 (ꢂ2), 60.9, 55.8 (ꢂ2); HRMS (ESI): calcd for
4.1.6.9. 3-(4-bromophenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-
selenadiazole (3l). White solid; yield: 40%; M.p.: 112e115 ꢀC; 1H
C
21H19N2O3Se [M þ H]þ, C21H18N2O3Se [M þ Na]þ: 427.0561,
449.0380, found: 427.0590, 449.0376.
NMR (300 MHz, CDCl3):
J ¼ 8.4 Hz, 2H, Ph-H), 6.62 (s, 2H, Ph-H), 3.89 (s, 3H, OCH3), 3.69 (s,
6H, OCH3). 13C NMR (150 MHz, CDCl3):
164.3, 163.6, 153.0 (ꢂ2),
d
7.52 (d, J ¼ 8.4 Hz, 2H, Ph-H), 7.35 (d,
4.1.6.5. 3-[3-(benzyloxy)phenyl]-4-(3,4,5-trimethoxyphenyl)-1,2,5-
d
selenadiazole (3e). Oil; yield: 45%; 1H NMR (300 MHz, CDCl3):
d
7.35
139.1, 135.1, 131.5 (ꢂ2), 130.7 (ꢂ2), 130.7, 123.7, 106.5 (ꢂ2), 60.9,
(m, 5H, Ph-H), 7.26 (m,1H, Ph-H), 7.10 (m,1H, Ph-H), 6.66 (s, 2H, Ph-
55.9 (ꢂ2); HRMS (ESI): calcd for
C
17H16BrN2O3Se [MþH]þ,

Q. Guan et al. / European Journal of Medicinal Chemistry 87 (2014) 1e9
7
C17H15BrN2NaO3Se [M
454.9502, 476.9277.
þ
Na]þ: 454.9510, 476.9329, found:
4.1.7.3. 3-(4-hydroxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-
selenadiazole (3o). White solid; yield: 85%; M.p.: 190e195 ꢀC; 1H
NMR (300 MHz, DMSO-d6):
J ¼ 8.6 Hz, 2H, Ph-H), 6.66 (s, 2H, Ph-H), 5.75 (s, 1H, Ph-OH), 3.69 (s,
3H, OCH3), 3.60 (s, 6H, OCH3); 13C NMR (150 MHz, CDCl3):
164.8,
d
7.21 (d, J ¼ 8.5 Hz, 2H, Ph-H), 6.78 (d,
4.1.6.10. 3-(4-methoxyphenyl)-4-(3,4-methylenedioxyphenyl)-1,2,5-
selenadiazole (4a). White solid; yield: 46%; M.p.: 98e101 ꢀC; 1H
d
164.4, 157.1, 152.9 (ꢂ2), 138.8, 131.3, 130.8 (ꢂ2), 128.4, 115.2 (ꢂ2),
106.6 ( ꢂ 2), 61.0, 55.9 (ꢂ2); HRMS (ESI): calcd for C17H17N2O4Se
[M þ H]þ, C17H16N2NaO4Se [M þ Na]þ: 393.0354, 415.0173, found:
393.0331, 415.0159.
NMR (300 MHz, CDCl3):
6.76 (d, J ¼ 8.0 Hz, 1H, Ph-H), 5.99 (s, 2H, OCH2O), 3.83 (s, 3H,
OCH3); 13C NMR (150 MHz, CDCl3):
138.8, 137.5, 130.7 (ꢂ2), 129.6, 121.2 (ꢂ2), 116.5, 116.1, 106.6, 55.9;
HRMS (ESI): calcd for C16H13N2O3Se [M þ H]þ: 361.0091, found:
361.0086.
d 7.39 (m, 2H, Ph-H), 6.89 (m, 4H, Ph-H),
d
164.7, 164.3, 156.1, 152.7 (ꢂ2),
4.1.7.4. 3-(3-bromo-4-hydroxyphenyl)-4-(3,4,5-trimethoxyphenyl)-
1,2,5-selenadiazole (3p). White solid; yield: 81%; M.p.: 148e153 ꢀC;
1H NMR (300 MHz, CDCl3):
d
7.69 (d, J ¼ 2.0 Hz, 1H, Ph-H), 7.27 (dd,
4 . 1 . 6 . 11 . 3 - ( 4 - m e t h o x y - 3 - n i t r o p h e n y l ) - 4 - ( 3 , 4 -
J1 ¼ 8.4 Hz, J2 ¼ 2.1 Hz, 1H, Ph-H), 6.97 (d, J ¼ 8.5 Hz, 1H, Ph-H), 6.68
methylenedioxyphenyl)-1,2,5-selenadiazole (4c). Brown solid; yield:
44%; M.p.: 58e63 ꢀC; 1H NMR (300 MHz, CDCl3):
d 8.01 (d,
(s, 2H, Ph-H), 5.91 (s, 1H, Ph-OH), 3.89 (s, 3H, OCH3), 3.72 (s, 6H,
OCH3). 13C NMR (150 MHz, CDCl3):
d
164.2, 162.9, 153.4, 153.0 (ꢂ2),
139.2, 133.2, 130.9, 130.1, 129.8, 115.7, 110.0, 106.6 (ꢂ2), 61.0, 56.1
(ꢂ2); HRMS (ESI): calcd for 17H16BrN2O4Se [M
H]þ,
17H15BrN2NaO4Se [M
Na]þ: 470.9459, 492.9278, found:
470.9441, 492.9276.
J ¼ 2.3 Hz, 1H, Ph-H), 7.63 (dd, J1 ¼ 8.8 Hz, J2 ¼ 2.3 Hz, 1H, Ph-H),
7.06 (d, J ¼ 8.8 Hz, 1H, Ph-H), 6.92 (d, J ¼ 1.6 Hz, 1H, Ph-H), 6.87
(dd, J1 ¼ 7.9 Hz, J2 ¼ 1.6 Hz, 1H, Ph-H), 6.80 (d, J ¼ 7.9 Hz, 1H, Ph-H),
6.02 (s, 2H, OCH2O), 4.00 (s, 3H, OCH3); 13C NMR (150 MHz, CDCl3):
C
þ
C
þ
d
164.0, 161.7, 153.4, 148.8, 147.9, 139.3, 134.6, 129.3, 128.4, 126.6,
123.4, 113.2, 109.4, 108.4, 101.5, 56.6; HRMS (ESI): calcd for
C
427.9762, found: 405.9905, 427.9740.
16H12N3O5Se [M þ H]þ, C16H11N3NaO5Se [M þ Na]þ: 405.9942,
4 . 1. 7 . 5 . 3 - ( 3 - h y d r o x y - 4 - m e t h o x y p h e n y l ) - 4 - ( 3 , 4 -
methylenedioxyphenyl)-1,2,5-selenadiazole (4n). White solid; yield:
86%; M.p.: 89e94 ꢀC; 1H NMR (300 MHz, CDCl3):
d 7.03 (d,
J ¼ 2.1 Hz, 1H, Ph-H), 6.93 (m, 3H, Ph-H), 6.79 (q, J ¼ 8.3 Hz, 2H, Ph-
4.1.6.12. 3-(naphthalen-2-yl)-4-(3,4-methylenedioxyphenyl)-1,2,5-
H), 5.99 (s, 2H, OCH2O), 3.91 (s, 3H, OCH3); 13C NMR (150 MHz,
selenadiazole (4d). White solid; yield: 47%; M.p.: 138e142 ꢀC; 1H
CDCl3):
d
164.3, 164.2, 148.4, 147.5 ( ꢂ 2), 145.3, 130.0, 129.4, 123.6,
NMR (300 MHz, CDCl3): d 8.03 (s, 1H, Ph-H), 7.83 (m, 3H, Ph-H), 7.51
121.5, 115.4, 110.2, 109.6, 108.1, 101.3, 55.9; HRMS (ESI): calcd for
(m, 3H, Ph-H), 6.99 (d, J ¼ 1.4 Hz, 1H, Ph-H), 6.89 (dd, J1 ¼ 8.0 Hz,
C
16H13N2O4Se [M þ H]þ: 377.0041, found: 377.0027.
J2 ¼ 1.4 Hz, 1H, Ph-H), 6.71 (d, J ¼ 8.0 Hz, 1H, Ph-H), 5.98 (s, 2H,
OCH2O); 13C NMR (150 MHz, CDCl3):
d 164.7, 164.4, 148.5, 147.6,
4.1.8. General synthetic procedures for compounds 3q, 3r and 4q
The compound 3c, 3h or 4c (0.5 mmol) was slowly mixed with
an Fe(OH)2 solution [treatment of FeSO4 (5.0 mmol) in H2O (15 mL)
with a cone. NH4OH solution (5 mL)] and reluxed for 30 min.
Following addition of warm H2O (10 mL), the mixture was filtered,
the precipitate washed with warm H2O (10 mL) and the combined
filtrates acidified with 3 N H2SO4. Extraction of the resultant solu-
tion with EtOAc (20 mL ꢂ 3) and evaporation of the solvent yielded
the crude product, purified by column chromatography (n-hexane:
EtOAc ¼ 10:1 as eluent) on silica gel to afford pure products.
133.5, 133.3, 132.9, 129.8, 129.0, 128.4, 127.9, 127.7, 126.9, 126.5,
126.2, 123.6, 109.6, 108.1, 101.3; HRMS (ESI): calcd for C19H12N2O4Se
[M þ H]þ: 381.0142, found: 381.0124.
4.1.7. General synthetic procedures for compounds 3mep and 4n
AlCl3 (1.5 mmol) was added in one portion to a solution of
compound 3b, 3e, 3f, 3g or 4b (0.5 mmol) in CH2Cl2 (25 mL) at 0 ꢀC.
When the reaction was completed (TLC control), the mixture was
poured into water (50 mL)/HCl aq. (50 mL, 2 M) and extracted with
CH2Cl2 (20 mL ꢂ 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 silica gel
chromatography to afford pure product.
4.1.8.1. 3-(3-amino-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-
1,2,5-selenadiazole (3q). Brown solid; yield: 77%; M.p.: 116e119 ꢀC;
1H NMR (300 MHz, CDCl3):
d
6.89 (s, 1H, Ph-H), 6.72 (d, 4H, Ph-H),
3.88 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.71 (s, 6H, OCH3). 13C NMR
(150 MHz, CDCl3):
4.1.7.1. 3-(3-hydroxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-
d
165.0, 164.4, 152.8 (ꢂ2), 148.1, 138.9, 136.0,
selenadiazole (3m). White solid; yield: 87%; M.p.: 125e130 ꢀC; 1H
131.4, 129.0, 119.9, 115.5, 109.7, 106.6 (ꢂ2), 60.9, 56.0 (ꢂ2), 55.6;
HRMS (ESI): calcd for C18H20N3O4Se [M þ H]þ, C18H19N3NaO4Se
[M þ Na]þ: 422.0619, 444.0438, found: 422.0606, 444.0373.
NMR (300 MHz, CDCl3):
d
7.20 (t, J ¼ 8.0 Hz, 1H, Ph-H), 6.93 (d,
J ¼ 7.1 Hz, 2H, Ph-H), 6.87 (dd, J1 ¼ 8.3 Hz, J2 ¼ 2.1 Hz, 1H, Ph-H),
6.68 (s, 2H, Ph-H), 3.86 (s, 3H, OCH3), 3.65 (s, 6H, OCH3); 13C NMR
(150 MHz, CDCl3):
d
168.0, 164.4, 164.2, 160.3 (ꢂ2), 148.4, 147.5,
4.1.8.2. 3-(3-amino-4-ethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-
1,2,5-selenadiazole (3r). Brown solid; yield: 83%; M.p.: 97e101 ꢀC;
130.50, 123.5, 113.7, 109.5, 108.1, 101.3 (ꢂ2), 55.2 (ꢂ2), 52.6; HRMS
H]þ, C17H16N2NaO4Se
1H NMR (300 MHz, CDCl3):
d 6.90 (s, 1H, Ph-H), 6.71 (d, 4H, Ph-H),
(ESI): calcd for
C
17H17N2O4Se [M
þ
[M þ Na]þ: 393.0354, 415.0173, found: 393.0329, 415.0213.
4.07 (q, J ¼ 7.0 Hz, 2H, OCH2CH3), 3.88 (s, 3H, OCH3), 3.71 (s, 6H,
OCH3), 1.44 (t, J ¼ 7.0 Hz, 3H, OCH2CH3). 13C NMR (150 MHz, CDCl3):
4 .1. 7 . 2 . 3 - ( 3 - h y d r o x y - 4 - m e t h o x y p h e n y l ) - 4 - ( 3 , 4 , 5 -
d
165.1,164.4,152.8 (ꢂ2),147.4,138.9,136.1,131.4,128.9,119.8,115.6,
trimethoxyphenyl)-1,2,5-selenadiazole (3n). White solid; yield: 84%;
110.6, 106.6 (ꢂ2), 63.9, 60.9, 56.0 (ꢂ2), 14.8; HRMS (ESI): calcd for
M.p.: 115e118 ꢀC; 1H NMR (300 MHz, CDCl3):
d
7.10 (d, J ¼ 2.0 Hz,
C
19H22N3O4Se [M þ H]þ, C19H21N3NaO4Se [M þ Na]þ: 436.0776,
1H, Ph-H), 6.91 (dd, J1 ¼ 8.4 Hz, J2 ¼ 2.0 Hz, 1H, Ph-H), 6.82 (d,
J ¼ 8.4 Hz, 1H, Ph-H), 6.69 (s, 2H, Ph-H), 5.80 (s, 1H, Ph-OH), 3.91 (s,
3H, OCH3), 3.88 (s, 3H, OCH3), 3.70 (s, 6H, OCH3). 13C NMR
458.0595, found: 436.0770, 458.0594.
4 . 1 . 8 . 3 . 3 - ( 3 - a m i n o - 4 - m e t h o x y p h e n y l ) - 4 - ( 3 , 4 -
(150 MHz, CDCl3):
d
164.5, 164.4, 152.9 (ꢂ2), 147.5, 145.4, 139.0,
methylenedioxyphenyl)-1,2,5-selenadiazole (4q). Brown solid; yield:
131.2, 129.5, 121.5, 115.6, 110.1, 106.6 (ꢂ2), 60.9, 56.0 (ꢂ2), 53.4;
HRMS (ESI): calcd for C18H19N2O5Se [M þ H]þ, C18H18N2NaO5Se
[M þ Na]þ: 423.0459, 445.0279, found: 423.0463, 445.0269.
85%; M.p.: 71e78 ꢀC; 1H NMR (300 MHz, CDCl3):
d 6.97 (d,
J ¼ 1.6 Hz,1H, Ph-H), 6.93 (dd, J1 ¼8.0 Hz, J2 ¼ 1.7 Hz,1H, Ph-H), 6.87
(s, 1H, Ph-H), 6.75 (m, 3H, Ph-H), 5.99 (s, 2H, OCH2O), 3.87 (s, 3H,

8
Q. Guan et al. / European Journal of Medicinal Chemistry 87 (2014) 1e9
OCH3); 13C NMR (150 MHz, CDCl3):
d
164.9,164.3,148.4,148.2,147.4,
reader (FASCalibur, BD Biosciences, USA). The IC50 values were
136.0, 130.3, 128.9, 123.6, 119.8, 115.5, 109.8, 109.6, 108.1, 101.3, 55.5;
HRMS (ESI): calcd for C16H14N3O3Se [M þ H]þ: 376.0200, found:
376.0197.
calculated after 20 min using the SPSS software.
4.2.4. Immunofluorescence staining
Immunostaining was carried out to detect the microtubule-
associated tubulin protein after exposure to CA-4 (1a) and the
investigated compound 3n. HT-1080 cells were seeded at
1 ꢂ 104 cells per well on a 24-well plate and grown for 24 h. The
cells were treated with the vehicle or twice the IC50 concentration
of CA-4 (1a) or 3n 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 permeabilised with 0.1% (v/v) Triton X-100 in PBS for
5 min. The cells were then blocked with 5% bovine serum albumin
4.1.9. 3-[3-(N-methylamino)-4-methoxyphenyl]-4-(3,4,5-
trimethoxyphenyl)-1,2,5-selenadiazole (3s)
To a solution of iodomethane (0.002 mol) in DMF (5 mL) was
added dropwise the solution of compound 3q (0.002 mmol) in DMF
(0.5 mL) and the mixture was heated under stirring at 40 ꢀC for
24 h. The reaction was diluted with H2O (100 mL) then extracted
with EtOAc (3 ꢂ 100 mL) and the combined organic phases were
washed with H2O (3 ꢂ 100 mL) and brine (50 mL), dried over
Na2SO4 and concentrated in vacuo. The residue was purified by
flash column chromatography (n-hexane: EtOAc ¼ 10:1 as eluent).
Brown solid; yield: 92%; M.p.: 119e123 ꢀC; 1H NMR (300 MHz,
(BSA) in PBS for 10 min. The primary
a-tubulin 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 un-
bound primary antibody, and the cells were then incubated with
FITC-conjugated 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 4,6-diamino-2-phenolindol dihydrochloride (DAPI)
and immunofluorescence was then detected using a fluorescence
microscope (Olympus, Tokyo, Japan).
CDCl3):
3.86 (s, 3H, OCH3), 3.70 (s, 6H, OCH3), 2.75 (s, 3H, NHCH3). 13C NMR
(150 MHz, CDCl3):
d 6.71 (m, 5H, Ph-H), 4.68 (s, 1H, NHCH3), 3.87 (s, 3H, OCH3),
d
165.6, 164.6, 152.8 (ꢂ2), 147.7, 139.0, 138.8,
131.7, 129.1, 117.9, 110.1, 108.5, 106.7 (ꢂ2), 60.9, 56.0 (ꢂ2), 55.5, 30.2;
HRMS (ESI): calcd for C19H22N3O4Se [M þ H]þ, C19H21N3NaO4Se
[M þ Na]þ: 436.0776, 458.0595, found: 436.0772, 458.0530.
4.2. Biological evaluation
4.2.5. Cell cycle analysis
HT-1080 cells (8 ꢂ 104 cells) were incubated with various con-
centrations of CA-4 (1a), 3n or 0.05% DMSO for the indicated times.
The cells were collected by centrifugation, washed with PBS and
fixed in ice-cold 70% ethanol. The fixed cells were harvested by
4.2.1. Cell culture
The human gastric adenocarcinoma cell line SGC-7901, the hu-
man fibrosarcoma cell line HT-1080 and the human pulmonary
carcinoma cell line A-549 were cultured in RPMI-1640 medium
containing 10% FBS, 100 U/mL streptomycin and 100 U/mL peni-
cillin at 37 ꢀC in a humidified atmosphere containing 5% CO2. All cell
lines were purchased from the American Type Culture Collection
(ATCC, Manassas, VA).
centrifugation and resuspended in 500
mL RNase. After 30 min of incubation at 37 ꢀC, the cells were
stained with 50
g/mL propidium iodide (PI) at 4 ꢀC in the dark for
mL of PBS containing 1 mg/
m
30 min. The samples were then analysed by FACScan flow cytom-
etry (BectoneDickinson, Franklin Lakes, NJ, USA). The experiments
were repeated at least three times.
4.2.2. In vitro anti-proliferative activity
The in vitro anti-proliferative activities of CA-4 (1a) and all of the
target compounds were determined with an MTT (Sigma) assay.
Briefly, cells were seeded into 96-well plates at a density of
1e3 ꢂ 104/well (depending on the cell growth rate). Twenty-four
hours later, triplicate wells were treated with media and the
agents. After 72 h of incubation at 37 ꢀC in 5% CO2, the drug-
4.3. Molecular modelling
The molecular modelling studies were performed with Accelrys
Discovery Studio 3.0. The crystal structure of tubulin complexed
with DAMA-colchicine (PDB: 1SA0) was retrieved from the RCSB
Protein Data Bank (http://www.rcsb.org/pdb). In the docking pro-
cess, the protein protocol was prepared via several operations,
including the standardisation of atom names, insertion of missing
atoms in residues and removal of alternate conformations, inser-
tion of missing loop regions based on SEQRES data, optimisation of
short and medium sized loop regions with the Looper Algorithm,
minimisation 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 (DAMA-colchicine) as the
binding site. The CA-4 (1a) and 3n were drawn with Chemdraw and
fully minimised using the CHARMm force field. Finally, they were
docked into the binding site using the CDOCKER protocol with the
default settings.
containing medium was removed and replaced with 100
fresh medium containing 5 mg/mL MTT solution. After 4 h of in-
cubation, the medium with MTT was removed, and 100 L of
mL of
m
dimethyl sulphoxide (DMSO) was added to each well. The plates
were gently agitated until the purple formazan crystals were dis-
solved, 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.
4.2.3. Inhibition of tubulin polymerisation
The effects of compounds 3n and CA-4 (1a) on the polymeri-
sation of tubulin were determined by employing a fluorescence-
based tubulin polymerisation assay kit (Cytoskeleton-Cat.#
BK011P) according to the manufacturer's protocol. Tubulin was re-
suspended in ice-cold G-PEM buffer (80 mM PIPES, 2 mM MgCl2,
0.5 mM EGTA,1 mM GTP, 20% (v/v) glycerol) and added to wells on a
96-well plate containing the designated concentration of the drug
or vehicle. The samples were mixed well, and tubulin assembly was
monitored (emission wavelength of 420 nm; excitation wavelength
pf 360 nm) at 1 min intervals for 90 min at 37 ꢀC using a plate
Acknowledgements
We gratefully acknowledge the National S&T Major Project
(2012ZX09103101-060), the National Natural Science Foundation of
China (30973614), and the Excellent Talent Supporting Project of
Liaoning Province (LR20013046) for their generous financial sup-
port. 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.

Q. Guan et al. / European Journal of Medicinal Chemistry 87 (2014) 1e9
9
S.K. Srivastava, V.A. Mahajan, V.H. Jadhav, K. Dutta, K. Krishnan, A. Chaudhary,
S.K. Agarwal, R. Mukherjee, A.C. Burman, Synthesis and evaluation of 4/5-
hydroxy-2,3-diaryl(substituted)-cyclopent-2-en-1-ones as cis-restricted ana-
logues of combretastatin A-4 as novel anticancer agents, J. Med. Chem. 50
(2007) 1744e1753.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2014.09.
046. These data include MOL files and InChiKeys of the most
important compounds described in this article.
[14] M. Wu, Q. Sun, C. Yang, D. Chen, J. Ding, Y. Chen, L. Lin, Y. Xie, Synthesis and
activity of combretastatin A-4 analogues: 1,2,3-thiadiazoles as potent anti-
tumor agents, Bioorg. Med. Chem. Lett. 17 (2007) 869e873.
[15] G.C. Tron, F. Pagliai, E.D. Grosso, A.A. Genazzani, G. Sorba, Synthesis and
cytotoxic evaluation of combretafurazans, J. Med. Chem. 48 (2005)
3260e3268.
References
[16] E. Moreno, D. Plano, I. Lamberto, M. Font, I. Encío, J.A. Palop, C. Sanmartín,
Sulfur and selenium derivatives of quinazoline and pyrido[2,3-d]pyrimidine:
synthesis and study of their potential cytotoxic activity in vitro, Eur. J. Med.
Chem. 47 (2012) 283e298.
[1] L.B. Salum, W.F. Altei, L.D. Chiaradia, M.N.S. Cordeiro, R.R. Canevarolo,
C.P.S. Melo, E. Winter, B. Mattei, H.N. Daghestani, M.C. Santos-Silva,
T.B. Creczynski-Pasa, R.A. Yunes, J.A. Yunes, A.D. Andricopulo, B.W. Day,
R.J. Nunes, A. Vogt, Cytotoxic 3,4,5-trimethoxychalcones as mitotic arresters
and cell migration inhibitors, Eur. J. Med. Chem. 63 (2013) 501e510.
[2] K.H. Downing, E. Nogales, Tubulin and microtubule structure, Curr. Opin. Cell.
Biol. 10 (1998) 16e22.
[3] A. Wienecke, G. Bacher, Indibulin, a novel microtubule inhibitor, discriminates
between mature neuronal and nonneuronal tubulin, Cancer Res. 69 (2009)
171e177.
ꢀ
[17] E. Domínguez-Alvarez, D. Plano, M. Font, A. Calvo, C. Prior, C. Jacob, J.A. Palop,
C. Sanmartín, Synthesis and antiproliferative activity of novel selenoester
derivatives, Eur. J. Med. Chem. 73 (2014) 153e166.
[18] M.A. Fernandez-Herrera, J. Sandoval-Ramírez, L. Sanchez-Sanchez, H. Lopez-
ꢀ
ꢀ
ꢀ
ꢀ
~
ꢀ
Munoz, M.L. Escobar-Sanchez, Probing the selective antitumor activity of 22-
oxo-26-selenocyanocholestane derivatives, Eur. J. Med. Chem. 74 (2014)
451e460.
[4] M.A. Jordan, L. Wilson, Microtubules as a target for anticancer drugs, Nat. Rev.
Cancer 4 (2004) 253e265.
[19] G. Mugesh, W.W.D. Mont, H. Sies, Chemistry of biologically important syn-
thetic organoselenium compounds, Chem. Rev. 101 (2001) 2125e2179.
[5] C. Dumontet, M.A. Jordan, Microtubule-binding agents: a dynamic field of
cancer therapeutics, Nat. Rev. Drug. Discov. 9 (2010) 790e803.
[6] Y. Lu, J. Chen, M. Xiao, W. Li, D.D. Miller, An overview of tubulin inhibitors that
interact with the colchicine binding site, Pharm. Res. 29 (2012) 2943e2971.
[7] G.R. Pettit, S.B. Singh, E. Hamel, C.M. Lin, D.S. Alberts, D. Garcia-Kendall,
Isolation and structure of the strong cell growth and tubulin inhibitor com-
bretastatin A-4, Experientia 45 (1989) 209e211.
[8] G.R. Pettit, S.B. Singh, M.R. Boyd, E. Hamel, R.K. Pettit, J.M. Schmidt, F. Hogan,
Antineoplastic agents. 291. Isolation and synthesis of combretastatins A-4, A-
5, and A-6(1a), J. Med. Chem. 38 (1995) 1666e1672.
[9] J. Zhou, J. Jin, Y. Zhang, Y. Yin, X. Chen, B. Xu, Synthesis and antiproliferative
evaluation of novel benzoimidazole-contained oxazole-bridged analogs of
combretastatin A-4, Eur. J. Med. Chem. 68 (2013) 222e232.
[10] Z.Y. Liu, Y.M. Wang, Y.X. Han, L. Liu, J. Jin, H. Yi, Z.R. Li, J.D. Jiang, D.W. Boykin,
Synthesis and antitumor activity of novel 3,4-diaryl squaric acid analogs, Eur.
J. Med. Chem. 65 (2013) 187e194.
[11] R. Romagnoli, P.G. Baraldi, M.D. Carrion, O. Cruz-Lopez, M. Tolomeo,
S. Grimaudo, A.D. Cristina, M.R. Pipitone, J. Balzarini, A. Brancale, E. Hamel,
Substituted 2-(30,40,50-trimethoxybenzoyl)-benzo[b]thiophene derivatives as
potent tubulin polymerization inhibitors, Bioorg. Med. Chem. 18 (2010)
5114e5122.
[12] Z.Y. Liu, Y.M. Wang, Z.R. Li, J.D. Jiang, D.W. Boykin, Synthesis and anticancer
activity of novel 3,4-diarylthiazol-2(3H)-ones (imines), Bioorg. Med. Chem.
Lett. 19 (2009) 5661e5664.
[20] E. Napolitano, R. Fiaschi, Marsili,
a-Aryl-a-(2-tetrahydropyranyloxy)meth-
anephosphonates as reagents in the Horner reaction. A simple novel synthesis
of ( )-combretastatin, Gazz. Chim. Ital. 118 (1988) 415e416.
[21] G.L. Regina, T. Sarkar, R.L. Bai, M.C. Edler, R. Saletti, A. Coluccia, F. Piscitelli,
L. Minelli, V. Gatti, C. Mazzoccoli, V. Palermo, C. Mazzoni, C. Falcone,
A.I. Scovassi, V. Giansanti, P. Campiglia, A. Porta, B. Maresca, E. Hamel,
A. Brancale, E. Novellino, R. Silvestri, New arylthioindoles and related bio-
isosteres at the sulfur bridging group. 4. Synthesis, tubulin polymerization,
cell growth inhibition, and molecular modeling studies, J. Med. Chem. 52
(2009) 7512e7527.
[22] Vincenzo Bertini, 1,2,5-selenadiazoles, Gazz. Chim. Ital. 97 (1967) 1870e1884.
€
[23] H.C. Nickel, P. Schmidt, K.J. Bohm, S. Baasner, K. Müller, M. Gerlach, E. Unger,
E.G. Günther, H. Prinz, Synthesis, antiproliferative activity and inhibition of
tubulin polymerization by 1,5- and 1,8-disubstituted 10H-anthracen-9-ones
bearing a 10-benzylidene or 10-(2-oxo-2-phenylethylidene) moiety, Eur. J.
Med. Chem. 45 (2010) 3420e3438.
[24] A.P.N. Burger, E.V. Brandt, D.G. Roux, O-(dihydrobenzofuranyl)-dibenzo-
pyrones from Umtiza listerana, Phytochemistry 22 (1983) 2813e2817.
a-
[25] R.B.G. Ravelli, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel,
M. Knossow, Insight into tubulin regulation from a complex with colchicine
and a stathmin-like domain, Nature 428 (2004) 198e202.
ꢀ
[26] A. Dorleans, B. Gigant, R.B.G. Ravelli, P. Mailliet, V. Mikol, M. Knossow, Vari-
ations in the colchicine-binding domain provide insight into the structural
switch of tubulin, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 13775e13779.
[13] M.K. Gurjar, R.D. Wakharkar, A.T. Singh, M. Jaggi, H.B. Borate, P.D. Shinde,
R. Verma, P. Rajendran, S. Dutt, G. Singh, V.K. Sanna, M.K. Singh,