Combretastatin A-4 analogues with benzoxazolone scaffold: Synthesis, structure and biological activity

Article abstract of DOI:10.1016/j.ejmech.2016.05.012

In order to design and synthesize a new class of heterocyclic analogues of natural combretastatin A-4 and its synthetic derivative AVE8062, the benzoxazolone ring was selected as a scaffold for a bioisosteric replacement of the ring B of both molecules. A library of 28 cis- and trans-styrylbenzoxazolones was obtained by a modified Wittig reaction under Boden's conditions. Structures of the newly synthesized compounds bearing the 3,4,5-trimethoxy-, 3,4-dimethoxy-, 3,5-dimethoxy-, and 4-methoxystyryl fragment at position 4, 5, 6 or 7 of benzoxazolone core were determined on the basis of spectral and X ray data. The in vitro cytotoxicity of styrylbenzoxazolones against different cell lines was examined. Stilbene derivative 16Z, (Z)-3-methyl-6-(3,4,5-trimethoxystyryl)-2(3H)-benzoxazolone, showed highest antiproliferative potential of the series, with IC₅₀ of 0.25 μM against combretastatin resistant cell line HT-29, 0.19 μM against HepG2, 0.28 μM against EA.hy926 and 0.73 μM against K562 cells. Furthermore, the results of flow cytometric analysis confirmed that 16Z induced cell cycle arrest in G2/M phase in the cell lines like combretastatin A-4. This arrest is followed by an abnormal exit of cells from mitosis without cytokinesis into a pseudo G1-like multinucleate state leading to late apoptosis and cell death. Accordingly, synthetic analogue 16Z was identified as the most promising potential anticancer agent in present study, and was selected as lead compound for further detailed investigations.

Full text of DOI:10.1016/j.ejmech.2016.05.012

Accepted Manuscript
Combretastatin A-4 analogues with benzoxazolone scaffold: Synthesis, structure and
biological activity
Mariana S. Gerova, Silviya R. Stateva, Elena M. Radonova, Rositsa B. Kalenderska,
Rusi I. Rusew, Rositsa P. Nikolova, Christo D. Chanev, Boris L. Shivachev, Margarita
D. Apostolova, Ognyan I. Petrov
PII:
S0223-5234(16)30389-0
10.1016/j.ejmech.2016.05.012
EJMECH 8601
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 February 2016
Revised Date: 27 April 2016
Accepted Date: 5 May 2016
Please cite this article as: M.S. Gerova, S.R. Stateva, E.M. Radonova, R.B. Kalenderska, R.I. Rusew,
R.P. Nikolova, C.D. Chanev, B.L. Shivachev, M.D. Apostolova, O.I. Petrov, Combretastatin A-4
analogues with benzoxazolone scaffold: Synthesis, structure and biological activity, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.05.012.
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ACCEPTED MANUSCRIPT
Graphical abstract
Combretastatin A-4 analogues with benzoxazolone scaffold: synthesis, structure and
biological activity
a
b
b
b
Mariana S. Gerova, Silviya R. Stateva, Elena M. Radonova, Rositsa B. Kalenderska, Rusi
c
c
a
c
I. Rusew, Rositsa P. Nikolova, Christo D. Chanev, Boris L. Shivachev, Margarita D.
b
a,
Apostolova, Ognyan I. Petrov *
a
University of Sofia "St. Kliment Ohridski", Faculty of Chemistry and Pharmacy, Department
of Pharmaceutical and Applied Organic Chemistry, 1 James Bourchier Blvd, 1164 Sofia,
Bulgaria
b
Bulgarian Academy of Sciences, Institute of Molecular Biology, Acad. G. Bonchev Str., bl.
2
1, 1113 Sofia, Bulgaria
c
Bulgarian Academy of Sciences, Institute of Mineralogy and Crystallography, Acad. G.
Bonchev Str., bl. 107, 1113 Sofia, Bulgaria

ACCEPTED MANUSCRIPT
Combretastatin A-4 analogues with benzoxazolone scaffold: synthesis, structure and
biological activity
a
b
b
b
Mariana S. Gerova, Silviya R. Stateva, Elena M. Radonova, Rositsa B. Kalenderska, Rusi
c
c
a
c
I. Rusew, Rositsa P. Nikolova, Christo D. Chanev, Boris L. Shivachev, Margarita D.
b
a,
Apostolova, Ognyan I. Petrov *
a
University of Sofia "St. Kliment Ohridski", Faculty of Chemistry and Pharmacy, Department
of Pharmaceutical and Applied Organic Chemistry, 1 James Bourchier Blvd, 1164 Sofia,
Bulgaria
b
Bulgarian Academy of Sciences, Institute of Molecular Biology, Acad. G. Bonchev Str., bl.
2
1, 1113 Sofia, Bulgaria
c
Bulgarian Academy of Sciences, Institute of Mineralogy and Crystallography, Acad. G.
Bonchev Str., bl. 107, 1113 Sofia, Bulgaria
*
Corresponding author. Tel.: +359 2 8161 275; fax: +359 2 9625 438. E-mail address:
opetrov@chem.uni-sofia.bg (Prof. O. Petrov)

ACCEPTED MANUSCRIPT
ABSTRACT
In order to design and synthesize a new class of heterocyclic analogues of natural
combretastatin A-4 and its synthetic derivative AVE8062, the benzoxazolone ring was
selected as a scaffold for a bioisosteric replacement of the ring B of both molecules. A library
of 28 cis- and trans-styrylbenzoxazolones was obtained by a modified Wittig reaction under
Boden’s conditions. Structures of the newly synthesized compounds bearing the 3,4,5-
trimethoxy-, 3,4-dimethoxy-, 3,5-dimethoxy-, and 4-methoxystyryl fragment at position 4, 5,
6
or 7 of benzoxazolone core were determined on the basis of spectral and X ray data. The in
vitro cytotoxicity of styrylbenzoxazolones against different cell lines was examined. Stilbene
derivative 16Z, (Z)-3-methyl-6-(3,4,5-trimethoxystyryl)-2(3H)-benzoxazolone, showed
highest antiproliferative potential of the series, with IC of 0.25 µM against combretastatin
5
0
resistant cell line HT-29, 0.19 µM against HepG2, 0.28 µM against EA.hy926 and 0.73 µM
against K562 cells. Furthermore, the results of flow cytometric analysis confirmed that 16Z
induced cell cycle arrest in G2/M phase in the cell lines like combretastatin A-4. This arrest is
followed by an abnormal exit of cells from mitosis without cytokinesis into a pseudo G1-like
multinucleate state leading to late apoptosis and cell death. Accordingly, synthetic analogue
1
6Z was identified as the most promising potential anticancer agent in present study, and was
selected as lead compound for further detailed investigations.
Keywords: Combretastatin A-4, Stilbene, Benzoxazolone, Anticancer agents, Tubulin binding
agents, X-ray.

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1
. Introduction
The microtubule network is an important target for cancer therapy because of its crucial role
in cell proliferation. Composed by polymerization of α,β-tubulin heterodimers, microtubules
are responsible for the formation of the mitotic spindle and the segregation of chromosomes
during cell division [1-3]. Destruction of microtubules can elicit cell cycle arrest in G2/M
phase followed by apoptotic cell death [1,4]. Therefore, compounds that disrupt the
tubulin/microtubule equilibrium can be examined as potent anticancer agents. Indeed, a
number of clinically useful drugs such as paclitaxel, docetaxel, vinblastin and vincristine exert
their antimitotic effect by binding to the taxane or the vinca domains of tubulin. The
colchicine binding site of tubulin is the third well-characterized and important target for new
chemotherapeutic products [5]. Amongst them, the natural cis-stilbene combretastatin A-4
(CA-4, 1, Fig. 1) stands out as powerful inhibitor of tubulin polymerization and promising
anticancer agent. By binding to the colchicine pocket of β-tubulin, CA-4 inhibits the
formation of the mitotic spindle and causes subsequent mitotic catastrophe in cancer cells
[
6,7]. In addition, CA-4 leads to rapid and selective vascular dysfunction in solid tumors by
attacking endothelial cells within the tumor vasculature [8,9]. A water soluble CA-4 prodrug,
combretastatin A-4 phosphate (CA-4P, 2, Fosbretabulin, Fig. 1) is in clinical development for
treatment of ovarian and thyroid cancers.
The therapeutic efficacy of 1 has inspired the synthesis of various stilbene derivatives,
including modifications in the main structural features of CA-4 – ring A and B, and the
ethylene bridge between them [10-14]. One of the most important transformations was
achieved by Ohsumi et al. [15] through meta OH – NH bioisosteric replacement in ring B
2
leading to aminocombretastatin AC7739 (3, Fig. 1). Compound 3 showed significant
antitumor activity and greater water solubility in comparison to natural CA-4. Based on 3, a
serinamide prodrug AVE8062 (4, Ombrabulin, Fig. 1) was developed as vascular disrupting
candidate for the treatment of soft tissue sarcoma [16,17].

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Fig. 1.
In an attempt to design new biomimetic analogues of natural CA-4 and its synthetic amino-
derivatives, we selected benzoxazolone heterocycle as a scaffold for a bioisosteric
replacement of the ring B. 2(3H)-Benzoxazolones are largely used in medicinal chemistry as
an important building block because of their wide range of biological activities such as
anticonvulsant [18], analgesic, anti-inflammatory [19,20], antiulcer [21] as well as
cytotoxicity against various cancer cell lines [22-24]. Further, benzoxazolone heterocycles are
considered to be good bioisosteres of phenols and amides in drug discovery and development
[
25]. Following the main strategy of bioisosterism, we proffer the synthesis of novel CA-
/benzoxalone hybrids 9Z-22Z (Fig. 2) with potential antitumor effects. Depending on the
4
position of the methoxystyryl fragment in the benzoxazolone moiety, we present four
isomeric structures of styrylbenzoxazolones.
Fig. 2.
The target compounds 9Z-22Z possess the main pharmacophores of CA-4 molecule: a
methoxy-substituted ring A, introduced in position 4, 5, 6 or 7 of benzoxazolone moiety as
ring B connected via cis-oriented double bond. The structures of 9Z-22Z as well as their
trans-isomers 9E-22E were confirmed by NMR spectroscopy and by X-ray crystallography
for 13Z, 14Z, 16Z and 16E. The heterocyclic cis-stilbene derivatives 9Z-22Z were tested in
vitro for cell growth inhibition and the ability to induce apoptosis in HepG2, EA.hy926 and
K562 cell lines. A possible mechanism of action for the most active compound 16Z is
discussed.
2
. Results and discussion
2
.1. Chemistry
The aim of this work was the preparation of heterocyclic stilbenes as novel structural
analogues of CA-4. Among the various synthetic methods leading to the formation of stilbene

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scaffold [14, 26-29], the Wittig olefination reaction is the most utilized because of its
simplicity and efficiency.
The synthesis of target compounds was based on the Wittig reaction under Boden’s conditions
[
30] using potassium carbonate as a base and 18-crown-6 as phase transfer catalyst. To
evaluate the effect of substitution in ring A on the biological activity of the stilbenes, various
methoxybenzaldehydes were olefinated with the corresponding heterocyclic phosphonium
salts 5-8 (Scheme 1). The resulting mixtures of E- and Z-isomers (Table 1) were separated by
column chromatography. In some cases, the E-stilbenes could be isolated by recrystallization
of the crude reaction mixture from ethanol. The isolation and purification of the Z-isomer was
always accomplished by column chromatography. The E- or Z-configuration of stilbene
1
derivatives was determined by H NMR spectroscopy on the basis of coupling constants of
the olefinic proton signals. As a general rule, the olefinic proton signals of the Z-isomers
presented as doublets in the 6.33 – 6.75 ppm range with a coupling constant of 12 Hz, while
the olefinic protons of the E-isomers appeared at 6.94 – 7.46 ppm as doublets with J constant
of 16 Hz. The olefinic protons of 13Z, 13E and 16Z, containing 3,4,5-trimethoxyphenyl
fragment as ring A, appeared as a singlets in deuterochloroform. However, splitting with J
constants of 12.2, 16.3 and 12.2 Hz respectively was observed in hexadeuteroacetone, which
allowed determination of the stereochemistry of the products.
Scheme 1.
Table 1.
The heterocyclic phosphonium salts 5-8 were easily prepared in three-steps from the
corresponding 4-, 5-, 6- or 7-methyl-2(3H)-benzoxazolones (23-26, Scheme 2). N-
Methylation with methyl iodide or dimethyl sulfate followed by bromination in the benzylic
position with N-bromosuccinimide (NBS) in tetrachloromethane led to compounds 31-34,
which were converted to the desired phosphonium salts in high yields.
Scheme 2.

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Benzoxazolones 23-26, which were the starting materials for the synthesis of the key
phosphonium salts, were prepared by us as follows. Compounds 24 and 25 were obtained
from commercially available o-aminophenols and urea following the classical procedure of
condensation. The access to compounds 23 and 26 was realized by methods developed in our
laboratory (Schemes 3 and 4). Scheme 3 outlines the synthetic approach for preparation of 4-
methyl-2(3H)-benzoxazolone (23). o-Toluidine was used as a commercially available and
inexpensive reagent for the preparation of 7-methylizatin (37), using a Sandmeyer method. In
turn, compound 37 was treated with potassium persulfate in the presence of sulfuric acid to
provide benzoxazindione 38 through a Baeyer-Villiger oxidation. This compound served as a
precursor for the formation of the key 2-amino-3-methylphenol (39), which was submitted to
cyclization with 1,1'-carbonyldiimidazole (CDI) to yield 23.
Scheme 3.
Scheme 4 illustrates our strategy for the preparation of 7-methyl-2(3H)-benzoxazolone (26) in
three steps and 50% overall yield from 3-methylsalicylic acid (40). The key step in this
approach was the Lossen rearrangement of the hydroxamic acid 42 that took place in the
presence of formamide and led to the formation of benzoxazolone derivative 26.
Scheme 4.
2
.2. X-Ray crystallography
To verify the E/Z configurations and the relative stability of the stilbene derivatives, single
crystal experiments were envisaged. Crystals suitable for X-ray investigation were obtained
with compounds 13Z, 14Z, 16Z and 16E by the vapor diffusion method from
DMSO/(ethanol:water, 1:1 v/v) solutions. The expected E or Z conformation observed by
NMR was also confirmed here (Fig. 3), thus no rapid E/Z isomerism is expected for these
compounds.

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The bond lengths and angles of the four molecules are comparable to those of similar
compounds [31-35] (Table 2). Rings A and B are essentially planar (rms not greater than 0.2
Å). Overall, the Z isomers are not planar, while 16E is almost planar (rms of 0.38 Å for the
mean plane for the whole molecule, Fig. 4). The angle between the mean planes of rings A
and B rings opens up from 50.08, 59.46 to 67.76° for 13Z, 14Z, 16Z respectively (Fig. S2.1,
Supplementary data). No typical hydrogen bonding interactions could be detected in the four
structures, but several CH …O interactions were identified (Table S2.1, Supplementary data).
3
π… π stacking interactions are noticeable in 13Z, 14Z, and 16Z, but not 16E.
Table 2.
Fig. 4.
To examine the potential tubulin-binding modes of the synthesized compounds [36,37], we
conducted docking simulations of 13Z, 14Z, 16Z, 16E and CA-4 onto the tubulin colchicine
complex (1SA0 [38], Brookhaven Protein Data Bank) in Molegro Virtual Docker (version
2
4
011.4.3.0.) [39]. Docking of CA-4 suggests that the 3,4,5-trimethoxyphenyl ring (A) of CA-
superimposes with the colchicine 1,2,3-trimethoxybenzene moiety (Fig. S2.2,
Supplementary data), while CA-4 ring B overlaps with the carbonyl/methoxy moieties of
colchicine. The predicted protein-ligand interactions suggest that CA-4 ring B hydroxyl and
colchicine carbonyl oxygen atoms participate in hydrogen bonding interactions (as acceptors)
with Val181 (Fig. S2.2, Supplementary data). At the other end of the molecules, hydrogen
bonds between the methoxy oxygen atoms and the Cys241 sulfohydryl group are predicted.
The predicted orientations of 13Z and 16Z fit well with the colchicine orientation, both in
terms of overall shape and pivotal chemical features (Fig. 5). According to the predicted
protein-ligand interactions, hydrogen bonding with Cys241 is conserved, but interaction with
Val181 is absent. 16Z participates in hydrogen bonding with Lys352 (D…A of 3.37 Å), while
no suitable donors are close enough to the corresponding 13Z carbonyl group (Fig. 5).
Docking of 14Z predicted an "inversion" of rings A and B with respect to 13Z, 16Z, CA-4 and

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colchicine (Fig. S2.3, Supplementary data). Accordingly, no hydrogen-bonding is observed to
Val181 and Cys241. The docking results may suggest a structural rationale for the observed
higher biological activity of 16Z (see Table 3).
Fig. 5.
2
.3. Biology
All synthesized compounds were tested in a cytotoxicity assay on HepG2, EA.hy 926 and
K562 cells. 16Z, containing 3,4,5-trimethoxystyryl fragment in position 6 of benzoxazolone
ring exhibited the highest activity in the tests. In the range of 50 to 750 nM 16Z exhibited
potent growth inhibition (IC ) on HepG2, EA.hy 926 and K562 cells (Table 3). With
5
0
increasing of the 16Z concentration up to 25 µM a cytostatic effect was observed on all cell
lines studied. Overall, Z-stilbenes were the biologically active isomers, with the E-isomers
showing little or no activity. The 16Z positional isomers 9Z, 13Z and 19Z also demonstrated
loss of activity, suggesting that the position of the styryl fragment on the benzoxazolone ring
plays an important role in this series of compounds.
Table 3.
Compound 16Z was further selected for investigating its antiproliferative effect on additional
cell lines (HT-29, Colon-26, A-549, MCF-7, MDA-MD-231, MCF-10A, HaCaT, NHEK).
The A549, MDA-MB-231 and HT-29 cells are known to be relatively resistant to anticancer
agents and CA-4 [40-44]. The newly synthesized compound 16Z was found to exhibit more
than 10 times stronger cytotoxic effect on colorectal adenocarcinoma cells than CA-4. When
we treated HT-29 with CA-4 (0.01 – 100 µM), the IC was 2.62 µM (Table S3.1, Fig. S3.1,
5
0
Supplementary data), while 16Z showed IC 0.25 µM. We observed a similar toxic effect of
5
0
1
6Z on mouse colon carcinoma cells (Colon-26) and human adenocarcinomic alveolar basal
epithelial cells (A-549), where 16Z was 9 and 50 times more effective, respectively, in
comparison to CA-4 (Table S3.1, Supplementary data).

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In the other cell lines studied (MCF-7, MDA-MB-231, HaCaT and NHEK) CA-4 had
predominantly cytostatic effects up to 25 µM (Fig. S3.1, Supplementary data), except with the
control cell line MCF-10A, where the IC was 1.17 µM. In contrast, it was observed that 16Z
5
0
has a low therapeutic window between 0.06 µM to 0.168 µM for MCF-7 and MDA-MB-231
cells, indicating better antiproliferative effect when compared to the CA-4, and suggesting a
possible
benzoxazolone. In addition, 16Z presented lowered toxicity for control cells, such as MCF-
0A (30%) and NHEK (nontoxic), while it exhibited higher ability to kill the more aggressive
functional
advantage
of
(Z)-3-methyl-6-(3,4,5-trimethoxystyryl)-2(3H)-
1
tumor cell line, including high resistant to CA-4 or chemotherapeutic ones (HT-29, A-549,
MCF-7, MDA-MB-231).
Since the HepG2 cell line is widely accepted as a standardized experimental model with
biological properties of human liver carcinoma cells, we chose these cells to conduct further
studies to gain insights into cellular and molecular mechanisms of 16Z action. According to
our in silico model 16Z can be reasonably expected to bind to tubulin at the same site as
colchicine and CA-4. Next we examined the effect of 16Z on the cell cycle. It is well known
that colchicine and CA-4 are the agents that inhibit microtubule polymerization by binding to
tubulin [37] causing block of the cell cycle in mitosis. To compare the effect on cell cycle of
1
6Z with those of CA-4 we exposed unsynchronized HepG2 cells to equitoxic concentrations
corresponding to their respective IC50 values (Table 3). We examined time-effects on cell
cycle progression of HepG2 cells flowcytometrically (Fig. 6). The cellular DNA content and
the relative percentages of cells in the G0/G1 (2N DNA content), S (between 2N and 4N),
G2/M (4N DNA content) stages, and polyploidity (>4N DNA content) were also calculated.
FACS measurements in HepG2 cells showed that the percentage of cells in G2/M
significantly increased within 8 h treatment with 16Z – 39% compared to 28% in the control
cultures (p < 0.031). Quantitation of the data also showed that 16Z-treated cells accumulate in

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the G2/M phase at 8 h post-treatment approximately 6% higher than that treated with CA-4
(33%).
Fig. 6.
At 24 h of treatment the HepG2 cells showed further accumulation with 4N DNA content
16Z: 80%; CA-4: 77%). The increased population of cells with 4N DNA correlated with
(
concomitant losses from G0/G1- and S-phases. A small population of cells (around 9%)
showed polyploidy with DNA content greater than diploid complement (Fig. 6 A and 6C,
>4N). This population entered another round of cell-cycle suggesting that a sub-population of
mitotically slipped-out cells continue the cell-cycle by entering another round of DNA
replication and mitosis. Several outcomes are associated with the application of anti-mitotic
chemotherapeutic agents [45,46]: 1) Cells can undergo sustained or chronic mitotic arrest
until the drug is cleared [45], thus cell population can survive and continue dividing as diploid
cells; 2) Adaptation might happen, when cells exit mitosis without engaging in anaphase or
cytokinesis, producing tetraploid (4N) multinucleated G1 cells without chromosomal
segregation [47]. Adapted cells can survive and continue dividing as tetraploid (4N) cells or
can exit G1 undergoing senescence or apoptosis as tetraploid (4N) cells. Cells can escape to
G1 overriding mitotic checkpoint signaling leading to apoptosis in interphase, possibly as a
result of treatment with 16Z, since we observed a quite similar perturbation of cell cycle
progression in the HepG2 cells after 48h treatment with selected concentration of 16Z and
CA-4 showing no additional accumulation of cells in G2/M phase. At 72 h of treatment, the
number of arrested cells in G2/M-phase decline and there was appearance of a hypodiploid (<
2
N) sub-G1 population, suggesting extensive DNA fragmentation, and indicative of cell death
(Fig. 6) directly from mitotic arrest.
It is well known that the G2/M cell cycle arrest could be strongly associated with inhibition of
tubulin polymerization [48] and it was shown that the compound 16Z is most effective in
causing G2/M cell cycle arrest. To evaluate the microtubule inhibitory activity of compound

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1
6Z, we employed ex vivo biochemical tubulin polymerization assay, where inhibition was
shown as decrease in optical density (O.D) (Fig. 7). The microtubule-polymerization
inhibition effects of 16Z suggested the possibility that it interacted directly with tubulin to
inhibit assembly. 16Z inhibited the rate and overall extent of purified tubulin assembly in a
concentration-dependent manner (Fig.7). A 10 µM concentration of 16Z inhibited the extent
of tubulin polymerization by 40% at 40 min, similar to 10 µM CA-4, while at 50 µM 16Z was
more active than CA-4. The plateau in the assembly reaction was reached at 23 min for 10
µM CA-4 and vehicle control (Tubulin), and at 20 min for 16Z. Complete inhibition of
tubulin polymerization was achieved with 50 µM CA-4 and 16Z. The effect of 50 µM 16Z
inhibition on tubulin assembly was better than that of 50 µM CA-4.
Fig. 7.
To detect cellular events perturbed by drug action, we next visualized cellular microtubules
and the spindle apparatus that is composed of microtubules in the absence and presence of
0
.19 µM 16Z. Figure 8A represents immunofluorescent micrographs of HepG2 cells that were
treated with vehicle (0.01% DMSO). Interphase cells showed normal radial arrays of
microtubules (green, Fig. 8, panel A). The mitotic population, a small percentage (4-10%) of
the total number of cells in a cell-cycle at any given time, displayed hallmark features of a
typical mitotic process. Congression of chromosomes at the metaphase plate followed by
anaphase onset, a characteristic telophase and cytokinesis were evident (Fig. 8, panel A).
Following 24 h treatment with 0.19 µM 16Z large proportions of the HepG2 cells showed
mitotic abnormalities. Typical mitotic defects included: a failure of a number of chromosomes
to align properly on the metaphase plate and the absence of two bipolar spindles with the
centromeres of individual chromosomes randomly attached to either of the spindle poles (Fig.
8
, panel B and C). Some percentage of cells with small pieces of fragmented DNA (apoptotic
bodies) was also seen at this time of treatment suggesting initiation of cell death (Fig. 8, panel
B, arrow). At 24 hours post-treatment, there was also an emergence of G1-like interphase

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cells that were multinucleated. These multinucleated cells resulted from a mitotic exit from
the cells slipped out of an abnormal multipolar mitosis without cell division (Fig. 8, panel D,
E and F). However, a small percentage of these mitotically slipped cells entered another round
of cell-cycle and thus accumulated massive DNA amounts that can trigger apoptosis due to
genotoxic stress. Indications of genotoxc stress – the presence of micronuclei was also
observed (Fig. 8, panel D, E). The micronuclei can result from a large spectrum of
mechanisms, both genetic and epigenetic. They may also result from exposure to clastogens
or aneugens, or be formed spontaneously as a defense mechanism.
Fig. 8.
To evaluate the possible ways of death after drug treatment, HepG2 and EA.hy 926 cells were
stained with Annexin-V and propidium iodide (PI). The results show that the number of
necrotic (upper left quadrant) and apoptotic cells (lower right + upper right quadrant)
significantly increased following 48 h treatment compare to the vehicle (Fig. 9).
Fig. 9.
Separate Annexin-V staining of adherent and non-adherent cells confirmed that most of the
apoptotic cells were confined to the detached fraction, probably entering apoptosis after
mitotic arrest (data not shown). However, for the eqitoxic concentrations of 16Z (0.19; 0.28
µM) and CA-4 (0.11 µM), no clear population of early apoptosis cells was observed, and
instead, dead cells appeared as double positive (Annexin-V positive, and PI positive),
indicating about 20% necrosis for both cell lines and drugs. We can conclude that the
apoptosis was a relatively late finding, occurring after arrest in mitosis in HepG2 and EA.hy
9
26 cells treated with 0.19 or 0.28 µM 16Z or 0.11 µM CA-4.
Next we examined the effects of the 16Z on matrigel-based tube formation to clarify the
possible effect of 16Z on angiogenesis. The provision of exogenous endothelial supplements
with EBM2 media promoted microtubule formation within the matrigel (Fig. S3.2,
Supplementary data). However, when equitoxic concentrations of CA-4 and 16Z were

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applied, microtubule formation was significantly impaired supporting the hypothesis that 16Z
inhibits the angiogenesis of EA.hy 926 cells in a way similar to CA-4 at 72h.
3
. Conclusions
We designed and synthesized a series of styrylbenzoxazolones as new biomimetic analogues
of combretastatin A-4 with potential anticancer properties. The analysis of our results showed
that the crucial role for the biological activity of the obtained compounds 9Z-22Z were both
the number of methoxy-groups in ring A as well as the position of the styryl fragment on
benzoxazolone heterocycle (4-, 5-, 6- or 7). Among the reported CA-4 bioisosteres,
compound 16Z, (Z)-3-methyl-6-(3,4,5-trimethoxystyryl)-2(3H)-benzoxazolone, exhibited
potent anti-proliferative and proapoptotic effects in liver cancer cells, being similar or better
compared to CA-4. The inhibition of cellular proliferation is due to induction of mitotic arrest.
This is followed by an abnormal exit of HepG2 cells from mitosis without cytokinesis into a
pseudo G1-like multinucleate state leading to late apoptosis and cell death. These results
suggest that the bioisosteric replacement of 3-hydroxy-4-methoxyphenyl moiety of CA-4
(ring B) with benzoxazolone scaffold would be a useful approach in searching for new
anticancer agents. Currently, we are expanding our work with the synthesis and biological
evaluation of a series of other heterocyclic analogues of combretastatin A-4.
4
4
4
. Experimental
.1. Chemistry
.1.1. General
All commercially obtained reagents and solvents were used as received. Reactions were
monitored by thin-layer chromatography (TLC) on silica gel plates (Kieselgel 60 F ,) using
2
54
hexane/acetone (2:1 v/v) or heptane/ethyl acetate (7:3 v/v) as eluent. Column chromatography
was performed with Merck 60 silica gel (0.040-0.063 mm, 230-400 mesh) or Merck

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1
aluminiumoxid 90 (0.063-0.200 mm, 70-230 mesh). H NMR spectra were acquired on a
Bruker DRX250 or Bruker DRX400 spectrometers in CDCl as solvent (unless otherwise
3
stated). Chemical shifts were reported in parts per million (ppm, δ) relative to the solvent peak
(
CDCl , 7.26 ppm). Coupling constants (J) were measured in hertz (Hz). IR spectra were
3
recorded on a Specord 71 spectrometer. Elemental analyses (C, H, N) were carried out by a
Vario III microanalyzer. Obtained results were within 0.4% of theoretical values. Melting
points were determined on a Boetius hot-stage microscope.
4
.1.2. General procedure for the synthesis of phosphonium salts 5-8
Triphenylphosphine (5.25 g, 20 mmol) was added to a solution of the corresponding
bromomethyl derivative 31-34 (4.84 g, 20 mmol) in chlorobenzene (30 mL). The reaction
mixture was heated to reflux for 15 min and then allowed to cool to room temperature. The
obtained crystals were filtered off and washed with chlorobenzene. The phosphonium salts
were used in the next stage without further purification.
4
.1.2.1. [(3-Methyl-2(3H)-benzoxazolone-4-yl)methyl]triphenylphosphonium bromide (5)
Starting from bromomethyl derivative 31, compound 5 was obtained as light yellow crystals.
-1
1
Yield: 65% (6.75 g). Mp: 265–267 °C. IR (nujol, cm ): 1770 (C=O). H NMR (CDCl , 400
3
MHz): δ 2.99 (s, 3H, NCH ), 5.81 (d, 2H, J = 13.6 Hz, PCH ), 6.76 (t, 1H, J = 7.9 Hz, ArH),
3
2
6
.98 (dd, 1H, J = 2.0, 7.8 Hz, ArH), 7.13 (dd, 1H, J = 2.0, 7.8 Hz, ArH), 7.59–7.63 (m, 6H,
ArH), 7.70–7.78 (m, 9H, ArH). Calcd. for C H BrNO P: C 64.30; H 4.60; N 2.78. Found: C
2
7
23
2
6
3.98; H 4.83; N 2.83.
4
.1.2.2. [(3-Methyl-2(3H)-benzoxazolone-5-yl)methyl]triphenylphosphonium bromide (6)
Starting from bromomethyl derivative 32, compound 6 was obtained as light yellow crystals.
-1
1
Yield: 88% (8.88 g). Mp: 283–285 °C. IR (nujol, cm ): 1780 (C=O). H NMR (CDCl , 250
3

ACCEPTED MANUSCRIPT
MHz): δ 3.15 (s, 3H, NCH ), 5.58 (d, 2H, J = 14.3 Hz, PCH ), 6.81–6.83 (m, 2H, ArH), 7.29–
3
2
7
.30 (m, 1H, ArH), 7.55–7.63 (m, 6H, ArH), 7.71–7.81 (m, 9H, ArH). Anal. Calcd. for
C H BrNO P: C 64.30; H 4.60; N 2.78. Found: C 63.90; H 4.63; N 2.97.
27
23
2
4
.1.2.3. [(3-Methyl-2(3H)-benzoxazolone-6-yl)methyl]triphenylphosphonium bromide (7)
Starting from bromomethyl derivative 33, compound 7 was obtained as white crystals. Yield:
-
1
1
7
3
1
2
2% (7.26 g). Mp: 308–311 °C. IR (nujol, cm ): 1780 (C=O). H NMR (CDCl , 250 MHz): δ
3
.28 (s, 3H, NCH ), 5.65 (d, 2H, J = 14.3 Hz, PCH ), 6.70–6.73 (m, 2H, ArH), 7.22–7.23 (m,
3
2
H, ArH), 7.57–7.82 (m, 15H, ArH). Anal. Calcd. for C H BrNO P: C 64.30; H 4.60; N
2
7
23
2
.78. Found: C 64.29; H 4.47; N 2.85.
4
.1.2.4. [(3-Methyl-2(3H)-benzoxazolone-7-yl)methyl]triphenylphosphonium bromide (8)
Starting from bromomethyl derivative 34, compound 8 was obtained as light yellow crystals.
-1
1
Yield: 68% (6.86 g). Mp: 264–265 °C. IR (nujol, cm ): 1780 (C=O). H NMR (CDCl , 400
3
MHz): δ 3.24 (s, 3H, NCH ), 5.46 (d, 2H, J = 14.2 Hz, PCH ), 6.90 (d, 1H, J = 7.9 Hz, ArH),
3
2
6
7
6
.99 (t, 1H, J = 7.9 Hz, ArH), 7.20 (d, 1H, J = 7.4 Hz, ArH), 7.57–7.62 (m, 6H, ArH), 7.71–
.77 (m, 9H, ArH). Anal. Calcd. for C H BrNO P: C 64.30; H 4.60; N 2.78. Found: C
2
7
23
2
4.58; H 4.84; N 2.62.
4
.1.3. General procedure for the synthesis of (E/Z)-styrylbenzoxazolones 9-22
To a stirred solution of the corresponding phosphonium salt 5-8 (1.51 g, 3 mmol) in
THF/CH Cl (15 mL, 2:1 v/v), powdered potassium carbonate (1.38 g, 10 mmol) and 18-
2
2
crown-6 (0.01 g) were added, followed by methoxy-substituted benzaldehyde (3 mmol). The
reaction mixture was refluxed for 2-6 h (monitored by TLC). After cooling, the inorganic salts
were filtered off and the filtrate was concentrated under reduced pressure. A mixture of the
corresponding E- and Z-stilbene and triphenylphosphine oxide was obtained. Both isomers

ACCEPTED MANUSCRIPT
were isolated by column chromatography on silica gel or aluminium oxide. The Z-stilbenes
were eluted first, followed by the E-isomers. The products were additionally purified by
recrystallyzation from ethanol.
4
.1.3.1. (E/Z)-3-Methyl-4-(3,4,5-trimethoxystyryl)-2(3H)-benzoxazolone (9)
Following the general procedure 4.1.3., diastereomers 9Z and 9E were obtained by reaction of
,4,5-trimethoxybenzaldehyde and phosphonium salt 5. Separation by column
chromatography (aluminium oxide, petroleum ether/acetone, 10:1 v/v) afforded pure stilbenes
Z (0.48 g, 47% yield) and 9E (0.38 g, 37% yield). Compound 9Z: white powder. Mp: 87–90
3
9
-1
1
°
C. IR (nujol, cm ): 1780 (C=O). H NMR (CDCl , 250 MHz): δ 3.46 (s, 3H, NCH ), 3.56 (s,
3 3
6
1
5
H, OCH ), 3.82 (s, 3H, OCH ), 6.33 (s, 2H, ArH), 6.66 (d, 1H, J = 12.1 Hz, =СН), 6.75 (d,
3 3
H, J = 12.1 Hz, =СН), 7.04–7.14 (m, 3H, ArH). Anal. Calcd. for C H NO : C 66.85; H
1
9
19
5
.61 N 4.10. Found: C 66.89; H 5.32; N 3.85. Compound 9E: white crystals. Mp: 166–167
-1
1
°
C. IR (nujol, cm ): 1760 (C=O). H NMR (CDCl , 250 MHz): δ 3.67 (s, 3H, NCH ), 3.88 (s,
3 3
3
H, OCH ), 3.92 (s, 6H, OCH ), 6.72 (s, 2H, ArH), 6.94 (d, 1H, J = 15.9 Hz, =СН), 7.09–7.14
3 3
(
m, 3H, ArH), 7.37 (d, 1H, J = 15.9 Hz, =СН). Anal. Calcd. for C H NO : C 66.85; H 5.61
19 19 5
N 4.10. Found: C 67.05; H 5.47; N 3.89.
4
.1.3.2. (E/Z)-4-(3,5-Dimethoxystyryl)-3-methyl-2(3H)-benzoxazolone (10)
Following the general procedure 4.1.3., diastereomers 10Z and 10E were obtained by reaction
of 3,5-dimethoxybenzaldehyde and phosphonium salt 5. Separation by column
chromatography (aluminium oxide, petroleum ether/acetone, 12:1 v/v) afforded pure stilbenes
1
0Z (0.35 g, 38% yield) and 10E (0.26 g, 28% yield). Compound 10Z: colourless oil. IR
-1
1
(
capillary film, cm ): 1780 (C=O). H NMR (CDCl , 250 MHz): δ 3.45 (s, 3H, NCH ), 3.56
3 3
(
s, 6H, OCH ), 6.24 (d, 2H, J = 2.3 Hz, ArH), 6.29 (t, 1H, J = 2.3 Hz, ArH), 6.69 (d, 1H, J =
3
1
2.2 Hz, =СН), 6.79 (d, 1H, J = 12.1 Hz, =СН), 7.01–7.12 (m, 3H, ArH). Anal. Calcd. for

ACCEPTED MANUSCRIPT
C H NO : C 69.44; H 5.50 N 4.50. Found: C 69.24; H 5.45 N 4.45. Compound 10E: white
18
17
4
-1
1
crystals. Mp: 189–190 °C. IR (nujol, cm ): 1770 (C=O). H NMR (CDCl , 250 MHz): δ 3.67
3
(
s, 3H, NCH ), 3.84 (s, 6H, OCH ), 6.45 (t, 1H, J = 2.2 Hz, ArH), 6.66 (d, 2H, J = 2.2 Hz,
3 3
ArH), 6.95 (d, 1H, J = 15.9 Hz, =СН), 7.09–7.14 (m, 2H, ArH), 7.32–7.36 (m, 1H, ArH), 7.46
(
d, 1H, J = 15.9 Hz, =СН). Anal. Calcd. for C H NO : C 69.44; H 5.50 N 4.50. Found: C
18 17 4
6
9.38; H 5.60 N 4.52.
4
.1.3.3. (E/Z)-4-(3,4-Dimethoxystyryl)-3-methyl-2(3H)-benzoxazolone (11)
Following the general procedure 4.1.3., diastereomers 11Z and 11E were obtained by reaction
of 3,4-dimethoxybenzaldehyde and phosphonium salt 5. Separation by column
chromatography (aluminium oxide, petroleum ether/acetone, 12:1 v/v) afforded pure stilbenes
1
1Z (0.32 g, 34% yield) and 11E (0.21 g, 23% yield). Compound 11Z: colourless oil. IR
-1
1
(
capillary film, cm ): 1780 (C=O). H NMR (CDCl , 250 MHz): δ 3.45 (s, 3H, NCH ), 3.48
3
3
(
s, 3H, OCH ), 3.83 (s, 3H, OCH ), 6.57 (br s, 1H, ArH), 6.66 (d, 1H, J = 12.3 Hz, =СН),
3 3
6
.70 (d, 1H, J = 12.3 Hz, =СН), 6.71–6.72 (m, 2H, ArH), 7.03–7.13 (m, 3H, ArH). Anal.
Calcd. for C H NO : C 69.44; H 5.50 N 4.50. Found: C 69.64; H 5.68 N 4.70. Compound
1
8
17
4
-1
1
1
1E: white crystals. Mp: 183–185 °C. IR (nujol, cm ): 1750 (C=O). H NMR (CDCl , 250
3
MHz): δ 3.68 (s, 3H, NCH ), 3.92 (s, 3H, OCH ), 3.94 (s, 3H, OCH ), 6.89 (d, 1H, J = 8.3 Hz,
3
3
3
ArH), 6.96 (d, 1H, J = 16.0 Hz, =СН), 7.01 (d, 1H, J = 2.0 Hz, ArH), 7.05–7.11 (m, 3H,
ArH), 7.32–7.35 (m, 1H, ArH), 7.34 (d, 1H, J = 16.0 Hz, =СН). Anal. Calcd. for C H NO :
1
8
17
4
C 69.44; H 5.50 N 4.50. Found: C 69.72; H 5.55 N 4.60.
4
.1.3.4. (E/Z)-4-(4-Methoxystyryl)-3-methyl-2(3H)-benzoxazolone (12)
Following the general procedure 4.1.3., diastereomers 12Z and 12E were obtained by reaction
of 4-methoxybenzaldehyde and phosphonium salt 5. Separation by column chromatography
(aluminium oxide, petroleum ether/acetone, 15:1 v/v) afforded pure stilbenes 12Z (0.29 g,

ACCEPTED MANUSCRIPT
3
4% yield) and 12E (0.30 g, 36% yield). Compound 12Z: white powder. Mp: 133–135 °C. IR
-1
1
(
nujol, cm ): 1750 (C=O). H NMR (CDCl , 250 MHz): δ 3.45 (s, 3H, NCH ), 3.76 (s, 3H,
3
3
OCH ), 6.64 (d, 1H, J = 12.0 Hz, =СН), 6.69–6.73 (m, 3H, ArH, =СН), 6.99–7.05 (m, 4H,
3
ArH), 7.09–7.12 (m, 1H, ArH). Anal. Calcd. for C H NO : C 72.58; H 5.37 N 4.98. Found:
17
15
3
-1
C 72.38; H 5.29 N 4.72. Compound 12E: white crystals. Mp: 180–181 °C. IR (nujol, cm ):
1
1
2
780 (C=O). H NMR (CDCl , 250 MHz): δ 3.67 (s, 3H, NCH ), 3.85 (s, 3H, OCH ), 6.93 (d,
3
3
3
H, J = 8.8 Hz, ArH), 6.98 (d, 1H, J = 15.8 Hz, =СН), 7.08–7.10 (m, 2H, ArH), 7.33–7.38
(
m, 2H, ArH, =СН), 7.44 (d, 2H, J = 8.6 Hz, ArH). Anal. Calcd. for C H NO : C 72.58; H
17 15 3
5
.37 N 4.98. Found: C 72.44; H 5.35 N 4.85.
4
.1.3.5. (E/Z)-3-Methyl-5-(3,4,5-trimethoxystyryl)-2(3H)-benzoxazolone (13)
Following the general procedure 4.1.3., diastereomers 13Z and 13E were obtained by reaction
of 3,4,5-trimethoxybenzaldehyde and phosphonium salt 6. Recrystallization of the crude
reaction mixture from ethanol afforded pure trans-stilbene 13E (0.27 g, 26% yield). Column
chromatography of the mother liquors (silica gel, petroleum ether/acetone, 5:1 v/v) gave pure
stilbenes 13Z (0.32 g, 31% yield) and 13E (0.13 g, 13% yield). Compound 13Z: white
-1
1
crystals. Mp: 144–147 °C. IR (nujol, cm ): 1780 (C=O). H NMR (CDCl , 250 MHz): δ 3.29
3
(
s, 3H, NCH ), 3.67 (s, 6H, OCH ), 3.83 (s, 3H, OCH ), 6.47 (s, 2H, ArH), 6.55 (s, 2H, =СН),
3 3 3
1
6
.86 (br s, 1H, ArH), 7.07–7.08 (m, 2H, ArH). H NMR (acetone-d , 250 MHz): δ 3.31 (s, 3H,
6
NCH ), 3.63 (s, 6H, OCH ), 3.71 (s, 3H, OCH ), 6.56 (d, 1H, J = 12.2 Hz, =СН), 6.57 (s, 2H,
3
3
3
ArH), 6.63 (d, 1H, J = 12.2 Hz, =СН), 7.07–7.11 (m, 2H, ArH), 7.17 (dd, 1H, J = 1.0, 7.8 Hz,
ArH). Anal. Calcd. for C H NO : C 66.85; H 5.61 N 4.10. Found: C 66.45; H 5.53; N 4.38.
1
9
19
5
-1
1
Compound 13E: white crystals. Mp: 174–176 °C. IR (nujol, cm ): 1770 (C=O). H NMR
(
CDCl , 250 MHz): δ 3.44 (s, 3H, NCH ), 3.88 (s, 3H, OCH ), 3.92 (s, 6H, OCH ), 6.74 (s,
3 3 3 3
2
H, ArH), 7.01 (s, 2H, =СН), 7.12 (d, 1H, J = 1.3 Hz, ArH), 7.17 (d, 1H, J = 8.3 Hz, ArH),
1
7
.23 (dd, 1H, J = 1.3, 8.3 Hz, ArH). H NMR (acetone-d , 250 MHz): δ 3.44 (s, 3H, NCH ),
6
3

ACCEPTED MANUSCRIPT
3
7
7
6
.74 (s, 3H, OCH ), 3.88 (s, 6H, OCH ), 6.92 (s, 2H, ArH), 7.18 (d, 1H, J = 16.3 Hz, =СН),
3
3
.19–7.23 (m, 1H, ArH), 7.26 (d, 1H, J = 16.3 Hz, =СН), 7.30 (dd, 1H, J = 1.7, 8.3 Hz, ArH),
.46 (d, 1H, J = 1.7 Hz, ArH). Anal. Calcd. for C H NO : C 66.85; H 5.61 N 4.10. Found: C
1
9
19
5
6.65; H 5.59; N 4.50.
4
.1.3.6. (E/Z)-5-(3,4-Dimethoxystyryl)-3-methyl-2(3H)-benzoxazolone (14)
Following the general procedure 4.1.3., diastereomers 14Z and 14E were obtained by reaction
of 3,4-dimethoxybenzaldehyde and phosphonium salt 6. Recrystallization of the crude
reaction mixture from ethanol afforded pure trans-stilbene 14E (0.22 g, 24% yield). Column
chromatography of the mother liquors (silica gel, petroleum ether/acetone, 7:1 v/v) gave pure
stilbenes 14Z (0.33 g, 35% yield) and 14E (0.12 g, 13% yield). Compound 14Z: white
-1
1
crystals. Mp: 139–142 °C. IR (nujol, cm ): 1760 (C=O). H NMR (CDCl , 400 MHz): δ 3.27
3
(
(
(
s, 3H, NCH ), 3.64 (s, 3H, OCH ), 3.85 (s, 3H, OCH ), 6.50 (d, 1H, J = 12.2 Hz, =СН), 6.54
3 3 3
d, 1H, J = 12.2 Hz, =СН), 6.73 (d, 1H, J = 8.3 Hz, ArH), 6.75 (d, 1H, J = 1.7 Hz, ArH), 6.79
dd, 1H, J = 1.7, 8.3 Hz, ArH), 6.85 (br s, 1H, ArH), 7.02–7.07 (m, 2H, ArH). Anal. Calcd.
for C H NO : C 69.44; H 5.50; N 4.50. Found: C 69.21; H 5.53; N 4.43. Compound 14E:
1
8
17
4
-1
1
white crystals. Mp: 169–170 °C. IR (nujol, cm ): 1770 (C=O). H NMR (CDCl , 400 MHz): δ
3
3
6
7
.42 (s, 3H, NCH ), 3.89 (s, 3H, OCH ), 3.93 (s, 3H, OCH ), 6.86 (d, 1H, J = 8.3 Hz, ArH),
3 3 3
.96 (d, 1H, J = 16.4 Hz, =СН), 7.00 (d, 1H, J = 16.4 Hz, =СН), 7.03–7.05 (m, 2H, ArH),
.09 (d, 1H, J = 1.3 Hz, ArH), 7.14 (d, 1H, J = 8.3 Hz, ArH), 7.20 (dd, 1H, J = 1.6, 8.3 Hz,
ArH). Anal. Calcd. for C H NO : C 69.44; H 5.50; N 4.50. Found: C 69.46; H 5.35; N 4.51.
1
8
17
4
4
.1.3.7. (E/Z)-5-(4-Methoxystyryl)-3-methyl-2(3H)-benzoxazolone (15)
Following the general procedure 4.1.3., diastereomers 15Z and 15E were obtained by reaction
of 4-methoxybenzaldehyde and phosphonium salt 6. Recrystallization of the crude reaction
mixture from ethanol afforded pure trans-stilbene 15E (0.15 g, 18% yield). Column

ACCEPTED MANUSCRIPT
chromatography of the mother liquors (silica gel, petroleum ether/acetone, 7:1 v/v) gave pure
stilbenes 15Z (0.28 g, 33% yield) and 15E (0.10 g, 12% yield). Compound 15Z: white
-1
1
powder. Mp: 121–122 °C. IR (nujol, cm ): 1760 (C=O). H NMR (CDCl , 400 MHz): δ 3.25
3
(
s, 3H, NCH ), 3.77 (s, 3H, OCH ), 6.48 (d, 1H, J = 12.2 Hz, =СН), 6.54 (d, 1H, J = 12.2 Hz,
3 3
=СН), 6.75 (d, 2H, J = 8.8 Hz, ArH), 6.82 (br s, 1H, ArH), 7.00 (dd, 1H, J = 1.4, 8.4 Hz,
ArH), 7.04 (d, 1H, J = 8.4 Hz, ArH), 7.14 (d, 2H, J = 8.8 Hz, ArH). Anal. Calcd. for
C H NO : C 72.58; H 5.37; N 4.98. Found: C 72.36; H 5.42; N 4.94. Compound 15E: white
17
15
3
-1
1
crystals. Mp: 183–185 °C. IR (nujol, cm ): 1790 (C=O). H NMR (CDCl , 400 MHz): δ 3.42
3
(
s, 3H, NCH ), 3.82 (s, 3H, OCH ), 6.89 (d, 2H, J = 8.8 Hz, ArH), 6.95 (d, 1H, J = 16.2 Hz,
3 3
=СН), 7.00 (d, 1H, J = 16.2 Hz, =СН), 7.08 (d, 1H, J = 1.4 Hz, ArH), 7.13 (d, 1H, J = 8.4 Hz,
ArH), 7.19 (dd, 1H, J = 1.4, 8.4 Hz, ArH), 7.43 (d, 2H, J = 8.8 Hz, ArH). Anal. Calcd. for
C H NO : C 72.58; H 5.37; N 4.98. Found: C 72.56; H 5.53; N 4.83.
17
15
3
4
.1.3.8. (E/Z)-3-Methyl-6-(3,4,5-trimethoxystyryl)-2(3H)-benzoxazolone (16)
Following the general procedure 4.1.3., diastereomers 16Z and 16E were obtained by reaction
of 3,4,5-trimethoxybenzaldehyde and phosphonium salt 7. Recrystallization of the crude
reaction mixture from ethanol afforded pure trans-stilbene 16E (0.30 g, 29% yield). Column
chromatography of the mother liquors (silica gel, petroleum ether/acetone, 5:1 v/v) gave pure
stilbene 16Z (0.36 g, 35% yield) and 16E (0.12 g, 12% yield). Compound 16Z: white crystals.
-1
1
Mp: 118–120 °C. IR (nujol, cm ): 1760 (C=O). H NMR (CDCl , 400 MHz): δ 3.39 (s, 3H,
3
NCH ), 3.69 (s, 6H, OCH ), 3.85 (s, 3H, OCH ), 6.48 (s, 2H, ArH), 6.53 (s, 2H, =СН), 6.83
3
3
3
1
(
(
(
d, 1H, J = 8.2 Hz, ArH), 7.14 (dd, 1H, J = 1.2, 8.2 Hz, ArH), 7.17 (br s, 1H, ArH). H NMR
acetone-d , 250 MHz): δ 3.38 (s, 3H, NCH ), 3.64 (s, 6H, OCH ), 3.71 (s, 3H, OCH ), 6.55
6
3
3
3
d, 1H, J = 12.2 Hz, =СН), 6.57 (s, 2H, ArH), 6.60 (d, 1H, J = 12.2 Hz, =СН), 7.11 (dd, 1H, J
0.4, 8.0 Hz, ArH), 7.15–7.16 (m, 1H, ArH), 7.20 (dd, 1H, J = 1.5, 8.0 Hz, ArH). Anal.
Calcd. for C H NO : C 66.85; H 5.61; N 4.10. Found: C 67.21; H 5.49; N 4.02. Compound
=
1
9
19
5

ACCEPTED MANUSCRIPT
-1
1
1
6E: white crystals. Mp: 159–160 °C. IR (nujol, cm ): 1770 (C=O). H NMR (CDCl , 400
3
MHz): δ 3.40 (s, 3H, NCH ), 3.85 (s, 3H, OCH ), 3.90 (s, 6H, OCH ), 6.71 (s, 2H, ArH), 6.92
3
3
3
(
d, 1H, J = 8.0 Hz, ArH), 6.95 (d, 1H, J = 16.4 Hz, =CH), 6.97 (d, 1H, J = 16.4 Hz, =CH),
.29 (dd, 1H, J = 1.2, 8.0 Hz, ArH), 7.38 (d, 1H, J = 1.2 Hz, ArH). Anal. Calcd. for
C H NO : C 66.85; H 5.61; N 4.10. Found: C 67.15; H 5.54; N 4.00.
7
19
19
5
4
.1.3.9. (E/Z)-6-(3,4-Dimethoxystyryl)-3-methyl-2(3H)-benzoxazolone (17)
Following the general procedure 4.1.3., diastereomers 17Z and 17E were obtained by reaction
of 3,4-dimethoxybenzaldehyde and phosphonium salt 7. Separation by column
chromatography (silica gel, petroleum ether/acetone, 8:1 v/v) afforded pure stilbenes 17Z
(0.32 g, 34% yield) and 17E (0.23 g, 25% yield). Compound 17Z: white crystals. Mp: 125–
-
1
1
1
3
27 °C. IR (nujol, cm ): 1760 (C=O). H NMR (CDCl , 400 MHz): δ 3.36 (s, 3H, NCH ),
3 3
.66 (s, 3H, OCH ), 3.85 (s, 3H, OCH ), 6.47 (d, 1H, J = 12.1 Hz, =СН), 6.51 (d, 1H, J = 12.1
3
3
Hz, =СН), 6.72 (d, 1H, J = 8.2 Hz, ArH), 6.75 (d, 1H, J = 1.6 Hz, ArH), 6.77–6.81 (m, 2H,
ArH), 7.09–7.12 (m, 2H, ArH). Anal. Calcd. for C H NO : C 69.44; H 5.50; N 4.50. Found:
18
17
4
-1
C 69.82; H 5.83; N 4.39. Compound 17E: white crystals. Mp: 200–201 °C. IR (nujol, cm ):
1
1
3
1
760 (C=O). H NMR (CDCl , 400 MHz): δ 3.41 (s, 3H, NCH ), 3.91 (s, 3H, OCH ), 3.95 (s,
3
3
3
H, OCH ), 6.88 (d, 1H, J = 8.4 Hz, ArH), 6.92 (d, 1H, J = 8.0 Hz, ArH), 6.96 (d, 1H, J =
3
6.4 Hz, =СН), 6.98 (d, 1H, J = 16.4 Hz, =СН), 7.04–7.06 (m, 2H, ArH), 7.29 (d, 1H, J = 8.0
Hz, ArH), 7.38 (br s, 1H, ArH). Anal. Calcd. for C H NO : C 69.44; H 5.50; N 4.50. Found:
18
17
4
C 69.80; H 5.80; N 4.39.
4
.1.3.10. (E/Z)-6-(4-Methoxystyryl)-3-methyl-2(3H)-benzoxazolone (18)
Following the general procedure 4.1.3., diastereomers 18Z and 18E were obtained by reaction
of 4-methoxybenzaldehyde and phosphonium salt 7. Recrystallization of the crude reaction
mixture from ethanol afforded pure trans-stilbene 18E (0.15 g, 18% yield). Column

ACCEPTED MANUSCRIPT
chromatography of the mother liquors (silica gel, petroleum ether/acetone, 10:1 v/v) gave
pure stilbenes 18Z (0.29 g, 34% yield) and 18E (0.03 g, 4% yield). Compound 18Z: light
-1
1
yellow crystals. Mp: 92–94 °C. IR (nujol, cm ): 1760 (C=O). H NMR (CDCl , 400 MHz): δ
3
3
.38 (s, 3H, NCH ), 3.79 (s, 3H, OCH ), 6.48 (d, 1H, J = 12.0 Hz, =СН), 6.54 (d, 1H, J = 12.0
3 3
Hz, =СН), 6.77 (d, 2H, J = 8.8 Hz, ArH), 6.82 (d, 1H, J = 8.4 Hz, ArH), 7.09–7.11 (m, 2H,
ArH), 7.15 (d, 2H, J = 8.8 Hz, ArH). Anal. Calcd. for C H NO : C 72.58; H 5.37; N 4.98.
1
7
15
3
Found: C 72.52; H 5.40; N 4.97. Compound 18E: white crystals. Mp: 187–189 °C. IR (nujol,
-1
1
cm ): 1760 (C=O). H NMR (CDCl , 400 MHz): δ 3.41 (s, 3H, NCH ), 3.84 (s, 3H, OCH ),
3
3
3
6
7
.91–6.92 (m, 3H, ArH), 6.96 (d, 1H, J = 16.4 Hz, =СН), 6.99 (d, 1H, J = 16.4 Hz, =СН),
.28 (dd, 1H, J = 1.0, 8.0 Hz, ArH), 7.38 (br s, 1H, ArH), 7.44 (d, 2H, J = 8.7 Hz, ArH). Anal.
Calcd. for C H NO : C 72.58; H 5.37; N 4.98. Found: C 72.18; H 5.29; N 4.34.
1
7
15
3
4
.1.3.11. (E/Z)-3-Methyl-7-(3,4,5-trimethoxystyryl)-2(3H)-benzoxazolone (19)
Following the general procedure 4.1.3., diastereomers 19Z and 19E were obtained by reaction
of 3,4,5-trimethoxybenzaldehyde and phosphonium salt 8. Separation by column
chromatography (silica gel, petroleum ether/acetone, 12:1 v/v) afforded pure stilbenes 19Z
(0.29 g, 28% yield) and 19E (0.33 g, 32% yield). Compound 19Z: white powder. Mp: 140–
-
1
1
1
3
6
42 °C. IR (nujol, cm ): 1770 (C=O). H NMR (CDCl , 250 MHz): δ 3.40 (s, 3H, NCH ),
3
3
.66 (s, 6H, OCH ), 3.84 (s, 3H, OCH ), 6.49 (s, 2H, ArH), 6.62 (d, 1H, J = 12.1 Hz, =СН),
3
3
.71 (d, 1H, J = 12.1 Hz, =СН), 6.80 (dd, 1H, J = 1.4, 7.4 Hz, ArH), 7.01 (t, 1H, J = 7.4 Hz,
ArH), 7.09 (dd, 1H, J = 1.2, 7.9 Hz, ArH). Anal. Calcd. for C H NO : C 66.85; H 5.61 N
1
9
19
5
4
.10. Found: C 67.21; H 5.92; N 4.11. Compound 19E: white crystals. Mp: 218–219 °C. IR
-1
1
(
nujol, cm ): 1780 (C=O). H NMR (CDCl , 250 MHz): δ 3.43 (s, 3H, NCH ), 3.88 (s, 3H,
3
3
OCH ), 3.93 (s, 6H, OCH ), 6.79 (s, 2H, ArH), 6.84 (dd, 1H, J = 1.6, 7.3 Hz, ArН), 7.05 (d,
3
3
1
H, J = 16.3 Hz, =СН), 7.18 (t, 1H, J = 7.5 Hz, ArH), 7.22–7.25 (m, 1H, ArН), 7.40 (d, 1H, J

ACCEPTED MANUSCRIPT
=
16.3 Hz, =СН). Anal. Calcd. for C H NO : C 66.85; H 5.61 N 4.10. Found: C 67.25; H
1
9
19
5
5
.85; N 4.20.
4
.1.3.12. (E/Z)-7-(3,5-Dimethoxystyryl)-3-methyl-2(3H)-benzoxazolone (20)
Following the general procedure 4.1.3., diastereomers 20Z and 20E were obtained by reaction
of 3,5-dimethoxybenzaldehyde and phosphonium salt 8. Separation by column
chromatography (aluminium oxide, petroleum ether/acetone, 7:1 v/v) afforded pure stilbenes
2
0Z (0.23 g, 25% yield) and 20E (0.25 g, 27% yield). Compound 20Z: white powder. Mp:
-1
1
1
00–103 °C. IR (nujol, cm ): 1780 (C=O). H NMR (CDCl , 250 MHz): δ 3.39 (s, 3H,
3
NCH ), 3.66 (s, 6H, OCH ), 6.34 (t, 1H, J = 2.3 Hz, ArH), 6.41 (d, 2H, J = 2.3 Hz, ArH), 6.66
3
3
(d, 1H, J = 12.2 Hz, =СН), 6.74 (d, 1H, J = 12.1 Hz, =СН), 6.79 (dd, 1H, J = 1.7, 7.2 Hz,
ArН), 6.98 (t, 1H, J = 7.8 Hz, ArН), 7.04 (dd, 1H, J = 1.7, 8.1 Hz, ArН). Anal. Calcd. for
C H NO : C 69.44; H 5.50 N 4.50. Found: C 69.70; H 5.61; N 4.65. Compound 20E: white
18
17
4
-1
1
crystals. Mp: 160–162 °C. IR (nujol, cm ): 1780 (C=O). H NMR (CDCl , 250 MHz): δ 3.43
3
(
s, 3H, NCH ), 3.84 (s, 6H, OCH ), 6.43 (t, 1H, J = 2.3 Hz, ArH), 6.71 (d, 2H, J = 2.3 Hz,
3 3
ArH), 6.85 (dd, 1H, J = 1.5, 7.5 Hz, ArН), 7.13 (d, 1H, J = 16.5 Hz, =СН), 7.18–7.23 (m, 2H,
ArН), 7.39 (d, 1H, J = 16.5 Hz, =СН). Anal. Calcd. for C H NO : C 69.44; H 5.50 N 4.50.
18
17
4
Found: C 69.22; H 5.64; N 4.64.
4
.1.3.13. (E/Z)-7-(3,4-Dimethoxystyryl)-3-methyl-2(3H)-benzoxazolone (21)
Following the general procedure 4.1.3., diastereomers 21Z and 21E were obtained by reaction
of 3,4-dimethoxybenzaldehyde and phosphonium salt 8. Separation by column
chromatography (aluminium oxide, petroleum ether/acetone, 7:1 v/v) afforded pure stilbenes
2
1Z (0.45 g, 48% yield) and 21E (0.34 g, 36% yield). Compound 21Z: white powder. Mp:
-1
1
1
09–110 °C. IR (nujol, cm ): 1780 (C=O). H NMR (CDCl , 250 MHz): δ 3.40 (s, 3H,
3
NCH ), 3.62 (s, 3H, OCH ), 3.86 (s, 3H, OCH ), 6.57 (d, 1H, J = 12.1 Hz, =СН), 6.73 (d, 1H,
3
3
3

ACCEPTED MANUSCRIPT
J = 12.1 Hz, =СН), 6.75 (d, 1H, J = 8.2 Hz, ArН), 6.78–6.82 (m, 3H, ArH), 6.99 (t, 1H, J =
7
5
.8 Hz, ArН), 7.07 (dd, 1H, J = 1.6, 8.1 Hz, ArН). Anal. Calcd. for C H NO : C 69.44; H
18 17 4
.50 N 4.50. Found: C 69.80; H 5.78; N 4.56. Compound 21E: white crystals. Mp: 192–193
-1
1
°
C. IR (nujol, cm ): 1750 (C=O). H NMR (CDCl , 250 MHz): δ 3.43 (s, 3H, NCH ), 3.92 (s,
3 3
3
H, OCH ), 3.96 (s, 3H, OCH ), 6.82 (dd, 1H, J = 1.5, 7.6 Hz, ArH), 6.88 (d, 1H, J = 8.8 Hz,
3 3
ArH), 7.03 (d, 1H, J = 16.5 Hz, =СН), 7.09–7.14 (m, 2H, ArH), 7.18 (d, 1H, J = 7.4 Hz,
ArH), 7.22–7.25 (m, 1H, ArH), 7.40 (d, 1H, J = 16.5 Hz, =СН). Anal. Calcd. for C H NO :
1
8
17
4
C 69.44; H 5.50 N 4.50. Found: C 69.75; H 5.84; N 4.60.
4
.1.3.14. (E/Z)-7-(4-Methoxystyryl)-3-methyl-2(3H)-benzoxazolone (22)
Following the general procedure 4.1.3., diastereomers 22Z and 22E were obtained by reaction
of 4-methoxybenzaldehyde and phosphonium salt 8. Separation by column chromatography
(aluminium oxide, petroleum ether/acetone, 10:1 v/v) afforded pure stilbenes 22Z (0.31 g,
3
7% yield) and 22E (0.36 g, 42% yield). Compound 22Z: white powder. Mp: 138–139 °C. IR
-1
1
(
nujol, cm ): 1780 (C=O). H NMR (CDCl , 250 MHz): δ 3.40 (s, 3H, NCH ), 3.79 (s, 3H,
3
3
OCH ), 6.55 (d, 1H, J = 12.0 Hz, =СН), 6.71–6.82 (m, 4H, ArH, =СН), 6.99–7.02 (d, 2H,
3
ArH), 7.17 (d, 2H, J = 8.5 Hz, ArH). Anal. Calcd. for C H NO : C 72.58; H 5.37 N 4.98.
1
7
15
3
Found: C 72.66; H 5.64; N 5.22. Compound 22E: white crystals. Mp: 201–203 °C. IR (nujol,
-1
1
cm ): 1750 (C=O). H NMR (CDCl , 250 MHz): δ 3.42 (s, 3H, NCH ), 3.84 (s, 3H, OCH ),
3
3
3
6
.81 (dd, 1H, J = 1.5, 7.5 Hz, ArH), 6.92 (d, 2H, J = 8.8 Hz, ArН), 7.02 (d, 1H, J = 16.5 Hz,
CH), 7.16 (t, 1H, J = 7.5 Hz, ArН), 7.22 (dd, 1H, J = 1.5, 8.0 Hz, ArН), 7.41 (d, 1H, J =
6.5 Hz, =CH), 7.50 (d, 2H, J = 8.8 Hz, ArН). Anal. Calcd. for C H NO : C 72.58; H 5.37
=
1
1
7
15
3
N 4.98. Found: C 72.88; H 5.57; N 4.67.
4
4
.2. X-Ray crystallography
.2.1. General

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All crystal were mounted on glass capillary and data were collected with Enraf Nonius CAD4
Diffractometer equipped with an with graphite monochromated detector using Mo-Kα
radiation (λ = 0.71073 Å) at 290 K. The determinations of the unit cell parameters, data
collection were performed using CAD4 Express [49] and reduction was carried out using
2
XCAD4 [50]. The crystal structures were solved by direct methods ShelxS and refined on F
by the full-matrix least-squares method with the ShelxL-2014 programs [51]. All non-
hydrogen atoms were located successfully from Fourier maps and were refined
anisotropically. H atoms on C atoms were generated geometrically with C—H = 0.9600 Å
and their positional parameters were refined as riding to the neighboring C atom with U (H)
iso
=
1.2U (C atoms). Most important crystallographic parameters and refinement indicators are
eq
presented in Table 4 and Ortep view of the asymmetric unit of structures 13Z, 14Z, 16Z and
6E is shown on Fig. 3. Complete crystallographic data for the structure reported in this paper
1
have been deposited in the CIF format with the Cambridge Crystallographic Data Center as
supplementary publication No. CCDC, 823149, 823151, 823151 and 1431461. Copies of the
data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2
1
EZ, UK (fax: (44) 1223336-033; e-mail: deposit@ccdc.cam.ac.uk).
Table 4.
4
.2.2. General Molecular Docking of the 13Z, 14Z, 16Z derivatives in the colchicine binding
site of tubulin
The starting molecular models of the styrylbenzoxazolones 13Z, 14Z, 16Z were taken from
the X-ray structures and minimization was performed through the Amber force field and the
Polak-Ribiere conjugate gradient method. The colchicine pocket of the α,β-tubulin–colchicine
complex (PDB code 1SA0 [38],) was employed in the docking studies. Ligand-protein
docking was performed with the molecular docking algorithm MolDock51 via Molegro

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Virtual Docker [39] (MVD) software, version 4.3.0. MolDock uses a heuristic search
algorithm (termed guided differential evolution) which is a combination of differential
evolution and a cavity-prediction algorithm. The docking scoring function is an extension of
the piecewise linear potential (PLP). After the ligands and the protein coordinates were
imported, all structural parameters including bond type, hybridization, explicit hydrogen,
charges, and flexible torsions were assigned using the automatic preparation function in MVD
software [39]. Due to the availability of the conformation of a structurally related compound,
colchicine, we used the template docking available in the MVD. Template docking is based
on extracting the chemical properties (e.g. pharmacophore elements of a ligand bound in the
active site) and using that information for docking structurally similar analogs. For each
compound, 50 docking runs were performed. MVD allows side chain conformational changes
by softening the potentials (steric, hydrogen bonds, and electrostatic force) used during a
docking simulation. The residues (considered flexible) were those close enough to the
reference ligand (colchicine) so that interaction can occur (6 Å around the colchicine). After
each ligand had been docked, the side chains chosen were energy-minimized by MVD with
respect to the conformation found, using the standard nonsoftened potentials. Only torsion
angles in the side chains were modified during the minimization; all other properties
(including bond lengths and backbone atom positions) were held fixed. The poses
representing the lowest value of the scoring function (MolDockScore) were further analyzed
in order to identify which of the ligand configurations most likely describe the correct binding
mode.
4
4
.3 Biology
.3.1. Cell cultures and cytotoxicity assay
Human hepatocellular carcinoma (HepG2, HB-8065), human chronic myelogeneous leukemia
K562, CCL-243), and human adenocarcinomic alveolar basal epithelial (A-549, CCL-185)
(

ACCEPTED MANUSCRIPT
cells were obtained from ATCC, HaCaT cell line was received from CLS-Germany, adult
normal human epidermal keratinocytes (NHEK, 192627) were received from Lonza,
(Belgium), and all cell lines were cultured according to the manufacturer’s instruction.
Human endothelial cells (EA.hy 926) were a gift from Dr. C-J.S. Edgell (University of North
Carolina). Human colorectal adenocarcinoma cells (HT-29) were a gift from Assoc. Prof.
Radostina Alexandrova (IEMPAM – Bulgarian Academy of Sciences). Human breast
adenocarcinoma cell lines (MCF-7, MDA-MB-231), mouse colon carcinomas cells (Colon-
2
6) and human epithelial breast control cell line (MCF-10A) were kindly provided by Prof.
Iana Tsoneva (IBPhBME – Bulgarian Academy of Sciences). The EA.hy 926, HT-29, Colon-
6, MCF-7, MDA-MB-231, and MCF-10A cells were maintained in Dulbecco's Modified
2
Eagle Medium (DMEM, Lonza, Switzerland) containing 10% fetal bovine serum (FBS), 100
U/mL penicillin, and 100 µg/mL streptomycin. All cell lines were maintained in a humidified
CO atmosphere at 37 °C.
2
All tested compounds were dissolved in DMSO at 2 mmol/L and diluted with medium to
obtain the desired concentration. DMSO concentration in the medium was kept constant at
0
1
.01%. To test the effect of the drugs on cell growth, the cells were seeded at a density of
4
x10 cells/100 µL in 96 well plates and 24h after plaiting cells were incubated up to 96 h
with different drug concentrations. The number of cells was count with Countess™
Automated Cell Counter (Invitrogen, USA) and the cytotoxic effect was assayed by MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test (Sigma, M-0283). Ten µL
MTT solution (5 mg/mL) was added to each well and further incubated for 3 h at 37 °C. To
dissolve the formazan product 100 µL/well 0.1 N HCl in anhydrous isopropanol was added.
The optical density was measured at 550/630 nm in a DTX880 spectrophotometer (Beckman
Coulter, USA). Experiments were performed in eight plicate for each compound in each
experiment. Results, expressed as concentrations that inhibit 50% of cell growth (IC ), were
5
0

ACCEPTED MANUSCRIPT
calculated by the OriginLab program. Each experiment was conducted from a minimum of
three different syntheses.
4
.3.2. Cell cycle and apoptosis
For cell cycle analysis, the cells were centrifuged and fixed with ice-cold methanol/acetone
solution (3:1 v/v) overnight at 4 °C. After two washes with phosphate buffered saline (PBS)
the cells were stained with a solution containing 1 mg/mL propidium iodide (PI) and 1 mg/mL
RNaseA/PBS for 30 min at 37 °C. The samples were measured by FACS Calibur flow
cytometer (Becton Dickinson, USA) equipped with Cellquest software. The cell doublets
were removed by gating the left area of FL2-W/FL2-A plot for analyses. The distribution of
cells in the cell cycle was analyzed using FlowJo software.
For detection of apoptosis we applied surface exposure of phosphatidylserine measured with
FITC-Annexin V (Apoptosis Detection kit, Cat. N: 556547, BD Pharmingen, USA) following
the manufacturer’s instruction.
4
.3.3. Immunocytochemistry
To assess the effect of 16Z and CA-4 on β-tubulin in HepG2 and EA.hy 926 cells an indirect
immunofluorescence was employed. Cells grown on coverslips were washed with PBS before
fixation with 3.7 % buffered paraformaldehyde. Single or double fluorescence cell labeling
was performed as described by Apostolova et al [52]. In the final step, cells were washed
three times with PBS for 5 minutes and incubated for 40 minutes with an appropriate
secondary antibody labeled with AlexaFluor-488 (Invitrogen, USA). F-actin was detected
using AlexaFlour 568 Phalloidin (Invitrogen, USA). Following three washes with PBS and
two in water, the slides were mounted in UltraCruz fluorescence mounting medium with
DAPI (CantaCruz Biotechnology, USA). Fluorescence microscopy was performed with a Carl
Zeiss AM240 microscope equipped with Andor (iXon+) camera.