Synthesis and biological evaluation of 3-functionalized 2-phenyl- and 2-alkylbenzo[b]furans as antiproliferative agents against human melanoma cell line

Article abstract of DOI:10.1016/j.bioorg.2019.102930

The key function of microtubules and mitotic spindle in cell division make them attractive targets in anticancer therapy. In the present study, functionalized in 3 position 2-phenyl- and 2-alkylbenzo[b]furans were synthesized and evaluated as antitumor ag

Full text of DOI:10.1016/j.bioorg.2019.102930

Accepted Manuscript
Synthesis and biological evaluation of 3-functionalized 2-phenyl- and 2-alkyl-
benzo[b]furans as antiproliferative agents against human melanoma cell line
Halina Kwiecień, Magdalena Perużyńska, Karolina Stachowicz, Katarzyna
Piotrowska, Joanna Bujak, Patrycja Kopytko, Marek Droździk
PII:
S0045-2068(18)30946-5
https://doi.org/10.1016/j.bioorg.2019.102930
102930
DOI:
Article Number:
Reference:
YBIOO 102930
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 August 2018
4 April 2019
15 April 2019
Please cite this article as: H. Kwiecień, M. Perużyńska, K. Stachowicz, K. Piotrowska, J. Bujak, P. Kopytko, M.
Droździk, Synthesis and biological evaluation of 3-functionalized 2-phenyl- and 2-alkylbenzo[b]furans as
antiproliferative agents against human melanoma cell line, Bioorganic Chemistry (2019), doi: https://doi.org/
10.1016/j.bioorg.2019.102930
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Synthesis and biological evaluation of 3-functionalized 2-phenyl- and 2-
alkylbenzo[b]furans as antiproliferative agents against human melanoma cell
line
Halina Kwiecieńa, Magdalena Perużyńskab, Karolina Stachowicza Katarzyna Piotrowskac,
Joanna Bujakc, Patrycja Kopytkoc, Marek Droździkb
a West Pomeranian University of Technology in Szczecin, Department of Organic Synthesis
and Drug Technology, Al. Piastów 42, 71-065 Szczecin, Poland
b Pomeranian Medical University, Department of Experimental & Clinical Pharmacology,
Powstańców Wlkp. 72, 70–111 Szczecin, Poland
c Pomeranian Medical University, Department of Physiology, Powstańców Wlkp. 72, 70–111
Szczecin, Poland
E-mail: halina.kwiecien@zut.edu.pl
Abstract
The key function of microtubules and mitotic spindle in cell division make them
attractive targets in anticancer therapy. In the present study, functionalized in 3 position 2-
phenyl- and 2-alkylbenzo[b]furans were synthesized and evaluated as antitumor agents.
Among the synthesized derivatives 13a, 13b and 14 exhibited the most potent
antiproliferative activity against human melanoma A375 cell line with IC50 values of 2.85
µM, 0.86 µM, 0.09 µM, respectively. The most promising compound defined was 14 with
three methoxy groups in the 3-aroyl substituent and 7-methoxy group in 2-
phenylbenzo[b]furan skeleton. Tubulin polymerization assay, confocal microscopy imaging
and flow cytometry analysis revealed that 2-phenyl-3-aroylbenzo[b]furans (13a, 13b and 14)
inhibited tubulin polymerization leading to disruption of mitotic spindle formation, cell cycle
arrest in G2/M phase and apoptosis.
Keywords
benzo[b]furans; acylation; anti-tubulin agents; antitumor agents; melanoma A375 cell line

1. Introduction
The majority of naturally derived anticancer drugs inhibit cell proliferation by acting on
spindle microtubules. Microtubules are protein biopolymers formed through polymerization
of heterodimer subunit of  and tubulin. The polymerization is reversible, and the
microtubule/tubule dynamics are involved in a number of cell functions, including cell
division, migration, and shape change. Rapid microtubule dynamics are especially prominent
in mitosis, and are essential for proper spindle assembly and function [1]. Disruption of
microtubule dynamics leads to cell apoptosis. A number of known antimitotic agents act by
binding to the protein tubulin, an -heterodimer that forms the core of the microtubule.
Vinica alkaloids, including vinblastine and vincristine as well as colchicine inhibit
microtubule polymerization. In contrast, taxanes, including paclitaxel, as well as laulimaide
bind to polymerized microtubules at the inner surface of the  subunit and promote tubulin
stabilization [2, 3]. The recent literature reports show that tubulin dynamics is still a
promising target for new anticancer agents [4-8]. Most of antimiotic agents interact with
tubulin through at least four major binding sites: the laulimalide, vinica alkaloid, taxane, and
colchicine site [9-11]. In contrast to targeting the taxane or vinca alkaloid binding sites, more
attention has been drawn to develop inhibitors of colchicine site, due to its potential
antimitotic and vascular disrupting properties. Such vascular disrupting agents take advantage
of the significant differences that exist between the normal, healthy tissues and tumour
vasculature [12].
Colchicine (1, Fig.1) inhibits microtubule assembly (antimitotic properties have been
known for over 50 years), but the therapeutic value of colchicine activity against cancer is
restrained by its toxicity and low therapeutic index. The significant toxicity of colchicine is
attributed to its very slow dissociation from tubulin, which leads to its persistence in sensitive
tissues for several days [13]. Although colchicine is not used as anticancer agent, there have
been multiple efforts to clinically develop colchicine binding site agents. Most known agents
that bind to the colchicine site often have shorter half-lives in vivo and are much better
tolerated. Combretastatin (2, Fig.1) and phenstatin (3) (combretastatin-analog with the double
bond of being replaced by a carbonyl group) are examples of reversible binders to the
colchicine site that have short half-lives in vivo and that are much better tolerated than
colchicine [14-16]. BNC-105 (5), phenstatin analogue with the benzene ring of phenstatin
replaced by benzofuran, exhibits selectivity for growth factor activated endothelial cells [17].
Phenstatin (3) and BNC-105 (5) have entered clinical trials, where they were administered as
2

the soluble disodium phosphate ester prodrugs 4 and 6 which were rapidly cleaved in vivo to
the active agents [15,16]. A clinical study for BNC105P (6) has shown to be generally well
tolerated [17,18]. It was found that the presence of both C7-OH and C2-substituents that
sterically interact with the trimethoxybenzoyl (cisoid-coformer) in BNC105 structure are
required for optimal potency of 5 as an antimimotic agent. The C7-OH group enables an
additional hydrogen bonding interaction with Asn β258 to be achieved. Only when both
effects are engendered (C7-OH + C2-substituent), the compound 5 exhibited good selectivity
toward activated endothelial cells [17].
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HN
OH
HO
O
1
2
3
Colchicine
Combretastatin
Phenstatin
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
O
O
O
Na2O2(O)P
P(O)O2Na2
6
5
4
BNC-105
Phenstatin Prodrug
BNC-105P
Figure 1. Chemical structures of some established drugs (2-6) and clinical trials agents
interacting with colchicine (1) binding site.
Heterocyclic benzo[b]furan is a structural unit of compounds with important
pharmacological properties, including anticancer agents [19, 20]. Both, naturally derived
benzo[b]furans such as moracins [21-22], as well as synthetically obtained benzo[b]furan
derivatives [23-28] bearing methoxy and/or hydroxy groups in their structures showed tubulin
destabilizing activity. Herein, we report the synthesis and antiproliferative activity of 2-
phenylbenzo[b]furan derivatives, functionalized with acetyl (12b), 3,4,5-trimethoxybenzoyl
(14) and 4-hydroxy-3,5-dimethoxybenzoyl groups at 3-position (13a, 13b), as well as some 2-
alkylbenzo[b]furan derivatives bearing bromine atom and functionalized with phenylacetyl
substituent (15, 16). The compounds were evaluated against A375 melanoma cell line. The
3

effects on tubulin polymerization, cell cycle and apoptosis were also evaluated. The aim of
this study was established the influence of C4-OH substituent in the benzoyl group or phenyl
group at C-2 position as well as an extra methylene between the carbonyl and phenyl group
on the antiproliferative activities.
2. Results and discussion
2.1. Chemistry
2-Phenylbenzo[b]furan (11a) and 7-methoxy-2-phenybenzo[b]furans (11b) were obtained
from 2-hydroxybenzaldehyde (7a) and 2-hydroxy-3-methoxybenzaldehyde (7b),
correspondingly, through three stage synthesis (Scheme 1). O-Alkylation of the 2-
hydroxybenzaldehydes with methyl 2-bromo-2-phenylacetate (8) in the presence of potassium
bicarbonate in dimethylformamide gave the corresponding methyl 2-(2-formylphenoxy)-2-
phenylacetates 9a and 9b in high yields. The desired 2-bromo-2-phenylacetate (8) was
prepared in quantitative yield via sequential, one-pot process involves chlorination,
bromination and esterification of phenylacetic acid. Hydrolysis of the methyl 2-(2-
formylphenoxy)-2-phenylacetates (9a and 9b) to the 2-(2-formylphenoxy)-2-phenylacetic
acids (10a and 10b), followed by cyclization of the acids gave benzo[b]furans 11a and 11b in
good yields.
Studies on the environmental friendly method for acylation of 2-phenylbenzo[b]furans
with acetic anhydride and 3,4,5-trimethoxybenzoic acid chloride with the use of ion-exchange
Amberylst-15 resin as a catalyst were carried out. The optimal reaction conditions for the
acetylation of 2-phenylbenzo[b]furan with acetic anhydride, using different ratio resin and
acetic anhydride to the benzo[b]furans as well as different reaction time, were established. As
result,
1-(2-phenylbenzo[b]furan-3-yl)ethan-1-one
(12a)
and
1-(7-methoxy-2-
phenylbenzo[b]-furan-3-yl)ethan-1-one (12b) were obtained in highest yields (69-72%) when
2.5 equivalents of acetic anhydride to 1.0 equivalent of 2-phenylbenzo[b]furan and dry
Amberlyst 15 resin were used and the reaction was carried out in anhydrous 1,2-
dichloroethane under reflux for 6 hours. The optimal reaction conditions for 7-methoxy-2-
phenylbenzo[b]furan with 3,4,5-trimethoxybenzoic acid chloride were also established (1.1
equivalent of acid chloride, dry Amberlyst 15 resin and reaction times 6.5 hours). The target
2-phenylbenzo[b]furan-3-yl)(3,4,5-trimethoxyphenyl)methanone (14) were obtained in good
yield (69%) under these conditions.
4

A selective synthesis of (4-hydroxy-3,5-dimethoxyphenyl)-(2-phenylbenzo[b]furan-3-
yl)methanone (13a) and (4-hydroxy-3,5-dimethoxyphenyl)(7-methoxy-2-
phenylbenzo[b]furan-3-yl)-methanone (13b) was achieved by acylation of adequate 2-
phenylbenozo[b]furan with 3,4,5-trimethoxybenzoyl chloride using an excess aluminium
chloride catalyst to the 2-phenylbenzo[b]furan. When the reaction was carried out in 1,2-
dichloroethane under reflux for 2-4 hours the desired hydroxy product was obtained in yield
of 68-76%.
The structural assignment of C4-OH group in 13a and 13b were achieved by comparative
analysis of 1HNMR spectra of adequate compounds. 1HNMR spectra of compounds 11b, 12b,
13b and 14 show the singlet peaks at 4.03, 4.09, 4.10 and 4.13 ppm, respectively that derived
from the 3 protons of C-7 methoxy group; whereas the 6 protons derived from C-3 and C-5
methoxy group of 13a and 13b are observed at 3.71 and 3.92 ppm, respectively. Since in the
1
structure of 13a in position C7 is only aromatic proton, thus the HNMR spectra
unambiguously indicate for the selective demethylation of the C-4 methoxy group. The
selective demethylation of C-4 methoxy group in 3,4,5-trimethoxyphenyl derivatives has
been described in the literature [29, 30].
5

Scheme 1 Synthesis and acylation of 2-phenylbenzo[b]furans. Reagents and conditions: (I) 7
(1 equiv), K2CO3 (2 equiv), DMF, 92-94 °C, 4 h; (II) 10% aq KOH, 80 °C, 2.5 h; (III) 10% aq
HCl, 0 °C to rt; (IV) Ac2O, AcONa, 120-125 °C, 4 h, (V) 2-phenylbenzo[b]furan (1 equiv),
acetic anhydride (2.5 equiv), dry Amberlyst 15 resin, 1,2-dichloroethane, reflux, 6.5h (VI)
3,4,5-trimethoxybenzoyl chloride, AlCl3 (2.5 equiv), 1,2-dichloroethane, 3.5 h, (VII) acid
chloride (1,5 equiv), dry Amberlyst 15 resin, 1,2-dichloroethane, reflux, 6.5 h.
To establish structure – proliferative activity relationship, 3-phenylcetyl functionalized
2-alkylbenzo[b]furans (15 and 16) were also prepared (Scheme 2). 1-(5-Bromo-2-
ethylbenzo[b]furan-3-yl)-2-(4-hydroxyphenyl)ethanone (15) was obtained from 5-bromo-2-
ethylbenzo[b]furan by acylation with 4-methoxyphenylacetyl chloride followed by
demethylation of the methoxy group to hydroxy according to the procedure described
previously [31]. 2-(3,5-Dibromo-4-hydroxyphenyl)-1-(2-butylbenzo[b]furan-3-yl)ethanone
(16) was prepared from 2-butylbenzo[b]furan by sequential reactions: acylation with 4-
methoxyphenylacetyl chloride, demethylation methoxy group and bromination, according to
the procedure described previously [32, 33].
Scheme 2 Synthesis of 15 and 16. Conditions: (I) 2-(4-methoxyphenyl)acetyl chloride (1 eq),
AlCl3 (1,5 eq), 1,2-dichloroethane, 0-15 °C, 6 h), (II) pyridine hydrochloride (10 eq), reflux,
10-12 min, (III) aq HBr (20%), NaClO3, 3.6 eq, rt, 2h [31-33].
2.2. Biological evaluation
6

2.2.1. In vitro antiproliferative activity
The antiproliferative activity of the compounds:12b, 13a, 13b, 14, 15 and 16 was assessed
using Cell Proliferation Reagent WST-1 assay. As shown in Tab. 1 and Fig. 2, assuming the
benzo[b]furan inhibitory effects against A375 cells, the compounds 13a, 13b and 14 revealed
the highest antiproliferative activity, with IC50 values of 2.85±0.82 µM, 0.86±0.37 µM,
0.09±0.03 µM, respectively. The obtained results showed low sensitivity of A375 cells to
12b, 15 and 16 compounds, with IC50 yielding >100 µM.
Table 1. The IC50 (half maximal inhibitory concentration) values of benzo[b]furan
derivatives against A375 cells, determined using WST-1 assay. The data indicate the mean of
IC50 values (μM)±SD of at least three independent experiments.
Compound
IC50 (µM)
>100
Compound
IC50 (µM)
0.09±0.03
12b
14
2.85±0.82
>100
13a
15
0.86±0.37
>100
13b
16
7

Figure 2. The proliferation of A375 cells after 48 h exposure to the benzo[b]furan derivatives
measured by WST-1 assay. *- p<0.05 statistical significance between treated and control
cells.
2.2.2. Structure-activity relationships
The structure-activity relationship (SAR) was analysed with respect to the
antiproliferative activity and IC50 value. Our data on 2-phenylbenzo[b]furans revealed that 3-
aroyl group in 3 position is necessary to produce anticancer activity (13a, 13b, 14) since the
analogue with 3-acetyl group (12b) was markedly less active (IC50>100 µM). Moreover, from
three the most active 3-aroyl benzo[b]furan derivatives (13a, 13b and 14) the most promising
compound defined was 14 (IC50=0.09±0.03 µM), with three methoxy group in the 3-aroyl
substituent and 7-methoxy group on benzo[b]furan skeleton. It should be pointed that the
differences between the three compounds become more distinct at 1 µM, where 13a lost
activity was seen (cell proliferation was equal to 95.79%, 54.36% and 10.51% for 13a, 13b
and 14, respectively). This observation is in keeping with findings of Romagnoli et al. who
indicated that antitumor activity of benzo[b]furan derivatives appeared to be dependent on
substitution at the heterocyclic furan ring rather than at the benzene moiety [34]. Moreover,
the replacement of the phenyl group at the 2-position to the alkyl group and introducing of an
extra methylene between the carbonyl group and the phenyl group at the 3-position
(compounds 15 and 16) caused a dramatic reduction in antiproliferative activity (IC50>100
µM) This can be explained by both reduction of the van der Waals forces after change phenyl
to alkyl group and significant conformational changes after replacing of the rigid carbonyl-
phenyl structure with carbonyl-methylene-phenyl structure.
8

Our findings are in line with other authors’ observations, which emphasize the role of
methoxy group in benzofuran anticancer activity. Moreover, Romagnoli et al. show that not
only presence of a methoxy group but also its localization in benzo[b]furan skeleton is
important. Namely, compounds with a methoxy substituent at the C-6 position of the
benzofuran ring, exhibited the best antiproliferative activity in the 3-amino and 3-
dimethylamino benzo[b]furan series, respectively, while shifting the methoxy group to the C-
3, C-5 or C-7 position resulted in major loss in activity [35]. Romagnoli et al. revealed also
that a methoxy group located at the C-6 position of the 2-(3′,4′,5′-trimethoxybenzoyl)-3-
methylbenzo[b]furan derivative yields the most active compound, whereas changing its
position to C-4, C-5 or C-7 lead to a reduction in potency [36].
2.2.3 Cell morphology
Cytotoxicity of benzo[b]furan derivatives was also determined during cell culture
optical microscopy imaging. The differences in A375 cells morphology were the most
significant at the highest concentration of the tested compounds. As shown in Fig. 3 the
cancer cells lost their characteristic shape and morphology, became spherical and ragged after
treatment with 13a and 13b derivatives (100 µM) (Fig. 3 C, D). It should be pointed that in 14
exposed cells (Fig. 3E) the similar effect was observed at even lower concentration 50 µM
(14 precipitates at higher concentration). As expected after exposure to less active 12b, 15
and 16 compounds only the cell density was lower, whereas cell morphology was preserved
(Fig. 3 B, F-G).
A
9

10

Figure 3. Optical microscopy images of A375 cells, controls (A) and after 48 h incubation
with 100 µM of compounds: 12b (B), 13a (C), 13b (D), 15 (F), 16 (G); 50 µM of 14 (E).
2.2.4. Apoptosis/necrosis detection - flow cytometry analysis
The FITC Annexin V Apoptosis Detection Kit II was used to detect apoptosis and
necrosis of the cells after 48 h incubation with benzo[b]furan derivatives. Simultaneous
staining with two dyes allows separation of four cell populations: viable (Annexin V-FITC−
and PI-), early apoptotic state (Annexin V-FITC+ and PI−), late apoptotic or already dead
(Annexin V-FITC+ and PI+) and necrotic (Annexin V-FITC− PI+). The flow cytometry
analysis revealed negligible cytotoxicity of 12b, 15 and 16 - the percentage of live cells was
high (about 90 %) and comparable with the control. However, the late apoptosis level in 12b,
15 and 16 treated cells was higher than in the control but did not exceed 8%. Simultaneously,
high anticancer activity of 3-aroyl 2-phenylbenzo[b]furan derivatives was confirmed. Namely
as shown in Fig. 4, after 13a, 13b and 14 treatment, the percentage of late apoptotic cells
increased to 57.63±13.40%, 71.21±15.03% and 58.52±14.90%, respectively. Representative
individual experiments are shown in Supplementary materials (Fig.S30).
Figure 4. The effect of benzo[b]furan derivatives (at concentration of 100 µM, except for 14:
50 µM) on the induction of apoptosis in A375 cells after 48 h treatment. The population of
live and apoptotic/necrotic cells was determined using flow cytometry. Two controls were
included: untreated cells growing in medium with DMSO (negative control) and treated with
doxorubicine 11.41 µM (positive control). Means of at least three independent experiments
are shown.
11

2.2.5. Cell cycle analysis
As known tubulin destabilizing agents cause preferential G2–M blockade, therefore
A375 cell populations were examined by flow cytometry to quantify cellular DNA content
and cell cycle distribution. As expected 3-aroyl 2-phenylbenzo[b]furan compounds caused
accumulation of A375 in a tetraploid (4N) state decreasing the percentage of cells in G0/G1
phase (Fig. 5). The percentage of A375 cells in G2/M phase was 66.34±3.26 %, 58.86±5.11
%, 63.62± 9.16 % for 13a (100 µM), 13b (100 µM) and 14 (50 µM), respectively. In contrast,
after exposure to compounds 12b (100 µM), 15 (100 µM) and 16 (100 µM) percentage of
A375 cells in G0/G1 and G2/M phase was comparable and similar to the control (untreated)
cells. Our data are in line with Kamal et al. data showing G2/M cell cycle arrest as common
mechanism leading to cancer cell apoptosis after benzofuran treatment [37].
Figure 5. Flow cytometric analysis of A375 cells cycle using Orange dye staining after
exposure to benzo[b]furan derivatives for 7 h (means of at least two independent experiments
are shown).
2.5. Confocal microscopy imaging
To confirm that the aforementioned cell cycle arrest is related to an interaction with
the microtubule system a confocal microscopy analysis was performed. After 7 h-incubation
of the cells with the tested compounds, the cells were fixed and α-tubulin and chromosomes
were stained. The control cells in mitosis showed bipolar spindle formation with
chromosomes arrangement in the central metaphase plate or anaphase distribution (Fig.6 A,
B). Similarly, after 12b treatment most of dividing cells were regular (single uncommon
12

mitosis was presented at Fig.S32 (A, B). However, in 3-aroyl 2-phenylbenzo[b]furans
treatment cells different phenotypes with enormous nuclei, diffused chromosomes and no
mitotic spindle were observed (Fig.6 C-H). Especially, in 14 binuclear or huge nuclei are
common. Additional graphs with scale are presented in Supplementary materials (Fig. S31).
In 15 and 16 treated cells normal bipolar spindles and single abnormal spindles (with
monoastral, tangled microtubules, diffused chromosomes or two asymmetric asters close to
each other) were detected. Examples of abnormal spindles detected in 15 and 16 treated cells
are shown in Fig. S32 (C-F).
The differences between cells treated with the tested compounds and untreated are
noticeable only during mitosis, suggesting mitotic-specific activity of the compounds. Our
data are in contrary to Kamal et al. study, where the most active compounds: (6-methoxy-5-
((4-methoxyphenyl)ethynyl)-3-methylbenzo[b]furan-2-yl)(3,4,5trimethoxyphenyl)methanone
and (E)-3-(6-methoxy-3-methyl-2-(1-(3,4,5-trimethoxyphenyl)vinyl)benzo[b]furan-5-yl)prop-
2-en-1-ol disrupted microtubule organization in all cells [37]. Cell-permeable small molecules
that perturb mitosis without effects on microtubule/cytoskeleton in interphase may be useful
in anticancer therapy and contribute to decrease in side effects.
13

Figure 6. Confocal microscopy imaging of A375 cells: the control (A, B) and after 7 h
incubation with compound 13a (C, D), 13b (E, F) at concentration of 100 µM and 14 (G, H)
at 10 µM, with stained α-tubulin (green) and chromosomes (blue); magnification 400×.
Abnormal spindles were marked with arrows.
2.2.6. Cell free tubulin polymerization assay
The effects of benzo[b]furan derivatives on tubulin polymerization in cell free
conditions were evaluated using a fluorescence-based tubulin polymerization assay. The
activity of the tested compounds was compared with paclitaxel (PTX), vinblastine (VBL) and
0.2% DMSO as the control and reference. As shown in Fig.7, DMSO in the control sample
had no direct effect on tubulin polymerization and generated three typical phases of
spontaneous microtubule formation, namely nucleation, growth and steady state inhibition.
The references compounds, like PTX and VBL, interacted with tubulin, and altered normal
polymerization curve. The overlapping VBL, 13a, 13b (100µM) and 14 (50 µM) curves
confirm that these three the most active compounds, inhibit tubulin polymerization causing
decrease in Vmax (maximal slope values for the growth phase) and reduction in final polymer
mass protein. These data are in line with confocal microscopy imaging and flow cytometry
analysis, since inhibition of tubulin polymerization prevent mitotic spindle formation leading
to polyploidy nuclei and cell cycle arrest in 4N. In comparison with these compounds 15 and
16 showed moderate impact on microtubule formation. Because of high auto-florescent signal
of compound 12b, it was not evaluated in the fluorescence based tubulin polymerization
assay.
14

Figure 7. Effects of benzo[b]furan derivatives on tubulin polymerization in cell free
conditions. Paclitaxel (PTX) and vinblastine (VBL) were used as references and DMSO as a
vehicle control. The final DMSO concentration did not exceed 0.2% in all samples. Means of
at least three independent experiments are shown.
3. Conclusion
We described efficient synthesis of 2-phenylbenzo[b]furans via corresponding 2-(2-
formylphenoxy)-2-phenylacetic acids and their functionalization at 3-positon. 3-Acetyl and 3-
trimethoxybenzoyl derivatives of 2-phenylbenzo[b]furan can be obtained in good yields by a
selective, green acylation method with the use of Amberlyst 15 ion exchange resin as a
catalyst. The reaction conditions for selective synthesis of (4-hydroxy-3,5-
dimethoxyphenyl)(2-phenylbenzo[b]furan-3-yl)methanones were also presented. The
functionalized benzo[b]furans as well as other 3-acylated 2-alkylbenzo[b]furans were
evaluated as antitumor agents.
Among the tested compounds, 2-phenyl derivatives 13a and 13b containing 4-hydroxy-3,5-
dimethoxybenzoyl group as well as 14 bearing 3,4,5-trimethoxybenzoyl group exhibited the
most potent antiproliferative activity against human melanoma A375 cell line. The change of
the phenyl substituent with alkyl at C2-furan ring as well as introducing of an extra methylene
between the carbonyl and the phenyl group at the 3-position caused a dramatic reduction in
antiproliferative activity. It was also found that 7-methoxy group in 2-phenylbenzo[b]furan
15

skeleton contribute to increase anticancer activity. Tubulin polymerization assay, confocal
microscopy imaging and flow cytometry analysis revealed that 3-aroyl-2-
phenylbenzo[b]furans inhibited tubulin polymerization leading to disruption of mitotic
spindle formation, cell cycle arrest in G2/M phase and apoptosis. Further studies to develop
synthesis and antiproliferative activity evaluation for the 3-functionalized 2-
arylbenzo[b]furan derivatives including these containing the 7-hydroxy group are in progress
in our laboratory and will be reported in due course.
4. Experimental section
4.1.Chemistry
4.1.1. General
NMR spectra were recorded with TM Bruker DPX-400 (400 MHz) spectrometer. Chemical
1
shifts (δ) are given in ppm from TMS (0 ppm), as an internal standard for H NMR, and
CDCl3 (77.0 ppm), for 13C NMR (100 MHz). Coupling constants (J) in hertz (Hz).
Abbreviations are as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
br.(broad).
GC/MS analysis were performed using an Agilent Technologies 6890N apparatus , equipped
with a mass detector 5973 Network and 3.0 m x 0.25 mm capillary column, filled with a 0.25-
m film of 5 % MePh silicate. Injector temperature was 250°C. A gradient of temperature
was 60°C for 3 min., then an increase of 10°C/min., to 300 ºC.
Fourier transform infrared-attenuated total reflection spectroscopy (FTIR–ATR) was
performed using a Nexus spectrometer with Golden Gate (ATR) (Thermo Nicolet Corp.).
Samples were dried at 60 °C under vacuum for 24 h, and 32 scans were averaged across the
spectral range of 400–4000 cm-1. All melting points were determined using a Boetius
apparatus and are uncorrected.
Most of the reagents and solvents were purchased in commercially available grade purity.
1,2-Dichloroethane was dried with calcium hydride before the use. Commercially Amberlyst
15 was dried before the use by azeotropic evaporation of water with chloroform.
Methylbromo-2-phenylacetate 8 was obtained from phenylacetic acid by sequential
processes chlorination, bromination and esterification (Supporting materials) although it is
commercially available.
4.1.2. General procedure for synthesis of compounds 9a and 9b
16

A mixture of methyl 2-bromo-2-phenylacetate (8) (10.8 g, 47 mmol), 2-hydroxybnezaldehyde
7a or 7b (47 mmol), K2CO3 (7.6 g, 55 mmol) and DMF (100 mL) was stirred and heated at
92-94 °C for 4.5 h. Then, the mixture was cooled, poured onto ice/H2O (150 mL) and the
mixture left to stand at 4 °C for 12 h. After this, the precipitate was filtered off, washed with
H2O and dried to give crude product as a solid.
4.1.2.1. Methyl 2-(2-formylphenoxy)-2-phenylacetate (9a)
Yield: 9.8 g (76%); a pale brown solid; mp. 103-105 °C; GC/MS  =19.84, MS m/z (%) 270
(M+, 13), 238 (21), 211(51), 194 (40), 181(49), 165 (48), 152 (13), 133 (100), 121 (29), 105
(57), 89 (14), 77 (62), 59 (13), 51 (19).
4.1.2.2. Methyl 2-(2-formyl-6-methoxyphenoxy)-2-phenylacetate (9b)
Yield: 4.01 g (60.7 %); a pale beige solid; mp 79-82 °C; GC/MS  = 21.48, MS m/z (%) 300
(M+, 56) 240 (52),224 (35), 211 (54), 197 (36), 181 (37), 167 (15), 163 (100),151 (48), 141
1
(17), 121 (20), 105 (39), 89 (14), 77 (54), 59 (23), 51 (17). H NMR (400 MHz, CDCl3) δ
10.50 (s, 1H, CHO), 7.53 – 7.48 (m, 2H, 2xAr-CH), 7.43 – 7.35 (m, 4H, 4xAr-CH), 7.17 –
7.09 (m, 2H, 2xAr-CH), 5.93 (s, 1H, CH), 3.90 (s, 3H, OCH3), 3.72 (s, 3H, OCH3). 13C NMR
(101 MHz, CDCl3) δ 190.6, 170.4, 151.9, 149.1, 135.3, 130.0, 129.4, 128.9, 127.6, 124.4,
121,4, 119.2, 117.9, 82.0, 56.2, 52.4.
4.1.3. General Procedure for synthesis of compounds 10a and 10b
A mixture of methyl 2-(2-formylphenoxy)-2-phenylacetate (9a or 9b) (7 mmol) 10% aq KOH
(30 mL) and methanol (2 mL) was stirred and heated at 75-80 °C for 2 h. After the mixture
was cooled to rt, 10% aq HCl was added under stirring and cooling with ice. The precipitate
was filtered off, washed several times with H2O and dried. The crude product was obtained as
a solid.
4.1.3.1 2-(2-Formylphenoxy)-2-phenylacetic acid (10a)
Yield (70%); white solid, mp 140-141 °C, lit. 140-142 °C [38]; Anal. Calcd for C15H12O4: C,
70.31; H, 4.72. Found: C, 70.18; H, 4.92.
4.1.3.2. 2-(2-Formyl-6-methoxyphenoxy)-2-phenylacetic acid (10b)
Yield: 2.3 g (72%); a pale beige solid mp 55-56 °C. 1H NMR (400 MHz, CDCl3) δ 10.27 (s,
1H, CHO), 7.50 – 7.43 (m, 2H, Ar), 7.40 – 7.31 (m, 4H, Ar), 7.19 – 7.09 (m, 2H, Ar), 6.51
(bs, 1H, COOH), 5.93 (s, 1H, CH), 3.85 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ
191.5, 173.6, 151.8, 148.1, 134.8, 129.6, 129.5, 129.0, 127.8, 124.7, 121.2, 118.3, 82.2, 56.1.
4.1.4 General procedure for synthesis of compounds 11a and 11b .
A mixture of 2-(2-formylphenoxy)-2-phenylacetic acid (10a or 10 b) (3.3 mmol), anhydrous
AcONa (33 mmol, 10 equiv) and Ac2O (35 mL) was stirred and heated at 125-130 °C for 4.5
17

h. Then, the mixture was cooled and poured onto ice/H2O (130 mL) and left at refrigerator for
12 h. The precipitate was filtered off, washed several times with cooled H2O and dried. The
crude product was recrystallized from n-hexane.
4.1.4.1 2-Phenylbenzo[b]furan (11a)
Yield: 90%; a pale beige solid, mp 112-115 °C; GC/MS  = 17.14 min, MS m/z (%) 194 (M+,
100), 165 (63), 139 (10), 126 (3%), 115 (5%), 97 (5%), 77 (4%), 63 (9), 51 (6%).
1H NMR (400 MHz, CDCl3) δ 7.87 (dt, J = 3.0, 1.7 Hz, 2H, Ar), 7.61 – 7.56 (m, 1H, Ar),
7.56 – 7.50 (m, 1H, Ar), 7.48 – 7.42 (m, 2H, Ar), 7.39 – 7.32 (m, 1H, Ar), 7.31 – 7.20 (m,
13
2H, Ar), 7.03 (d, J = 0.8 Hz, 1H, CH (furan). C NMR (101 MHz, CDCl3) δ 155.9, 154.9,
130.5, 129.2, 128.8, 128.5, 124.9, 124.2, 122.9, 120.9, 111.2, 101.3.
4.1.4.2. 7-Methoxy-2-phenylbenzo[b]furan (11b);
Yield: 1.21 g (96.8%); a pale beige solid; mp 65-68 °C. GC/MS  = 19.66 min, MS m/z (%)
224 (M+, 100), 181 (30), 153 (40), 152 (50), 127 (8), 112 (4), 102 (4), 87 (3), 76 (7), 63 (4),
51 (5). 1H NMR (400 MHz, CDCl3) δ 7.91 – 7.86 (m, 2H, Ar), 7.46 – 7.39 (m, 2H, Ar), 7.36
– 7.30 (m, 1H, Ar), 7.20 – 7.11 (m, 2H, Ar), 7.00 (s, 1H, CH, furan), 6.79 (dd, J = 7.4, 1.4
Hz, 1H, Ar), 4.03 (s, 3H, OCH3). 13C NMR (101 MHz, CDCl3) δ 156.0, 145.3, 144.1, 130.9,
130.3, 128.7, 128.5, 125.0, 123.6, 113.3, 106.7, 101.6, 56.1.
4.1.5 General procedure for synthesis 12a-b
A mixture of benzo[b]furan (11a or 11b) (22 mmol) Ac2O (0.3 mL, 33 mmol, 1.5 equiv),
Amberlyst 15 (1 g) and 1,2-dichloroethane (20 mL) was stirred at reflux for 6h. The catalyst
was filtered, the filtrate neutralized by 5% aq sodium hydroxide. The organic layer was
separated and solvent removed. The crude product was recrystallized from n-hexane.
4.1.5.1 1-(2-Phenylbenzo[b]furan-3-yl)ethan-1-one (12a) Yield: 69%, a semi-solid.
1H NMR (400 MHz, CDCl3) δ 8.15 – 8.07 (m, 1H, Ar-CH), 7.77 – 7.71 (m, 2H, Ar), 7.54 –
7.48 (m, 4H, Ar), 7.38 – 7.32 (m, 2H, Ar), 2.37 (s, 3H, COCH3). 13C NMR (101 MHz,
CDCl3) δ 195.6, 160.5, 154.0, 130.6, 130.2, 129.8, 128.7, 126.9, 125.5, 124.4, 122.5, 118.5,
111.2, 30.6.
4.1.5.2 1-(7-Methoxy-2-phenylbenzo[b]furan-3-yl)ethan-1-one (12b) Yield (72%), a pale
brown solid; mp. 118-121 °C. GC/MS = 23.62 min, MS m/z (%) 266 (M+, 58), 251 (100),
1
223 (12), 165 (22), 152 (26), 126 (8), 102 (5), 76 (4), 51 (2). H NMR (400 MHz, CDCl3) δ
7.94 - 7.92 (m, 2H), 7.83 (s, 1H), 7.77 (d, J = 8.44 Hz, 1H), 7.45 (t, J = 7.45, 2H), 7.39 - 7.36
(m, 1H), 6.78 (d, J = 8.43 Hz, 1H), 4.09 (s, 3H, OCH3), 2.63 (s, 3H, COCH3). 13C NMR (400
18

MHz, CDCl3) 13C NMR (400 MHz, CDCl3) δ 197.3, 158.1, 148.9, 143.9, 130.5, 129.8, 129.0,
128.8, 127.9, 125.2, 123.1, 105.5, 103.3, 56.3, 27.0. . IR (KBr) vmax/cm-1 1654, 1620,1573.
4.1.6 General procedure for synthesis of compounds 13a and 13b
To a stirring mixture of benzo[b]furan (11a or 11b) (22 mmol), 3,4,5-trimethoxybenzoyl
chloride (0.5 g, 2.2 mmol) and dry 1,2-dichloroethane (30 mL) was added AlCl3 (0.88 g, 6.6
mmol, 3 equiv) in portion, at 45 °C for 1 h. The heating was continued at 45-50 °C for 2.5 h.
The mixture was cooled to rt and poured into solution of ice/water (20 mL) and 35% HCl
(1mL) and the layers was separated. Organic layer was washed with water (2x15 mL), 2% aq
NaOH (2 x 15 mL) and water (2 x 15 mL). The organic layer was dried (Na2SO4)separated
and solvent removed under vacuo. The crude product was recrystallized from n-hexane.
4.1.6.1. (4-Hydroxy-3,5-dimethoxyphenyl)(2-phenylbenzo[b]furan-3-yl)methanone (13a)
Yield: 68 %, a dark beige solid mp 107-109 °C. GC/MS  = 28,99 min, MS m/z (%) 374 (M+,
100), 359 (10), 343 (12), 271 (10), 241 (10) 221 (47), 181 (23), 165 (55), 139 (15), 123 (10),
108 (6), 67 (8), 53 (3). 1H NMR (400 MHz, CDCl3) δ 7.74 -7.65 (m, 3H, Ar), 7.60 -7.57 (m,
1H, Ar), 7.39 – 7.22 (m, 5H, Ar), 7.19 (s, 2H, Ar), 6.0 (1H, OH), 3.71 (6H, OCH3). 13C NMR
(101 MHz, CDCl3) δ 190.5, 156.8, 154, 146.8, 139.9, 129.8, 129.7, 128.7, 128.6, 128.4,
125.5, 123.9, 121.6, 116.0, 111.3, 107.5, 56.4, 43.6. IR (KBr) vmax/cm-1 3367, 1638, 1602,
1579.
4.1.6.2. (4-Hydroxy-3,5-dimethoxyphenyl)(7-methoxy2-phenylbenzo[b]furan-3-yl)methanone
(13b)
Yield: 76%, a dark beige solid mp 67-69 °C. 1H NMR (400 MHz, CDCl3) δ 7.92 (dd, J = 5.2,
3.4 Hz, 2H, Ar), 7.59 (d, J = 8.4 Hz, 1H, Ar), 7.49 (s, 1H, Ar), 7.48 – 7.41 (m, 2H, Ar), 7.40 –
7.34 (m, 1H, Ar), 7.15 (s, 2H, Ar), 6.82 (d, J = 8.4 Hz, 1H, Ar), 6.05 (bs, 1H, OH), 4.10 (s,
13
3H, OCH3), 3.91 (s, 6H, OCH3). C NMR (101 MHz, CDCl3) δ 194.9, 157.8, 148.4, 146.6,
144.1, 138.8, 131.8, 130.0, 129.8, 129.1, 128.8, 128.8, 125.3, 123.2, 107.5, 105.4, 102.6,
56.5, 56.3. IR (KBr) vmax/cm-1 3351, 1638, 1613, 1579.
4.1.7. Synthesis of (7-Methoxy-2-phenylbenzo[b]furan-3-yl)(3,4,5-trimethoxyphenyl)
methanone (14)
A mixture of benzo[b]furan (11b) (0.5 g, 2.2 mmol), 3,4,5-trimethoxybenzoyl chloride (0.5 g,
2.2 mmol), Amberlyst 15 (0.3 g) and1,2-dichloroethane (20 mL) was stirred at reflux for 6.5
h. The catalyst was filtered, the filtrate neutralized by 5% aq sodium hydroxide. The organic
layer was separated, dried ((Na2SO4) and solvent removed under vacuo. The crude product
was recrystallized from n-hexane.
19

Yield: 69 %; a pale brown solid mp 64-66; GC/MS,  = 34.9 min, MS m/z (%) 418.2 (M+,
1
100), 387.1 (10), 375.1 (15), 251.1 (60), 165 (15), 152 (15). H NMR (400 MHz, CDCl3) δ
7.93 (dd, J = 5.2, 3.4 Hz, 2H, Ar), 7.61 (d, J = 8.4 Hz, 1H, Ar), 7.57 (s, 1H, Ar), 7.50 – 7.43
(m, 2H, Ar), 7.41 – 7.34 (m, 1H, Ar), 7.09 (s, 2H, Ar), 6.81 (d, J = 8.4 Hz, 1H, Ar), 4.13 (s,
3H, OCH3), 3.96 (s, 3H, OCH3), 3.89 (s, 6H, 2xOCH3). 13C NMR (101 MHz, CDCl3) δ 194.5,
158.0, 152.9, 148.6, 144.1, 141.5, 134.2, 131.9, 129.8, 129.3, 129.1, 128.8, 125.4, 122.8,
107.4, 105.4, 102.7, 61.0, 56.4, 56.3. IR (KBr) vmax/cm-1 1638, 16013, 1573.
4.1.8 1-(5-Bromo-2-ethylbenzo[b]furan-3-yl)-2-(4-hydroxyphenyl)ethanone (15)
1
A white solid, mp 144-146 °C, H NMR (400 MHz, CDCl3/DMSO-d6) δ 8.95 (s, 1H, OH),
8.14 (s, 1H, Ar), 7.49 – 7.27 (m, 2H, Ar), 7.03 (d, J = 8.4 Hz, 2H, Ar), 6.79 (d, J = 8.4 Hz,
2H, Ar), 4.15 (s, 2H, CH2), 3.17 (q, J = 7.5 Hz, 2H, CH2), 1.35 (t, J = 7.5 Hz, 3H, CH3).
13C NMR (101 MHz, CDCl3/DMSO-d6) δ 194.0, 168.3, 156.3, 152.1, 130.4, 128.0, 127.2,
124.4, 124.0, 116.9, 115.8, 115.5, 112.5, 48.2, 22.5, 11.9.
4.1.9 2-(3,5-Dibromo-4-hydroxyphenyl)-1-(2-butylbenzo[b]furan-3-yl)ethanone (16)
A white solid mp, 107-109 °C; 1H NMR (400 MHz, CDCl3) δ 7.92 – 7.85 (m, 1H, Ar), 7.53 –
7.46 (m, 1H, Ar), 7.38 – 7.29 (m, 4H, Ar), 5.94 (bs, 1H, OH), 4.19 (s, 2H, CH2), 3.20 – 3.11
(m, 2H, CH2), 1.76 (dt, J = 12.8, 7.6 Hz, 2H, CH2), 1.49 – 1.35 (m, 2H, CH2), 0.95 (t, J = 7.4
Hz, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 193.0, 167.9, 153.7, 148.5, 133.3, 128.4, 125.4,
124.6, 124.1, 121.1, 116.2, 111.4, 109.8, 47.6, 29.9, 28.8, 22.6, 13.8.
4.2. Biological evaluation
4.2.1.Cell culture
Human malignant melanoma cells (A375, catalogue no. 88113005) were purchased
from European Collection of Authenticated Cell Cultures (ECACC). Cells were cultured in a
humidified incubator (5% CO2, 37°C) in culture medium (Dulbecco's Modified Eagle
Medium, Sigma-Aldrich, Germany) supplemented with glucose (4.5 g/L), sodium pyruvate
(0.11 g/L), 10% heat-inactivated fetal bovine serum (HyClone, South America), L-glutamine
(2 mM, Sigma-Aldrich, Germany) and 0.4% penicillin-streptomycin (Sigma-Aldrich,
Germany).
4.2.2. Antiproliferative activity and cell morphology
The antiproliferative activity of the compounds was evaluated using the Cell
Proliferation Reagent WST-1 assay (Sigma-Aldrich, Germany). The WST-1 test is based on
the reduction of the tetrazolium salt WST-1 to a soluble red formazan by mitochondrial
20

dehydrogenase. The amount of formazan dye is directly correlated to the number of
metabolically active cells. In the present study, A375 cells were seeded in 96-well plate
(2×103 cells/well) and then cultured in 100 µL medium in standard conditions. After 24 h the
culture medium was removed and the cells were treated with tested compounds at final
concentrations in the medium: 0.01, 0.1, 1, 10, 50, 100 µM for 48 h. All the tested
compounds were dissolved in DMSO. In the final concentrations the amount of DMSO did
not exceed 0.2%. The cells without the tested compounds were used as the controls. After 48
h cell morphology was recorded using Smart Fluorescent Cell Analyzer Microscope JuLi™
(Korea). Next the WST-1 reagent was added (10 µl), incubated with the cells for 30 min and
then the absorbance was measured at 450 nm (with 620 nm background correction), using a
spectrophotometric microplate reader (Infinite 200 Pro, Tecan, Switzerland). The interaction
between compounds (without cells) and WST-1 reagents was also determined (Ablank). The
results were normalized to the control cells, and the cell viability was calculated using the
following formula: number of viable cells (% of control) = [(Atest – Ablank)/(Acontrol −
Ablank)] × 100%. The readings were acquired from three independent experiments (each
conducted in triplicate). For the most active compounds (13a, 13b, 14) three additional WST-
1 experiments (each conducted in triplicate) with additional dilution factors (10; 5; 1; 0.5; 0.1;
0.05; 0.01 µM) were performed. Statistical analysis was carried out using Statistica 12
(StatSoft Inc., Tulsa, Oklahoma, USA). Results are expressed as mean ± standard deviation.
Experimental data were assessed using the Student’s t-test. A p-value level of <0.05 was
considered statistically significant.
4.2.3. Apoptosis/necrosis detection - flow cytometry analysis
FITC Annexin V Apoptosis Detection Kit II (BD Pharmingen, Belgium) was used to
distinguish apoptotic and necrotic cells after tested compounds treatment. A375 cells were
plated in 6-well plates (5×104 cells/well). After 24 h the culture medium was removed and the
cells were treated with tested compounds at final concentrations of 100 µM, except for 14 (50
µM) and incubated for another 48 h. After treatment the cells were washed with PBS (both
the cell medium and PBS were collected) and harvested by trypsinization. After
centrifugation, the supernatant was discarded, the cells were washed with PBS and again spin.
After centrifugation, the supernatant was discarded, pellet was resuspended in 1X Binding
Buffer and incubated with Annexin V-FITC and propidium iodide (PI) for 15 min at room
temperature in the dark. Next, stained A375 cells were diluted by 1X Binding Buffer and
directly analyzed by Navios flow cytometer (Beckman Coulter, USA). The positive Annexin
V-FITC indicates the loss of plasma membrane, which characterizes the early stage of
21

apoptosis. The positive PI indicates the damage of cell membrane, which occurs either in the
terminal stage of cell apoptosis or necrosis. Therefore, the apoptotic cells were identified as
Annexin V-FITC+ and PI−, the nonviable cells were identified as Annexin V-FITC+ and PI+,
and the viable cells as Annexin V-FITC− and PI−. Two controls were used: untreated cells
growing in medium with DMSO (negative control) and treated with doxorubicine 11.41 µM
(positive control). The readings were acquired from three independent experiments.
4.2.4. Cell cycle analysis
For cell cycle analysis, A375 cells were seeded in 12-well plates (2×104 cells/well),
cultured for about 41 h and next additional 7 h [39] with 100 µM of the compounds: 12 b,
13a, 13b, 15, 16 and 50 µM of compound 14. Next, medium was collected, cells were washed
with PBS and collected by trypsynization. After centrifugation, supernatant was discarded
and pellet was resuspended in medium (1 ml) with Vybrant® DyeCycle™ Orange Stain (10
µM, Thermofishern Scientific), incubated for 30 min at 37°C, and DNA content was
measured by Navios flow cytometer (Beckman Coulter, USA). The cell proportions in
G0/G1, S and G2/M phases were analyzed using an appropriate software (Kaluza Flow
Cytometry Analysis Software, Beckman Coulter, ModFit LT 4.1, Verity Software House,
USA). The readings were acquired from three independent experiments.
4.2.5. Confocal microscopy imaging
To visualize the microtubule and DNA, A375 cells were seeded on coverslips at
density of 5×104 cells, and cultured as above for about 41 h. Afterwards, 100 µM of the
compounds: 12 b, 13a, 13b, 15, 16 and 10 µM of compound 14 were added, and incubated
for additional 7 h. Next, the cells were fixed in 4% buffered formalin for 5 min, at 37°C,
washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 10 min at RT.
Nonspecific antibody binding was blocked for 10 min (RT) and subsequently incubated with
monoclonal anti-α-tubulin antibody produced in mouse (Sigma) diluted 1:2000 for overnight
(4°C). The coverslips were then washed tree times with 0.05% Triton X-100 in PBS and the
cells were incubated with secondary antibodies to mouse IgG conjugated with FITC (Sigma)
diluted 1:64 for 1 h at RT, washed and counterstained with DAPI (Sigma). Finally, cells were
washed with PBS (twice) and mounted on glass slides. The slides were examined with a
FV1000 confocal microscope (Olympus, Germany) in two separated channels: for DAPI (405
nm laser), FITC (488 nm laser).
4.2.6. Cell free tubulin polymerization assay
22

Tubulin polymerization assay was conducted using a fluorescence-based tubulin
polymerization kit (BK011P, Cytoskeleton, USA) according to the manufacturer’s protocol.
The compounds 13a, 13b, 15, 16 (100 µM) and 14 (50 µM) were evaluated for their effect on
tubulin polymerization (in cell free conditions). Because of high auto-florescent signal of
compound 12b, it was not evaluated in the fluorescence based tubulin polymerization assay.
Paclitaxel and vinblastine (3 µM) were used as references and DMSO as a vehicle control.
The finally DMSO concentration did not exceed 0.2% in all samples. After incubation of the
tested compounds at 37°C for 1 min, the icy tubulin reaction mixture (2 mg/mL tubulin, 1.0
mM GTP in 80 mM PIPES, 2.0 mM MgCl2, 0.5 mM EGTA and 15% glycerol, pH 6.9) was
added. The samples were mixed and tubulin assembly was monitored (excitation: 360 nm,
emission: 450 nm) at 1 min intervals for 1 h at 37°C using a spectrophotometric microplate
reader (Infinite 200 Pro, Tecan, Switzerland). The readings were acquired from at least three
independent experiments.
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Graphical abstract
O
OH
O
O
Ph
O
O
27

Highlights
This manuscript presents an efficient synthesis of 2-phenylbenzo[b]furans via corresponding
2-(2-formylphenoxy)-2-phenylacetic acids and their functionalization at 3-positon. 3-Acetyl
and 3-trimethoxybenzoyl derivatives of 2-phenylbenzo[b]furan can be obtained in good
yields by a selective, green acylation method with the use of Amberlyst 15 ion exchange resin
as a catalyst.
The functionalized benzo[b]furans as well as other 3-acylated 2-alkylbenzo[b]furans were
evaluated as antitumor agents. Among the tested compounds, 2-phenyl derivatives and
containing 4-hydroxy-3,5-dimethoxybenzoyl group as well as benzo[b]furan derivative
bearing 3,4,5-trimethoxybenzoyl group exhibited the most potent antiproliferative activity
against human melanoma A375 cell line.
Tubulin polymerization assay, confocal microscopy imaging and flow cytometry analysis
revealed that 3-aroyl-2-phenylbenzo[b]furans inhibited tubulin polymerization leading to
disruption of mitotic spindle formation, cell cycle arrest in G2/M phase and apoptosis.
28