Design, synthesis, and biological evaluation of a new class of benzo[b]furan derivatives as antiproliferative agents, with in silico predicted antitubulin activity

Article abstract of DOI:10.1111/cbdd.13052

A new series of 3-benzoylamino-5-(1H-imidazol-4-yl)methylaminobenzo[b]furans were synthesized and screened as antitumor agents. As a general trend, tested compounds showed concentration-dependent antiproliferative activity against HeLa and MCF-7 cancer cell lines, exhibiting GI₅₀ values in the low micromolar range. In most cases, insertion of a methyl substituent on the imidazole moiety improved the antiproliferative activity. Therefore, methyl-imidazolyl-benzo[b]furans compounds were tested in cell cycle perturbation experiments, producing cell cycle arrest with proapoptotic effects. Their core similarity to known colchicine binding site binders led us to further study the structure features as antitubulin agents by in silico protocols.

Full text of DOI:10.1111/cbdd.13052

DR. ANNAMARIA MARTORANA (Orcid ID : 0000-0002-9560-1882)
Article type : Research Article
Design, synthesis, and biological evaluation of a new class
of benzo[b]furan derivatives as antiproliferative agents,
with in silico predicted antitubulin activity
a
a
b
a
Antonino Lauria , Carla Gentile , Francesco Mingoia , Antonio Palumbo Piccionello ,
a
a,b
a
a,
Roberta Bartolotta , Riccardo Delisi , Silvestre Buscemi , Annamaria Martorana *
a
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche “STEBICEF”–
Università di Palermo, Via Archirafi 32 -90123 Palermo, Italy.
b
Istituto per lo Studio dei Materiali Nanostrutturati, (ISMN), Consiglio nazionale delle
Ricerche (CNR), Via U. La Malfa 153 - 90146 Palermo, Italy.
Corresponding Author:
Dr Annamaria Martorana
Via Archirafi 32, - 90123 Palermo (Italy)
Email_Address: annamaria.martorana@unipa.it
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13052
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A new series of 3-benzoylamino-5-(1H-imidazol-4-yl)methylaminobenzo[b]furans were
synthesized and screened as antitumor agents. The MTT test (HeLa and MCF-7) underlined a
significant growth inhibition effect of the derivatives 7a-f. Compounds 7b-d,f, tested in cell
cycle perturbation experiments on HeLa cells produced a cell cycle arrest in G2/M phase. In
addition, the structure similarity of these benzo[b]furans with known colchicine binding sites
binders led us to investigate their potent interference with the microtubule system by
computational analysis as a support of the observed biological effects.
Design, synthesis, and biological evaluation of a new class
of benzo[b]furan derivatives as antiproliferative agents,
with in silico predicted antitubulin activity
Running title:
Benzo[b]furan derivatives with antiproliferative activity
a
a
b
a
Antonino Lauria , Carla Gentile , Francesco Mingoia , Antonio Palumbo Piccionello ,
a
a,b
a
a,
Roberta Bartolotta , Riccardo Delisi , Silvestre Buscemi , Annamaria Martorana *
a
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche “STEBICEF”–
Università di Palermo, Via Archirafi 32 -90123 Palermo, Italy.
This article is protected by copyright. All rights reserved.

b
Istituto per lo Studio dei Materiali Nanostrutturati, (ISMN), Consiglio nazionale delle
Ricerche (CNR), Via U. La Malfa 153 - 90146 Palermo, Italy.
Corresponding Author:
Dr Annamaria Martorana
Via Archirafi 32, - 90123 Palermo (Italy)
Email_Address: annamaria.martorana@unipa.it
Tel: +39-091-2389-6821
ABSTRACT
A new series of 3-benzoylamino-5-(1H-imidazol-4-yl)methylaminobenzo[b]furans were
synthesized and screened as antitumor agents. As a general trend, tested compounds showed
concentration-dependent antiproliferative activity against HeLa and MCF-7 cancer cell lines,
exhibiting GI50 values in the low micromolar range. In most cases, insertion of a methyl
substituent on the imidazole moiety improved the antiproliferative activity. Therefore,
methyl-imidazolyl-benzo[b]furans compounds were tested in cell cycle perturbation
experiments, producing cell cycle arrest with proapoptotic effects. Their core similarity to
known colchicine binding site binders led us to further study the structure features as
antitubulin agents by in silico protocols.
KEYWORDS
3
-benzoylamino-5-(1H-imidazol-4-yl)methylaminobenzo[b]furans, antitumor agents,
HeLa and MCF-7 cell lines, G2/M phase, colchicine binding site, VLAK protocol, induced fit
docking studies, antitubulin agents.
The discovery of more potent, clinically safe, and commercially attractive new targeted drugs
is the driving force at the base of medicinal chemistry research. Since a long time, our
research work focused the attention on the design, synthesis, and biological evaluation of new
[
1-4]
heterocyclic compounds able to halt tumor cell proliferation (Figure 1 compounds 1-4).
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FIGURE 1.
[
5]
In silico approach, performed through the Virtual Lock-and-Key (VLAK) protocol, allowed
us to select new annelated thieno- series of type 2, 3 and 4 as novel antitumor agents (Figure
[
6]
1
). These compounds, featured by a sulfur atom in the scaffold and functionalized with
[1]
suitable side chains, similar to that used for the precursor series 1, demonstrated in vitro
[
7-9]
antiproliferative activity, exhibiting very low toxicity and high potency.
Therefore, as a part of our ongoing search for biologically active small molecules, keeping
our attention on heterocyclic ring systems, herein, we propose the synthesis, the biological
screening, and the in silico insights of a new series of benzo[b]furan derivatives 5, 6, and 7
(Figure 2).
FIGURE 2.
The pharmaceutical interest toward these new molecules is supported by several experimental
[
10]
data described in literature,
which assigned to the benzo[b]furan scaffold peculiar
biological activities. Romagnoli and co-workers reported an interesting series of 3-
arylaminobenzo[b]furans 8 as result of accurate structure-activity relationships (SAR)
[
11]
studies. These compounds, acting on the colchicine binding site of tubulin, showed high
[
12]
antiproliferative activity, comparable to that of the thiophene derivatives 9a,b (Figure 3).
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FIGURE 3.
1
1
.
EXPERIMENTAL SECTION
.1. Chemistry
1
13
Compounds 5-7 were completely characterized in terms of mp, H and C NMR, IR and
HRMS. Full details of the syntheses are provided in the SI. Unless otherwise indicated, all
reagents and solvents were purchased from commercial sources and used without further
purification. All melting points (°C) were determined on a Büchi Tottoli capillary apparatus
and are uncorrected; IR spectra were determined in bromoform with a Jasco FT/IR 5300
1
13
spectrophotometer. H NMR, C NMR spectra were respectively recorded, at 200 and 50.3
MHz in CDCl or DMSO-d6 solution, using a Bruker AC-E series 200 MHz spectrometer.
3
Chemical shifts values are given in ppm and referred as the internal standard to
tetramethylsilane (TMS). The following abbreviations are used: brs= broad signal, s= singlet,
d= doublet, t= triplet, q= quartet, m= multiplet. The purity of all compounds screened in
biological assays was determined to be >95% by HPLC/MS analysis. HRMS were obtained
on an Agilent 6540 UHD accurate-mass Q-TOF spectrometer equipped with a Dual AJS ESI
source working in positive mode. Microanalyses were in agreement with theoretical values ±
0
.4%. Thin layer chromatography was performed on precoated (0.25 mm) silica gel GF254
plates, compounds were detected with a 254 nm UV lamp. Column chromatography was
performed with Merck silica gel ASTM (230 and 400 mesh), or with a Biotage FLASH40i
chromatography module (prepacked cartridge system). 2-fluorobenzonitrile (10) is
commercially available. 5-nitro-2-fluorobenzonitrile (11) was obtained following the
[13]
procedure reported in literature.
.1.1. Synthesis of ethyl 3-amino-5-nitro-benzo[b]furan-2-carboxylate 12
1
To a solution of 2-fluoro-5-nitrobenzonitrile (11) (0.3 g, 1.8 mmol) in dry DMF (5 mL), ethyl
glycolate (0.3 mL, 3.2 mmol) and potassium carbonate (0.95 g, 6.8 mmol) were added at
room temperature, and the mixture was heated at 100°C for 12h. After the reaction was
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completed, the reaction mixture was cooled and poured in water/ice. The precipitate was
collected by filtration and dried to give orange-red needles of 12.
1
.1.2. General procedure for the synthesis of ethyl 3-benzoylamino-5-
nitrobenzo[b]furan-2-carboxylates 14a-f
To a suspension of amine 12 (92 mg, 0.37 mmol) and pyridine (0.56 mmol) was added the
appropriate benzoyl chloride 13 (0.56 mmol). The reaction mixture was stirred at room
temperature over about 12h, and then poured onto stirred water/ice. The precipitate was
collected by filtration, dried overnight. The crude was crystallized from ethyl acetate.
1
.1.3. General procedure for the synthesis of ethyl 5-amino-3-benzoylamino-
benzo[b]furan-2-carboxylates 5a-f
A suspension of 5-nitro compound 14a-f (1 mmol), 10% Pd/C (0.05 g) in ethanol was
subjected to hydrogenation using a Parr Hydrogenation apparatus at 500 psi for 2h. The
catalyst was removed by filtration and the filtrate was concentrated under reduced pressure.
The crude was crystallized from ethanol.
1
4
.1.4. General procedure for the synthesis of ethyl 3-benzoylamino-5-[(1H-imidazolyl-
-yl-methyl)-amino]-benzo[b]furan-2-carboxylates 6a-f and 7a-f
To a suspension of the 5a-f (1.25 mmol) and the appropriate aldehyde 15 (1.6 mmol) in dry
ethanol (10 mL) was added acetic acid up to pH 4. The mixture was stirred for 30 min, and
3
then NaBH CN (1.6 mmol) was added continuing the stirring at room temperature for further
6
-24h. The reaction solvent was removed under reduced pressure and the crude product was
purified by chromatography column using dichloromethane/methanol 98:2 as eluent.
Recrystallization from ethanol. In the reactions for the synthesis of the methyl-imidazolyl
side-chain compounds 7a-f, it was possible to isolate derivative 16 in variable yields (10-
2
3%).
1
.2. Biology
Propidium iodide, ribonuclease A (RNAse A), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT), and DMSO were obtained from Sigma Aldrich (St. Louis, MO,
USA). RPMI, fetal bovine serum (FBS), phosphate buffered saline (PBS), L-glutamine
solution (200 mM), trypsin-EDTA solution (170.000 U/l trypsin and 0.2 g/l EDTA) and
penicillin-streptomycin solution (10.000 U/ml penicillin and 10 mg/ml streptomycin) were
purchased from Lonza (Verviers, Belgium).
1
.2.1. Cell culture
The tumor cell lines HeLa (human epithelial cervical cancer) and MCF-7 (human epithelial
breast cancer), were obtained from American Type Culture Collection (ATCC) (Rockville,
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MD, USA). The cells were cultured as monolayers and maintained in a humidified
atmosphere with 5% CO at 37°C. MCF-7 cells were grown in DMEM while HeLa cells in
2
RPMI. Both medium were supplemented with 5% FBS, 2 mM L-glutamine, 50 IU/ml
2
penicillin, and 50 µg/ml streptomycin. The cells were routinely cultured in 75 cm culture
flasks and were trypsinized using trypsin-EDTA when the cells reached approximately 80%
confluence. Exponentially growing cells were used for experiments.
1
.2.2. Antiproliferative Evaluation Assay
Antiproliferative activity of 3-benzoylamino-benzo[b]furan derivatives 5a-f, 6a-f and 7a-f
was evaluated by MTT assay against HeLa and MCF-7 tumor cell lines. . The MTT assay is
[
14]
based on the protocol first described by Mossmann. It is a measurement of cell metabolic
activity, and correlates quite well with cell proliferation. The assay was performed as
[9]
previously described.
.2.3. Cell-cycle analysis
1
Effects of the 3-benzoylamino-benzo[b]furan derivatives 7a-f exposure on cell-cycle were
evaluated by DNA staining with propidium iodide and flow cytometry analysis. HeLa cells
were seeded on 6 well plates at a density of 130000 cells/well, and treated 24 hours after
seeding with or without various concentrations of test compounds. Following the treatments,
cells were collected, washed in PBS, fixed in ice-cold 70% ethanol and kept at -20°C. Fixed
cells were centrifuged, resuspended in PBS and stained with staining solution (20 µg/ml
propidium iodide, 200 µg/ml RNAse A and Triton X-100 in PBS). for 30 min at 37°C. The
DNA contents of more than 10000 cells were subjected to fluorescence-activated cell sorting
®
®
TM
(
FACS) analysis (Coulter Epics XL , Beckman) and the percentage of cells belonging to
the different compartments of the cell cycle was determined. All experiments were performed
in duplicate and reproduced at least two times.
1
.3. In silico studies
[11]
The structure similarity of the benzo[b]furans 6 and 7 with colchicine binding site binders
and the evidence of cell cycle arrest in G2/M phase, that is known to be a characteristic of
these ligands, led to approach the in silico methods by two ways: the VLAK approach, a
ligand based method developed by us, and the IFD (Induced Fit Docking), a structure based
one.
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[
5]
VLAK protocol:
The protocol, described in supporting information, start to define the “lock model” for the
studied target. A set of 5 ligands known colchicine binding site binders was selected (Figure
4
).
O
N
H
NH
O
O
H
O
N
NH
O
N
O
N
N
O
N
N
N
H
O
O
H
O
Lexibulil
Tivantinib
Colchicine
O
S
O
N
NH
HN
O
N
N
HN
H
N
H
O
Plinabulil O
Nocodazole
FIGURE 4.
The “lock model” was used as template in the virtual screening of the title compounds. At the
end the affinity score (A%) to each tested compound is assigned. As reported in Table 1 the
A% values are in the range 0.63-0.87, assigning them a quite good affinity to colchicine
binding site.
Table 1.
Cmd A% Cmd A%
6
a
0.63
0.70
0.73
0.87
0.62
0.64
7a
7b
7c
7d
7e
7f
0.57
0.64
0.77
0.63
0.59
0.64
6
b
6
c
6
d
6
e
6
f
[
15-17]
Induced fit docking (IFD):
The crystal structure of colchicine-bound tubulin was downloaded from the PDB database
PDB code: 4O2B) and the tubulin dimer with bound colchicine (chains A and B) was
(
extracted from the protein model. The protein was edited using Schrödinger protein
preparation wizard, by adding of hydrogen for protonation of its ionisable residues,
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modification of tautomeric forms and repositioning of reorientable hydrogen to yield the
optimized protein-ligand complex. All the synthesized derivatives were drawn using
ChemDraw. The geometry was optimized by adding hydrogen atoms, generating ionization
states, neutralizing the charged groups, generating tautomers, and low-energy ring
conformers. The energy minimized structures were further used for IFD. Moreover the
crystallized ligand colchicine was re-docked with the aim to evaluate the ability of IFD
protocol to reproduce the experimental conformation (RMSD = 1.5 Å).
2
2
. RESULTS AND DISCUSSION
.1. Chemistry
In Scheme 1 is reported the general synthetic route for the preparation of 3-benzoyl-amino-
benzo[b]furan derivatives 5-7.
1
) preparation of the benzofuran core: the 2-fluoro-5-nitrobenzonitrile (11) was obtained
through nitration of (10), using a mixture of concentrated nitric and sulfuric acids, under inert
[13]
atmosphere. The reactivity of the ortho-fluorine atom on substrate (11) is improved by the
nearby presence of both cyano and nitro substituents, leading to nucleophilic displacement by
2 3
ethyl glycolate, in the presence of K CO and N,N-dimethyl-formamide (DMF). The
following in situ intramolecular cyclization brought about the formation benzo[b]furan
central core, allowing isolation of the 3-amino-benzo[b]furan derivative 12.
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SCHEME 1.
) amide bond formation at C3 position: in order to insert the benzoyl moiety, the key
2
intermediate 3-amino-benzo[b]furan 12 was treated with benzoyl chlorides 13a-f substituted
with electron-withdrawing and electron-releasing groups. The nucleophilic acyl substitution
was carried out employing pyridine both as base and solvent. The use of other reaction media
gave a significant decrease of the yields.
3
) NO
hydrogenation with 10% Pd/C in ethanol, leading to the 5-amino-benzo[b]furans 5a-f.
) imidazolyl moiety installation: in the end, the isolation of ethyl 3-benzoylamino-5-[(1H-
2
reduction: nitro group reduction on derivatives 14a-f was performed by
4
imidazolyl-4-yl-methyl)-amino]-benzo[b]furan-2-carboxylates 6a-f and 7a-f was achieved
through a reductive amination with imidazole-4-carbaldehydes 15 and sodium
cyanoborohydride, as selective reducing agent. In this synthetic step, when the carbaldehyde
1
5” was used, it was possible to isolate compound 16, where hydrolysis of the amide function
was observed.
2
2
.2. Biology
.2.1. Antiproliferative activity
The antiproliferative activity of the synthesized 3-benzoylamino-benzo[b]furan derivatives
a-f, 6a-f, and 7a-f was evaluated on HeLa and MCF-7 tumour cell lines for 48h, using MTT
5
based cell viability assay. In Table 2 were displayed the GI50 values of all tested compounds,
which, for an easier analysis, were divided into three main subgroups: 5-amino-
benzo[b]furans 5a-f, imidazolyl side-chain compounds 6a-f, and methyl-imidazolyl side-
chain derivatives 7a-f. The 5-amino-benzo[b]furan compounds 5a-f were not active or
showed low activity, with GI50 values higher than 30 μM.
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TABLE 2.
R
1
R1
R1
R
O
2
R2
R3
R2
R3
N
N
R3
HN
HN
O
O
O
NH
NH
NH
H
2
N
HN
HN
O
O
O
O
O
O
O
O
5
6
7
HeLa
MCF-7
HeLa
MCF-7
HeLa
MCF-7
[
GI50 ± SE (µM)] [GI50 ± SE (µM)] [GI50 ± SE (µM)]
[GI50 ± SE (µM)]
[GI50 ± SE (µM)]
[GI50 ± SE (µM)]
a) R
1
=R
=R
=R
=R
=R
= Cl; R
2
=R
= H; R
= H; R
=R = OCH
= H; R
3
= H
35.09 ± 3.52 >50
>50 >50
35.32 ± 3.92 30.84 ± 4.09 13.65 ± 1.02 18.22 ± 1.28
41.59 ± 3.08 49.65± 4.91
7.69 ± 0.46
12.27 ± 1.46
b) R
1
3
2
= OCH
3
7.98 ± 0.67 13.36 ± 1.94
14.31 ± 1.97
9.20 ± 0.34
12.19 ± 1.59
2.01 ± 0.13
2.14 ± 0.35
17.35 ± 1.78
15.53 ± 0.98
9.21 ± 1.02
14.16 ± 2.02
1.55 ± 0.14
c) R
1
3
2
= CH
3
d) R
1
2
3
3
>50
>50
>50
>50
12.18 ± 1.19 3.88 ± 0.40
>50 >50
e) R
1
3
= CF
2 3
f) R
1
2
3
= F; R = H
42.05 ± 5.08 35.63 ± 4.30 13.61 ± 0.86 11.25 ± 1.11
As clearly highlighted by the data reported in Table 2, the insertion of the imidazolyl- side
chain moiety exerted a weighty influence on the antiproliferative activity. In fact, with the
exception of derivatives 6a and 6e, where the benzoyl moiety is respectively unsubstituted or
bears a 4-trifluoromethyl substituent, the inhibition growth activity of benzo[b]furan
compounds of type 6 were promising, showing GI50 values in the low micromolar range.
Within this subgroup, remarkable was the activity of the 3,4,5-trimethoxy-benzoyl-
benzo[b]furan 6d on MCF-7 cell line, with a GI50 value in the low micromolar range (3.88
M).
On the other hand, insertion of a methyl group on the imidazole resulted in a strong increase
of activity for the derivatives 7a,e,f, where the benzoyl moiety was respectively unsubstituted
(
7a vs 6a), or functionalized with 4-trifluoromethyl- (7e vs 6e) and 3-chloro-4-fluoro- (7f vs
f) substituents. The cytotoxic activity was worthily interesting in the case of derivative 7f,
6
the most active of the benzo[b]furan series, where GI50 values on HeLa and MCF-7 tumour
cell lines were respectively at 2.14 and 1.55 μM.
Generally, the biological activity of tested benzo[b]furan derivatives was less influenced by
the nature of substituents (both with electron-withdrawing and electron-releasing effects)
located on the benzoyl moiety than by the imidazolyl side chains. However, it was evident
that the biological activity of the unsubstituted (5a, 6a, and 7a) and of the 4-trifluoromethyl-
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(
5e, 6e, and 7e) derivatives, with respect to the other cases, varied in peculiar way along the
progression of the synthesis, from the 5-amino-benzo[b]furans to the methyl-imidazolyl side-
chain derivatives. Finally, the biological screening of derivative 3-amino 16, tested in the 50–
0
.1 μM concentration range on both tumour cell lines, showed no activity, highlighting the
crucial importance of the benzoyl moiety for the antiproliferative effect.
2
.2.2. Cell Cycle analysis
The methyl-imidazolyl side-chain compounds 7a-f were also tested on HeLa cells, in cell
cycle perturbation experiments, to evaluate their potential influence on cell-cycle distribution.
The flow cytometric analysis was performed after 24h of incubation, in order to detect the
shifts in cell cycle distribution before a significant amount of cells undergoing apoptosis. The
working concentrations were chosen taking into account the GI50 values measured at 48h.
FIGURE 5.
In Figure 5 the percentage of cells in the respective cell cycle phase (G1, S, and G2/M) were
shown, along with the percentage of cells in the sub-G0/G1 (dead cells) detected by flow
cytometry. Untreated HeLa cells displayed a normal diploid distribution presenting fast
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proliferation characteristics, with S+G2/M cell phases accounting for about 40% of the total
cells (ctrl). Significant suppression of the G0/G1 phase with accumulation of cells in the
G2/M was observed after cell exposure to 1xGI50 μM of methyl-imidazolyl- side-chain
derivatives 7b-d,f. The effect was concentration-dependent and, in our experimental
conditions, treatment with a concentration of about 2xGI50 produced deep alterations in the
distribution profile. In fact, it was possible to observe a significant reduction of the cell
number in all phases and a simultaneous increase of the cell population in subG0/G1, which
are indicative of apoptotic cells. Interestingly, the unsubstituted 7a and the 4-trifluoromethyl-
7
e derivatives, assayed at 1x and 2xGI50, failed to produce significant cell cycle alterations.
On the contrary, cell exposure to 2.5xGI50 and 5xGI50 μM of derivatives 7a,e, induced G0/G1
arrest associated with reduction of the cell number in other phases.
2
.3. In silico insights
The structure similarity of the benzo[b]furans 6, 7 with colchicine binding site binders 8 and
9
7
, and the evidence of cell cycle arrest in G2/M phase induced by cell exposure to derivatives
b-d,f, led to hypothesize the interference of these novel compounds with the microtubule
system during the cancer cell division processes. This hypothesis was studied by in silico
approach, by using ligand based and structure based methods.
In the first case the VLAK protocol was applied with the aim to confirm the hypothesis by
molecular descriptors matching through fingerprint virtual screening. As described above the
method confirmed that the titled structure posses the chemical-physical characteristic to make
them potential tubulin modulators. Moreover, to investigate the binding modes of each
derivative in the colchicine binding site of tubulin, in silico structure based method was also
[
15]
approached. In particular Induced Fit Docking protocol (IFD) was approached. It combines
molecular dynamics, docking, and MM-GBSA scoring to predict the binding capabilities of
ligands versus biological targets. The main aim of IFD protocol was to study the different
binding modes of the benzofuran derivatives, taking into account the amino acid residues that
are involved in the binding.
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TABLE 3.
[
a]
Title Prime Energy Glide gscore IFDScore
6
a
-34437
-34462
-34487
-34496
-34480
-34395
-34463
-34481
-34478
-34545
-34371
-34475
-34392
-34316
-7.17
-10.51
-11.96
-12.23
-10.45
-11.78
-8.16
-1729
-1734
-1736
-1737
-1734
-1732
-1731
-1735
-1734
-1737
-1729
-1735
-1730
-1725
6
b
6
c
6
d
6
e
6
f
7
a
7
b
-11.04
-10.38
-10.11
-10.53
-11.28
-10.38
-9.17
7
c
7
d
7
e
7
f
COL
CA4
[a]
defined as: Glide Score + 0.05 Prime Energy
The analysis of calculated values reported in Table 3 displayed that all ligand-tubulin
complexes have quite good IFD scores. Particularly, it is evident as the derivatives 6 and 7
display affinity to the binding site similar to colchicine (COL) and higher than
combretastatin A4 (CA4). However when the amino acids involved in the binding complexes
were analyzed, significant differences were observed.
Considering the colchicine binding site as a deep pocket placed at the α-β interface of tubulin
heterodimers it is possible to indentify three main sub-zones involved in the binding
[
18,19]
complexes.
The zone 2 is located on the β subunit and accommodate the amino acids
mainly involved in the colchicine binding complex. The other two zones (zone 1 and zone 3)
are accessory and respectively located in the α subunit interface and in a deeper cavity of the
[18]
β subunit.
In Table 4 the amino acid residues pivotal for the inhibition of tubulin
functionality are correlated with the derivatives 6a-f, 7a-f and with the two standards COL,
CA4. The orange squares indicate the ligand-tubulin interactions with a distance cut-off of
3
Å.
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TABLE 4.
ZONE 1
ZONE 2
ZONE 3
COL
CA4
✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔
✔ ✔ ✔ ✔ ✔ ✔ ✔
✔ ✔ ✔ ✔ ✔ ✔ ✔
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6f
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7f
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[a]
Aminoacids involved in all binding poses
The tested benzofuran compounds showed good interactions with the amino acids ALAα180,
SERα178, ILEβ318, ALAβ316, LEUβ255, Leu β248 and LYSβ254, in agreement with the
IFD scores. Unlike derivatives 7a,e, the benzo[b]furans 7b-d,f present an aromatic group in
contact with the CYSβ241, this feature is generally considered to be the most relevant for the
identification of new antitubulin molecules, and indeed, the presence of this interaction is
[
18]
often used as a filtering method in docking results.
The above mentioned analysis are
clearly showed in Figure 6, where the ligand interaction maps of the most representative
derivatives 7b and 7e are showed and of the reference standard COL (see supplementary
material for the full reported ligand interaction maps).
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FIGURE 6.
The analysis of the binding modes of the ligands, in the colchicine binding site, showed two
different poses. In fact, as depicted in Figure 7 (right, red circle) the the derivatives 7b-d,f
have been characterized by the position of imidazolyl- side chain buried in the binding cavity,
interacting with the amino acids residues critical for the inhibition of tubulin functionality.
On the contrary, the analysis of the derivatives 7a,e shows this structural moiety projected
outside the cavity (Figure 7, left, red circle), involving, in the ligand-protein complex, amino
acids, which are not essential for the antitubulin effect. This behaviour seems consistent with
the biological data. In fact, cell cycle perturbation experiments results, that could suggest an
antitubulin effect for the derivatives 7b-d,f, were not compatible with an antimitotic
mechanism in the other cases. The alternative behaviour of benzofuran compounds could be
justified by the presence of different substituents on the benzoyl moiety, which is the only
point of diversity among the chemical moieties of the benzofuran derivatives, and for this
reason determinant for the orientation in the binding poses.
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FIGURE 7.
. CONCLUSION
3
In this work, the synthesis and the biological evaluation of the new benzo[b]furan series of
type 5, 6, and 7 were reported. As a general trend, the antiproliferative screening on HeLa
and MCF-7 tumour cell lines underlined a significant growth inhibition effect of final
imidazolyl- side chain compounds 6a-f and 7a-f. Methyl insertion on the imidazole ring
resulted in a notable increase of activity. The cytotoxic effect is remarkably interesting in the
case of derivative 7f, where GI50 values on both HeLa and MCF-7 tumour cell lines are
respectively at 2.14 and 1.55 μM. No differences were observed in the biological activity by
changing the substituents on the benzoyl portion, even though the presence of this moiety
was crucial for the antiproliferative effects. Indeed, the lack of activity observed through the
biological screening of derivative 16 confirmed the importance of the benzoyl moiety in the
benzo[b]furan series. To evaluate their potential influence on cell-cycle distribution, the
methyl-imidazolyl- side-chain compounds 7a-f were also tested on HeLa cells, in cell cycle
perturbation experiments, producing cell cycle arrest with proapoptotic effects. Their core
similarity to known colchicine binding site binders led us to further study by in silico
protocols the structure features as antitubulin agents. The results gained from the in silico
analysis suggested that other biological targets are involved in the observed biological
effects.
ACKNOWLEDGMENTS
This work was in part supported by an FFR2012 – University of Palermo grant
(CUP:B78C12000380001) and by Italian MIUR within the “FIRB-Futuro in Ricerca 2012”
Program - Project RBFR12SIPT.
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CONFLICT OF INTEREST
The authors declare no conflict of interest.
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SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supporting information tab for
this article.
FIGURE 1. Heterocyclic derivatives with outstanding antiproliferative activities.
FIGURE 2. Chemical structures of ethyl 5-amino-3-benzoylamino-benzo[b]furan-2-
carboxylates 5, and ethyl 3-benzoylamino-5-[(1H-imidazolyl-4-yl-methyl)-amino]-
benzo[b]furan-2-carboxylates 6, 7.
FIGURE 3. Chemical structures of 2-(alkoxycarbonyl)-3-(3,4,5-
trimethoxyanilino)benzo[b]thiophene 8a, 2-(alkoxycarbonyl)-3-(3,4,5-
trimethoxyanilino)thieno[2,3-b]pyridine 8b, 2-(alkoxycarbonyl)-3-(3,4,5-
trimethoxyanilino)benzo[b]furan 9, and respectively IC50 ranges against a panel of seven
cancer cell lines.
FIGURE 4. Structures of known colchicine binding site binders.
FIGURE 5. Effects of compounds 7a-f on the cell cycle distribution of HeLa cells following
2
4h treatment. The cells were cultured without any compound (ctrl) or with compound used
at the indicated concentrations. Cell cycle distribution was analyzed after standard DNA-
staining with propidium iodide as described in methods. The histograms represent the
percentages of cells in the respective cell cycle phases (G0/G1, S, and G2/M), along with the
percentage of cells in the subG1 (dead cells) obtained by flow cytometry. Results are
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expressed as the mean of two independent experiments, performed in duplicate ±SE.
Statistical analyses were performed using the Student’s t test to determine the differences
between the datasets. *p < 0.05 denote significant differences from untreated control cells.
FIGURE 6. Ligand interaction maps of representative derivatives and colchicine.
FIGURE 7. Docking poses of derivatives 7a,e (left) and 7b-d,f (right) in the colchicine
binding site (the imidazolyl- side chain moieties are red circled).
SCHEME 1. Synthesis of ethyl 3-benzoylamino-benzo[b]furan-2-carboxylates 5a-f, 6a-f and
7
a-f.
TABLE 1. Affinity scores (A%) calculated by VLAK protocol for the colchicine binding
site.
TABLE 2. Antiproliferative activity at 48h of 3-benzoylamino-benzo[b]furan derivatives 5a-
f, 6a-f, and 7a-f against HeLa and MCF-7 cell lines expressed as GI50 values [GI50 ± SE
(µM)].
TABLE 3. IFD scores of benzofuran derivatives 6, 7, and the reference compounds
colchicine (COL) and combretastatin A4 (CA4).
TABLE 4. Summary of the amino acids residues of the colchicine binding site involved in
the binding.
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