Concise synthesis and biological evaluation of 2-Aryl-3-Anilinobenzo[b]thiophene derivatives as potent apoptosis-inducing agents

Article abstract of DOI:10.1016/j.bioorg.2021.104919

Many clinically used agents active in cancer chemotherapy exert their activity through the induction of cell death (apoptosis) by targeting microtubules, altering protein function or inhibiting DNA synthesis. The benzo[b]thiophene scaffold holds a pivotal place as a pharmacophore for the development of anticancer agents, and, in addition, this scaffold has many pharmacological activities. We have developed a flexible method for the construction of a new series of 2-aryl-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophenes as potent antiproliferative agents, giving access to a wide range of substitution patterns at the 2-position of the 6-methoxybenzo[b]thiophene common intermediate. In the present study, all the synthesized compounds retained the 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophene moiety, and the structure–activity relationship was examined by modification of the aryl group at its 2-position with electron-withdrawing (F) or electron-releasing (alkyl and alkoxy) groups. We found that small substituents, such as fluorine or methyl, could be placed in the para-position of the 2-phenyl ring, and these modifications only slightly reduced antiproliferative activity relative to the unsubstituted 2-phenyl analogue. Compounds 3a and 3b, bearing the phenyl and para-fluorophenyl at the 2-position of the 6-methoxybenzo[b]thiophene nucleus, respectively, exhibited the greatest antiproliferative activity among the tested compounds. The treatment of both Caco2 (not metastatic) and HCT-116 (metastatic) colon carcinoma cells with 3a or 3b triggered a significant induction of apoptosis as demonstrated by the increased expression of cleaved-poly(ADP-ribose) polymerase (PARP), receptor-interacting protein (RIP) and caspase-3 proteins. The same effect was not observed with non-transformed colon 841 CoN cells. A potential additional effect during mitosis for 3a in metastatic cells and for 3b in non-metastatic cells was also observed.

Full text of DOI:10.1016/j.bioorg.2021.104919

Bioorganic Chemistry 112 (2021) 104919
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Concise synthesis and biological evaluation of 2-Aryl-3-Anilinobenzo[b]
thiophene derivatives as potent apoptosis-inducing agents
,
,d
Romeo Romagnolia *, Delia Pretia, Ernest Hamelb, Roberta Bortolozzic, Giampietro Violac
,
Andrea Brancalee, Salvatore Ferlaf, Giampaolo Morcianog, Paolo Pintong
a
`
Dipartimento di Scienze Chimiche, Farmaceutiche ed Agrarie, Via Luigi Borsari 46, Universita degli Studi di Ferrara, 44121 Ferrara, Italy
b Molecular Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick National Laboratory for Cancer
Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA
c
`
Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Universita di Padova, 35131 Padova, Italy
d Istituto di Ricerca Pediatrica (IRP), Corso Stati Uniti 4, 35128 Padova, Italy
e School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, UK
f Swansea University Medical School, Institute of Life Sciences 2, Swansea University, Swansea SA2 8PP, UK
g Department of Medical Sciences, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, 44121 Ferrara, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
Many clinically used agents active in cancer chemotherapy exert their activity through the induction of cell death
(apoptosis) by targeting microtubules, altering protein function or inhibiting DNA synthesis. The benzo[b]
thiophene scaffold holds a pivotal place as a pharmacophore for the development of anticancer agents, and, in
addition, this scaffold has many pharmacological activities. We have developed a flexible method for the con-
struction of a new series of 2-aryl-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophenes as potent anti-
proliferative agents, giving access to a wide range of substitution patterns at the 2-position of the 6-
methoxybenzo[b]thiophene common intermediate. In the present study, all the synthesized compounds retained
the 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophene moiety, and the structure–activity relationship
was examined by modification of the aryl group at its 2-position with electron-withdrawing (F) or electron-
releasing (alkyl and alkoxy) groups. We found that small substituents, such as fluorine or methyl, could be
placed in the para-position of the 2-phenyl ring, and these modifications only slightly reduced antiproliferative
activity relative to the unsubstituted 2-phenyl analogue. Compounds 3a and 3b, bearing the phenyl and para-
fluorophenyl at the 2-position of the 6-methoxybenzo[b]thiophene nucleus, respectively, exhibited the greatest
antiproliferative activity among the tested compounds. The treatment of both Caco2 (not metastatic) and HCT-
116 (metastatic) colon carcinoma cells with 3a or 3b triggered a significant induction of apoptosis as demon-
strated by the increased expression of cleaved-poly(ADP-ribose) polymerase (PARP), receptor-interacting protein
(RIP) and caspase-3 proteins. The same effect was not observed with non-transformed colon 841 CoN cells. A
potential additional effect during mitosis for 3a in metastatic cells and for 3b in non-metastatic cells was also
observed.
Microtubules
Benzo[b]thiophene
Apoptosis
Alteration of mitochondrial function
1. Introduction
Microtubules are cylindrical structures of great length formed by the
polymerization of αβ-tubulin heterodimers [8], and these structures are
Among different strategies used for cancer chemotherapy, the iden-
tification of compounds that induce apoptosis (programmed cell death)
in cancer cells is recognized as an important approach for the discovery
of new anti-cancer agents [1–3]. Molecules that interfere with the
microtubule system usually cause a major increase in apoptosis in cancer
cells sensitive to these agents [4–7].
dynamic components of the cytoskeleton of eukaryotic cells [9]. Func-
tions of microtubules include the formation of the mitotic spindle,
required for separation of duplicated chromosomes before cell division,
determination and maintenance of cell shape, regulation of motility and
signaling, secretion and organelle transport inside the cell [10]. The
microtubule spindle of dividing cells is especially remarkable in that it
* Corresponding author.
E-mail address: rmr@unife.it (R. Romagnoli).
https://doi.org/10.1016/j.bioorg.2021.104919
Received 10 March 2021; Received in revised form 12 April 2021; Accepted 14 April 2021
Available online 20 April 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

R. Romagnoli et al.
Bioorganic Chemistry 112 (2021) 104919
forms and disappears rapidly during the 10–15 min of the M phase of the
cell cycle. The biological importance of microtubules makes them an
interesting target for the development of anticancer drugs [11–14].
Numerous chemically diverse substances disrupt the formation of the
mitotic spindle, and these can be divided into microtubule stabilizing
and destabilizing agents [15,16]. While both groups cause mitotic arrest
and subsequent apoptosis, the destabilizing agents specifically inhibit
tubulin assembly into microtubules and can cause the disassembly of
pre-existing microtubules [17,18].
position of 3-(3,4,5-trimethoxybenzoyl)-benzo[b]thiophene skeleton
[26]. While compound 2a is a weak TPI, with IC50 > 40
μ
M, derivative
M. The two
2c is able to inhibit tubulin assembly with a IC50 of 3.5
μ
compounds 2a and 2c are also equipotent in inhibiting the growth of
MCF-7 human breast carcinoma cells, with IC50 values of 0.64 and 0.52
μ
M, respectively, while compound 2c is four-fold more potent than 2a
against human Burkitt lymphoma CA46 cells (IC50 = 0.5 and 2.0 μM,
respectively). As with 2a, derivative 2c shows a modest inhibition (21%)
of the binding of [3H]colchicine to tubulin at a concentration of 5
μM.
Among the natural microtubule depolymerizing agents, com-
bretastatin A-4 (CA-4, 1a, Fig. 1) is one of the more studied antimitotic
agents [19]. CA-4, isolated from the bark of the South African tree
Combretum caffrum, strongly and reversibly inhibits the polymerization
of tubulin through a strong interaction with the colchicine site of tubulin
[20].
The activity of compounds 2a-c appears to be highly dependent upon
the presence of the 6-methoxy group on the benzo[b]thiophene ring.
In our work on anticancer agents based on the benzo[b]thiophene
skeleton, we described a series of compounds based on the 2-alkox-
ycarbonyl-3-(3,4.5-trimethoxyanilino)-benzo[b]thiophene system, with
2-methoxy
and
2-ethoxycarbonyl-3-(3,4,5-trimethoxyanilino)-6-
Despite the synthesis of many tubulin inhibitors, there remains a
need to identify novel small molecules that target microtubules [21–23].
Limited examples of tubulin polymerization inhibitors (TPIs) based on
the chemical structure of the benzo[b]thiophene molecular structure
have been reported. Among them, Pinney and co-workers reported
several TPIs with general structure 2 characterized by the presence of
the 2-aryl-3-substituted-6-methoxybenzo[b]thiophene structural motif
as its core structure [24].
methoxybenzo[b]thiophenes derivatives 2d and 2e, respectively. Both
compounds display potent antiproliferative activity in the nanomolar
range as inhibitors of the growth of cancer cell lines [27]. These com-
pounds interact strongly with tubulin by binding to the colchicine site
with activity comparable with that of CA-4. Although the presence of an
ester function at the 2-position of the 3-(3,4,5-trimethoxyanilino)-6-
methoxybenzo[b]thiophene system plays a pivotal role for potent anti-
tumor activity, it represents a point of metabolic instability. Such esters
can be degraded by human liver microsomes, arguing against the po-
tential of this class of compounds as chemotherapeutic agents. This
suggested the preparation of a new series of derivatives obtained by
replacing the 2-alkoxycarbonyl group of compounds 2d-e with the
potentially more stable phenyl ring to overcome this potential metabolic
instability.
Compound 2a, based on the 2-(4-methoxyphenyl)-3-(3,4,5-trime-
thoxybenzoyl)-benzo[b]thiophene ring system, has unusual inhibitory
effects on tubulin polymerization, with inhibition of the rate of assembly
up to about 3–4
μM and a partial effect on tubulin polymerization at a
concentration of 40
μ
M. At a concentration of 50 μM, this derivative
shows a modest inhibition (28%) of the binding of [3H]colchicine to
tubulin. Replacing the carbonyl moiety of the 3,4,5-trimethoxybenzoyl
group of the compound 2a with an oxygen atom furnished derivative
2b. This compound was evaluated against the NCI 60 cell line panel and
We were intrigued by the idea of replacing the bridged carbonyl
moiety of the 3,4,5-trimethoxybenzoyl group at the 3-position of the
benzo[b]thiophene derivatives 2a and 2c with an anilinic nitrogen (NH)
moiety to furnish a new series of 2-aryl-3-(3,4,5-trimethoxyanilino)-6-
methoxybenzo[b]thiophene analogues with general structure 3 pre-
pared by a flexible method, giving access to a wide range of substitution
patterns at the 2-position of the 6-methoxybenzo[b]thiophene common
has an average IC50 value of 0.49 μM and an IC50 of 0.88 μM in the
tubulin polymerization assay [25]. Compound 2c, an analogue of de-
rivative 2a, is characterized by the presence of the 4′-methoxy-3′-
hydroxyphenyl nucleus (corresponding to the B-ring of CA-4) at the 2-
Fig. 1. Chemical structure of CA-4 (1) and representative examples of benzo[b]thiophene derivatives (2a-e).
2

R. Romagnoli et al.
Bioorganic Chemistry 112 (2021) 104919
intermediate (Fig. 2).
2. Chemistry
The 2-aryl-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thio-
In the present study, all the synthesized compounds were charac-
terized by a common 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]
thiophene moiety, which characterized compounds 2d and 2e, and the
modifications were focused on variation of the substituents on the
phenyl ring located at the 2-position of the 3-(3,4,5-trimethoxybenzoyl/
phenoxy)-6-methoxybenzo[b]thiophene derivatives 2a-c (Fig. 2). Thus,
once the 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophene
scaffold of compound 3p was identified as the minimum structural
requirement for activity in this series of compounds, our strategy for
further development of active antimitotic agents was to perform modi-
fications at the 2-position of the 6-methoxybenzo[b]thiophene ring. For
compounds 3a-o, the first attempt at optimization included the intro-
duction of a phenyl ring at the 2-position of the benzo[b]thiophene
molecular skeleton (compound 3a). Modifications were also focused on
varying the aryl moiety by adding electron-withdrawing (F) or electron-
releasing (alkyl or alkoxy) groups at either the para- or meta-position of
the 2-phenyl ring.
phene derivatives 3a-o were synthesized by a three-step procedure
shown in Scheme 1, starting from the common intermediate 6-
methoxybenzo[b]thiophene 6. This latter compound was synthesized by
a two-step, previously reported procedure [28], starting from the
commercially available 3-methoxybenzenethiol 4, which, after S-alkyl-
ation with bromoacetaldehyde diethyl acetal to afford derivative 5, was
cyclicized with boron trifluoride etherate. Compound 6 was subjected to
a regioselective C-2 arylation with different substituted iodobenzene
derivatives [29], mediated by a catalytic amount of palladium (II) ace-
tate in the presence of silver trifluoroacetate and trifluoroacetic acid as
additives, in mild conditions with water as solvent with the addition of
Tween 80 (2% w/w), to furnish derivatives 7a-o.
The subsequent chemoselective C-3 bromination of compounds 7a-o
and derivative 6 with N-bromosuccinimide (NBS) in THF furnished the
2-aryl-3-bromo-6-methoxybenzo[b]thiophene derivatives 8a-o and 3-
Fig. 2. Design strategy for conjugates 3a-p obtained by the combination of pharmacophores of compounds 2a and 2c-e.
3

R. Romagnoli et al.
Bioorganic Chemistry 112 (2021) 104919
Scheme 1. Reagents and conditions. a: BrCH2CH(OEt)2, K2CO3, acetone, rt; b: BF3 Et2O, CH2Cl2, rt; c: ArI, Pd(OAc)2 (5 mol%), CF3COOAg, CF3COOH, Tween 80/
H2O (2% w/w), rt, 48 h; d: NBS, THF; e: 3,4,5-trimethoxyaniline, Pd2dba3, xanthphos, sodium t-butoxide, toluene, 100 ◦C.
bromo-6-methoxybenzo[b]thiophene 9, respectively. Finally, these
against the human cervix carcinoma (HeLa) and colon adenocarcinoma
(HT-29) tumor cell lines, with CA-4 (1) as reference compound.
Compound 3a, characterized by the presence of a phenyl ring at the
2-position of the 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thio-
phene skeleton, was the most active compound of the series, with IC50
–
latter compounds were submitted to the C N Buchwald-Hartwig
palladium catalyzed cross-coupling protocol, by reaction with 3,4,5-tri-
methoxyaniline in the presence of tris(dibenzylideneacetone)dipalla-
dium(0) (Pd2dba3), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(xanthphos) and sodium tert-butoxide (t-BuONa) (as catalyst, ligand and
values of 0.23 and 0.06
μM against HeLa and HT-29 cancer cells,
◦
base, respectively) in toluene at 100 C, to yield the novel derivatives
respectively. Although this derivative was less active than CA-4 against
HeLa cells, it was very potent against the HT-29 cell line, which is
resistant to CA-4, with an IC50 value of 60 nM. Along with compound 3a,
derivatives 3b (p-F), 3c (p-Me), 3 h (p-OMe) and 3j (p-OEt) were also
more active than CA-4 against the HT-29 cell line. All the tested com-
pounds were much less active than CA-4 against the HeLa cell line.
The substitution pattern on the phenyl moiety at the 2-position of the
benzo[b]thiophene ring played an important role for antiproliferative
activity. The introduction of the electron-withdrawing fluorine atom at
the para-position of the 2-phenyl ring (3b) led to a 2- and 7-fold
3a-p.
3. Biological results and discussion
3.1. In vitro antiproliferative activities against human HeLa and HT-29
cell lines.
In Table 1, we summarize the growth inhibitory effects of the sixteen
novel 3-(3,4,5-trimethoxyanilino)benzo[b]thiophene derivatives 3a-p
4

R. Romagnoli et al.
Bioorganic Chemistry 112 (2021) 104919
was not tolerated, being detrimental for antiproliferative activity, with
10–20- and 8–36-fold reduction of activity against HeLa and HT-29 cells,
respectively, relative to compound 3j. These compounds thus revealed
the importance of the steric effect of the substituent at the para-position
of the 2′-phenyl on antiproliferative activity
Table 1
In vitro inhibitory effects of compounds 3a-p and reference derivative CA-4 (1)
on proliferation of HeLa and HT-29 human tumor cell lines.
Compound
IC50
(μM)
HeLa
HT-29
Finally, removing the 2-phenyl group of compound 3a, to furnish
derivative 3p, caused a 7- and 100-fold reduction of activity against
HeLa and HT-29 cells, respectively, thus demonstrating that the pres-
ence at the 2-position of the 3-(3,4,5-trimethoxyanilino)-6-methox-
ybenzo[b]thiophene molecular skeleton of a phenyl group without
substituents or with a small electronegative fluorine atom at its para-
position (derivatives 3a and 3b, respectively) was essential for potent
antiproliferative activity, while the introduction of electron-releasing
alkyl or alkoxy substituents was not as well tolerated and reduced po-
tency. Moreover, the position of the substituent on the phenyl ring
influenced antiproliferative activity, with the p-substituted phenyl de-
rivatives 3c (methyl), 3 h (methoxy) and 3j (ethoxy) being more potent
than the isomeric m-substituted phenyl analogues 3d, 3i and 3 k,
respectively.
3a
0.23 ± 0.05
0.5 ± 0.05
0.5 ± 0.04
21.5 ± 3.1
11.0 ± 2.3
26.7 ± 0.9
14.1 ± 3.2
1.5 ± 0.05
8.2 ± 2.1
0.06 ± 0.03
0.4 ± 0.05
1.4 ± 0.7
12.0 ± 2.0
9.2 ± 2.7
25.7 ± 1.2
5.6 ± 1.1
1.7 ± 07
3b
3c
3d
3e
3f
3 g
3 h
3i
6.2 ± 0.7
1.0 ± 0.2
36.0 ± 2.6
16.6 ± 0.5
7.9 ± 05
3j
1.8 ± 03
3 k
3 l
30.1 ± 1.2
37.1 ± 3.3
18.9 ± 2.3
28.3 ± 2.2
22.9 ± 3.6
1.7 ± 0.2
3 m
3n
21.0 ± 0.7
12.9 ± 5.0
6.2 ± 0.9
3.1 ± 0.1
3o
3p
CA-4 (1)
0.004 ± 0.001
aIC50 = compound concentration required to inhibit tumor cell proliferation by
50%. Data are expressed as the mean ± SE from the dose–response curves of at
least three independent experiments carried out in triplicate.
3.2. Molecular modeling studies
To evaluate the influence of the new structural modifications that
characterize compounds with general structure 3 on binding in the
colchicine site of
αβ-tubulin, preliminary docking studies were per-
reduction in activity against HeLa and HT-29 cells, respectively, in
formed. There was significant overlap with the co-crystallized colchi-
cine, with the trimethoxyphenyl ring in proximity to βCys241, a key
interaction point for tubulin polymerization inhibition (Fig. 3A, 2-(4-
fluorophenyl)-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thio-
phene derivative 3b). The benzo[b]thiophene portion pointed towards
comparison with the parent unsubstituted 2-phenyl analogue 3a. De-
rivative 3b, with IC50 values of 0.5 and 0.4 μM, against HeLa and HT-29
cells, respectively, along with compound 3a, were the only compounds
of the series active at sub-micromolar concentrations against both can-
cer cell lines. Replacing fluorine with the weak-electron releasing
methyl group, to furnish derivative 3c, yielded a compound that main-
tained activity against HeLa cells but that had 3.5-fold reduced potency
against HT-29 cells. Moving the methyl group from the para- to the meta-
position had a negative effect in maintaining antiproliferative activities,
with the meta-tolyl derivative 3d 43- and 9-fold less active than the
isomeric para-tolyl 3c against HeLa and HT-29 cells, respectively.
Replacement of the methyl moiety of 3d with the strong electron-
withdrawing trifluoromethyl group, to furnish derivative 3f, main-
tained the antiproliferative activity on HeLa cells and caused a 2-fold
reduction in potency on HT-29 cells. Starting from compound 3c, ho-
mologation of the alkyl chain from methyl to ethyl (to yield derivative 3
g) had a detrimental effect on activity, with a 28- and 4-fold reduction of
potency relative to 3c on HeLa and HT-29 cells, respectively. A similar
reduction in activity was observed with an additional methyl group at
the meta-position of para-tolyl derivative 3c, to furnish the meta, para-
xylyl analogue 3e.
the tubulin
at an optimal distance of 3.1 Å, permitting an interaction with atoms of
the polypeptide backbone of Thr179. The differently para-substituted
α-subunit, with the sulfur atom interacting with βLys352 and
α
phenyl rings could be tolerated and are placed in a small hydrophobic
area near βMet259, where the colchicine methoxy group is positioned,
and the latter is involved in different interactions than the former with
the surrounding residues. Interestingly, all the docked derivatives
occupy the colchicine site in a comparable manner (Fig. 3B, 2-(4-
ethoxyphenyl)-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thio-
phene derivative 3j). The potential binding mode for this new family of
compounds, as determined in molecular modeling studies, was consis-
tent with an interaction at the colchicine site. To prove this hypothesis,
selected compounds were tested for their in vitro inhibitory activities
both of tubulin assembly and colchicine binding to tubulin.
3.3. In vitro inhibition of tubulin polymerization and colchicine binding
Replacing the weak electron-releasing methyl with the stronger
methoxy group at the para position of the 2′-phenyl group (to furnish
compound 3 h) caused a 3-fold reduction in activity against HeLa cells,
while the two compounds had similar potency against HT-29 cells.
Moving the methoxy group from the para- to the meta-position (com-
pounds 3 h and 3i, respectively) caused a 5- and 4-fold reduction in
activity against HeLa and HT-29 cells, respectively, a reduction that was
less dramatic than that observed for the two isomeric para- and meta-
tolyl derivatives 3c and 3d.
To investigate whether the antiproliferative activities of the most
potent compounds of the series derived from an interaction with mi-
crotubules, derivatives 3a-c, 3h and 3j were selected to determine their
ability to inhibit tubulin polymerization and for effects on the binding of
[3H]colchicine to tubulin. For comparison, CA-4 was examined in
contemporaneous experiments and strongly inhibited tubulin polymer-
ization with an IC50 value of 0.54 μM. In contrast to the molecular
modeling studies reported in the introduction section that predicted that
these compounds should be characterized by an excellent affinity in the
binding site of colchicine in tubulin, none of the compounds examined
Unlike what was observed for the para-methyl and ethyl homologues
3c and 3 g, respectively, increasing the length of the alkoxy chain from
para-methoxy (3i) to ethoxy (3j) resulted in a 2-fold increase in anti-
proliferative activity against HT-29 cells, while the two compounds were
equipotent against HeLa cells. A dramatic drop of activity (17- and 36-
fold against HeLa and HT-29 cells, respectively) was observed by mov-
ing the ethoxy group from the para- to the meta-position (derivatives 3j
and 3 k, respectively). Starting from para-ethoxy derivative 3j, a further
homologation of the alkyl chain of the alkoxy moiety from ethyl to n-
propyl (3 l) to n-butyl (3 m) to end with n-pentyl (3n) or the presence of
branched isopropyl chain (3o) at the para-position of the phenyl ring
significantly affected tubulin assembly, as all had an IC50 > 20 μM.
Moreover, in the colchicine binding studies, only modest inhibition
(30%) of the binding of [3H]colchicine to tubulin was observed with 5
μ
M compound 3b, in comparison with CA-4, which in these experiments
inhibited colchicine binding by 97%. Inhibition of colchicine binding by
derivatives 3a, 3c, 3 h and 3j was even lower (7–24% inhibition was
observed with these agents).
The results of the in vitro inhibition of tubulin polymerization and
colchicine-binding assays did not support the preliminary data of the
5

R. Romagnoli et al.
Bioorganic Chemistry 112 (2021) 104919
Fig. 3. A) Proposed binding for compound 3b in the colchicine site. The trimethoxyphenyl ring is in proximity to βCys241, while the benzo[b]thiophene portion is
pointing towards the tubulin -subunit. The p-fluoro phenyl ring is placed in a small hydrophobic area near βMet259. B) Proposed binding mode for 3j. In both
panels, co-crystallized colchicine is shown in cyan, and the carbon atoms of -tubulin residues are shown in blue, those of the β-subunit are in dark green. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
α
α
computational-based docking studies reported in the previous section
and are consistent with the conclusion that the tested compounds may
exert their antiproliferative effects by mechanisms other than inhibition
of tubulin assembly at the colchicine site. In this regard, replacing the
sp2 centre of the carbonyl in the 3,4,5-trimethoxybenzoyl moiety of
derivatives 2a and 2c with an anilinic nitrogen (NH) reduced anti-
tubulin activity.
apoptotic stimuli than are non-transformed cells [31]. The three cell
lines (Caco2, HCT-116 and 841 CoN) were treated with 60 nM 3a or 400
nM 3b for 48 h, and these treatments triggered a significant induction of
apoptosis as suggested by the increased expression of cleaved poly(ADP-
ribose) polymerase (PARP), receptor-interacting protein (RIP) and cas-
pase 3 proteins and by the intensity of annexin-V staining in both met-
astatic and non-metastatic cells (Fig. 4A and 4B). Not to be
underestimated in anticancer strategies are the side effects of drugs in
the “healthy” cells surrounding the cancer cells [32]. Thus, the same
analyses were performed on non-transformed 841 CoN cells. These cells
showed no differences between vehicle and 3a and 3b treatments,
highlighting the absence of apoptotic effects on normal cells following a
therapeutic treatment with compound 3a or 3b (Fig. 4A and 4B).
3.4. Antitumoral properties of compounds 3a and 3b in colorectal cancer
cells
Because of their initial activity in HT-29 colon cancer cells, we
examined the effects of 3a and 3b in other colon cell lines. This seemed
pertinent because of the major impact of colorectal cancer on human
health. In 2019, the Global Cancer Observatory (GLOBOCAN) reported
colorectal cancer as the second most deadly and the third most common
cancer diagnosed in the world [30]. For this reason, and taking into
consideration the promising antiproliferative activities of compounds 3a
and 3b in HT-29 cells, we undertook an in-depth analysis of their in vitro
effects on three different types of colon cells: Caco2 (not metastatic),
HCT-116 (metastatic) and 841 CoN (non-tumor cells), all with
epithelial-like morphology. In most of the following studies, we used the
IC50 values obtained with the HT-29 cells (60 nM for 3a and 400 nM for
3b).
3.4.2. Compounds 3a and 3b induce cell cycle arrest
Cancer cells almost always display abnormalities in the control of
cell cycle checkpoints, and thus strategies able to affect this process are
crucial for counteracting tumor growth [33]. To investigate whether
compounds 3a and 3b affected cell cycle progression, and thus prolif-
eration, two important assays were performed. First, we measured the
percentages of cells in the G0/G1, S and G2/M phases, via fluorescence
and propidium iodide (PI) incorporation (which quantifies cellular DNA
content); the second and more specific assay explored the expression of
key proteins involved in the cell cycle, such as cyclins (Cyc.), p27
(marker of G1 phase) and pHH3 (marker of mitosis). The first method
unveiled a strong effect of 3a and 3b in Caco2 and HCT-116 cells con-
cerning cell accumulation in the G0/G1 phase, but not the G2/M phase,
without any appreciable effects on 841 CoN cells (Fig. 5A). The second
3.4.1. Derivatives 3a and 3b trigger apoptotic cell death
First, we investigated the ability of compounds 3a and 3b to induce
apoptotic cell death, as cancer cells are significantly more resistant to
Fig. 4. (A) Cleaved PARP, RIP and caspase 3 protein expression evaluated to determine the extent of cell death occurring in the Caco2 and HCT-116 cell lines, but not
in 841 CoN cells, following treatment with 3a or 3b. (B) Detection of apoptosis by annexin-V staining quantification in 841 CoN, Caco2 and HCT-116 cell lines
following 3a (grey bar) or 3b (black bar) treatment at 60 or 400 nM, respectively. N.s. = not significant; *** = p < 0.001.
6

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Bioorganic Chemistry 112 (2021) 104919
Fig. 5. (A) Calculation in percentage of the amount of 841 CoN, Caco2 and HCT-116 cells in G0/G1, S and G2/M phases in basal condition and after 3a (grey bar) or
3b (black bar) treatments at 60 or 400 nM, respectively, on the basis of the PI uptake. (B) Cell cycle marker investigation under the same conditions as were used in
Panel A by using selective antibodies against markers of the cell cycle: Cyc. D1 and p27 for G1 phase; Cyc. A for S phase; Cyc. F and B1 for G2/M; pHH3 for mitosis.
Cyc. = Cyclin; n.s. = not significant; *** = p < 0.001.
assay confirmed the preliminary data obtained in Fig. 5A, showing
increased expression of G1 phase markers (in accord with the cell
accumulation in G1), such as Cyc. D1 and p27 for both the compounds in
HCT-116 and Caco2 cells, and a concomitant decrease of proteins
involved in the S (Cyc. A) and G2/M (Cyc. B1 and F) phases (Fig. 5B).
The cell cycle marker evaluation revealed also a significant accumula-
tion of pHH3 in Caco2 following 3b administration and in HCT-116 for
3a, not detected by the previous method. These findings not only
confirmed cell cycle arrest in G1 phase, but also indicated a potential
additional effect during mitosis for 3a in metastatic cells and for 3b in
non-metastatic cells (Fig. 5B). Also, in this case, no effects were detected
in the 841 CoN control cells.
3.4.3. Derivatives 3a and 3b impact mitochondrial morphology and
mitochondrial dynamics
Mitochondria, organelles of bacterial origin and responsible for
producing energy for the whole body, are physically and functionally
associated with the intrinsic apoptotic pathway that often is disrupted
during cancer cell transformation [34,35]. Therefore, we evaluated
several mitochondrial parameters that might have been altered as a
Fig. 6. (A) Mitochondrial morphology investigation. Representative images in 841 CoN, Caco2 and HCT-116 cells using the mtDSRed plasmid. Number of objects
index shows the number of mitochondria inside cells in each experimental condition, and the volume of the total mitochondrial network is quantitated in terms of µ3.
(B) Evaluation of the expression of proteins involved in mitochondria fusion (OPA1, MFN1 and MFN2). Reaction conditions were as used in the experiments shown in
Panel A. For Panel A: n.s. = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001.
7

R. Romagnoli et al.
Bioorganic Chemistry 112 (2021) 104919
consequence of the apoptotic process.
mitochondrial calcium homeostasis using a specific organelle-target
aequorin (AEQ) probe, one targeted to mitochondria (mtAEQ). First,
we transfected mtAEQ into 841 CoN, Caco2 and HCT-116 cells, and,
second, we treated them with vehicle or compound 3a or 3b. Under all
conditions we investigated, the ATP agonist caused a significant increase
in calcium uptake only in the metastatic HCT-116 cell line following 3a
or 3b treatment. No differences were detected in the control 841 CoN or
non-metastatic Caco2 cells (Fig. 7B).
Current opinion is that mitochondrial fragmentation occurs during
apoptosis, yielding an increased number of smaller and dysfunctional
mitochondria [36]. To determine whether 3a and 3b caused this effect
while they induced apoptosis, we examined mitochondrial morphology
with the mitochondria-targeted red fluorescent protein mtDsRed, which
was transfected into cells in the same manner in all experimental con-
ditions. Cells were treated with 3a or 3b at 60 or 400 nM, respectively,
and images of treated, living cells (HCT-116 and Caco2) demonstrated
increased numbers of mitochondria (designated as n◦ of objects),
without changes in the total mitochondrial network (Fig. 6A) when
compared to untreated cells. As a consequence, mitochondria appeared
smaller following compound treatment, in terms of the volume of an
individual mitochondrion. In nontumoral cells, no appreciable changes
were detected. Taken together, these findings indicated a significant
organelle fragmentation following 3a or 3b treatment without changes
in the overall mitochondrial mass.
4. Conclusions
Previously [27], we discovered a new class of structurally simple
synthetic inhibitors of tubulin polymerization, based on the 3-(3,4,5-
trimethoxyanilino)-6-methoxybenzo[b]thiophene moiety of compounds
with general structure 2d-e. Here, we have synthesized a new series of
related compounds in a concise three-step synthetic procedure starting
from a 6-methoxybenzo[b]thiophene common intermediate. Modifica-
tions were focused on varying the aryl moiety at the C-2 position of the
6-methoxybenzo[b]thiophene nucleus by adding electron-withdrawing
(F) or electron-releasing (Me, Et and alkoxy) groups The 2-phenyl-3-
(3,4′5-trimethoxyanilino)-6-methoxybenzo[b]thiophene derivative 3a
displayed the greatest antiproliferative activity among the new com-
Moreover, mitochondria are dynamic structures that fuse and divide
continuously [36,37]. This phenomenon is ensured by a pool of GTPase
proteins with a prevalent mitochondrial localization. In humans, these
proteins include Mfn1, Mfn2 and Opa1, and they are involved in mito-
chondrial fusion events. To understand the cause of mitochondrial
fragmentation, we tested OPA1, MFN1 and MFN2 expression levels
before and after treatments with 3a or 3b (Fig. 6B). In agreement with
the morphology findings, the 841 CoN colon cells did not show any
change in protein expression, in contrast to the two colon cancer cell
lines, where their expression was reduced (Fig. 6B). These data
demonstrated that 3a and 3b induced mitochondrial fragmentation by
reducing expression of proteins that ensure mitochondrial fusion.
pounds, with IC50 values of 0.23 and 0.06 μM against HeLa and HT-29
cells, respectively. We observed that the insertion of a substituent at
the para-position of the phenyl at the 2-position of the 6-methoxybenzo
[b]thiophene nucleus reduced antiproliferative activity in comparison
with the unsubstituted phenyl analogue 3a. This effect was less pro-
nounced by the introduction of an electron-withdrawing fluorine atom
(compound 3b), and it was more evident for electron releasing alkyl and
alkoxy groups, suggesting that the presence of the unsubstituted phenyl
moiety at the 2-position of 6-methoxybenzo[b]thiophene nucleus was
best for antiproliferative activity. Five of the synthesized compounds,
3a, 3b (p-F), 3c (p-Me), 3 h (p-OMe) and 3j (p-OEt), had the best anti-
proliferative activities against the CA-4 resistant HT-29 cell line and
were 3–50-fold more active than CA-4. Mechanism studies demon-
strated that 3a and 3b arrested cell cycle progression predominantly in
the G0/G1 phase and induced cell apoptosis. Although molecular
modeling studies predicted that these compounds could act through
inhibition of tubulin polymerization, fitting well into the colchicine site,
the in vitro assays demonstrated that for the most active derivatives
antiproliferative activity was not related to inhibition of tubulin as-
sembly. This was also confirmed based on the failure to observe the
usual G2/M arrest with either 3a or 3b. For all these reasons, we think it
unlikely that tubulin plays a significant role in the cytotoxicity of these
compounds, especially in view of their weak inhibition of colchicine
3.4.4. Derivatives 3a and 3b impact mitochondrial membrane potential
Dissipation of mitochondrial membrane potential is an early event of
apoptosis, and we therefore measured it by the tetramethylrhodamine
methyl ester (TMRM) probe. Treatment of cells with 3a or 3b led to a
significant reduction of mitochondrial potential when compared to the
untreated condition (Fig. 7A). In the control 841 CoN cells, compound
3a did not affect mitochondrial membrane potential, but 3b showed a
negative effect (Fig. 7A).
3.4.5. Mitochondrial calcium handling
Mitochondria are organelles that exert most of their functions by
controlling intracellular calcium, which is derived either from such in-
ternal stores as the endoplasmic reticulum or from the extracellular
milieu [34,38]. Increased mitochondrial calcium uptake may be a signal
for cell death via the intrinsic pathway [39]. We therefore measured
Fig. 7. (A) Mitochondrial membrane potential in 841 CoN, Caco2 and HCT-116 cells measured by using the TMRM probe. Values on the ordinate are expressed as
arbitrary fluorescence ratio between TMRM fluorescence in basal conditions and upon FCCP administration. (B) Mitochondrial calcium uptake upon agonist (ATP)
administration measured by transfecting cells with the luminescent mitochondria-targeted aequorin. Reaction conditions were the same as used in the experiments
shown in Panel A. n.s. = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001.
8

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Bioorganic Chemistry 112 (2021) 104919
binding.
for 48 h. After this time, the reaction mixture was diluted with EtOAc
(10 mL), filtered on a pad of celite and the filtrate washed sequentially
with water (5 mL) and brine (5 mL), dried over Na2SO4 and evaporated
under reduced pressure. The crude residue was purified by flash column
chromatography on silica gel using a mixture of ethyl ether and petro-
leum ether to afford the title compound.
The good activity of several derivatives in the HT-29 colon cancer
cells led us to perform a detailed analysis in two additional colon cancer
cell lines and in a non-transformed colon cell line. These studies
confirmed the antiproliferative properties of compounds 3a and 3b
against colon cancer cells and showed that the compounds strongly
induced cell death and cell cycle arrest in the G0/G1 phase. Our data
demonstrated that apoptosis was a mechanism of cell death, but other
types of cell death cannot yet be excluded. As apoptosis can be triggered
by the intrinsic pathway, we explored the “state of health” of the
mitochondrial pool by measuring the morphology, membrane potential
and calcium homeostasis of mitochondria following 3a and 3b treat-
ment. Both derivatives triggered mitochondrial fragmentation by
negatively modulating OPA1, MFN1 and MFN2, and they lowered
mitochondrial membrane potential. We observed that these effects
occurred in the two colon cancer cell lines, but not in the non-
transformed 841 CoN colon cells. Thus, based on these in vitro find-
ings, 3a and 3b could be considered as safe for the tumor-surrounding
healthy tissues as they had no or limited effects on 841 CoN control
colon cells. In these cells, our only negative finding was a loss of mito-
chondrial membrane potential with 3b treatment, perhaps indicating
toxicity with prolonged treatment. A second issue concerns possible
differential affinities towards metastatic and non-metastatic tumors.
Derivatives 3a and 3b showed greater effectiveness for HCT-116 cells,
which displayed a greater extent of cell death and decrease in mito-
chondrial membrane potential as compared with the non-metastatic
Caco2 cells. Moreover, 3a and 3b caused increased calcium uptake in
mitochondria, an important signal of cell death, in HCT-116 cells but not
in Caco2 cells. A further difference between the two colon cancer cell
lines was an additional block in mitosis caused by 3a (but not 3b) in
HCT-116 cells and by 3b (but not 3a) in Caco2 cells. Further studies of
the molecular mechanism of action of 3a and 3b are underway.
5.1.2.1. 6-Methoxy-2-phenylbenzo[b]thiophene (7a). Following general
procedure A, the crude residue purified by flash chromatography, using
Et2O:petroleum ether 0.5:9.5 (v:v) for elution, furnished compound 7a
as a yellow oil. Yield: 75%. 1H NMR (DMSO‑d6) δ: 3.84 (s, 3H), 7.04 (dd,
J = 9.0 and 2.4 Hz, 1H), 7.41 (dd, J = 8.6 and 1.8 Hz, 1H), 7.48–7.51 (m,
1H), 7.54–7-62 (m, 2H), 7.60–7.62 (m, 2H), 7.64 (d, J = 2.4 Hz, 1H),
7.73 (d, J = 9.0 Hz, 1H). MS (ESI): [M + 1]+=241.3.
5.1.2.2. 2-(4-Fluorophenyl)-6-methoxybenzo[b]thiophene (7b). Follow
ing general procedure A, the crude residue purified by flash chroma-
tography, using petroleum ether for elution, furnished compound 7b as
a white solid. Yield: 62%, mp 63 ◦C. 1H NMR (DMSO‑d6) δ: 3.84 (s, 3H),
7.03 (dd, J = 9.0 and 2.4 Hz, 1H), 7.29–7.39 (m, 4H), 7.59–7.66 (m,
2H), 7.69 (d, J = 9.0 Hz, 1H). MS (ESI): [M + 1]+=259.3.
5.1.2.3. 6-Methoxy-2-(p-tolyl)benzo[b]thiophene (7c). Following gen-
eral procedure A, the crude residue purified by flash chromatography,
using Et2O:petroleum ether 0.1:9.9 (v:v) for elution, furnished com-
pound 7c as a colorless oil. Yield: 85%. 1H NMR (DMSO‑d6) δ: 2.40 (s,
3H), 3.82 (s, 3H), 6.97 (dd, J = 8.8 and 2.4 Hz, 1H), 7.33–7.38 (m, 6H),
7.61 (d, J = 2.4 Hz, 1H). MS (ESI): [M + 1]+=255.3.
5.1.2.4. 6-Methoxy-2-(m-tolyl)benzo[b]thiophene (7d). Following gen-
eral procedure A, the crude residue purified by flash chromatography,
using Et2O:petroleum ether 0.1:9.9 (v:v) for elution, furnished com-
pound 7d as a colorless oil. Yield: 58%. 1H NMR (DMSO‑d6) δ: 2.39 (s,
3H), 3.84 (s, 3H), 7.04 (dd, J = 9.0 and 2.4 Hz, 1H), 7.21–7.24 (m, 2H),
7.36–7.41 (m, 2H), 7.57 (s, 1H), 7.63 (d, J = 2.4 Hz, 1H), 7.73 (d, J =
8.8 Hz, 1H). MS (ESI): [M + 1]+=255.3.
5. Experimental protocols
5.1. Chemistry
5.1.1. Materials and methods
5.1.2.5. 2-(3,4-Dimethylphenyl)-6-methoxybenzo[b]thiophene
(7e).
1H experiments were recorded on either a Bruker AC 200 or a Bruker
Avance III 400 spectrometer, and 13C NMR spectra were recorded on a
Varian 400 Mercury Plus or a Bruker Avance III 400 spectrometer.
Chemical shifts (δ) are given in ppm upfield, and the spectra were
recorded in appropriate deuterated solvents, as indicated. Mass spectra
were recorded by an ESI single quadrupole mass spectrometer Waters
ZQ 2000 (Waters Instruments, UK), and the values are expressed as [M
+ 1]+. Melting points (mp) were determined on a Buchi-Tottoli appa-
ratus and are uncorrected. All products reported showed 1H and 13C
NMR spectra in agreement with the assigned structures. The purity of
tested compounds was determined by combustion elemental analyses
conducted by the Microanalytical Laboratory of the Chemistry Depart-
ment of the University of Ferrara with a Yanagimoto MT-5 CHN recorder
elemental analyzer. All tested compounds yielded data consistent with a
purity of at least 95% as compared with the theoretical values. Reaction
courses and product mixtures were routinely monitored by TLC on silica
gel (precoated F254 Merck plates), and compounds were visualized with
aqueous KMnO4. Flash chromatography was performed using 230–400
mesh silica gel and the indicated solvent system. Organic solutions were
dried over anhydrous Na2SO4.
Following general procedure A, the crude residue purified by flash
chromatography, using petroleum ether for elution, furnished com-
pound 7e as a colorless oil. Yield: 56%. 1H NMR (DMSO‑d6) δ: 2.82 (s,
3H), 2.98 (s, 3H), 3.84 (s, 3H), 7.01 (dd, J = 8.8 and 2.4 Hz, 1H),
7.27–7.32 (m, 3H), 7.51 (s, 1H), 7.62 (d, J = 2.4 Hz, 1H), 7.72 (d, J =
8.8 Hz, 1H). MS (ESI): [M + 1]+=269.4.
5.1.2.6. 6-Methoxy-2-(3-(trifluoromethyl)phenyl)benzo[b]thiophene
(7f). Following general procedure A, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.1:9.9 (v:v) for elution,
furnished compound 7f as a colorless oil. Yield: 53%. 1H NMR
(DMSO‑d6) δ: 3.85 (s, 3H), 7.11 (dd, J = 8.8 and 2.4 Hz, 1H), 7.67 (d, J
= 2.6 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.77–7.80 (m, 3H), 7.87–7.94
(m, 2H). MS (ESI): [M + 1]+=309.3.
5.1.2.7. 2-(4-Ethylphenyl)-6-methoxybenzo[b]thiophene (7g). Following
general procedure A, the crude residue purified by flash chromatog-
raphy, using petroleum ether for elution, furnished compound 7 g as a
colorless oil. Yield: 53%. 1H NMR (DMSO‑d6) δ: 1.23 (t, J = 7.6 Hz, 3H),
2.65 (q, J = 7.6 Hz, 2H), 3.84 (s, 3H), 7.02 (dd, J = 8.8 and 2.4 Hz, 1H),
7.32 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 7.55 (s, 1H), 7.63 (d, J
= 2.4 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H). MS (ESI): [M + 1]+=269.4.
5.1.2. General procedure a for the synthesis of compounds 7a-o
To a suspension of 6-methoxybenzo[b]thiophene 6 (164 mg, 1
mmol), CF3COOAg (331 mg, 1.5 mmol), trifluoroacetic acid (228 mg,
0.15 mL, 2 mmol), the appropriate iodobenzene derivative (2.0 mmol)
and Tween-80/H2O (2 mL, 2 wt%) was degassed under a stream of argon
over 10 min. Pd(OAc)2 (11.2 mg, 0.05 mmol, 5 mol %) was added and
the mixture degassed again for 10 min, then stirred at room temperature
5.1.2.8. 6-Methoxy-2-(4-methoxyphenyl)benzo[b]thiophene
(7h).
Following general procedure A, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.5:9.5 (v:v) for elution,
furnished compound 7 h as a colorless oil. Yield: 60%. 1H NMR
9

R. Romagnoli et al.
Bioorganic Chemistry 112 (2021) 104919
(DMSO‑d6) δ: 3.82 (s, 3H), 3.84 (s, 3H), 7.02 (dd, J = 9.0 and 2.4 Hz,
1H), 7.06 (d, J = 8.8 Hz, 2H), 7.47 (s, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.62
(d, J = 2.4 Hz, 1H), 7.70 (d, J = 9.0 Hz, 1H). MS (ESI): [M + 1]+=271.1.
5.1.3. General procedure b for the preparation of compounds 8a-o and 9
To a solution of 6-methoxybenzo[b]thiophene 6 or the appropriate 6-
methoxy-2-arylbenzo[b]thiophene 7a-o (1 mmol) in THF (5 mL) was
added NBS (0.196 g, 1.1 mmol) in small portions at 0 ◦C. After 1 h at
◦
0 C, the reaction mixture was allowed to warm to room temperature
5.1.2.9. 6-Methoxy-2-(3-methoxyphenyl)benzo[b]thiophene (7i). Follow
ing general procedure A, the crude residue purified by flash chroma-
tography, using Et2O:petroleum ether 0.1:9.9 (v:v) for elution, furnished
compound 7i as a colorless oil. Yield: 54%. 1H NMR (DMSO‑d6) δ: 3.83
(s, 3H), 3.84 (s, 3H), 6.97–6.99 (m, 1H), 7.00–7.17 (m, 1H), 7.04 (dd, J
= 8.8 and 2.2 Hz, 1H), 7.10–7.17 (m, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.62
(s, 1H), 7.63 (d, J = 2.2 Hz, 1H), 7.75 (d, J = 9.0 Hz, 1H). MS (ESI): [M
+ 1]+=271.3.
and stirred for an additional 1 h. The reaction mixture was quenched
with 10% aqueous sodium thiosulfate solution at 0 ◦C (10 mL). The
resulting solution was extracted with dichloromethane (3x10 mL), and
the combined organic solvent was washed with brine (10 mL), dried and
evaporated. The residue was purified by column chromatography on
silica gel.
5.1.3.1. 3-Bromo-6-methoxy-2-phenylbenzo[b]thiophene (8a). Following
general procedure B, the crude residue purified by flash chromatog-
raphy, using Et2O:petroleum ether 0.25:9.75 (v:v) for elution, furnished
compound 8a as a clear oil. Yield: 67%. 1H NMR (DMSO‑d6) δ: 3.84 (s,
3H), 7.04 (dd, J = 8.8 and 2.4 Hz, 1H), 7.38 (dd, J = 8.6 and 1.8 Hz, 1H),
7.45–7.50 (m, 1H), 7.54–7-60 (m, 1H), 7.61–7.64 (m, 2H), 7.62 (d, J =
1.8 Hz, 1H), 7.75 (d, J = 9.0 Hz, 1H). MS (ESI): [M]+=318.7, [M +
2]+=320.9.
5.1.2.10. 2-(4-Ethoxyphenyl)-6-methoxybenzo[b]thiophene (7j). Follow
ing general procedure A, the crude residue purified by flash chroma-
tography, using Et2O:petroleum ether 0.25:9.75 (v:v) for elution, fur-
nished compound 7j as a white solid. Yield: 54%, mp 82–84 ◦C. 1H NMR
(DMSO‑d6) δ: 1.36 (t, J = 7.2 Hz, 3H), 3.84 (s, 3H), 4.07 (q, J = 7.2 Hz,
2H), 7.03–7.07 (m, 3H), 7.48 (s, 1H), 7.52 (d, J = 8.6 Hz, 2H), 7.62 (d, J
= 2.2 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H). MS (ESI): [M + 1]+=285.4.
5.1.3.2. 3-Bromo-2-(4-fluorophenyl)-6-methoxybenzo[b]thiophene (8b).
Following general procedure B, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.25:9.75 (v:v) for elution,
furnished compound 8b as a white solid. Yield: 92%, mp 97–98 ◦C. 1H
NMR (DMSO‑d6) δ: 3.82 (s, 3H), 6.99 (dd, J = 8.8 and 2.4 Hz, 1H), 7.32
(s, 1H), 7.34–7.40 (m, 2H), 7.43–7.55 (m, 2H), 7.62 (d, J = 2.4 Hz, 1H).
MS (ESI): [M]+=337.2, [M + 2]+=339.4.
5.1.2.11. 2-(3-Ethoxyphenyl)-6-methoxybenzo[b]thiophene (7k). Follow
ing general procedure A, the crude residue purified by flash chroma-
tography, using Et2O:petroleum ether 0.1:9.99 (v:v) for elution, fur-
nished compound 7 k as a clear oil. Yield: 61%. 1H NMR (DMSO‑d6) δ:
1.35 (d, J = 7.0 Hz, 3H), 3.84 (s, 3H), 4.08 (q, J = 7.0 Hz, 2H), 6.96 (ddd,
J = 8.4, 2.6 and 0.8 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H), 7.07–7.09 (m, 1H),
7.11 (dt, J = 7.6 and 1.6 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.61–7.64 (m,
2H), 7.75 (d, J = 9.0 Hz, 1H). MS (ESI): [M + 1]+=285.4.
5.1.3.3. 3-Bromo-6-methoxy-2-(p-tolyl)benzo[b]thiophene (8c). Follow
ing general procedure B, the crude residue purified by flash chroma-
tography, using Et2O:petroleum ether 0.1:9.9 (v:v) for elution, furnished
5.1.2.12. 6-Methoxy-2-(4-propoxyphenyl)benzo[b]thiophene (7l). Foll
owing general procedure A, the crude residue purified by flash chro-
matography, using Et2O:petroleum ether 0.5:9.5 (v:v) for elution, fur-
nished compound 7 l as a clear oil. Yield: 50%. 1H NMR (DMSO‑d6) δ:
1.04 (t, J = 7.4 Hz, 3H), 1.72–1.78 (m, 2H), 3.84 (s, 3H), 4.02 (q, J = 7.2
Hz, 2H), 7.03–7.08 (m, 3H), 7.48 (s, 1H), 7.49 (d, J = 7.6 Hz, 2H), 7.62
(d, J = 2.2 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H). MS (ESI): [M + 1]+=299.4.
1
compound 8c as a pale yellow oil. Yield: 63%. H NMR (DMSO‑d6) δ:
2.38 (s, 3H), 3.84 (s, 3H), 6.92 (dd, J = 8.2 and 2.4 Hz, 1H), 7.34–7.38
(m, 5H), 7.60 (d, J = 2.2 Hz, 1H). MS (ESI): [M]+=333.3, [M +
2]+=335.4.
5.1.3.4. 3-Bromo-6-methoxy-2-(m-tolyl)benzo[b]thiophene (8d). Follow
ing general procedure B, the crude residue purified by flash chroma-
tography, using Et2O:petroleum ether 0.1:9.9 (v:v) for elution, furnished
5.1.2.13. 2-(4-Butoxyphenyl)-6-methoxybenzo[b]thiophene (7m). Follo
wing general procedure A, the crude residue purified by flash chroma-
tography, using Et2O:petroleum ether 0.25:9.75 (v:v) for elution, fur-
nished compound 7 m as a clear oil. Yield: 58%. 1H NMR (DMSO‑d6) δ:
0.95 (t, J = 7.4 Hz, 3H), 1.44–1.48 (m, 2H), 1.69–1.74 (m, 2H), 3.84 (s,
3H), 4.03 (t, J = 6.6 Hz, 2H), 7.02–7.08 (m, 3H), 7.49–7.52 (m, 3H),
7.62 (d, J = 2.2 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H). MS (ESI): [M +
1]+=313.4.
1
compound 8d as a colorless oil. Yield: >95%. H NMR (DMSO‑d6) δ:
2.40 (s, 3H), 3.82 (s, 3H), 6.98 (dd, J = 9.0 and 2.4 Hz, 1H), 7.23–7.27
(m, 2H), 7.33–7.39 (m, 3H), 7.61 (d, J = 2.6 Hz, 1H). MS (ESI):
[M]+=333.3, [M + 2]+=335.4.
5.1.3.5. 3-Bromo-2-(3,4-dimethylphenyl)-6-methoxybenzo[b]thiophene
(8e). Following general procedure B, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 2.5:7.5 (v/v) for elution,
furnished compound 8e as a colorless oil. Yield: 89%. 1H NMR
(DMSO‑d6) δ: 2.30 (s, 6H), 3.82 (s, 3H), 6.98 (dd, J = 8.8 and 2.4 Hz,
1H), 7.14–7.22 (m, 2H), 7.28–7.37 (m, 2H), 7.59 (d, J = 2.2 Hz, 1H). MS
(ESI): [M]+=347.3, [M + 2]+=349.4.
5.1.2.14. 6-Methoxy-2-(4-(pentyloxy)phenyl)benzo[b]thiophene
(7n). Following general procedure A, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.25:9.75 (v:v) for elution,
furnished compound 7n as a clear oil. Yield: 51%. 1H NMR (DMSO‑d6) δ:
0.88 (t, J = 7.2 Hz, 3H), 1.33–1.40 (m, 4H), 1.71–1.74 (m, 2H), 3.80 (s,
3H), 3.99 (t, J = 6.4 Hz, 2H), 7.01–7.04 (m, 3H), 7.46–7.49 (m, 3H),
7.60 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 9.2 Hz, 1H). MS (ESI): [M +
1]+=327.2.
5.1.3.6. 3-Bromo-6-methoxy-2-(3-(trifluoromethyl)phenyl)benzo[b]thio-
phene (8f). Following general procedure B, the crude residue purified
by flash chromatography, using Et2O:petroleum ether 0.1:9.9 (v:v) for
elution, furnished compound 8f as a colorless oil. Yield: 91%. 1H NMR
(DMSO‑d6) δ: 3.83 (s, 3H), 7.00 (dd, J = 9.0 and 2.4 Hz, 1H), 7.53 (d, J
= 8.8 Hz, 1H), 7.65 (d, J = 2.6 Hz, 1H), 7.79–7.82 (m, 3H), 7.86–7.92
(m, 1H). MS (ESI): [M]+=387.3, [M + 2]+=389.4.
5.1.2.15. 2-(4-Isopropoxyphenyl)-6-methoxybenzo[b]thiophene
(7o).
Following general procedure A, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.5:9.5 (v:v) for elution,
furnished compound 7o as a white solid. Yield: 57%, mp 93–94 ◦C. 1H
NMR (DMSO‑d6) δ: 1.29 (d, J = 5.8 Hz, 6H), 3.84 (s, 3H), 4.63–4.67 (m,
1H), 7.06–7.07 (m, 3H), 7.46–7.51 (m, 3H), 7.61 (d, J = 2.2 Hz, 1H),
7.71 (d, J = 9.0 Hz, 1H). MS (ESI): [M + 1]+=299.4.
5.1.3.7. 3-Bromo-2-(4-ethylphenyl)-6-methoxybenzo[b]thiophene (8g).
Following general procedure B, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.25–9.75 (v/v) for
1
elution, furnished compound 8 g as a pale yellow oil. Yield: 92%. H
10

R. Romagnoli et al.
Bioorganic Chemistry 112 (2021) 104919
NMR (DMSO‑d6) δ: 1.26 (t, J = 7.6 Hz, 3H), 2.68 (q, J = 7.6 Hz, 2H),
3.82 (s, 3H), 6.97 (dd, J = 8.8 and 2.4 Hz, 1H), 7.35 (m, 5H), 7.63 (d, J
= 2.4 Hz, 1H). MS (ESI): [M]+=347.3, [M + 2]+=349.4.
5.1.3.15. 3-Bromo-2-(4-isopropoxyphenyl)-6-methoxybenzo[b]thiophene
(8o). Following general procedure B, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.25:9.75 (v:v) for elution,
furnished compound 8o as a white solid. Yield: 75%, mp 83–85 ◦C. 1H
NMR (DMSO‑d6) δ: 1.32 (d, J = 5.8 Hz, 6H), 3.82 (s, 3H), 4.67–4.72 (m,
1H), 6.97 (dd, J = 8.6 and 2.4 Hz, 1H), 7.05 (d, J = 8.8 Hz, 1H),
7.34–7.38 (m, 3H), 7.59 (d, J = 2.2 Hz, 1H). MS (ESI): [M]+=377.3, [M
+ 2]+=379.4.
5.1.3.8. 3-Bromo-6-methoxy-2-(4-methoxyphenyl)benzo[b]thiophene (8
h). Following general procedure B, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.25:9.75 (v:v) for elution,
furnished compound 8 h as a white solid. Yield: 67%, mp 114–116 ◦C.
1H NMR (DMSO‑d6) δ: 3.80 (s, 3H), 3.83 (s, 3H), 7.00 (dd, J = 9.0 and
2.4 Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H), 7.51 (d, J = 8.8 Hz, 2H), 7.60 (d, J
= 2.4 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H). MS (ESI): [M]+=348.8, [M +
2]+=350.7.
5.1.3.16. 3-Bromo-6-methoxybenzo[b]thiophene (9). Following general
procedure B, the crude residue purified by flash chromatography, using
Et2O:petroleum ether 0.2:9.8 (v:v) for elution, furnished compound 9 as
a clear oil. Yield: 65%. 1H NMR (DMSO‑d6) δ: 3.80 (s, 3H), 6.97 (dd, J =
8.8 and 2.4 Hz, 1H), 7.51 (s, 1H), 7.54 (d, J = 2.4 Hz, 1H), 7.66 (d, J =
8.8 Hz, 1H). MS (ESI): [M]+=242.0, [M + 2]+=244.1.
5.1.3.9. 3-Bromo-6-methoxy-2-(3-methoxyphenyl)benzo[b]thiophene
(8i). Following general procedure B, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.5:9.5 (v:v) for elution,
furnished compound 8i as a colorless oil. Yield: 94%. 1H NMR
(DMSO‑d6) δ: 3.81 (s, 3H), 3.82 (s, 3H), 6.98–7.05 (m, 3H), 7.06–7.19
(m, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.43 (t, J = 8.6 Hz, 1H), 7.61 (d, J =
2.4 Hz, 1H). MS (ESI): [M]+=349.3, [M + 2]+=351.4.
5.1.4. General procedure C for the preparation of compounds 3a-p
In a dry glass pressure vessel charged with Ar, a mixture of the
appropriate 3-bromo-6-methoxy-2-aryl[b]thiophene derivative 8a-o (1
mmol) or 3-bromo-6-methoxybenzo[b]thiophene 9 (243 mg, 1 mmol),
3,4,5-trimethoxyaniline (220 mg, 1.2 mmol, 1.2 equiv.), xanthphos (23
mg, 0.04 mmol, 4 mol%, 30 mg), sodium tert-butoxide (0.134 g, 1.40
mmol), and Pd2dba3 (18.2 mg, 0.02 mmol, 2 mol%) in dry toluene (5
mL) were added under Ar, and the mixture was heated with stirring at
100 ◦C for 18 h. After cooling to room temperature, EtOAc was added (5
mL), the mixture was filtered through celite under vacuum and the
filtrate diluted with EtOAc (10 mL) and water (5 mL). The aqueous
phase was separated and extracted with ethyl acetate (5 mL). The
combined organic phases were washed with brine (5 mL), dried over
Na2SO4 and concentrated under reduced pressure to give a residue,
which was purified by chromatography on silica gel.
5.1.3.10. 3-Bromo-2-(4-ethoxyphenyl)-6-methoxybenzo[b]thiophene
(8j). Following general procedure B, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.5:9.5 (v:v) for elution,
furnished compound 8j as a white solid. Yield: 95%, mp 91–93 ◦C. 1H
NMR (DMSO‑d6) δ: 1.37 (t, J = 6.8 Hz, 3H), 3.82 (s, 3H), 4.09 (q, J = 6.8
Hz, 2H), 6.97 (dd, J = 9.0 and 2.4 Hz, 1H), 7.06 (J = 9.0 Hz, 2H), 7.48 (s,
1H), 7.34 (dd, J = 8.8 and 2.0 Hz, 2H), 7.59 (d, J = 2.2 Hz, 1H). MS
(ESI): [M]+=363.3, [M + 2]+=365.4.
5.1.3.11. 3-Bromo-2-(3-ethoxyphenyl)-6-methoxybenzo[b]thiophene (8
k). Following general procedure A, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.25:9.75 (v:v) for elution,
furnished compound 8 k as a clear oil. Yield: 86%. 1H NMR (DMSO‑d6) δ:
1.35 (d, J = 6.8 Hz, 3H), 3.82 (s, 3H), 4.06 (q, J = 6.8 Hz, 2H), 6.95–6.99
(m, 4H), 7.37–7.43 (m, 2H), 7.61 (d, J = 9.0 Hz, 1H). MS (ESI):
[M]+=363.3, [M + 2]+=365.4.
5.1.4.1. 6-Methoxy-2-phenyl-N-(3,4,5-trimethoxyphenyl)benzo[b]thio-
phen-3-amine (3a). Following general procedure C, the crude residue
was purified by flash chromatography, using ethyl acetate:petroleum
ether 2.5:7.5 (v:v) as eluent, to furnish 3a as a yellow solid. Yield: 57%,
mp 112–114 ◦C. 1H NMR (DMSO‑d6) δ: 3.55 (s, 3H), 3.62 (s, 6H), 3.81 (s,
3H), 6.17 (s, 2H), 6.94 (dd, J = 8.8 and 2.4 Hz, 1H), 7.22–7.28 (m, 2H),
7.39 (d, J = 8.8 Hz, 1H), 7.47–7.49 (m, 4H), 8.05 (s, 1H). 13C NMR
(DMSO‑d6) δ: 55.44 (3C), 60.02, 92.12 (2C), 105.71, 113.91, 122.18,
126.02, 127.11, 128.57 (2C), 129.28 (2C), 130.42, 131.66, 134.07,
5.1.3.12. 3-Bromo-6-methoxy-2-(4-propoxyphenyl)benzo[b]thiophene (8
l). Following general procedure B, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.25:9.75 (v:v) for elution,
furnished compound 8 l as a clear oil. Yield: 75%. 1H NMR (DMSO‑d6) δ:
1.01 (t, J = 7.4 Hz, 3H), 1.72–1.76 (m, 2H), 3.82 (s, 3H), 3.98 (q, J = 7.2
Hz, 2H), 6.97 (dd, J = 8.8 and 2.4 Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H),
7.35–7.39 (m, 3H), 7.61 (d, J = 2.2 Hz, 1H). MS (ESI): [M]+=377.3, [M
+ 2]+=379.4.
135.83, 139.12, 142.26, 153.21 (2C), 156.73. MS (ESI): [M
+
1]+=422.0. Anal. calcd for C24H23NO4S: C, 68.39; H, 5.50; N, 3.32;
found: C, 68.04; H, 5.26; N, 3.21.
5.1.4.2. 2-(4-Fluorophenyl)-6-methoxy-N-(3,4,5-trimethoxyphenyl)benzo
[b]thiophen-3-amine (3b). Following general procedure C, the crude
residue was purified by flash chromatography, using ethyl acetate:pe-
troleum ether 2.5:7.5 (v:v) as eluent, to furnish 3b as a yellow solid.
Yield: 69%, mp 143–145 ◦C. 1H NMR (DMSO‑d6) δ: 3.53 (s, 3H), 3.62 (s,
6H), 3.79 (s, 3H), 6.15 (s, 2H), 6.93 (dd, J = 8.8 and 2.4 Hz, 1H),
7.26–7.30 (m, 2H), 7.35 (d, J = 9.0 Hz, 1H), 7.37–7.49 (m, 3H), 8.04 (s,
1H). 13C NMR (DMSO‑d6) δ: 55.53 (3C), 60.11, 92.32 (2C), 105.83,
114.02, 115.45, 115.66, 122.09, 124.79, 130.41, 130.62, 131.33,
131.40, 131.67, 135.78, 139.46, 142.17, 153.30 (2C), 156.83, 160.07
and 162.50 (1JC-F = 243 Hz). MS (ESI): [M + 1]+=440.4. Anal. calcd for
5.1.3.13. 3-Bromo-2-(4-butoxyphenyl)-6-methoxybenzo[b]thiophene (8
m). Following general procedure B, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.2:9.8 (v:v) for elution,
furnished compound 8 m as a clear oil. Yield: 86%. 1H NMR (DMSO‑d6)
δ: 0.96 (t, J = 7.4 Hz, 3H), 1.45–1.49 (m, 2H), 1.67–1.73 (m, 2H), 3.82
(s, 3H), 4.02 (t, J = 6.4 Hz, 2H), 7.02 (dd, J = 8.8 and 2.4 Hz, 1H), 7.07
(d, J = 8.8 Hz, 2H), 7.35–7.39 (m, 3H), 7.59 (d, J = 2.4 Hz, 1H). MS
(ESI): [M]+=391.3, [M + 2]+=393.3.
C
24H22FNO4S: C, 65.59; H, 5.05; N, 3.19; found: C, 65.38; H, 4.89; N,
5.1.3.14. 3-Bromo-6-methoxy-2-(4-(pentyloxy)phenyl)benzo[b]thiophene
(8n). Following general procedure B, the crude residue purified by flash
chromatography, using Et2O:petroleum ether 0.25:9.75 (v:v) for elution,
furnished compound 8n as a clear oil. Yield: 85%. 1H NMR (DMSO‑d6) δ:
0.89 (t, J = 7.2 Hz, 3H), 1.34–1.41 (m, 4H), 1.72–1.75 (m, 2H), 3.83 (s,
3H), 3.99 (t, J = 6.4 Hz, 2H), 6.96 (dd, J = 8.8 and 2.4 Hz, 1H), 7.06 (dd,
J = 6.4 and 2.0 Hz, 2H), 7.33–7.36 (m, 3H), 7.58 (d, J = 2.0 Hz, 1H),
7.69 (d, J = 9.2 Hz, 1H). MS (ESI): [M]+=405.3, [M + 2]+=407.3.
3.02.
5.1.4.3. 6-Methoxy-2-(p-tolyl)-N-(3,4,5-trimethoxyphenyl)benzo[b]thio-
phen-3-amine (3c). Following general procedure C, the crude residue
was purified by flash chromatography, using ethyl acetate:petroleum
ether 2.5:7.5 (v:v) as eluent, to furnish 3c as a yellow solid. Yield: 78%,
mp 76–77 ◦C. 1H NMR (DMSO‑d6)δ: 2.32 (s, 3H), 3.31 (s, 3H), 3.64 (s,
6H), 3.79 (s, 3H), 6.14 (s, 2H), 6.91 (dd, J = 8.8 and 2.4 Hz, 1H), 7.24 (d,
11

R. Romagnoli et al.
Bioorganic Chemistry 112 (2021) 104919
J = 7.6 Hz, 2H), 7.34–7.39 (m, 3H), 7.45 (d, J = 2.4 Hz, 1H), 7.97 (s,
1H). 13C NMR (DMSO‑d6) δ: 21.30, 55.97 (3C), 60.57, 92.54 (2C),
106.24, 114.36, 122.78, 126.70, 129.68 (4C), 130.89, 131.61, 132.27,
136.40, 136.87, 139.23, 142.88, 153.74 (2C), 157.23. MS (ESI): [M +
1]+=436.1. Anal. calcd for C25H25NO4S: C, 68.94; H, 5.79; N, 3.22;
found: C, 68.72; H, 5.59; N, 2.98.
petroleum ether 3:7 (v:v) as eluent, to furnish 3 h as a light brown solid.
Yield: 78%, mp 63–65 ◦C. 1H NMR (DMSO‑d6) δ: 3.55 (s, 3H), 3.63 (s,
6H), 3.78 (s, 3H), 3.81 (s, 3H), 6.15 (s, 2H), 6.93 (dd, J = 8.8 and 2.4 Hz,
1H), 7.02 (d, J = 6.4 Hz, 2H), 7.39–7.41 (m, 3H), 7.47 (d, J = 2.4 Hz,
1H), 7.96 (s, 1H). 13C NMR (DMSO‑d6) δ: 55.10, 55.52 (3C), 60.12,
92.03 (2C), 105.79, 113.91, 114.12 (2C), 122.40, 126.24, 130.41,
130.52 (2C), 131.95, 135.98, 138.40, 142.54, 153.32 (2C), 156.81,
158.41, 188.31. MS (ESI): [M + 1]+=452.1. Anal. calcd for C25H25NO5S:
C, 66.50; H, 5.58; N, 3.10; found: C, 66.36; H, 5.32; N, 2.89.
5.1.4.4. 6-Methoxy-2-(m-tolyl)-N-(3,4,5-trimethoxyphenyl)benzo[b]thio-
phen-3-amine (3d). Following general procedure C, the crude residue
was purified by flash chromatography, using ethyl acetate:petroleum
ether 2:8 (v:v) as eluent, to furnish 3d as a yellow solid. Yield: 59%, mp
58–60 ◦C. 1H NMR (DMSO‑d6) δ: 2.32 (s, 3H), 3.53 (s, 3H), 3.63 (s, 6H),
3.79 (s, 3H), 6.16 (s, 2H), 6.91 (dd, J = 8.8 and 2.4 Hz, 1H), 7.14 (d, J =
7.6 Hz, 1H), 7.22–7.27 (m, 1H), 7.31–7.34 (m, 2H), 7.36 (d, J = 8.8 Hz,
1H), 7.45 (d, J = 2.4 Hz, 1H). 7.99 (s, 1H). 13C NMR (DMSO‑d6) δ: 21.11,
55.51 (3C), 60.10, 92.37 (2C), 105.75, 113.93, 122.27, 125.83, 126.51,
127.84, 128.54, 129.89, 130.55, 131.89, 134.10, 135.74, 137.65,
139.18, 142.25, 153.25 (2C), 156.74. MS (ESI): [M + 1]+=436.4. Anal.
calcd for C25H25NO4S: C, 68.94; H, 5.79; N, 3.22; found: C, 68.76; H,
5.61; N, 3.01.
5.1.4.9. 6-Methoxy-2-(3-methoxyphenyl)-N-(3,4,5-trimethoxyphenyl)
benzo[b]thiophen-3-amine (3i). Following general procedure C, the
crude residue was purified by flash chromatography, using ethyl acetate:
petroleum ether 2.5:7.5 (v:v) as eluent, to furnish 3i as a yellow solid.
Yield: 71%, mp 58–60 ◦C. 1H NMR (DMSO‑d6) δ: 3.55 (s, 3H), 3.64 (s,
6H), 3.73 (s, 3H), 3.81 (s, 3H), 6.17 (s, 2H), 6.94 (t, J = 7.6 Hz, 1H),
6.98–7.05 (m, 2H), 7.01–7.06 (m, 1H), 7.36–7.38 (m, 1H), 7.43 (d, J =
8.6 Hz, 1H), 7.48 (d, J = 1.6 Hz, 1H), 8.04 (s, 1H). 13C NMR (DMSO‑d6)
δ: 54.87, 55.39, 55.44 (2C), 60.02, 92.19 (2C), 105.68, 112.59, 113.89,
114.89, 121.52, 122.27, 125.74, 129.57, 130.44, 131.64, 135.32,
135.75, 139.20, 142.27, 153.20 (2C), 156.70, 159.26. MS (ESI): [M +
1]+=452.1. Anal. calcd for C25H25NO5S: C, 66.50; H, 5.58; N, 3.10;
found: C, 66.40; H, 5.34; N, 2.93.
5.1.4.5. 2-(3,4-Dimethylphenyl)-6-methoxy-N-(3,4,5-trimethoxyphenyl)
benzo[b]thiophen-3-amine (3e). Following general procedure C, the
crude residue was purified by flash chromatography, using ethyl acetate:
petroleum ether 2:8 (v:v) as eluent, to furnish 3e as a yellow solid. Yield:
5.1.4.10. 6-Methoxy-2-(4-ethoxyphenyl)-N-(3,4,5-trimethoxyphenyl)
benzo[b]thiophen-3-amine (3j). Following general procedure C, the
crude residue was purified by flash chromatography, using ethyl acetate:
petroleum ether 2.5:7.5 (v:v) as eluent, to furnish 3j as a light brown
solid. Yield: 67%, mp 158–160 ◦C. 1H NMR (DMSO‑d6) δ: 1.34 (t, J = 6.8
Hz, 3H), 3.54 (s, 3H), 3.63 (s, 6H), 3.81 (s, 3H), 4.04 (q, J = 6.8 Hz, 2H),
6.15 (s, 2H), 6.93 (dd, J = 8.8 and 2.4 Hz, 1H), 7.00 (d, J = 8.8 Hz, 2H),
7.37–7.41 (m, 3H), 7.47 (d, J = 2.4 Hz, 1H), 7.96 (s, 1H). 13C NMR
(DMSO‑d6) δ: 14.61, 55.42 (3C), 60.02, 62.90, 91.93 (2C), 105.68,
109.63, 113.81, 114.44 (2C), 122.31, 125.99, 126.21, 130.40 (2C),
131.85, 135.88, 138.26, 142.44, 153.20 (2C), 156.71, 157.59. MS (ESI):
[M + 1]+=466.0. Anal. calcd for C26H27NO5S: C, 67.08; H, 5.85; N, 3.01;
found: C, 66.89; H, 5.69; N, 2.89.
1
◦
74%, mp 73–75 C. H NMR (DMSO‑d6) δ: 2.25 (s, 6H), 3.55 (s, 3H),
3.63 (s, 6H), 3.80 (s, 3H), 6.17 (s, 2H), 6.93 (dd, J = 9.0 and 2.0 Hz, 1H),
7.18–7.24 (m, 2H), 7.25 (s, 1H), 7.37 (d, J = 9.2 Hz, 1H), 7.46 (d, J =
2.2 Hz, 1H), 7.96 (s, 1H). 13C NMR (DMSO‑d6) δ: 19.06, 19.40, 55.41
(3C), 60.02, 92.14 (2C), 105.65, 113.76, 122.26, 126.06, 126.72,
129.64, 130.26 (2C), 131.49, 131.91, 135.08, 135.72, 136.19, 138.64,
142.30, 153.18 (2C), 156.64. MS (ESI): [M + 1]+=450.2. Anal. calcd for
C
26H27NO4S: C, 69.46; H, 6.05; N, 3.12; found: C, 69.27; H, 5.89; N,
2.97.
5.1.4.6. 6-Methoxy-2-(3-(trifluoromethyl)phenyl)-N-(3,4,5-trimethox-
yphenyl)benzo[b]thiophen-3-amine (3f). Following general procedure C,
the crude residue was purified by flash chromatography, using ethyl
acetate:petroleum ether 2.5:7.5 (v:v) as eluent, to furnish 3f as a yellow
solid. Yield: 73%, mp 63–64 ◦C. 1H NMR (DMSO‑d6) δ: 3.54 (s, 3H), 3.63
(s, 6H), 3.81 (s, 3H), 6.18 (s, 2H), 6.96 (dd, J = 8.8 and 2.0 Hz, 1H), 7.36
(d, J = 8.8 Hz, 1H), 7.50 (d, J = 2.0 Hz, 1H), 7.69–7.78 (m, 4H), 8.23 (s,
1H). 13C NMR (DMSO‑d6) δ: 55.42 (3C), 59.99, 92.74 (2C), 105.85,
114.12, 121.48, 122.87, 123.68, 125.62, 129.15, 129.47, 129.72,
130.77, 131.28, 133.37, 135.20, 135.42, 140.81, 141.45, 153.18 (2C),
156.73. MS (ESI): [M + 1]+=490.3. Anal. calcd for C25H22F3NO4S: C,
61.34; H, 4.53; N, 2.86; found: C, 61.11; H, 4.36; N, 2.72.
5.1.4.11. 6-Methoxy-2-(3-ethoxyphenyl)-N-(3,4,5-trimethoxyphenyl)
benzo[b]thiophen-3-amine (3 k). Following general procedure C, the
crude residue was purified by flash chromatography, using ethyl acetate:
petroleum ether 2.5:7.5 (v:v) as eluent, to furnish 3 k as a yellow solid.
Yield: 64%, mp 129–131 ◦C. 1H NMR (DMSO‑d6) δ: 1.29 (d, J = 6.8 Hz,
3H), 3.55 (s, 3H), 3.64 (s, 6H), 3.81 (s, 3H), 3.98 (q, J = 6.8 Hz, 2H),
6.18 (s, 2H), 6.84–7.01 (m, 4H), 7.34 (t, J = 6.8 Hz, 1H), 7.43–7.47 (m,
4H), 8.03 (s, 1H). 13C NMR (DMSO‑d6) δ: 14.52, 55.39, 55.45 (2C),
60.03, 62.82, 92.21 (2C), 105.68, 113.15, 113.89, 115.25, 121.39,
122.29, 125.83, 129.56, 130.45, 131.66, 135.30, 139.16, 141.25,
142.29, 153.22 (2C), 156.71, 158.54. MS (ESI): [M + 1]+=466.4. Anal.
calcd for C26H29NO5S: C, 67.08; H, 5.85; N, 3.01; found: C, 66.85; H,
5.72; N, 2.89.
5.1.4.7. 2-(4-Ethylphenyl)-6-methoxy-N-(3,4,5-trimethoxyphenyl)benzo
[b]thiophen-3-amine (3 g). Following general procedure C, the crude
residue was purified by flash chromatography, using ethyl acetate:pe-
troleum ether 2:8 (v:v) as eluent, to furnish 3 g as a yellow solid. Yield:
69%, mp 52–54 ◦C. 1H NMR (DMSO‑d6) δ: 1.21 (t, J = 7.6 Hz, 3H), 2.63
(q, J = 7.6 Hz, 2H), 3.55 (s, 3H), 3.64 (s, 6H), 3.81 (s, 3H), 6.18 (s, 2H),
6.93 (dd, J = 8.8 and 2.4 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H), 7.39–7.42
(m, 3H), 7.47 (d, J = 2.4 Hz, 1H), 8.01 (s, 1H). 13C NMR (DMSO‑d6) δ:
15.41, 27.86, 55.38, 55.43 (2C), 60.02, 92.07 (2C), 105.69, 113.81,
122.28, 126.17, 127.95 (2C), 129.18 (2C), 130.39, 131.34, 131.74,
135.86, 138.74, 142.39, 142.58, 153.22 (2C), 156.70. MS (ESI): [M +
1]+=450.4. Anal. calcd for C26H27NO4S: C, 69.46; H, 6.05; N, 3.12;
found: C, 69.28; H, 5.89; N, 3.01.
5.1.4.12. 6-Methoxy-2-(4-propoxyphenyl)-N-(3,4,5-trimethoxyphenyl)
benzo[b]thiophen-3-amine (3 l). Following general procedure C, the
crude residue was purified by flash chromatography, using ethyl acetate:
petroleum ether 2.5:7.5 (v:v) as eluent, to furnish 3 l as a yellow solid.
Yield: 66%, mp 71–73 ◦C. 1H NMR (DMSO‑d6) δ: 0.98 (t, J = 7.2 Hz, 3H),
1.74–1.80 (m, 2H), 3.55 (s, 3H), 3.63 (s, 6H), 3.81 (s, 3H), 3.95 (q, J =
7.2 Hz, 2H), 6.15 (s, 2H), 6.94 (dd, J = 8.4 and 2.4 Hz, 1H), 7.01 (d, J =
8.4 Hz, 2H), 7.37–7.41 (m, 3H), 7.47 (d, J = 2.4 Hz, 1H), 7.96 (s, 1H).
13C NMR (DMSO‑d6) δ: 10.89, 22.50, 55.96 (3C), 60.56, 69.35, 92.49
(2C), 106.23, 114.34, 115.03 (2C), 122.84, 126.53, 126.71, 130.94
(2C), 132.40, 136.41, 138.81, 142.97, 141.25, 153.74 (2C), 157.24,
158.28. MS (ESI): [M + 1]+=480.1. Anal. calcd for C27H29NO5S: C,
67.62; H, 6.09; N, 2.92; found: C, 67.45; H, 5.82; N, 2.77.
5.1.4.8. 6-Methoxy-2-(4-methoxyphenyl)-N-(3,4,5-trimethoxyphenyl)
benzo[b]thiophen-3-amine (3 h). Following general procedure C, the
crude residue was purified by flash chromatography, using ethyl acetate:
12

R. Romagnoli et al.
Bioorganic Chemistry 112 (2021) 104919
5.1.4.13. 2-(4-Butoxyphenyl)-6-methoxy-N-(3,4,5-trimethoxyphenyl)
benzo[b]thiophen-3-amine (3 m). Following general procedure C, the
crude residue was purified by flash chromatography, using ethyl acetate:
petroleum ether 2.5:7.5 (v:v) as eluent, to furnish 3 m as a yellow solid.
Yield: 56%, mp 41–42 ◦C. 1H NMR (DMSO‑d6) δ: 0.90 (t, J = 7.4 Hz, 3H),
1.41–1.44 (m, 2H), 1.67–1.70 (m, 2H), 3.52 (s, 3H), 3.61 (s, 6H), 3.79 (s,
3H), 3.96 (t, J = 6.4 Hz, 2H), 6.13 (s, 2H), 6.91 (dd, J = 8.8 and 2.4 Hz,
1H), 6.98 (d, J = 8.8 Hz, 2H), 7.35–7.39 (m, 3H), 7.44 (d, J = 2.4 Hz,
1H), 7.94 (s, 1H). 13C NMR (DMSO‑d6) δ: 13.69, 18.76, 30.76, 55.51
(3C), 60.10, 67.09, 92.03 (2C), 105.77, 113.88, 114.56 (2C), 122.39,
126.07, 126.26, 130.48 (3C), 131.93, 135.96, 138.35, 142.51, 153.29
(2C), 156.79, 157.84. MS (ESI): [M + 1]+=494.1. Anal. calcd for
incubated at 37 ◦C in a humidified 5% CO2 incubator for 18 h prior to the
experiments. After medium removal, 100 μL of the drug solution, dis-
solved in complete medium at different concentrations, was added to
each well and incubated at 37 ◦C for 72 h. Cell viability was assayed by
the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) test as previously described [27]. The IC50 was defined as the
compound concentration required to inhibit cell proliferation by 50%.
5.2.2. Effects on tubulin polymerization and colchicine binding to tubulin
Bovine brain tubulin was purified as described previously [40]. To
evaluate the effects of the compounds on tubulin assembly in vitro [41],
varying concentrations were preincubated with 10 □M tubulin in 0.8 M
monosodium glutamate (pH 6.6 with HCl in 2 M stock solution) at 30˚C
and then cooled to 0˚C. After addition of GTP, the mixtures were
transferred to 0 ◦C cuvettes in a recording spectrophotometer and
C
28H31O5S: C, 68.13; H, 6.33; N, 2.84; found: C, 68.01; H, 6.21; N, 2.58.
5.1.4.14. 6-Methoxy-2-(4-(pentyloxy)phenyl)-N-(3,4,5-trimethox-
yphenyl)benzo[b]thiophen-3-amine (3n). Following general procedure C,
the crude residue was purified by flash chromatography, using ethyl
acetate:petroleum ether 2:8 (v:v) as eluent, to furnish 3n as a light
brown solid. Yield: 56%, mp 117–119 ◦C. 1H NMR (DMSO‑d6) δ: 0.86 (t,
J = 7.2 Hz, 3H), 1.33–1.37 (m, 4H), 1.70–1.74 (m, 2H), 3.52 (s, 3H),
3.61 (s, 6H), 3.79 (s, 3H), 3.95 (t, J = 6.4 Hz, 2H), 6.13 (s, 2H), 6.91 (dd,
J = 8.8 and 2.4 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 7.35–7.39 (m, 3H),
7.45 (d, J = 2.4 Hz, 1H), 7.94 (s, 1H). 13C NMR (DMSO‑d6) δ: 13.94,
21.89, 27.74, 28.40, 55.51 (3C), 60.11, 67.39, 92.03 (2C), 105.77,
113.89, 114.56 (2C), 122.39, 126.08, 126.26, 130.48 (2C), 131.94,
135.96, 138.35, 142.51, 153.29 (2C), 156.79, 156.80, 157.84. MS (ESI):
[M + 1]+=508.2. Anal. calcd for C29H33NO5S: C, 68.61; H, 6.55; N, 2.76;
found: C, 68.46; H, 6.35; N, 2.58.
◦
warmed to 30 C, and the assembly of tubulin was observed turbidi-
metrically. The IC50 value was defined as the compound concentration
that inhibited the extent of assembly by 50% after a 20 min incubation.
Inhibition of colchicine binding to tubulin was measured as described
before [42,43], except that the reaction mixtures contained 0.5
μM
tubulin, 5
μ
M [3H]colchicine and 5
μM test compound. Only one DEAE-
cellulose filter was used per sample, and filtration was by gravity.
5.2.3. Molecular modeling.
All molecular docking studies were performed on a Viglen Genie
Intel®CoreTM i7-3770 vPro CPU@ 3.40 GHz × 8 running Ubuntu 18.04.
Molecular Operating Environment (MOE) 2019.10 [44] and Maestro
¨
(Schrodinger Release 2020–3) [45] were used as molecular modeling
software. The tubulin structure was downloaded from the PDB data bank
(http://www.rcsb.org/; PDB code 4O2B). The protein was pre-processed
5.1.4.15. 2-(4-Isopropoxyphenyl)-6-methoxy-N-(3,4,5-trimethoxyphenyl)
benzo[b]thiophen-3-amine (3o). Following general procedure C, the
crude residue was purified by flash chromatography, using ethyl acetate:
petroleum ether 2.5:7.5 (v:v) as eluent, to furnish 3o as a yellow solid.
Yield: 73%, mp 64–65 ◦C. 1H NMR (DMSO‑d6) δ: 1.27 (d, J = 6.0 Hz,
6H), 3.55 (s, 3H), 3.63 (s, 6H), 3.81 (s, 3H), 4.62–4.65 (m, 1H), 6.16 (s,
2H), 6.93 (dd, J = 8.8 and 2.4 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H),
7.36–7.42 (m, 3H), 7.46 (d, J = 2.4 Hz, 1H), 7.97 (s, 1H). 13C NMR
(DMSO‑d6) δ: 21.79 (2C), 55.37, 55.42 (2C), 60.01, 68.94, 91.99 (2C),
105.62, 113.78, 115.44 (2C), 122.31, 125.82, 126.05, 130.42 (3C),
131.84, 135.85, 138.26, 142.42, 153.19 (2C), 156.51, 156.68. MS (ESI):
[M + 1]+=480.2. Anal. calcd for C27H29NO5S: C, 67.62; H, 6.09; N, 2.92;
found: C, 67.39; H, 5.89; N, 2.71.
¨
using the Schrodinger Protein Preparation Wizard by assigning bond
orders, adding hydrogens and performing a restrained energy minimi-
zation of the added hydrogens using the OPLS_2005 force field. Ligand
structures were built with MOE and then prepared using the Maestro
LigPrep tool by energy minimizing the structures (OPLS_2005 force
field), generating possible ionization states at pH 7 ± 2, and generating
tautomers and low-energy ring conformers. After isolating a tubulin
dimer structure, a 12 Å docking grid (inner-box 10 Å and outer-box 22
Å) was prepared using as centroid the co-crystallized colchicine. Mo-
lecular docking studies were performed using Glide SP precision keeping
the default parameters and setting 5 as the number of output poses per
input ligand to include in the solution. The output database was saved as
a mol2 file. The docking results were visually inspected for the quality of
binding to the active site.
5.1.4.16. 6-Methoxy-N-(3,4,5-trimethoxyphenyl)benzo[b]thiophen-3-
amine (3p). Following general procedure C, the crude residue was pu-
rified by flash chromatography, using ethyl acetate:petroleum ether 3:7
(v:v) as eluent, to furnish 3p as a red solid. Yield: 62%, mp 68–70 ◦C. 1H
NMR (DMSO‑d6) δ: 3.54 (s, 3H), 3.80 (s, 6H), 3.82 (s, 3H), 6.20 (s, 2H),
6.98 (dd, J = 8.8 and 2.4 Hz, 1H), 7.51 (s, 1H), 7.54 (d, J = 2.4 Hz, 1H),
7.66 (d, J = 8.8 Hz, 1H), 8.75 (s, 1H). 13C NMR (DMSO‑d6) δ: 51.83,
55.88 (3C), 94.36 (2C), 114.76, 118.91, 123.76, 125.22, 126.93,
128.46, 131.20, 134.43, 140.25, 153.52 (2C), 162.57. MS (ESI): [M +
1]+=346.1. Anal. calcd for C18H19NO4S: C, 62.59; H, 5.54; N, 4.06;
found: C, 62.45; H, 5.38; N, 3.87.
5.2.4. Cell death and mitochondrial parameters following 3a and 3b
treatments
In all experimental assays, 841 CoN, Caco2 and HCT-116 cells were
plated onto 35-mm-well plates and treated for 48 h with vehicle
(DMSO), 60 nM 3a or 400 nM 3b. Where transfection was required, it
was performed before treatment with compounds.
5.2.5. Immunoblot assay
For immunoblotting, cells were lysed in RIPA buffer, protein was
quantified by the Lowry method, and 10 µg of protein was loaded into
each slot of a 4–20% precast gel. After electrophoretic separation, pro-
teins were transferred onto nitrocellulose membranes that were incu-
bated overnight with the following primary antibodies: actin (as loading
marker), cleaved RIP, cleaved caspase 3 and cleaved PARP (for cell
death), cyclins A, F, B1 and D1, p27, pHH3 (for cell cycle), MFN1/2 and
OPA1 (for mitochondrial dynamics). Protein expression was assessed by
specific HRP-labeled secondary antibodies, followed by detection by
chemiluminescence using a ChemiDoc™ Touch Gel Imaging System.
5.2. Biological assays and computational studies
5.2.1. Initial antiproliferative assays
Human cervix carcinoma (HeLa) and human colorectal adenocarci-
noma (HT-29) cells were grown in DMEM medium (Gibco, Milano,
Italy). This medium was supplemented with 115 units/mL of penicillin G
(Gibco, Milano, Italy), 115 μg/mL streptomycin (Invitrogen, Milano,
Italy) and 10% fetal bovine serum (Invitrogen, Milano, Italy). Individual
wells of a 96-well tissue culture microtiter plate were inoculated with
5.2.6. Annexin-V assay
L of complete medium containing 8x103 cells. The plates were
Cells were gently harvested, processed with buffers, and incubated
100
μ
13

R. Romagnoli et al.
Bioorganic Chemistry 112 (2021) 104919
with annexin V-Cy3 solution according to the manufacturer’s protocol
(Biovision, Milpitas, CA, USA). The green fluorescence signal was
quantified under all conditions on a Tali image-based cytometer
(Invitrogen).
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The authors declare no conflict of interest. This research was sup-
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vision of Cancer Treatment and Diagnosis of the National Cancer
Institute, which includes federal funds under Contract No.
HHSN261200800001E. The content of this publication does not neces-
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