Synthesis, antiproliferative activity, and in silico insights of new 3-benzoylamino-benzo[ b ]thiophene derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.12.002

A new series of 3-benzoylamino-5-imidazol-5-yl-benzo[b]thiophenes and the parent amino derivatives were synthesized and screened as antitumor agents. All tested compounds showed concentration-dependent antiproliferative activity profile against HeLa cell

Full text of DOI:10.1016/j.ejmech.2014.12.002

European Journal of Medicinal Chemistry 90 (2015) 537e546
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiproliferative activity, and in silico insights of new
-benzoylamino-benzo[b]thiophene derivatives
3
a
a
a
a
Annamaria Martorana , Carla Gentile , Ugo Perricone , Antonio Palumbo Piccionello ,
a
a
a
b
Roberta Bartolotta , Alessio Terenzi , Andrea Pace , Francesco Mingoia ,
a
a, *
Anna Maria Almerico , Antonino Lauria
a
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche “STEBICEF” e Universit aꢀ di Palermo, Via Archirafi 32, 90123 Palermo, Italy
Istituto per lo Studio dei Materiali Nanostrutturati, (ISMN), Consiglio nazionale delle Ricerche (CNR), Via U. La Malfa 153, 90146 Palermo, Italy
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 October 2014
Received in revised form
A new series of 3-benzoylamino-5-imidazol-5-yl-benzo[b]thiophenes and the parent amino derivatives
were synthesized and screened as antitumor agents. All tested compounds showed concentration-
dependent antiproliferative activity profile against HeLa cell line, exhibiting GI50 values in the low
micromolar range. The most active compounds were tested in cell cycle perturbation experiments. A
rapid accumulation of cells in the G2/M phase, with a concomitant reduction of cells in both the S and
G0/G1 phases, was observed, suggesting that cell exposure to selected derivatives produces mitotic
failure. To rationalize the biological results, the 3-benzoylamino-benzo[b]thiophenes were analyzed
through the in silico VLAK protocol. Compounds presenting the 3,4,5-trimethoxy-benzoyl moiety were in
silico classified as potential antimitotic agents or topoisomerase II inhibitors, in good agreement with the
biological studies.
29 November 2014
Accepted 1 December 2014
Available online 2 December 2014
Keywords:
3-Benzoylamino-5-imidazol-4-yl-benzo[b]
thiophenes
Antitumor agents
HeLa cell line
© 2014 Elsevier Masson SAS. All rights reserved.
G2/M phase
VLAK protocol
Mechanism of action
1. Introduction
antiproliferative activities against a wide range of human cancer
cell lines (Fig. 1).
Heterocycles are heavily involved in life processes. DNA, RNA,
proteins, and coenzymes are classical examples of fundamental
biological macromolecules structured by heterocyclic moieties. The
biomolecular standpoint of drug action confirms the broad
biocompatibility and the versatile aptitude of heterocycles to mimic
endogenous molecules and consequently to interact with biological
entities [1]. These features make heterocyclic chemistry one of the
major pivots for the development of new drugs, justifying the
intense attention of the scientific community on this field.
Since long time, our research work has been focused on the
design, the synthesis, and the biological evaluation of novel het-
erocyclic ring systems able to halt tumor cell proliferation [2e5].
Several of our tested molecules exhibited outstanding
In this regard, the indolo[3,2-e]-1,2,3-triazolo[1,5-a]pyrimidine
derivatives of type 1, designed as DNA-interacting drugs, showed a
relevant antiproliferative effect on the renal and CNS human tumor
cell lines [6]. In an analogous fashion, the isoindolo[2,1-a]qui-
noxalines 2 emerged as high promising antitumor scaffold, with
GI50 values in the low micromolar to nanomolar concentrations.
After treatment with 2, the analysis of the cell cycle demonstrated
an arrest in G2/M phase, in addition to cell apoptosis, displayed by
mitochondrial depolarization, and activation of caspase-3, and
caspase-9. Moreover, isoindole-quinoxaline derivatives inhibited
tubulin polymerization similarly to colchicine and vinblastine in a
concentration-dependent manner [7]. Recently, the in silico
approach, performed through the Virtual Lock-and-Key (VLAK)
protocol [8], allowed us to select new annelated thieno-triazolo and
triazine series of type 3, 4, and 5 as new antitumor agents. These
compounds, featuring by a sulfur atom in their scaffold and func-
tionalized with proper side chains, demonstrated excellent in vitro
and in vivo antiproliferative activity, exhibiting very low toxicity
and high potency [9e11]. On the basis of these data, keeping our
attention on the promising thieno heterocyclic ring systems, we
Abbreviations: TMB, 3,4,5-trimethoxy-benzoyl; VLAK, Virtual Lock-and-Key;
DMSO, dimethyl sulfoxide; MA, mechanism of action; NCI, National Cancer Insti-
tute; ACAM, Anti-cancer Agent Mechanism.
*
Corresponding author.
E-mail address: antonino.lauria@unipa.it (A. Lauria).
http://dx.doi.org/10.1016/j.ejmech.2014.12.002
223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
0

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A. Martorana et al. / European Journal of Medicinal Chemistry 90 (2015) 537e546
Fig. 1. Heterocyclic ring system series with outstanding antiproliferative activities.
decided to investigate a set of new benzo[b]thiophene derivatives
of type 6 as anticancer compounds (Fig. 2). Several published works
assigned to this scaffold a peculiar biological activity, suggesting
the proposed molecules as potential bioisosteres of major micro-
tubules assembly inhibitors. In fact, in the past few years, an ever
increasing number of thieno derivatives as potent tubulin poly-
merization inhibitors have been proposed [12e14]. In this contest,
Romagnoli and co-workers reported an interesting series of 2-
(
alkoxycarbonyl)-3-anilinobenzo[b]thiophenes of type 7. These
compounds, acting on the colchicine binding site of tubulin,
showed an excellent antiproliferative activity, interfering with
microtubules dynamics during cancer cell division processes
Fig. 2. Chemical structures of ethyl 3-benzoylamino-5-[(1H-imidazol-4-yl-methyl)-
amino]-benzo[b]thiophene-2-carboxylates (6), 2-(alkoxycarbonyl)-3-anilino-benzo[b]
thiophenes and thieno[2,3-b]pyridines (7).
[15,16].
2
. Results and discussion
2.1. Chemistry
2.2. Biology
As shown in Scheme 1, the general synthetic route for the
2.2.1. Antiproliferative activity
preparation of 3-benzoylamino-benzo[b]thiophene derivatives
The antiproliferative activity of 3-benzoylamino-5-imidazol-
4yl-benzo[b]thiophene derivatives 6ael and their corresponding
precursors 13aef was evaluated on HeLa tumor cell line for 48 h
using MTT based cell viability assay. All tested compounds showed
concentration-dependent antiproliferative activity, but did not
affect the cell membrane integrity in the concentration range
6
2
ael involved five steps, starting from the commercially available
-fluorobenzonitrile (8). Pale yellow needles of 2-fluoro-5-
nitrobenzonitrile (9) were obtained, in quantitative yield, by
nitration of 8, using a mixture of concentrated nitric and sulfuric
acids, under inert atmosphere [17]. The presence of both cyano and
nitro substituents on substrate 9 enhanced the reactivity of the
ortho-fluorine atom, which readily underwent nucleophilic
displacement by ethyl thioglycolate, in the presence of NaH (60% in
oil dispersion) and dimethyl sulfoxide (DMSO). Subsequent intra-
molecular cyclization in situ led to the formation of the central core,
and the isolation of the key intermediate 3-amino-benzo[b]thio-
phene 10.
To introduce the benzoyl moiety, amino derivative 10 was
treated with substituted benzoyl chlorides 11aef. 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. Reduction of nitro group on compounds
1e20 mM, as determined preliminary by the Trypan Blue exclusion
method. For an easier analysis of the biological results, the tested
molecules have been divided into three main subgroups: 5-amino-
benzo[b]thiophenes 13aef, imidazole side-chain compounds
6aef, and methyl-imidazole side-chain derivatives 6gel (Table 1).
The majority of our benzo[b]thiophene compounds proved a good
growth inhibition effect, exhibiting GI50 values in the low
micromolar range. As a general trend, 5-amino-benzo[b]thio-
phenes 13aef showed lower activity than the corresponding
compounds with the imidazole side-chain 6. Only when the
3
benzoyl moiety was functionalized with 4-CF , the activity of the
amino intermediate 13e was more potent than imidazole de-
rivatives 6e and 6k. The presence of the methyl moiety on the
imidazole ring resulted in a significant increase of activity for the
3,4,5-trimethoxy (6d vs 6j) and the 3-chloro-4-fluoro-benzoyla-
mino-benzo[b]thiophene derivatives (6f vs 6l), while it was irrel-
evant in the other cases. Relatively to the influence of the
substituents on the benzoyl moiety there is no clear difference on
activity between electron-withdrawing and electron-releasing
substituents. Anyway, 3,4,5-trimethoxy benzoyl (TMB) de-
rivatives 13d, 6d, and 6j stand out as the most active of each
subgroup.
12aef was realized through hydrogenation with 10% Pd/C in
ethanol. After removal of the catalyst, amino derivatives 13aef
were easily isolated as pure needles, in good yields. The last syn-
thetic step provided the imidazole moiety insertion via a reductive
amination in the presence of imidazole-4-carbaldehydes 14a,b.
The reaction of the amino group with the aldehydes led to the
formation of the corresponding imines, which, by mean of sodium
cyanoborohydride addition, provided to a tightly selective reduc-
tion affording ethyl 3-benzoylamino-5-[(1H-imidazol-4-yl-
methyl)-amino]-benzo[b]thiophene-2-carboxylates 6ael.

A. Martorana et al. / European Journal of Medicinal Chemistry 90 (2015) 537e546
539
Scheme 1. Synthesis of ethyl 3-benzoylamino-5-[(1H-imidazol-4-yl-methyl)-amino]-benzo[b]thiophene-2-carboxylates 6ael. Reagents and conditions: (i): HNO
3
:H
2
SO
4
(1:1), N
2
,
ꢁ
0
2 3
C, 2 h; (ii): ethyl thioglycolate/NaH 60%, anhydrous DMSO; (iii): pyridine, rt, 12 h; (iv): 10% Pd/C, H , ethanol, rt 2 h; (v): NaCNBH , ethanol/AcOH, 2 h.
2
.2.2. Cell cycle analysis
The derivatives 13d, 6j, and 6l, which showed the highest
VLAK protocol [18]. Mostly developed in the last years, as well as
the recent protocol BIOTA [19], this protocol was employed in the
design and optimization of lead compounds as anticancer drugs.
The interesting results, concerning a wide set of heterocyclic de-
rivatives, confirmed this in silico methodology as powerful
approach in prediction capability of biological activities [9,11,20].
The VLAK protocol is focused on the search of molecular de-
scriptors, which could be considered as the “pins of the lock”. The
lock represents the biological target, or a specific mechanism of
action (MA), which need a key (the molecule) to be tuned [20].
The first step of the VLAK protocol is the conversion of the
biological targets, or the MAs, in “lock models” in which the keys
(designed ligands) fit to modulate the biological activity [18]. In this
study, the VLAK protocol was applied to predict the MA of 3-
benzoylamino-benzo[b]thiophene derivatives by using the Na-
tional Cancer Institute (NCI) Anti-cancer Agent Mechanism (ACAM)
database as training set. Drug screening data are available for each
structure as measurement of their growth inhibition ability over a
panel of about 60 human tumor cell lines, and all tested molecules
are explicitly designed as training set for neural network and
multivariate analysis [21e23]. In particular, this database is
constituted by 114 antitumor drugs ranked according to their MA
(supporting information, Table 1) that can be considered as the
locks in this protocol [24].
Thus, for each class of drugs (Alkylating Agents, Antimitotic
Agents, Topoisomerase I Inhibitors, Topoisomerase II Inhibitors,
RNA/DNA Antimetabolites, and DNA Antimetabolites), a “lock
model” was generated starting from available known compounds.
A set of 2D and 3D molecular descriptors was selected in order to
best differentiate ACAM drugs database by MA. Once defined the
lock model for each MA, compounds have been classified in terms
of structure affinity to lock models (see computational details for
protocol specifications). In Table 2 the percentages of affinity (A%)
for the screened derivatives are reported.
antiproliferative effect, were tested in cell cycle perturbation ex-
periments to evaluate their potential influence on cell-cycle dis-
tribution. The flow cytometric analysis was performed after 24 and
4
8 h of incubation in order to detect the shifts in cell cycle distri-
bution before a significant amount of cells underwent apoptosis.
The working concentrations were fixed taking into account the
GI50 values measured at 48 h. The histograms show the percentage
of cells in the respective cell cycle phase (G1, S, and G2/M), along
with the percentage of cells in the sub-G0/G1 (apoptotic cells)
obtained by flow cytometry (Fig. 3). Untreated HeLa cells revealed a
normal diploid distribution, presenting fast proliferation charac-
teristics, with S þ G2/M phase cells accounting for about 40% of the
total cells (ctrl). After cell exposure to selected 3-benzoylamino-
benzo[b]thiophene derivatives 13d, 6j, and 6l, a rapid accumulation
of cells in the G2/M phase, with a concomitant reduction of cells in
both the S and G0/G1 phases, was observed. These changes
occurred in a concentration-dependent manner. In fact, while in
the experimental conditions a treatment with 1ꢀGI50 produced
only slight variations, a concentration of about 2ꢀGI50 induced
significant effects on cell cycle distribution. The time-dependent
tendency of benzo[b]thiophenes effects was confirmed, after 48 h
of treatment, by the deep alterations in the cell cycle distribution
profile, with significant reduction of the cells number in all phases,
and by the simultaneous increase of the cells population in subG0/
G1, which is indicative of apoptotic cells (data not shown).
2
.3. In silico insights
In attempt to rationalize biological results of selected hits on
cell-cycle distribution and considering the structural features of the
proposed derivatives of type 6 and 13, similar to known antimitotic
agents, the benzothiophenes were analyzed through the in silico

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A. Martorana et al. / European Journal of Medicinal Chemistry 90 (2015) 537e546
Table 1
Cytotoxicity at 48 h of 3-benzoylamino-benzo[b]thiophene derivatives 13aef, 6aef, and 6gel against HeLa cell line expressed as GI50 values [GI50 ± SE (
m
M)].
1
3aef
6aef
a 17.89 ± 2.03
b 12.92 ± 0.94
c 11.72 ± 1.17
d 6.79 ± 0.33
e 16.84 ± 0.98
f 10.65 ± 1.32
6gel
g 23.32 ± 2.91
h 8.22 ± 0.75
i 11.42 ± 1.03
j 2.23 ± 0.04
k 12.82 ± 1.09
l 2.91 ± 0.24
R
R
R
R
R
R
1
1
1
1
1
1
]R
]R
]R
]R
]R
2
3
3
2
3
]R
]H; R
]H; R
]R
]H; R
3
]H
a >50
b >50
2
]OCH
]CH
3
2
3
c 24.44 ± 2.37
d 4.66 ± 0.31
e 6.68 ± 0.57
f 12.29 ± 1.69
3 3
]OCH
]CF
2 3
]Cl; R
2 3
]F; R ]H
Fig. 3. Effects of compounds 13d, 6j, and 6l on the cell cycle distribution of HeLa cells following 24 h treatment. The cells were cultured without any compound (ctr) or with
compound used at the indicated concentrations. Cell cycle distribution was analyzed by the standard propidium iodide procedure as described in Methods. The histograms
represent the percentages of cells in the respective cell cycle phase (G0/G1, S, and G2/M), along with the percentage of cells in the subG1 (dead cells) obtained by flow cytometry.
Results are 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.
Analyzing the results for compounds that showed the most
significant biological effect (13d, 6j, and 6l) it is noteworthy that
TMB derivatives 13d, 6j represent the most significant compounds
in terms of affinity for the class of antimitotic agents (B), showing
good values of A%. The structural features of these compounds,
similar to the well-known antimitotic agents such as colchicine and
combretastatin A-4, could justify these results (Fig. 4).
3. Conclusions
In this work, the synthesis and the biological evaluation of the
new benzo[b]thiophene series of type 6 and the related amino
precursors 13aef are reported. Generally, all tested compounds
showed a good antiproliferative effect with GI50 values in a low
micromolar range. Antiproliferative screening on HeLa tumor cell
line underlined a significant growth inhibition effect of final
imidazole-side chain compounds 6ael. Methyl insertion on the
imidazole ring resulted in a notable increase of activity for the TMB
(6d vs 6j) and the 3-chloro-4-fluoro-benzoylamino-benzo[b]thio-
phene derivatives (6e vs 6k), while it was irrelevant in the other
cases.
No differences were observed in the biological activity by
changing the substituents on the benzoyl moiety (both electron-
withdrawing and electron-releasing substituents), and the TMB
derivatives 13d, 6d, and 6j resulted the most active of each sub-
group. To evaluate their potential influence on cell-cycle distribu-
tion, derivatives 13d, 6j, and 6l were also tested in cell cycle
perturbation experiments. After cell exposure to benzo[b]thio-
phene derivatives, a rapid accumulation of cells in the G2/M phase,
with a concomitant reduction of cells in both the S and G0/G1
phases, was observed.
Molecule 6l did not show high affinity percentage towards the
antimitotic mechanism of action group (42.98), maybe because of
the structural modifications involving the benzoyl moiety. Thus, in
terms of structural analogies with known antimitotic compounds,
these results highlight the importance of the TMB moiety and
underline that the presence of the imidazole ring as substituent,
essential for the biological activity, does not affect the structural
analogy with known antimitotic agents. The difference in the af-
finity toward antimitotic agents for compound 6l could be justified
by the different functionalization of the benzoyl moiety rather
than the presence of the methyl group on the imidazole ring.
Another important aspect to be underlined is the good affinity
percentage for topoisomerase II inhibitors showed by 3-
benzoylamino-benzo[b]thiophene derivatives 6b,d,h,j. It is worth
noting that these findings are in agreement with the biological
insights. In particular, the in silico studies make possible to hy-
pothesize that observed arrest in the G2/M phase, after cell
exposure to selected derivatives, involving mitotic failure can be
associated, with both an antimitotic mechanism by interference
with mitotic spindle formation and the DNA-Topoisomerase-II
inhibition.
To rationalize biological results of selected hits on cell-cycle
distribution, considering the structural features of the proposed
derivatives of type 6 and 13, similar to known antimitotic agents,
the benzothiophenes were analyzed through the in silico VLAK
protocol. Compounds presenting the TMB moiety were classified as

A. Martorana et al. / European Journal of Medicinal Chemistry 90 (2015) 537e546
541
Table 2
Percentage of affinity (A%) calculated for the tested compounds.
Compound Alkylating agents Antimitotic agents DNA anti metabolites Topoisomerase I inhibitors Topoisomerase II inhibitors RNA-DNA anti metabolites
1
1
1
1
1
1
6
6
6
6
6
6
6
6
6
6
6
3a
3b
3c
3d
3e
3f
b
c
d
e
f
g
h
i
j
k
66.27
64.59
66.37
46.27
53.87
60.85
41.37
43.16
35.25
40.22
42.25
44.48
41.62
43.40
35.20
41.46
42.23
44.94
53.90
47.50
60.57
40.77
40.22
55.29
48.52
56.39
38.92
41.95
45.58
54.88
49.57
56.74
42.30
42.98
41.35
38.99
38.31
23.35
33.44
43.58
31.03
29.74
22.62
31.11
34.67
30.19
28.07
26.70
20.07
28.77
32.02
38.57
53.55
45.36
56.25
43.74
45.92
52.66
48.83
33.77
38.89
51.70
46.60
50.26
47.40
31.21
41.64
53.42
26.95
36.60
29.59
56.32
34.65
30.79
60.69
52.80
62.67
45.41
46.64
47.89
61.26
54.34
61.32
44.37
48.74
46.99
50.29
47.35
45.25
57.40
54.95
42.63
40.57
35.61
49.06
48.70
40.24
37.03
38.56
33.64
44.63
44.10
l
In bold are reported the highest A% values for the most active compounds (13d, 6d, and 6j) as antimitotic and topoisomerase II inhibitors classes.
Fig. 4. TMB derivatives (13d and 6j) with good percentage of affinity (A%) and well-known antimitotic agents (Conchicine and Combretastatin A-4).
potential antimitotic agents, showing high affinity percentage for
this mechanism of action class. The presence of the imidazole ring
does not seem to affect the mechanism of action as previously
observed by biological assays. This is also underlined by the pres-
ence of several imidazole derivatives as the best ranked antimitotic
agents in Table 2. Another important aspect to be underlined is the
good percentage of affinity for topoisomerase II inhibitors showed
by some other 3-benzoylamino-benzo[b]thiophene derivatives as
screened in biological assays was determined to be >95% by HPLC/
MS analysis. Mass spectra were performed using a GC and MS
Shimadzu QP5050 with EI (75 ev). Microanalyses were in agree-
ment with theoretical values ±0.4%. Thin layer chromatography
was performed on precoated (0.25 mm) silica gel GF254 plates,
compounds were detected with 254 nm UV lamp. Column chro-
matography was performed with Merck silica gel ASTM (230 and
400 mesh), or with a Biotage FLASH40i chromatography module
(prepacked cartridge system). 2-fluorobenzonitrile (8) is commer-
cially available. 2-fluoro-5-nitro-benzonitrile (9) was obtained
following the procedure reported in literature [17].
6
b,d,h,j. These findings could be in accordance with biological as-
says where a possible antimitotic mechanism or a potential inhi-
bition of the topoisomerase II enzyme could justify the cell cycle
arrest at G2/M checkpoint.
4.1.1. Synthesis of ethyl 3-amino-5-nitro-benzo[b]thiophene-2-
4
. Experimental
carboxylate (10)
According to the literature procedure [9], ethyl thioglycolate
(1.2 mL, 11 mmol) was added to a stirred mixture of NaH 60%
dispersion in mineral oil (0.36 g, 15 mmol) in dry DMSO (10 mL).
After stirring at room temperature for 20 min, a solution of 2-
fluoro-5-nitrobenzonitrile (9) (1.66 g, 10 mmol) in dry DMSO
(20 mL) was added dropwise. The reaction was stirred at room
temperature for further 3 h and then was poured onto stirred
water/ice. The precipitate was collected by filtration and dried to
4
.1. Chemistry
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 spec-
trophotometer. H NMR, C NMR spectra were recorded, at 200 and
0.3 MHz respectively, in CDCl or DMSO-d solution, using a
1
13
ꢁ
give orange-red needles of 10, 2.48 g. Yield 93%. Mp 207e208 C. IR
ꢂ1 1
5
3
6
n
max: 3491, 3367 (NH
3H, J ¼ 8.0 Hz, CH
exchange with D O, NH
J ¼ 2.0, 8.8 Hz, H-6), 8.59 (d, 1H, J ¼ 2.0 Hz, H-4); C NMR (DMSO-
: 14.3 (q), 60.2 (t), 96.9 (s), 119.4 (d), 122.0 (d), 124.3 (d), 131.4
(s), 144.5 (s), 144.9 (s), 149.7 (s), 163.9 (s). Anal. Calcd. (%) for
2
), 1657 (CO) cm . H NMR (CDCl
3
)
d
: 1.41 (t,
), 6.03 (br s, 2H,
), 7.84 (d, 1H, J ¼ 8.8 Hz, H-7), 8.29 (dd, 1H,
Bruker AC-E series 200 MHz spectrometer. Chemical shifts values
are given in ppm and referred as the internal standard to tetra-
methylsilane (TMS). The following abbreviations are used: br
s ¼ broad signal, s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet,
m ¼ multiplet, rt ¼ room temperature. The purity of all compounds
3
), 4.42 (q, 2H, J ¼ 8.0 Hz, CH
2
2
2
13
6
d ) d

542
A. Martorana et al. / European Journal of Medicinal Chemistry 90 (2015) 537e546
0
C
11
H
10
N
2
O
4
S: C, 49.62; H, 3.79; N, 10.52; found: C, 49.64; H, 3.81; N,
H-6 ), 8.36 (dd, 1H, J ¼ 2.0, 8.0 Hz, H-6), 9.27 (d, 1H, J ¼ 2.0 Hz, H-4),
13
10.48.
10.83 (s,1H, NH). C NMR (CDCl
s), 121.9 (d), 123.5 (s), 124.1 (d), 125.0 (s), 126.1 (d), 128.4 (d), 130.6
(d), 132.7 (s), 140.5 (s), 144.4 (s), 151.5 (s), 164.0 (s), 164.2 (s). Anal.
Calcd. (%) for C19 S: C, 52.06; H, 2.99; N, 6.39; found: C,
52.10; H, 2.93; N, 6.42.
3
) d: 14.2 (q), 62.5 (t),117.7 (s),120.9
(
4
5
.1.2. General procedure for the synthesis of ethyl 3-benzoylamino-
-nitro-benzo[b]thiophene-2-carboxylates 12aef
13 3 2 5
H F N O
To a suspension of amine 10 (0.37 mmol) and pyridine
(
0.56 mmol) was added the appropriate benzoyl chloride 11
(
0.56 mmol). The reaction mixture was stirred at room temperature
4
.1.2.6. Ethyl 3-(3-chloro-4-fluoro-benzoylamino)-5-nitro-benzo[b]
for about 12 h, and then poured onto stirred water/ice. The pre-
cipitate was collected by filtration, dried over a night. The crude
was crystallized from ethyl acetate.
ꢁ
thiophene-2-carboxylate (12f). Yield 57.4%. Mp 218e219 C. IR
3
n
max
:
ꢂ1
1
620 (NH), 1693, 1684 (CO) cm . H NMR (CDCl
3
)
d
: 1.47 (t, 3H,
J ¼ 8.0 Hz, CH
3
), 4.49 (q, 2H, J ¼ 8.0 Hz, CH ), 7.36 (t, 1H, J ¼ 10.0 Hz,
2
0
H-5 ), 7.94 (d, 1H, J ¼ 8.0 Hz, H-7), 8.03 (dq, 1H, J ¼ 2.0, 10.0 Hz, H-
4
.1.2.1. Ethyl 3-benzoylamino-5-nitro-benzo[b]thiophene-2-
0
0
6
2
), 8.23 (dd,1H, J ¼ 2.0, 8.0 Hz, H-6), 8.37 (dd,1H, J ¼ 2.0,10.0 Hz, H-
ꢁ
13
carboxylate (12a). Yield 95%. Mp 200e201 C. IR
1
4
n
max: 3606 (NH),
),
2
), 7.48e7.69 (m, 3H, H-3 , H-4 , H-5 ), 7.89
), 9.24 (d,1H, J ¼ 2.0 Hz, H-4),10.83 (s, 1H, NH). C NMR (CDCl
3
) d:
ꢂ1 1
687,1679 (CO) cm . H NMR (CDCl
3
)
d
: 1.43 (t, 3H, J ¼ 8.0 Hz, CH
3
1
4.2 (q), 62.5 (t),117.3 (d, J ¼ 5 Hz),117.4 (s),121.8 (d),122.5 (s),123.4
0
0
0
0
0
.44 (q, 2H, J ¼ 8.0 Hz, CH
(
(
s), 123.9 (d), 128.1 (d), 130.3 (s), 131.4 (s), 132.6 (d), 140.5 (s), 144.4
s), 145.2 (s), 161.0 (d, J ¼ 30 Hz), 163.2 (s), 164.2 (s). Anal. Calcd. (%)
(
(
1
d, 1H, J ¼ 8.8 Hz, H-7), 8.11 (dd, 2H, J ¼ 2.0, 8.0 Hz, H-2 , H-6 ), 8.32
dd, 1H, J ¼ 2.0, 8.8 Hz, H-6), 9.24 (d, 1H, J ¼ 2.0 Hz, H-4), 10.69 (s,
2 5
for C18H12ClFN O S: C, 51.13; H, 2.86; N, 6.63; found: C, 51.16; H,
H, NH). 13C NMR (CDCl
) d: 14.2 (q), 62.3 (t), 117.3 (s), 121.7 (d),
3
23.3 (d), 124.2 (d), 127.9 (d), 128.6 (s), 129.0 (d), 132.8 (d), 132.9 (s),
41.0 (s), 144.5 (s), 145.1 (s), 164.1 (s), 165.3 (s). Anal. Calcd. (%) for
S: C, 58.37; H, 3.81; N, 7.56; found: C, 58.34; H, 3.84; N,
2
.90; N, 6.65.
1
1
4
.1.3. General procedure for the synthesis of ethyl 5-amino-3-
18 14 2 5
C H N O
7
benzoylamino-benzo[b]thiophene-2-carboxylates 13aef
.53.
A suspension of 5-nitro compound 12aef (1 mmol), 10% Pd/C
(
0.05 g) in ethanol was subjected to hydrogenation using a Parr
4
.1.2.2. Ethyl
3-(4-methoxy-benzoylamino)-5-nitro-benzo[b]thio-
Hydrogenation apparatus at 500 psi for 2 h. After then, the catalyst
was removed by filtration and the filtrate was concentrated under
reduced pressure. The crude was recrystallized from ethanol.
ꢁ
phene-2-carboxylate (12b). Yield 82%. Mp 191e192 C. IR
max
n :
: 1.43 (t, 3H,
ꢂ1
1
3
601 (NH), 1696, 1672 (CO) cm . H NMR (CDCl
J ¼ 8.0 Hz, CH ), 3.91 (s, 3H, OCH ), 4.44 (q, 2H, J ¼ 8.0 Hz, CH
d, 2H, J ¼ 8.0 Hz, H-3 ,H-5 ), 7.89 (d,1H, J ¼ 9.0 Hz, H-7), 8.07 (d, 2H,
3
)
d
3
3
2
), 7.05
0
0
(
0
0
4.1.3.1. Ethyl 5-amino-3-benzoylamino-benzo[b]thiophene-2-
J ¼ 8.0 Hz, H-2 , H-6 ), 8.32 (dd, 1H, J ¼ 2.0, 9.0 Hz, H-6), 9.25 (d, 1H,
ꢁ
13
carboxylate (13a). Yield 91%. Mp 184e185 C. IR
n
max: 3612, 3356
: 1.42 (t,
), 4.83 (br s, 2H, NH
.96 (dd, 1H, J ¼ 2.0, 8.0 Hz, H-6), 7.49 (d, 1H, J ¼ 2.0 Hz, H-4),
), 8.10 (d, 1H, J ¼ 8.0 Hz, H-7), 10.48 (s, 1H,
: 14.3 (q), 61.6 (t), 111.0 (d), 119.0 (d), 123.2
d), 127.8 (d), 128.9 (d), 130.4 (s), 131.1 (s), 132.4 (d), 133.9 (s), 139.1
(s), 143.6 (s), 146.6 (s), 161.2 (s), 165.9 (s). Anal. Calcd. (%) for
J ¼ 2.0 Hz, H-4), 10.64 (s, 1H, NH). C NMR (CDCl
q), 62.3 (t),114.1 (d), 114.2 (d), 121.2 (d), 121.6 (s), 121.8 (s), 126.8 (s),
32.3 (d), 132.8 (d), 141.3 (s), 141.8 (s), 144.5 (s), 145.0 (s), 164.2 (s),
64.8 (s). Anal. Calcd. (%) for C19 S: C, 56.99; H, 4.03; N, 7.00;
found: C, 56.95; H, 4.07; N, 7.03.
3
) d: 14.2 (q), 55.6
ꢂ1 1
2 3
(NH ), 3293 (NH), 1688, 1664 (CO) cm . H NMR (CDCl ) d
(
1
1
3
6
7
H, J ¼ 8.0 Hz, CH
3
), 4.41 (q, 2H, J ¼ 8.0 Hz, CH
2
2
)
16 2 6
H N O
6 5
.52e7.63 (m, 5H, C H
13
3
NH). C NMR (CDCl ) d
(
4.1.2.3. Ethyl
3-(4-methyl-benzoylamino)-5-nitro-benzo[b]thio-
ꢁ
phene-2-carboxylate (12c). Yield 98%. Mp 231e232 C. IR
max
n :
ꢂ1
1
C
8
18 16 2 3
H N O S: C, 63.51; H, 4.74; N, 8.23; found: C, 63.53; H, 4.72; N,
.21.
3
607 (NH), 1700, 1677 (CO) cm . H NMR (CDCl
J ¼ 8.0 Hz, CH ), 2.47 (s, 3H, CH ), 4.44 (q, 2H, J ¼ 8.0 Hz, CH
d, 2H, J ¼ 8.8 Hz, H-3 ,H-5 ), 7.90 (d,1H, J ¼ 8.0 Hz, H-7), 8.00 (d, 2H,
3
)
d
: 1.43 (t, 3H,
3
3
2
), 7.37
0
0
(
0
0
4.1.3.2. Ethyl 5-amino-3-(4-methoxy-benzoylamino)-benzo[b]thio-
phene-2-carboxylate (13b). Yield 98%. Mp 160e161
J ¼ 8.8 Hz, H-2 , H-6 ), 8.33 (dd, 1H, J ¼ 2.0, 8.0 Hz, H-6), 9.25 (d, 1H,
13
ꢁ
C. IR nmax
:
J ¼ 2.0 Hz, H-4), 10.66 (s, 1H, NH). C NMR (CDCl
3
)
d
: 14.2 (q), 21.6
q), 62.3 (t), 118.5 (s), 121.7 (d), 123.3 (d), 124.2 (d), 128.0 (d), 129.7
d), 130.3 (s), 132.9 (s), 141.2 (s), 143.6 (s), 144.5 (s), 145.1 (s), 164.1
s), 165.3 (s). Anal. Calcd. (%) for C19 S: C, 59.37; H, 4.20; N,
ꢂ1
1
3
(
2
7
613, 3360 (NH
DMSO-d
: 1.19 (t, 3H, J ¼ 8.0 Hz, CH
H, J ¼ 8.0 Hz, CH ), 5.08 (br s, 2H, NH
2
), 3296 (NH), 1683, 1660 (CO) cm
), 3.91 (s, 3H, OCH
) 6.94 (d, 1H, J ¼ 2.0 Hz, H-4),
.
H NMR
(
(
(
6
)
d
3
3
), 4.24 (q,
16 2 5
H N O
2
2
0
0
0
.09e7.17 (m, 3H, H-6, H-3 , H-5 ), 7.50 (d, 1H, J ¼ 8.0 Hz, H-7), 8.07
7.29; found: C, 59.40; H, 4.17; N, 7.32.
0
13
(
d, 2H, J ¼ 8.8 Hz, H-2 , H-6 ), 10.04 (s, 1H, NH). C NMR (DMSO-d
: 13.9 (q), 55.6 (q), 60.8 (t), 113.5 (d), 113.7 (d), 118.8 (d), 119.2 (d),
126.3 (s), 127.2 (s), 129.6 (d), 130.2 (s), 134.2 (s), 138.8 (s), 139.7 (s),
161.7 (s), 161.9 (s), 164.9 (s). Anal. Calcd. (%) for C19H18N O S: C,
6
)
d
4.1.2.4. Ethyl 3-(3,4,5-trimethoxy-benzoylamino)-5-nitro-benzo[b]
ꢁ
thiophene-2-carboxylate (12d). Yield 99%. Mp 211e213 C. IR
3
n
max
:
ꢂ1
1
607 (NH), 1680, 1668 (CO) cm . H NMR (CDCl
3
)
d
: 1.46 (t, 3H,
), 4.45 (q,
), 7.38 (s, 2H, H-2 ,H-6 ), 7.93 (d, 1H, J ¼ 8.0 Hz, H-
), 8.36 (dd, 1H, J ¼ 2.0, 8.0 Hz, H-6), 9.32 (d, 1H, J ¼ 2.0 Hz, H-4),
: 14.2 (q), 56.4 (q), 61.0 (t), 62.3
2 4
6
1.61; H, 4.90; N, 7.56; found: C, 61.59; H, 4.93; N, 7.52.
J ¼ 8.0 Hz, CH
3
), 3.98 (s, 3H, OCH
3
), 4.02 (s, 6H, 2ꢀOCH
3
0
0
2
H, J ¼ 8.0 Hz, CH
2
7
1
4.1.3.3. Ethyl
5-amino-3-(4-methyl-benzoylamino)-benzo[b]thio-
0.72 (s, 1H, NH). 13C NMR (CDCl
)
d
phene-2-carboxylate (13c). Yield 70%. Mp 208e209 C. IR n :
ꢁ
ꢂ1
3
max
H NMR
), 4.23 (q,
1
(
(
(
q), 105.3 (d), 117.0 (s), 117.5 (d), 121.7 (d), 121.9 (d), 128.4 (s), 131.3
s), 132.8 (s), 141.1 (s), 145.1 (s), 145.6 (s), 153.4 (s), 164.2 (s), 165.0
s). Anal. Calcd. (%) for C21
3613, 3360 (NH
(DMSO-d ):
1.18 (t, 3H, J ¼ 8.0 Hz, CH
2H, J ¼ 8.0 Hz, CH ), 5.32 (br s, 2H, NH
6), 7.38 (d, 2H, J ¼ 8.0 Hz, H-3 , H-5 ), 7.64 (d, 1H, J ¼ 8.0 Hz, H-7),
2
), 3297 (NH), 1682, 1665 (CO) cm
), 2.42 (s, 3H, CH
) 6.90e6.95 (m, 2H, H-4, H-
.
6
d
3
3
20
H N
2
O
8
S: C, 54.78; H, 4.38; N, 6.08;
2
2
0
0
0
found: C, 54.76; H, 4.40; N, 6.10.
0
13
7
.97 (d, 2H, J ¼ 8.8 Hz, H-2 , H-6 ), 10.20 (s, 1H, NH). C NMR
4.1.2.5. Ethyl 3-(4-trifluoromethyl-benzoylamino)-5-nitro-benzo[b]
(DMSO-d : 14.5 (q), 21.5 (q), 61.3 (t), 105.8 (d), 118.3 (d), 123.0 (d),
125.9 (s), 127.8 (d), 129.0 (d), 131.0 (s), 135.5 (s), 137.1 (s), 141.9 (s),
146.5 (s), 161.9 (s), 163.0 (s), 165.2 (s). Anal. Calcd. (%) for
6
) d
ꢁ
thiophene-2-carboxylate (12e). Yield 74%. Mp 236e237 C. IR
3
n
max
:
ꢂ1
1
3
616 (NH), 1695, 1684 (CO) cm . H NMR (CDCl ) d: 1.44 (t, 3H,
J ¼ 8.0 Hz, CH
3
), 4.46 (q, 2H, J ¼ 8.0 Hz, CH
2
), 7.85 (d, 2H, J ¼ 8.0 Hz,
19 18 2 3
C H N O S: C, 64.39; H, 5.12; N, 7,90; found: C, 64.42; H, 5.15; N,
7.93.
0
0
0
H-3 , H-5 ), 7.92 (d, 1H, J ¼ 8.0 Hz, H-7), 8.23 (d, 2H, J ¼ 8.0 Hz, H-2 ,

A. Martorana et al. / European Journal of Medicinal Chemistry 90 (2015) 537e546
543
4
.1.3.4. Ethyl 5-amino-3-(3,4,5-trimethoxy-benzoylamino)-benzo[b]
4.1.4.2. Ethyl 5-[(1H-imidazol-4-yl-methyl)-amino]-3-(4-methoxy-
benzoylamino)-benzo[b]thiophene-2-carboxylate (6b). Yield 62%.
ꢁ
thiophene-2-carboxylate (13d). Yield 93%. Mp 182e183 C. IR
n
max
:
ꢂ1
1
ꢁ
ꢂ1 1
3
(
6
404, 3316 (NH
DMSO-d
: 1.20 (t, 3H, J ¼ 8.0 Hz, CH
), 4.22 (q, 2H, J ¼ 8.0 Hz, CH
2
), 3213 (NH), 1696, 1655 (CO) cm
), 3.76 (s, 3H, OCH
), 5.34 (br s, 2H, NH
.
H NMR
), 3.88 (s,
) 6.87
Mp 124e125 C. IR
n
max: 3306, 3243 (NH), 1682, 1667 (CO) cm . H
), 3.86 (s, 3H, OCH ),
) 5.34 (t,
6
)
d
3
3
NMR (DMSO-d
4.20 (q, 2H, J ¼ 8.0 Hz, CH
6
)
d: 1.15 (t, 3H, J ¼ 8.0 Hz, CH
3
3
H, 2ꢀOCH
3
2
2
2
), 4.28 (d, 2H, J ¼ 2.0 Hz, NCH
2
0
0
0
0
(
d, 1H, J ¼ 2.0 Hz, H-4), 6.93 (dd, 1H, J ¼ 2.0, 8.0 Hz, H-6), 7.41 (s, 2H,
1H, J ¼ 2.0 Hz, NH), 6.91 (s, 1H, H-5 ), 7.04e7.16 (m, 4H, H-3 , H-5 ,
0
0
13
00
H-2 , H-6 ), 7.65 (d, 1H, J ¼ 8.0 Hz, H-7), 10.22 (s, 1H, NH). C NMR
DMSO-d : 14.0 (q), 56.1 (q), 60.1 (q), 60.8 (t), 99.5 (s), 105.3 (d),
05.7 (d), 118.3 (s), 123.0 (d), 125.8 (d), 128.9 (s), 135.2 (s), 137.1 (s),
40.5 (s), 146.5 (s), 152.7 (s), 161.8 (s), 164.6 (s). Anal. Calcd. (%) for
H-2 , H-6), 7.54 (d, 1H, J ¼ 8.0 Hz, H-7), 7.58 (d, 1H, J ¼ 2.0 Hz, H-4),
0
0
(
1
1
6
)
d
8.06 (d, 2H, J ¼ 8.0 Hz, H-2 , H-6 ) 10.06 (s, 1H, NH), 11.96 (br s, 1H,
13
6
NH). C NMR (DMSO-d ) d: 14.0 (q), 40.1 (t), 55.4 (q), 60.8 (t), 99.5
(s), 103.5 (d), 113.6 (d), 113.7 (s), 117.7 (d), 122.0 (s), 122.5 (s), 122.9
(d), 126.2 (d), 129.7 (d), 134.8 (d), 135.6 (s), 137.0 (s), 146.6 (s), 161.9
(s), 162.0 (s), 164.9 (s). Anal. Calcd. (%) for C23H N O S: C, 61.32; H,
22 4 4
C
6
21 22 2 6
H N O S: C, 58.59; H, 5.15; N, 6.51; found: C, 58.63; H, 5.17; N,
.55.
4.92; N, 12.44; found: C, 61.31; H, 4.94; N, 12.48.
4
.1.3.5. Ethyl 5-amino-3-(4-trifluoromethyl-benzoylamino)-benzo[b]
4
.1.4.3. Ethyl 5-[(1H-imidazol-4-yl-methyl)-amino]-3-(4-methyl-
ꢁ
thiophene-2-carboxylate (13e). Yield 80%. Mp 198e199 C. IR
3
n
max
:
benzoylamino)-benzo[b]thiophene-2-carboxylate (6c). Yield 58%.
ꢂ1
1
612, 3356 (NH
2
), 3293 (NH), 1688, 1664 (CO) cm
: 1.18 (t, 3H, J ¼ 8.0 Hz, CH ), 4.22 (q, 2H, J ¼ 8.0 Hz,
), 5.34 (br s, 2H, NH
) 6.89 (d, 1H, J ¼ 2.0 Hz, H-4), 6.93 (dd, 1H,
. H NMR
ꢁ
ꢂ1 1
Mp 138e139 C. IR
NMR (DMSO-d
: 1.18 (t, 3H, J ¼ 8.0 Hz, CH
d, 2H, J ¼ 2.0 Hz, NCH ), 4.23 (q, 2H, J ¼ 8.0 Hz, CH
J ¼ 2.0 Hz, NH), 6.91e6.93 (m, 2H, H-4, H-5 ), 7.06 (dd, 1H, J ¼ 2.0,
n
max: 3307, 3253 (NH), 1680, 1669 (CO) cm . H
), 2.42 (s, 3H, CH ), 4.14
), 6.11 (t, 1H,
(
DMSO-d
6
)
d
3
6
)
d
3
3
CH
2
2
(
2
2
J ¼ 2.0, 8.0 Hz, H-6), 7.66 (d, 1H, J ¼ 8.0 Hz, H-7), 7.98 (d, 2H,
0
0
0
0
0
0
J ¼ 8.8 Hz, H-3 , H-5 ), 8.25 (d, 2H, J ¼ 8.8 Hz, H-2 , H-6 ), 10.54 (s,
H, NH). 13C NMR (DMSO-d
: 14.0 (q), 60.9 (t), 119.1 (s), 118.3 (d),
20.8 (s), 123.1 (d), 123.7 (s), 125.5 (d), 126.9 (s), 128.6 (d), 129.1 (d),
34.4 (s), 137.1 (s), 146.6 (s), 147.9 (s), 161.7 (s), 164.3 (s). Anal. Calcd.
%) for C19 S: C, 55.88; H, 3.70; N, 6.86; found: C, 55.84; H,
0
0
00
8
.0 Hz, H-6), 7.38 (d, 2H, J ¼ 8.0 Hz, H-3 , H-5 ), 7.56 (s, 1H, H-2 ),
1
1
1
(
6
) d
0
0
7
.68 (d, 1H, J ¼ 8.0 Hz, H-7), 7.97 (d, 2H, J ¼ 8.0 Hz, H-2 , H-6 ), 10.23
13
(
(
s, 1H, NH), 11.93 (br s, 1H, NH). C NMR (DMSO-d
q), 40.4 (t), 60.8 (t), 103.4 (d), 117.4 (d), 117.7 (d), 122.7 (s), 122.9 (d),
26.1 (s), 127.8 (d), 129.0 (d), 131.3 (s), 134.8 (d), 135.3 (s), 137.0 (s),
41.8 (s), 146.6 (s), 161.9 (s), 165.3 (s), 172.0 (s). Anal. Calcd. (%) for
S: C, 63.58; H, 5.10; N, 12.89; found: C, 63.60; H, 5.12; N,
6
) d: 14.0 (q), 21.0
15 3 2 3
H F N O
.73; N, 6.82.
1
1
3
23 22 4 3
C H N O
4
.1.3.6. Ethyl 5-amino-3-(3-chloro-4-fluoro-benzoylamino)-benzo[b]
12.85.
ꢁ
thiophene-2-carboxylate (13f). Yield 52%. Mp 226e227 C. IR
3
n
max
:
ꢂ1
1
615, 3360 (NH
2
), 3290 (NH), 1689, 1667 (CO) cm
), 4.23 (q, 2H, J ¼ 8.0 Hz, CH
), 6.87 (d,1H, J ¼ 2.0 Hz, H-4), 6.93 (dd,1H, J ¼ 2.0,
.
H NMR
4.1.4.4. Ethyl 5-[(1H-imidazol-4-yl-methyl)-amino]-3-(3,4,5-
trimethoxy-benzoylamino)-benzo[b]thiophene-2-carboxylate
(
CDCl
3
)
d
: 1.18 (t, 3H, J ¼ 8.0 Hz, CH
3
2
),
(6d).
ꢁ
5
.35 (br s, 2H, NH
2
Yield 94%. Mp 165e166 C. IR: 3301, 3245 (NH), 1684, 1668 (CO)
0
ꢂ1 1
1
5
0.0 Hz, H-2 ), 7.65 (d, 1H, J ¼ 8.0 Hz, H-7), 7.66 (t,1H, J ¼ 10.0 Hz, H-
cm . H NMR (DMSO-d
6
)
d
: 1.20 (t, 3H, J ¼ 7.0 Hz, CH
), 4.34 (d, 2H, J ¼ 1.8 Hz, NCH
), 6.14 (t,1H, J ¼ 1.8 Hz, NH), 6.89 (d,1H, J ¼ 1.8 Hz,
3
), 3.76 (s, 3H,
0
0
), 8.08 (dq, 1H, J ¼ 2.0, 10.0 Hz, H-6 ), 8.29 (dd, 1H, J ¼ 2.0, 8.0 Hz,
OCH
2H, J ¼ 7.0 Hz, CH
H-4) 6.95 (s, 1H, H-5 ), 7.07 (dd, 1H, J ¼ 1.8, 8.8 Hz, H-6), 7.40 (s, 2H,
3
), 3.89 (s, 6H, 2ꢀOCH
3
2
), 4.24 (q,
13
H-6), 10.34 (s, 1H, NH). C NMR (DMSO-d
6
)
d
: 14.0 (q), 61.1 (t), 99.5
2
0
0
(
(
(
s), 117.3 (d), 118.5 (s), 120.0 (d), 122.6 (s), 123.2 (s), 125.8 (d), 128.9
d), 131.5 (s), 134.3 (d), 137.0 (s), 141.1 (s), 146.6 (s), 161.6 (d), 163.2
s), 164.3 (s). Anal. Calcd. (%) for C18 14ClFN S: C, 55.03; H, 3.59;
0
0
00
H-2 , H-6 ), 7.58 (s, 1H, H-2 ), 7.70 (d, 1H, J ¼ 8.8 Hz, H-7), 10.25 (s,
13
H
2
O
3
1H, NH), 12.22 (br s, 1H, NH). C NMR (DMSO-d
(t), 56.0 (q), 60.1 (q), 60.2 (t), 99.5 (s), 103.3 (s), 105.3 (d), 117.7 (d),
22.9 (d), 126.1 (d), 129.1 (s), 129.5(s), 134.8 (s), 135.2 (d), 137.0 (s),
40.4 (s), 146.6 (s), 152.6 (d), 156.9 (s), 161.8 (s), 164.9 (s). Anal.
Calcd. (%) for C25 S: C, 58.81; H, 5.13; N, 10.97; found: C,
6
) d: 14.0 (q), 40.2
N, 7.13; found: C, 55.04; H, 3.60; N, 7.22.
1
1
4
5
.1.4. General procedure for the synthesis of ethyl 3-benzoylamino-
-[(1H-imidazol-4-yl-methyl)-amino]-benzo[b]thiophene-2-
26 4 6
H N O
58.83; H, 5.14; N, 10.95.
carboxylates 6a-l
To a suspension of the 13aef (1.25 mmol) and the appropriate
aldehyde (1.6 mmol) in dry ethanol (10 mL) was added acetic acid
4
.1.4.5. Ethyl 5-[(1H-imidazol-4-yl-methyl)-amino]-3-(4-
trifluoromethyl-benzoylamino)-benzo[b]thiophene-2-carboxylate
up to pH 4. The mixture was stirred for 30 min, and then NaCNBH
1.6 mmol) was added continuing the stirring at room temperature
3
ꢁ
(6e). Yield 84%. Mp 192e193 C. IR
n
d
max: 3298, 3233 (NH), 1679,
: 1.21 (t, 3H, J ¼ 6.0 Hz, CH ),
), 4.53e4.56 (m, 3H, NCH , NH), 6.90 (1,
(
ꢂ1 1
1665 (CO) cm . H NMR (DMSO-d
6
)
3
for further 6e24 h. The reaction solvent was removed under
reduced pressure and the crude product was purified by chroma-
tography column using dichloromethane/methanol 98:2 as eluant.
Recrystallized from ethanol.
4
1
.25 (q, 2H, J ¼ 6.0 Hz, CH
2
2
00
0
0
H, H-5 ), 7.19 (d, 1H, J ¼ 2.0 Hz, H-4), 7.57 (s, 1H, H-2 ), 7.74 (d, 1H,
0
0
J ¼ 8.0 Hz, H-7), 7.99 (d, 2H, J ¼ 10.0 Hz, H-3 , H-5 ), 8.22 (d, 2H,
0
0
13
J ¼ 10.0 Hz, H-2 , H-6 ), 10.56 (s, 1H, NH), 11.93 (br s, 1H, NH).
C
NMR (DMSO-d : 14.0 (q), 40.0 (t), 60.9 (t), 99.5 (s), 113.0 (d), 117.9
6
) d
4
.1.4.1. Ethyl 3-benzoylamino-5-[(1H-imidazol-4-yl-methyl)-amino]-
(d), 121.1 (d), 125.4 (s), 125.5 (s), 126.2 (d), 128.6 (d), 129.0 (s), 130.6
(d), 134.4 (d), 134.8 (s), 136.9 (s), 137.8 (s), 146.7 (s), 161.7 (s), 164.5
ꢁ
benzo[b]thiophene-2-carboxylate (6a). Yield 76%. Mp 184e185 C.
ꢂ1 1
IR
: 1.19 (t, 3H, J ¼ 8.0 Hz, CH
m, 3H, NH, NCH ) 6.89 (s, 1H, H-5 ), 7.19 (s, 1H, H-2 ), 7.29 (dd, 1H,
J ¼ 2.0, 8.0 Hz, H-6), 7.56e7.65 (m, 5H, C ), 7.73 (d, 2H, J ¼ 8.0 Hz,
H-7), 8.04 (d, 1H, J ¼ 2.0 Hz, H-4) 10.39 (s, 1H, NH), 12.10 (br s, 1H,
NH). 13C NMR (DMSO-d
: 13.5 (q), 46.1 (t), 60.4 (t),100.3 (d), 119.7
s), 122.8 (s), 122.9 (d), 125.9 (s), 128.4 (d), 129.5 (d), 131.1 (s), 132.5
s), 134.4 (d), 135.1 (d), 135.7 (d), 137.0 (d), 146.2 (s), 149.5 (s), 161.6
s), 165.9 (s). Anal. Calcd. (%) for C22 S: C, 62.84; H, 4.79; N,
3.32; found: C, 62.86; H, 4.80; N, 13.35.
n
max: 3301, 3245 (NH), 1684, 1668 (CO) cm . H NMR (DMSO-d
6
)
(s), 166.3 (s). Anal. Calcd. (%) for C23
19 3 4 3
H F N O S: C, 56.55; H, 3.92; N,
d
3
), 4.24 (q, 2H, J ¼ 8.0 Hz, CH
2
), 4.52e4.54
11.47; found: C, 56.53; H, 3.91; N, 11.49.
00
00
(
2
6
H
5
4.1.4.6. Ethyl 5-[(1H-imidazol-4-yl-methyl)-amino]-3-(3-chloro-4-
fluoro-benzoylamino)-benzo[b]thiophene-2-carboxylate
(6f).
ꢁ
6
)
d
Yield 43%. Mp 162e163 C. IR
n
max: 3306, 3253 (NH), 1690, 1669
ꢂ1 1
(
(
(
(CO) cm . H NMR (CDCl
3
)
d
: 1.34 (t, 3H, J ¼ 8.0 Hz, CH
3
), 4.26e4.30
(m, 3H, NH, NCH
2
), 4.32 (q, 2H, J ¼ 8.0 Hz, CH
2
), 6.82 (dd, 1H, J ¼ 2.0,
0
0
0
H
20
N
4
O
3
8.0 Hz, H-6), 6.96 (s, 1H, H-5 ), 7.24 (t, 1H, J ¼ 8.0 Hz, H-5 ), 7.43 (d,
0
0
1
1H, J ¼ 8.0 Hz, H-7), 7.47 (s, 1H, H-2 ), 7.73 (d, 1H, J ¼ 2.0 Hz, H-4),

544
A. Martorana et al. / European Journal of Medicinal Chemistry 90 (2015) 537e546
0
0
7
9
4
1
1
.89 (dq, 1H, J ¼ 2.0, 8.0 Hz, H-6 ), 8.07 (dd, 1H, J ¼ 2.0, 8.0 Hz, H-2 ),
4.1.4.11. Ethyl 5-[(5-methyl-1H-imidazol-4-yl-methyl)-amino]-3-(4-
trifluoromethyl-benzoylamino)-benzo[b]thiophene-2-carboxylate
13
.73 (s, 1H, NH), 10.45 (br s, 1H, NH). C NMR (DMSO-d
0.6 (t), 60.9 (t), 103.0 (d), 117.0 (d), 117.9 (d), 119.6 (s), 120.0 (s),
23.0 (d), 123.4 (s), 126.1 (s), 129.0 (d), 129.1 (d), 130.3 (d), 131.7 (s),
34.4 (s), 134.8 (d), 136.9 (s), 146.8 (s), 161.7 (s), 163.3 (s), 172.2 (s).
Anal. Calcd. (%) for C22 18ClFN S: C, 55.87; H, 3.84; N, 11.85;
found: C, 55.91; H, 3.81; N, 11.88.
6
)
d
: 14.0 (q),
ꢁ
(6k). Yield 34%. Mp 171e173 C. IR
n
max: 3612, 3294 (NH), 1691,
ꢂ1 1
1644 (CO) cm . H NMR (DMSO-d
6
)
d: 1.18 (t, 3H, J ¼ 7.0 Hz, CH
3
),
),
2.08 (s, 3H, CH
3
), 4.05 (br s, 2H, CH
2
), 4.22 (q, 2H, J ¼ 7.0 Hz, CH
2
H
4
O
3
6.03 (br s, 1H, NH), 6.88 (d, 1H, J ¼ 2.0 Hz, H-4), 7.06 (dd, 1H, J ¼ 2.0,
0
0
8.6 Hz, H-6), 7.44 (s, 1H, H-2 ), 7.68 (d, 1H, J ¼ 8.6 Hz, H-7), 7.97 (d,
0
0
0
0
2
H, J ¼ 8.2 Hz, H-3 , H-5 ), 8.20 (d, 2H, J ¼ 8.2 Hz, H-2 , H-6 ), 10.53
13
4.1.4.7. Ethyl
3-benzoylamino-5-[(5-methyl-1H-imidazol-4-yl-
6
(s, 1H, NH), 11.67 (br s, 1H, NH). C NMR (DMSO-d ): d 10.3 (q), 14.0
methyl)-amino]-benzo[b]thiophene-2-carboxylate (6g). Yield 49%.
(q), 39.1 (t), 60.9 (t), 99.5 (s), 102.4 (d), 118.1 (d), 122.9 (d), 123.6 (s),
125.4 (s), 125.5 (d), 126.1 (s), 128.7 (d), 131.9 (s), 133.1 (d), 134.5 (s),
137.0 (s), 137.9 (s), 146.8 (s), 161.7 (s), 164.5 (s), 172.0 (s). Anal. Calcd.
ꢁ
ꢂ1 1
Mp 169e171 C. IR
n
max: 3607, 3336 (NH), 1671, 1543 (CO) cm . H
: 1.22 (t, 3H, J ¼ 6.0 Hz, CH ), 2.24 (s, 3H, CH ),
.22e4.33 (m, 4H, OCH , NCH
), 5.27 (t, 1H, J ¼ 4.0 Hz, NH), 7.27 (d,
H, J ¼ 8.0 Hz, H-7), 7.52 (s, 1H, H-2 ), 7.60e7.66 (m, 5H, C
NMR (DMSO-d
6
)
d
3
3
4
1
2
2
21 3 4 3
(%) for C24H F N O S: C, 57.36; H, 4.21; N, 12.15; found: C, 57.46; H,
00
6 5
H ), 8.12
4.31; N, 12.25.
(
d,1H, J ¼ 2.0 Hz, H-4), 8.15 (dd,1H, J ¼ 2.0, 8.0 Hz, H-6),10.25 (s,1H,
13
NH), 11.96 (br s, 1H, NH). C NMR (DMSO-d
9.0 (t), 60.8 (t), 102.8 (d), 117.9 (d), 122.9 (s), 123.5 (d), 125.0 (s),
25.9 (s), 127.8 (d), 128.4 (d), 129.2 (d), 131.0 (s), 132.3 (s), 132.7 (d),
46.5 (s), 147.5 (s), 160.8 (s), 165.4 (s), 171.9 (s). Anal. Calcd. (%) for
S: C, 63.58; H, 5.10; N, 12.89; found: C, 63.54; H, 5.06; N,
6
)
d: 10.0 (q), 14.0 (q),
4.1.4.12. Ethyl 5-[(5-methyl-1H-imidazol-4-yl-methyl)-amino]-3-(3-
chloro-4-fluoro-benzoylamino)-benzo[b]thiophene-2-carboxylate
3
1
1
C
ꢁ
(6l). Yield 35%. Mp 184.7e186 C. IR
n
max: 3607, 3332 (NH), 1701,
ꢂ1 1
1655 (CO) cm . H NMR (DMSO-d
2.15 (s, 3H, CH ), 4.11 (br s, 2H, CH
3 2
6
)
d
: 1.24 (t, 3H, J ¼ 7.1 Hz, CH
3
),
),
H
23 22
N
4
O
3
), 4.28 (q, 2H, J ¼ 7.1 Hz, CH
2
12,86.
6.10 (br s, 1H, NH), 6.92 (d, 1H, J ¼ 1.7 Hz, H-4), 7.12 (dd, 1H,
0
0
0
J ¼ 8.8 Hz, H-6), 7.47 (s, 1H, H-2 ), 7.66e7.75 (m, 2H, H-7, H-2 ), 8.13
0
0
4
.1.4.8. Ethyl 5-[(5-methyl-1H-imidazol-4-yl-methyl)-amino]-3-(4-
(dd, 1H, J ¼ 2.1, 13.3 Hz, H-6 ), 8.34 (dd, 1H, J ¼ 2.0, 7.1 Hz, H-5 ),
13
methoxy-benzoylamino)-benzo[b]thiophene-2-carboxylate
(6h).
6
10.50 (s,1H, NH) 11.96 (br s,1H, NH). C NMR (DMSO-d ) d: 10.5 (q),
ꢁ
Yield 34%. Mp 165e1667 C. IR
n
max: 3603, 3328 (NH), 1670, 1611
: 1.18 (t, 3H, J ¼ 6.2 Hz, CH ), 2.09 (s,
), 4.06 (br s, 2H, CH ), 4.22 (q, 2H,
), 6.07 (br s, 1H, NH), 6.87 (s, 1H, H-4), 7.00e7.18 (m,
14.0 (q), 39.1 (t), 60.9 (t), 99.5 (s), 102.4 (d), 116.9 (d), 117.4 (d), 118.1
(d), 119.6 (s), 119.9 (s), 123.0 (d), 123.5 (s), 126.1 (s), 129.2 (d), 130.3
(d), 131.7 (s), 134. 5 (s), 137.0 (s), 146.8 (s), 161.7 (s), 163.3 (s), 172.2
(s). Anal. Calcd. (%) for C23H20ClFN O S: C, 56.73; H, 4.14; N, 11.50;
4 3
found: C, 56.77; H, 4.10; N, 11.54.
ꢂ1 1
(
CO) cm . H NMR (DMSO-d
6
)
d
3
3
H, CH
3
), 3.87 (s, 3H, OCH
3
2
J ¼ 6.2 Hz, CH
2
0
0
00
3
8
H, H-6, H-3 , H-5 ), 7.51 (s, 1H, H-2 ), 7.67 (d, 1H, J ¼ 8.2 Hz, H-7),
0
0
.06 (d, 2H, J ¼ 8.0 Hz, H-2 , H-6 ), 10.17 (s, 1H, NH), 11.36 (s,1H, NH).
1
3
C NMR (DMSO-d
6
) d: 10.0 (q), 14.0 (q), 40.1 (t), 55.4 (q), 60.7 (t),
4.2. Biology
9
9.5 (s), 102.9 (d), 110.5 (s), 113.6 (s), 117.9 (d), 121.3 (s), 122.5 (d),
122.8 (s), 126.1 (d), 129.5 (s), 129.7 (d), 133.1 (d), 135.6 (s), 137.0 (s),
4.2.1. Chemicals and reagents
1
46.5 (s), 162.0 (s), 164.8 (s). Anal. Calcd. (%) for C24 S: C,
H
24
N
4
O
4
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
6
2.05; H, 5.21; N, 12.06; found: C, 62.09; H, 5.24; N, 12.10.
4
.1.4.9. Ethyl 5-[(5-methyl-1H-imidazol-4-yl-methyl)-amino]-3-(4-
bovine serum (FBS), phosphate buffered saline (PBS), L-glutamine
methyl-benzoylamino)-benzo[b]thiophene-2-carboxylate
(6i).
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).
ꢁ
Yield 32%. Mp 164e166 C. IR
n
max: 3615, 3360 (NH), 1689, 1667
: 1.18 (t, 3H, J ¼ 6.2 Hz, CH ), 2.09
), 4.06 (br, s, 2H, CH ), 4.22 (q, 2H,
ꢂ1 1
(
(
CO) cm . H NMR (DMSO-d
6
)
d
3
s; 3H, CH
3
), 2.42 (s, 3H, CH
3
2
J ¼ 6.2 Hz, CH
2
), 6.05 (br s, 1H, NH), 6.88 (d, 1H, J ¼ 2.0 Hz, H-4), 7.06
0
0
(
dd, 1H, J ¼ 2.0, 8.6 Hz, H-6), 7.38 (d, 2H, J ¼ 8.0 Hz, H-3 , H-5 ), 7.44
4.2.2. Cell culture
0
0
(
2
s, 1H, H-2 ), 7.67 (d, 1H, J ¼ 8.6 Hz, H-7), 8.98 (d, 2H, J ¼ 8.0 Hz, H-
The cancer cell line HeLa (human epithelial cervical cancer) was
obtained from American Type Culture Collection (ATCC) (Rockville,
MD, USA). The cells were cultured in RPMI supplemented with 5%
0
0
13
, H-6 ), 10.23 (s, 1H, NH), 11.84 (br s, 1H, NH). C NMR (DMSO-d )
6
d
: 10.1 (q), 14.0 (q), 21.0 (q), 39.0 (t), 60.8 (t), 102.8 (d), 117.9 (d),
22.8 (d), 126.1 (s), 127.8 (d), 128.9 (d), 129.6 (s), 131.2 (s), 133.1 (d),
35.4 (s), 137.0 (s), 141.8 (s), 146.6 (s), 161.9 (s), 165.3 (s), 171.1 (s),
72.1 (s). Anal. Calcd. (%) for C24 S: C, 64.27; H, 5.39; N,
1
1
1
FBS, 2 mM
L-glutamine, 50 IU/ml penicillin, and 50
m
g/ml strepto-
mycin and maintained in a humidified atmosphere with 5% CO
2
at
ꢁ
2
24
H N
4
O
3
37 C. 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.
12.49; found: C, 64.31; H, 5.36; N, 12.51.
4.1.4.10. Ethyl
5-[(5-methyl-1H-imidazol-4-yl-methyl)-amino]-3-
(
(
1
2
2
3,4,5-trimethoxy-benzoylamino)-benzo[b]thiophene-2-carboxylate
ꢁ
6j). Yield 56%. Mp 167e168 C. IR
n
d
max: 3617, 3363 (NH), 1690,
: 1.25 (t, 3H, J ¼ 6.2 Hz, CH ),
), 3.94 (s, 6H, OCH x2), 4.35 (d,
), 4.29 (q, 2H, J ¼ 7.0 Hz, CH ), 6.23 (t, 1H,
4.2.3. Antiproliferative evaluation assay
ꢂ1 1
665 (CO) cm . H NMR (DMSO-d
6
)
3
3-benzoylamino-benzo[b]thiophene derivatives 6ael and
13aef were submitted to the MTT assay to assess the growth in-
hibition activity against HeLa cells. The MTTassay is a measurement
of cell metabolic activity, quite effective in estimating cell prolif-
eration, which is based on the protocol first described by Moss-
mann [25]. The assay was performed as previously described [26].
The cells were seeded into a series of standard 96-well plates in
.16 (s; 3H, CH
3
), 3.81 (s, 3H, OCH
3
3
H, J ¼ 1.6 Hz, CH
2
2
J ¼ 1.6 Hz, NH), 6.85 (d, 1H, J ¼ 1.6 Hz, H-4), 7.11 (dd, 1H, J ¼ 2.0,
0 0 00
.8 Hz, H-6), 7.46 (s, 2H, H-2 , H-6 ), 7.62 (s, 1H, H-2 ), 7.74 (d. 1H,
8
13
J ¼ 8.8 Hz, H-7), 10.27 (s, 1H, NH), 12.09 (br s, 1H, NH). C NMR
DMSO-d : 14.0 (q), 21.0 (q), 40.1 (t), 56.0 (q), 60.1 (q), 60.8 (t),
9.5 (s), 102.7 (s), 105.3 (d), 117.9 (s), 123.0 (d), 126.1 (s), 129.1 (s),
33.2 (d), 135.2 (s), 137.0 (s), 140.3 (s), 140.4 (s), 146.6 (s), 152.6 (d),
61.8 (s), 164.8 (s), 172.0 (s). Anal. Calcd. (%) for C26 S: C,
9.53; H, 5.38; N, 10.68; found: 59.56; H, 5.40; N, 10.66.
(
9
1
1
6
) d
4
2
100
m
l of complete culture medium at 1.0 ꢀ 10 cells/cm . Cells
ꢁ
were incubated for 24 h under 5% CO
2
at 37 C and the medium was
H
28
N
4
O
6
then replaced with 100
ml of fresh medium supplemented by 5% (v/
5
v) FBS containing the treatments. Benzothiophene derivatives were

A. Martorana et al. / European Journal of Medicinal Chemistry 90 (2015) 537e546
545
previously dissolved in DMSO to obtain 20 mM stock solutions.
Working solutions were freshly prepared on the day of testing by
dilutions of the stock solutions in the complete culture medium. For
Database entries containing cations or consisting of a mix of two
structures were excluded. Thus the starting database was consti-
tuted from 114 compounds (Table 1 in supporting information)
classified in six mechanism of action: 30, Alkylating Agents; 13,
Antimitotic Agents; 24, Topoisomerase I Inhibitors; 15, Topoisom-
erase II Inhibitors; 16, RNA/DNA Antimetabolites; 16, DNA Anti-
metabolites (Table 2). 2D and 3D molecular descriptors were
calculated for all structures to achieve all the structural information
useful for building a lock model database. Each descriptor value
the experiments a concentration range from 50 to 0.05
mM was
used. 24 h after seeding aliquots of 100 l of different solutions at
m
the appropriate concentrations were added to the appropriate
wells and the cells were incubated for 48 h without renewal of the
medium. In each experiment, DMSO concentration never exceeded
0
.25% and culture medium with 0.25% DMSO was used as control.
After the incubation time, cells were washed and 100 L FBS-free
m
range is delimited by
mechanism of action,
m
m
Dj(MA) ±
sDj(MA), where (MA) is a specific
medium containing 0.5 mg/mL of MTT was added. The medium
Dj(MA) is the molecular descriptor j average
ꢁ
was discarded after a 4 h incubation at 37 C and formazan blue
value and
the molecular descriptor value Dj of a tested structure X falls within
the defined range ( Dj ± Dj)
¼ 1 (i.e. D1, D3, and Dj), otherwise
sDj(MA) is the standard deviation of descriptor j. When
formed in the cells was dissolved in DMSO. The absorbance (optical
density, OD) at 570 nm of MTT-formazan was measured in a
microplate reader. As the absorbance is directly proportional to the
number of living, metabolically active cells, the percentage of
growth (PG) to with respect to untreated cell control for each drug
concentrations was calculated according to one of the following
two expressions:
m
s
a
a
¼ 0. Therefore, after processing descriptor values as mentioned
above, every training set database structure is converted into a
binary sequence. Then all of the binary values are summed, so the
higher is the number of fitted pins (descriptors with
a
¼ 1), the
higher will be the affinity of the investigated compound to a spe-
cific class with potential anticancer mechanism. Thus, the per-
centage of affinity A% (Eq. (1)) for each compound belonging to
training set database has been defined for each class (MA) as:
if ðODtest ꢂ ODtzeroÞ ꢃ 0; then
PG ¼ 100 ꢀ ðODtest ꢂ ODtzeroÞ=ðODctr ꢂ ODtzeroÞ
if ðODtest ꢂ ODtzeroÞ < 0; then
X
.
PG ¼ 100 ꢀ ðODtest ꢂ ODtzeroÞ=ODtzero
A% ¼
ai; jðMAÞ Dtot*100
(1)
where ODtzero is the average of optical density measurements
before exposure of cells to the test compound, ODtest is the average
of optical density measurements after the desired period of time,
and ODctr is the average of optical density measurements after the
desired period of time with no exposure of cells to the test com-
pound. The concentration necessary for 50% of growth inhibition
where Sai, j (MA) is the sum of all fitted molecular descriptors for
the MA class and Dtot is the total of the molecular descriptors used
in the VLAK protocol.
Acknowledgments
(
GI50) for each derivative was calculated from concen-
trationeresponse curves using linear regression analysis by fitting
the test concentrations that give PG values above and below the
reference value (50%). Each result was the mean value of three
separate experiments performed in quadruplicate.
This work was in part supported by an FFR2012 e University of
Palermo grant (CUP:B78C12000380001).
Appendix A. Supplementary data
4
.2.4. Cell-cycle analysis
Effects of 3-benzoylamino-benzo[b]thiophene derivatives 13d,
j, and 6l exposure on cell-cycle were performed by DNA staining
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.12.002.
6
with propidium iodide (PI) and flow cytometry analysis. HeLa cells
References
4
2
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and treated 24 h after seeding without or with indicated concen-
trations of the test compound for 24 h. Following the treatments,
cells were collected, washed in PBS and stained with staining so-
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m
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