In Vitro Activity of Organochalcogen Compounds: II. Cytotoxic Effect of 2-Aminobenzo[b]thiophenes Against K562 and HeLa Tumor Cell Lines

Article abstract of DOI:10.1134/S107036322011033X

Abstract: In vitro antitumor activity of some benzo[b]thiophenes with a tertiary amino group in the second position was studied against erythroleukemia (K562) and cervical carcinoma (HeLa) cell lines.

Full text of DOI:10.1134/S107036322011033X

ISSN 1070-3632, Russian Journal of General Chemistry, 2020, Vol. 90, No. 11, pp. 2214–2218. © Pleiades Publishing, Ltd., 2020.
SHORT
COMMUNICATIONS
In Vitro Activity of Organochalcogen Compounds:
II.1 Cytotoxic Effect of 2-Aminobenzo[b]thiophenes Against
K562 and HeLa Tumor Cell Lines
E. A. Popovaa,*, A. A. Kornevb, S. V. Shmakovb, G. D. Nepochatyia,
O. A. Kotyuninaa, M. L. Petrova, V. M. Boitsovb,**, and A. V. Stepakovc
a St. Petersburg State Institute of Technology (Technical University), St. Petersburg, 190013 Russia
b St. Petersburg Academic University – Nanotechnology Research and Education Centre, Russian Academy of Sciences,
St. Petersburg, 194021 Russia
c St. Petersburg State University, St. Petersburg, 199034 Russia
e-mail: *ekaterinaapopova@yandex.ru; **bovitali@yandex.ru
Received November 12, 2020; revised November 14, 2020; accepted November 14, 2020
Abstract—In vitro antitumor activity of some benzo[b]thiophenes with a tertiary amino group in the second posi-
tion was studied against erythroleukemia (K562) and cervical carcinoma (HeLa) cell lines.
Keywords: benzo[b]thiophenes, 1,2,3-thiadiazoles, in vitro antitumor activity, erythroleukemia (K562) cell line,
cervical carcinoma (HeLa) cell line
DOI: 10.1134/S107036322011033X
Benzo[b]thiophene and its derivatives occupy a
special place among sulfur-containing heterocycles,
since the compounds containing this fragment have a
surprisingly wide spectrum of biological activity [2].
Among the beneficial properties found for benzo[b]-
thiophene derivatives are anti-cancer [3–8], anti-diabetic
[9], anti-tuberculosis [10], anti-malarial [11], anti-fungal
[12], anti-depressant [13], anti-convulsant [14], anti-
hyperglycemic [15], anti-angiogenic [16], antimitotic
[17], anti-inflammatory, and analgesic [18] deserves
special attention.Also on the basis of benzo[b]thiophene,
inhibitors of acetyl-CoAcarboxylase [19, 20] and tubulin
polymerization [21, 22] were synthesized. It should
be noted that 2-aminobenzo[b]thiophenes have been
successfully used to create selective estrogen receptor
modulator (SERM) raloxifene and its analogues [23, 24].
Based on the above information, we have determined
the purpose of this work in assessing in vitro antitumor
activity of some benzo[b]thiophenes, which have a
tertiary amino group in the second position. Also we
would like to emphasize here that our research group is
actively developing original synthetic methods for the
preparation of polysubstituted benzothiophenes with
amino-, alkoxy- and alkylthio-substituents in the second
position based on reactions involving 1,2,3-thiadiazoles
[25–29].
In this work, substituted benzo[b]thiophenes 1a–1e
obtained according to previously developed by us
protocols were selected as objects for in vitro study
(Fig. 1).
The synthesis of compounds 1a–1e is presented
in Scheme 1. Initially, the reaction of acetophenones
2a–2c with ethyl hydrazinecarboxylate yielded the
corresponding ethoxycarbonylhydrazones 3a–3c. At the
next stage, hydrazones 3a–3c were treated with thionyl
chloride to form 4-aryl-1,2,3-thiadiazoles 4a–4c (Hurd–
Mori reaction) [30–32]. At the final stage, compounds
4a, 4b reacted with various secondary amines such as
pyrrolidine, 2-ethylpiperidine, and morpholine in DMF in
the presence of copper(I) iodide and potassium carbonate
to form 2-aminobenzo[b]thiophenes 1a–1d in 60–82%
[25]. The interaction of 4-(2-chloro-5-nitrophenyl)-1,2,3-
thiadiazole (4c) with morpholine in DMF medium at
1 For communication I, see [1].
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IN VITRO ACTIVITY OF ORGANOCHALCOGEN COMPOUNDS: II.
2215
Scheme 1. Synthesis of benzo[b]thiophenes 1a–1e.
80°Cproceedswithoutacatalysttoform4-(4-nitrobenzo[b]-
thiophen-2- yl)morpholine (4e) in 12% yield [32].
antiproliferative activity against two cancer cell lines.
Among them, only 1-(benzo[b]thiophen-2-yl)-2-
ethylpiperidine (1b) was the most promising compound
against both K562 and HeLa cell lines tested. The
obtained experimental data indicate the expediency of
searching for pharmacologically active substances among
2-aminobenzo[b]thiophene derivatives.
Next, the cytotoxic effect of the synthesized
2-aminobenzo[b]thiophenes 1a–1e was investigated
against human chronic myeloid leukemia (K562)
and cervical carcinoma (HeLa) cell lines by MTS or
AlamarBlue assay. The results are summarized in
Figs. 2, 3. As shown in Figs. 2, 3 one of compounds
exhibited potent antiproliferative activity against these
two cell lines in an incubation time- and dose-dependent
manner. First cytotoxic effects of the 2-substituted
benzo[b]thiophenes 1a–1e were tested against K562 cell
line and then a short test for their activity against HeLa cell
line was performed. Among the screened compound 1b
bearing 2-ethylpiperidine substituent showed significant
cell growth inhibition against both tumor cell lines (IC50
2 and 10 μg/mL for K562 and HeLa respectively).
Melting points were measured on a Boetius melting
point apparatus. 1H and 13C NMR spectra were recorded
on a Bruker DPX-400 spectrometer at 400.13 and
100.16 MHz, respectively. High-resolution mass spectra
(ESI) were taken on a Micromass 70-VSE instrument. The
reaction progress was monitored by TLC on Silica gel
60 F254 plates; spots were visualized under UV light or
by treatment with iodine vapor. The solvents used were
purified and dried according to standard procedures. All
chemicals were used as purchased.
In summary, a series of 2-substituted benzo[b]thio-
phenes 1a–1e were prepared and tested for their potent
1-(Benzo[b]thiophen-2-yl)pyrrolidine (1a) was
prepared in accordance with the developed by us method
as described in [25]. Yield 78%.
1-(Benzo[b]thiophen-2-yl)-2-ethylpiperidine (1b)
was prepared in accordance with the developed by us
method as described in [25]. Yield 60%.
4-(Benzo[b]thiophen-2-yl)morpholine (1c) was
prepared in accordance with the developed by us method
as described in [33]. Yield 82%.
Ethyl 2-(1-(2-bromo-5-methoxyphenyl)ethyliden)-
hydrazinocarboxylate (3b) was prepared in accordance
with the developed by us method as described in [34].
Yield 41.5%.
4-(2-Bromo-5-methoxyphenyl)-1,2,3-thiadiazole
(4b). The substance 3b (3.9 g, 0.013 mol) was placed in
a 50 mL two-necked flask equipped with a reflux con-
Fig. 1. Structures of benzo[b]thiophenes 1a–1e.
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POPOVA et al.
Fig. 2. Cytotoxicity of compounds 1a–1e against the human leukemia K562 cell line (MTS assay): (a) 24 h and (b) 72 h.
denser, magnetic stirrer, calcium chloride tube, and a
system for removing gaseous HCl. While cooling to
0–5°C, 20 mL of cooled thionyl chloride was added to
3b. Then the cooling bath was removed and the reaction
mixture was heated to 50°C (until the beginning of
the evolution of gaseous HCl). The reaction mixture
was stirred with heating for 8 h. Thionyl chloride was
evaporated under reduced pressure, the residue was
poured into 20 mL of cold water. The formed precipitate
was filtered off, washed with water until neutral, and
recrystallized from ethanol. Yield 2.05 g (75.6%), mp
extracted with chloroform (3×10 mL), the extract was
washed with saturated NaCl solution (2×20 mL), water
(2×20 mL), dried over Na2SO4, the solvent was removed
in vacuo. The residue was purified on SiO2 column
(10×3 cm) (eluent hexane/ethyl acetate, 4 : 1). Yield
0.220 g (88.3%), white solid, mp 118–119°С. 1H NMR
spectrum (СDCl3), δ, ppm: 3.26 m (2H, J = 4.9 Hz), 3.86 s
(3H), 3.88 m (2H, J = 4.9 Hz), 6.19 s (1H), 6.77 d.d (1Н,
Jortho = 8.8 Hz, Jmeta = 2.2 Hz), 7.01 d (1Н, J = 2.2 Hz),
7.49 d (1Н, J = 8.8 Hz). 13С NMR spectrum (СDCl3),
δС, ppm: 50.9, 55.5, 66.3, 99.4, 104.5, 110.6, 122.3,
124.8, 141.4, 157.8, 159.0. Mass spectrum (HRMS-ESI),
m/z: 234.0907 [М + H]+ (calculated for C13H15NO2S:
250.0896).
1
118–119°С. H NMR spectrum (СDCl3), δ, ppm: 3.88
(3H), 6.91 d.d (1H, Jmeta = 3.2 Hz, Jortho = 8.8 Hz), 7.57 d
(1H, Jmeta = 3.2 Hz), 7.62 d (1H, Jortho = 8.8 Hz), 9.07 s
(1H). 13С NMR spectrum (СDCl3), δС, ppm: 55.7,
112.5, 116.9, 117.4, 132.2, 134.6, 134.9, 159.1, 160.5.
Mass spectrum (HRMS-ESI), m/z: 270.9543 [М + H]+
(calculated for C13H15NO2S: 270.9535).
4-(4-Methoxybenzo[b]thiophen-2-yl)morpholine
(1d). The 1,2,3-thiadiazole 4b (0.271 g, 1.00 mmol),
K2CO3 (0.420 g, 3.00 mmol), morpholine (0.175 g,
2.00 mmol), anhydrous copper(II) acetate (0.032 g,
0.20 mmol) and 3 mL of DMF were placed in a
10 mL single-necked round-bottom flask equipped with
a magnetic stirrer, reflux condenser and bubble counter.
The reaction mixture was degassed three times (3–4 min
each, vacuum/argon). The reaction mixture was heated
in an argon atmosphere at 80°C for 4 h. The color of the
reaction mixture was observed to change from blue-green
to yellow. After completion of the reaction, the reaction
mixture was cooled and poured into 50 mL of saturated
ammonium chloride solution. The resulting solution was
Fig. 3. Cytotoxicity of compounds 1a–1e against the human
cervical carcinoma HeLa cell line (AlamarBlue assay): 24 h
(1а, 1c–1d), 24 and 72 h (1b).
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IN VITRO ACTIVITY OF ORGANOCHALCOGEN COMPOUNDS: II.
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4-(4-Nitrobenzo[b]thiophen-2-yl)morpholine (1e)
was prepared in accordance with the developed by us
method as described in [33]. Yield 12 %.
reagent was added into each well and incubated at 37°C
for 2 h. Finally the absorbance was recorded at 570 and
600 nm using 96 well plate reader “Multiskan GO”
(Thermo Fisher Scientific, USA). For coloured solutions
the protocol was modified as followed: the absorbance
was recorded directly before the addition of alamarBlue
reagent and measured values were further subtracted from
final absorbance.
Bioassay details. Tumor cell line. The erythro-
leukemia (K562) and cervical carcinoma (HeLa) cell
lines were obtained from the Bank of Cell Cultures of the
Institute of Cytology of the RussianAcademy of Sciences.
K562 cells were cultured in the RPMI-1640 medium
(Thermo scientific, USA) with the addition of 10% fetal
calf serum (FCS) (Thermo scientific, USA) and 40 μg/mL
gentamicin (Sigma, USA). HeLa cells were cultured in
the DMEM medium (Thermo scientific, USA) with the
addition of 10% fetal calf serum (FCS) (Thermo scientific,
USA) and 40 μg/mL gentamicin (Sigma, USA).
FUNDING
This work is supported by the Russian Science Foundation
under grant 20-15-00332.
CONFLICT OF INTEREST
No conflict of interest was declared by the authors.
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