Synthesis and in vitro anti-breast cancer activity of some novel 1,5-benzothiazepine derivatives

Article abstract of DOI:10.2298/JSC110715219A

The title compounds 3a-j, substituted 1,5-benzothiazepines, were synthesized by the condensation of variously substituted chalcones 1 and 2-aminothiophenol 2 via conventional as well as non-conventional inorganic solid support microwave irradiation method

Full text of DOI:10.2298/JSC110715219A

J. Serb. Chem. Soc. 77 (6) 725–731 (2012)
UDC 547.53–304.9+547.78:615277–188
JSCS–4303
Original scientific paper
Synthesis and in vitro anti-breast cancer activity of
some novel 1,5-benzothiazepine derivatives
*
K. L. AMETA , NITU S. RATHORE and BIRESH KUMAR
Department of Chemistry, FASC, Mody Institute of Technology and Science,
Deemed University, Lakshmangarh, Rajasthan-332311, India
(Received 15 July, revised 19 December 2011)
Abstract: The title compounds 3a–j, substituted 1,5-benzothiazepines, were
synthesized by the condensation of variously substituted chalcones 1 and
2-aminothiophenol 2 via conventional as well as non-conventional inorganic
solid support microwave irradiation methods. The non-conventional protocol
offers several advantages, such as simple procedure, fast reaction rate, mild
reaction conditions and improved yields, compared to conventional methods.
The structures of the products 3a–j were established by elemental analysis,
1
FTIR, H-NMR, 13C-NMR and mass spectroscopic studies. The synthesized
compounds were also evaluated for their cytotoxicity against the human breast
cancer cell line MDA-MB-435, with some exhibiting in vitro anti-breast cancer
activity.
Keywords: 1,5-benzothiazepines; 2-aminothiophenol; chalcones; anti-breast
cancer activity.
INTRODUCTION
Disease poses a major threat to human beings and scientists are fighting to
find solutions in the form of various medications. When the era of synthetic
drugs began, it opened thousand doors for the development of various synthetic
molecules with potential action. Chalcones are the well known intermediates for
the synthesis of various differently sized bioactive heterocycles, such as isoxazo-
les, pyrimidines, pyrazoles, etc., which have been reported to possess various
1,2
3
4
biological activities, such as antimicrobial, anti-inflammatory, antimalarial,
antioxidant and antitubercular.
5
6
Among these chalcone-derived heterocycles, benzothiazepines are well known
important nitrogen and sulphur containing compounds that possess a broad spec-
7,8
9
trum of biological activities, such as antimicrobial,
-HIV, anti-cytotoxic, anticancer,
anti-convulsant, anti-
10
11
12,13
14
DPPH free radical scavenger and
inhibition of cholinesterases, ureases and α-glycosidases. Due to biological acti-
*Corresponding author. E-mail: klameta77@yahoo.co.in
doi: 10.2298/JSC110715219А
725
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AMETA, RATHORE and KUMAR
15–21
22–24
vities, various conventional
and non-conventional
approaches have been
developed for the synthesis of 1,5-benzothiazepines derivatives.
Cancer is one of the most dangerous, fast propagating diseases of the present
century with quite high mortality rates even in developed countries. The situation
is even worse in undeveloped countries due to a lack of knowledge, poverty and
25
the non-availability of quality drugs.
As part of our continuing research in the development of new bioactive he-
terocycles containing nitrogen and sulphur atoms, simple and convenient me-
thods for the transformation of chalcones into 1,5-benzothiazepines by conven-
tional and non-conventional methods are described herein. The non-conventional
approaches were based on the observation that organic solvents in classical pro-
cedures are used in much larger quantities than the solutes they carry. Further-
26–30
more, the employment of solid supports in conjugation with microwaves
leads to high yields, remarkable reaction rate enhancements, and high catalytic
activities with the optimum utilisation of energy.
The anti-breast cancer activity of the synthesised compounds was also inves-
tigated.
EXPERIMENTAL
Materials, method and instrumentation
All the chemicals were of AR grade and were obtained from Sigma–Aldrich and Merck.
Melting points (m.p.) were determined in open capillaries on a Veego (VMP-PM) melting
point apparatus and are uncorrected. The microwave-assisted reactions were realised using a
microwave synthesizer model CATA-R (Catalyst Systems, Pune, India), operating at 700 W,
generating 2450 MHz frequency. The purity of the compounds was routinely checked by thin
layer chromatography (TLC) with silica gel-G (Merck). The instruments used for obtaining
1
the spectroscopic data were: IR – FTIR spectrophotometer Bruker Alpha-Zn-Se; H-NMR
(CDCl3, 500 MHz) and 13C-NMR (CDCl3, 125 MHz), FT–NMR spectrometer Bruker AV III.
The elemental analysis was realised on a Carlo Erba 1108 analyzer and the obtained values
were within the ±0.5 % of the theoretical values. Column chromatography was performed on
silica gel (Merck, 60–120 mesh).
General procedure for the preparing of 1,5-benzothiazepines 3a–j
Condensation of the required chalcone (1) and 2-aminothiophenol (2) afforded the
desired products 3a–j in good yield, as shown in Scheme 1.
Conventional solution phase method. Compound 1 (0.010 mol) was dissolved in the
minimum quantity of ethanol. To this, 2-aminothiophenol 2 (0.010 mol) was added and the
resulting reaction mixture was refluxed for 3 h at 60–70 °C. Then, the mixture was acidified
with 5–6 drops of glacial acetic acid and heating was continued for a further 4–5 h. After
cooling, the content was poured onto crushed ice. The mixture was filtered and the solid was
purified by recrystallization from methanol to afford compound 3a–j.
Non-conventional solid phase method. Compound 1 (0.010 mol) was dissolved in the
minimum quantity of DMF. To this, 2-aminothiophenol 2 (0.010 mol) and different inorganic
solid supports (4.0 g) was added. The resulting mixture was uniformly mixed with a glass rod
and air dried to remove the solvent. The absorbed material was irradiated inside the micro-
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SYNTHESIS AND ANTI-CANCER ACTIVITY OF 1,5-BENZOTHIAZEPINES
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wave synthesizer for a specific time at medium power level (700 W). After completion of re-
action (monitored by TLC), the reaction mixture was cooled to room temperature and the
product was extracted with methanol (2×20 mL). Removal of the solvent and subsequent
recrystallization with methanol resulted in analytical samples of 3a–j. For the biological eva-
luation, the experimental drugs were solubilised in DMF solvent at 400-fold the desired final
maximum test concentration and stored frozen prior to use.
Data of synthesized compounds are are given in Supplementary material.
Scheme 1. Preparation route of novel substituted 1,5-benzothiazepines (3a–j) from variously
substituted chalcones (1) and 2-aminothiophenol (2) via conventional and non-conventional
methods. The conventional method involved the 7–8 h reflux whereas the non-conventional
method involved 5–7 min under microwave irradiation.
Biological evaluation
Protocol for in vitro anti-cancer screening. Human tumour breast cancer cell line
(MDA-MB-435) was used in this study. The cytotoxic activity was measured in vitro for the
newly synthesized compounds using the sulforhodamine B stain (SRB) assay method.31,32 The
cell lines were grown in RPMI 1640 medium containing 10 % foetal bovine serum and 2 mM
L-glutamine. For the present screening experiment, cells were inoculated into 96-well microtiter
plates. For the present screening experiment, cells were inoculated into 96 well microtiter
plates in 90 µL at plating densities according to the SRB assay method. After cell inoculation,
the microtiter plates were incubated at 37 °C, 5 % CO2, 95 % air and 100 % relative humidity
for 24 h prior to the addition of the experimental drugs. After 24 h, one plate of the cell line
was fixed in situ with trichloroacetic acid TCA, to represent a measurement of the cell
population for the cell line at the time of drug addition (Tz). At the time of drug addition, an
aliquot of frozen concentrate was thawed and diluted to 10 times the desired final maximum
test concentration with complete medium containing test compound at a concentration of 10-3
M. Additional three 10-fold serial dilutions were made to provide a total of four drug concen-
trations plus control. Aliquots of 10 µl of these different drug dilutions were added to the
appropriate microtiter wells already containing 90 µl of medium, resulting in the required final
drug concentrations. Different concentrations of the compound under test (10-7, 10-6, 10-5 and
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AMETA, RATHORE and KUMAR
10-4 M) were added to the cell monolayer. Triplicate wells were prepared for each individual
concentration.
After compound addition, end point measurement was performed using the “Elisa” test
under standard conditions for taking the absorbance at particular wavelength (690 nm refe-
rence wavelength). The percent growth was calculated on a plate-by-plate basis for the test
wells relative to the control wells. The percent growth was expressed as the ratio of the ave-
rage absorbance of the test well to the average absorbance of the control wells times 100. Using
the six absorbance measurements (time zero (Tz), control growth (C), and test growth in the
presence of drug at the four concentration levels (Ti)), the percentage growth was calculated at
each of the drug concentration levels. Percentage growth inhibition was calculated as:
100((Ti–Tz)/(C–Tz)) for concentrations for which Ti ≥ Tz, i.e., (Ti–Tz) is positive or zero, or
100 [(Ti–Tz)/Tz] for concentrations for which Ti < Tz, i.e., (Ti–Tz) is negative.
The dose response parameters were calculated for each test compound. The growth inhi-
bition of 50 % (GI50) was calculated from 100((Ti–Tz)/(C–Tz)) = 50, which is the drug concen-
tration resulting in a 50 % reduction in the net protein increase (as measured by SRB staining)
compared to the control cells during the drug incubation.
RESULTS AND DISCUSSION
The aim of the present work was to design and synthesize some new 1,5-
-benzothiazepine derivatives carrying biologically active thiazepine moieties that
were expected to have anti-breast cancer activity. Substituted chalcones 1 were
reacted with 2-aminothiophenol 2 to give 1,5-benzothiazepine derivatives 3a–j.
The characterization data of the synthesized compounds are tabulated in Table I.
The structures of the compounds 3a–j were determined on the basis of analytical
and spectroscopic data (given in the Supplementary material to this paper). Thus,
the IR spectra showed bands at around 1606 (C=N), 665 (C–S) and 862 (C–Br).
1
The H-NMR spectra revealed the presence of a doublet at around δ 3.32 ppm,
corresponding to the CH group, and a triplet at around δ 4.98 ppm, corres-
2
ponding to the CH group.
In view of the immense utility of eco-friendly synthetic approaches, im-
proved syntheses of the 1,5-benzothiazepines were performed under microwave
TABLE I. Characterization data of the synthesized compounds
Reaction time
MW, min Conventional, h MW Conventional
Yield, %
Entry Molecular formula M.p., °C
3a
3b
3c
3d
3e
3f
3g
3h
3i
C21H15Br2NO2S
C21H14Br2ClNO2S 82–83
C21H13Br2Cl2NO2S 89–90
C21H14Br2FNO2S
C21H14Br2FNO2S
C21H14Br2ClNO2S 90–91
C22H17Br2NO3S
C22H17Br2NO2S
C22H17Br2NO4S
C21H14Br3NO2S
80–81
5.0
6.0
5.5
5.0
6.0
6.0
5.0
7.0
7.0
6.0
6.0
6.5
7.0
7.0
5.0
6.0
6.5
7.5
6.0
7.5
78.0
82.0
84.0
83.0
86.0
80.0
89.0
81.0
79.0
90.0
64.0
69.0
69.0
71.0
71.0
69.0
68.0
68.0
68.0
72.0
81–82
87–88
82–83
85–86
78–79
93–94
3j
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SYNTHESIS AND ANTI-CANCER ACTIVITY OF 1,5-BENZOTHIAZEPINES
729
irradiation. In this context, the suitability of different solid supports, i.e., acidic
alumina, basic alumina, neutral alumina, montmorillonite K 10 and K CO , were
2
3
examined. From the results given in Table II, it is clear that basic alumina was
the most adaptable and simplest catalyst and gave good yields in the synthesis of
the 1,5-benzothiazepines 3a–j. Finally in order to evaluate the use of microwave
irradiation, the 1,5-benzothiazepines were also synthesised under conventional
conditions. The yields and reaction times of the two preparation methods are
compared in Table I.
TABLE II. Comparative study for the synthesis of 3a–j by non-conventional MW using dif-
ferent solid supports
Exp. No.
Medium
MW Power, W Time, min Temp., °C Yield, %
1
2
3
4
5
Alumina acidic
Alumina basic
Alumina neutral
700
700
700
700
700
9–10
5–7
11–12
9–11
12–13
83
92
81
80
72
57
80
64
73
69
Montmorillonite K 10
Potassium carbonate
The newly synthesized compounds were evaluated for their in vitro cyto-
toxicity against a human breast cancer cell line (MDA-MB-435) with adriamycin
(ADR) as the reference drug. The relationship between control in % growth and
molar drug concentration was plotted to obtain the control growth of the breast
cancer cell line (MDA-MB-435). The response parameter calculated was the
GI value, which corresponds to the concentration required for a 50 % inhibition
50
of cell viability (Table III).
TABLE III. In vitro anticancer screening of the synthesised compounds against human breast
cell line (MDA-MB-435); growth relative to the control (%), each value is the mean ± SD of
three experiments
Molar drug concentration, mol dm-3
Compound
GI50 / μM
10-7
10-6
10-5
10-4
3a
3b
3c
3d
3e
3f
3g
3h
3i
100.00±0.00
99.66±0.39
100.00±0.00
90.95±8.00
97.64±2.68
99.88±0.20
99.43±1.00
98.58±2.45
95.26±4.56
100.00±0.00
100.00±0.00
97.44±2.60
96.72±3.18
99.99±0.01
95.62±4.60
98.75±2.01
98.97±1.96
96.74±3.26
91.80±7.10
100.00±0.00
37.54±4.95
68.84±6.13
33.62±3.67
88.24±8.34
77.61±7.35
79.60±4.71
66.40±3.14
84.74±7.44
78.39±3.18
95.98±3.56
–41.23±16.63
–46.19±4.42
–26.90±5.27
80.39±7.35
–42.53±4.99
–38.30±6.27
–33.50±6.83
–41.03±10.39
–47.86±5.12
–15.75±18.88
28.0
32.2
29.5
>100
33.9
36.1
34.8
35.7
31.9
45.7
<0.1
3j
ADR
–22.63±8.93 –39.32±16.81 –48.07±19.26 –57.02±13.25
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AMETA, RATHORE and KUMAR
CONCLUSIONS
By comparing different inorganic solid supports, it was concluded that basic
alumina was the most suitable support for the synthesis of 1,5-benzothiazepines
via a non-conventional method, giving the products in good yields. It was ob-
served from the obtained results that most of the tested compounds showed some
anti-breast cancer activity, as compared to the reference drug. These preliminary
results of biologically screening of the tested compounds give an indication of
the possible importance of the thiazepine moiety in anti-breast cancer compounds
and give an encouraging framework in this field that may lead to the discovery of
potent anticancer agents.
SUPPLEMENTARY MATERIAL
Аnalytical and spectral data of synthesized compounds are available electronically from
http://www.shd.org.rs/JSCS/, or from the corresponding author on request.
Acknowledgements. The authors are thankful to Prof. B. L. Verma, retd. professor of che-
mistry, M. L. S. University Udaipur and the Dean, FASC, MITS University, India, for their
constant encouragement during this work. Authors are also thankful to the Head, Sophisti-
cated Analytical Instrument Facility, Indian Institute of Technology, Madras, India, for spec-
tral analysis, and the Director, Tata Memorial-ACTRC (The Advanced Centre for Treatment, Re-
search and Education in Cancer) Mumbai, India, for anti-breast cancer activity determinations.
И З В О Д
СИНТЕЗА НОВИХ ДЕРИВАТА 1,5-БЕНЗОТИАЗЕПИНА И ЊИХОВА
IN VITRO АКТИВНОСТ ПРЕМА КАНЦЕРУ ДОЈКЕ
K. L. AMETA, NITU S. RATHORE и BIRESH KUMAR
Department of Chemistry, FASC, Mody Institute of Technology and Science, Deemed University,
Lakshmangarh, Rajasthan-332311, India
Супституисани деривати 1,5-бензотиазепина (3а–ј) синтетисани су реакцијом конден-
зације супституисаних халкона (1) и 2-аминофенола (2) класичним и новим поступцима под
условима микроталасног озрачивања. Нова метода има неколико предности у поређењу са
класичном, као што су једноставнији поступак, краће реакционо време, блажи реакциони
услови, побољшан принос. Структуре производа 3а–ј утврђене су на основу елементалне
1
анализе FT-ИЦ, H-NMR и 13C-NMR спектроскопије и масене спектрометрије. Испитана је
цитотоксична активност синтетисаних једињења према ћелијској линији канцера дојке
MDA-MB-435.
(Примљено 15. јула, ревидирано 19. децембра 2011)
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