P-tert-Butylcalix[8]arene catalysed synthesis of 3,5-dinitrothiophene scaffolds: Antiproliferative effect of some representative compounds on selective anticancer cell lines

Article abstract of DOI:10.1016/j.tetlet.2013.12.068

A new efficient protocol for the synthesis of 3,5-dinitrothiophene scaffolds was developed by using simple p-tert-butylcalix[8]arene in aqueous medium. Biological activities of some representative compounds were also studied to inhibit the cell growth on selective anticancer cell lines.

Full text of DOI:10.1016/j.tetlet.2013.12.068

Tetrahedron Letters 55 (2014) 996–1001
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
p-tert-Butylcalix[8]arene catalysed synthesis of 3,5-dinitrothiophene
scaffolds: antiproliferative effect of some representative compounds
on selective anticancer cell lines
Piyali Sarkar a, Samares Maiti b, Krishnendu Ghosh b, Sumita Sengupta (Bandyopadhyay) b, Ray J. Butcher c,
Chhanda Mukhopadhyay a,
⇑
a Department of Chemistry, University of Calcutta, 92 APC Road, Kolkata 700009, India
b Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, 92 APC Road, Kolkata 700009, India
c Department of Chemistry, Howard University, Washington, DC 20059, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new efficient protocol for the synthesis of 3,5-dinitrothiophene scaffolds was developed by using sim-
ple p-tert-butylcalix[8]arene in aqueous medium. Biological activities of some representative compounds
were also studied to inhibit the cell growth on selective anticancer cell lines.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 14 September 2013
Revised 14 December 2013
Accepted 17 December 2013
Available online 25 December 2013
Keywords:
3,5-Dinitrothiophene scaffolds
p-Tertbutylcalix[8]arene
Aqueous medium
Chromatography free
Antiproliferative effect
Organocatalysis has recently acquired an escalating interest
because of lower toxicity, ease of handling and higher ecological
compatibility compared to metal catalyst or organometallic
catalyst.1,2 Moreover, organocatalysts overcome the problems of
catalyst recovery (in case of co-solvent) for an organic reaction
taking place in aqueous medium.3
enabled us to avoid hazardous organic solvents (benzene, metha-
nol etc.), used in conventional pathways. This type of SNAr of the
thiophene
H
H
H
H
H
O
O
H
O
H
H
O
O
O
O
O
Congruent with our focus on the calixarenes in the context of
catalysis, it has been seen that calixarenes have a great impact as
a catalyst to promote organic reactions in aqueous as well as in
organic solvents for their specific architecture.4 The interesting
vase-like structure of calixarenes (having both hydrophobic as well
as hydrophilic groups) lends themselves in a plethora of organoc-
atalysis.5 Strategic introduction of functional substituents at the
upper or lower rim of calixarenes is needed to dissolve them in
water in order to catalyse an organic reaction in aqueous medium.
In contrast to acidic or basic group incorporation in a simple calix-
arene to convert it to a surfactant or a phase transfer catalyst,4,5 we
discovered an efficient and expeditious ligand-free and metal-free
method of synthesis of 3,5-dinitrothiophene scaffolds employing a
strategy where modification of p-tert-butylcalix[8]arene (1) was
not at all obligatory to be an organocatalyst in water. The approach
p-Tert-butylcalix[8]arene
Step 1:
Step 2:
Ac2O, AcOH,
CH3
CH3
Br
NBS, 50 °C,
1 hr, stirring
S
S
O
O
2
3
Conc. H2SO4
Fuming HNO3
(i) 0 C, 1 hr stirring
(ii) 25 C, 2 hr, stirring
O2N
CH3
Br
°
°
S
Br
NO2
S
O
3
4
⇑
Corresponding author. Tel.: +91 9433019610.
E-mail address: cmukhop@yahoo.co.in (C. Mukhopadhyay).
Scheme 1. Synthesis of 2-bromo-3,5-dinitrothiophene.
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.12.068

P. Sarkar et al. / Tetrahedron Letters 55 (2014) 996–1001
997
compounds was not frequently reported and the reaction condi-
tions were not standardized in most of the cases.6 Here we have
developed a viable high throughput synthesis of the 2-substi-
tuted-3,5-dinitrothiophene derivatives at room temperature
O2N
NO2
1 (10 mol%),
+
RNH2
HN
R
NO2
Br
NO2
H2O (2 ml),
S
S
25
°C, 2-2.5 h
stirring
5a (aromatic)
5b (aliphatic)
4
6a, 6b
NO2
NO2
NO2
NO2
HN
NO2
HN
NO2
HN
NO2
HN
NO2
S
S
S
S
H3C
H3C
CH3
6aa (87%)
OCH3
6ab (88%)
CH3
6ad (87%)
NO2
6ac (85%)
NO2
NO2
NO2
HN
NO2
HN
NO2
HN
NO2
HN
NO2
S
S
S
S
I
H3C
Cl
Cl
Br
6ae (85%)
6ag (81%)
6ah (80%)
NO2
6af (82%)
NO2
NO2
NO2
HN
NO2
HN
NO2
HN
S
NO2
HN
NO2 H3C
H3C
S
S
S
Cl
OCH3
6al (88%)
6ak (84%)
Cl
6ai (85%)
6aj (80%)
NO2
NO2
NO2
NO2
HN
S
NO2
HN
NO2
HN
NO2
HN
NO2
S
S
S
H3C
N
N
+_
(
)
N
6ap (85%)
6am (84%)
6an (87%)
6ao (87%)
NO2
NO2
NO2
NO2
HN
NO2
HN
NO2
HN
NO2
HN
NO2
S
S
S
S
N
6ar (85%)
6aq (86%)
6as (84%)
NO2
6at (83%)
NO2
NO2
NO2
HN
NO2
N
S
NO2
S
N
NO2
HN
S
NO2
S
O
6ba (83%)
6bb (81%)
Cl
6au (81%)
6av (86%)
NO2
NO2
NO2
NO2
N
NO2
N
NO2
N
S
NO2
HN
NO2
S
S
S
N
H3C
6bf (80%)
6bc (80%)
6bd (82%)
6be (87%)
Scheme 2. Calixarene 1 catalysed synthesis of some N-substituted products.

998
P. Sarkar et al. / Tetrahedron Letters 55 (2014) 996–1001
in 1H NMR as
J = 12.3 Hz), 3.51 (d, 8H, J = 12.9 Hz), 1.27 (s, 72H).
d 9.64 (s, 8H), 7.19 (s, 16H), 4.38 (d, 8H,
Table 1
Optimization of reaction conditions for the SNAr reactiona
NO2
NH
Efforts to date lean on the forwarding steps of privileged organ-
ic compounds towards medicinal chemistry. Hence our investiga-
tion throws light on the impact of these synthesized products on
this field.
NH2
NO2
Br
NO2
catalyst
S
+
NO2
S
solvent
25 ºC, stirring
In the present world, breast cancer is the major health concern
for women.7 Presently, some drugs are in use for treatment of
breast cancer,8 but they have several side effects. Till today, inven-
tion of a therapeutic drug for proper prevention and cure of breast
cancer is one of the most challenging problems. Nitrothiophene
derivatives possess cytotoxic activities against a variety of micro-
organisms like, bacteria (Escherichia coli, mycobacterium tuberculo-
sis),6a,9 fungus (Micrococcus luteus, Aspergillus niger),6a,9 as well as
parasites. Thus, slight amendment of functional groups of nitrothi-
ophene could lead to an anticancer drug. This influenced us to
study the antiproliferative effect of the nitrothiophene derivatives
on human breast adenocarcinoma, MCF7 cell line. The effect of
these compounds on MCF-7 cell line was compared with the effect
of some of them on A549 cell line which is adenocarcinoma of ba-
sal epithelial cells of the lung.
Primarily, our investigation was focussed on the synthesis of
the main building block, 2-bromo-3,5-dinitrothiophene (4). Nitra-
tion of 2-bromothiophene with acetyl nitrate followed by treat-
ment of nitrating mixture is the most prevalent approach for this
synthesis.6a,10 Overall yields of those previous methodologies were
too low for large scale production. So, to increase reaction effi-
ciency we adopted a new route, two step synthesis of 4. In the first
step 1-(5-bromothiophene-2-yl)ethanone (3) was prepared in high
yields (84%) from commercially available 2-acetylthiophene mod-
ifying previously reported procedure11 (Scheme 1) utilizing a facile
bromination with N-bromosuccinimide (NBS) in the presence of
excess acetic anhydride and 6–8 drops of acetic acid. The resulting
compound 3 was allowed to react with fuming nitric acid and
CH3
4
6aa
5a
CH3
Entry
Solvent
Catalyst (10 mol %)
Time (h)
Yieldb (%)
1
2
3
4
5
6
7
8
MeOH
MeOH
EtOH
Benzene
Benzene
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
—
—
—
—
—
—
—
—
12
24
24
12
24
12
24
48
60
24
24
24
24
24
24
48
1
28
38
39
19
36
17
35
36
36
36
37
38
36
43
47
47
62
86
87
87
9
—
10
11
12
13
14
15
16
17
18
19
20
Tetraethylene glycol
Polyethylene glycol
Pyrogallol
4-tert-Butylphenol
p-tert-Butylcalix[4]arene
p-tert-Butylcalix[6]arene
p-tert-butylcalix[6]arene
p-tert-Butylcalix[8]arene
p-tert-Butylcalix[8]arene
p-tert-Butylcalix[8]arene
p-tert-Butylcalix[8]arene
2
H2O
H2O
2.5
3
a
Reaction conditions: 4 and 5a were mixed in equivalent amount (mmol scale)
and stirred in 2 ml solvent.
b
Isolated yield.
(25 °C). In addition, this chromatography-free method saves both
solvent and time. The catalyst 1 showed its characteristic peaks
Figure 1. SEM image of nano ranged catalyst 1.
CATALYST 1
90
80
70
60
50
40
30
20
0
2
4
6
8 10 12 14 16
CATALYST LOADING (MOL%)
(b)
(a)
Figure 2. (a) Intensity distribution plot with particle size, (b) yields at different catalyst loading.

P. Sarkar et al. / Tetrahedron Letters 55 (2014) 996–1001
999
concentrated sulfuric acid mixture, to afford complete conversion
to give 91% isolated yield (overall yield of two steps being 76%).12
Then as the facile evolution of HBr had been an important is-
sue in substitution step (Scheme 2), percentage of conversion
might be increased by the addition of any inorganic acid or base
(like NaOH, HCl, K2CO3 etc.) in water medium. But the major dis-
advantage was that compound 4 could not display tolerance to a
broad range of inorganic acids or bases. In case of organic acids or
bases catalyst recovery was either quite impossible or needed
stringent legislation. So use of neutral catalyst in aqueous med-
ium was the key goal at the time of optimization of the reaction
conditions. p-Toluidine (5a) was used as the model substrate
during screening of reaction parameters (catalyst and solvent).
The results are summarized in Table 1. Use of general organic sol-
vents (MeOH, EtOH and benzene) could not give more than 40%
yields and same was reflected in case of water. Any mentionable
change was not observed upon stirring with compounds having a
number of aliphatic –OH (tetraethylene glycol, polyethylene gly-
col) or phenolic –OH (pyrogallol, 4-tert-butylphenol) groups for
24 h. To obtain a facile route towards the synthesis, calixarenes
were used. Noticeably, when the reaction proceeded with
p-tert-butylcalix[4] or [6]arene a slight increase in yield (43%
and 47%, respectively), was observed even though they contained
cavities. Under the same conditions, p-tert-butylcalix[8]arene
was successfully employed to furnish the corresponding products
in excellent yield (87%) in just 2 h.
This excellent yield is attributable to the hydroxyl group along
with its hydrophobic cavity. These results illustrate that the signif-
icant influence of the cumulative cavity size is readily apparent for
the reaction and catalyst 1 offers the optimal cavity size.
Catalyst 1 has 300–400 nm particle size estimated by SEM
image (Fig. 1). Slight coagulation changed the particle size to
999–1972 nm after 30 min stirring in water. It was measured from
the DLS data (Fig. 2 (a)) of that aqueous suspension given above.
The appropriate effective catalytic amount of 1 was determined
by the study of addition of different amounts of catalyst in reaction
medium. According to Figure 2 (b) there was a significant increase
in yield up to 10 mol % of catalyst but no increment was observed
after further increase in catalyst concentration. So, the optimized
effective catalytic amount was 10 mol %.
Figure 3. Crude NMR of reaction mixture in case of ‘6aa’ [‘S’ for starting material (S1 for 4 and S2 for 5a) and ‘P’ for product] (a) after 5 h without catalyst, (b) after 30 min with
10 mol % catalyst, (c) after 1 h with 10 mol % catalyst, (d) after 2 h with 10 mol % catalyst.
O2N
NO2
Br
1 (10 mol%),
+
RSH
S
R
NO2
NO2
H2O (2 ml),
25 °C, 2-2.5 h
stirring
S
S
4
7
8
NO2
NO2
NO2
NO2
S
NO2
S
NO2
S
NO2
S
NO2
S
S
S
S
CH3
Cl
8c (85%)
8b (83%)
8a (85%)
8d (82%)
Scheme 3. Calixarene 1 catalysed synthesis of some S-substituted products.

1000
P. Sarkar et al. / Tetrahedron Letters 55 (2014) 996–1001
The catalytic activity of 1 has been proved from the stacking
from the reactions of amines and the results are summarized in
diagram of crude 1H NMR spectra (Fig. 3).
Scheme 2. Use of electron rich aromatic amines including
quinoline-amines gave the best results whereas in the presence
of electron withdrawing groups (Cl, Br etc.) a slight decrease in
yield was observed. Reactions with aliphatic amines and benzylic
amines were also examined.
Catalyst recovery was possible by solvation of crude product in
ethyl acetate. The residue after filtration gave the catalyst which
was also reusable at least 5 times without hampering percentage
of yields.
Further investigations on diversification of the reaction using
different nucleophiles (amines, thiophenols and phenols) were
performed. Good (80%) to excellent yields (88%) were obtained
Introduction of thiophenols as nucleophile also provided the
dinitro thiophene derivatives in excellent yields (Scheme 3).
O
NH2
CH3
NO2
N
O
+
Br
NO2
O
HO
S
OH
N
OH
S
Br
O
HO
HO
H
H
OH
N
OH
OH
OH
H
O
OH
OH
OH
OH
OH
OH
CH3
NO2
NH
NO2
S
EtOAc
#
CH3
OH
O
N
O
NO2
OH
OH
OH
O
NO2
Br
N
OH
S
S
H
NH
O
H
H
H
H
H
H
OH
OH
H
N
O
O
O
H
OH
H
O
O
O
O
O
CH3
CH3
probable transition state
(H-bonds are not shown due to simplicity)
Figure 4. Probable mechanism of the SNAr reaction.
Table 2
IC50 values of 6bc, 6bf, 6be, 6aa, 6bd, 6ab, 6ah, 6ai, 8c & 8a
C
IC50
(lM) SE MCF-7
C
IC50
(l
M) SE MCF-7
C
IC50 (lM) SE A549
6bc
6bf
6be
6aa
6bd
109.27 9.49
88.70 0.55
66.75 6.95
10.50 0.68
78.69 14.49
6ab
6ah
6ai
8c
4.62 0.18
3.94 0.2
2.54 0.14
6.3 0.3
6ab
6ah
6ai
8c
27.38 2.07
45.06 1.81
38.87 3.88
28.89 0.86
32.03 0.93
8a
4.47 0.2
8a
C = Synthesized compounds.

P. Sarkar et al. / Tetrahedron Letters 55 (2014) 996–1001
1001
Considering reactions of phenols (p-cresol, p-tert-butylphenol
and 2-naphthol) though 30–35% of conversion took place in the
absence of base in water at 25 °C in 3–3.5 h, the product could
not be isolated by this chromatography-free method.
A549 cell line. Some compounds with aromatic moiety on N or
S-centre having a quite low IC50 value could behave as potential
drugs after further studies.
A plausible mechanism has been shown (Fig. 4) with the same
model substrate as in optimization table (Table 1). It would be ex-
pected that catalyst 1 forms host–guest type complex with both
the starting materials which favours the nucleophilic attack.
The hydrophobic part and hydrophilic phenolic –OH group cre-
ate such an environment which may impose proximity of the
nucleophile towards electrophilic centre (C-Br). Also the nitro
group of the thiophene ring seemed to be H-bonded with phenolic
–OH of 1.
Now to spotlight the biological activity, the anti-proliferative ef-
fect of the representative thiophene compounds (6bc, 6bf, 6be, 6aa,
6bd, 6ab, 6ah, 6ai, 8c & 8a) on human breast carcinoma cell line
MCF-7 was studied. To determine the anti-tumorogenic activity of
these above mentioned synthesized thiophene compounds on hu-
man breast cancer cell-line, MCF-7, Trypan blue exclusion assays
were performed and in accordance to that MCF-7 cells were treated
with different concentrations of the compounds for 24 h. The dose
dependent cytotoxicity of these compounds on MCF-7 cell line,
showing anti-proliferative effect resulting in a decrease in cell
viability in a dose dependent manner as compared to control, is
depicted as the bar diagram in Supplementary section (Fig. S3).
The chief findings of our ex vivo studies demonstrated that the
Acknowledgments
One of the authors (P.S.) thanks the Council of Scientific &
Industrial Research, New Delhi for her fellowship (JRF).
Supplementary data
Supplementary data (experimental procedure, spectral data and
crystallographic data in CIF) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.
2013.12.068.
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In conclusion, use of a simple calix[8]arene produced a number
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3, 5-dinitro-2-sulfanyl-thiophene derivatives with efficient yields
just in 2–2.5 h in eco-friendly solvent water. These compounds
showed antiproliferative effect on MCF-7 cell line as well as on
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