Inhibitors of apoptosis in testicular germ cells: Synthesis and biological evaluation of some novel IBTs bearing sulfonamide moiety

Article abstract of DOI:10.1016/j.ejmech.2012.11.015

Pifithrin-α, a known p53 inactivator, inhibits p53-dependant mitochondrial cell death induced by toxins or γ-radiation. It has been found that aromatic IBT analogues of PFT-α are more cytoprotective and nonpeptide-based, isatin sulfonamides selectively in

Full text of DOI:10.1016/j.ejmech.2012.11.015

European Journal of Medicinal Chemistry 59 (2013) 203e208
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Inhibitors of apoptosis in testicular germ cells: Synthesis and biological evaluation
of some novel IBTs bearing sulfonamide moiety
,
a,
Navneet Chandak a, Jitender K. Bhardwaj b, Rajnesh K. Sharma b , Pawan K. Sharma
*
*
a Department of Chemistry, Kurukshetra University, Kurukshetra 136119, Haryana, India
b Reproductive Physiology Laboratory, Department of Zoology, Kurukshetra University, Kurukshetra 136119, Haryana, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2012
Received in revised form
9 November 2012
Accepted 12 November 2012
Available online 20 November 2012
Pifithrin-
a
, a known p53 inactivator, inhibits p53-dependant mitochondrial cell death induced by toxins
are more cytoprotective and
or -radiation. It has been found that aromatic IBT analogues of PFT-
g
a
nonpeptide-based, isatin sulfonamides selectively inhibit caspases 3 and 7, responsible for mitochondrial
mediated apoptosis. Therefore, we envisioned the synthesis of novel IBTs 4 and 5 bearing sulfonamide
moiety and observed the mitigating effects of these IBTs in rescue of malathion induced apoptosis in
testicular germ cells of goat. Two IBTs (4b; R ¼ CH3, 5b; R1 ¼ Cl) showed very high survival rate of cells
whereas IBT 4f (R ¼ NO2) showed some exceptional behaviour by increasing the apoptosis. These IBTs
nullify the cytotoxic effect of malathion on mitochondria, following p53-independent pathway.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Imidazobenzothiazoles (IBTs)
Sulfonamide
Inhibitors of apoptosis
Testicular germ cells
1. Introduction
apoptosis, mitochondria releases a number of pro-apoptotic
proteins such as Apoptois Inducing Factor (AIF), Smac/DIABLO,
Cytochrome C etc. which are involved in apoptosome formation
and its formation leads to activation of caspases, also called caspase
cascade, responsible for induction of apoptosis [6e8]. Of late, p53
inactivators have gained much attention of chemists as well as
oncologists due to their possible applications in neurodegenerative
disorders, cancer chemotherapy as well as diseases related to sig-
Apoptosis is a programmed cell death that affects the single
scattered cells in the midst of a normal living tissue and plays a key
role in regulating development, homeostasis, and immune defence
by clearing redundant or abnormal cells in organisms. A delicate
balance between pro-apoptotic and anti-apoptotic mechanisms
determines whether a cell death signal can activate the execution of
the apoptotic program. In humans, hypo-apoptosis as well as
hyper-apoptosis, both can lead to severe pathological conse-
quences. For example, suppression of the apoptotic machinery is
responsible for severe autoimmune diseases and is a hallmark of
cancer [1,2] whereas high rate of apoptosis sometimes leads to
tissue as well as nerve degeneration resulting into severe neuro-
logical disorders such as Alzheimer’s disease, Parkinson’s disease,
stroke etc [3,4]. Apoptosis sometimes enhances due to over-
expression of genes, such as p53, which are involved in controlling
the whole apoptotic mechanism in our body or can be induced
by toxins or ionizing radiations [5]. Mitochondria plays an
important role in controlling cell death and follows either
p53-dependent or -independent pathway of apoptosis. During
nalling pathways [3,9,10]. Recently, a small molecule, Pifithrin-
also known as PFT- , was originally identified as a leading
compound of the known p53 inactivators after broad screening of
10,000 compounds which inhibits cell death from -radiation as
a,
a
g
well as protecting mice from lethal genotoxic stress, focal cortical
ischaemic injury, and neuronal excitotoxic damage [9,11,12]. This
protection by PFT-a has been attributed to inhibition of p53
transactivation via diminishing p53-dependent and -independent
mitochondria-mediated cell death in vivo and in vitro [9,13]. It’s
a matter of great debate from a long time within chemists and
biologists that whether ring opened PFT-
form. NMR experiments have proved many times this fact that PFT-
as well as its ring opened analogues cyclize in situ in protic
solution and it has recently been reported that cyclized analogues
of PFT- , known as imidazo[2,1-b]benzothiazoles (IBTs) are more
a is active or its ring closed
a
a
* Corresponding authors. Tel.: þ91 9466066674, þ91 9416457355; fax: þ91 1744
238277.
E-mail addresses: rkszookuk@yahoo.co.in (R.K. Sharma), pksharma@kuk.ac.in,
talk2pawan@gmail.com (P.K. Sharma).
potent p53 inactivators than reference as well as opened analogues
[14,15]. Another report confirmed that the aromatic IBT analogues
are more protective against dexamethasone as well as g-radiation-
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.015

204
N. Chandak et al. / European Journal of Medicinal Chemistry 59 (2013) 203e208
induced apoptosis [5] but at the same time, they were found to be
unable to modulate the transcriptional activity of p53, indicating
the role of these aromatic IBTs towards p53-independent apoptosis
[16]. It is well known in the literature that malathion exerts its
cytotoxic effects by the induction of apoptosis via direct effect on
mitochondria [17]. Thus, the chemoprotection of cells from
apoptosis induced by toxins or ionizing radiation can be important
for biodefense and in the treatment of acute injuries. It has been
discovered that targeting only two capsases 3 and 7 alone are
sufficient for blocking apoptosis and first time reported
nonpeptide-based caspase inhibitors, the isatin sulfonamides are
finding their selectivity towards caspases 3 and 7 by interacting
primarily with the S2 subsite, and not binding in the caspase
primary aspartic acid binding pocket (S1), suggesting a big role of
sulfonamide group [18,19]. Appreciating these findings, in the
present investigation, we synthesized two series of small hetero-
cycles, imidazobenzothiazoles (IBTs, 4 and 5) bearing sulfonamide
bromoacetylcoumarin 3 in refluxing 2-methoxyethanol followed
by neutralization using aqueous ammonia yielded corresponding
imidazobenzothiazoles, IBTs 4 and5 respectively [5,22]. Spectral data
(1H NMR, 13C NMR, IR and mass) of the newly synthesized
compounds were in full agreement with the proposed structures. In
general, both IBTs 4 and 5 were identified by the presence of char-
acteristic imidazo proton (C4eH) which appeared during cyclization
resonating in the range
d
8.76e9.11 as a singlet in 1H NMR. All
coumarin IBTs (5aec) were showing characteristic C]O stretch of
lactone nature at 1720 cmꢀ1 in FT-IR.
2.2. Apoptosis inhibition studies
All the newly synthesized IBTs (4ae4f and 5ae5c) were evalu-
ated for their in vitro inhibition of apoptosis induced by malathion
in testicular germ cells. Results obtained are reported in Table 1. Out
of IBTs (4ae4f) with phenyl analogues, 4b having a methyl
substituent in the phenyl ring was found to be the most potent
moiety, structurally related to PFT-a (Fig. 1) and observed the
mitigating effects of novel IBTs in rescue of malathion induced
apoptosis in testicular germ cells of goat (Capra hircus). To the best
of our knowledge, this is in itself the first study of IBTs towards
inhibition of apoptosis in testicular germ cells, particularly on
a domestic animal.
inhibitor exhibiting 64
ꢁ
4.35% average cell survival when
compared with controlled % average cell survival (CACV; 67 ꢁ 4.58)
and malathion tested % average cell survival (41 ꢁ 6.24). Three
compounds namely 4a (R ¼ H), 4d (R ¼ Cl) and 4e (R ¼ Br) showed
mild activity exhibiting % average cell survival 50 ꢁ 5.29, 56 ꢁ 3.00
and 52 ꢁ 4.58 respectively. Compound 4c (R ¼ F) showed weaker
protective ability exhibiting 43 ꢁ 4.00% average cell survival.
Surprisingly, compound 4f (R ¼ NO2) was found to be promoter of
apoptosis exhibiting 39 ꢁ 2.00% average cell survival. Out of IBTs
(5ae5c) with coumarin analogues, 5b (R0 ¼ Cl) was found to be the
most potent inhibitor exhibiting 61 ꢁ 6.00% average cell survival
whereas compounds 5a (R0 ¼ H) and 5c (R0 ¼ Br) showed weaker
cytoprotective activity exhibiting 46 ꢁ 4.35 and 48 ꢁ 5.56% average
cell survival, respectively. Figs. 2 and 3 are the fluorescent photo-
graphs of testicular germ cells in control group showing pinkish
normal germ cells with their intact cell membranes and round
nuclei, taken at ꢂ 100 and ꢂ 400 resolution respectively. Fig. 4
is the fluorescent photograph of malathion treated testicular
germ cells showing apoptotic germ cells with bright green
condensed nuclei, taken at ꢂ 400 resolution. Figs. 5 and 6 are the
fluorescent photographs of malathion treated testicular germ cells
2. Results and discussion
2.1. Chemistry
The synthetic route used to synthesize the target imidazo-
benzothiazoles, IBTs 4 and 5 is outlined in Scheme 1. The starting
material, 2-aminobenzothiazole-6-sulfonamide 1 was synthesized
in two steps because the conventional method of preparing 2-
aminobenzothiazoles in a single step from appropriate anilines by
treatment with Br2/KSCN unfortunately failed to give 1 [20]. There-
fore, the required starting material was prepared from sulfanilamide
by first converting into 4-aminosulfonylphenylthiourea using
ammonium thiocyanate in acidic medium followed by its oxidative
cyclization with Br2 in chloroform [21]. Reaction of 1 with either
appropriate p-substituted phenacyl bromide 2 or 6-substituted-3-
S
NH
S
N
O
N
N
PFT-α
Active Form
H2NO2S
H2NO2S
S
S
N
N
N
N
R'
O
O
R
4
5
Fig. 1. Structure of Pifithrin-a (PFT-a) and the general structure of related analogues bearing sulfonamide moiety.

N. Chandak et al. / European Journal of Medicinal Chemistry 59 (2013) 203e208
205
H2NO2S
S
NH2
N
1
O
O
R'
R
Br
Br
2
O
O
3
2-Methoxyethanol,
reflux, 4h
2-Methoxyethanol,
reflux, 4h
H2NO2S
H2NO2S
S
S
N
N
N
N
R'
O
O
R
IBTs 4
IBTs 5
Compound 2 & 4
a
b
c
d
e
f
R
H
CH3
F
Cl
Br
NO2
Compound 3 & 5
a
b
c
R'
H
Cl
Br
Scheme 1. Synthesis of novel imidazobenzothiazoles (IBTs) 4 & 5.
supplemented with compound 4b and 5b respectively showing
decrease in number of apoptotic germ cells, taken at ꢂ 400 reso-
lution. Results are summarized graphically in Fig. 7.
cell death. Notably, all of the compounds that were tested for anti-
apoptotic activity were analogous to the cyclized form of PFT-
a
.
Critical features to make an IBT suitable for better anti-apoptotic
activity were aromatization, position as well as nature of the
substituent introduced whether electron releasing or withdrawing,
and surface area of newly designed IBT. We shall discuss each point
one by one in the same order as written above. It has already been
reported in the literature that aromatic analogues of IBTs are highly
active [5]. Therefore, we synthesized sulfonamide bearing aromatic
3. Discussion and conclusions
All the newly synthesized IBTs (4ae4f and 5ae5c) were evalu-
ated for their in vitro inhibition of apoptosis. It has been reported
that PFT-a does not remain in uncyclized state in protic solution
and get converted into ring closed form, well known as IBT, after
few hours as confirmed by NMR experiments and exhibits anti-
apoptotic activity [14,15]. Therefore, additional compounds were
synthesized to optimize the IBT scaffold for making other isosteric
analogues for anti-apoptotic activity in the search of a more potent
inhibitor as well as to suppress mitochondrial mediated cell death
induced by toxins. As a primary screen for biological activity, each
compound was tested for its ability to prevent malathion induced
Table 1
Malathion induced inhibition of apoptosis in testicular germ cells.
Compound no.
Group R or R’
Average cell survival
ꢁ standard deviation (%)a
4a
4b
4c
4d
4e
4f
5a
5b
H
CH3
F
50 ꢁ 5.29*
64 ꢁ 4.35*
43 ꢁ 4.00*
56 ꢁ 3.00*
52 ꢁ 4.58*
39 ꢁ 2.00
46 ꢁ 4.35*
61 ꢁ 6.00*
48 ꢁ 5.56*
41 ꢁ 6.24*
67 ꢁ 4.58
Cl
Br
NO2
H
Cl
Br
5c
MACVb
CACVc
*p < 0.05 (t test).
Fig. 2. Fluorescent photograph of testicular germ cells showing pinkish normal germ
cells with their intact cell membranes and round nuclei in control group
(arrow). ꢂ 100. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
a
Compound tested at 10 mM. Malathion toxic at 10 mM, tested at 5 mM.
MACV: Malathion tested % average cell survival.
CACV: Control % average cell survival.
b
c

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N. Chandak et al. / European Journal of Medicinal Chemistry 59 (2013) 203e208
Fig. 3. Fluorescent photograph of testicular germ cells showing pinkish normal germ
cell with their intact cell membranes and round nuclei in control group (arrow). ꢂ 400.
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
Fig. 5. Fluorescent photograph of malathion treated testicular germ cells supple-
mented with compound 4b showing decrease in number of apoptotic germ
cells. ꢂ 400.
inactivators [14,15]. As reported in the literature that larger the
surface area of an IBT, better would it show anti-apoptotic activity
[5]. Therefore, we also tried to observe the role of surface area in
controlling the anti-apoptotic activity of IBT by making some
coumarin analogues (5ae5c) and compared the results with its
phenyl analogues (4a, 4d and 4e) respectively. The results do not
show a general trend as the anti-apoptotic activity of 5b was
significant while that of 5a and 5c was very weak. This indicates
that replacement of a phenyl ring with a coumarin moiety under-
standably involves varying electronic effects besides increasing the
surface area. After observing and comparing the anti-apoptotic
results, we concluded that IBTs 4b and 5b exhibited significant
protection against malathion induced apoptosis and may be
promising for further development. It is reported in the literature
analogues and found significant results. However, in our case, we
noticed that nature of the substituent plays a big role in deciding
the anti-apoptotic activity. In general, more the electron releasing
nature of the substituent, better the anti-apoptotic activity of that
IBT analogue. The trend with respect to substituent on the phenyl
ring of IBTs 4 and coumarin ring of IBTs 5 was found to be
CH3 > Cl > Br > H > F > NO2. In case of NO2, this trend became so
enormous that our IBT 4f showed some unusual behaviour by
increasing the apoptosis suggesting that R and R0 should not be
strongly electron withdrawing in nature to make an IBT suitable for
apoptotic inhibition. Literature survey shows that NO2 substituted
IBTs don’t serve as better anti-apoptotic [5] but they are better p53
Fig. 4. Fluorescent photograph of malathion treated testicular germ cells showing
apoptotic germ cells with bright green condensed nuclei (arrow). X 400. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
Fig. 6. Fluorescent photograph of malathion treated testicular germ cells supple-
mented with compound 5b showing decrease in number of apoptotic germ
cells. ꢂ 400.

N. Chandak et al. / European Journal of Medicinal Chemistry 59 (2013) 203e208
207
4.1.1. 2-Phenylimidazo[2,1-b][1,3]benzothiazole-7-sulfonamide
80
70
60
50
40
30
20
10
0
Average Cell Survival (%)
(4a)
Yield: 70%; m.p. 294e296 ꢃC (d); IR (KBr) cmꢀ1: 3302 & 3148 (m,
NeH stretch), 1589 (s, C]N stretch), 1543 (m, NeH bend), 1497 (s,
C]C stretch), 1335 & 1165 (s, SO2 stretch); 1H NMR (DMSO-d6,
300 MHz): d 8.82 (s, 1H, imidazo C4eH), 8.55 (s, 1H, Ar), 8.15 (d, 1H,
J ¼ 8.4 Hz, Ar), 8.00 (d, 1H, J ¼ 8.4 Hz, Ar), 7.88 (d, 2H, J ¼ 7.2 Hz, Ar),
7.49 (s, ex, 2H, SO2NH2), 7.44 (m, 2H, Ar), 7.32 (m, 1H, Ar); 13C NMR
(DMSO-d6, 75.5 MHz): d 148.5, 147.3, 141.0, 134.1, 133.8, 130.3, 129.2,
128.0, 125.2, 124.9, 123.3, 114.0, 109.7; DART-MS: m/z 330.07
(M þ H)þ, C15H11N3O2S2Hþ calcd. 330.02.
4.1.2. 2-(4-Methylphenyl)imidazo[2,1-b][1,3]benzothiazole-7-sulfo-
namide (4b)
Yield: 62%; m.p. 300e302 ꢃC (d); IR (KBr) cmꢀ1: 3356 & 3148 (s,
NeH stretch), 1589 (s, C]N stretch), 1543 (m, NeH bend), 1497 (s,
C]C stretch), 1335 & 1157 (s, SO2 stretch); 1H NMR (DMSO-d6,
Compound/Control
Fig. 7. Inhibition of malathion induced apoptosis.
300 MHz): d 8.76 (s, 1H, imidazo C4eH), 8.54 (s, 1H, Ar), 8.13 (d, 1H,
J ¼ 8.4 Hz, Ar), 8.00 (d, 1H, J ¼ 8.4 Hz, Ar), 7.77 (d, 2H, J ¼ 7.8 Hz, Ar),
7.49 (s, ex, 2H, SO2NH2), 7.25 (d, 2H, J ¼ 7.8 Hz, Ar), 2.33 (s, 3H, CH3);
that apoptosis induced by irradiations is p53-dependent while
dexamethasone induced apoptosis is not [23]. Data suggests that
these IBTs nullify the apoptotic (cytotoxic) effect of malathion on
mitochondria which is not following p53-dependant pathway,
confirming a recent report [16] because IBT 4f (p-NO2) which is
expected to be better p53 inactivator, is not showing promising
anti-apoptotic activity. Thus, these novel IBTs will provide deeper
insight in understanding the mechanism of apoptosis via mito-
chondrial pathway in a much better way and may thus influence
therapeutic strategy.
13C NMR (DMSO-d6, 75.5 MHz):
d 148.3, 147.4, 141.0, 137.2, 134.1,
131.2, 130.2, 129.8, 125.2, 124.9, 123.4, 113.9, 109.2, 21.2 (CH3);
DART-MS: m/z 344.08 (M þ H)þ, C16H13N3O2S2Hþ calcd. 344.04.
4.1.3. 2-(4-Fluorophenyl)imidazo[2,1-b][1,3]benzothiazole-7-sulfo-
namide (4c)
Yield: 65%; m.p. 256e258 ꢃC (d); IR (KBr) cmꢀ1: 3325 & 3155 (m,
NeH stretch), 1597 (s, C]N stretch), 1551 (m, NeH bend), 1497 (s,
C]C stretch), 1327 & 1165 (s, SO2 stretch); 1H NMR (DMSO-d6,
300 MHz): d 8.80 (s, 1H, imidazo C4eH), 8.55 (s, 1H, Ar), 8.13 (d, 1H,
J ¼ 8.4 Hz, Ar), 8.00 (d,1H, J ¼ 8.4 Hz, Ar), 7.88e7.93 (m, 2H, Ar), 7.49
(s, ex, 2H, SO2NH2), 7.29 (t, 2H, J ¼ 8.7 Hz, Ar); 13C NMR (DMSO-d6,
4. Experimental protocols
1
75.5 MHz):
d
162.0 (d, JCF ¼ 244.6 Hz), 148.5, 146.1, 140.7, 134.0,
3
All reactions were carried out under atmospheric pressure.
Melting points were determined on glass slide using MR-VIS
LABINDIA VISUAL Melting Range Apparatus and are uncorrected.
The infrared (IR) spectra were recorded on ABB MB 3000 DTGS FT-
IR instrument using the KBr pellet technique. 1H NMR and 13C NMR
spectra were recorded in pure DMSO-d6 on Bruker NMR spec-
trometers at 300 MHz and 75.5 MHz respectively using tetrame-
thylsilane (TMS) as internal standard. Chemical shifts are expressed
130.2, 130.0, 127.1 (d, JCF ¼ 8.3 Hz), 124.8, 123.1, 115.9 (d,
2JCF ¼ 22.6 Hz), 113.9, 109.2; DART-MS: m/z 348.06 (M þ H)þ,
C
15H10FN3O2S2Hþ calcd. 348.01.
4.1.4. 2-(4-Chlorophenyl)imidazo[2,1-b][1,3]benzothiazole-7-sulfo-
namide (4d)
Yield: 75%; m.p. 312e314 ꢃC (d); IR (KBr) cmꢀ1: 3348 & 3256 (m,
NeH stretch), 1589 (s, C]N stretch), 1543 (m, NeH bend), 1497 (s,
C]C stretch), 1327 & 1157 (s, SO2 stretch); 1H NMR (DMSO-d6,
in
d, ppm. Mass spectra (DART-MS) were recorded on a JEOL-
AccuTOF JMS-T100LC Mass spectrometer having a DART (Direct
Analysis in Real Time) source in ESþ mode. The purity of the
compounds was checked by 1H NMR and thin layer chromatog-
raphy (TLC) on silica gel plates using a mixture of chloroform and
methanol as eluent. Iodine or UV lamp was used as a visualizing
agent. Abbreviations ‘s’ for singlet, ‘d’ for doublet, ‘m’ for multiplet,
‘ex’ for exchangeable proton are used for NMR assignments and ‘s’
for strong, ‘m’ for medium for IR assignments. ‘d’ stands for
decomposition in melting point data.
300 MHz): d 8.87 (s, 1H, imidazo C4eH), 8.56 (s, 1H, Ar), 8.12 (d, 1H,
J ¼ 6.6 Hz, Ar), 8.01 (d, 1H, J ¼ 6.6 Hz, Ar), 7.88 (d, 2H, J ¼ 6.3 Hz, Ar),
7.50e7.55 (m, 4H, SO2NH2, Ar); 13C NMR (DMSO-d6, 75.5 MHz):
d
148.7, 146.0, 141.2, 134.1, 132.9, 132.3, 130.3, 129.3, 126.9, 125.0,
123.5, 114.0, 110.2; DART-MS: m/z 364.03 (M
H)þ,
15H10ClN3O2S2Hþ calcd. 363.99.
þ
C
4.1.5. 2-(4-Bromophenyl)imidazo[2,1-b][1,3]benzothiazole-7-sulfo-
namide (4e)
Yield: 60%; m.p. 298e300 ꢃC (d); IR (KBr) cmꢀ1: 3333 & 3240 (m,
NeH stretch), 1643 (s, C]N stretch), 1558 (m, NeH bend), 1528 (s,
C]C stretch), 1311 & 1157 (s, SO2 stretch); 1H NMR (DMSO-d6,
4.1. General procedure for the synthesis of imidazobenzothiazoles
(IBTs), 4aee and 5aec
300 MHz): d 8.88 (s, 1H, imidazo C4eH), 8.55 (s, 1H, Ar), 8.13 (d, 1H,
A mixture of 2-aminobenzothiazole-6-sulfonamide (1, 4 mmol)
and appropriate p-substituted phenacyl bromide or 6-substituted-
3-bromoacetylcoumarin (2 or 3, 4.8 mmol) was refluxed in 2-
methoxyethanol (15 mL) for 4 h. The reaction mixture was cooled
to room temperature and neutralized by adding cold aqueous
ammonia (10%, 40 mL) slowly with vigorous stirring till yellow or
orange coloured solid precipitated out which was filtered, washed
with excess of water followed by cold ethanol (20 mL) and crys-
tallized either from chloroformemethanol (3 : 1; 4aee) or from
DMF-ethanol (1 : 1; 4f, 5aec) to afford the target IBTs.
J ¼ 8.1 Hz, Ar), 8.01 (d, 1H, J ¼ 8.1 Hz, Ar), 7.83 (d, 2H, J ¼ 7.8 Hz, Ar),
7.64 (d, 2H, J ¼ 7.8 Hz, Ar), 7.50 (s, ex, 2H, SO2NH2); 13C NMR
(DMSO-d6, 75.5 MHz): d 148.7, 146.1, 141.2, 134.0, 133.2, 132.1, 130.3,
127.2, 125.0, 123.4, 120.8, 114.0, 110.3; DART MS m/z 407.99
(M þ H)þ, C15H10BrN3O2S2Hþ calcd. 407.93.
4.1.6. 2-(4-Nitrophenyl)imidazo[2,1-b][1,3]benzothiazole-7-sulfon-
amide (4f)
Yield: 82%; m.p. > 330 ꢃC; IR (KBr) cmꢀ1: 3348 & 3140 (s, NeH
stretch), 1597 (s, C]N stretch), 1543 (m, NeH bend), 1504 (s, C]C

208
N. Chandak et al. / European Journal of Medicinal Chemistry 59 (2013) 203e208
stretch), 1335
&
1165 (s, SO2 stretch); 1H NMR (DMSO-d6,
with antibiotics (200-unit having concentration of penicillin
100 IU/mL and streptomycin 100 IU/mL) in CO2 incubator (5% CO2,
95% humidity, 38 ꢃC). The cells were plated at a density of 105 cells/
300 MHz): d 9.11 (s, 1H, imidazo C4eH), 8.57 (s, 1H, Ar), 8.31 (d, 2H,
J ¼ 8.1 Hz, Ar), 8.16 (d, 1H, J ¼ 8.4 Hz, Ar), 8.11 (d, 2H, J ¼ 8.1 Hz, Ar),
8.02 (d, 1H, J ¼ 8.4 Hz, Ar), 7.53 (s, ex, 2H, SO2NH2); 13C NMR
mL and pre-incubated with 10
DMSO for 30 min before induction of apoptosis. Then the apoptosis
was induced with 5 M malathion in DMSO. The cell apoptosis was
mM of each tested compound in
(DMSO-d6, 75.5 MHz):
d 149.5, 146.6, 145.0, 141.5, 140.4, 133.9,
130.5, 125.8, 125.0, 124.7, 123.5, 114.3, 112.6; DART-MS: m/z 375.06
m
(M þ H)þ, C15H10N4O4S2Hþ calcd. 375.01.
assayed by Acridine Orange staining after 6 h of culture duration
under the fluorescence microscope (Olympus, Japan) using 500e
525 nm filters [5,24]. Normal cells were identified by their intact
cell membranes and round nucleus with scanty chromatin. Cells
with bright green condensed nuclei (intact or fragmented) were
interpreted as apoptotic.
4.1.7. 2-(2-Oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3]benzothia-
zole-7-sulfonamide (5a)
Yield: 85%; m.p. > 330 ꢃC; IR (KBr) cmꢀ1: 3271 & 3140 (m, NeH
stretch), 1720 (s, lactone C]O stretch), 1605 (s, C]N stretch), 1497
(s, C]C stretch), 1311 & 1157 (s, SO2 stretch); 1H NMR (DMSO-d6,
300 MHz): d 8.87 (s, 1H, imidazo C4eH), 8.62 (s, 1H, Ar), 8.51 (s, 1H,
Acknowledgements
coumarin C4eH), 8.35 (d, 1H, J ¼ 8.4 Hz, Ar), 7.94 (d, 1H, J ¼ 8.4 Hz,
Ar), 7.80 (d, 1H, J ¼ 7.5 Hz, coumarin), 7.49e7.56 (m, 3H, SO2NH2,
coumarin), 7.37 (d, 1H, J ¼ 8.1 Hz, coumarin), 7.30 (t, 1H, J ¼ 7.2 Hz,
Defence Research and Development Organization (DRDO), New
Delhi is thankfully acknowledged for financial support in the form
of a research project. Navneet Chandak is grateful to the Council of
Scientific and Industrial Research (CSIR), New Delhi for the award of
senior research fellowship. The authors are thankful to Sophisti-
cated Analytical Instrument Facility, Central Drug Research Insti-
tute, Lucknow for Mass spectra.
coumarin); 13C NMR (DMSO-d6, 75.5 MHz):
d 158.9, 152.7, 141.4,
140.5, 137.3, 134.1, 131.9, 130.3,129.1, 125.2, 124.9, 123.3,120.4,119.7,
116.3,114.6, 114.3; DART-MS: m/z 398.12 (M þ H)þ, C18H11N3O4S2Hþ
calcd. 398.01.
4.1.8. 2-(6-Chloro-2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3]
benzothiazole-7-sulfonamide (5b)
Yield: 80%; m.p. > 330 ꢃC; IR (KBr) cmꢀ1: 3348 & 3163 (m, NeH
stretch), 1720 (s, lactone C]O stretch), 1597 (s, C]N stretch), 1497
(s, C]C stretch), 1327 & 1157 (s, SO2 stretch); 1H NMR (DMSO-d6,
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.11.015.
300 MHz): d 8.93 (s, 1H, imidazo C4eH), 8.64 (s, 1H, Ar), 8.53 (s, 1H,
coumarin C4eH), 8.39 (d, 1H, J ¼ 8.4 Hz, Ar), 7.94e7.98 (m, 2H, Ar,
coumarin C5eH), 7.57 (d, 1H, J ¼ 8.7 Hz, coumarin), 7.51 (s, ex, 2H,
SO2NH2), 7.44 (d, 1H, J ¼ 8.4 Hz, coumarin); 13C NMR (DMSO-d6,
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