Synthesis and antiproliferative activity of imidazo[2,1-b][1,3,4]thiadiazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2014.08.032

A series of 2,5,6-substituted imidazo[2,1-b][1,3,4]thiadiazole derivatives have been prepared and were tested for antiproliferative activity on cancer cells at the National Cancer Institute. Results showed that molecules with a benzyl group at position 2, exhibited an increase in activity for the introduction of a formyl group at the 5 position. The compound 2-benzyl-5-formyl-6-(4-bromophenyl)imidazo[2,1-b][1,3,4]thiadiazole 22 has been chosen for understanding the mechanism of action by various molecular and cellular biology studies. Results obtained from cell cycle evaluation analysis, analysis of mitochondrial membrane potential and Annexin V-FITC by flow cytometric analysis, ROS production and expression of apoptotic and DNA-repair proteins suggested that compound 22 induced cytotoxicity by activating extrinsic pathway of apoptosis, however, without affecting cell cycle progression.

Full text of DOI:10.1016/j.bmcl.2014.08.032

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiproliferative activity of
imidazo[2,1-b][1,3,4]thiadiazole derivatives
Sujeet Kumar a,1, Vidya Gopalakrishnan b,1, Mahesh Hegde b, Vivek Rana a, Sharad S. Dhepe a,
Sureshbabu A. Ramareddy a, Alberto Leoni c, Alessandra Locatelli c, Rita Morigi c, Mirella Rambaldi c,
Mrinal Srivastava b, Sathees C. Raghavan b, Subhas S. Karki a,
⇑
a Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy, Bangalore 560 010, Karnataka, India
b Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, Karnataka, India
c Dipartimento di Farmacia e Biotecnologie, Università di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2,5,6-substituted imidazo[2,1-b][1,3,4]thiadiazole derivatives have been prepared and were
tested for antiproliferative activity on cancer cells at the National Cancer Institute. Results showed that
molecules with a benzyl group at position 2, exhibited an increase in activity for the introduction of a
Received 29 April 2014
Revised 9 August 2014
Accepted 12 August 2014
Available online xxxx
formyl group at the
5 position. The compound 2-benzyl-5-formyl-6-(4-bromophenyl)imidazo
[2,1-b][1,3,4]thiadiazole 22 has been chosen for understanding the mechanism of action by various
molecular and cellular biology studies. Results obtained from cell cycle evaluation analysis, analysis of
mitochondrial membrane potential and Annexin V-FITC by flow cytometric analysis, ROS production
and expression of apoptotic and DNA-repair proteins suggested that compound 22 induced cytotoxicity
by activating extrinsic pathway of apoptosis, however, without affecting cell cycle progression.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Antiproliferative agents
Imidazo[2,1-b][1,3,4]thiadiazoles
Cell-based screening assay
Hollow fiber assay
Acute toxicity
Apoptosis
Cancer is a disease of uncontrolled cell growth and is one of the
prime diseases to be cured at the present generation.1 Among
different cancers, leukemia, and lymphoma account for 8% of all
cancer.
Development of novel small molecule inhibitors against differ-
ent cancers, particularly leukemia and lymphoma is an area of
active investigation.2 Kinases, a target involved also in chronic
myelogenous leukemia, have been widely studied, leading to the
development of several inhibitors.3,4 Besides, novel small molecule
inhibitors against DNA repair is also investigated by us and others.
Recently, we have shown that small molecule inhibitor, SCR7, can
interfere with the binding of Ligase IV to the broken DNA and
prevent nonhomologous DNA end joining.5
Although there are many drugs currently under trial against can-
cer and particularly haematological malignancies, only few mole-
cules have emerged as promising drugs. To overcome this, it is
important to develop potent anticancer drugs with novel chemical
backbones. Gadad et al. in 1999 reported on the antitumor effects
of imidazo[2,1-b][1,3,4]thiadiazoles6 and in 2003 Terzioglu et al.
reported some hydrazone derivatives of 2,6-dimethyl imidazo
[2,1-b][1,3,4]thiadiazole-5-carbohydrazide as anticancer agents
against ovarian cancer cell line OVCAR.7 Moreover, a large number
of imidazothiadiazole derivatives have been reported to possess
diverse pharmacological properties such as antitubercular,8 anti-
bacterial,9 antifungal,10 anticonvulsant, analgesic,11 antisecretory,12
anti-inflammatory,13 cardiotonic,14 diuretic15 and herbicidal16
activities.
Considering the importance of this scaffold, we focused our
research on the synthesis of imidazo[2,1-b][1,3,4]thiadiazole
derivatives endowed with anticancer activity.17 Among them, com-
pound 4a (Chart 1) exhibited maximum cytotoxicity by inducing
extrinsic pathway of apoptosis without affecting the cell cycle.
On the basis of these results, using 4a as lead compound, we
synthesized a series of analogs in order to investigate their antipro-
liferative activity. Most of the planned compounds bear the same
substituent as compound 4a at the 2 position (benzyl group), and
at the 5 position (formyl group), whereas other derivatives bear
different groups such as cyclohexylethyl or ethyl at the 2 position,
Br and SCN at the 5 position. Furthermore, the substituent at the
para position of the 6-phenyl ring was changed. In order to study
the SAR, the antiproliferative activity of previously reported com-
pounds was also investigated.
⇑
Corresponding author.
E-mail address: subhasskarki@gmail.com (S.S. Karki).
Equal first authors.
We have synthesized a series of derivatives of imidazo[2,1-b]
[1,3,4]thiadiazole bearing the above mentioned substituents at 2,
1
http://dx.doi.org/10.1016/j.bmcl.2014.08.032
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
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2
S. Kumar et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
1683–1671 cmÀ1 for C@O stretching. Compounds 25–29 showed
CHO
N
S
vibration bands at 2164–2159 cmÀ1 for SCN in their respective IR
spectra.
N
F
N
In 1H NMR, the presence of singlet between d 8.64 and 7.88 ppm
for imidazole proton (C5AH) confirmed the cyclization of 2-amino-
5-substituted-1,3,4-thiadiazole 1–3 with respective phenacyl
bromide. All 5-substituted derivatives showed the absence of
C5AH in their respective spectra confirmed the substitution at
5th position. Compounds 19–24 showed a singlet between d
10.20 and 10.03 ppm for CHO proton. All the compounds showed
prominent signals for aromatic protons around d 8.64–6.94 ppm.
Bridge headed methylene proton at C2 appeared between d 4.48
and 4.21 ppm for 13–16, 19–22, 25–29 derivatives. For the com-
pounds 10, 17, and 23 alkyl protons appeared between d 1.80
and 0.93 ppm. The compounds 12 and 27 showed OCH3 proton
between d 4.33 and 3.87 ppm. Compounds 11, 12, 18 and 24
showed ethyl protons between d 1.49 and 1.31 ppm for CH3 as trip-
let and d 3.21–3.05 ppm for CH2 as quartet, respectively.
As a preliminary test, the compounds were tested at a single
high concentration (10À5 M) in the full NCI 60 cell panel (NCI 60
Cell One-Dose Screen). This panel is organized into subpanels
representing leukemia, melanoma and cancers of lung, colon,
kidney, ovary, breast, prostate and central nervous system. Only
compounds with pre-determined threshold inhibition criteria in
a minimum number of cell lines progress to the full 5-concentra-
tion assay. These criteria were selected to efficiently capture com-
pounds with anti-proliferative activity based on careful analysis of
historical Developmental Therapeutics Program (DTP) screening
data. The one-dose data is a mean graph of the percent growth of
treated cells (unpublished results).
17
Chart 1. Cytotoxic agent described as 4a in Ref.
.
5
and 6 positions. The 2-aralkyl/alkyl/-6-aryl-imidazo[2,1-b]
[1,3,4]thiadiazole derivatives 5–12, reported in Scheme 1, were
prepared by reaction of 2-amino-5-substituted-1,3,4-thiadiazoles
1–3, with the appropriate phenacyl bromide (4). The compounds
5–9 were previously reported in the literature18,19 and compound
12 reported in literature20 without spectral data. For the com-
pound 11, although commercial, we set up a procedure for its
synthesis.
The starting 2-amino-5-substituted-1,3,4-thiadiazoles were
prepared according to the literature,21–23 whereas the various
phenacyl bromides are commercially available or prepared by
bromination of the corresponding ketones in glacial acetic acid.
The 2-aralkyl/alkyl/-6-aryl-imidazo[2,1-b][1,3,4]thiadiazole deriv-
atives (5–8, 10 and 11) were subjected to electrophilic substitution
at position 5 with bromine in the presence of sodium acetate in
acetic acid to obtain the 5-bromo derivatives 13–18 in good yield.
The 2-benzyl-6-phenylimidazo[2,1-b][1,3,4]thiadiazole-5-carbal-
dehyde (19) was reported in a patent24 without spectral values.
For the aldehydes 20, 21, 23 and 24 although commercial, we
synthesized by means of the Vilsmeier reaction. Introduction of
thiocyanate functional group at the 5 position was carried out by
reaction with potassium thiocyanate in glacial acetic acid by drop
wise addition of bromine in glacial acetic acid to get 25–29 in good
yield.
Compounds 6, 15, 21, 22, 23, 25 and 27 were active at a high
concentration (10À5 M) and therefore entered the 5-concentration
test. They were dissolved in DMSO and evaluated using five con-
centrations at 10-fold dilutions, the highest being 10À4 M. Table 1
shows the results obtained, expressed as micromolar concentra-
tion at three assay endpoints: the 50% growth inhibitory power
(GI50), the cytostatic effect (TGI = Total Growth Inhibition) and
Structures of the synthesized compounds were established on
the basis of IR, 1H NMR and mass spectra and CHN data. All synthe-
sized compounds showed absorption bands ranging from 3172 to
3019 cmÀ1 for CAH aromatic stretching, 2999–2825 cmÀ1 for
CAH aliphatic stretching. Compounds 19–24 showed peaks at
O
N
EtOH
N
N
N
R'
+
R'
R
R
Na2CO3
S
NH2
Br
N
S
5a: R = benzyl, R' = H
1: R = benzyl
4
6b: R = benzyl, R' = Cl
2
: R = cyclohexylethyl
3: R = ethyl
7b
8b
9b
: R = benzyl, R' = NO2
: R = benzyl, R' = Br
: R = benzyl, R' = OCH3
10
: R = cyclohexylethyl, R' = F
11: R = ethyl, R' = Cl
12: R = ethyl, R' = OCH3
iii
i
ii
N
O
N
Br
S
N
N
N
N
N
N
N
R'
R'
R'
R
R
R
N
S
S
S
13
: R = benzyl, R' = H
19
25
: R = benzyl, R' = H
: R = benzyl, R' = H
20: R = benzyl, R' = Cl
21: R = benzyl, R' = NO2
14: R = benzyl, R' = Cl
15: R = benzyl, R' = NO2
26: R = benzyl, R' = Cl
27: R = benzyl, R' = OCH3
16
: R = benzyl, R' = Br
22
28
:
R = benzyl, R' = Br
: R = benzyl, R' = NO2
17: R = cyclohexylethyl, R' = F
18: R = ethyl, R' = Cl
23: R = cyclohexylethyl, R' = F
24: R = ethyl, R' = Cl
29: R = benzyl, R' = Br
Scheme 1. Synthesis of 2,5,6-substituted imidazo[2,1-b][1,3,4]thiadiazole derivatives. aRef. 18; bRef. 19. Reagents and conditions: (i) Br2, CH3COOH. (ii) DMF, POCl3,
80–90 °C—Na2CO3. (iii) KSCN, Br2, CH3COOH.
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S. Kumar et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 1
Nine subpanels at five concentrations: growth inhibition, cytostatic and cytotoxic activity (
l
M) of selected compounds
Compda
Modes
Leukemia
NSCLC
Colon
CNS
Melanoma
Ovarian
Renal
Prostate
Breast
MGMIDb
6
GI50
TGI
LC50
2.1
23.4
95.5
7.6
20.9
57.5
2.2
5.3
14.1
5.0
13.8
34.7
3.7
8.9
21.9
4.6
10.5
30.9
2.6
5.9
15.1
2.9
8.1
23.4
4.0
12.0
49.0
3.7
11.0
31.6
15
21
GI50
TGI
69.2
—
12.6
56.2
52.5
83.2
3.4
47.9
40.7
—
6.3
83.2
3.7
64.6
42.7
—
17.4
79.4
16.2
75.9
GI50
TGI
LC50
5.9
26.3
69.2
2.9
45.7
95.5
3.1
31.6
85.1
2.9
26.9
83.2
3.7
42.7
—
4.1
39.8
—
3.7
58.9
—
3.0
50.1
—
2.8
26.3
83.2
3.5
38.0
91.2
22
GI50
TGI
LC50
1.41
13.5
—
4.2
30.2
95.5
2.2
10.0
81.3
2.2
7.4
64.6
3.2
19.5
81.3
4.2
41.7
—
3.2
20.0
93.3
3.0
24.6
—
3.5
28.2
—
2.9
19.5
89.1
23
25
GI50
TGI
10.2
70.8
13.2
75.9
5.9
63.1
7.1
67.6
21.9
87.1
12.3
97.7
14.1
93.3
7.4
—
11.8
61.7
11.5
79.4
GI50
TGI
LC50
13.5
57.5
—
6.0
75.9
97.7
11.2
91.2
—
4.4
77.6
—
20.9
85.1
—
9.8
79.4
97.7
15.1
70.8
95.5
15.5
40.7
—
7.4
75.9
—
10.5
75.9
97.7
27
GI50
TGI
LC50
3.6
72.4
—
4.6
52.5
83.2
4.1
61.7
—
3.9
55.0
—
5.8
77.6
89.1
5.6
56.2
—
7.9
70.8
—
16.2
-
—
4.8
60.3
—
5.1
64.6
95.5
Std
GI50
3.2
14.5
10.0
21.4
15.5
18.6
15.9
10.0
16.6
12.6
The GI50 and TGI values are reported only when <100 M.
The compound exposure time was 48 h.
Std = Rifamycin SV.
a
Highest concd tested = 10À4
lM.
Mean Graph MIDpoint: average value for all cell lines tested; that is, mean GI50
b
.
A
Control
5 µM
1 µM
0.1 µM
0.5 µM
B
C
G1
G1
100
50
0
100
50
0
G2/M
S
SUB G1
G2/M
S
SUB G1
control
0.1
0.5
1
5
control 0.1
0.5
1
5
Concentration (µM)
Concentration (µM)
Figure 1. Cell cycle analysis of Reh and Nalm6 cells following 22-treatment. Reh cells were treated with 22 (0.1, 0.5, 1 and 5 lM) for 48 h, harvested, stained with propidium
iodide and subjected to flow cytometry. (A) Histogram obtained after FACS analysis for Reh cells. Histograms show the percentage of cells in the sub G1, G1, S and G2 phases
of the cell cycle. In the histogram M1 is G1, M2 is G2, M3 is S and M4 is sub G1 phase (B and C). Bar diagram representing the cell cycle progression of Reh (B) and Nalm6 (C)
cells. The data presented is derived from three independent experiments for REH and two independent experiments for Nalm6 and error bars are indicated. For each sample,
10,000 cells were used for sorting.
the cytotoxic effect (LC50). Rifamycin SV has been selected as the
reference drug since in the COMPARE analysis (http://dtp.nci.nih.
gov/docs/compare/compare.html) showed significant correlation
with these compounds. Moreover antitumor effects of Rifamycins
on a variety of human cancer-derived cells have been reported.25
For compounds 6, 15, 21 and 25, the 5-concentration test was
repeated and no significant differences were found. For these com-
pounds the data reported in Table 1 are the mean values between
the two experiments. Compound 6 was submitted to BEC (Biolog-
ical Evaluation Committee) for a possible future development.
The tested compounds showed a mean pGI50 range between
16.2 and 2.9 lM. The biological activity of the compounds is not
correlated with their calculated logP (Supplementary Table).
In light of the NCI-60 results, the following considerations may
be done: the benzyl substituent at the 2 position, chosen for most
of the derivatives synthesized, proved to be very interesting in fact
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S. Kumar et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
A
B
100
EARLY APOPTOSIS
LATE APOPTOSIS
80
60
40
20
0
0.38
0.03
5.51
0.50
0.52
0.42
51.32
98.60
6.05
5.23
88.41
43.03
C
XEF (1µM)
Cells alone
Cells (Annexin V-FITC + PI)
Cells + XEF (1 µm)
(Annexin V-FITC + PI)
Figure 2. Detection of apoptosis induced by 22 in Reh cells. After 22-treatment (1 lM) for 48 h, Reh cells were stained with annexin V-FITC and PI, and analyzed by flow
cytometry. (A) In each panel, the lower left quadrant shows cells which are negative for both annexin V-FITC and PI, lower right shows annexin V positive cells which are in
the early stage of apoptosis, upper left shows only PI positive cells which are dead, and upper right shows both annexin V/PI positive, which are in the stage of late apoptosis
or necrosis. The values mentioned in the quadrants show the percentage of early apoptotic cells or late apoptotic cells and necrotic cells in control as well as treated cases. (B)
Bar diagram showing percentage of cells in different apoptotic phases.
A
Cells alone
Cells + dye
2,4-DNP
0.1 µm
1 µM
5 µM
0.5 µm
B
100
Red
Green
80
60
40
20
0
C
2,4-DNP 0.1
0.5
1
5
Concentration (µM)
Figure 3. Detection of loss of mitochondrial membrane potential followed by 22-treatment. Reh cells were treated with 22 (0.1, 0.5, 1 and 5 lM) for 48 h, harvested, stained
with JC-1 dye, and analyzed by flow cytometry. 2,4-DNP treated Reh cells were served as positive control. (A) Dot plot representing JC-1 stained cells at different
concentration of 22. (B) Bar diagram showing ratio of red versus green fluorescent cells.
the introduction of an ethyl group gave compounds inactive in the
single concentration test and the cyclohexylethyl group was useful
only when a formyl group was present at the 5 position (23).
As far as the 5 position is concerned, Br is not favorable since
among the compounds with this substituent only compound 15
was active (GI50 16.2 lM) whereas there is an increase in activity
with the introduction of the SCN group since two compounds
entered the 5-concentration screen and compound 27 showed
GI50 = 5.1 lM with low toxicity (LC50 95.5 lM).
kg for the compound administered IP in DMSO. In light of this
result, BEC decided to submit 6 to the hollow fiber test.26 The com-
pound was not active enough to warrant further testing.
Effect on growth inhibition of leukemic cells: Based on above
results, cytotoxic effects of the most active compounds on leuke-
mic cell lines 6 and 22, were further investigated. Firstly, leukemic
cell lines, Reh (pre B-cell leukemia), K562 (chronic myelogenous
leukemia) and Nalm6 (pre B-cell leukemia), were treated with
increasing concentrations of compounds 6 (10, 50, 100 and
The formyl group gave good results: Besides the aforementioned
compound 23, even the 2-benzyl derivatives 21 and 22 deserve a
mention for their high growth inhibitory activity (mean GI50 3.5
250 lM) or 22 (0.1, 0.5, 1, 5 and 10 lM) and cell viability was
determined by trypan blue assay, after 24, 48 and 72 h of addition
of compounds (Fig. S1A and C).27 The DMSO treated cells served as
vehicle control as the compounds were dissolved in DMSO. Inter-
estingly, compound 22 showed a time-dependent inhibition on cell
viability in all the three leukemic cell lines, while the effect was
less in the case of compound 6 (Fig. S1A and C). The compound
and 2.9
lM, respectively) and low toxicity (mean LC50 91.2 and
89.1 M).
l
Further studies continued at the NCI where the maximum toler-
ated dose (MTD) was determined for 6. It was found to be 400 mg/
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S. Kumar et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
22 exhibited increased cell death at concentrations of 5
l
M and
stained with propidium iodide and subjected to FACS analysis. His-
togram of vehicle control showed a standard cell cycle pattern,
which includes M1 (G1) and M2 (G2) separated by M3 (S) phase.
The M4 (Sub G1) phase (mostly dead cells) was not prominent.
Upon addition of compound, a concentration dependent change
was observed in the cell cycle pattern. Most of the cells were accu-
mulated in subG1, while number of cells in G2/M, G1 and S phases
were reduced indicating cell death in presence of 22 (Fig. 1A–C).
The results were comparable in both the cell lines used. However,
there was no evidence for cell cycle arrest. Hence, these results fur-
ther confirm that 22 induce cytotoxicity in leukemic cells, without
leading to a cell cycle arrest.
above in the cell lines, Reh and Nalm6 (Fig. S1C and D). The growth
of control cells and DMSO treated cells were comparable (data not
shown). Thus results suggest that 22 induce cytotoxicity in human
leukemic cells in a dose- and time-dependent manner. The effect of
6 and 22 on leukemic cells was further verified using MTT assay. In
order to do this, cells treated with compounds 6 (10, 50, 100 and
250 lM) or 22 (0.1, 0.5, 1, 5 and 10 lM) were harvested after 48
and 72 h, and subjected to the assay. The results showed that com-
pound 6 was less effective even at higher concentrations in the cell
lines tested, while 22 showed significant cytotoxic effect at 5 and
10
lM concentrations (Fig. S1B and D).
Cytotoxic effect of 22 (3 M) on leukemic cells, Reh, and normal
l
The induction of apoptosis was further studied and quantified
by performing annexin V-FITC/PI double-staining. Reh cells were
cell 293T (human kidney epithelial cells) were compared using
live-dead cell assay. Interestingly, results showed that 22 induced
more cell death in Reh than 293T cells in both 24 and 48 h time
points (Fig. S1E and F). Trypan blue assay further confirmed the
above observation (data not shown). Thus our results suggest that
the cytotoxic effect of 22 is less in normal cells compared to that in
cancerous cells.
Since compound 22 inhibited the cell proliferation, we per-
formed Fluorescence-Activated Cell Sorting (FACS) analysis to
determine its effect on cell cycle progression. Reh and Nalm6 cells
harvested after treatment with 22 (1
double stained and subjected for flowcytometric analysis. Dot plot
results showed that at 1 M, 43.03% of cells were in early apoptotic
lM for 48 h) were then
l
stage (stained only by annexin V-FITC), while 51% were in late
apoptotic stage, which was 10 fold higher than the control
(Fig. 2A and B). Comparable results were obtained when the exper-
iment was repeated. These results showed that 22 induce translo-
cation of phosphatidyl serine leading to apoptosis.
Mitochondrial membrane potential plays an important role in
induction of apoptosis. Apoptosis results in the loss of potential
were harvested after 48 h of treatment with 22 (0.1, 0.5, 1, 5 lM),
A
0.09%
0.36%
0.09%
Negative Control
Control
10 min
15 min
0.06%
0.12%
45.02%
Positive Control
30 min
60 min
0.77%
0.86%
0.33%
B
Negative control
100 nM
Control
500 nM
0.62%
0.41%
0.28%
27.26%
Positive control
1 µM
5 µM
10 µM
Figure 4. Determination of intracellular ROS production in Reh cells following treatment with 22. (A) Reh cells treated with 22 (1 lM) for different time points were used for
testing the formation of intracellular ROS by flow cytometry. (B) Reh cells were treated with 22 at different concentrations (0.1, 0.5, 1, 5 and 10 lM) for 10 min and assayed for
ROS production. H2O2 treated cells were used as positive control. Cells alone were used as negative control.
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6
S. Kumar et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
which can be detected by staining with JC-1 dye.28 JC-1 is a fluores-
cent cation that incorporates into mitochondrial membrane and
form aggregates in healthy nonapoptotic cells. This aggregation
changes the fluorescence properties of JC-1 dye leading to a shift
from green to red fluorescence which has absorption/emission
maxima of 585/590 nm. However, in apoptotic cells, the mitochon-
drial membrane potential collapses and the JC-1 cannot accumu-
late in the mitochondria and remains in the cytoplasm, resulting
in a green fluorescent monomeric form which has absorption/
emission maxima of 510/527 nm. Result showed that most of the
DMSO treated cells exhibited red fluorescence. Interestingly 22
analysis showed that cleavage of PARP in a concentration depen-
dent manner, suggesting its activation (Fig. 5). Since we could
observe PARP cleavage, we were interested in testing the activation
of CASPASE 3, 9 and 8. Immunoblot analysis showed cleavage of
caspase 3 in a concentration dependent manner. Besides, we also
observed reduction in the expression of BID, a pro-apoptotic
protein, particularly at 1 lM of compound 22 (Fig. 5). CASPASE 3
is activated in apoptotic cells both during extrinsic and intrinsic
pathways. Since there was no distinct change in the expression of
CASPASE 9 (involved in intrinsic pathway of apoptosis), activation
of CASPASE 8 (involved in extrinsic pathway of apoptosis) was
probed. Results showed cleavage of CASPASE 8 (Fig. 5) and up-reg-
ulation of another death receptor signaling protein FAS. Thus, our
data suggest that compound 22 induce cell death by activating
death receptor pathway of apoptosis.
In this Letter, we report the synthesis and antiproliferative
activity of a series of substituted imidazo[2,1-b][1,3,4]thiadiazoles.
Compound 22 in NCI-60 test showed a remarkable antiproliferative
activity and selected for further studies. Data obtained in different
leukemia cell lines revealed that compound 22 induces cytotoxic-
ity in dose- and time-dependent manner, induces translocation
of phosphatidyl serine of cells to outer membrane, and alters mito-
chondrial membrane potential and the expression of apoptotic and
DNA-repair proteins. These results together with the activation of
caspase 3 and 8 indicate that compound 22 induced cytotoxicity
through extrinsic pathway of apoptosis.
treated cells emitted green fluorescence in
a
concentration-
M most of
dependent manner (from 1 M onwards) and at 5
l
l
the cells showed green fluorescence which was similar to that of
positive control 2,4-DNP. Thus, our results indicate that 22 altered
mitochondrial membrane potential in a dose-dependent manner
which further suggests the activation of apoptosis (Fig. 3A and B).
The over production of ROS is an indicator of cellular response
leading to DNA damage and apoptosis. Since the compound 22
treatment resulted in apoptosis, we wondered whether there is
ROS production in cell upon treatment with 22 in order to address
this question FACS analysis was performed to examine ROS pro-
duction after treating with 22 either at different time points
(Fig. 4A) or at different concentrations of the compound (Fig. 4B).
Result showed that there was no ROS generation in both conditions
tested.
Since the above experiments suggested activation of apoptosis
we were interested in studying expression of different apoptosis
and DNA damage repair proteins by western blot analysis. The cell
lysate was prepared from Reh cells after treating with compound 22
Acknowledgments
We gratefully acknowledge the support for this research effort
from All India Council for Technical Education (AICTE), New Delhi,
India (Ref. No. 8023/BOR/RID/RPS-169/2008-09). The authors are
also grateful to NMR research centre, Indian Institute of Science,
Bangalore, India for recording NMR spectra for our compounds.
We are grateful to the National Cancer Institute (Bethesda, MD)
for the antitumor tests.
(0.5
lM and 1 lM) for 48 h. Western blot analysis showed that at
1
lM of 22, KU70 is downregulated (Fig. 5). KU70 is a DNA end
binding protein involved in nonhomologous end joining, one of
the DNA double-strand break repair pathways. KU70 also has
cytoprotective functions that suppresses apoptosis.29 Hence the
reduced expression of KU70 promotes apoptosis. Poly (ADP-ribosyl)
polymerase (PARP) is a single strand break repair enzyme known to
be cleaved by caspase 3 upon apoptotic activation. Immunoblotting
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.08.
032.
0
0.5
1
μM
KU 70
kDa 116
86
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