Synthesis of imidazo[2,1-b][1,3,4]thiadiazole-chalcones as apoptosis inducing anticancer agents

Article abstract of DOI:10.1039/c4md00228h

A series of new imidazo[2,1-b][1,3,4]thiadiazole-chalcones were synthesized by Claisen-Schmidt condensation and evaluated for their cytotoxic activity against various human cancer cell lines. These compounds showed moderate to appreciable antiproliferativ

Full text of DOI:10.1039/c4md00228h

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Synthesis of imidazo[2,1-b][1,3,4]thiadiazole–
chalcones as apoptosis inducing anticancer
Cite this: DOI: 10.1039/c4md00228h
agents†
Ahmed Kamal,*ac Vangala Santhosh Reddy, Karnewar Santosh, G. Bharath Kumar,
a
b
a
a
a
c
a
Anver Basha Shaik, Rasala Mahesh, Sumit. S. Chourasiya, Ibrahim Bin Sayeed
and Srigiridhar Kotamraju*b
A
series of new imidazo[2,1-b][1,3,4]thiadiazole–chalcones were synthesized by Claisen–Schmidt
condensation and evaluated for their cytotoxic activity against various human cancer cell lines. These
compounds showed moderate to appreciable antiproliferative activities. Interestingly, compounds like
11a and 11b exhibited significant cytotoxic activity with IC50 values ranging from 0.65 to 2.25 mM in
certain cancer cell lines. The structure–activity relationship (SAR) studies reveal that 3,4,5-trimethoxy
group containing compounds showed superior cytotoxic activity against selected cancer cell lines
Received 3rd June 2014
Accepted 14th August 2014
0 1
compared to other chalcones. These compounds showed G /G phase arrest, apart from activation of
caspase-3 and 8 in DU-145 cells. The growth inhibitory effect of these compounds was associated with
a decrease in cell cycle regulatory protein cyclin D1 and increase in cyclin dependent kinase inhibitors
like Cip1/p21 and Kip1/p27.
DOI: 10.1039/c4md00228h
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aryl ring were carried out extensively. Moreover, chalcone
derivatives in which the ring B is replaced by a heterocyclic ring
Chalcones represent an important class of natural products, have been systematically investigated.
Introduction
18,19
which are intermediate precursors of all avonoid based
1–4
compounds. Chalcones are interesting simple molecules due
to their varied biological activities including anti-inamma-
5
6,7
8
9
tory, anti-malarial,
antituberculosis, anti–HIV, anti-
and inhibition of several enzymes like
aromatase, topoisomerase, and certain protein-tyrosine kinases
10–13
proliferative,
14
like cyclin-dependent kinases. They display signicant bio-
logical activities due to their potential interactions with
different proteins related to cell proliferation and apoptosis.
Recent results show that chalcone derivatives can induce
15–17
apoptosis in cancer cells,
and most of them contain either
hydroxy or methoxy groups in both the rings (A and B). The
structure–activity relationship (SAR) studies of trimethoxy
chalcones (TMC) (II) reveal that the 3,4,5-trimethoxyphenyl
group in ring A is thought to be essential for the anticancer
activity. Some of the recent studies towards the improvement of
their cytotoxicity and the impact of different substituents in the
a
Medicinal Chemistry & Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500007, India. E-mail: ahmedkamal@iict.res.in
b
Centre for Chemical Biology, Indian Institute of Chemical Technology, Hyderabad,
Telangana-500607, India
c
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education
Fig. 1 Structures of anticancer agent doxorubicin (I), trimethoxy-
chalcone (TMC) (II), imidazothiadiazole–oxindole (III), imidazothiazole
guanylhydrazone derivative (IV) and imidazothiadiazole–chalcones
(7a–m and 11a–d).
and Research, Hyderabad, Telangana-500034, India
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4md00228h
1
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Imidazo[2,1-b]thiadiazoles (III and IV) (Fig. 1) are familiar
Whereas, imidazothiadiazole–chalcone derivatives (11a–d)
compounds and their derivatives are known to exhibit a wide were obtained by the Claisen–Schmidt condensation of appro-
20
spectrum of biological activities like antimicrobial, antibac- priate substituted imidazothiadiazole ethanones (9a–d) upon
21
22
23
terial, antitubercular and anticancer activity. Several treatment with 3,4,5-trimethoxybenzaldehyde (10) in the pres-
representative chalcones and imidazothiadiazole derivatives are ence of NaOH (10%) as shown in Scheme 2. Imidazothiadiazole
shown in Fig. 1. By taking into account these structural aspects ethanones (9a–d) were prepared by Grignard reaction of the
of chalcones and imidazothiadiazoles, we have designed and corresponding imidazo[2,1-b]thiodiazole aldehydes (5a–d) fol-
synthesized a new class of imidazothiadiazole linked chalcones lowed by oxidation with IBX in DMSO.
(7a–m, 11a–d) and evaluated their anticancer potential. Some of
these conjugates showed excellent antiproliferative effects in
certain cancer cell lines. Moreover, to understand the possible
mechanisms involved in mediating the antiproliferative effect,
we have studied the cell cycle alterations and apoptotic markers
in DU-145 prostate cancer cells.
Evaluation of biological activity
Anticancer activity. The imidazothiadiazole–chalcones
(7a–m and 11a–d) were evaluated for their cytotoxic activity
Results and discussion
Chemistry
Imidazothiadiazole–chalcones (7a–m) were prepared by the
24
Claisen–Schmidt condensation of appropriate substituted
acetophenones (6a–d) upon treatment with imidazo[2,1-b]thia-
diazole aldehydes (5a–d) in the presence of NaOH (10%) as
25
shown in Scheme 1. Imidazo[2,1-b]thiadiazole aldehydes were
obtained by means of the Vilsmeier reaction on the corre-
sponding imidazo[2,1-b]thiadiazole (4a–d), wherein 4a–c were
prepared from the appropriate 5-methyl-1,3,4-thiadiazol-2-
amine (2) and bromoketones (1), however, 4d is prepared from 2
and 2-bromoacetic acid as illustrated in Scheme 1.
ꢀ ꢀ
3
Scheme 2 (i) CH MgBr, THF, 5 C 6–8 h; (ii) IBX, DMSO, 0 C, 2 h, 55–
65%; (iii) 10% aq. NaOH, 12 h, rt, 75–85%.
Scheme 1 (i) Acetone, reflux, 6–8 h; (ii) 2N HCl, reflux, 1 h, 85–95%; (iii) C
for 3 h, 70–80%; (v) 10% aq. NaOH, 12 h, rt, 75–85%.
2 5 3 3 3
H OH, POCl , reflux, 7–9 h, 20% NH , 70–80%; (iv) POCl , DMF, reflux
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23
against various human cancer cell lines of prostate, breast, and activity. In addition, intermediates 5a–b and 9a–b exhibited
26,27
lung by using the MTT assay method.
All the chalcone poorer cytotoxic activities than 7a–m and 11a–d. Interestingly,
derivatives showed moderate to good cytotoxic activity with IC50 compounds 11a and 11b showed better antiproliferative activity
values ranging from 0.65 to 18.64 mM. Interestingly, compounds with IC50 values ranging from 0.65 to 2.25 mM than doxorubicin
1
1a and 11b showed signicant growth inhibitory/cytotoxic (2.45 to 3.41 mM) against tested cell lines and the results are
activity against a number of cancer cell lines tested in this study summarized in Table 1.
Table 1). The structure–activity relationship (SAR) studies Effect on cell cycle arrest. To examine the mechanism
reveal that 3,4,5-trimethoxy group containing compounds underlying the antiproliferative effect of these imidazothiadia-
7a, 7b, 7c, 11a, and 11b) showed superior cytotoxic activity zole–chalcones, we analyzed the cell cycle alteration phase
(
(
against selected cancer cell lines compared to the other chal- distribution and the DNA content of the cell by treating DU-145
cone derivatives. Compounds having a pyridine moiety (7e, 7f, cells with them at 3 mM concentration for 24 h by ow cytom-
7g and 7h) showed less activity in comparison with other chal- etry. The effect of these compounds (11a and 11b) and doxo-
cones. Moreover, compounds 7d, 7h, 7l and 11d exhibited a rubicin (employed as a standard) on cell cycle events was
reduced amount of activity, due to the replacement of the analyzed. Treatment of DU-145 cells with compounds 11a and
substituted phenyl ring by a simple chlorine atom. Similarly, 11b caused an accumulation of 83% and 81% cells in the G
compounds 7c and 11c containing chlorine (Cl) substituted on phase respectively as compared to 65% in untreated cells. On
the phenyl ring showed less cytotoxic activity than 7a–b and the other hand, doxorubicin has shown 92% cells in the G /G
0 1
/G
0
1
11a–b (OCH3 and F substituted phenyl ring). In addition, phase (Fig. 2 and Table 2).
compound 7m (has two uorine atoms on phenyl rings) also
Effect on chromatin condensation. Apoptosis is an impor-
showed the lowest activity. It is observed that the potent tant process of cell death of undesirable cells during develop-
compounds 11a and 11b possess a trans double bond beside the ment or homeostasis in multicellular organisms and during
28
aryl group however in compounds 7a and 7b, the double bond is apoptosis, chromatin condensation takes place. To see
attached to the imidazothiadiazole ring. The cytotoxic activity of whether imidazothiadiazole–chalcone conjugate (11a and 11b)
these imidazothiadiazole–chalcone derivatives is comparable to induced cytotoxicity occurs through apoptosis, DU-145 prostate
that of doxorubicin (I), a well known chemotherapeutic drug cancer cells were treated with 3 mM concentration of these
and more potent than trimethoxy chalcone (II). Moreover, the compounds for a period of 24 h. Hoechst 33258 staining was
imidazothiadiazole–oxindole derivative showed moderate used to visualize nuclear condensation. It was found that both
a
Table 1 IC50 values (in mM) for compounds 7a–m and 11a–d in
selected human cancer cell lines
a
IC50 values (mM)
b
c
c
d
Compound
DU 145
MDA MB-231
MCF-7
A549
5
5
9
9
7
7
7
7
7
7
7
7
7
7
7
7
7
1
1
1
1
a
b
a
b
a
b
c
d
e
f
g
h
i
j
k
l
m
1a
1b
1c
1d
47.23
59.46
> 100
> 100
3.56
—
—
39.43
38.69
98.07
—
—
—
—
4.24
6.68
> 100
4.32
4.65
2.46
11.85
5.32
15.96
11.32
11.35
6.36
4.36
6.75
9.25
16.02
0.92
1.32
3.39
12.34
3.41
—
2.78
5.82
4.24
5.91
9.67
12.65
9.89
8.95
4.95
5.78
6.92
12.32
18.64
1.34
1.27
2.64
19.34
3.12
4.67
3.25
5.89
6.68
9.54
15.38
10.25
15.32
13.58
10.32
4.24
4.89
5.32
15.39
17.67
0.65
0.89
2.91
15.39
2.45
4.07
14.62
14.65
17.68
14.87
5.34
5.92
5.65
19.32
17.68
2.25
1.90
4.28
24.36
2.10
2.95
Fig. 2 Effect of compounds 11a and 11b on cell cycle arrest. DU-145
cells were treated with compounds 11a, 11b and doxorubicin (3 mM) for
Doxo (I)
TMC (II)
24 h. Untreated cells and DMSO-treated cells served as controls. Cell-
cycle analysis was performed with propidium iodide as indicated in the
Experimental section. The cell-cycle phase distribution was deter-
a
Values are the mean of three independent experimental
b
c
d
determinations. Prostate cancer. Breast cancer. Lung cancer cell mined by FACS and the percentage of cells in each phase was analyzed
lines.
by FCS express 4 plus.
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Table 2 Cell cycle distribution of compounds 11a, 11b and doxoru-
bicin in DU145 cells
G
0
/G
1
S
G2/M
Control
65
83
81
92
10
8
11
5
25
9
8
1
1
1a
1b
Doxo (I)
3
these compounds caused a signicant nuclear condensation as
shown in Fig. 3. Doxorubicin, a known inducer of cell death by
apoptosis, was used as a positive control at the same
concentration.
Caspases-3 and 8 activation. We measured the activation of
caspase-3 and caspase 8 for the conrmation of the chromatin
29–31
condensation results.
DU-145 cells were treated with
compounds (11a and 11b) along with doxorubicin at 3 mM
concentration for 24 h. It was found that both the compounds
signicantly activated the caspase-3 and 8, as in the case of
doxorubicin (I). These results reveal that imidazothiadiazole–
chalcone conjugates (11a and 11b) induce DU-145 cell death by
apoptosis (Fig. 4).
Fig. 4 Effect of compounds 11a and 11b on caspase-3 and 8 activities
as a measure of apoptosis. DU-145 cells were treated with compounds
Effect of imidazothiadiazole–chalcone derivatives on cell-
11a, 11b and doxorubicin at 3 mM concentration for 24 h and cell
cycle regulatory proteins. As cell cycle progression from G0 lysates were incubated with the fluorogenic caspase-3 and
8
ꢀ
through G phase involves activation of the cell cycle regula- substrates DEVD-AFC and IETD-AFC respectively for 1 h at 37 C and
1
3
2–36
released AFC fluorescence was measured using a multimode reader
using an excitation/emission of 400/500 nm.
tory proteins like cyclin D1 and E,
we investigated the
effect of compounds 11a and 11b on the cell cycle proteins. It
was observed that these conjugates along with doxorubicin
down-regulated cyclin D1 and cyclin dependent kinase (CDK)
4
in cells treated with 3 mM concentration of the respective
compounds for a period of 24 h. Cyclin D1 levels decreased by
Fig. 5 Effect of compounds 11a and 11b on cell cycle protein alter-
ations. DU-145 cells were treated with 11a, 11b and doxorubicin (I) at
Fig. 3 Imidazothiadiazole–chalcone compounds cause apoptosis in
DU-145 cells. Cells were treated with 11a, 11b and doxorubicin at 3 mM
concentration for 24 h, washed with PBS, and incubated with
3
mM concentration for 24 h. Cell cycle proteins (CDK4, cyclinD1,
cyclin E, p21and p27) and GAPDH were measured by western blot
analysis as described in the Experimental section.
À1
Hoechst-33258 stain (4 mg mL ) for 20 min to measure chromatin
condensation. Images were photographed using fluorescence
microscopy equipped with a DAPI filter.
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both 11a and 11b, however, the levels of cyclin E were down-
regulated with only 11a. In tune with the altered cyclin D1 and
E expression proles, the levels of p21 and p27 cyclin
9 S.
Cheenpracha,
C.
Karalai,
C.
Ponglimanont,
S. Subhadhirasakul and S. Tew-trakul, Bioorg. Med. Chem.,
2006, 14, 1710.
dependent kinase inhibitors were signicantly increased 10 E. Bombardelli, P. Valent, U.S. Patent, 6423740, 2003.
(Fig. 5).
11 J. Quintin, J. Desrivot, S. Thoret, P. Menez, J. Cresteil and
G. Lewin, Bioorg. Med. Chem. Lett., 2009, 19, 167.
1
2 J. Aponte, M. Verastegui, E. Malaga, M. Zimil, M. Quiliano,
A. J. Vaisberg, R. H. Gilman and G. B. Hammond, J. Med.
Chem., 2008, 51, 6230.
Conclusion
In conclusion, a new class of imidazothiadiazole–chalcones
were synthesized and evaluated for their cytotoxic activities
against four representative human cancer cell lines. All the
modied chalcone derivatives showed moderate to good
cytotoxic activity against the cancer cell lines tested in this
study. Among them compounds 11a and 11b exhibited
signicant anti-cancer potency with IC50 values ranging from
13 A. Modzelewska, C. Pettit, G. Achanta, N. E. Davidson,
P. Huang and S. R. Khan, Bioorg. Med. Chem., 2006, 14,
3491.
1
1
1
4 O. Nerya, R. Musa, S. Khatib, S. Tamir and J. Vaya,
Phytochemistry, 2004, 65, 1384.
5 T. Takahashi, N. Takasuka, M. Iigo, M. Baba, H. Nishino,
H. Tsuda and T. Okuyama, Cancer Sci., 2004, 95, 448.
6 C. Claude-Alain, L. Jean-Chritophe, T. Patrick, P. Christelle,
H. Gerard, C. Albert-Jose and D. Jean-Lue, Anticancer Res.,
0.65 to 2.25 mM. The results revealed that compounds 11a
and 11b caused cell cycle arrest with a majority of population
of cells accumulated in the sub-G1 phase suggesting that
these compounds have the capability to induce cell death by
apoptosis. Further, the role of cell cycle regulatory proteins
provides the mechanism for the cell growth inhibitory prop-
erties of these compounds demonstrating the down-regula-
tion of CDK4 and cyclin D1 proteins because of the increase
in G1/S checkpoint-associated tumor suppressor proteins p21
and p27. Overall, the present study shows that these new
heterocyclic chalcones exhibit promising cytotoxic activity and
has the potential to act as new leads for the treatment of
cancer.
2001, 21, 3949.
1
7 B. L. Wei, C. H. Teng, J. P. Wang, S. J. Won and C. N. Lin, Eur.
J. Med. Chem., 2007, 42, 660.
8 Q. C. Meng, L. Ni, K. J. Worsencro, J. Ye, M. D. Weingarten,
J. M. Simpson, A. Sounder, R. B. Howard, C. L. Sundell,
M. A. Wasserman and J. A. Sikorski, J. Med. Chem., 2007,
1
50, 1304.
19 S. K. Kumar, H. Erin, P. Catherine, G. Halluru,
N. E. Davidson and S. R. Khan, J. Med. Chem., 2003, 46,
2813.
20 F. A. Ashour, N. S. Habib, M. Taibbi, S. el Dane and A. S. el
Dine, Farmaco, 1990, 45, 1341.
Acknowledgements
21 A. A. Fawzia, N. S. Habib, S. Nargues, M. Taibbi, S. Dine and
A. S. el Dine, Chem. Abstr., 1991, 114, 207141q.
The authors V. S. R., G. B. K., A. B. S., R. M., and I. B. S. are
thankful to the CSIR, New Delhi, for the award of research
fellowships. SK is thankful to ICMR, New Delhi, India, for the
award of Senior Research Fellowship. We also acknowledge
the CSIR for nancial support under the 12 Five Year plan
projects “Affordable Cancer Therapeutics (ACT)” (CSC0301)
and “Small Molecules in Lead Exploration (SMiLE)”
22 T. D. Onkol, D. S. Uzun, L. Adak, S. Ozkan and S. Ahin, J.
Enzyme Inhib. Med. Chem., 2008, 23, 277.
2
3 A. Andreani, M. Granaiola, A. Locatelli, R. Morigi,
M. Rambaldi, L. Varoli, N. Calonghi, C. Cappadone,
G. Farruggia, C. Stefanelli, L. Masotti, T. L. Nguyen,
E. Hamel and R. H. Shoemaker, J. Med. Chem., 2012, 55,
th
2078.
(CSC0111).
2
4 F. Herencia, M. L. Ferr ´a ndiz, A. Ubeda, J. N. Dom ´ı nguez,
J. E. Charris, G. Lobo and M. J. Alcaraz, Bioorg. Med. Chem.
Lett., 1998, 8, 1169.
Notes and references
25 A. U. Siddiqui, V. U. Maheshwar Roa, M. Maimirani and
A. H. Siddiqui, J. Heterocycl. Chem., 1995, 32, 353.
1
2
3
A. W. Stu and T. J. Marby, Phytochemistry, 1972, 10, 2812.
A. M. Abdula, Eur. J. Chem., 2013, 4, 207.
H. P. Avila, E. F. Smania, F. D. Minache and A. Jr Smania,
Bioorg. Med. Chem., 2008, 16, 9790.
26 N. Jain, D. Yada, T. B. Shaik, G. Vasantha, P. S. Reddy,
S. V. Kalivendi and B. Sreedhar, ChemMedChem, 2011, 6, 859.
27 A. Kamal, V. S. Reddy, S. Karnewar, S. S. Chourasiya,
A. B. Shaik, G. B. Kumar, C. Kishor, M. K. Reddy,
M. P. N. Rao, A. Nagabhushana, K. V. S. Ramakrishna,
A. Addlagatta and S. Kotamraju, ChemMedChem, 2013, 8,
2015.
4
5
Z. Nowakowska, Eur. J. Med. Chem., 2007, 42, 125.
H. M. Yang, H. R. Shin, S. H. Cho, S. C. Bang, G. Y. Song and
J. H. Ju, Bioorg. Med. Chem., 2007, 15, 104.
6
J. N. Domnguez, C. Len, J. Rodrigues, N. Gamboa de
Domnguez, J. Gut and P. J. Rosenthal, J. Med. Chem., 2005, 28 Y. Zhang, X. Wang, W. Fang, X. Cai, F. Chu, X. Liao and J. Lu,
8, 3654.
Bioinorg. Chem. Appl., 2013, 1, 437134.
M. Liu, P. Wilairat and M. L. Go, J. Med. Chem., 2001, 44, 29 L. J. Browne, C. Gude, H. Rodriguez, R. E. Steele and
443. A. Bhatnager, J. Med. Chem., 1991, 34, 725.
Y. Lin, Y. Zhou, M. Flavin, L. Zhou, W. Niea and F. Chen, 30 S. Iyer, D. J. Chaplin, D. S. Rosenthal, A. M. Boulares, L. Li
Bioorg. Med. Chem. Lett., 2002, 10, 2795. and M. E. Smulson, Cancer Res., 1998, 58, 4510.
4
7
8
4
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View Article Online
MedChemComm
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31 H. Zhu, J. Zhang, N. Xue, Y. Hu, B. Yang and Q. He, Invest. 34 S. Y. Shieh, J. Ahn, K. Tamai, Y. Taya and C. Prives, Genes
New Drugs, 2010, 28, 493.
Dev., 2000, 14, 289.
32 D. O. Morgan, Nature, 1995, 374, 131.
35 A. J. Levine, Cell, 1998, 8, 21.
33 T. Taguchi, Y. Kato, Y. Baba, Y. G. Nishimura, Y. Tanigaki, 36 S. B. Parker, G. Eichele, P. Zhang, A. Rawls, A. T. Sands,
C. Horiuchi, I. Mochmatsu and M. Tsukuda, Oncol. Rep.,
004, 11, 421.
A. Bradley, E. N. Olson, J. W. Harper and S. J. Elledge,
Science, 1995, 267, 1024.
2
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2014