Design, synthesis and biological evaluation of imidazopyridine / pyrimidine - chalcone derivatives as potential anticancer agents

Ahmed Kamal; J. Surendranadha Reddy; M. Janaki Ramaiah; D. Dastagiri; E. Vijaya Bharathi; M. Victor Prem Sagar; S. N. C. V. L. Pushpavalli; Paramita Ray; Manika Pal-Bhadra

doi:10.1039/C0MD00116C

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DOI: 10.1039/C0MD00116C (Concise Article) Med. Chem. Commun., 2010, 1, 355-360

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Design, synthesis and biological evaluation of imidazopyridine/pyrimidine-chalcone derivatives as potential anticancer agents†

Ahmed Kamal *, J. Surendranadha Reddy , M. Janaki Ramaiah , D. Dastagiri , E. Vijaya Bharathi , M. Victor Prem Sagar , S. N. C. V. L. Pushpavalli , Paramita Ray and Manika Pal-Bhadra *
Chemical Biology Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India. E-mail: ahmedkamal@iict.res.in; Fax: +91-40-27193189; Tel: +91-40-27193157

Received 20th July 2010 , Accepted 14th August 2010

First published on 27th October 2010

Abstract

A new series of imidazo[2,1-b]pyridine/pyrimidine chalcone derivatives were synthesized and evaluated for their anticancer activity. These chalcone derivatives showed promising activity with GI₅₀ values ranging from 0.28 to 30.0 μM. The detailed biological aspects of one of the promising compound 3f on the MCF-7 cell line were studied. Interestingly, compound 3f induced G1 cell cycle arrest, down regulation of G1 phase cell cycle regulatory proteins such as cyclin D1, E1, and CDK2. Moreover, compound 3f showed the characteristic features of apoptosis such as enhancement in the levels of p27 and TNFR1 proteins with concomitant down regulation of procaspase-9. One of the representative compound of this series 3f could be considered as the potential lead for its development as a new anticancer agent.

Introduction

Chalcones (1,3-diaryl-2-propen-1-ones) represent an important group of natural products belonging to the flavonoids family.^1,2 Chemically, these are open-chained molecules bearing two aromatic rings that are joined by a three-carbon enone fragment. These molecules possess interesting biological activities including cytotoxic^3,4 antimalarial,⁵ antileishmanial,⁶ anti-inflammatory,⁷ anti-HIV,⁸ antifungal⁹ and as tyrosine kinase inhibitors.¹⁰ Natural and synthetic chalcones have been reported to possess strong antiproliferative effects in primary as well as established ovarian cancer cells¹¹ and in gastric cancer (HGC-27) cells.¹² Hydroxyl chalcones and isoliquiritigenin have been shown to be potent inhibitors of skin carcinogenesis.¹³ The remarkable biological potential of these chalcones is due to their possible interactions with various proteins related to cell apoptosis and proliferation. Recent studies have shown that these chalcones induce apoptosis in a variety of cell types, including breast cancers.^14–16 The 3,4,5-trimethoxyphenyl ring is thought to be essential for retaining the anticancer activity of chalcones. Some of the recent advances in the development of anticancer agents involve structural modification of chalcones to improve their bioavailability and to study the role of various substituents on aryl or heteroaryl rings.¹⁷ In addition, chalcone derivatives wherein the B-ring is replaced by a heterocyclic ring have been systematically investigated.¹⁸ Imidazopyridine/pyrimidine are also well known compounds and many derivatives of this fused ring system have been evaluated for potential biological activity particularly for antitumor activity.^19,20 Some representative trimethoxychalcone (1), imidazopyrimidine guanylhydrazone (2) and imidazopyridine/pyrimidine derivatives (3) are illustrated in Fig. 1.


	Fig. 1 Structures of trimethoxychalcone (1), imidazopyrimidine guanyl hydrazones (2), imidazo pyridine/pyrimidine-chalcone derivatives (3).

Considering the potent bioactivities of compounds possessing an imidazopyridine/pyrimidine core, we became interested to synthesize new chalcone derivatives incorporating an imidazopyridine/pyrimidine skeleton and evaluated their anticancer activity. The promising activity obtained, prompted us to investigate their role in the cell proliferation and apoptosis of human breast cancer cell line (MCF-7). Further, it was considered of interest to investigate the effect of compound 3f on some of the proteins that regulate the cell cycle progression.

Results and discussion

Chemistry

The imidazo[1,2-a]pyridine and pyrimidine chalcone derivatives (3a–i) were prepared by the Claisen-Schmidt condensation of 3,4,5-trimethoxyacetophenone (9a) or 3,4-dimethoxyacetophenone (9b) with substituted imidazo[1,2-a]pyridine and pyrimidine aldehydes (8a–h) in the presence of KOH (50%). The imidazo[1,2-a]pyridine and pyrimidine aldehydes (8a–h) were obtained by means of the Vilsmeier reaction on the corresponding imidazo[1,2-a] pyridine and pyrimidine (7a–h), that were obtained from compounds 6a–h. The intermediate compounds 6a–h were obtained by the reaction of appropriate 2-aminopyridine/pyrimidine (4a,b) and bromoketones (5a–e) as shown in Scheme 1.


	Scheme 1 Reagents and conditions: (a) acetone, reflux, 6–8 h; (b) 2 N HCl, reflux, 1 h, 85–92%; (c) POCl₃, DMF, reflux, 1 h, 75–80%; (d) 50% aq. KOH, 12 h, rt, 78–82%.

Biological evaluation

(A) Antiproliferative activity. The representative compounds 3a and 3c–h were evaluated for in vitro antiproliferative activity in the standard 60 human cancer cell lines derived from nine cancer types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate, and breast cancers). For each compound, dose–response curves for each cell line were measured at a minimum of five concentrations at 10-fold dilutions. A protocol of 48 h continuous drug exposure is used, and a sulforhodamine B (SRB) protein assay was used to estimate cell viability or growth. These compounds (3a and 3c–h) showed significant anticancer activity in a large number of cell lines with GI₅₀ values ranging from 0.28 to 30.0 μM. Specifically, compound 3f exhibited promising antiproliferative activity with GI₅₀ values less than 1 μM particularly against CCRF-CEM (0.44), K-562 (0.36), SR (0.44), RPMI-8226 (0.29) in leukemia cancer, NCI-H460 (0.68) in non small cell lung cancer, HCC-2298 (0.48), HCT-116 (0.84), KM12 (0.57) in colon cancer, IGROV1 (0.44) in ovarian cancer, DU-145 (0.65) in prostate cancer, MCF-7 (0.56) in breast cancer and LOXMVI (0.95), UACC-257 (0.62) in melanoma cancer cell lines as shown in Table 1. Compounds 3b (NSC: 750138) and 3i (NSC: 750339) did not exhibit significant activity in the one dose screening of the NCI.

Table 1 Antiproliferative activity of compounds 3a and 3c–h and in selected cancer cell lines^a

Cancer panel/cell line	GI₅₀/μM
Cancer panel/cell line	3a	3c	3d	3e	3f	3g	3h
a Data obtained from NCI's in vitro anticancer activity cells screen.
Leukemia
CCRF-CEM	18.8	1.54	1.66	4.15	0.44	4.01	4.05
HL-60(TB)	34.0	2.53	2.01	4.12	1.52	2.52	3.33
K-562	13.1	2.99	1.51	2.83	0.36	2.63	3.03
MOLT-4	32.9	2.18	1.77	3.73	1.03	3.10	3.75
SR	3.71	1.90	1.23	2.91	0.44	1.53	2.28
RPMI-8226	7.50	0.39	1.34	1.98	0.29	2.21	2.56
Non-small cell lung
A549/ATCC	7.96	3.86	2.04	4.04	2.80	2.71	3.34
EKVX	7.42	1.71	3.12	2.37	3.74	1.61	2.71
HOP-62	3.62	4.42	2.88	2.97	3.01	2.83	3.12
HOP-92	2.36	—	0.28	2.65	—	1.43	1.77
NCI-H226	24.1	6.10	2.04	3.55	4.55	2.28	3.08
NCI-H23	3.41	3.78	2.20	1.51	2.37	1.35	1.58
NCI-H322M	4.63	2.08	3.02	1.66	3.15	1.76	2.07
NCI-H460	4.09	2.99	1.42	2.01	0.68	1.43	1.59
NCI-H522	3.12	2.74	1.10	1.77	5.73	1.66	2.87
Colon
COLO 205	3.50	3.92	1.77	2.11	1.92	1.70	1.70
HCC-2998	8.27	3.80	1.96	1.83	0.48	1.58	1.77
HCT-116	2.26	2.62	1.63	1.26	0.84	1.17	1.53
HCT-15	4.85	3.73	1.60	2.10	1.13	1.38	2.11
HT29	4.80	3.20	2.09	1.93	1.48	2.19	2.52
KM12	4.99	2.52	1.32	1.73	0.57	1.61	1.69
SW-620	4.16	2.78	—	1.68	—	1.75	2.09
CNS
SF-268	4.00	3.58	2.37	1.64	1.57	2.36	1.90
SF-295	15.2	1.85	2.24	1.99	3.15	2.39	2.43
SF-539	2.39	2.71	1.87	1.63	1.65	1.36	1.55
SNB-19	25.2	4.91	2.10	1.93	1.59	3.01	3.12
SNB-75	5.49	3.92	2.68	1.76	2.35	1.89	2.39
U251	3.00	2.91	1.66	1.29	1.10	1.29	1.68
Ovarian
IGROV1	8.86	3.03	1.89	2.23	0.44	2.20	1.93
OVCAR-3	2.88	2.47	2.44	1.83	1.98	1.75	1.40
OVCAR-4	5.72	2.43	3.13	4.10	2.49	2.91	2.93
OVCAR-5	9.33	7.38	2.64	1.98	3.79	1.53	2.46
OVCAR-8	3.92	2.83	1.68	3.39	1.31	2.72	2.69
NCI/ADR-RES	3.36	2.18	1.89	1.41	1.57	1.56	1.58
SK-OV-3	14.8	4.92	3.01	4.04	2.49	2.50	2.64
Renal
786-0	3.90	3.74	2.31	1.72	2.87	1.84	2.76
A498	10.4	2.93	0.98	2.40	2.45	2.13	2.20
ACHN	3.26	3.22	2.09	1.95	2.85	1.57	2.21
CAKI-1	8.33	1.39	2.31	1.82	2.50	1.58	2.06
RXF 393	2.94	—	—	1.87	—	1.11	1.44
SN12C	11.4	4.10	2.44	2.09	1.91	2.56	2.26
TK-10	4.16	3.28	3.78	2.09	3.78	2.88	3.25
UO-31	1.31	0.55	1.07	1.49	1.44	0.51	0.73
Prostate
PC-3	19.8	3.71	2.63	3.80	2.73	4.32	4.35
DU-145	7.21	2.63	1.90	1.61	0.65	2.55	1.63
Breast
MCF7	2.38	2.76	1.12	1.44	0.56	1.14	1.61
MDA-MB-231/ATCC	5.22	3.28	3.08	4.59	3.06	2.73	2.99
HS 578T	2.89	1.85	—	3.27	—	1.91	2.01
BT-549	3.27	0.89	1.86	1.64	2.59	1.45	1.80
T-47D	2.59	3.01	1.72	3.52	1.43	2.53	2.09
MDA-MB-468	11.9	2.28	1.72	2.12	1.90	1.81	2.75
Melanoma
LOX IMVI	6.05	2.26	1.87	1.66	0.95	1.46	1.57
MALME-3M	1.56	2.36	3.65	1.57	6.69	1.81	2.84
M14	9.80	4.48	2.08	1.82	3.63	1.53	2.16
MDAMB-435	5.76	1.78	2.49	2.26	2.04	2.28	2.36
SK-MEL-2	4.95	2.37	2.27	2.43	1.72	1.87	2.38
SK-MEL-28	7.10	3.30	—	1.93	—	1.65	3.68
SK-MEL-5	2.72	1.50	1.33	1.62	1.63	1.52	2.22
UACC-62	5.58	4.86	1.61	2.05	2.22	1.88	1.74
UACC-257	5.85	3.24	1.88	1.66	0.62	1.82	3.73

MTT assay was also carried out to identify the cytotoxic effect of some of these chalcone derivatives (3a and 3d–h) on MCF-7 cells at 4 μM concentration. Moreover, the anticancer activity of compound 1 (3,4,5-trimethoxy chalcone) and positive control doxorubicin was examined to substantiate the anticancer activity of these chalcone derivatives (3a and 3d–h) and it is interesting to observe that these have shown a higher cytotoxicity than 1. Amongst all the derivatives the compound 3f was found to be the most significant one as shown in Fig. 2.


	Fig. 2 Effect of chalcone derivatives (3a and 3d–h) on cell viability. MCF-7 cells were treated with 4 μM concentration of compounds for 24 h in 96 well plate seeded with 10,000 cells per well. OD readings were taken at 420 nm. Doxorubicin (Doxo) and trimethoxychalcone (1) were used as positive controls.

(B) Cell cycle effects. To investigate the mechanism underlying the antiproliferative effect of chalcone derivatives, the cell-cycle distribution was analyzed by treating MCF-7 cells at 4 μM concentration for 24 h by flow cytometry. Results indicated 85.55% (3a), 92.41% (3d), 95.24% (3e), 97.86% (3f), 90.04% (3g), 74.16% (3h) of cells in G0/G1 phase respectively, whereas, positive control doxorubicin (Doxo) and control cells showed 94.85% and 64.04% respectively in G0/G1 phase. Among all the compounds tested compound 3f could be considered as the most effective derivative to produce cell cycle arrest (Fig. 3a,b and supplementary Tables 1 and 2†).


	Fig. 3 (a) FACS analysis of cell cycle distribution of MCF-7 cells after treatment with compounds 3a and 3d–h at 4 μM concentration for 24 h. Doxorubicin (Doxo) was used as positive control. (b) The histogram depicting the percentage of cells in sub G1 phase, an indicator of percentage of apoptosis for the chalcone derivatives (3a and 3d–h).

(C) Effect on cell cycle regulatory proteins. The interesting results obtained from cell cycle distribution, prompted us to investigate the cell cycle arrest at G1 with respect to the cell cycle regulatory proteins such as cyclin D1, cyclin E1, cyclin A, CDK2 and E2F-1, that are essential for the cell cycle progression from G1 to S phase.²¹ Thus MCF-7 cells were treated with compound 3f and the positive control doxorubicin at 4 μM concentration for 24 h, and Western blot analysis was carried out. As demonstrated in Fig. 4, compound 3f down regulated the protein levels of cyclin D1, which is required for the early G1 as well as cyclin E and CDK2 that regulate the late G1 phase with no change in the levels of cyclin A and E2F-1 was observed. The results indicate that compound 3f induced cell cycle arrest at G1 phase.


	Fig. 4 Effect of chalcone derivatives on the expression of cyclin and associated proteins. MCF-7 cells were treated with compound 3f and doxorubicin (Doxo) at 4 μM concentration. Western blot analysis was carried out with antibodies against (cyclin D1, cyclin-A and cyclin E1); CDK2, E2F-1 and β-actin was used as loading control.

(D) Effect on protein expression of CDK inhibitors p21and p27. Previous studies have shown that CDK inhibitors regulate the progression of cells in G0/G1 phase and induction of CDKIs causes a blockade of G1/S transition thereby resulting in cell cycle arrest.²² The cip/kip family of CDK inhibitors, p21 and p27 impede cell cycle progression by inhibiting cyclin E-CDK2.²³ Hence, we investigated the effect of compound 3f on the expression of p21 waf1/cip and p27 kip1 proteins in MCF-7 cells. Cells were treated over a period of 24 h and Western blot analysis was conducted with anti-p21 and p27 antibodies as shown in Fig. 5. Protein levels of p27 were upregulated, whereas p21 protein levels were not increased in relation to the control. In order to confirm this aspect promoter studies on p21 gene (P21 promoter −145/+8 with luciferase gene cloned downstream as reporter gene) were carried out. Ectopic expression of p21 promoter in MCF-7 cells by transfection method followed by compound treatment for 24 h resulted in no change in the levels of promoter activity in compound 3f whereas positive control doxorubicin there was some upregulation of promoter activity (Fig. 6). This data clearly showed that compound 3f follows p27 (CDK inhibitor) dependent apoptotic pathway.


	Fig. 5 Effect of compound 3f on the expression of tumor suppressor proteins (p21and p27). MCF-7 cells were treated with 4 μM concentration of compound 3f and doxorubicin (Doxo) was used as the positive control. Cell lysates were collected and Western blot analysis was carried out with above mentioned antibodies and β-actin was used as the loading control.


	Fig. 6 Effect of compound 3f and doxorubicin (Doxo) on the P21 promoter activity. The MCF-7 cells were transiently transfected with p21 (−145/+7) promoter with luciferase gene cloned downstream of the p21 promoter and after 24 h the compound treatments were carried out at 4 μM concentration for 24 h. Cell lysates were isolated and luciferase activity was observed and is the indicator of promoter activity.

(E) Effect on procaspase-9 and TNFR1 levels. Compound 3f showed apoptosis in Sub G1 phase and this prompted us to observe the caspase mediated pathway, as the induction of apoptotic cell death is mediated by caspases (intrinsic or extrinsic pathway). The extrinsic pathway of apoptosis originates at the membrane and engages membrane bound death-receptors. Tumor necrosis factor receptor, (TNFR1) is death receptor that transducers both death and survival signals. The extrinsic and intrinsic pathways unite²⁴ in the activation caspase-3²⁵ but MCF-7 cells lack endogenous caspase-3, caspase-9 functions in the process of apoptosis. So MCF-7 cells were treated with compound 3f at 4 μM concentration for 24 h and Western blot analysis was carried out and doxorubicin was used as the positive control. It is observed that there was decrease in procaspase-9 level and an increase in TNFR1 protein level when treated with compound 3f as shown in Fig. 7. This data indicated that compound 3f follows extrinsic apoptotic pathway.


	Fig. 7 Effect of compound 3f on the expression of TNFR1 and procaspase 9. MCF-7 cells were treated with 4 μM concentration of compound 3f and doxorubicin (Doxo) was used as the positive control. Cell lysates were collected and Western blot analysis was carried out with above mentioned antibodies and β-actin was used as the loading control.

Conclusion

In conclusion, we have synthesized a new class of chalcones, wherein the B-ring has been replaced by an imidazo[2,1-b]pyridine/pyrimidine moiety. Compound 3f showed significant antiproliferative activity in the cell line panel assay of National Cancer Institute (NCI). From the MTT proliferation assay, it was observed that compound 3f is the most effective antiproliferative agent than the other compounds of the series in MCF-7 cells at 4 μM concentration. The FACS analysis also showed more population in sub-G1 phase indicating that these compounds have apoptotic inducing ability. From the results of the detailed biological assays, the down regulation of G1 phase cell cycle regulatory proteins such as cyclin D1, E1, and CDK2 was observed and G1/S check point associated tumor suppressor protein p27 was upregulated, thus suggesting that these proteins are responsible for cell cycle blockade at G1 phase in compound 3f. Moreover, procaspase-9 level was decreased and TNFR1 protein level was increased, thus indicating that compound 3f followed extrinsic apoptotic pathway. In this study an insight in the cell cycle regulatory role as well as apoptotic inducing ability of the compound 3f was elucidated. These studies suggest that compound 3f has the potential to be taken up for further in vivo studies, particularly against breast cancer.

Acknowledgements

We thank the National Cancer Institute, Bethesda, for in vitro anticancer assay in human cell lines. J.S.N.R., D.D.G., E.V.B and M.V.P.S are thankful to CSIR, New Delhi, for the award of research fellowships.

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Footnote

† Electronic supplementary information (ESI) available: Spectral data of compounds 3a–i, 7a–h and 8a–h and experimental procedures for synthesis and biological evaluations. See DOI: 10.1039/c0md00116c