Synthesis of pyrazolo[1,5-a]pyrimidine linked aminobenzothiazole conjugates as potential anticancer agents

Accepted Manuscript Synthesis of pyrazolo[1,5-a]pyrimidine linked aminobenzothiazole conjugates as potential anticancer agents Ahmed Kamal, Jaki R. Tamboli, V. Lakshma Nayak, S.F. Adil, M.V.P.S. Vishnuvardhan, S. Ramakrishna PII: DOI: Reference:

S0960-894X(13)00469-1 http://dx.doi.org/10.1016/j.bmcl.2013.03.129 BMCL 20365

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

27 November 2012 14 March 2013 30 March 2013

Please cite this article as: Kamal, A., Tamboli, J.R., Lakshma Nayak, V., Adil, S.F., Vishnuvardhan, M.V.P.S., Ramakrishna, S., Synthesis of pyrazolo[1,5-a]pyrimidine linked aminobenzothiazole conjugates as potential anticancer agents, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl. 2013.03.129

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Synthesis of pyrazolo[1,5-a]pyrimidine linked aminobenzothiazole conjugates as potential anticancer agents Ahmed Kamal,*a Jaki R. Tamboli,a V. Lakshma Nayak,b S. F. Adil,c M.V.P.S. Vishnuvardhan ,b S. Ramakrishna b a

Division of Organic Chemistry, bPharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India c Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.

Abstract: A series of pyrazolo[1,5-a]pyrimidine linked 2-aminobenzothizole conjugates (6a-t) were synthesized and evaluated for their anticancer activity against five human cancer cell lines. Among them two compounds 6p and 6m showed significant anticancer activity with IC50 values ranging from 2.01-7.07 and 1.94-3.46 µM respectively. Moreover, cell cycle arrest in G2/M and reduction in Cdk1 expression level were observed upon treatment of these compounds and they also induced caspase-3 dependent apoptosis. This was further confirmed by Hoechst staining as well as DNA fragmentation analysis.

Keywords: Aminobenzothiazole conjugates, Anticancer activity, Cell cycle arrest, Cdk inhibitor, Caspase-3, DNA Fragmentation, Hoechst staining.

* Corresponding authors. Tel.: +91-40-27193157; fax: +91-40-27193189; E-mail addresses: [email protected]

Cytotoxicity and genotoxicity of anticancer drugs to the normal cells are major problems in cancer therapy and cause the risk of inducing secondary malignancy.1 A dose of anticancer drug sufficient to kill tumor cells is often toxic to the normal tissue and leads to many side effects, which in turn, limits its treatment efficacy. Thus, a massive search for new anticancer agents has been fueled by various academics and industries to unveil the new molecular targets and mechanisms based on the lead candidates of different classes of compounds. The cyclindependent kinases (CDKs) are a family of serine/threonine kinases that function as critical regulators of the mammalian cell cycle which integrates extracellular signaling, DNA synthesis and mitosis.2 Deregulation of cell cycle progression is a universal characteristic of cancer, and the majority of human cancers have abnormalities in some component of CDK activity, frequently through elevated and/or inappropriate CDK activation. Examples include cyclin overexpression, for example, cyclin D1 over expression is commonly observed in breast cancer,3 and loss of expression of CDK inhibitory proteins (CKI) through mutational or epigenetic alterations, for example, p16 loss has been observed in skin, lung, breast, and colorectal cancer.4 Synthetic inhibitors of CDK activity therefore present as a logical approach in the development of new cancer therapies. A large number of pyrazolo[1,5-a]pyrimidine derivatives are reported to exhibit a broad spectrum of biological activities such as antitumor5, anxiolytic6 and antimicrobial.7 But pyrazolo[1,5-a]pyrimidine derivatives are widely used as inhibitors of cyclin-dependent kinases (CDKs), that are involved in mediating the transmission of mitogenic signals and numerous other cellular events8-14 including cell proliferation, migration, differentiation, metabolism and immune response. It was also observed that many of these derivatives may block proliferation of various cancer cell lines.15 A number of selective CDK inhibitors have been described in the literature, and some of them that are undergoing clinical trials are roscovitine (1),16 SCH-727965 (2),17 and BMS 387032 (3),18 as shown in Figure 1. However, opportunities exist to identify and develop additional novel CDK inhibitors that may possess superior biological profile in comparison to the presently known candidates. Hence, there is a great challenge that exists in this area towards the identification of new conjugates comprising of pharmacophores of known antitumor agents for enhancing the selectivity as well as antitumour activity.



Benzothiazoles are the important class of heterocycles, that have attracted considerable attention19-29 and are known to exhibit various biological properties including antimicrobial, anticancer, antiamyloid, and antirheumatic.30–33 Arylbenzothiazoles and aminobenzothiazoles are known to possess promising anticancer activity both in vivo and in vitro models.34–38 Substituted benzothiazoles such as 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (PMX 610) (4) exhibit exquisitely potent (GI50 < 0.1 nM) and selective in vitro antitumor properties in human cancer cell lines (e.g., colon, non smallcell lung and breast subpanels) in the 60 human cancer cell line screen39 of the National Cancer Institute (NCI), apart from exhibiting significant anticancer activity against malignant cell lines.40 Moreover 2-(4-aminophenyl)-benzothiazole (CJM 126) (5) and its analogs comprise of a novel mechanistic class of antitumor agents41,42 as shown in Figure 1. Our previous efforts towards the synthesis of a variety of hybrid molecules led to the development of efficient anticancer agents. We envisaged that pyrazolo[1,5-a]pyrimidine and aminobenzothiazoles subunits within a single molecule could enhance the cytotoxic effect. These consideration

and

encouraging

anticancer

profile

of

pyrazolo[1,5-a]pyrimidine

and

aminobenzothiazoles led us to synthesize these new conjugates. In the present study, we report the synthesis of 2-aminobenzothiazole linked pyrazolo[1,5-a]pyrimidine conjugates having an amide linkage as outlined in Scheme 1. The most versatile approach available for the synthesis of pyrazolo[1,5-a]pyrimidines consists of the condensation of bifunctional nucleophile 3-amino-5phenyl-1H-pyrazoles with bifunctional electrophiles such as 1,3-diketones. A variety of substituted acetophenones (7a-d) were oxalylated by dimethyl oxalate in the presence of sodium methoxide, to give β-diketoester (8a-d). Treatment of the aryl β-diketoesters with 3-amino-5phenyl-1H-pyrazole affords the cyclodehydrated methyl 2,7-diphenylpyrazolo[1,5-a]pyrimidine5-carboxylates (9a-d) which was then hydrolyzed to 2,7-diphenylpyrazolo[1,5-a]pyrimidine-5carboxylic acids43 (10a-d) as shown in Scheme 1. Then 2-aminobenzothiazoles (11a-e) upon treatment with different 2,7-diarylpyrazolo[1,5-a]pyrimidine-5-carboxylic acids (10a-d) form an amide linkage using EDCI/HOBt to afford the required pyrazolo[1,5-a]pyrimidine linked aminobenzothiazole conjugates (6a-t) in good yields as shown in Scheme 1.



Some representative conjugates from this series were screened by the National Cancer Institute (NCI), USA for their anticancer activity. After preliminary screening on the tumor cell lines, two compounds 6m and 6p were tested for five dose concentration on a panel of 60 human tumor cell lines derived from nine different cancer types: leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and breast. The results expressed as GI50 values for the test compounds are illustrated in Table 1. These compounds exhibited promising activity with GI50 values ranging 1.7–21 and 0.7– 85 µM respectively. Particularly compound 6p exhibited GI50 value of 0.7 µM against lung cancer cell line. The significant antiproliferative activity showed by the representative compounds like 6m and 6p prompted us to evaluate the anticancer activity of this series against a panel of five human cancer cell lines, A549 (lung), DU-145 (Prostate), MCF-7 (breast), ACHN (renal) and Hela (Cervical) by employing MTT assay44 using roscovitine as the reference drug. The results are summarized in Table 2 and expressed as IC50 values. The in vitro screening results revealed that some of these compounds exhibited promising anticancer activity with IC50 values ranging from 1.94 to 41.68 µM against different cancer cell lines. The most active compounds 6p and 6m showed IC50 values ranging from 2.01-7.07 and 1.94-3.46 µM, respectively. From the structure activity relationship (SAR) studies of these compounds, some of the interesting trends have been observed. The substitution pattern on the phenyl ring of aminobenzothiazole subunit not showed any considerable effect on the activity of such analogues. On the other hand, it was observed that the substitution pattern (R1, R2, R3) on the 7phenyl ring of the pyrazolo[1,5-a]pyrimidine subunit is playing an important role in the overall activity profile of these compounds. The presence of 3,4,5-trimethoxy, 4-fluoro and 4-methoxy substitutions on the 7-phenyl ring of the pyrazolo[1,5-a]pyrimidine subunit increased the cytotoxicity of these compounds. Particularly, the compounds 6m and 6p having 3,4,5trimethoxy and 4-fluoro substitution pattern displayed higher anticancer potential as compared to other substitution on the same phenyl ring. Whereas the other compounds like 6l, 6n, 6s and 6t shows moderate anticancer activity and the remaining substitutions on the phenyl ring decreases the anticancer potential of such analogues. These aspects shows that the presence of 3,4,5trimethoxy substitution and 4-fluoro substitution on the 7-phenyl ring of pyrazolo[1,5a]pyrimidines subunit is important to retain the anticancer activity. These interesting preliminary

results prompted us to investigate their detailed biological effects in the A549 cell line for 6p and 6m. The tumor suppressor gene p53 is a multifunctional protein responsible for maintaining genomic integrity and its mutation is known to cause tumors in humans.45 P53 gene activates signaling for DNA repair initially, and in failure of DNA repair, induces apoptosis or cell cycle arrest.46-48 To investigate the mechanism underlying the anticancer activity of these potent compounds (6p and 6m) the cell cycle distribution in A549 cancer cell line was analyzed by flow cytometry. In this study A549 cells were treated with these compounds at 1 and 2 µM concentrations for 48 h. The data obtained clearly indicated that these compounds show G2/M cell cycle arrest in comparison to the untreated control. These conjugates showed 40% and 44% of cell accumulation in G2/M phase at 1 µM concentration, whereas they exhibited 52% and 64% of cell accumulation at 2 µM concentration, respectively in 6p and 6m as depicted in Figure 2. The positive control, roscovitine showed 40% of cell accumulation in G2/M phase at 2 µM concentration whereas in control (untreated cells) 13% of G2/M phase was observed. As discussed earlier pyrazolo[1,5-a]pyrimidine derivatives are widely used as inhibitors of cyclin-dependent kinases (CDKs), have the potential to induce cell cycle arrest and apoptosis in cancer cells. Further, to understand the molecular events involved in G2/M cell cycle arrest, the effect on the expression level of CDKs particularly Cdk1,49,50 was investigated. For this purpose, the active conjugates, 6m and 6p were treated with A549 cells and the Western blot analysis was carried out at 2 µM concentration. It was observed that there was a significant reduction of Cdk1 expression level as shown in Figure 3.



Apoptosis is one of the major pathways that lead to the process of cell death. Chromatin condensation and fragmented nuclei are known as the classic characteristics of apoptosis. Hence it was considered of interest to investigate the apoptotic inducing effect of these two potential compounds (6p and 6m) by Hoechst staining (H 33258) in A-549 cancer cell line. Therefore cells were treated with 6p and 6m at 2 µM concentration for 24 h, and roscovitine was used as the reference compound. Manual field quantification of apoptotic cells based on cytoplasmic condensation, presence of apoptotic bodies, nuclear fragmentation of the tested compounds revealed that there was significant increase in the percentage of apoptotic cells as seen from Figure 4. It is reported that the cell cycle arrest at G2/M phase takes place by the induction of cellular apoptosis.53-55 Hence, it was considered of interest to understand the correlation of cytotoxicity with that to apoptosis by these two compounds 6p and 6m.

Moreover

caspases

play a crucial role in the induction of apoptosis and amongst them caspase-3 happens to be one of the effector caspase. Hence, A-549 cells were treated with 6p and 6m, and compared to roscovitine for the activation of caspase-3. The results indicated that there was significant induction of caspase-3 activity in the cells treated with these compounds in comparison to the control as shown in Figure 5. The reference compound, roscovitine also induced 4 fold induction at 2 µM concentration. Therefore activation of caspase-3 by 6p and 6m indicate that they were capable of inducing apoptosis in A-549 cells. DNA laddering was carried out in order to elucidate the mode of action of these compounds especially for their ability to induce oligonucleosomal DNA fragmentation (DNA ladder), which is a characteristic feature of the programmed cell death or apoptosis.56, 57 From the in vitro anticancer studies it was observed that compounds 6p and 6m significantly inhibited the growth of human lung cancer cell line A-549. Therefore it was of interest to determine the mechanism of cell death in the same cell line. The DNA fragmentation analysis revealed that these compounds induced a discrete ladder pattern in A-549 cell line at 2 µM after 48 h of

incubation thereby showing significant fragmentation. Roscovitine also exhibited DNA fragmentation, however, no fragmentation was observed in untreated cells as shown in Figure 6.

In the conclusion, we have designed and synthesized a series of aminobenzothiazole linked pyrazolo[1,5-a]pyrimidine conjugates (6a-t) and evaluated their anticancer activity against five human cancer cell lines (A-549, HeLa, MCF-7, HT-29 and ACHN). Some of these compounds exhibited promising anticancer activity at micro molar (µM) concentration. The in vitro screening results revealed that these compounds exhibited promising anticancer activity with IC50 values ranging from 1.94 to 41.68 µM against different cancer cell lines. Two of the potent compounds 6m and 6p showed IC50 values ranging 1.94-3.46 µM and 2.01-7.07 µM respectively. Studies also showed that 6m and 6p caused the G2/M cell cycle arrest in A-549 cancer cell line and reduction in Cdk1 expression level. Hoechst 33258 staining and DNA fragmentation assay revealed that this compound induces cell death by apoptosis. Further, activation of caspase-3 also suggested that these compounds induce apoptotic cell death. Based on the studies, it can be deduced that coupling of pyrazolo[1,5-a]pyrimidine carboxylic acid moieties to aminobenzothiazole scaffold through an amide functionality has shown promising anticancer activity and could be considered as a lead for the effective treatment against cancer.

Acknowledgment J. R. T. is thankful to DST (India) for the award of research fellowship.

References and notes 1. Munoz, N.; Bosch, F. X.; de Sanjose, S.; Shah, K. V. Mutat. Res. 1994, 305, 293. 2. Murray, A. W. Cell. 2004, 116, 221. 3.

Fu, M.; Wang, C.; Li, Z.; Sakamaki, T.; Pestell, R. G. Endocrinology. 2004, 145, 5439.

4.

Malumbres, M.; Barbacid, M. Nature Rev. Cancer. 2001, 1, 222.

5. (a) Li, J.; Zhao, Y. F.; Zhao, X.L.; Yuan, X. Y.; Gong, P. Arch. Pharm. 2006, 339, 593; (b) Ahmed, O. M.; M.A. Mohamed, Ahmed, R.R.; Ahmed, S. A. Eur. J. Med. Chem. 2009, 44, 3519. 6. (a) Chen, Y. L.; J.P. Patent 2000502723; Chem. Abstr. 1998, 17, 204905; (b) Selleri, S.; Bruni, F.; Costagli, C.; Costanzo, A.; Guerrini, G.; Ciciani, G.; Gratteri, P.; Besnard, F.; Costa, B.; Montali, M.; Martini, C.; Fohlin, J.; Siena, G. D.; Aiello, P. M. J. Med. Chem. 2005, 48, 6756; (c) Popik, P.; Kostakis, E.; Krawczyk, M.; Nowak, G.; Szewczyk, B.; Krieter, P.; Chen, Z.; Russek, S. J.; Gibbs, T. T.; Farb, D. H.; Skolnick, P.; Lippa, A. S.; Basile, A. S. J. Pharmacol. Exp. Ther. 2006, 319, 1244. 7.

Bondock, S.; Fadaly, W.; Metwally, M. A. Eur. J. Med. Chem. 2010, 45, 3692.

8. Shenone, S.; Bruno, O.; Bondavalli, F.; Ranise, A.; Mosti, L.; Menozzi, G.; Fossa, P.; Donnini, S.; Santoro, A.; Ziche, M.; Manetti, F.; Botta, M. Eur. J. Med. Chem. 2004a, 39, 939. 9. Kim, D. C.; Lee, Y. R.; Yang, B.; Shin, K. J.; Kim, D. J.; Chung, B. Y.; Yoo, K. H. Eur. J. Med. Chem. 2008, 38, 525. 10. Shenone, S.; Bruno, O.; Ranise, A.; Bondavalli, F.; Fossa, P.; Mosti, L.; Menozzi, G.; Carraro, F.; Naldini, A.; Bernini, C.; Manetti, F.; Botta, M. Bioorg. Med. Chem. Lett. 2004b, 14, 2511. 11. Morisi, R.; Celano, M.; Tosi, E.; Schenone, S.; Navarra, M.; Ferretti, E.; Costante, G.; Durante, C.; Botta, G.; Agostino, M. D.; Brullo, C.; Filetti, S.; Botta, M.; Russo, D. J.Endocrinol. Invest. 2007, 10, 31. 12. Angelucci, A.; Schenone, S.; Gravina, G. L.; Muzi, P.; Festuccia, C.; Vicentini, C.; Botta, M.; Bologna, M. Eur. J. Cancer. 2006, 42, 2838. 13. Farley, M. E.; Hoffman, W. F.; Rubino, R. S.; Hungate, R. W.; Tebben, A. J.; Rutledge, R. Z. R. Z.; McFall, R. C.; Huckle, W. R.; Kendall, R. L.; Coll, K. E.; Thomas, K. A. Bioorg. Med. Chem. Lett. 2002, 12, 2767.

14. Farley, M. E.; Rubino, R. S.; Hoffman, W. F.; Hambaugh, S. R.; Arrington, K. L.; Hungate, R. W.; Bilodeau, M. T.; Tebben, A. J.; Rutledge, R. Z.; Kendall, R. L.; McFall, R. C.; Huckle, W. R.; Coll, K. E.; Thomas, K. A. Bioorg.Med.Chem. Lett. 2002, 12, 3537. 15. Krystof, V.; Moravcova, D.; Paprskarova, M.; Barbier, P.; Peyrot, V.; Hlobikova, A.; Havlicek, L.; Stramd, M. Eur. J. Med. Chem. 2006, 41, 1405. 16. McClue, S. J.; Blake, D.; Clarke, R.; Cowan, A.; Cummings, L.; Fischer, P. M.; MacKenzie, M.; Melville, J.; Stewart, K.; Wang, S.; Zhelev, N.; Zheleva, D.; Lane, D. P. Int. J. Cancer. 2002, 102, 463. 17. Parry, D.; Guzi, T.; Seghezzi, W.; Paruch, K.; Doll, R.; Nomeir, A.; Wang, Y.; Oft, M.; Kirschmeier, P.; Chen, T.; Lees, E. Poster no. 4371, American Association Cancer Research, Los Angeles, CA, April 14–18, 2007. 18. Misra, R. N.; Xiao, H.; Kim, K. S.; Lu, S.; Han, W. C.; Barbosa, S. A.; Hunt, J. T.; Rawlins, D. B.; Shan, W.; Ahmed, S. Z.; Qian, L.; Chen, B. C.; Zhao, R.; Bednarz, M. S.; Kellar, K. A.; Mulheron, J. G.; Batorsky, R.; Roongta, U.; Kamath, A.; Marathe, P.; Ranadive, S. A.; Sack, J. S.; Tokarski, J. S.; Pavletich, N. P.; Lee, F. Y. F.; Webster, K. R.; Kimball, S. D. J. Med. Chem. 2004, 47, 1719. 19. Yamazaki, K.; Kaneko, Y.; Suwa, K.; Ebara, S.; Nakazawa, K.; Yasuno, K. Bioorg. Med. Chem. 2005, 13, 2509. 20. Brantley, E.; Antony, S.; Kohlhagen, G.; Meng, L. H.; Agama, K.; Stinson, S. F.; Sausville, E. A.; Pommier, Y. Cancer Chemother. Pharmacol. 2006, 58, 62. 21. Lion, C. J.; Matthews, C. S.; Wells, G.; Bradshaw, T. D.; Stevens, M. E. G.; Westwell, A. D. Bioorg. Med. Chem. Lett. 2006, 16, 5005. 22. Huang, S. T.; Hsei, I. J.; Chen, C. Bioorg. Med. Chem. 2006, 14, 6106. 23. Yoshida, M.; Hayakawa, I.; Hayashi, N.; Agatsuma, T.; Oda, Y.; Tanzawa, F.; Iwasaki, S.; Koyama, K.; Furukawa, H.; Kurakata, S.; Sugano, Y. Bioorg. Med. Chem. Lett. 2005, 15, 3328. 24. Das, J.; Moquin, R.V.; Lin, J.; Liu, C.; Doweyko, A. M.; DeFex, H. F.; Fang, Q.; Pang, S.; Pitt, S.; Shen, D. R.; Schieven, G. L.; Barrish, J. C.; Wityak, J. Bioorg. Med. Chem. Lett. 2003, 13, 2587. 25. O’Brien, S. E.; Browne, H. L.; Bradshaw, T. D.; Westwell, A. D.; Stevens, M. F. G.; Laughton, C. A. Org. Biomol. Chem. 2003, 1, 493.

26. Hutchinson, I.; Jennings, S. A.; Vishnuvajjala, B. R.; Westwell, A. D.; Stevens, M. F. G. J. Med. Chem. 2002, 45, 744. 27. Saeed, S.; Rashid, N.; Jones, P. G.; Ali, M.; Hussain, R. Eur. J. Med. Chem. 2010, 45, 1323. 28. Elzahabi, H. S. A. Eur. J. Med. Chem. 2011, 46, 4025. 29. Hu, W. P.; Chen, Y. K.; Liao, C. C.; Yu, H. S.; Tsai,Y. M.; Huang, S. M.; Tsai, F. Y.; Shen, H. C.; Chang, L. S.; Wang, J. J. Bioorg. Med. Chem. 2010, 46, 6197. 30. Abdel-Rahman, H. M.; Morsy, M. A. J. Enzyme Inhib. Med. Chem. 2007, 22, 57. 31. Hutchinson, A.; Chua, M.; Browne, H. L.; Trapani, V.; Bradshaw, T. D.; Westwell, A. D.; Stevens, M. F. J. Med. Chem. 2001, 44, 1446. 32. Kashiyama, E.; Hutchinson, I.; Chua, M. S.; Stinson, S. F.; Phillips, L. R.; Kaur, G.; Sausville, E. A.; Bradshaw, T. D.; Westwell, A. D.; Stevens, M. F. J. Med. Chem. 1999, 42, 4172. 33. Mortimer, C. G..; Wells, G.; Crochard, J. P.; Stone, E. L.; Bradshaw, T. D.; Stevens, M. F.; Westwell, A. D. J. Med. Chem. 2006, 49, 179. 34. Chua, M. S.; Shi, D. F.; Wrigley, S.; Bradshaw, T. D.; Hutchinson, I.; Shaw, P. N.; Barrett, D. A.; Stanley, L. A.; Stevens, M F. J. Med. Chem. 1999, 42, 381. 35. Trapani, V.; Patel, V.; Leong, C. O.; Ciolino, H. P.; Yeh, G. C.; Hose, C.; Trepel, J. B.; Stevens, M. F.; Sausville, E. A.; Loaiza-Pérez, A. I. Br. J. Cancer. 2003, 8, 599. 36. Shi, D. F.; Bradshaw, T. D.; Wrigley, S.; McCall, C. J.; Lelieveld, P.; Fichtner, I.; Stevens, M.F. J. Med. Chem. 1996, 39, 3375. 37. Bradshaw, T. D.; Westwell, A. D. Curr. Med. Chem. 2004, 11, 1009. 38. Vullo, D.; Franchi, M.; Gallori, E.; Antel, J.; Scozzafava, A.; Supuran, C.T. J. Med.Chem. 2004, 47, 1272. 39. Catriona, G. M.; Geoffrey, W.; Jean, C. P.; Erica, L. S.; Tracey, D. B.; Malcolm, F. G. S.; Andrew, D. W. J. Med. Chem. 2006, 49, 179. 40. Al-Soud, Y. A. Arkivoc. 2008, 15, 225. 41. Kashiyama, E.; Hutchinson, I.; Chua, M. S.; Stinson, S. F.; Phillips, L.R.; Kaur, G.; Sausville, E. A.; Bradshaw, T. D.; Westwell, A. D.; Stevens, M.F.G. J. Med. Chem. 1999, 42 4172. 42. Bradshaw, T. D.; Wrigley, S.; Shi, D. F.; Schultz, R. J.; Paull, K. D.; Stevens,M. F.G. Br. J. Cancer. 1998, 77, 745.

43. (a) Kamal, A.; Tamboli, J. R.; Ramaiah, M. J.; Adil, S. F.; Rao, G. K.; Viswanath, A.; Reddy, A. M.; Pushpavalli, S.N.C.V.L.; Pal-Bhadra, M. Chem Med Chem , 2012, 7, 1453. 44. Samanta, K.; Chakravarti, B.; Mishra, J. K.; Dwivedi, S. K. D.; Nayak, V. L.; Choudhry, P.; Bid, H. K.; Konwar, R.; Chattopadhyay, N.; Panda, G. Bioorg. Med. Chem. Lett. 2010, 20, 283. 45. Pirollo, K. F.; Bouker, K. B.; Chang, E. H. Anticancer Drugs. 2000, 11, 419. 46. Nylader, K.; Dabelsteen, E.; Hall, P. A. J. Oral Pathol. Med. 2000, 29, 413. 47. Kaul, R.; Mukherjee, S.; Ahmed, F.; Bhat, M. K.; Chhipa, R.; Galande, S.; Chattopadhyay, S. Int. J. Cancer. 2003, 103, 606. 48. Gartel, A. L.; Feliciano, I. L.; Tyner, A. L. Oncol. Res. 2003, 13, 405. 49. O’Connor, P. M.; Ferris, D. K.; Pagano, M.; Draetta, G.; Pines, J.; Hunter, T.; Longo, D. L.; Kohn, K. W. J. Biol. Chem. 1993, 268, 8298. 50. Marko, D.; Schatzle, S.; Friedel, A.; Genzlinger, A.; Zanki, H.; Meijer, L.; Eisenbrand, G. Br. J. Cancer. 2001, 84, 283. 51. General procedure for the synthesis of compounds 6a-t: To a solution of compounds (10a-d) (0.6 mmol) in dichloromethane (20 ml) was added 1-(3-dimethylaminopropyl)-3ethylcarbodiimide (EDCI) (1.2 mmol) and 1-hydroxy-1,2,3-benzotriazole (HOBt) (0.1 mmol). Then added corresponding 2-aminobenzothiazoles 11a-e (0.6 mmol) at 0o C. After 10 minute, the reaction mixture was stirred at room temperature for 8 h. The reaction was monitored by TLC. After completion of reaction, water was added to reaction mixture and the aqueous phase was extracted with dichloromethane (2 × 30 ml). The organic layer was dried with Na2SO4 and evaporated under reduced pressure to afford the crude product which was further purified by column chromatography on silica gel using ethyl acetate and hexane as solvent system to obtain the pure products as yellow solids. N-(6-chlorobenzo[d]thiazol-2yl)-7-(4-fluorophenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-5-carboxamide

(6a):

The

compound 6a was prepared according to the general procedure. Yield 81%; mp: 248-249 oC; 1

H NMR (300 MHz, CDCl3): δ 6.99 (d, 1H, J = 8.5 Hz), 7.07 (d, 3H, J = 8.3 Hz), 7.29 (d,

1H, J = 8.1 Hz), 7.34-7.48 (m, 3H), 7.67 (d, 1H, J = 8.8 Hz), 7.78 (s, 1H), 7.98 (d, 2H, J = 7.1 Hz); 8.28 (d, 2H, J = 8.5 Hz); 13C NMR (300 MHz, CDCl3): δ 95.3, 104.2, 115.8, 116.1, 121.3, 121.4, 124.2, 126.3, 126.6, 128.8, 129.5, 131.9, 132, 132.3, 145.6, 146.3, 148.7, 149.3, 157, 157.6, 161.3, 162.8; MS (ESI): 500 [M+H]+; HRMS (ESI) calcd for C26H16ON5ClFS

[M+H]+

500.0748.;

found:

500.0754.

N-(6-chlorobenzo[d]thiazol-2-yl)-7-(4-methoxy

phenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-5-carbox amide (6b): The compound 6b was prepared according to the general procedure. Yield 85%; mp: 299-300 oC; 1H NMR (300 MHz, CDCl3): δ 3.95 (s, 3H), 7.16 (t, 3H, J = 7.7 Hz), 7.42-7.55 (m, 4H), 7.78 (d, 1H, J = 8.6 Hz), 7.86 (d, 1H, J = 1.8 Hz), 7.89 (s, 1H), 8.07 (d, 2H, J = 6.6 Hz); 8.36 (d, 2H, J = 8.8 Hz);

13

C NMR (300 MHz, CDCl3): δ 55.6, 94.9, 103.5, 114.1, 121.2, 121.5, 122.5, 124.2,

125, 126.4, 126.7, 128.8, 129.4, 131.5, 132.2, 132.5, 145.5, 147.1, 148.7, 149.5, 157.2, 157.4, 161.6, 162.3; MS (ESI): 512 [M+H]+; HRMS (ESI) calcd for C27H19O2N5ClS [M+H]+ 512.0948.; found: 512.0957. N-(6-chlorobenzo[d]thiazol-2-yl)-7-(3,4-dimethoxyph enyl)-2phenylpyrazolo[1,5-a]pyrimidine-5-carboxamide (6c):

The compound 6c was prepared

according to the general procedure. Yield 80%; mp: 249-250 oC; 1H NMR (300 MHz, CDCl3): δ 4.01 (s, 3H), 4.03 (s, 3H ), 7.09 (d, 1H, J = 7.9 Hz), 7.16 (s, 1H), 7.40-7.51 (m, 4H), 7.76 (d, 1H, J = 7.9 Hz), 7.84 (s, 1H), 7.88 (s, 1H), 7.94 (d, 1H, J = 7.9 Hz), 8.04 (d, 3H, J = 5.9 Hz); MS (ESI): 542 [M]+. N-(6-chlorobenzo[d]thiazol-2-yl)-2-phenyl -7-(3,4,5trimethoxy phenyl)pyra zolo[1,5-a]pyrimi dine-5-carboxamide (6d): The compound 6d was prepared according to the general procedure. Yield 72%; mp: 312-313 oC; 1H NMR (300 MHz, CDCl3): δ 4.01 (s, 9H), 7.09 (dd, 1H, J = 2.2, 8.3 Hz), 7.21 (d, 1H, J = 4.5 Hz),7.36 (d, 1H, J = 3.0 Hz), 7.42-7.58 (m, 3H), 7.63 (s, 2H), 7.7 (d, 1H, J = 9.0 Hz),7.92 (s, 1H), 8.07 (dd, 2H, J = 1.5, 8.3 Hz);

13

C NMR (300 MHz, CDCl3): δ 56.8, 61.4, 95.5, 104.4, 107.7,

121.7, 121.8, 124.6, 125.6, 126.7, 126.9, 129.2, 129.9, 132.5, 132.7, 141.5, 145.9, 147.4, 149.1,

149.9,

153.5,

157.3,

157.9,

161.8;

MS

(ESI):

573

[M+H]+.

N-(6-

fluorobenzo[d]thiazol-2-yl)-7-(4-fluorophenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-5-carbo xamide (6e): The compound 6e was prepared according to the general procedure. Yield 79%; mp: 269-270 oC; 1H NMR (300 MHz, CDCl3): δ 7.22 (t, 1H, J = 3.7 Hz), 7.34 (t, 2H, J = 6.7 Hz), 7.44-7.63 (m, 5H), 7.81 (dd, 1H, J = 4.5, 8.3 Hz) 7.88 (s, 1H), 8.05 (d, 2H, J = 6.7 Hz), 8.34 (dd, 2H, J = 5.2, 9.0 Hz); MS (ESI): 484 [M+H]+; HRMS (ESI) calcd for C26H16ON5F2S [M+H]+ 484.1041.; found: 484.1052. N-(6-fluorobenzo[d]thiazol-2-yl)-7-(4-methoxy phe nyl)-2-phenylpyrazolo[1,5-a]pyrimidine-5-carbox amide (6f): The compound 6f was prepared according to the general procedure. Yield 84%; mp: 294-295 oC; 1H NMR (300 MHz, CDCl3): δ 3.96 (s, 3H), 7.12-7.24 (m, 4H), 7.41-7.61 (m, 4H), 7.81 (dd, 1H, J = 4.7, 8.8 Hz), 7.89 (s, 1H), 8.08 (d, 2H, J = 6.9 Hz), 8.37 (d, 2H, J = 8.8 Hz); MS (ESI): 496

[M+H]+; HRMS (ESI) calcd for C27H19O2N5FS [M+H]+ 496.1197.; found: 496.1204. 7-(3,4dimethoxyphenyl)-N-(6-fluorobenz[d]thiazol-2-yl)-2-phenylpyrazolo[1,5-a]pyri

midine-5-

carboxamide (6g): The compound 6f was prepared according to the general procedure. Yield 80%; mp: 246-247 oC; 1H NMR (300 MHz, CDCl3): δ 4.03 (s, 3H), 4.05 (s, 3H), 7.10 (dd, 1H, J = 3.7, 8.3 Hz), 7.17-7.26 (m, 2H), 7.43-7.60 (m, 4H), 7.81 (dd, 1H, J = 4.5, 9.0 Hz), 7.90 (d, 1H, J = 3.7 Hz), 7.96 (dd, 1H, J = 1.5, 8.3 Hz), 8.06 (t, 3H, J = 4.5 Hz); 13C NMR (300 MHz, CDCl3): δ 56.1, 56.2, 95, 103.5, 106.1, 107.9, 110.9, 115.2, 121.5, 122.3, 122.6, 123.6, 124.1, 125.4, 126.6, 128.8, 129.5, 132.2, 133.3, 144.8, 147.1, 148.5, 149.6, 152.0, 157.5, 161.6; MS (ESI): 548 [M+Na]+. N-(6-fluorobenzo[d]thiazol-2-yl) -2-phenyl -7-(3,4,5trimetho xyphenyl)pyrazolo[1,5-a]pyrimi dine-5-carboxamide (6h): The compound 6f was prepared according to the general procedure. Yield 78%; mp: 300-301 oC; 1H NMR (300 MHz, CDCl3): δ 4.01 (s, 9H), 7.23 (s, 2H), 7.44-7.61 (m, 4H), 7.63 (s, 2H), 7.8 (dd, 1H, J = 4.5, 9.0 Hz), 7.92 (s, 1H), 8.07 (dd, 2H, J = 1.5, 8.3 Hz); MS (ESI): 556 [M+H]+; HRMS (ESI) calcd for C29H23O4N5FS [M+H]+ 556.1452.; found: 556.1465. 7-(4-fluorophenyl)-N(6-methoxbenzo [d]thiazol-2-yl)-2-phenylpyrazolo[1,5-a]pyrimidine-5-carboxamide (6i): The compound 6f was prepared according to the general procedure. Yield 77%; mp: 198-199 o

C; 1H NMR (500 MHz, CDCl3): δ 4.05 (s, 3H), 5.83 (dd, 1H, J = 2.9, 8.9 Hz), 5.94 (s, 1H),

6.05-6.11 (m, 3H), 6.16-6.27 (m, 3H), 6.50 (d, 1H, J = 8.9 Hz), 6.60 (d, 1H, J = 7.9 Hz), 6.79 (d, 2H, J = 6.9 Hz), 7.08 (dd, 2H, J = 5.9, 8.9 Hz);

C NMR (300 MHz, CDCl3): δ 56.1,

13

95.6, 104.5, 104.6, 115.8, 116.1, 116.4, 122.2, 126.8, 127, 129.2, 129.5, 129.9, 132.2, 132.4, 143.2, 146, 146.5, 149.7, 155.3, 157.3, 157.9, 161.4, 163.2; MS (ESI): 496 [M+H]+; HRMS (ESI)

calcd

for

C27H19O2N5FS

[M+H]+

496.1238.;

found:

496.1248.

N-(6-

methoxybenzo[d]thiazol-2-yl)-7-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-5carboxamide (6j): The compound 6j was prepared according to the general procedure. Yield 84%; mp: 230-231 oC; 1H NMR (300 MHz, CDCl3): δ 3.90 (s, 3H), 3.95 (s, 3H), 7.09 (dd, 1H, J = 2.4, 8.8 Hz), 7.12-7.18 (m, 3H), 7.36 (d, 1H, J = 2.2 Hz), 7.41-7.54 (m, 3H), 7.76 (d, 1H, J = 8.8 Hz), 7.88 (s, 1H), 8.07 (d, 2H, J = 6.9 Hz), 8.36 (d, 2H, J = 8.8 Hz);

13

C NMR

(300 MHz, CDCl3): δ 55.5, 55.7, 94.8, 103.4, 104.1, 114, 115.3, 121.8, 122.5, 126.6, 128.7, 129.3, 131.5, 132.2, 133.5, 142.9, 145.5, 147, 149.4, 154.9, 156.9, 157.3, 161.3, 162.2; MS (ESI): 508 [M+H]+; HRMS (ESI) calcd for C28H22O3N5S [M+H]+ 508.1437.; found: 508.1445.

7-(3,4-dimethoxyphenyl)-N-(6-methoxybenzo[d]thiazol-2-yl)-2-phenylpyrazolo

[1,5-a]pyrimi dine-5-carboxamide (6k): The compound 6f was prepared according to the general procedure. Yield 83%; mp: 240-241 oC; 1H NMR (300 MHz, CDCl3): δ 3.90 (s, 3H), 4.03 (s, 6H), 7.05-7.20 (m, 3H), 7.33-7.57 (m, 4H), 7.76 (d, 1H, J = 8.3 Hz), 7.90 (s, 1H), 7.96 (d, 1H, J = 8.3 Hz), 8.06 (d, 3H, J = 6.0 Hz); MS (ESI): 538 [M+H]+; HRMS (ESI) calcd for C29H24O4N5S [M+H]+ 538.1521.; found: 538.1528. N-(6-methoxybenzo[d]thiazol2-yl)-2-phenyl-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidine-5-carboxamide

(6l):

The compound 6l was prepared according to the general procedure. Yield 79%; mp: 245-246 o

C; 1H NMR (300 MHz, CDCl3): δ 3.82 (s, 3H), 3.87 (s, 6H), 4.01 (s, 3H), 6.78 (dd, 1H, J =

4.5, 7.5 Hz), 6.86 (d, 1H, J = 6.7 Hz), 7.07 (d, 1H, J = 8.3 Hz), 7.36 (s, 1H), 7.40-7.52 (m, 3H), 7.8 (dd, 1H, J = 2.2, 9.0 Hz), 8.02 (dd, 2H, J = 1.5, 8.3 Hz), 8.21 (d, 1H, J = 5.2 Hz), 8.30 (dd, 1H, J = 2.2, 5.2 Hz);

C NMR (300 MHz, CDCl3): δ 55.7, 56.4, 61, 95.1, 104,

13

104.1, 107.2, 115.4, 121.9, 122.2, 125.2, 126.5, 128.8, 129.5, 132,1, 133.5, 142.9, 145.6, 146.9, 149.5, 153.1, 154.8, 157, 157.4, 161.2; MS (ESI): 568 [M+H]+; HRMS (ESI) calcd for C30H26O5N5S [M+H]+ 568.1649.; found: 568.1658. N-(benzo[d] thiazol-2-yl)-7-(4fluorophenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-5-carbox amide (6m): The compound 6m was prepared according to the general procedure. Yield 81%; mp: 216-217 oC; 1H NMR (300 MHz, CDCl3): δ 7.21 (s, 1H), 7.30-7.41 (m, 3H), 7.44-7.54 (m, 4H), 7.87-7.92 (m, 3H), 8.05 (dd, 2H, J = 1.5, 8.3 Hz), 8.31-8.37 (m, 2H);

13

C NMR (300 MHz, CDCl3): δ 94.8,

103.8, 115.2, 115.5, 120.8, 120.9, 123.7, 125.8, 126.1, 128.3, 129, 131.4, 131.6, 131.8, 145.2, 145.7, 148.2, 148.8, 156.4, 157, 160.8, 162.3; MS (ESI): 466 [M+H]+; HRMS (ESI) calcd for C26H17ON5S

[M+H]+

466.1132.;

found:

466.1141.

N-(benzo[d]

thiazol-2-yl)-7-(4-

methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-5-carboxamide (6n): The compound 6n was prepared according to the general procedure. Yield 76%; mp: 235-236 oC; 1H NMR (300 MHz, CDCl3): δ 3.95 (s, 3H), 7.16 (t, 3H, J = 6.0 Hz), 7.33-7.54 (m, 5H), 7.88 (t, 3H, J = 4.9 Hz), 8.07 (d, 2H, J = 8.3 Hz), 8.36 (d, 2H, J = 9.0 Hz); 13C NMR (300 MHz, CDCl3): δ 55.5, 94.9, 103.4, 114, 121.2, 121.4, 122.4, 124.1, 126.3, 126.6, 128.7, 129.4, 131.5, 132.2, 132.3, 145.4, 147, 148.6, 149.4, 157.1, 157.3, 161.5, 162.3; MS (ESI): 478 [M+H]+; HRMS (ESI) calcd for C27H20O2N5S [M+H]+ 478.1332.; found: 478.1340. N-(benzo[d]thiazol-2-yl)7-(3,4-dimethoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-5-carbo xamide (6o): The compound 6o was prepared according to the general procedure. Yield 78%; mp: 210-211 oC; 1

H NMR (300 MHz, CDCl3): δ 4.02 (s, 6H), 7.02 (d, 1H, J = 2.2 Hz), 7.06 (dd, 3H, J = 2.2,

9.0 Hz), 7.24 (t, 1H, J = 4.5 Hz), 7.46-7.54 (m, 5H), 7.65 (s, 1H), 7.84 (dd, 1H, J = 4.5, 9.0 Hz), 7.94 (s, 1H), 8.09 (d, 1H, J = 6.7 Hz); MS (ESI): 508 [M+H]+; HRMS (ESI) calcd for C28H22O3N5S [M+H]+ 508.1411.; found: 508.1423. N-(benzo[d]thiazol-2-yl)-2-phenyl-7(3,4,5-trimethoxy phenyl)pyrazolo [1,5-a]pyrimidine-5-carboxamide (6p): The compound 6p was prepared according to the general procedure. Yield 80%; mp: 187-188 oC; 1H NMR (300 MHz, CDCl3): δ 4.01 (s, 9H), 7.21 (s, 1H), 7.34-7.55 (m, 5H), 7.63 (s, 2H), 7.86-7.94 (m, 3H), 8.07 (d, 2H, J = 8.1 Hz); 13C NMR (300 MHz, CDCl3): δ 56.3, 61, 95.1, 104, 107.2, 121.3, 121.4, 124.2, 125.2, 126.3, 126.5, 128.8, 129.5, 132.1, 132.3, 141, 145.5, 147, 148.7, 149.5, 153.1, 156.9, 157.4, 161.4; MS (ESI): 538 [M+H]+; HRMS (ESI) calcd for C29H24O4N5S [M+H]+ 538.1543.; found: 538.1545. N-(5,6-dimethylbenzo[d]thiazol-2-yl)-7(4-fluoro phenyl)-2-phenylpyrazolo [1,5-a]pyrimidine-5-carboxamide (6q): The compound 6q was prepared according to the general procedure. Yield 76%; mp: 253-254 oC; 1H NMR (300 MHz, CDCl3): δ 2.40 (s, 3H), 2.42 (s, 3H), 7.21 (s, 1H), 7.34 (t, 2H, J = 8.4 Hz), 7.427.55 (m, 3H), 7.64 (d, 2H, J = 2.6 Hz), 7.88 (s, 1H), 8.06 (d, 2H, J = 7.3 Hz), 8.34 (dd, 2H, J = 4.9, 8.6 Hz); MS (ESI): 494 [M+H]+. N-(5,6-dimethylbenzo[d]thiazol-2-yl)-7-(4-methoxy phenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-5-carboxamide (6r): The compound 6r was prepared according to the general procedure. Yield 84%; mp: 290-291 oC; 1H NMR (300 MHz, CDCl3): δ 2.39 (s, 3H), 2.40 (s, 3H), 3.95 (s, 3H), 7.12-7.16 (m, 3H), 7.41-7.53 (m, 3H), 7.63 (d, 2H, J = 3.0 Hz), 7.87 (s, 1H), 8.07 (dd, 2H, J = 1.5, 8.3 Hz), 8.33-8.38 (m, 2H); C NMR (300 MHz, CDCl3): δ 20, 20.1, 55.4, 94.8, 103.4, 114, 121.3, 121.6, 122.5, 126.6,

13

128.7, 129.3, 129.7, 131.5, 132.3, 133.5, 135.4, 145.6, 146.9, 147.3, 149.4, 156.1, 157.2, 161.3,

162.2;

MS

(ESI):

506

[M+H]+.

7-(3,4-dimethoxyphenyl)-N-(5,6-

dimethylbenzo[d]thiazol-2-yl)-2-phenylpyrazolo[1,5-a]pyrimi dine-5-carboxamide (6s): The compound 6s was prepared according to the general procedure. Yield 82%; mp: 252-253 oC; 1

H NMR (300 MHz, CDCl3): δ 2.39 (s, 3H), 2.40 (s, 3H), 4.02 (s, 3H), 4.03 (s, 3H), 7.10 (d,

1H, J = 8.6 Hz), 7.16 (s, 1H), 7.39-7.53 (m, 3H), 7.63 (d, 2H, J = 3.5 Hz), 7.89 (s, 1H), 7.96 (dd, 1H, J = 2.0, 8.4 Hz), 8.06 (d, 3H, J = 6.4 Hz);

C NMR (300 MHz, CDCl3): δ 20, 20.2,

13

56, 56.1, 94.8, 103.4, 110.8, 112.5, 121.3, 121.5, 122.6, 123.5, 126.5, 128.8, 129.3, 129.5, 132.2, 133.5, 135.5, 145.4, 146.7, 147, 148.6, 149.5, 151.9, 156.2, 157.1, 161.3; MS (ESI): 536 [M+H]+; HRMS (ESI) calcd for C30H26O3N5S [M+H]+ 536.1757.; found: 536.1768. N(5,6-dimethylbenzo[d]thiazol-2-yl)-2-phenyl-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyri

midine-5-carboxamide (6t): The compound 6t was prepared according to the general procedure. Yield 75%; mp: 282-283 oC; 1H NMR (300 MHz, CDCl3): δ 2.40 (s, 3H), 2.41 (s, 3H), 4.00 (s, 9H), 7.21 (d, 1H, J = 5.2 Hz), 7.43-7.55 (m, 3H), 7.64 (d, 4H, J = 3.7 Hz), 7.92 (s, 1H), 8.06 (dd, 2H, J = 1.5, 8.3 Hz);

C NMR (300 MHz, CDCl3): δ 20, 20.2, 56.3, 61,

13

95.1, 104, 105.7, 107.2, 121.3, 121.6, 125.2, 126.5, 128.8, 129.4, 129.6, 132.1, 133.6, 135.5, 145.6, 146.9, 147.2, 149.5, 153, 156.1, 157.3, 161.3, 162.5; MS (ESI): 566 [M+H]+; HRMS (ESI) calcd for C31H28O4N5S [M+H]+ 566.1856.; found: 566.1863. 52. Evaluation of in vitro anti-cancer activity: The cytotoxicity of the compounds was determined using MTT assay. 1×106 cells/well were seeded in 200 µl DMEM, supplemented with 10% FBS in each well of 96-well microculture plates and incubated for 24 h at 370C in a CO2 incubator. Compounds, diluted to the desired concentrations in culture medium, were added to the wells with respective vehicle control. After 48 h of incubation, 10 µl MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) (5 mg/ml) was added to each well and the plates were further incubated for 4 h. Then the supernatant from each well was carefully removed, formazon crystals were dissolved in 100 µl of DMSO and absorbance at 540 nm wavelength was recorded. Cell cycle analysis: To determine the effect of compound on the stages of cell cycle, A549 cells (1×106) were seeded in six-well plates and incubated for 24 h. After 24 h incubation cells were treated with compounds 6p and 6m at 1 and 2 µM for 48 h along with standard compound Roscovitine. After 48 h treatments, both floating and trypsinized adherent cells were collected and fixed with 70% ethanol. After fixation cells were washed with PBS and stained with 50 µg/mLpropidium iodide in hypotonic lysis buffer (0.1% sodium citrate,0.1% Triton X-100) containing DNase-free RNase-A for 20 min.Stained cells were analyzed using fluorescence-activated cell sorter caliber (Becton Dickinson). Protein extraction and Western blot analysis: 5×105 A549 cells were seeded in 60 mm dish and were allowed to grow for 24 h, 2 µM concentration of compounds 6m and 6p were added to the culture media, and the cells were incubated for an additional 48 h. Total cell lysates from cultured A549 cells were obtained by lysing the cells in ice-cold RIPA buffer (1×PBS, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) and containing 100 lg/mL PMSF, 5 lg/mL Aprotinin, 5 lg/mL leupeptin, 5 lg/mL pepstatin and 100 lg/mL NaF. After centrifugation at 12,000 rpm for 10 min, the protein in supernatant was quantified by Bradford method (BIO-RAD) using Multimode varioskan instrument (Thermo-Fischer

Scientifics). Fifty micrograms of protein per lane was applied in 12% SDS–polyacrylamide gel. After electrophoresis, the protein was transferred to polyvinylidine difluoride (PVDF) membrane (Amersham Biosciences). The membrane was blocked at room temperature for 2 h in 1×TBS + 0.1% Tween20 (TBST) containing 5% blocking powder (Santacruz). The membrane was washed with TBST for 5 min, and primary antibody was added and incubated at 40C overnight. Mouse monoclonal antibodies Cdk1 and β-actin were purchased from cell singnaling technology. Membranes were washed with TBST three times for 15 min and the blots were visualized with chemiluminescence reagent (Thermo Fischer Scientifics Ltd).Images were captured using Chemi Doc UVP (BIO-RAD). Morphological analysis for apoptosis with Hoechst staining: Cells were seeded at a density of 10,000 cells over 18-mm cover slips and incubated for 24 h. Then, the medium was replaced, and cells were treated with compound 6p and 6m at 2 µM for 48 h. Cells treated with vehicle (0.001% DMSO) were included as controls for all experiments. After overnight treatment, Hoechst 33258 (Sigma Aldrich) were added to medium at a concentration of 0.5 mg/mL containing 4% Para formaldehyde. After incubation for 30 min, cells from each dish were captured from randomly selected fields under fluorescent microscope (Leica, Germany) to qualitatively determine the proportion of viable and apoptotic cells based on their relative fluorescence and nuclear fragmentation. Caspase 3 activity: Caspase-3 assay was conducted for detection of apoptosis in Lung cancer cell line (A549). The commercially available apoptosis detection kit (Sigma-Caspase-3 Assay kit, Colorimetric) was used. A549 cells treated with compounds 6p and 6m for 48 h. After 48 h of treatment, cells were collected by centrifugation, washed once with PBS, and cell pellets were collected. Suspended the cell pellet in lysis buffer and incubated for 15 min. After incubation, cells were centrifuge at 20,000 rpm for 15 min and collected the supernatant. Supernatants were used for measuring caspase 3 activity using an ELISA-based assay, according to the manufacturer’s instructions. DNA fragmentation assay: Cells were seeded (1×106) in six-well plates. After incubation of 24 h cells were treated with compound 6p, 6m and Roscovitine at 2 µM concentration. After 48 h of treatment, cells were collected and centrifuged at 2500 rpm for 5 min at 40C. Pellet was collected and washed with Phosphate buffered saline (PBS). Then added 100 µl of Lysis buffer centrifuged at 3000 rpm for 5 min at 40C and collected supernant. And added 10 µl of 10% SDS and 10 µl of (50 mg/ml) RNase-A and incubated for 2 h at 56 0C. After incubation

Proteinase K (25 mg/ml) was added and further incubated at 37 0C for 2 h. Then added 65 µl of 10 M Ammonium acetate and 500 µl of ice cold ethanol and mixed well. And these samples were incubated at -800C for 1 h. After incubatation samples were centrifuged at 12000 rpm for 20 min at 40C. After centrifuge pellet was washed with 80% ethanol and air dried for 10 min at room temperature. Dissolved the pellet in 50 µl of TE buffer and DNA laddering was determined by using 2% agarose gel electrophoresis in TE Buffer. 53. Iyer, S.; Chaplin, D. J.; Rosenthal, S. S.; Boulares, A. M.; Li, L.; Smulson, M. E. Cancer Res. 1998, 58, 4510. 54. Zhu, H.; Zhang, J.; Xue, N.; Hu, Y.; Yang, B.; He, Q. Invest. New Drugs. 2010, 28, 493. 55. Weir, N. M.; Selvendiran, K.; Kutala,V. K.; Tong, L.; Vishwanath, S.; Rajaram, M.; Tridandapani, S.; Anant, S.; Kuppusamy, P. Cancer Biol. Ther. 2007, 6, 178. 56. Konstantinov, S. M.; Berger, M. R. Cancer Lett. 1999, 144, 153. 57. Henkels, P. M.; Turchi, J. J. Cancer Res. 1999, 59, 3077.

.

Figures

Figure 1. Structure of roscovitine (1), SCH-727965 (2), BMS 387032 (3) PMX610 (4), 2-(4aminophenyl)benzothiazole (CJM 126) conjugates

(5) Benzothiazole linked pyrazolo[1,5-a]pyrimidine

A

C

E

B

D

F

Figure 2. Flow cytometric analysis of compounds 6p, 6m and roscovitine in A-549 lung cancer cell line. A: Control cells, B: Roscovitine (2 µM), C: 6m (1 µM), D: 6m (2 µM), E: 6p (1 µM) and F: 6p (2 µM).

Figure 3: Effect of compounds on Cdk1 level. A549 cells were treated with compounds 6m and 6p at 2 µM concentration for 48 h. The cell lysates were collected and expression level of Cdk1 were determined by Western blot analysis. β-Actin was used as a loading control.

A

B

C

D

Figure 4: Hoechst staining in A-549 lung cancer cell line, A: A-549 control cells, B: roscovitine at 2 µM, C: 6p at 2 µM and D: 6m at 2 µM.

Figure 5: Effect of compounds 6p and 6m on caspase-3 activity: A-549 cells were treated with compounds 6p and 6m at 1 and 2 µM concentrations for 48 h. Roscovitine is used as a positive control. Values indicated are the mean ± SD of two different experiments performed in triplicates.

Figure 6: DNA Fragmentation analysis of compounds 6m and 6p. Lane 1: A549 cells (un treated), Lane 2: 6p (2 µM) and Lane 3: 6m (2 µM), Lane 4: Roscovitine (2 µM) and Lane 5: 50 bp marker.

Tables Table 1. Anticancer activity of benzothiazole-pyrazolo[1,5-a]pyrimidine conjugates in human cancer cell lines Cancer panel/cell lines

GI50 µM 6m[b]

Cancer panel/cell lines

6p[c]

Leukamia CCRF-CEM K-562 MOLT-4 RPMI-8226 SR HL-60(TB)

1.8 4.2 21 5.5 3.9 NT[e]

54 25 NT[e NT[e] 19.7

Non-small-cell-lung A549/ATCC EKVX HOP-62 HOP-92 NCI-H226 NCI-H23 NCI-H322M NCI-H460 NCI-H522

2.7 6.6 2.2 1.6 6.3 2.8 5.8 2.1 2.5

NT[e] 42 9.8 0.7 27 11.5 67.5 29 1.9

Colon COLO 205 HCC-2998 HCT-116 HCT-15 HT29 KM12 SW-620

NT[e] NT[e] 2.7 NT[e] NT[e] NT[e] NT[e]

NT[e] NT[e] 4.5 NT[e] NT[e] 22 NT[e]

CNS SF-268 SF-295 SF-539 SNB-19 SNB-75 U251

3.8 4.1 2.6 5.2 1.7 2.0

4.5 10.6 1.3 16.7 2.7 5.1

Melanoma LOX IMVI MALME-3M M14 MDA-MB-435 SK-MEL-2

NT[e] 3.7 3.1 NT[e] 3.1

5.5 12 2.3 2.6 11.2

GI50 µM 6m[b]

Ovarian IGROV1 OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-8 NCI/ADR-RES SK-OV-3 Renal 786-0 A498 ACHN CAKI-1 RXF 393 SN12C TK-10 UO-31

Prostate PC-3 DU-145

Breast MCF7 MDA-MB231/ATCC HS 578T BT-549 T-47D MDA-MB-468 Melanoma SK-MEL-28 SK-MEL-5 UACC-257 UACC-62

6p[c]

4.3 1.9 1.8 NT[e] 2.1 NT[e] 2.3

56 13.3 NT[e NT[e 5.3 19.6 12.5

2.1 1.8 2.2 NT[e] 2.5 6.4 2.4 1.9

2.5 9.2 45.7 53.7 6.2 85 11.9 NT[e

NT[e] 4.9

NT[e 65

5.2 2.3 2.2 2.0 3.3 5.1

22.4 5.6 4.7 6.3 21.4 13.9

NT[e] NT[e] NT[e] 3.7

3.3 13.8 NT[e] 14.4

[a] Compound concetration required to decrease cell growth to half that of untreated cells. [b] 6m (NSC 763669). [c] 6p (NSC 763635). [e]Not tested.

Table 2: IC50 a values (expressed in µM) of compounds b

c

d

e

f

Compound

A549

6a

6.60

17.64

19.77

13.80

9.77

6b

3.09

5.88

4.89

7.24

5.88

6c

6.16

9.33

6.91

7.76

9.12

6d

18.19

38.90

30.19

41.68

22.38

6e

14.79

22.9

22.90

35.48

29.51

6f

11.74

31.38

35.48

39.27

41.68

6g

6.91

18.62

22.38

8.12

8.31

6h

22.38

25.70

27.54

32.35

25.11

6i

23.98

15.42

24.54

26.30

17.33

6j

17.35

7.58

19.05

38.33

24.54

6k

15.84

14

18.72

13.26

10.96

6l

2.81

3.98

2.95

5.24

8.70

6m

1.94

2.08

2.29

3.46

2.63

6n

1.54

2.95

2.23

1.69

4.67

6o

5.49

19.07

14.65

7.94

8.91

6p

2.01

3.16

2.88

4.36

7.07

6q

12.58

12.88

16.98

33.0

18.19

6r

13.18

12.33

19.66

22.44

14.12

6s

2.95

6.91

2.51

8.70

6.76

6t

2.34

3.71

2.29

3.80

8.91

Roscovitine

2.18

1.90

3.98

2.88

1.81

a

DU-145

MCF-7

ACHN

Hela

50% Inhibitory concentration and the values are average of three individual experiments. Lung cancer c Prostate cancer d Breast cancer e Renal cell carcinome f Cervical cancer b

Table 3. Cell cycle distribution of A549 cell line with roscovitine, 6m and 6p SubG1 G0/G1 S

G2/M

A: Control (A549)

1.02

81.33

4.48

13.17

B: Roscovitine-2 µM

7.30

46.75

5.26

40.69

C: 6m-1 µM

4.01

35.34

6.10

44.54

D: 6m-2 µM

3.75

28.75

6.80

64.70

E: 6p-1 µM

11.77

42.29

5.90

40.05

F: 6p-2 µM

9.05

32.18

6.32

52.45

Schemes 6 5 1 1

3

3

2

1

2

3

3

7a-d

8a-d

3

2 3

7a: R1 = H; R2 = F; R3 = H 7b: R1 = H; R2 = OCH3; R3 = H 7c: R1 = OCH3; R2 = OCH3; R3 = H 7d: R1 = OCH3; R2= OCH3; R3 = OCH3

9a-d

6 5

6 5

1

1

2 2

2 3

6a-t

6a: R1 = R3 = R = H; R2 = F; R' = Cl 6b: R1 = R3 = H, R = H; R2 = OCH3; R' = Cl 6c: R1 = R2 = OCH3; R3 = R = H; R' = Cl 6d: R1 = R2 = R3 = OCH3, R = H; R' = Cl 6e: R1 = R3 = R = H; R2 = R' = F 6f: R1 = R3 = R = H; R2 = OCH3; R' = F 6g: R1 = R2 = OCH3; R3 = R = H; R' = F 6h: R1 = R2 = R3 = OCH3, R = H; R' = F 6i: R1 = R3 = R = H; R2 = F; R' = OCH3 6j: R1 = R3 = R = H; R2 = R' = OCH3 6k: R1 = R2 = R' = OCH3; R3 = R = H 6l: R1 = R2= R3 = R' = OCH3, R = H 6m: R1 = R3 =R = R' = H; R2 = F 6n: R1 =R3 = R = R' = H ; R2 = OCH3 6o: R1 = R2 = OCH3; R3 = R= R'= H 6p: R1 =R2= R3 = OCH3, R = R' = H 6q: R1 = R3 = H; R2 = F; R = R'= CH3 6r: R1 = R3 = H; R2 = OCH3; R =R' = CH3 6s: R1 = R2 = OCH3; R3 = H, R =R' = CH3 6t: R1 =R2= R3 = OCH3, R =R' = CH3

3

11a-e

10a-d

3 3

3

Scheme 1: Reagents and conditions: (i) dimethyl oxalate, NaOMe, THF, rt, 8h (ii) 3-amino-5phenyl-1H-pyrazole, cat. HCl, ethanol, reflux, 2h (iii) 2N NaOH, MeOH, reflux, 2h (iv) EDCI/HOBt, CH2Cl2, 0 oC-rt, 8h.

Graphical Abstract Synthesis of pyrazolo[1,5-a]pyrimidine linked aminobenzothiazole conjugates as potential anticancer agents Ahmed Kamal,*a Jaki R. Tamboli,a V. Lakshma Nayak,b S. F. Adil,c M.V.P.S. Vishnuvardhan ,b S. Ramakrishna b