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

Article abstract of DOI:10.1016/j.bmcl.2013.03.129

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 to 7.07 and 1.94-3.46 μM, respectively. Moreover, cell cycle arrest in G ₂/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 staining as well as DNA fragmentation analysis.

Full text of DOI:10.1016/j.bmcl.2013.03.129

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:
S0960-894X(13)00469-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.03.129
BMCL 20365
Reference:
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
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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
b
aDivision of Organic Chemistry, Pharmacology Division, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007, India
cDepartment 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: ahmedkamal@iict.res.in

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 cyclin-
dependent 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.

<Insert Figure 1>
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-5-
phenyl-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-5-
phenyl-1H-pyrazole affords the cyclodehydrated methyl 2,7-diphenylpyrazolo[1,5-a]pyrimidine-
5-carboxylates (9a-d) which was then hydrolyzed to 2,7-diphenylpyrazolo[1,5-a]pyrimidine-5-
carboxylic 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.

<Insert 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 7-
phenyl 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 3,4,5-
trimethoxy 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 3,4,5-
trimethoxy substitution and 4-fluoro substitution on the 7-phenyl ring of pyrazolo[1,5-
a]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.
<Insert Table 1>
<Insert Table 2>
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.
<Insert Figure 2>
<Insert Table 3>
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.
<Insert 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.
<Insert 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.
<Insert Figure 5>
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.
<Insert 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)-3-
ethylcarbodiimide (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-2-
yl)-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;
1H 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
o
1
prepared according to the general procedure. Yield 85%; mp: 299-300 C; H 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
13
Hz); 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)-2-
phenylpyrazolo[1,5-a]pyrimidine-5-carboxamide (6c): The compound 6c was prepared
o
1
according to the general procedure. Yield 80%; mp: 249-250 C; H 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-(3,4,5-
trimethoxy phenyl)pyra zolo[1,5-a]pyrimi dine-5-carboxamide (6d): The compound 6d was
o
1
prepared according to the general procedure. Yield 72%; mp: 312-313 C; H 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
13
(dd, 2H, J = 1.5, 8.3 Hz); 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
o
1
prepared according to the general procedure. Yield 84%; mp: 294-295 C; H 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-(3,4-
dimethoxyphenyl)-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
o
1
80%; mp: 246-247 C; H 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),
13
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); C 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-(3,4,5-
trimetho xyphenyl)pyrazolo[1,5-a]pyrimi dine-5-carboxamide (6h): The compound 6f was
o
1
prepared according to the general procedure. Yield 78%; mp: 300-301 C; H 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
oC; 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); 13C NMR (300 MHz, CDCl3): δ 56.1,
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-5-
carboxamide (6j): The compound 6j was prepared according to the general procedure. Yield
o
1
84%; mp: 230-231 C; H 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); 13C 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]thiazol-
2-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
oC; 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); 13C NMR (300 MHz, CDCl3): δ 55.7, 56.4, 61, 95.1, 104,
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-(4-
fluorophenyl)-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); 13C 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
o
1
6n was prepared according to the general procedure. Yield 76%; mp: 235-236 C; H 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;
1H 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
o
1
6p was prepared according to the general procedure. Yield 80%; mp: 187-188 C; H 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
o
1
6q was prepared according to the general procedure. Yield 76%; mp: 253-254 C; H 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), 7.42-
7.55 (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
o
1
prepared according to the general procedure. Yield 84%; mp: 290-291 C; H 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);
13C NMR (300 MHz, CDCl3): δ 20, 20.1, 55.4, 94.8, 103.4, 114, 121.3, 121.6, 122.5, 126.6,
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;
1H 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); 13C NMR (300 MHz, CDCl3): δ 20, 20.2,
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); 13C NMR (300 MHz, CDCl3): δ 20, 20.2, 56.3, 61,
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-(4-
aminophenyl)benzothiazole (CJM 126) (5) Benzothiazole linked pyrazolo[1,5-a]pyrimidine
conjugates

B
A
D
C
F
E
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
triplicates.
SD of two different experiments performed in

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
Cancer panel/cell
lines
GI50 µM
6p[c]
6m[b]
6p[c]
6m[b]
Leukamia
CCRF-CEM
K-562
MOLT-4
RPMI-8226
SR
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
1.8
4.2
21
5.5
3.9
NT[e]
54
4.3
1.9
56
25
13.3
NT[e
NT[e
5.3
19.6
12.5
NT[e
NT[e]
19.7
1.8
NT[e]
2.1
HL-60(TB)
NT[e]
2.3
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
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
1.9
Colon
Prostate
PC-3
DU-145
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
NT[e]
NT[e]
2.7
NT[e]
NT[e]
4.5
NT[e]
4.9
NT[e
65
NT[e]
NT[e]
NT[e]
NT[e]
NT[e]
NT[e]
22
KM12
SW-620
NT[e]
CNS
Breast
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
MCF7
5.2
2.3
2.2
2.0
3.3
5.1
22.4
5.6
4.7
6.3
21.4
13.9
MDA-MB-
231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
Melanoma
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
NT[e]
3.7
5.5
12
2.3
2.6
11.2
NT[e]
NT[e]
NT[e]
3.7
3.3
13.8
NT[e]
14.4
3.1
NT[e]
3.1
[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
DU-145
17.64
5.88
MCF-7
19.77
4.89
ACHN
13.80
7.24
Hela
9.77
5.88
9.12
6a
6.60
6b
3.09
6c
6.16
9.33
6.91
7.76
6d
18.19
14.79
11.74
6.91
38.90
22.9
30.19
22.90
35.48
22.38
27.54
24.54
19.05
18.72
2.95
41.68
35.48
39.27
8.12
22.38
29.51
41.68
8.31
6e
6f
31.38
18.62
25.70
15.42
7.58
6g
6h
22.38
23.98
17.35
15.84
2.81
32.35
26.30
38.33
13.26
5.24
25.11
17.33
24.54
10.96
8.70
6i
6j
6k
14
6l
3.98
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
3.16
14.65
2.88
7.94
8.91
6p
2.01
4.36
7.07
6q
12.58
13.18
2.95
12.88
12.33
6.91
16.98
19.66
2.51
33.0
18.19
14.12
6.76
6r
22.44
8.70
6s
6t
2.34
3.71
2.29
3.80
8.91
Roscovitine
2.18
1.90
3.98
2.88
1.81
a 50% Inhibitory concentration and the values are average of three individual experiments.
bLung cancer
c Prostate cancer
d Breast cancer
e Renal cell carcinome
f Cervical cancer

Table 3. Cell cycle distribution of A549 cell line with roscovitine, 6m and 6p
SubG1
G0/G1
81.33
46.75
35.34
28.75
42.29
32.18
S
G2/M
13.17
40.69
44.54
64.70
40.05
52.45
A: Control (A549)
1.02
4.48
5.26
6.10
6.80
5.90
6.32
B: Roscovitine-2 µM 7.30
C: 6m-1 µM
D: 6m-2 µM
E: 6p-1 µM
F: 6p-2 µM
4.01
3.75
11.77
9.05

Schemes
6
5
1
1
3
3
2
1
2
3
3
3
8a-d
2
7a-d
3
1 2 3
R = H; R = F; R = H
7a:
9a-d
1
7b
: R = H; R = OCH ; R = H
2
3
3
1
2
7c
: R = OCH ; R = OCH ; R = H
3
3
3
1
2
3
7d
: R = OCH ; R = OCH ; R = OCH
3
3
3
6
5
6
5
1
1
2
2
2
3
11a-e
6a-t
3
10a-d
1 3 2
R = R = R = H; R = F; R' = Cl
6a:
1
6b
: R = R = H, R = H; R = OCH ; R' = Cl
3
2
3
3
1 2 3
: R = R = OCH ; R = R = H; R' = Cl
3
6c
3
3
1
2
3
6d
6e:
: R = R = R = OCH R = H; R' = Cl
3,
1 3 2
R = R = R = H; R = R' = F
1 3 2
: R = R = R = H; R = OCH ; R' = F
3
6f
1
2
3
6g
6h
6i:
: R = R = OCH ; R = R = H; R' = F
3
1
2
3
: R = R = R = OCH , R = H; R' = F
3
1 3 2
R = R = R = H; R = F; R' = OCH
3
1
3
2
6j
: R = R = R = H; R = R' = OCH
3
1 2 3
: R = R = R' = OCH ; R = R = H
3
6k
1
2
3
6l
: R = R = R = R' = OCH , R = H
3
1 3 2
R = R =R = R' = H; R = F
6m:
1
3
2
6n
: R =R = R = R' = H ; R = OCH
3
1 2 3
: R = R = OCH ; R = R= R'= H
3
6o
1 2 3
: R =R = R = OCH , R = R' = H
3
6p
1 3 2
R = R = H; R = F; R = R'= CH
6q:
3
1
6r
: R = R = H; R = OCH ; R =R' = CH
3
2
3
3
1
6s
: R = R = OCH ; R = H, R =R' = CH
2
3
3
3
1
2
6t
: R =R = R = OCH , R =R' = CH
3
3
3
Scheme 1: Reagents and conditions: (i) dimethyl oxalate, NaOMe, THF, rt, 8h (ii) 3-amino-5-
phenyl-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