untitled European Journal of Chemistry 2 (3) (2011) 331‐336 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2011 EURJCHEM DOI:10.5155/eurjchem.2.3.331‐336.319 European Journal of Chemistry Journal homepage: www.eurjchem.com Synthesis and antitumor activity of novel pyrazolo[1,5‐a]pyrimidine derivatives Mervat Mostafa El‐Enanya, Mona Monir Kamelb, Omneya Mahmoud Khalilb and Hala Bakr El‐Nassanb,* a Pharmacology Chemistry Department, Faculty of Pharmaceutical Science and Pharmaceutical Industries, Future University, Cairo, 11562, Egypt** b Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt *Corresponding author at: Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt. Tel.: +00202.23632245; fax: +00202.23635140. E‐mail address: hala_bakr@hotmail.com (H.B. El‐Nassan). ** Sabbatical leave from Cairo University. ARTICLE INFORMATION ABSTRACT Received: 14 November 2010 Received in revised form: 21 February 2011 Accepted: 30 March 2011 Online: 30 September 2011 KEYWORDS A novel series of pyrazolo[1,5‐a]pyrimidine‐3‐carbonitriles substituted with 7‐amino, 7‐substituted amino and 5‐substituted amino groups was synthesized. Some of the newly synthesized compounds were tested in vitro on human colon tumor cell line (HCT116). Compound 14a displayed the highest activity among the tested compounds with IC50 that equals to 0.0020 μM. Pyrazole 5‐Aminopyrazole Pyrazolo[1,5‐a]pyrimidine Antitumor activity Cytotoxic activity HCT116 1. Introduction Cancer is defined as malignant growth of cells. Most tumors arise from a combination of genetic mutations in the cell. These genetic changes lead to activation of oncogenes and suppression or deletion of tumor suppressor genes. As a result, there is unregulated cell proliferation and also a delay in programmed cell death, apoptosis [1]. Most of the clinically used antineoplastic drugs aim to suppress the proliferative process (e.g.: DNA replication or chromosome segregation) [1]. Pyrazolo[1,5‐a]pyrimidines are of considerable chemical and pharmacological importance as purine analogs and many derivatives of pyrazolo[1,5‐a]pyrimidines have been reported to exhibit cytotoxic activity [2‐10]. Different mechanisms account for the cytotoxic effect of this class of compounds, where they have been reported to act as vascular endothelial growth factor receptor inhibitor [2] and cyclin dependent kinase inhibitors [3,6‐8]. Several 7‐substituted aminopyrazolo[1,5‐a]pyrimidine derivatives were reported to have antiproliferative activity against HCT116 and other cell lines (e.g. compounds 1‐3) [3,6,8] (Figure 1). In the present study, several pyrazolo[1,5‐a]pyrimidine derivatives, bearing 2‐methylsulphanyl group, 3‐nitrile group and 7‐amino (6a, 6b, 7a‐d, 8 and 9) or 7‐substituted amino group (14a‐ c) were prepared. Besides, different substitutions were introduced at position 5 (aromatic ring, amino group or carbonyl group) (Scheme 1 and 2). Meanwhile, the study aimed to synthesize 7‐substituted amino‐ 5‐methylpyrazolo[1,5‐a]pyrimidine derivatives. Nevertheless, the product obtained using two different reaction conditions was the 7‐ methyl derivative. 2. Experimental 2.1. Instrumentation Melting points were determined using a Griffin apparatus and were uncorrected. IR spectra were recorded on Mattson Genesis II FT‐IR and values were represented in cm‐1. 1H NMR were carried out on Varian Gemini 200 MHz spectrophotometer, Microanalytical center, Cairo University, Cairo, Egypt, using TMS as an internal standard and chemical shifts were recorded in ppm on δ scale and coupling constants (J) are given in Hz. The electron impact (EI) mass spectra were recorded on Shimadzu QP‐2010 plus, Microanalytical center, Cairo University, Cairo, Egypt. Analytical thin layer chromatography (TLC) on silica gel plates containing UV indicator was employed routinely to follow the course of reactions and to check the purity of products. All reagents and solvents were purified and dried by standard techniques. Elemental microanalyses were performed at Microanalytical Center, Cairo University, Cairo, Egypt, and were within ±0.4%. 2.2. Synthesis 2.2.1. General procedure for the synthesis of 7‐amino‐2‐methyl sulphanyl‐5‐(substituted phenyl)‐4,5‐dihydropyrazolo [1,5‐a] pyrimidine‐3,6‐dicarbonitriles 6a,b and 7‐amino‐2‐methyl sulphanyl‐5‐(substituted phenyl)pyrazolo[1,5‐a]pyrimidine‐3,6‐ dicarbonitriles 7a‐d A mixture of 5‐amino‐3‐methylsulphanyl‐1H‐pyrazole‐4‐ carbonitrile (4) [11] (0.31 g, 0.002 mol), the appropriate substituted benzylidenemalononitrile 5a‐f [12‐15] (0.002 mol) and triethylamine (2 mL) in absolute ethanol (20 mL) was heated under reflux for 7 h. The precipitate formed was filtered, dried and crystallized from acetic acid. 7‐Amino‐5‐(2‐chlorophenyl)‐2‐methylsulphanyl‐4,5‐dihydro pyrazolo[1,5‐a]pyrimidine‐3,6‐dicarbonitrile (6a): Yield: 33%. M.p.: 246‐247 oC. FT‐IR (cm‐1): 3453, 3307, 3236 (NH/NH2), 2228, 2192 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.6 (s, 3H, SCH3), 5.7 (d, 332 El‐Enany et al. / European Journal of Chemistry 2 (3) (2011) 331‐336 Figure 1. Examples of 7‐substituted aminopyrazolo[1,5‐a]pyrimidines with antiproliferative activity. N H N SCH3 H2N NC 4 N NN NH2 CN SCH3 NC Ar N H NN NH2 CN SCH3 NC Ar 6a,b 7a-d a b c N NN NH2 CN SCH3 H2N 8 N H N N NH2 CN SCH3 O 9 Reagents: a) ArCH=CH(CN)2 5a-f, triethylamine, ethanol; b) CH2(CN)2, triethylamine, ethanol; c) NCCH2COOC2H5, Fusion at 160 oC. Compound 5a, 6a 5b, 6b 5c, 7a 2-ClC6H4 2-CH3OC6H4 2-OHC6H4 Compound 5d, 7b 5e, 7c 5f, 7d 3-NO2C6H4 4-ClC6H4 4-(CH3)2NC6H4 Ar Ar Scheme 1 1H, H5, J=2 Hz), 7.3 (s, 1H, NH, D2O exchangeable), 7.4‐7.5 (m, 4H, Ar‐H), 9.2 (s, 2H, NH2, D2O exchangeable). Anal. Calcd. for C15H11ClN6S: C, 52.55; H, 3.23; N, 24.51. Found: C, 52.62; H, 3.44; N, 24.27%. 7‐Amino‐5‐(2‐methoxyphenyl)‐2‐methylsulphanyl‐4,5‐dihydro pyrazolo[1,5‐a]pyrimidine‐3,6‐dicarbonitrile (6b): Yield: 48%. M.p.: 244‐245 oC. FT‐IR (cm‐1): 3425, 3277, 3219 (NH/NH2), 2218, 2189 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.6 (s, 3H, SCH3), 3.7 (s, 3H, OCH3), 5.4 (s, 1H, H5), 7.2 (s, 1H, NH, D2O exchangeable), 6.9‐7.3 (m, 4H, Ar‐H), 9.0 (s, 2H, NH2, D2O exchangeable). MS (m/z (%)): 338 [M+, 1.12%]. Anal. Calcd. for C16H14N6OS: C, 56.79; H, 4.17; N, 24.83. Found: C, 56.50; H, 3.84; N, 24.97%. 7‐Amino‐5‐(2‐hydroxyphenyl)‐2‐methylsulphanylpyrazolo[1,5‐a] pyrimidine‐3,6‐dicarbonitrile (7a): Yield: 22%. M.p.: 224‐225 oC. FT‐ IR (cm‐1): 3436, 3344 (NH2), 2200 (CN). 1H NMR (200 MHz, DMSO‐ d6, δ ppm): 2.7 (s, 3H, SCH3), 7.1‐8.2 (m, 4H, Ar‐H), 8.9 (s, 2H, NH2, D2O exchangeable), 9.3 (s, 1H, OH, D2O exchangeable). Anal. Calcd. for C15H10N6OS: C, 55.89; H, 3.12; N, 26.07. Found: C, 56.02; H, 3.59; N, 26.23%. 7‐Amino‐2‐methylsulphanyl‐5‐(3‐nitrophenyl)pyrazolo[1,5‐a] pyrimidine‐3,6‐dicarbonitrile (7b): Yield: 44%. M.p.: 287‐288 oC. FT‐ IR (cm‐1): 3308, 3273 (NH2), 2214, 2131 (CN), 1528, 1345 (NO2). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.7 (s, 3H, SCH3), 7.8 (t, 1H, J=8 Hz, Ar‐H), 8.3 (d, 1H, J=7.8 Hz, Ar‐H), 8.4 (d, 1H, J=8 Hz, Ar‐H), 8.6 (s, 1H, Ar‐H), 9.4 (br s, 2H, NH2, D2O exchangeable). Anal. Calcd. for C15H9N7O2S: C, 51.27; H, 2.58; N, 27.90. Found: C, 51.71; H, 2.30; N, 27.80%. 7‐Amino‐5‐(4‐chlorophenyl)‐2‐methylsulphanylpyrazolo[1,5‐a] pyrimidine‐3,6‐dicarbonitrile (7c): Yield: 45%. M.p.: 273‐274 oC. FT‐ IR (cm‐1): 3439, 3297 (NH2), 2222 (CN). 1H NMR (200 MHz, DMSO‐ d6, δ ppm): 2.7 (s, 3H, SCH3), 7.6 (d, 2H, J=7.6 Hz, Ar‐H), 7.8 (d, 2H, J=7.6 Hz, Ar‐H), 9.3 (br s, 2H, NH2, D2O exchangeable). MS (m/z (%)): 342 [(M+2)+, 38.96%], 340 [M+, 100%]. Anal. Calcd. for C15H9ClN6S: C, 52.86; H, 2.66; N, 24.65. Found: C, 52.70; H, 2.30; N, 24.86. 7‐Amino‐5‐(4‐dimethylaminophenyl)‐2‐methylsulphanylpyrazolo [1,5‐a]pyrimidine‐3,6‐dicarbonitrile (7d): Yield: 32%. M.p.: 280‐281 oC. FT‐IR (cm‐1): 3449, 3370 (NH2), 2209 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.7 (s, 3H, SCH3), 3.0 (s, 6H, ‐N(CH3)2), 6.7 (d, 2H, J=8.8 Hz, Ar‐H), 7.8 (d, 2H, J=9.2 Hz, Ar‐H), 9.0 (br s, 2H, NH2, D2O exchangeable). Anal. Calcd. for C17H15N7S: C, 58.43; H, 4.32; N, 28.05. Found: C, 58.73; H, 4.64; N, 28.30%. 2.2.2. 5,7‐Diamino‐2‐methylsulphanylpyrazolo[1,5‐a] pyrimidine‐3‐carbonitrile (8) A mixture of 5‐amino‐3‐methylsulphanyl‐1H‐pyrazole‐4‐ carbonitrile (4) (0.77 g, 0.005 mol), malononitrile (0.33 g, 0.005 mol) and triethylamine (2 mL) in absolute ethanol (30 mL) was heated under reflux for 10 h. The precipitate formed was filtered, dried and crystallized from acetic acid. Yield: 26%. M.p.: 254‐255 oC. FT‐IR (cm‐1): 3479, 3431, 3373, 3317 (NH2), 2203 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.6 (s, 3H, SCH3), 5.3 (s, 1H, H6), 6.7 (s, 2H, NH2, D2O exchangeable), 7.3 (s, 2H, NH2, D2O exchangeable). Anal. Calcd. for C8H8N6S: C, 43.62; H, 3.66; N, 38.15. Found: C, 43.99; H, 3.55; N, 38.60%. El‐Enany et al. / European Journal of Chemistry 2 (3) (2011) 331‐336 333 N H N SCH3 H2N NC 4 N NN CH3 CN SCH3 Cl N NN CH3 CN SCH3 RHN N NN NHR CN SCH3 10 12 13 14a-c 15a-d N H NN CN SCH3 O N NN Cl CN SCH3 N H NN CH3 CN SCH3 O 11 a b c c d d Reagents: a) C6H5COCH2COOC2H5, Fusion at 160 oC; b) CH3COCH2COOC2H5, Fusion at 170 oC (method A), acetic acid (method B); c) POCl3; d) RNH2, triethylamine, ethanol. Compound 14a, 15a 14b, 15b 4-CH3OC6H4 Compound 2-CH3C6H415d 14c, 15c R C6H11 C6H5 R Scheme 2 2.2.3. 7‐Amino‐2‐methylsulphanyl‐5‐oxo‐4,5‐dihydropyrazolo [1,5‐a]pyrimidine‐3‐carbonitrile (9) A mixture of 5‐amino‐3‐methylsulphanyl‐1H‐pyrazole‐4‐ carbonitrile (4) (2.31 g, 0.015 mol) and ethyl cyanoacetate (1.69 g, 1.70 mL, 0.015 mol) was heated at 160 oC in an oil bath for 2 h. The solid formed was triturated with ethanol (10 mL), filtered, dried and crystallized from DMF. Yield: 93%. M.p.: >300 oC. FT‐IR (cm‐1): 3433, 3319 (NH/NH2), 2220 (CN), 1650 (CO). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.6 (s, 3H, SCH3), 5.2 (d, 1H, H6, J=2.2 Hz), 7.6 (s, 2H, NH2, D2O exchangeable), 11.9 (br s, 1H, NH, D2O exchangeable). Anal. Calcd. for C8H7N5OS: C, 43.43; H, 3.18; N, 31.65. Found: C, 43.01; H, 3.36; N, 31.22%. 2.2.4. 2‐Methylsulphanyl‐5‐phenyl‐7‐oxo‐4,7‐dihydropyrazolo [1,5‐a]pyrimidine‐3‐carbonitrile (10) A mixture of 5‐amino‐3‐methylsulphanyl‐1H‐pyrazole‐4‐ carbonitrile (4) (2.31 g, 0.015 mol) and ethyl benzoylacetate (2.88 g, 2.60 mL, 0.015 mol) was heated at 160 oC in an oil bath for 2 h. The solid formed was triturated with ethanol (10 mL), filtered, dried and crystallized from acetic acid. Yield: 67%. M.p.: 268‐269 oC. FT‐IR (cm‐1): 3466 (NH), 2222 (CN), 1718 (CO). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.6 (s, 3H, SCH3), 6.2 (s, 1H, NH, D2O exchangeable), 7.5‐7.8 (m, 6H, Ar‐H+ H6). Anal. Calcd. for C14H10N4OS: C, 59.56; H, 3.57; N, 19.84. Found: C, 59.97; H, 3.57; N, 20.16%. 2.2.5. 7‐Methyl‐2‐methylsulphanyl‐5‐oxo‐4,5‐dihydropyrazolo [1,5‐a]pyrimidine‐3‐carbonitrile (11) Method A: A mixture of 5‐amino‐3‐methylsulphanyl‐1H‐ pyrazole‐4‐carbonitrile (4) (2.31 g, 0.015 mol) and ethyl acetoacetate (1.95 g, 1.90 mL, 0.015 mol) was heated at 170 oC in an oil bath for 2 h. The solid product was triturated with ethanol (10 mL), filtered, dried and crystallized from acetic acid. Method B: A mixture of 5‐amino‐3‐methylsulphanyl‐1H‐ pyrazole‐4‐carbonitrile (4) (2.31 g, 0.015 mol) and ethyl acetoacetate (1.95 g, 1.90 mL, 0.015 mol) in glacial acetic acid (50 mL) was heated under reflux for 15 h. The solid formed was filtered, dried and crystallized from acetic acid. Yield: 90% (Method A), 77% (Method B). M.p.: 300‐301 oC [16]. FT‐IR (cm‐1): 3470 (NH), 2225 (CN), 1661 (CO). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.2 (s, 3H, CH3), 2.6 (s, 3H, SCH3), 5.8 (s, 1H, H6), 13.2 (s, 1H, NH, D2O exchangeable). 2.2.6. General procedure for the synthesis of pyrazolo[1,5‐a] pyrimidines 12 and 13 A mixture of pyrazolo[1,5‐a]pyrimidine derivatives 10 or 11 (0.004 mol) and phosphorus oxychloride (20 mL) was heated under reflux for 7 h. The reaction mixture was cooled and poured gradually onto crushed ice. The resulting product was filtered, dried, and crystallized from ethanol. 7‐Chloro‐2‐methylsulphanyl‐5‐phenylpyrazolo[1,5‐a] pyrimidine‐ 3‐carbonitrile (12): Yield: 73%. M.p.: 196‐197 oC. FT‐IR (cm‐1): 2220 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.7 (s, 3H, SCH3), 7.5‐8.2 (m, 6H, Ar‐H + H6). Anal. Calcd. for C14H9ClN4S: C, 55.90; H, 3.01; N, 18.62. Found: C, 56.00; H, 3.23; N, 18.20%. 5‐Chloro‐7‐methyl‐2‐methylsulphanylpyrazolo[1,5‐a] pyrimidine‐3‐carbonitrile (13): Yield: 71%. M.p.: 220‐221 oC. FT‐IR (cm‐1): 2223 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.6 (s, 3H, CH3), 2.7 (s, 3H, SCH3), 7.5 (s, 1H, H6). Anal. Calcd. for C9H7ClN4S: C, 45.28; H, 2.95; N, 23.47. Found: C, 45.65; H, 2.96; N, 23.45%. 2.2.7. General procedure for the synthesis of 2‐methylsulphanyl‐ 5‐phenyl‐7‐(substituted amino)pyrazolo[1,5‐a]pyrimidine‐3‐ carbonitriles 14a‐c 334 El‐Enany et al. / European Journal of Chemistry 2 (3) (2011) 331‐336 A mixture of 7‐chloropyrazolo[1,5‐a]pyrimidine derivative 12 (0.60 g, 0.002 mol), the appropriate primary amine (0.002 mol) and triethylamine (0.50 mL) in absolute ethanol (20 mL) was heated under reflux for 9 h. The reaction mixture was concentrated under reduced pressure, and the solid formed upon cooling was filtered, dried and crystallized from the suitable solvent. 7‐Cyclohexylamino‐2‐methylsulphanyl‐5‐phenylpyrazolo[1,5‐a] pyrimidine‐3‐carbonitrile (14a): (Crystallized from ethanol). Yield: 60%. M.p.: 194‐195 oC. FT‐IR (cm‐1): 3383 (NH), 2213 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 1.2‐1.9 (m, 11H, aliphatic‐H), 2.7 (s, 3H, SCH3), 7.0 (s, 1H, H6), 7.5‐8.2 (m, 5H, Ar‐H), 7.8 (s, 1H, NH, D2O exchangeable). Anal. Calcd. for C20H21N5S: C, 66.08; H, 5.82; N, 19.26. Found: C, 66.20; H, 5.60; N, 19.39%. 7‐Anilino‐2‐methylsulphanyl‐5‐phenylpyrazolo[1,5‐a]pyrimidine‐ 3‐carbonitrile (14b): (Crystallized from acetic acid). Yield: 44%. M.p.: 254‐255 oC. FT‐IR (cm‐1): 3349 (NH), 2211 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.8 (s, 3H, SCH3), 6.7‐8.3 (m, 11H, Ar‐H), 10.3 (s, 1H, NH, D2O exchangeable). Anal. Calcd. for C20H15N5S: C, 67.20; H, 4.22; N, 19.59. Found: C, 67.05; H, 4.12; N, 19.84%. 7‐(4‐Methoxyanilino)‐2‐methylsulphanyl‐5‐phenylpyrazolo[1,5‐ a]pyrimidine‐3‐carbonitrile (14c): (Crystallized from ethanol). Yield: 70%. M.p.: 192‐193 oC. FT‐IR (cm‐1): 3305 (NH), 2211 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.8 (s, 3H, SCH3), 3.8 (s, 3H, OCH3), 6.5‐7.9 (m, 10H, Ar‐H), 8.2 (s, 1H, NH, D2O exchangeable). Anal. Calcd. for C21H17N5OS: C, 65.09; H, 4.42; N, 18.07. Found: C, 65.00; H, 4.23; N, 17.77%. 2.2.8. General procedure for the synthesis of 7‐methyl‐2‐ methylsulphanyl‐5‐(substituted amino)pyrazolo[1,5‐a] pyrimidine‐3‐carbonitriles 15a‐d A mixture of 5‐chloropyrazolo[1,5‐a]pyrimidine derivative 13 (0.48 g, 0.002 mol), the appropriate primary amine (0.002 mol) and triethylamine (0.50 mL) in absolute ethanol (20 mL) was heated under reflux for 11 h. The reaction mixture was concentrated under reduced pressure, and the product formed upon cooling was filtered, dried and crystallized from ethanol. 5‐Cyclohexylamino‐7‐methyl‐2‐methylsulphanylpyrazolo[1,5‐a] pyrimidine‐3‐carbonitrile (15a): Yield: 91%. M.p.: 180‐181 oC. FT‐IR (cm‐1): 3378 (NH), 2211 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 1.2‐1.9 (m, 11H, aliphatic‐H), 2.4 (s, 3H, CH3), 2.7 (s, 3H, SCH3), 6.4 (s, 1H, H6), 7.7 (br s, 1H, NH, D2O exchangeable). Anal. Calcd. for C15H19N5S: C, 59.77; H, 6.35; N, 23.23. Found: C, 59.55; H, 6.31; N, 22.92%. 5‐Anilino‐7‐methyl‐2‐methylsulphanylpyrazolo[1,5‐a]pyrimi‐ dine‐3‐carbonitrile (15b): Yield: 77%. M.p.: 209‐210 oC. FT‐IR (cm‐ 1): 3215 (NH), 2230 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.3 (s, 3H, CH3), 2.7 (s, 3H, SCH3), 6.2 (s, 1H, H6), 7.1‐7.5 (m, 5H, Ar‐H), 10.0 (s, 1H, NH, D2O exchangeable). Anal. Calcd/ for C15H13N5S: C, 60.99; H, 4.43; N, 23.71. Found: C, 60.86; H, 4.60; N, 23.45%. 5‐(4‐Methoxyanilino)‐7‐methyl‐2‐methylsulphanylpyrazolo[1,5‐ a]pyrimidine‐3‐carbonitrile (15c): Yield: 59%. M.p.: 168‐169 oC. FT‐ IR (cm‐1): 3362 (NH), 2217 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.3 (s, 3H, CH3), 2.7 (s, 3H, SCH3), 3.8 (s, 3H, OCH3), 6.0 (s, 1H, H6), 7.0 (d, 2H, J=8.7 Hz, Ar‐H), 7.3 (d, 2H, J=8.7 Hz, Ar‐H), 9.9 (s, 1H, NH, D2O exchangeable). MS (m/z (%)): 325 [M+, 100%]. Anal. Calcd. for C16H15N5OS: C, 59.06; H, 4.64; N, 21.52. Found: C, 59.39; H, 5.10; N, 21.11%. 5‐(2‐Methylanilino)‐7‐methyl‐2‐methylsulphanylpyrazolo[1,5‐a] pyrimidine‐3‐carbonitrile (15d): Yield: 53%. M.p.: 192‐193 oC. FT‐IR (cm‐1): 3368 (NH), 2214 (CN). 1H NMR (200 MHz, DMSO‐d6, δ ppm): 2.2 (s, 3H, CH3C6H4), 2.3 (s, 3H, CH3), 2.7 (s, 3H, SCH3), 5.7 (s, 1H, H6), 7.3‐7.4 (m, 4H, Ar‐H), 9.9 (s, 1H, NH, D2O exchangeable). Anal. Calcd. for C16H15N5S: C, 62.11; H, 4.88; N, 22.63. Found: C, 62.40; H, 4.55; N, 22.29%. 2.3. Biological testing 2.3.1. Materials and methods The human colon tumor cell line (HCT116) was obtained as a gift from National Cancer Institute NCI, Maryland MD, USA. All chemicals and solvents were purchased from Sigma–Aldrich. 2.3.2. Measurement of potential cytotoxicity The cytotoxic activity of some of the newly synthesized compounds was tested in vitro on human colon tumor cell line (HCT116) using Sulforhodamine‐B stain (SRB) assay according to the method of Skehan et al. [17]. Cells were plated in 96‐multiwell plate (104 cells/well) for 24 h before treatment with the tested compounds to allow attachment of the cells to the wall of the plate. The tested compounds were dissolved in DMSO and diluted with saline to the appropriate volume. Different concentrations of the tested compound (0, 1, 2.5, 5 and 10 μg/mL) were added to the cell monolayer. Triplicate wells prepared for each individual dose. Monolayer cells were incubated with the tested compound for 48 h at 37 oC in atmosphere of 5% CO2. After 48 h, cells were fixed with trichloroacetic acid, washed with water and stained for 30 min with 0.4% (w:v) Sulforhodamine‐B stain dissolved with 1% acetic acid. Excess stain was removed by four washes with 1% acetic acid and attached stain was recovered with Tris EDTA buffer. Colour intensity was measured in ELISA reader. The relation between surviving fraction and compound concentration was plotted and IC50 (the concentration required for 50% inhibition of cell viability) was calculated for each compound and results are given in Table 1. Figure 2 represents IC50 in μM of the synthesized compounds and doxorubicin against human colon tumor cell line (HCT116) Table 1. Results of in vitro cytotoxic activity of some of the synthesized compounds on human colon tumor cell line (HCT116). Compound no. IC50 in μM* Doxorubicin 0.0069 7b 0.0062 7c 0.0375 8 0.0433 9 0.0814 14a 0.0020 14b 0.0113 14c 0.0238 15c 0.0077 15d 0.0598 *The values given are means of three experiments. Figure 2. IC50 in μM of the synthesized compounds and doxorubicin against human colon tumor cell line (HCT116). 3. Results and discussion 3.1. Chemistry The synthesis of the target compounds is outlined in Schemes 1 and 2. The starting compound, 5‐amino‐3‐methylsulphanyl‐1H‐ pyrazole‐4‐carbonitrile (4) [11] was reacted with the appropriate substituted benzylidenemalononitrile 5a‐f [12‐15] to afford 7‐ amino‐2‐methylsulphanyl‐5‐(substituted phenyl)‐4,5‐dihydropyra‐ zolo[1,5‐a]pyrimidine‐3,6‐dicarbonitriles 6a,b or 7‐amino‐2‐ methylsulphanyl‐5‐(substituted phenyl)pyrazolo[1,5‐a]pyrimidine‐ 3,6‐dicarbonitriles 7a‐d. The reaction of the pyrazole derivative 4 with substituted benzylidenemalononitriles 5a‐f may proceed via initial nucleophilic attack by the exocyclic amino group of compound 4 on the activated double bond in 5a‐f to form a Micheal adduct. Intramolecular cyclization of the latter may result in the formation of the dihydro derivative 6 which may then aromatize into 7 (Scheme 3). El‐Enany et al. / European Journal of Chemistry 2 (3) (2011) 331‐336 335 Scheme 3 Trials to oxidize 6a,b into 7 (R=2‐Cl, 2‐CH3O) via the use of two molar equivalents of benzylidenemalononitriles 5a,b or by increasing the time of the reaction (up to 15 h) were unsuccessful. Compound 4 was also reacted with malononitrile to give 5,7‐ diaminopyrazolo[1,5‐a]pyrimidine 8. 1H NMR spectrum of compound 8 revealed the presence of a singlet signal at δ 5.3 ppm corresponding to H6 and two exchangeable singlet signals at δ 6.7 and δ 7.3 ppm corresponding to protons of the two NH2 groups at positions 5 and 7, respectively. Furthermore, 1H‐pyrazole derivative 4 was reacted with ethyl cyanoacetate, ethyl benzoylacetate and ethyl acetoacetate to give one of two possible products; the 5‐oxopyrazolo[1,5‐a]pyrimidine or the 7‐oxo isomer (Scheme 4). The assignment of the product as 5‐oxo or 7‐oxopyrazolo[1,5‐a]pyrimidine depends on 1H NMR study. Scheme 4 The reaction of compound 4 with ethyl cyanoacetate and ethyl acetoacetate afforded 5‐oxopyrazolo[1,5‐a]pyrimidine derivatives 9 and 11, respectively. Both compounds showed an exchangeable singlet signal at δ 11.9 ppm and δ 13.2 ppm, respectively, corresponding to NH proton due to tautomerism with the adjacent carbonyl group. The 7‐oxo isomer, lacking such tautomerism, should give that signal at δ 5‐7 ppm. On the other hand, the reaction of compound 4 with ethyl benzoylacetate afforded 7‐oxopyrazolo[1,5‐a]pyrimidine 10. Herein, 1H NMR of compound 10 revealed the presence of an exchangeable singlet signal at δ 6.2 ppm corresponding to NH proton. Chlorination of compounds 10 and 11 with POCl3 followed by nucleophilic substitution with primary amines afforded 7‐ (substituted amino) and 5‐(substituted amino)pyrazolo[1,5‐ a]pyrimidine derivatives 14a‐c and 15a‐d, respectively. 3.2. In vitro anticancer screening Some of the newly synthesized compounds were evaluated for their in vitro cytotoxic activity against human colon tumor cell line, HCT116. Doxorubicin which is one of the most effective anticancer agents was used as the reference drug in this study. The response parameter calculated was the IC50 value, which corresponds to the concentration required for 50% inhibition of cell viability. The IC50 in μM of the tested compounds compared to the reference drug are shown in Table 1 and represented graphically in Figure 2. From the results in Table 1, it was found that all the tested compounds exhibited good antitumor activity against HCT116 with IC50 between 0.0020 and 0.0814 μM. Two of the tested compounds (7b and 14a) showed antitumor activity superior to doxorubicin with IC50 that equals to 0.0069 and 0.0020 μM, respectively. 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