Materials and methods Chemistry Phenyl hydrazine, malononitrile, triethylorthoester and ammoniac were purchased from Sigma Chemical (Berlin, AZD8931 manufacturer Germany). Analytical grade GW3965 solubility dmso solvents (ethanol, HCl, ethyl acetate, chloroform) were obtained from Merck. Melting points (mp) were determined on a Buchi capillary apparatus and were uncorrected. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 300 spectrometer (1H at 300 MHz and 13C at 75 MHz) with deuterio-dimethylsulphoxide (d-DMSO) as solvent and tetramethylsilane as internal standard reference.

Infra-red (IR) spectra were recorded on a Bio-rad FTS-6000 spectrometer. Solvents used in reactions were dried and distilled before use. The purity of all synthesized compounds was controlled by thin layer chromatography (TLC; Merck silica gel plates 60F-254). High resolution Barasertib order masses were recorded on a spectrometer JEOL JMS-Gcmate II is composed of a GC/MS system from compounds dissolved in dichloromethane. Synthesis and spectral data of compounds 2–5 5-Amino-4-cyano-N 1-phenyl pyrazoles (2) 5-Amino-4-cyano-1-N 1-phenyl pyrazoles prepared via a standard addition

of hydrazine derivatives to ketene ethoxymethylene compounds following the reported procedure. Recrystallization from ethanol afforded pure 2 in good yields. 4-Cyano-N 1-phenyl pyrazolo-5-imidates (3) The required 5-amino-4-cyano-N 1 -phenyl pyrazole (1.0 mmol) was treated with triethylorthoester 6.0 mmol) and a catalytic amount of acetic acid and the mixture was refluxed for 24 h. After cooling, the reaction mixture was evaporated. The product was filtered, washed with diethyl ether then purified by recrystallisation (ethanol) Morin Hydrate (Gupta et al., 2008; Allouche et al., 2013). 4-Amino-N 1-phenyl pyrazolo[3,4-d]pyrimidine (4) A solution of imidate 3 (1.0 mmol)

in dry ethanol (5 ml) was treated with ammoniac (2.0 mmol) and a catalytic amount of acetic acid. The reaction mixture was refluxed for 6 h, and the formed solid was collected by filtration, dried and recrystallized from ethanol to give compound 4. a) 4-Amino-N 1 -phenyl-1H-pyrazolo[3,4-d]pyrimidine 4a Yield 83 %; mp 228 °C; IR (cm−1); ν NH2 3100, 3283; ν C=N 1480, 1500, 1590 cm−1; RMN 1H (δ ppm, DMSO): 4.69 (2H, s, NH2), 7.36 (1H, t, J = 7.3 Hz, ArH4), 7.48 (2H, t, J = 7.3 Hz, ArH3 and ArH5), 7.52 (2H, d, J = 7.3 Hz, ArH2 and ArH6), 7.60 (1H, s, H3), 7.72 (1H, s, H6), 13C RMN (δ ppm, DMSO): 114.14 (C-3a), 124.27 (C-2′ and C-6′), 129.00 (C-4′), 129.58 (C-3′ and C-5′), 130.04 (C-3), 136.94 (C-1′), 141.36 (C-7a), 149.83 (C-6), 156.84 (C-4); HRMS Calcd. for C11H9N5: 211.0858, found: 211.0859.   b) 4-Amino-3-methyl-N 1 -phenyl-1H-pyrazolo[3,4-d]pyrimidine 4b Yield 68 %; mp 192 °C; IR (cm−1); ν NH2 3083, 3317; ν C=N 1626, 1647, 1665; RMN 1H (δ ppm, DMSO): 2.76 (3H, s, CH3), 5.97 (2H, s, NH2), 7.