PRODRUGS OF PYRAZOLO[3,4-d]PYRIMIDINES: FROM LIBRARY SYNTHESIS TO EVALUATION AS POTENTIAL ANTICANCER AGENTS IN AN ORTHOTOPIC GLIOBLASTOMA MODEL

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1 PRODRUGS OF PYRAZOLO[3,4-d]PYRIMIDINES: FROM LIBRARY SYNTHESIS TO EVALUATION AS POTENTIAL ANTICANCER AGENTS IN AN ORTHOTOPIC GLIOBLASTOMA MODEL Giulia Vignaroli,,ǁ Giulia Iovenitti,,ǁ Claudio Zamperini,,ǁ Federica Coniglio,,ǁ Pierpaolo Calandro, Alessio Molinari, Anna Lucia Fallacara, Andrea Sartucci, Alessia Calgani, David Colecchia, Andrea Mancini, Claudio Festuccia, Elena Dreassi, Massimo Valoti, Francesca Musumeci, Mario Chiariello, Adriano Angelucci, Maurizio Botta,*, ǁ, Silvia Schenone Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via Aldo Moro 2, Siena, Italy ǁ Lead Discovery Siena S.r.l., via Vittorio Alfieri 31, 53019, Castelnuovo Berardenga, Siena (IT) Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università dell Aquila, Via Vetoio, Coppito, L Aquila, Italy. Consiglio Nazionale delle Ricerche, Istituto di Fisiologia Clinica and Istituto Toscano Tumori, Core Research Laboratory, Via Fiorentina 1, 53100, Siena (IT) Dipartimento di Scienze della Vita, Università degli Studi di Siena, Via Aldo Moro 2, Siena, Italy Dipartimento di Scienze Farmaceutiche, Università degli Studi di Genova, Viale Benedetto XV 3, Genova, Italy Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife Science Building, Suite 333, 1900 N 12 th Street, Philadelphia, Pennsylvania

2 INDEX 1. IN VITRO ADME ASSAYS 3 2. ENZYMATIC ASSAYS 4 3. SPECTRA 5 2

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4 1. IN VITRO ADME ASSAYS Metabolic stability with human liver microsomes Compounds 1a, 4a and 9a and their parent drugs 1, 4 and 9 were incubated for one hour at 37 C with a solution of man-pooled HLM. After this time the percentage of compounds and metabolites was determined by HPLC MS analysis. Table S1 displays the percentage of unmodified prodrug/drug recovered after the incubation and the percentage and the type of metabolites. Table S1. Compound Metab. Stability a Metabolites (%) (%) M1= M (14.5%) M2= M (4.5%) M3= M-90 (2.6%) M4= M+16(<0.1 %) 1a 86.4 M1= M (4.3%) M2= M (1.1%) M3= M-260 (3.1%) M4= M+16 (5.0%) M1= M (1.7%) M4= M+16 (1.1%) 4a 93.6 M1= M (3.1%) M2= M (1.5%) M4= M+16(1.8 %) M1=M+16 (5.0%) 9a 99.0 M1= M+16 (<0.1%) a Expressed as percentage of unmodified drug 4

5 2. BIOLOGICAL ASSAYS Enzymatic Assays. Active recombinant kinases were purchased from ProQuinase (Germany). Specific peptide substrates (Abltide, cat ; Src Substrate Peptide, cat ) were purchased from Merk-Millipore. Kinase assays were performed as follow: active Src and Abl wt reactions were performed in presence of 100 µm ATP and 50 µm peptide substrate. All inhibition assays were conducted with 0.01 μg active kinase, 0.33 pmol [ γ32 P]ATP, 10 mm magnesium acetate, 8 mm 3-(N-morpholino)propanesulfonic acid (MOPS)/sodium hydroxide (ph 7), 1 mm ethylenediaminetetraacetic acid (EDTA), and 10 % DMSO in a final volume of 10 μl. After 5 min at 30 C, the reaction was stopped by adding 5 μl of 4 % phosphoric acid. Aliquots (10 μl) were then transferred into a P30 Filtermat (PerkinElmer), washed five times with 75 mm phosphoric acid and once with acetone for 5 mins. The filter was dried and transferred to a sealable plastic bag, and scintillation cocktail (4 ml) was added. Spotted reactions were read in a scintillation counter. The ID 50 values were obtained according to Equation (1), where v is the measured reaction velocity, V is the apparent maximal velocity in the absence of inhibitor, I is the inhibitor concentration, and the ID 50 is the 50% inhibitory dose. (1) ν=v/(1+(i/id 50 ) K i values, expressed as a concentration (μm), toward recombinant human Pim-1 were calculated according to Equation (2) for competitive inhibition toward ATP and peptide substrates, where [S ATP ] and [S pep ] are the concentration of competing substrate (ATP and peptide, respectively). Curve fitting was performed with the program GraphPad Prism version (2) K i =(ID 50 /(1+K matp /[S ATP ]))/(1+K mpep /[S pep ]) Human carboxylesterase 1 (hce 1) assay. Enzymatic hydrolysis of compound 4a to 4 was examined under the following conditions. Increasing concentrations (10, 50 and 100 μm) of 4a were incubated with a pre-warmed (37 C) mixture, containing the purified enzyme hce1 (CES1) (25 U/reaction) and 50 mm HEPES buffer ph 7.4, for 1h. All reactions were terminated by addition of equal volume of acetonitrile. The supernatant from the above reactions were analysed by reversephase HPLC-UV-MS, using the gradient method described into the manuscript. Percentage of hydrolysis was determined compared to the control (Fig. S1). All the experiments were conducted in triplicate. 5

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7 3. SPECTRA 1 H NMR 7

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14 13 C NMR 14

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