3-Amino-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles: A new class of CDK2 inhibitors Paolo Pevarello, a, * Daniele Fancelli, a Anna Vulpetti, a Raffaella Amici, a Manuela Villa, a Valeria Pittala `, a,  Paola Vianello, a Alexander Cameron, a Marina Ciomei, b Ciro Mercurio, b James R. Bischoff, b,à Fulvia Roletto, b Mario Varasi a and Maria Gabriella Brasca a a Department of Chemistry, Nerviano Medical Science, BU-Oncology, Via Pasteur 10, 20014 Nerviano MI, Italy b Department of Biology, Nerviano Medical Science, BU-Oncology, Via Pasteur 10, 20014 Nerviano MI, Italy Received 1 September 2005; revised 12 October 2005; accepted 20 October 2005 Abstract—We have recently reported about a new class of Aurora-A inhibitors based on a bicyclic tetrahydropyrrolo[3,4-c]pyrazole scaffold. Here we describe the synthesis and early expansion of CDK2/cyclin A–E inhibitors belonging to the same chemical class. Synthesis of the compounds was accomplished using a solution-phase protocol amenable to rapid parallel expansion. Compounds with nanomolar activity in the biochemical assay and able to efficiently inhibit CDK2-mediated tumor cell proliferation have been obtained. Ó 2005 Elsevier Ltd. All rights reserved. Uncontrolled cell growth and proliferation are hall- marks of all cancers and are directly linked to cell cycle dysregulation. 1 Cyclin-dependent kinase 2 (CDK2) in complex with cyclins E and/or A is a key cell cycle reg- ulator and continues to be an attractive target for the discovery of new antitumor agents. 2 In particular, inhib- itors of CDK2/cyclin A/E have already progressed into clinical trials with encouraging early results. 3 Recent findings in the biology of the CDKs show that CDK2 may be dispensable for tumor formation and mainte- nance 4 but compounds able to inhibit both CDK2 and CDK1 may still be regarded as potential anti-tumor agents. 5 We recently reported on a series of 3-aminopy- razoles as potent, orally available inhibitors of CDK2/ cyclin A/E and CDK1/cyclin B, displaying activity in vi- vo in a murine tumor xenograft model. 6 A limitation associated with the 3-aminopyrazole scaffold is that high-throughput synthesis can be applied easily only for placing substituents at position 3, that is, toward the solvent-accessible region when the ligand is placed in the ATP-pocket of the kinase. 7 Positions 4- and 5-, which could be exploited to gain accessibility to the bur- ied and phosphate binding regions to direct substituents are not synthetically amenable to rapid parallel medici- nal chemistry. We reasoned that by condensing a second heterocyclic ring onto the 3-aminopyrazole moiety we could gain ac- cess to the phosphate binding region of the ATP-kinase pocket. Thus, we would obtain a new chemical class that could be used to exploit an additional pocket in the ATP binding site (Fig. 1). The generation of this new chemi- cal class, 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles, was recently reported by us in a paper dealing with the char- acterization of potent Aurora-A inhibitors. 8 Here, we describe the solution-phase synthesis and early expan- sion of this class toward CDK2/cyclin A inhibitors. A general solution-phase synthetic methodology was need- ed to allow the generation of many derivatives using parallel medicinal chemistry methodologies. The synthe- sis of the scaffold 1,4,5,6-tetrahydropyrrolo[3,4-c]pyra- zole was accomplished as previously described. 8 Scheme 1 reports a general solution-phase synthesis for this class of compounds that was applied to obtain 0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2005.10.071 Keywords: CDK2; Cyclins; Kinase selectivity; Tumor cell proliferation inhibition. Abbreviations: CDK, cyclin-dependent kinase; ATP, adenosine tri- phosphate; FACS, fluorescence activated cell sorting; BrdU, bro- modesoxyuridine; WB, Western blots; pRb, retinoblastoma protein; ADME, absorption-disposition-metabolism-excretion. * Corresponding author. Tel.: +390331581927; fax: +390331581347; e-mail: paolo.pevarello@nervianoms.com   Present address: Istituto di Chimica Farmaceutica, Universita ` di Catania, Via Doria 9, 95125 Catania, Italy. à Present address: Centro Nacional de Investigaciones Oncolo ´ gicas (CNIO), Melchor Ferna ´ndez Almagro n°3, 28029 Madrid, Spain. Bioorganic & Medicinal Chemistry Letters xxx (2005) xxx–xxx ARTICLE IN PRESS