DOI: 10.1002/cmdc.200600302 6-Substituted PyrroloACHTUNGTRENNUNG[3,4-c]pyrazoles: An Improved Class of CDK2 Inhibitors Maria Gabriella Brasca,* [a] Clara Albanese, [b] Raffaella Amici, [d] Dario Ballinari, [b] Luca Corti, [a] Valter Croci, [b] Daniele Fancelli, [d] Francesco Fiorentini, [c] Marcella Nesi, [a] Paolo Orsini, [a] Fabrizio Orzi, [a] Wilma Pastori, [b] Ettore Perrone, [a] Enrico Pesenti, [b] Paolo Pevarello, [e] Federico Riccardi-Sirtori, [a] Fulvia Roletto, [b] Patrick Roussel, [f] Mario Varasi, [d] Anna Vulpetti, [g] and Ciro Mercurio [d] Dedicated to Professor S. V. Ley on the occasion of his 60 th birthday Introduction The cyclin dependent kinases (CDKs) are serine/threonine kin- ases intimately involved in the regulation of the cell cycle. CDK2 in particular, is a heterodimer composed of a catalytic subunit, CDK2, and one of two activating subunits, cyclin E or cyclin A. The two isoforms of the kinase have distinct roles during the cell cycle. CDK2/cyclin E is mainly involved in pro- gression through G1/S, centrosome duplication, and DNA repli- cation. CDK2/cyclin A is a key regulator of G2/M progression. Many past observations, such as the transient transfection of a catalytically inactive form of CDK2, [1] the inducible expression of a dominant negative form of CDK2 [2] and microinjection of antibodies against CDK2 and cyclin A [3] have suggested an es- sential role for CDK2 in cell proliferation. Recent data from the CDK2 and cyclin E knockout mice [4] indicating that CDK2 is not necessary for normal development has opened up an interest- ing debate. Some support the hypothesis that selective CDK2 inhibitors will not be toxic. Others argue that CDK2, not being essential for normal cell division, is unlikely involved in tumor cell proliferation. We must not forget, however, that inhibitors render CDK2 inactive rather than prevent its expression and it is not possible to exclude the activation of compensatory mechanisms to overcome the lack of CDK2 or cyclin E genes in mice. In this context, we believe that the pharmacological in- hibition of CDK2 activity remains a potential therapeutic strategy for anticancer therapies. Recently, we reported the design and solid- phase generation of potent Aurora-A inhibi- tors bearing a 1,4,5,6-tetrahydropyrroloACHTUNGTRENNUNG[3,4- c]pyrazole core structure. [5] This novel ade- nine mimetic scaffold, which is endowed with high versatility, has also been exploited for the solution-phase synthesis of a series of CDK2/cyclin A in- hibitors. [6] In this study we identified two compounds (1 and 2) with nanomolar activity against CDK2/cyclin A in the biochemical assay and able to efficiently inhibit CDK2-mediated cell prolif- eration although, because of both suboptimal physicochemical properties and an overall unfavorable early ADME profile, fur- ther optimization was required (Table1). In particular, as the low buffer solubility of these compounds prevented their use in vivo as injectables, the first objective in the optimization of this class of compounds was the improve- [a] Dr. M.G. Brasca, L. Corti, Dr. M. Nesi, Dr. P. Orsini, F. Orzi, Dr. E. Perrone, Dr. F. Riccardi-Sirtori Oncology Business Unit, Department of Chemistry, Nerviano Medical Scien- ces, Viale Pasteur 10, 20014 Nerviano MI (Italy) Fax:(+ 39)0331-58-1347 E-mail:gabriella.brasca@nervianoms.com [b] Dr. C. Albanese, Dr. D. Ballinari, V. Croci, W. Pastori, Dr. E. Pesenti, Dr. F. Roletto Oncology Business Unit, Department of Biology, Nerviano Medical Sciences, Viale Pasteur 10, 20014 Nerviano MI (Italy) [c] Dr. F. Fiorentini Preclinical Development, Nerviano Medical Sciences, Viale Pasteur 10, 20014 Nerviano MI (Italy) [d] Dr. R. Amici, Dr. D. Fancelli, Dr. M. Varasi, Dr. C. Mercurio Present address: Congenia S.r.l. (Italy) [e] Dr. P. Pevarello Present address: Centro Nacional de Investigaciones Oncologicas (CNIO) (Spain) [f] Dr. P. Roussel Present address: Basilea Pharmaceutica Ltd (Switzerland) [g] Dr. A. Vulpetti Present address: Novartis Pharma AG (Switzerland) We have recently reported a new class of CDK2/cyclin A inhibitors based on a bicyclic tetrahydropyrroloACHTUNGTRENNUNG[3,4-c]pyrazole scaffold. The introduction of small alkyl or cycloalkyl groups in position 6 of this scaffold allowed variation at the other two diversity points. Conventional and polymer-assisted solution phase chemistry pro- vided a way of generating compounds with improved biochemi- cal and cellular activity. Optimization of the physical properties and pharmacokinetic profile led to a compound which exhibited good efficacy in vivo on A2780 human ovarian carcinoma. ChemMedChem 2007,2,841–852 # 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 841