Discovery of 2‑(Cyclohexylmethylamino)pyrimidines as a New Class of Reversible Valosine Containing Protein Inhibitors Giovanni Cervi, † Paola Magnaghi, † Daniela Asa, † Nilla Avanzi, † Alessandra Badari, † Daniela Borghi, † Michele Caruso, † Alessandra Cirla, † Liviana Cozzi, † Eduard Felder, † Arturo Galvani, † Fabio Gasparri, † Antonio Lomolino, † Steven Magnuson, ‡ Beatrice Malgesini, † Ilaria Motto, † Maurizio Pasi, † Simona Rizzi, † Barbara Salom, † Graziella Sorrentino, † Sonia Troiani, † Barbara Valsasina, † Thomas O’Brien, ‡ Antonella Isacchi, † Daniele Donati, † and Roberto D’Alessio* ,† † Oncology, Nerviano Medical Sciences S.r.l., Viale Pasteur 10, 20014 Nerviano, Milan, Italy ‡ Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States * S Supporting Information ABSTRACT: Valosine-containing protein (VCP), also known as p97 or cdc48 in yeast, is a highly abundant protein belonging to the AAA ATPase family involved in a number of essential cellular functions, including ubiquitin-proteasome mediated protein degradation, Golgi reassembly, transcription activation, and cell cycle control. Altered expression of VCP has been detected in many cancer types sometimes associated with poor prognosis. Furthermore, VCP mutations are causative of some neurodegenerative disorders. In this paper we report the discovery, synthesis, and structure-activity relationships of substituted 2-aminopyrimidines, representing a new class of reversible VCP inhibitors. This class of compounds, identified in a HTS campaign against recombinant VCP, has been progressively expanded and manipulated to increase biochemical potency and gain cellular activity. ■ INTRODUCTION Valosine-containing protein (VCP/p97), known as cdc48 in yeast, is a member of the AAA (ATPases associated with various cellular activities) family of ATPases 1 and is an essential and highly abundant protein in cells. VCP may be considered an enzymatic machine that transfers, through adaptor proteins, the energy derived from ATP hydrolysis to the substrates to perform a wide range of cellular functions such as endoplasmic reticulum associated degradation (ERAD), Golgi reassembly, transcription regulation, and cell cycle control. 2 VCP is characterized by a highly flexible hexameric structure with subunits arranged as a ring around a central pore. Each subunit is composed of an N-terminal domain responsible for the binding to adaptor proteins and two AAA domains called D1 and D2 that sustain nucleotide binding and hydrolysis. 3,4 The structure and the role of the C-terminal extension still remain elusive. The pivotal role of this protein in many essential cellular functions suggests that altered expression or mutation of VCP may lead to pathological consequences. Consistent with this finding, several reports indicate that elevated levels of VCP have been detected in many cancer types sometimes associated with poor prognosis. 5,6 Moreover, several data demonstrate an involvement of VCP in neurodegenerative diseases. 7 The increasing interest around this new potential molecular target has prompted different groups to undertake screening programs in the attempt to identify small molecules able to inhibit VCP enzymatic activity. This would facilitate a better understanding of the effective relevance of this target in cancer therapeutics. A few molecules interfering with VCP functions were previously reported in literature. 8-11 Recently, a class of potent and specific allosteric inhibitors have been described by our group. 12 Here we report the synthesis and the structure-activity relationships of substituted 2-aminopyrimidines, a new class of reversible VCP inhibitors, acting through a different mechanism of action. The original hit 1, discovered after a HTS campaign against recombinant VCP, was the 2- alkylsulfanylpyrimidine shown in Figure 1. This compound inhibited VCP ATPase activity with an IC 50 of 4.8 μM in standard assay conditions. Serial dilution experiments indicated a reversible mode of binding and mass Received: August 28, 2014 Figure 1. Structure of 2-alkylsulfanylpyrimidine 1. Article pubs.acs.org/jmc © XXXX American Chemical Society A dx.doi.org/10.1021/jm501313x | J. Med. Chem. XXXX, XXX, XXX-XXX