Studies on the Enantiomers of RC-33 as Neuroprotective Agents: Isolation, Configurational Assignment, and Preliminary Biological Profile DANIELA ROSSI, 1 ALICE PEDRALI, 1 ANNAMARIA MARRA, 1 LUCA PIGNATARO, 2 DIRK SCHEPMANN, 3 BERNHARD WÜNSCH, 3 LIAN YE, 4 KRISTINA LEUNER, 4 MARCO PEVIANI, 5 DANIELA CURTI, 5 ORNELLA AZZOLINA, 1 AND SIMONA COLLINA 1 * 1 Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology section, University of Pavia, Pavia, Italy 2 Dipartimento di Chimica, Università degli Studi di Milano, Istituto di Scienze e Tecnologie Molecolari (ISTM) del CNR, Milan, Italy 3 Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Münster, Germany 4 Molecular & Clinical Pharmacy, FAU Erlangen/Nuremberg, Erlangen, Germany 5 Department of Biology and Biotechnology “L. Spallanzani”, Laboratory of Cellular and Molecular Neuropharmacology, University of Pavia, Pavia, Italy ABSTRACT In this study we addressed the role of chirality in the biological activity of RC-33, recently studied by us in its racemic form. An asymmetric synthesis procedure was the first experiment, leading to the desired enantioenriched RC-33 but with an enantiomeric excess (ee) not good enough for supporting the in vitro investigation. An enantioselective high-performance liquid chromatography (HPLC) procedure was then successfully carried out, yielding both RC-33 enantiomers in amounts and optical purity suitable for the pharmacological study. The absolute configuration of pure enantiomers was easily assigned exploiting the asym- metric synthesis previously devised. As emerged in the preliminary in vitro biological investiga- tion, (S)- and (R)-RC-33 possess a comparable affinity towards the σ 1 receptor and a very a similar behavior in the calcium influx assay, resulting in an equally effective σ 1 receptor agonist. Overall, the results obtained so far suggest that the interaction with the biological target is nonstereoselective and leads us to hypothesize that there is a lack of stereoselectivity in the biological activity of RC-33. Chirality 25:814–822, 2013. © 2013 Wiley Periodicals, Inc. KEY WORDS: absolute configuration assignment; enantioselective HPLC; asymmetric hydrogenation; neuroprotective agents; σ 1 receptor agonists; chemical correlation INTRODUCTION Sigma1 (σ 1 ) receptors are involved in several dysfunctions of the central nervous system (CNS), such as depression, anxiety, schizophrenia, acute and chronic neurodegenerative diseases, in pain control, as well as in the etiology of some types of cancer. 1 Moreover, a mutation in the σ 1 receptor gene was recently found to be associated with frontotemporal lobar degeneration (FTLD), which is the most common cause of dementia under the age of 65 years, with associated motor neuron disorders 2 and with a familial juvenile form of amyotrophic lateral sclerosis. 3 Additionally, the σ 1 receptor has been found implicated in neurite sprouting and elonga- tion in vitro, suggesting a role for the receptor in neuroplasticity. 4–7 Accordingly, the research field related to σ 1 receptors is highly attractive. This drove, and it is still driving, researchers of the medicinal chemistry and biology fields to the discovery of potent and selective σ 1 receptor ligands and to the development of cell models able to distinguish between agonists and antagonists. In the last 10 years our research group has conducted extensive studies aimed at discovering novel σ 1 receptor ligands as potential neuroprotective agents. 8–15 In this context, a drug discovery library based on arylalkenyl- and arylalkylaminic scaffolds was prepared (Fig. 1). In our compound library, the most promising molecule is rac- 1-[3-(1,1’-biphen)-4-yl]butyl-piperidine · HCl (rac-RC-33 · HCl, Fig. 1), showing excellent σ 1 receptor affinity (K i = 0.70 ± 0.3 nM), combined with high selectivity over various receptors expressed in the CNS. 10 Additionally, rac-RC-33 · HCl turned out to be a potent σ 1 receptor agonist in our validated PC12 cell model of neuronal differentiation 10 and showed high metabolic stability in several biological matrices (i.e., mouse and rat blood, rat, dog, and human plasma). 11 Accordingly rac-RC-33 · HCl was identified as the optimal candidate to be further investigated. It has to be noted that the enantiomers of a chiral drug must be considered as two different chemical entities, potentially possessing different behavior in a physiological envi- ronment (i.e., they can exhibit different pharmacological and/or toxicological profile). Accordingly, the racemate (1:1 mixture of the two enantiomers) could be defined as a mixture in which the eutomer (the more active and/or the less toxic enantiomer) is impure of the distomer (the less active and/or the more toxic enantiomer). Thus, in the case of a chiral molecule already investigated as a racemate, the first issue to be addressed is the study of the role of chirality in the biological activity by pre- paring the pure enantiomers and evaluating their pharmacolog- ical activity. Among the different approaches for the preparation of enantiopure compounds, chiral resolution of racemates, manipulation of chiral starting material (the so-called chiral pool), Additional Supporting Information may be found in the online version of this article. *Correspondence to: Simona Collina, Department of Drug Sciences, Medici- nal Chemistry and Pharmaceutical Technology section, University of Pavia, Viale Taramelli 12, 27100 Pavia (Italy). E-mail: simona.collina@unipv.it Received for publication 24 May 2013; Accepted 19 June 2013 DOI: 10.1002/chir.22223 Published online 7 September 2013 in Wiley Online Library (wileyonlinelibrary.com). © 2013 Wiley Periodicals, Inc. CHIRALITY 25:814–822 (2013)