Future Med Chem. 2016 Mar;8(3):287-95.

Toward the identification of neuroprotective agents: g-scale synthesis, pharmacokinetic evaluation and CNS distribution of (R)-RC-33, a promising Sigma1 receptor agonist.


Annamaria Marra,1 Daniela Rossi,1 Luca Pignataro,2 Chiara Bigogno,3 Annalisa Canta,4 Norberto Oggioni,4 Alessio Malacrida,4 Massimo Corbo,5 Guido Cavaletti,4 Daniela Curti,6 Giulio Dondio,3 Simona Collina1 

1Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia-I; 2Università degli Studi di Milano, Dipartimento di Chimica, Via C. Golgi 19, 20133 Milano-I; 3Aphad S.r.l., Via della Resistenza 65, 20090 Buccinasco Milan-I; 4Experimental Neurology Unit, Department of Surgery and Translational Medicine and Milan Center for Neuroscience, University of Milan Bicocca, Via Cadore 48, 20900 Monza-I; 5Department of Neurorehabilitation Sciences, Casa Cura Policlinico (CCP), via Dezza 48, 20144 Milan-I. 6Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Via Ferrata 9, 27100 Pavia-I.



Aim: Nowadays, there is a great interest in the therapeutic potential of sigma1 receptor ligands for treating different CNS pathologies. Our previous investigations led to identify (R)-RC-33 as a potent and selective S1R agonist. Results: Herein, we report the gram-scale synthesis, pharmacokinetic profile and CNS distribution of (R)-RC-33 in the mouse to determine the most suitable dosage schedule for in vivo administration. For comparative purposes, the same experiments were also performed with PRE-084, the most widely used S1R agonist commonly in pharmacological experiments. Discussion: (R)-RC-33 shows a similar pharmacokinetic profile and a better CNS distribution when compared with PRE-084. Conclusion: (R)-RC-33 may be a promising candidate for in vivo studies in animal models of neurodegenerative diseases.

KEYWORDS: CNS distribution; Parkinson’s disease; S1R agonist; amyotrophic lateral sclerosis; multiple sclerosis; neurological disorders; pharmacokinetic profile

PMID: 26898712



Sigma receptor (SR, Figure 1) was initially considered as opioid receptors and successively confused with phencyclidine (PCP) binding site in the N-methyl-D-aspartate (NMDA) receptor. Today, SR is recognized as an independent receptor class that modulates cell survival and excitability and serves many critical functions in the human body. [1] Two SR subtypes, having different tissue distributions and distinct physiological and pharmacological profiles, have been identified. S1Rs are overexpressed in the Central and Peripheral Nervous System (CNS and PNS). Very recently, its X-ray crystal structure was determined (PDB file available, PDB codes: 5HK1 and 5HK2). [2] It is a 223-aminoacid protein, showing no homology with any known protein. In detail, it contains three hydrophobic domains with two transmembrane-spanning helices connected by an extracellular loop, and intracellular C and N terminus. Recent pharmacological investigations localize S1R at the endoplasmic reticulum (ER), in close contact with the mitochondria, in the so called mitochondria-associated-ER membrane (MAM), where it regulates ER-mitochondrion signaling and ER-nucleus crosstalk. [3, 4] The receptor is considered to be an ER chaperone protein acting as inter-organelle signaling modulator since it can translocate to the plasma membrane or to other subcellular compartments under stressful conditions and/or pharmacological manipulation. The modulatory effect of S1R ligands depends on numerous cellular components they interact, e.g. different classes of ion channels, kinases and G-protein coupled receptors (GPCR). Moreover, it is well-know the neuromodulatory and neuroprotective role of S1R activation on cholinergic and glutamatergic system. [5] Nevertheless, the endogenous ligands of these receptors have not been unequivocally identified yet. Recent evidence has been presented in support of the hypothesis that DMT, N,N dimethyltryptamine, a well-known hallucinogen, may in fact be an endogenous S1R agonist. [6] To sum up, the knowledge about this molecular target is still quite poor.

Pharmacological studies have indicated that S1R play critical roles in the mammalian nervous system. To this aim, the S1R agonist PRE-084 was used as reference compound. Briefly, several in vitro and in vivo studies have indicated that S1R agonists may promote neurite outgrowth and trophic factors production, reduce excitotoxicity by multiple mechanisms, repress microglial activation and maintain mitochondrial integrity with reduced production of reactive oxygen. As consequence, it has been hypothesized that S1R agonists may be of great therapeutic interest for CNS pathologies, such as human Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS) and Parkinson disease (PD). [1]


Figure 1. Timeline of key events in the history of sigma receptor (SR) and their modulators.


Our interdisciplinary research group identified (R)-RC-33 as a novel selective S1R agonist with an excellent S1R affinity (Ki= 1.8 nM) and good in vitro metabolic stability. [1, 7] Since study the metabolism of bioactive candidates is an important step of the drug development process to determine the most appropriate dosage schedule for in vivo assays, here the pharmacokinetic (PK) profiles of (R)-RC-33 in the mouse has been investigated. For comparative purposes, the same experiments were also performed with PRE-084. Moreover, one of the big challenges in CNS drug development is achieving an efficacious concentration profile in the brain and a good drug candidate has to possess the right balance between distribution in plasma and brain. Therefore, the CNS penetration of both compounds has been also studied. Initially, to accomplish the in vivo PK profile and future in vivo biological investigation, a gram-scale synthesis of (R)-RC-33 was performed (Figure 2).




Figure 2. Work flow.



(R)-RC-33  has been prepared in amount (1 g scale) and with optical purity suitable for in vivo studies, exploring and comparing asymmetric synthesis, chiral chromatography and fractional crystallization. PK studies evidenced that the brain/plasma and spinal cord/plasma concentration ratios of (R)-RC-33 are about 15 and 20 fold, evidencing an excellent distribution in the CNS (Figure 3A). As regards PRE-084, the concentration in brain, spinal cord and plasma was almost the same (Figure 3B). Therefore, (R)-RC33 showed a similar pharmacokinetic profile and a better CNS distribution when compared with the gold standard S1R agonist PRE-084.




Figure 3. Plasma pharmacokinetic profile and CNS-distribution of (A) (R)-RC-33 and (B) PRE-084 in mouse following intraperitoneal (IP) injection at the dose of 10 mg/kg.


According to these results, it could be considered an optimal pharmacological tool for studying the therapeutic potential of S1R agonists. In this regard, studies are currently underway in our labs to test i) the systemic availability of (R)-RC-33 after administration using different routes of administration (e.g. oral), ii) the in vitro ADME in the human species to predict in vivo pharmacokinetic behavior in man, iii) the in vivo assessment of the most suitable protocol of treatment based on chronic tolerability/toxicity study in C57BL/6 mice, and iv) the therapeutic potential of (R)-RC-33 in animal model of CNS mediated diseases.


Importance of the study: Since 2006 an increasing number of reports demonstrated neuroprotective effects of S1R agonists in treating neuropathologies, such as ALS, MS and PD. Despite the intense scientific research, to date there is still an urgent need for the discovery novel chemical entities for the treatments of these pathologies. Our data suggest that the novel S1R agonist (R)-RC33 show a similar PK profile and a better CNS distribution when compared with the gold standard S1R agonist PRE-084, evidencing that it could be a promising drug candidate for further studies in animal models of human diseases aimed at exploiting its pharmacological/therapeutic potential.



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  3. Hayashi T, Rizzuto R, Hajnoczky G, Su TP. MAM: more than just a housekeeper. Trends Cell Biol. 19(2), 81-8 (2009).
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  5. Pabba M. The essential roles of protein-protein interaction in sigma-1 receptor functions. Front Cell Neurosci 7: 50 (2013).
  6. Cozzi NV, Gopalakrishnan A, Anderson LL, Feih JT, Shulgin AT, Daley PF, Ruoho AE. Dimethyltryptamine and other hallucinogenic tryptamines exhibit substrate behavior at the serotonin uptake transporter and the vesicle monoamine transporter. J. Neural Transm. 116, 1591–1599 (2009).
  7. Marra A, Rossi D, Pignataro L, Bigogno C, Canta A, Oggioni N, Malacrida A, Corbo M, Cavaletti G, Curti D, Dondio G, Collina S, Toward the identification of neuroprotective agents. G-scale synthesis, pharmacokinetic evaluation and CNS distribution of (R)-RC-33, a promising SIGMA1 receptor agonist. Future Medicinal Chemistry, 8(3), 287-95 (2016).



Simona Collina University of Pavia

Dept of Drug Sciences Viale Taramelli, 12

27100 Pavia-I simona.collina@unipv.it



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