Annamaria Marra

Sigma receptor modulators: a patent review

Introduction: Sigma receptors are involved in several central nervous system (CNS) disorders, including mood disorders (depression and anxiety), psychosis, schizophrenia, movement disorders (i.e., Parkinsons disease) and memory deficits (i.e., Alzheimers disease). Recently, the involvement of sigma receptors in neuropathic pain and cancer has also been observed. Areas covered: This review aims at highlighting the research advancements published in the patent literature between 1986 and 2012, dividing patents according to both their time frame and applicants. The review especially focuses on the development of sigma receptor modulators and their application over the years with respect to CNS diseases, neuropathic pain and neurodegenerative pathologies. The literature was sought through Espacenet, Orbit, ISI Web and PubMed databases. Expert opinion: In recent years, considerable progress in the knowledge of the biology and pharmacology of sigma receptors has encouraged research on the potential benefits of sigma modulators in a wide range of pathologies. So far, only few potent agonists and antagonists of sigma receptors are in clinical trial for acute and chronic neurodegenerative diseases (SA4503 and ANAVEX 2-73) or neuropathic pain (E-52862).

Chemical, Pharmacological, and in vitro Metabolic Stability Studies on Enantiomerically Pure RC‐33 Compounds: Promising Neuroprotective Agents Acting as σ1 Receptor Agonists

Our recent research efforts identified racemic RC‐33 as a potent and metabolically stable σ1 receptor agonist. Herein we describe the isolation of pure RC‐33 enantiomers by chiral chromatography, assignment of their absolute configuration, and in vitro biological studies in order to address the role of chirality in the biological activity of these compounds and their metabolic processing. The binding of enantiopure RC‐33 to the σ1 receptor was also investigated in silico by molecular dynamics simulations. Both RC‐33 enantiomers showed similar affinities for the σ1 receptor and appeared to be almost equally effective as σ1 receptor agonists. However, the R‐configured enantiomer showed higher in vitro hepatic metabolic stability in the presence of NADPH than the S enantiomer. Overall, the results presented herein led us to select (R)‐RC‐33 as the optimal candidate for further in vivo studies in an animal model of amyotrophic lateral sclerosis.

Identification of RC-33 as a potent and selective σ1 receptor agonist potentiating NGF-induced neurite outgrowth in PC12 cells. Part 2: g-scale synthesis, physicochemical characterization and in vitro metabolic stability.

Strong pharmacological evidences indicate that σ1 receptors are implicated in the pathophysiology of all major CNS disorders. In the last years our research group has conducted extensive studies aimed at discovering novel σ1 ligands and we recently selected (R/S)-RC-33 as a novel potent and selective σ1 receptor agonist. As continuation of our work in this field, here we report our efforts in the development of this new σ1 receptor agonist. Initially, we investigated the binding of (R) and (S) enantiomers of RC-33 to the σ1 receptor by in silico experiments. The close values of the predicted affinity of (R)-RC-33 and (S)-RC-33 for the protein evidenced the non-stereoselective binding of RC-33 to the σ1 receptor; this, in turn, supported further development and characterization of RC-33 in its racemic form. Subsequently, we set-up a scaled-up, optimized synthesis of (R/S)-RC-33 along with some compound characterization data (e.g., solubility in different media and solid state characterization by thermal analysis techniques). Finally, metabolic studies of RC-33 in different biological matrices (e.g., plasma, blood, and hepatic S9 fraction) of different species (e.g., rat, mouse, dog, and human) were performed. (R/S)-RC-33 is generally stable in all examined biological matrices, with the only exception of rat and human liver S9 fractions in the presence of NADPH. In such conditions, the compound is subjected to a relevant oxidative metabolism, with a degradation of approximately 65% in rat and 69% in human. Taken together, our results demonstrated that (R/S)-RC-33 is a highly potent, selective, metabolically stable σ1 agonist, a promising novel neuroprotective drug candidate.

Toward the identification of neuroprotective agents: g-scale synthesis, pharmacokinetic evaluation and CNS distribution of (R)-RC-33, a promising SIGMA1 receptor agonist.

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.

Studies on the enantiomers of RC-33 as neuroprotective agents: isolation, configurational assignment, and preliminary biological profile

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 asymmetric synthesis previously devised. As emerged in the preliminary in vitro biological investigation, (S)- and (R)-RC-33 possess a comparable affinity towards the σ1 receptor and a very a similar behavior in the calcium influx assay, resulting in an equally effective σ1 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.

Enantiomeric 4-Acylamino-6-alkyloxy-2 Alkylthiopyrimidines As Potential A3 Adenosine Receptor Antagonists: HPLC Chiral Resolution and Absolute Configuration Assignment by a Full Set of Chiroptical Spectroscopy.

The chiral separation of enantiomeric couples of three potential A3 adenosine receptor antagonists: (R/S)-N-(6-(1-phenylethoxy)-2-(propylthio)pyrimidin-4-yl)acetamide (), (R/S)-N-(2-(1-phenylethylthio)-6-propoxypyrimidin-4-yl)acetamide (), and (R/S)-N-(2-(benzylthio)-6-sec-butoxypyrimidin-4-yl)acetamide () was achieved by high-performance liquid chromatography (HPLC). Three types of chiroptical spectroscopies, namely, optical rotatory dispersion (ORD), electronic circular dichroism (ECD), and vibrational circular dichroism (VCD), were applied to enantiomeric compounds. Through comparison with Density Functional Theory (DFT) calculations, encompassing extensive conformational analysis, full assignment of the absolute configuration (AC) for the three sets of compounds was obtained. Chirality 28:434-440, 2016.

A step forward in the sigma enigma: a role for chirality in the sigma1 receptor–ligand interaction?

In our recent research racemic RC-33 was identified as a potent and highly promising σ1 receptor agonist, showing excellent σ1 receptor affinity and promoting NGF-induced neurite outgrowth in PC12 cells at very low concentrations. Surprisingly, both its interaction with the biological target and its effect on neurite sprouting proved to be non-stereoselective. Starting from the observation that a hydrogen bond center in the scaffold of a σ1 ligand is an important pharmacophoric element for receptor/ligand interaction, we hypothesized that the absence of such pharmacophoric feature in the structure of RC-33 could be also responsible for the lack of enantioselectivity in its interaction with the target receptor. To verify our hypothesis, in this paper we evaluated – both in silico and in vitro – the ability of a series of enantiomeric arylalkylaminoalcohols and arylpyrrolidinols 1–5 to interact with the receptor. All these compounds are structurally related to RC-33 and are characterized by the presence of an –OH group as the additional pharmacophore feature. Interestingly, the results of our study show that the σ1 receptor exhibits enantiopreference toward compounds characterized by (S)-configuration at the stereogenic center bearing the aromatic moiety only when the alcoholic group is also present at that chiral center, thus supporting our original hypothesis.

Beyond the affinity for protein kinase C: exploring 2-phenyl-3-hydroxypropyl pivalate analogues as C1 domain-targeting ligands

Over the past fifteen years, we reported the design and synthesis of different series of compounds targeting the C1 domain of protein kinase C (PKC) that were based on various templates. Out of the pivalate templates, 2-[4-(benzyloxy)phenyl]-3-hydroxypropyl pivalate (compound 1) emerged as the most potent and promising PKCα ligand, showing a Ki value of 0.7 μM. In the present contribution our efforts are aimed at better understanding which structural modifications of the pivalate template are allowed for its affinity to the C1 domain of PKC to be preserved or increased. To this aim, thirteen novel analogues of 1 were designed and their interaction with the target was evaluated in silico. Designed compounds were then prepared and fully characterized as well as their affinity for the α and δ isoforms of PKC evaluated. Additionally, in order to investigate the role of chirality in the ligand–target interaction, the pure enantiomers of the most interesting PKC ligands were prepared and their affinity for PKC isoforms was determined. Results from our study revealed that: i) the presence of the ester function seems to be essential for the ligand–target interaction; ii) only a few structural modifications at the ester group are allowed for the C1 domain affinity to be preserved; and iii) the [3H]PDBu replacement experiments showed that the C1 domain of PKC does not exhibit enantiopreference for the pure stereoisomers of tested compounds. Altogether our observations provide further insights into the ligand–target interactions of the PKC C1 domain and represent a step-forward in future development of more specific and effective PKC ligands.

Novel Enantiopure Sigma Receptor Modulators: Quick (Semi-)Preparative Chiral Resolution via HPLC and Absolute Configuration Assignment.

The identification of novel pan-sigma receptor (SR) modulators, potentially useful in cancer treatment, represents a new goal of our research. Here, we report on the preparation of novel chiral compounds characterized by a 3-C alkyl chain bridging an aromatic portion to a 4-benzyl-piperidine moiety. All of the studied compounds have been prepared both in racemic and enantiomerically-pure form, with the final aim to address the role of chirality in the SR interaction. To isolate and characterize enantiomeric compounds, high-performance liquid chromatography (HPLC) procedures were set up. A systematic analytical screening, involving several combinations of chiral stationary and mobile phases, allowed us to optimize the analytical resolution and to set up the (semi-)preparative chromatographic conditions. Applying the optimized procedure, the enantiomeric resolution of the studied compounds was successfully achieved, obtaining all of the compounds with an enantiomeric excess higher than 95%. Lastly, the absolute configuration has been empirically assigned to enantiopure compounds, combining the electronic circular dichroism (ECD) technique to the elution order study.

Development of easy‐to‐use reverse‐phase liquid chromatographic methods for determining PRE‐084, RC‐33 and RC‐34 in biological matrices. The first step for in vivo analysis of sigma1 receptor agonists

Over the years there has been a growing interest in the therapeutic potential for central nervous system pathologies of sigma receptor modulators. The widely studied PRE-084 and our compounds RC-33 and RC-34 are very potent and selective sigma 1 receptor agonists that could represent promising drug candidates for Amyotrophic Lateral Sclerosis (ALS). Herein, we develop and validate robust and easy-to-use reverse-phase chromatographic methods suitable for detecting and quantifying PRE-084, RC-33 and RC-34 in mouse blood, brain and spinal cord. An HPLC/UV/ESI-MS system was employed for analyzing PRE-084 and an HPLC/UV-PDA system for determining RC-33 and RC-34. Chromatographic separations were achieved on Waters Symmetry RP18 column (150 × 3.9 mm, 5 µm), eluting with water and acetonitrile (both containing 0.1% formic acid) in gradient conditions. The recovery of PRE-084, RC-33 and RC-34 was >95% in all the considered matrices. Their limits of quantitation and detection were also determined. Validation proved the methods be suitable for separating tested compounds from endogenous interferences, being characterized by good sensitivity, linearity, precision and accuracy. A preliminary central nervous system distribution study showed a high distribution of RC-33 in brain and spinal cord, with concentration values well above the determined limit of quantitation. The proposed methods will be used in future preclinical investigations.