Central Nervous System Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Central Nervous System Agents) - Volume 9, Issue 3, 2009

It was more than 15 years ago that we first reviewed the “sigma enigma” [1]. At that time sigma (??) receptors represented a novel binding protein for which agents from an assortment of therapeutic classes and drugs of abuse displayed affinity. Indeed, the receptors seemed quite promiscuous because they displayed (at least micromolar) affinity for a very large number of agents encompassing a wide variety of structure-types: from simple arylalkylamines to more structurally complex, polycyclic cyclopentanoperhydrophenanthrenes. The receptors were initially thought to be a type of opioid receptor. Today, sigma receptors still remain somewhat of an enigma because relatively little is known about their structural composition and/or functionality (e.g. transduction mechanisms or second messenger systems), and what little is known suggests they are very different from most other recognized receptor types. On the other hand, various agents with nanomolar, and even sub-nanomolar, affinity now have been identified and pharmacophore models have been proposed that are in the process of being further defined, or re-defined. Sigma receptors offer a window of understanding to totally unique types of receptor proteins. In 1976, examining the actions of various opiates in the chronic spinal dog, Martin and colleagues proposed the existence of several distinct types of opioid receptors [2]. The actions of morphine were attributed to activation of a set of receptors to which the name μ-opioid receptors (now, MORs) was applied, whereas those of ketocyclazocine were ascribed to a different class of opioid receptors termed κ-opioid receptors (now, KORs). The benzomorphan SKF-10,047 (N-allylnormetazocine; NANM), although displaying some antagonist properties at μ-opioid receptors, produced effects distinct from those of morphine and ketocyclazocine. To account for these actions, SKF-10,047 was designated as an early prototypic agonist ligand for “sig-opioid” receptors. During the following decade it gradually became apparent that various non-opiate ligands display high affinity for these receptors, that the stereochemistry of benzomorphans preferred by sig receptors was usually the reverse of what was preferred by μ and κ receptors, and that sig receptor pharmacology was quite distinct from that of the other opioid receptors. Eventually, the “sig-opioid” receptors were effectively divorced from the opioid receptor family. Subsequently, NANM and several related benzomorphans were found to bind at sites displaying high affinity for phencyclidine (PCP) and, for a while, these sites were termed PCP/sig (or sig/PCP) receptors. Due to differences in the brain localization of these binding proteins, and because the antipsychotic agent haloperidol possessed high affinity for some PCP/sig sites but not for others, terms such as “haloperidol-sensitive” and “haloperidol-insensitive” sig sites began to appear in the literature. It is now recognized that sig sites are distinct from PCP sites, and that haloperidol binds at sig receptors but displays lower affinity for PCP receptors. Another ligand found to differentiate these sites was di-o-tolylguanidine (DTG) which was introduced in the mid 1980s. This agent, and its tritiated version, [3H]DTG, are still in use today. Various antipsychotic agents (e.g. phenothiazines, thioxanthenes, butyrophenones) were found to bind at sig receptors (although not necessarily with very high affinity) leading to early speculation that selective sig ligands might represent a novel class of agents for the treatment of schizophrenia. Rimcazole (with micromolar affinity for sig receptors) was the first sig ligand to enter clinical trials, but studies were terminated shortly thereafter. Today it is realized that rimcazole is a weak sig receptor antagonist with high affinity for the dopamine transporter [3]. This, coupled with the use of different membrane preparations and animal species, different radioligands, lack of appropriate functional assays, and an initial lack of awareness of receptor heterogeneity (see below), temporarily dampened enthusiasm for sig receptor research.

Methods for addressing sigma receptor affinity and activity have been explored and although several protocols have been employed, only few procedures resulted reliable. Sigma-1 receptor affinity protocol using guinea-pig brain and (+)-[3H]-pentazocine and sigma-2 receptor affinity protocol employing rat liver and [3H]-DTG are usually reported by authors as standard procedures. By contrast, the intrinsic activity evaluation of sigma ligands has been performed in several manners: tumor cell lines, isolated organ bath, in vivo animal model. The last is not considered in the present paper because this method studied the physiological role of sigma receptors. The studies carried out in tumor cell lines involved the role of sigma receptors in tumors progression while, although isolated organ bath experiment employed physiological samples, the pharmacokinetic properties of ligands, a strictly requirement for the in vivo assays, did not affect the pharmacodynamic properties of tested compounds. The advances in the above mentioned assays have been reported.

A large number of therapeutic roles have been proposed for sig1 receptors but the involvement of sig1 receptor in non-acute pain had not been well explored up to now. sig1 receptor knock-out mice became available offering us the possibility to study the role of sig1 receptor in nociception, particularly in models where central sensitization processes play a significant role. Given the attractive therapeutic potential, we have developed a chemical program aimed at the discovery of novel and selective sig1 ligands. Herein we discuss the rational basis of this approach and report preliminary pharmacological results of several chemical series and aspects of their structure-activity relationship on sig1 receptor. Functional data in pain models are presented mainly on one series that provide evidence to consider selective sig1 receptor antagonists an innovative and alternative approach for treating neuropathic pain.

Chaperones are proteins that assist the correct folding of other protein clients either when the clients are being synthesized or at their functional localities. Chaperones are responsible for certain diseases. The sigma-1 receptor is recently identified as a receptor chaperone whose activity can be activated/deactivated by specific ligands. Under physiological conditions, the sigma-1 receptor chaperones the functional IP3 receptor at the endoplasmic reticulum and mitochondrion interface to ensure proper Ca2+ signaling from endoplasmic reticulum into mitochondrion. However, under pathological conditions whereby cells encounter enormous stress that results in the endoplasmic reticulum losing its global Ca2+ homeostasis, the sigma-1 receptor translocates and counteracts the arising apoptosis. Thus, the sigma-1 receptor is a receptor chaperone essential for the metabotropic receptor signaling and for the survival against cellular stress. The sigma-1 receptor has been implicated in many diseases including addiction, pain, depression, stroke, and cancer. Whether the chaperone activity of the sigma-1 receptor attributes to those diseases awaits further investigation.

Sigma1 receptors were imaged in living human brain by positron emission tomography (PET) using [11C] SA4503. A dynamic 90-min scan and kinetic analysis enabled quantification of receptor density in the brain. The sigma1 receptors were distributed throughout the brain in normal subjects, but decreased in the frontal, temporal, and occipital lobes, cerebellum and thalamus in patients with early Alzheimer's disease and in the putamen in patients with Parkinson's disease. In addition, rates of receptor occupancy by the neuroleptic haloperidol and the selective serotonin reuptake inhibitor fluvoxamine were evaluated by [11C]SA4503-PET and found to be high. [11C]SA4503-PET is useful for studying the pathophysiology of neurological and psychiatric disorders such as schizophrenia and for evaluation of the pharmacodynamics of psychiatric drugs.

Endoplasmic protein sigma-1 receptors represent unique binding sites in the brain, and they exert a potent influence on a number of neurotransmitter systems. Several lines of evidence suggest that sigma-1 receptors play roles in the pathophysiology of psychiatric diseases, as well as in the active mechanisms of some selective serotonin reuptake inhibitors (SSRIs). Interestingly, we reported that some SSRIs possess moderate to high affinities at sigma-1 receptors in the brain. Among them, the order of affinity for sigma-1 receptors was as follows: fluvoxamine > sertraline > fluoxetine > citalopram ≫ paroxetine. In a cell culture system, we demonstrated that fluvoxamine, but not sertraline or paroxetine, significantly potentiated nerve-growth factor (NGF)-induced neurite outgrowth in PC12 cells, and that the effect of fluvoxamine on NGF-induced neurite outgrowth was significantly antagonized by treatment with the selective sigma-1 receptor antagonist NE-100. Furthermore, we reported that phencyclidine (PCP)-induced cognitive deficits in mice were significantly improved by subsequent subchronic administration of fluvoxamine, but not sertraline and paroxetine, and that the effect of fluvoxamine on PCP-induced cognitive deficits was antagonized by co-administration of NE-100. Moreover, a recent study using the specific sigma-1 receptor ligand [11C] SA4503 and positron emission tomography (PET) have demonstrated that an oral administration of fluvoxamine, but not paroxetine, could bind to sigma-1 receptors in the healthy human brain, in a dose-dependent manner. These findings suggest that sigma-1 receptors might be implicated in the active mechanisms of fluvoxamine. In this article, the author would like to discuss the novel role of sigma-1 receptors in the active mechanisms of some SSRIs including fluvoxamine.

Herein the evolution in the development of new sigma (??) receptor ligands since the middle &90s by our research group is reported. In the effort to contribute to the identification of the structural features for high-affinity ligands selective versus serotonin, dopamine and other CNS-related receptors, two general classes of (naphthalene)alkylamine compounds were prepared and explored, with the aim of addressing the affinities toward the two recognized sig receptor subtypes. The common template of these compounds was mainly an unsubstituted or methoxy-substituted naphthalene or tetralin nucleus, linked by an alkyl spacer to a substituted piperazine or piperidine ring. The design of new ligands was thought keeping in mind their possible application as PET diagnostic tools and fluorescence tools. High-affinity sig2 receptor ligands were found among N-cyclohexylpiperazine derivatives, such as 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4- tetrahydronaphthalen-1-yl)propyl]piperazine (3) (PB 28), when they were assayed in radioligand binding with [3H]-DTG in rat liver. Unfortunately, these ligands were all devoid of a significant selectivity relative to sig1 receptor whose binding was assayed with (+)-[3H]-pentazocine in guinea pig brain. Nevertheless, compound 3 had previously shown to be 40-fold selective with a slightly different binding method in animals' tissues. Moreover, it demonstrated 46-fold and 59-fold sig2 versus sig1 receptor binding selectivity in MCF7 and MCF7 ADR tumor cell lines respectively. In the class of piperazines, also high-affinity sig1 receptor ligands were found, possibly due to the presence of a double N-atom and an additional reverse mode of binding. Piperidine derivatives were investigated as high-affinity and selective sig1 receptor ligands leading to some 3,3-dimethylpiperidines such as 3,3-dimethyl-1-[3-(6-methoxynaphthalen-1-yl)propyl]piperidine (69) which resulted to be highly selective relative to the sig2 receptor. For the best ligands, functional assays were conducted in order to investigate agonist/antagonist activity. The effect of chirality in the intermediate methyl-alkyl chain was explored for a class of 4-methylpiperidines linked to some (4-chlorophenoxy)alkyl moieties, and compound (-)-(S)-92 emerged as the most selective sig1 relative to sig2 receptor ligand.

It was postulated that N6-allyl bicyclic derivatives 1 bind with N-8 at the proton donor site of the sig1 receptor and that a substituent in 2-position of the bicyclic framework 1 results in unfavorable steric interactions with the sig1 receptor protein. In order to support this hypothesis both enantiomers of 6-allyl-8-(4-methoxybenzyl)-6,8-diazabicyclo[ 3.2.2]non-2-ene (2/ent-2) and 6-benzyl-8-(4-methoxybenzyl)-6,8-diazabicyclo[3.2.2]nonane 3/ent-3 were synthesized stereoselectively. The (S,S)-configured enantiomers 2 and 3 are the eutomers with eudismic ratios of 31 and 4.8, respectively. Therefore, these enantiomers are used in the sig1 pharmacophore model. The N6-allyl derivative 2 with a double bond in the three carbon bridge adopts the orientation 2c with N-8 interacting with the sig1 receptor proton donor site (Fig. 2) resulting in slightly reduced steric interactions of the small double bond in 2/3-position. The almost C2-symmetric benzyl derivative 3 can adopt both orientations 2c and 2d at the sig1 receptor (N-8 or N-6 interacts with the sig 1 receptor proton donor site) resulting in subnanomolar sig1 receptor affinity (Ki = 0.91 nM).

The sigma-2 (sig2) receptor is proving to be an important protein in the field of cancer biology. The observations that sig2 receptors have a 10-fold higher density in proliferating tumor cells than in quiescent tumor cells, and that sig2 receptor agonists are capable of killing tumor cells via apoptotic and non-apoptotic mechanisms, indicate that this receptor is an important molecular target for the development of radiotracers for imaging tumors using techniques such as Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) and for the development of cancer chemotherapeutic agents. In spite of recent promising results towards achieving these goals, research in this field has been hampered by the fact that the molecular identity of the protein sequence of the sig2 receptor is currently not known. Consequently, most of what is known about this protein has been obtained using either radiolabeled or fluorescent probes for this receptor, or biochemical analysis of the effect of sig2 selective ligands on cells growing under tissue culture conditions. This article provides a review of the development and use of sig2 receptor ligands, and how these ligands have been used with a variety of in vitro and in vivo models to gain a greater understanding of the role this receptor plays in cancer.

Several sig1 receptor ligands with sub-nanomolar affinity and excellent selectivity have been reported, but relatively few sig2-selective ligands are known. 1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl] piperazine (PB28; 1) has been reported by us as a high-affinity sig2 receptor ligand with significant sig2 selectivity, and several analogs of (1) now have been developed. Among these are the class of cyclohexylpiperazines that display a good compromise between affinity/activity and selectivity for sig2 receptors. Very little is currently known about the nature of sig2 receptors. In the absence of structure-based receptor information, we applied a comparative molecular field analysis (CoMFA) - a three-dimensional structure-activity relationship (3D-QSAR) method - to a set of cyclohexylpiperazine sig2 ligands to develop a predictive model that might provide information about the stereoelectronic nature of the receptor binding site. Two CoMFA models were generated from two different alignments: the first used an automated FlexS algorithm, and the second used a rationally-driven manual alignment. Significantly better predictivity was obtained with the manual alignment (TSET: q2 = 0.73, r2 = 0.95; PSET: r2 = 0.55/0.73) than from the automated alignment (TSET: q2 = 0.69, r2 = 0.98; PSET: r2 = 0.13/0.16). The resulting CoMFA maps account for observed structure-affinity relationships and suggest a possible anatomy for the sig2 receptor/cyclohexylpiperazine binding site.