[3H]N-[4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl]-2-methoxy-5-methylbenzamide: A novel sigma-2 receptor probe
Introduction
The sigma (σ) receptors are a distinct family of proteins capable of binding to a wide variety of drugs (Martin et al., 1976, Walker et al., 1990). Based on photoaffinity labeling studies with [3H] azido-1,3-di-o-tolylguanidine ([3H]azido-DTG), two different types of σ receptors have been defined, σ1 and σ2 receptors (Hellewell et al., 1994). The σ1 receptor has a molecular weight of 25 kDa, and the gene encoding this receptor has been cloned from guinea pig, human, mouse, and rat (Hanner et al., 1996, Seth et al., 1997, Kekuda et al., 1996, Mei and Pasternak, 2001). The gene for the σ1 receptor contains four exons and three introns and is approximately 7 kbp long. The σ2 receptor has not been cloned, but biochemical studies indicate that this protein has a molecule weight of 21.5 kDa. Colocalization of σ1 and σ2 receptors has been reported in many normal tissues and numerous drugs have been found to bind to σ1 and σ2 receptors with similar affinity (McCann et al., 1994, Hellewell et al., 1994).
A σ1 receptor splice variant missing exon 3 does not bind the σ1-selective radioligand, [3H](+)-pentazocine but retains affinity for the σ1/σ2 nonselective ligand, [3H]DTG (Wang et al., 2004). Based on this observation, it was suggested that the σ2 receptor may be the product of alternative gene splicing of σ1 receptor, namely the σ1β receptor. However, in vitro binding studies conducted on tissues obtained from a σ1 receptor knockout mouse demonstrated that although [3H](+)-pentazocine binding was significantly reduced in brain tissues, binding of σ1/σ2 radioligand [3H]DTG was unaffected (Langa et al., 2003). These data suggest that the σ2 receptor is encoded by different gene and may not simply represent a slice variant of the σ1 receptor.
Although the biological function of the σ receptors is not known, there is a large body of evidence to support that the σ1 receptor plays an important role in central nervous system function. A number of in vitro binding and in vivo behavioral studies have suggested that neuroactive steroids are endogenous ligands for the σ1 receptor (Su et al., 1988, Monnet et al., 1995, Maurice et al., 1999). Ligands binding to the σ1 receptor also modulate the release of neurotransmitters, and several of these compounds have shown promise as antipsychotic, antidepressants, and drugs blocking the reinforcing effect of psychostimulants (Takebayashi et al., 2002, Su and Hayashi, 2003, Matsumoto et al., 2003, Guitart et al., 2004).
Much less is known about the biological function of the σ2 receptor. However, a number of studies have reported that σ2 receptors over expressed in a wide variety of human and murine tumor cells grown in cell culture (Bem et al., 1991, Vilner et al., 1995, Mach et al., 1997). Furthermore, we have previously reported that the density of σ2 receptors is 10-fold higher in proliferative versus quiescent mouse mammary adenocarcinoma cells in vitro (Mach et al., 1997, Al-Nabulsi et al., 1999) and in vivo (Wheeler et al., 2000). Based on these data, we have proposed that the σ2 receptor may serve as a receptor-based biomarker of the proliferative status of solid tumors. Recent studies have also shown that σ2 receptor ligands can induce apoptosis in tumor cells, which raises the possibility that σ2 selective ligands may be useful as anticancer or chemosensitizing agents (Crawford and Bowen, 2002, Matsumoto et al., 2004). A potential role of the σ2 receptor in regulating tumor cell proliferation and apoptosis has led to a renewed interest in understanding the biological function of this receptor.
[3H](+)-pentazocine is a selective ligand for in vitro studies of the σ1 receptor. The radioligand most often used to study σ2 receptors in vitro is [3H]DTG. However, this radioligand binds with equal affinity to σ1 and σ2 receptors (Walker et al., 1990, Hellewell et al., 1994). In vitro binding studies aimed at measuring the density of σ2 receptors in tissue or tumor membrane homogenates requires the addition of unlabeled (+)-pentazocine to the assay in order to mask the binding of [3H]DTG to σ1 receptors (Hellewell et al., 1994). Furthermore, the relatively low affinity of [3H]DTG to σ2 receptors (Kd ∼ 25 nM) requires the use of a relatively high concentration of this compound in the binding assay. Therefore, the development of a novel radioligand with high affinity and specificity for σ2 receptors would represent an important improvement in the methodology used to assay σ2 receptors in vitro.
Our group has previously reported a number of compounds having a high affinity and selectivity for σ2 receptors compared to their affinity for σ1 receptors (Mach et al., 1999, Mach et al., 2001, Mach et al., 2002, Mach et al., 2004). Two of these compounds are the conformationally flexible benzamide analogs, N-[4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl]-2-methoxy-5-methyl-benzamide (RHM-1) and N-[2-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)ethyl]-2-methoxy-5-methylbenzamide (RHM-2), which have been shown to have a high affinity and selectivity for σ2 receptors (Mach et al., 2004). Their high σ2 receptor affinity (Ki < 15 nM) and selectivity (σ2/σ1 ratio > 300) suggests that they may be useful for assessing the σ2 receptor status of solid tumors and normal tissues when they are radiolabeled. Therefore, RHM-1 and RHM-2 were radiolabeled with tritium (specific activity = 80 Ci/mmol), and the binding of [3H]RHM-1 and [3H]RHM-2 to σ2 receptors of rat liver and breast tumor homogenates were evaluated in vitro. [3H]RHM-1 was found to have a higher affinity for σ2 receptors compared to [3H]RHM-2 and [3H]DTG. The results of our studies demonstrate that [3H]RHM-1 is a useful radioligand for assessing the σ2 receptor status of tumors and normal tissues.
Section snippets
Precursor synthesis and radiolabeling
The tritiated compounds were synthesized by American Radiolabeled Chemicals, Inc. (St. Louis, MO) via O-alkylation of the corresponding phenol precursor (Tu et al., 2005); the specific activity of the radioligands was 80 Ci/mmol. The chemical structures of [3H]RHM-1 and [3H]RHM-2, which vary in the length of the carbon spacer between the amide nitrogen and the tertiary amine group, are shown in Fig. 1. [3H]DTG (58.1 Ci/mmol) was purchased from PerkinElmer (Boston, MA).
Drugs and preparation
Chemical reagents and the
Radiochemical synthesis
The chemical structures of [3H]RHM-1 and [3H]RHM-2 are shown in Fig. 1. The specific activity of both radioligands is 80 Ci/mmol, with a radiochemical purity of > 98%, determined by high performance liquid chromatography (HPLC). The complete details of the synthesis and radiolabeling procedures will be published elsewhere. The primary difference between these two radioligands is the four carbon spacer for [3H]RHM-1 and the two carbon spacer for [3H]RHM-2. Because of this structural difference, [3
Discussion
The goal of the current study was to evaluate two tritiated conformationally flexible benzamide analogs, [3H]RHM-1 and [3H]RHM-2, as potential ligands for measuring σ2 receptor function in vitro. Our previous studies have shown that both compounds have a high selectivity for σ2 receptors and similar Ki values for displacing [3H]DTG from σ2 receptors in vitro (Mach et al., 2004). Although [3H]DTG is the traditional radioligand for studying σ2 receptors in vitro, this ligand has a relatively low σ
Acknowledgements
This research was funded by grants CA 102869 from the National Institutes of Health and DAMD17-01-0446 from the Department of Defense. We thank Dr. Carolyn Anderson and her group for assistance with MicroBeta counting experiments. We appreciate Dr. Wenhua Chu for coordinating the custom radiolabeling of these ligands.
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2015, Bioorganic and Medicinal ChemistryCitation Excerpt :All compounds were independently assayed at least two times. The σ receptor binding affinity studies were performed following our previously reported procedure.51,52 The σ1 receptor binding assays were performed in 96-well plates by incubating test compounds with approximately 300 μg protein of guinea pig brain membrane homogenates and 5 nM (+)-[3H]pentazocine (1.30 GBq/μmol, Perkin Elmer, Boston, MA).