Synthesis and biological evaluation of 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide stereoisomers as novel positive allosteric modulators of sigma-1 receptor

Novel positive allosteric modulators of sigma-1 receptor represented by 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide enantiomers were synthesised using an asymmetric Michael addition of 2-nitroprop-1-enylbenzene to diethyl malonate. Following the chromatographic separation of the methyl erythro- and threo-4-nitro-3R- and 3S-phenylpentanoate diastereoisomers, target compounds were obtained by their reductive cyclisation into 5-methyl-4-phenylpyrrolidin-2-one enantiomers and the attachment of the acetamide group to the heterocyclic nitrogen. Experiments with electrically stimulated rat vas deference contractions induced by the PRE-084, an agonist of sigma-1 receptor, showed that (4R,5S)- and (4R,5R)-2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamides with an R-configuration at the C-4 chiral centre in the 2-pyrrolidone ring were more effective positive allosteric modulators of sigma-1 receptor than were their optical antipodes.     

Assessment of sigma-1 receptor occupancy in mice with non-radiolabelled FTC-146 as a tracer

Abstract

The use of liquid chromatography coupled with mass spectrometry (LC-MS/MS) is advantageous in in-vivo receptor occupancy assays at pre-clinical drug developmental stages. Relatively, its application is effective in terms of high throughput, data reproducibility, sensitivity, and sample processing. In this perspective, we have evaluated the use of FTC-146 as a non-radiolabelled tracer to determine the sigma-1 receptor occupancy of test drugs in mice brain. Further, the brain and plasma exposures of test drug were determined at their corresponding occupancies. In this occupancy method, the optimized tracer treatment (sacrification) time after intravenous administration was 30?min. The tracer dose was 3?µg/kg and specific brain regions of interest were frontal cortex, pons and midbrain. Mice were pretreated orally with SA4503, fluspidine, haloperidol, and donepezil followed by tracer treatment. Among the test drugs, SA4503 was used as positive control group at its highest test dose (7?mg/kg, intraperitoneal). There was a dose-dependent decrease in brain regional FTC-146 binding in pretreated mice. From the occupancy curves of SA4503, fluspidine, haloperidol, and donepezil the effective dose (ED₅₀) value ranges are 0.74–1.45, 0.09–0.11, 0.11–0.12, and 0.07–0.09?mg/kg, respectively. Their corresponding brain effective concentration (EC₅₀) values are 74.3–132.5, 3.4–3.7, 122.5–139.5, and 8.8–11.0?ng/g and plasma EC₅₀ values are 34.3–53.7, 0.08–0.10, 7.8–9.5, and 0.6–0.7?ng/mL. Brain regional distribution and binding inhibition upon pretreatment were comparable with data reported with labeled [¹⁸F]FTC-146. Drug exposures were simultaneously determined and correlated with sigma-1 occupancy from the same experiment. Wide category drugs can be assayed for sigma-1 receptor engagement and their correlation with exposures aid in clinical development.     

Functional assays to define agonists and antagonists of the sigma-2 receptor

The sigma-2 receptor has been identified as a biomarker in proliferating tumors. To date there is no well-established functional assay for defining sigma-2 agonists and antagonists. Many sigma-2 ligands with diverse structures have been shown to induce cell death in a variety of cancer cells by triggering caspase-dependent and independent apoptosis. Therefore, in the current study, we used the cell viability assay and the caspase-3 activity assay to determine sigma-2 agonists and antagonists. Three classes of sigma-2 ligands developed in our laboratory were evaluated for their potency to induce cell death in two tumor cell lines, mouse breast cancer cell line EMT-6 and human melanoma cell line MDA-MB-435. The data showed that the EC₅₀ values of the sigma-2 ligands using the cell viability assay ranged from 11.4 μM to >200 μM, which were comparable with the EC₅₀ values obtained using the caspase-3 assay. Based on the cytotoxicity of a sigma-2 ligand relative to that of siramesine, a commonly accepted sigma-2 agonist, we have categorized our sigma-2 ligands into agonists, partial agonists, and antagonists. The establishment of functional assays for defining sigma-2 agonists and antagonists will facilitate functional characterization of sigma-2 receptor ligands and sigma-2 receptors.     

Characterization and evaluation of two novel fluorescent sigma-2 receptor ligands as proliferation probes

We synthesized and characterized two novel fluorescent sigma-2 receptor selective ligands, SW120 and SW116, and evaluated these ligands as potential probes for imaging cell proliferation. Both ligands are highly selective for sigma-2 receptors versus sigma-1 receptors. SW120 and SW116 were internalized into MDA-MB-435 cells, and 50% of the maximum fluorescent intensity was reached in 11 and 24 minutes, respectively. In vitro studies showed that 50% of SW120 or SW116 washed out of cells in 1 hour. The internalization of SW120 was reduced ≈30% by phenylarsine oxide, an inhibitor of endocytosis, suggesting that sigma-2 ligands are internalized, in part, by an endocytotic pathway. Subcellular localization studies using confocal and two-photon microscopy showed that SW120 and SW116 partially colocalized with fluorescent markers of mitochondria, endoplasmic reticulum, lysosomes, and the plasma membrane, suggesting that sigma-2 receptors localized to the cytoplasmic organelles and plasma membrane. SW120 did not colocalize with the nuclear dye 4',6-diamidino-2-phenylindole. In vivo studies showed that the uptake of SW120 in solid tumors and peripheral blood mononuclear cells of mice positively correlated with the expression level of the cell proliferation marker Ki-67, suggesting that sigma-2 fluorescent probes may be used to image cell proliferation in mice.     

New analogs of SYA013 as sigma-2 ligands with anticancer activity

Our previous study has revealed 4-(4-(4-chlorophenyl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)butan-1-one·2HCl (SYA013) 1 as a sigma ligand with moderate selectivity for the sigma-2 receptor. Given the overexpression of sigma receptors in solid tumors and reports of sigma ligands with anticancer activities, we selected 1 for evaluation in several solid tumor cell lines. In addition, we have synthesized new analogs of 1 and now report that several of them bind preferentially at the sigma-2 receptor and have shown inhibition of several cancer cell lines including MDA-MB-231, MDA-MB-486, A549, PC-3, MIA PaCa-2 and Panc-1 cells. In particular, compounds 1 and 12 have demonstrated sub-micromolar activity against the Panc-1 cell line. It has also been observed that several of these compounds demonstrate selective toxicity toward cancer cells, when compared to normal cells.     

Synthesis and biological evaluation of novel 4-benzylpiperazine ligands for sigma-1 receptor imaging

We report the synthesis and evaluation of 4-benzylpiperazine ligands (BP-CH(3), BP-F, BP-Br, BP-I, and BP-NO(2)) as potential σ(1) receptor ligands. The X-ray crystal structure of BP-Br, which crystallized with monoclinic space group P2(1)/c, has been determined. In vitro competition binding assays showed that all the five ligands exhibit low nanomolar affinity for σ(1) receptors (K(i)=0.43-0.91nM) and high subtype selectivity (σ(2) receptor: K(i)=40-61nM; K(i)σ(2)/K(i)σ(1)=52-94). [(125)I]BP-I (1-(1,3-benzodioxol-5-ylmethyl)-4-(4-iodobenzyl)piperazine) was prepared in 53±10% isolated radiochemical yield, with radiochemical purity of >99% by HPLC analysis after purification, via iododestannylation of the corresponding tributyltin precursor. The logD value of [(125)I]BP-I was found to be 2.98±0.17, which is within the range expected to give high brain uptake. Biodistribution studies in mice demonstrated relatively high concentration of radiolabeled substances in organs known to contain σ(1) receptors, including the brain, lung, kidney, heart, and spleen. Administration of haloperidol 5min prior to injection of [(125)I]BP-I significantly reduced the concentration of radioactivity in the above-mentioned organs. The accumulation of radiolabeled substance in the thyroid was quite low suggesting that [(125)I]BP-I is relatively stable to in vivo deiodination. These findings suggest that the binding of [(125)I]BP-I to σ(1) receptors in vivo is specific.     

N-arylalkylpiperidines as high-affinity sigma-1 and sigma-2 receptor ligands: phenylpropylamines as potential leads for selective sigma-2 agents

To delineate the differences between the structural requirements necessary for recognition at sigma-1 and sigma-2 receptors, a range of phenethyl- and phenylpropylpiperidines were evaluated in binding assays. Phenethylpiperidines were found to favor sigma-1 receptors, whereas phenylpropylpiperidines tend to favor sigma-2 receptors. It appears that phenylpropylamine is a potential pharmacophore for selective sigma-2 ligands.     

Radiosynthesis and evaluation of new PET ligands for peripheral cannabinoid receptor type 1 imaging

Cannabinoid receptor type 1 (CB1) is mainly expressed in the brain, as well as being expressed in functional relevant concentrations in various peripheral tissues. 1-(4-Chlorophenyl)-3-(3-(6-(pyrrolidin-1-yl)pyridin-2-yl)phenyl)urea (PSNCBAM-1, 1) was developed as a potent allosteric antagonist for CB1 and its oral administration led to reductions in the appetite and body weight of rats. Several analogs of 1 (compounds 2 and 3) were recently identified through a series of structure-activity relationship studies. Herein, we report the synthesis of radiolabeled analogs of these compounds using [¹¹C]COCl₂ and an evaluation of their potential as PET ligands for CB1 imaging using in vitro and in vivo techniques. [¹¹C]2 and [¹¹C]3 were successfully synthesized in two steps using [¹¹C]COCl₂. The radiochemical yields of [¹¹C]2 and [¹¹C]3 were 17 ± 8% and 20 ± 9% (decay-corrected to the end of bombardment, based on [¹¹C]CO₂). The specific activities of [¹¹C]2 and [¹¹C]3 were 42 ± 36 and 37 ± 13 GBq/μmol, respectively. The results of an in vitro binding assay using brown adipose tissue (BAT) homogenate showed that the binding affinity of 2 for CB1 (K_D = 15.3 µM) was much higher than that of 3 (K_D = 26.0 µM). PET studies with [¹¹C]2 showed a high uptake of radioactivity in BAT, which decreased in animals pretreated with AM281 (a selective antagonist for CB1). In conclusion, [¹¹C]2 may be a useful PET ligand for imaging peripheral CB1 in BAT.     

Sigma-1 受体的药理学作用及相关药物研究进展

常见的神经系统疾病如药物成瘾及其导致的神经毒性、神经精神疾病、神经退行性疾病等,严重影响人类健康与正常生活。而临 床上现有的相关治疗药物往往会导致锥体外系反应等副作用,且用药后治疗不够彻底,疾病易复发,因此,开发新靶点及新型有效、安 全的相关治疗药物,迫在眉睫。Sigma-1 受体是一种受体型分子伴侣,参与多种神经传导系统的调节,可与多种精神类药物结合,有望 成为神经系统调节药物的重要靶点。研究发现,有些小分子配体对 Sigma-1 受体具有良好的亲和力,在药物成瘾、精神分裂症、抑郁症、 阿尔茨海默病等疾病中显示出较好疗效。对 Sigma-1 受体的药理学作用以及相关小分子配体药物的研发作一综述。     

Synthesis and biological evaluation of 18F labeled fluoro-oligo-ethoxylated 4-benzylpiperazine derivatives for sigma-1 receptor imaging

We report the synthesis and evaluation of a series of fluoro-oligo-ethoxylated 4-benzylpiperazine derivatives as potential σ₁ receptor ligands. In vitro competition binding assays showed that 1-(1,3-benzodioxol-5-ylmethyl)-4-(4-(2-fluoroethoxy)benzyl)piperazine (6) exhibits low nanomolar affinity for σ₁ receptors (K_i = 1.85 ± 1.59 nM) and high subtype selectivity (σ₂ receptor: K_i = 291 ± 111 nM; K_iσ₂/K_iσ₁ = 157). [¹⁸F]6 was prepared in 30–50% isolated radiochemical yield, with radiochemical purity of >99% by HPLC analysis after purification, via nucleophilic ¹⁸F^? substitution of the corresponding tosylate precursor. The log D_{pH 7.4} value of [¹⁸F]6 was found to be 2.57 ± 0.10, which is within the range expected to give high brain uptake. Biodistribution studies in mice demonstrated relatively high concentration of radiotracers in organs known to contain σ₁ receptors, including the brain, lungs, kidneys, heart, and spleen. Administration of haloperidol 5 min prior to injection of [¹⁸F]6 significantly reduced the concentration of radiotracers in the above-mentioned organs. The accumulation of radiotracers in the bone was quite low suggesting that [¹⁸F]6 is relatively stable to in vivo defluorination. The ex vivo autoradiography in rat brain showed high accumulation of radiotracers in the brain areas known to possess high expression of σ₁ receptors. These findings suggest that [¹⁸F]6 is a suitable radiotracer for imaging σ₁ receptors with PET in vivo.     

Isosteric N-arylpiperazine replacements in a series of dihydropyridine NPY1 receptor antagonists

4-Amino-N-arylpiperidines serve as effective bioisosteres for N-arylpiperazines in the series of dihydropyridine NPY₁ receptor antagonists. These were prepared by a ZnCl₂-mediated reductive amination reaction between elaborated primary amines, 2 or 5, and 4-arylpiperidones.     

Overexpression of sigma-1 receptor inhibits ADAM10 and ADAM17 mediated shedding in vitro

The sigma-1 receptor is a molecular chaperone protein highly enriched in the brain. Recent studies linked it to many diseases, such as drug addition, Alzheimer’s disease, stroke, depression, and even cancer. Sigma-1 receptor is enriched in lipid rafts, which are membrane microdomains essential in signaling processes. One of those signaling processes is ADAM17- and ADAM10-dependent ectodomain shedding. By using an alkaline phosphatase tagged substrate reporter system, we have shown that ADAM10-dependent BTC shedding was very sensitive to both membrane lipid component change and sigma-1 receptor agonist DHEAS treatment while ADAM17-dependent HB-EGF shedding was not; and overexpression of sigma-1 receptor diminished ADAM17- and ADAM10-dependent shedding. Our results indicate that sigma-1 receptor plays an important role in modifying the function of transmembrane proteases.     

Syntheses and in vitro evaluation of decalinvesamicol analogues as potential imaging probes for vesicular acetylcholine transporter (VAChT)

A series of vesamicol analogues, o-iodo-trans-decalinvesamicol (OIDV) or o-bromo-trans-decalinvesamicol (OBDV), were synthesized and their affinities to vesicular acetylcholine transporter (VAChT) and σ receptors (σ-1, σ-2) were evaluated by in vitro binding assays using rat cerebral or liver membranes. OIDV and OBDV showed greater binding affinity to VAChT (K(i)=20.5±5.6 and 13.8±1.2nM, respectively) than did vesamicol (K(i)=33.9±18.1nM) with low affinity to σ receptors. A saturation binding assay in rat cerebral membranes revealed that [(125)I]OIDV had a single high affinity binding site with a K(d) value of 1.73nM and a B(max) value of 164.4fmol/mg protein. [(125)I]OIDV revealed little competition with inhibitors, which possessed specific affinity to each σ (σ-1 and σ-2), serotonin (5-HT(1A) and 5-HT(2A)), noradrenaline, and muscarinic acetylcholine receptors. In addition, BBB penetration of [(125)I]OIDV was verified in in vivo. The results of the binding studies indicated that OIDV and OBDV had great potential to be VAChT imaging probes with high affinity and selectivity.     

Pannexin-1 as a potentiator of ligand-gated receptor signaling

Pannexins are a class of plasma membrane spanning proteins that presumably form a hexameric, non-selective ion channel. Although similar in secondary structure to the connexins, pannexins notably do not form endogenous gap junctions and act as bona fide ion channels. The pannexins have been primarily studied as ATP-release channels, but the overall diversity of their functions is still being elucidated. There is an intriguing theme with pannexins that has begun to develop. In this review we analyze several recent reports that converge on the idea that pannexin channels (namely Panx1) can potentiate ligand-gated receptor signaling. Although the literature remains sparse, this emerging concept appears consistent between both ionotropic and metabotropic receptors of several ligand families.     

Pannexin-1 as a potentiator of ligand-gated receptor signaling

Pannexins are a class of plasma membrane spanning proteins that presumably form a hexameric, non-selective ion channel. Although similar in secondary structure to the connexins, pannexins notably do not form endogenous gap junctions and act as bona fide ion channels. The pannexins have been primarily studied as ATP-release channels, but the overall diversity of their functions is still being elucidated. There is an intriguing theme with pannexins that has begun to develop. In this review we analyze several recent reports that converge on the idea that pannexin channels (namely Panx1) can potentiate ligand-gated receptor signaling. Although the literature remains sparse, this emerging concept appears consistent between both ionotropic and metabotropic receptors of several ligand families.     

Design, synthesis and evaluation of bicyclic benzamides as novel 5-HT1F receptor agonists

Several fused bicyclic systems have been investigated to serve as the core structure of potent and selective 5-HT_1F receptor agonists. Replacement of the indole nucleus in 2 with indazole and ‘inverted’ indazole provided more potent and selective 5-HT_1F receptor ligands. Indoline and 1,2-benzisoxazole systems also provided potent 5-HT_1F receptor agonists, and the 5-HT_1A receptor selectivity of the indoline- and 1,2-benzisoxazole-based 5-HT_1F receptor agonists could be improved with modification of the benzoyl moiety of the benzamides. Through these studies, we found that the inherent geometries of the templates, not the nature of hybridization of the linking atom, were important for the 5-HT_1F receptor recognition.     

Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor

Chloroquine, a widely used anti-malarial and anti-rheumatoid agent, has been reported to induce apoptotic and non-apoptotic cell death. Accumulating evidence now suggests that chloroquine can sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis by inhibiting autophagy. However, chloroquine is reported to induce GM1 ganglioside accumulation in cultured cells at low μM concentrations and prevent damage to the blood brain barrier in mice. It remains unknown whether chloroquine has neuroprotective properties at concentrations below its reported ability to inhibit lysosomal enzymes and autophagy. In the present study, we demonstrated that chloroquine protected mouse hippocampal HT22 cells from glutamate-induced oxidative stress by attenuating production of excess reactive oxygen species. The concentration of chloroquine required to rescue HT22 cells from oxidative stress was much lower than that sufficient enough to induce cell death and inhibit autophagy. Chloroquine increased GM1 level in HT22 cells at low μM concentrations but glutamate-induced cell death occurred before GM1 accumulation, suggesting that GM1 induction is not related to the protective effect of chloroquine against glutamate-induced cell death. Interestingly, BD1047 and NE-100, sigma-1 receptor antagonists, abrogated the protective effect of chloroquine against glutamate-induced cell death and reactive oxygen species production. In addition, cutamesine (SA4503), a sigma-1 receptor agonist, prevented both glutamate-induced cell death and reactive oxygen species production. These findings indicate that chloroquine at concentrations below its ability to inhibit autophagy and induce cell death is able to rescue HT22 cells from glutamate-induced cell death by reducing excessive production of reactive oxygen species through sigma-1 receptors. These results suggest potential use of chloroquine, an established anti-malarial agent, as a neuroprotectant against oxidative stress, which occurs in a variety of neurodegenerative diseases.     

Synthesis of radioiodine-labeled 2-phenylethyl 1-thio-beta-D-galactopyranoside for imaging of LacZ gene expression

A potent inhibitor of beta-galactosidase (EC 3.2.1.23), 2-phenylethyl 1-thio-beta-D-galactopyranoside (PETG), was radioiodinated for noninvasive imaging of LacZ gene expression. In order to introduce radioiodine to the phenyl ring of PETG, 2-(4-bromophenyl)ethanethiol was prepared and attached to the C-1 position of beta-D-galactose pentaacetate under conditions that resulted in the exclusive formation of the beta anomer. The bromo group of PETG was converted to the tributylstannyl group where radioiododemetallation was carried out. Radioiodine-labeled PETG tetraacetate was purified by HPLC, which can be used as a prodrug for biological evaluation or hydrolyzed to 2-(4-[123I/125I]iodophenyl)ethyl 1-thio-beta-D-galactopyranoside ([123I/125I]7) under basic conditions. The resulting radioiodine-labeled PETG was obtained in overall 62% radiochemical yield (decay-corrected) and with specific activity of 46-74 GBq/micromol.     

Imatinib analogs as potential agents for PET imaging of Bcr-Abl and c-KIT expression at a kinase level

We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT receptor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [¹⁸F]-labeled STI-571 was prepared with high specific activity (75 GBq/μmol) by nucleophilic displacement and an average radiochemical yield of 12%. [¹³¹I]-labeled STI-571 was prepared with high purity (>95%) and an average radiochemical yield of 23%. The uptake rates of [¹⁸F]-STI-571 in K562 cells expressing Abl and in U87WT cells overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71 (confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [¹⁸F]-STI-571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast.     

Design,synthesis and evaluation of benzoisothiazolones as selective inhibitors of PHOSPHO1

We report the discovery and characterization of a series of benzoisothiazolone inhibitors of PHOSPHO1, a newly identified soluble phosphatase implicated in skeletal mineralization and soft tissue ossification abnormalities. High-throughput screening (HTS) of a small molecule library led to the identification of benzoisothiazolones as potent and selective inhibitors of PHOSPHO1. Critical structural requirements for activity were determined, and the compounds were subsequently derivatized and measured for in vitro activity and ADME parameters including metabolic stability and permeability. On the basis of its overall profile the benzoisothiazolone analogue 2q was selected as MLPCN probe ML086.