Synthesis, Characterization and HPLC Analysis of the (1S,2S,5R)-Diastereomer and the Enantiomer of the Clinical Candidate AR-15512.

AR-15512 (formerly known as AVX-012 and WS-12) is a TRPM8 receptor agonist currently in phase 2b clinical trials for the treatment of dry eye. This bioactive compound with menthol-like cooling activity has three stereogenic centers, and its final structure and absolute configuration, (1R,2S,5R), have been previously solved by cryo-electron microscopy. The route of synthesis of AR-15512 has also been reported, revealing that epimerization processes at the C-1 can occur at specific stages of the synthesis. In order to confirm that the desired configuration of AR-15512 does not change throughout the process and to discard the presence of the enantiomer in the final product due to possible contamination of the initial starting material, both the enantiomer of AR-15512 and the diastereomer at the C-1 were synthesized and fully characterized. In addition, the absolute configuration of the (1S,2S,5R)-diastereomer was determined by X-ray crystallographic analysis, and new HPLC methods were designed and developed for the identification of the two stereoisomers and their comparison with the clinical candidate AR-15512.

I2 imidazoline receptor modulation protects aged SAMP8 mice against cognitive decline by suppressing the calcineurin pathway.

Brain aging and dementia are current problems that must be solved. The levels of imidazoline 2 receptors (I2-IRs) are increased in the brain in Alzheimer's disease (AD) and other neurodegenerative diseases. We tested the action of the specific and selective I2-IR ligand B06 in a mouse model of accelerated aging and AD, the senescence-accelerated mouse prone 8 (SAMP8) model. Oral administration of B06 for 4 weeks improved SAMP8 mouse behavior and cognition and reduced AD hallmarks, oxidative stress, and apoptotic and neuroinflammation markers. Likewise, B06 regulated glial excitatory amino acid transporter 2 and N-methyl-D aspartate 2A and 2B receptor subunit protein levels. Calcineurin (CaN) is a phosphatase that controls the phosphorylation levels of cAMP response element-binding (CREB), apoptotic mediator BCL-2-associated agonist of cell death (BAD) and GSK3ß, among other molecules. Interestingly, B06 was able to reduce the levels of the CaN active form (CaN A). Likewise, CREB phosphorylation, BAD gene expression, and other factors were modified after B06 treatment. Moreover, phosphorylation of a target of CaN, nuclear factor of activated T-cells, cytoplasmic 1 (NFATC1), was increased in B06-treated mice, impeding the transcription of genes related to neuroinflammation and neural plasticity. In summary, this I2 imidazoline ligand can exert its beneficial effects on age-related conditions by modulating CaN pathway action and affecting several molecular pathways, playing a neuroprotective role in SAMP8 mice.

Rationally derived inhibitors of hepatitis C virus (HCV) p7 channel activity reveal prospect for bimodal antiviral therapy.

Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or 'viroporins', contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery. We describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.

Bicyclic α-Iminophosphonates as High Affinity Imidazoline I2 Receptor Ligands for Alzheimer's Disease.

Imidazoline I2 receptors (I2-IR), widely distributed in the CNS and altered in patients that suffer from neurodegenerative disorders, are orphans from a structural point of view, and new I2-IR ligands are urgently required for improving their pharmacological characterization. We report the synthesis and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of a new family of bicyclic α-iminophosphonates endowed with relevant affinities for human brain I2-IR. Acute treatment in mice with a selected compound significantly decreased Fas-associated protein with death domain (FADD) in the hippocampus, a key signaling mediator of neuroprotective actions. Additionally, in vivo studies in the familial Alzheimer's disease 5xFAD murine model revealed beneficial effects in behavior and cognition. These results are supported by changes in molecular pathways related to cognitive decline and Alzheimer's disease. Therefore, bicyclic α-iminophosphonates are tools that may open new therapeutic avenues for I2-IR, particularly for unmet neurodegenerative conditions.

Pharmacology and preclinical validation of a novel anticancer compound targeting PEPCK-M.

BACKGROUND: Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate. The mitochondrial isozyme, PEPCK-M is highly expressed in cancer cells, where it plays a role in nutrient stress response. To date, pharmacological strategies to target this pathway have not been pursued. METHODS: A compound embodying a 3-alkyl-1,8-dibenzylxanthine nucleus (iPEPCK-2), was synthesized and successfully probed in silico on a PEPCK-M structural model. Potency and target engagement in vitro and in vivo were evaluated by kinetic and cellular thermal shift assays (CETSA). The compound and its target were validated in tumor growth models in vitro and in murine xenografts. RESULTS: Cross-inhibitory capacity and increased potency as compared to 3-MPA were confirmed in vitro and in vivo. Treatment with iPEPCK-2 inhibited cell growth and survival, especially in poor-nutrient environment, consistent with an impact on colony formation in soft agar. Finally, daily administration of the PEPCK-M inhibitor successfully inhibited tumor growth in two murine xenograft models as compared to vehicle, without weight loss, or any sign of apparent toxicity. CONCLUSION: We conclude that iPEPCK-2 is a compelling anticancer drug targeting PEPCK-M, a hallmark gene product involved in metabolic adaptations of the tumor.

Behavioral and Cognitive Improvement Induced by Novel Imidazoline I2 Receptor Ligands in Female SAMP8 Mice.

As populations increase their life expectancy, age-related neurodegenerative disorders such as Alzheimer's disease have become more common. I2-Imidazoline receptors (I2-IR) are widely distributed in the central nervous system, and dysregulation of I2-IR in patients with neurodegenerative diseases has been reported, suggesting their implication in cognitive impairment. This evidence indicates that high-affinity selective I2-IR ligands potentially contribute to the delay of neurodegeneration. In vivo studies in the female senescence accelerated mouse-prone 8 mice have shown that treatment with I2-IR ligands, MCR5 and MCR9, produce beneficial effects in behavior and cognition. Changes in molecular pathways implicated in oxidative stress, inflammation, synaptic plasticity, and apoptotic cell death were also studied. Furthermore, treatments with these I2-IR ligands diminished the amyloid precursor protein processing pathway and increased Aß degrading enzymes in the hippocampus of SAMP8 mice. These results collectively demonstrate the neuroprotective role of these new I2-IR ligands in a mouse model of brain aging through specific pathways and suggest their potential as therapeutic agents in brain disorders and age-related neurodegenerative diseases.

Neuroprotective Effects of a Structurally New Family of High Affinity Imidazoline I2 Receptor Ligands.

The imidazoline I2 receptors (I2-IRs) are widely distributed in the brain, and I2-IR ligands may have therapeutic potential as neuroprotective agents. Since structural data for I2-IR remains unknown, the discovery of selective I2-IR ligands devoid of α2-adrenoceptor (α2-AR) affinity is likely to provide valuable tools in defining the pharmacological characterization of these receptors. We report the pharmacological characterization of a new family of (2-imidazolin-4-yl)phosphonates. Radioligand binding studies showed that they displayed a higher affinity for I2-IRs than idazoxan, and high I2/α2 selectivity. In vivo studies in mice showed that acute treatments with 1b and 2c significantly increased p-FADD/FADD ratio (an index of cell survival) in the hippocampus when compared with vehicle-treated controls. Additionally, acute and repeated treatments with 2c, but not with 1b, markedly reduced hippocampal p35 cleavage into neurotoxic p25. The present results indicate a neuroprotective potential of (2-imidazolin-4-yl)phosphonates acting at I2-IRs.

First diastereoselective [3 + 2] cycloaddition reaction of diethyl isocyanomethylphosphonate and maleimides.

Bicyclic α-iminophosphonates were prepared via the first diastereoselective silver catalyzed [3 + 2] cycloaddition reaction of diethyl isocyanomethylphosphonate and diversely N-substituted maleimides. The reduction of the resulting imine by catalytic hydrogenation led to cyclic α-aminophosphonates, which are α-aminoester surrogates. The relative stereochemistry of the adducts was confirmed by X-ray crystallographic analysis of . The diastereoselectivity of the cycloaddition reaction was rationalised by theoretical studies.