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

Abstract: 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

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

A bicyclic α-iminophosphonate improves cognitive decline in 5xFAD murine model of neurodegeneration

I2 receptors (I2-IR) are widely distributed in the central nervous system. I2-IR ligands are associated with a neuroprotective effect but, as I2-IR structure remains unknown, the discovery of better and more selective ligands is necessary to understand the pharmacological and molecular implications of I2-IR. Recently, we described a new imidazoline-structure family which showed high affinity and selectivity for I2-IR. In vivo studies in mice indicated a neuroprotective role and revealed beneficial effects in behaviour and cognition with a murine model of neurodegeneration, senescence-accelerated prone mouse (SAMP8). Herein, we report a novel non-imidazoline-structure of bicyclic α-iminophosphonates family with high affinities for I2-IR. In vivo studies in 5X-FAD mice (a transgenic representative model of AD) and SAMP8 mice (a model of neurodegeneration linked to aging) showed an improvement in behaviour and cognition, a reduction of AD hallmarks and of neuroinflammation markers for the mice treated with the lead compound B06. After evaluating several pathways associated with neurodegeneration, we demonstrated that CaN pathway plays a critical role on the neuroprotective effects of I2-IR ligands on SAMP8 mice model. To rule out warnings of the novel family, we calculated DMPK and physicochemical properties for the novel bicyclic α-iminophosphonates. As well, we carried out drug metabolism, safety studies and in vivo pharmacokinetics for lead compound B06. In summary, we present a novel family of I2-IR ligands, its effectiveness in in vivo models and the possible neuroprotective molecular mechanism mediated by them. This highlights that the modulation of I2-IR by bicyclic α-iminophosphonates may open a new therapeutic venue for unmet neurodegenerative conditions

Bicyclic alfa-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 suffered from neurodegenerative disorders, are orphan from the structural point of view and new I2-IR ligands are urgently required for improving their pharmacological characterization. We report the synthesis and 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 the FADD protein in the hippocampus, a key marker in 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

Isocyanide as a key functional group for the synthesis of biologically active compounds

[eng] The distinctive reactivity of isocyanides has been recognised as an advantageous characteristic for the formation of heterocyclic compounds. In the present manuscript new synthetic possibilities are explored involving the isocyanide group as the key point for the development of new reactions that allow the access to unpublished compounds. In particular, some of them show very promising pharmacological properties. According to the different final products accessed, the pharmacological studies and the resulting publications, this dissertation has been classified into six sections. Synthesis of DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepines (PBDs). In all the synthesis of C2-endo-unsaturated PDBs published so far, the C2-endo double bond is installed either when the pre-PDB synthon is already synthesized or when the tricyclic core is already built. Both approaches involve several synthetic steps that, unfortunately, in the most part of the examples constitute a considerable drawback. In order to avoid the cumbersome installation of the double bond after the formation of the tricyclic core, we investigate a new synthetic strategy that relies on the unprecedented use of enantiopure 3-acyl-5-alcoxycarbonyl-2-pyrrolines, synthesized by our group, as key starting materials. Interestingly, the methylketone appendage at the 3-position and the ester substituent at the 5 position are the most suitable to give access to the PBD compounds through known organic transformations. In this section, objectives, discussion and experimental details to access enantiomerically pure PBDs will be commented. First diastereoselective [3+2]cycloaddition reaction of diethyl isocyanomethylphosphonate and maleimides published in Org. Biomol. Chem. 2013, 11, 1640-1649. 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 one of the compounds. The diastereoselectivity of the cycloaddition reaction was rationalised by theoretical studies. Easy Access to (2-imidazolin-4-yl)phosphonates by a microwave assisted multicomponent reaction, published in Tetrahedron, 2015, 71, 2872-2881. An efficient and user-friendly synthetic process involving the combination of multicomponent reaction methodology and microwave heating generates unprecedented (2-imidazolin-4-yl)phosphonates. This strategy presents a silver-catalysed, operationally simple and environmentally friendly transformation without the need of anhydrous atmosphere or additional solvents. Neuroprotective effects of a structurally new family of high affinity imidazoline I2 receptor ligands published in ACS Chem. Neurosci. 2017, 8, 737-742. 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 two of the compounds 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 one compound 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. Novel compounds as high affinity ligands under revision for protection as a patent and potent transference of technology.[cat] La peculiar reactivitat dels isocianoacetats ha estat reconeguda com una avantatjosa característica en la formació de compostos heterocíclics. En aquest manuscrit es presenten noves possibilitat sintètiques utilitzant isocianoacetats com a punt clau per desenvolupar noves reaccions per accedir a nous compostos amb interès farmacològic. L’accés a aquests nous compostos s’ha realitzat a través de dues reaccions principals; les reaccions de cicloaddició i les reaccions multicomponent. En les reaccions de cicloaddició s’ha descrit la preparació de les pirrolbenzodiazepines (PBD), gràcies a la utilització d’un precursor obtingut a través d’una cicloaddició formal [3+2], sota catàlisis cooperativa d’alcaloides de cincona i sals de plata i amb posterior enriquiment de l’excés enantiomèric per self-disproportonation of enantiomers via sublimació. També s’ha descrit la cicloadició [3+2] entre el isocianometilfosfonat de dietil (PhosMic) i maleimides per donar lloc a α-aminofosfonats bicíclics de manera diastereoselectiva, amb la confirmació de la seva estructura relativa per anàlisi de raig X. Pel que fa a les reaccions multimponent s’ha estudiat la reacció entre PhosMic, amines i cetones per donar lloc a les (2-imidazolin-4-il)fosfonats en condicions respectuoses amb el medi ambient, ja que la reacció presenta una elevada economia del àtom, és eficient i transcorre en unes condicions suaus gracies al us del microones. Finalment, la caracterització farmacològica d’aquest compostos en front dels receptor d’imidazolina del tipus 2 (I2-IRs), receptors àmpliament distribuïts en el cervell i relacionats amb activitat neuroprotectora, ha proporcionat nomes eines per poder estudiar-los

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 Aβ 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. Keywords Imidazoline I2 receptors (2-imidazolin-4-yl)phosphonates Behavior Cognition Neurodegeneration Neuroprotection Agin