An LPAR 5 -antagonist that reduces nociception and increases pruriception.

Introduction: The G-protein coupled receptor LPAR5 plays a prominent role in LPA-mediated pain and itch signaling. In this study we focus on the LPAR5-antagonist compound 3 (cpd3) and its ability to affect pain and itch signaling, both in vitro and in vivo. Methods: Nociceptive behavior in wild type mice was induced by formalin, carrageenan or prostaglandin E2 (PGE2) injection in the hind paw, and the effect of oral cpd3 administration was measured. Scratch activity was measured after oral administration of cpd3, in mice overexpressing phospholipase A2 ( sPLA 2 tg ), in wild type mice (WT) and in TRPA1-deficient mice (Trpa1 KO). In vitro effects of cpd3 were assessed by measuring intracellular calcium release in HMC-1 and HEK-TRPA1 cells. Results: As expected, nociceptive behavior (induced by formalin, carrageenan or PGE2) was reduced after treatment with cpd3. Unexpectedly, cpd3 induced scratch activity in mice. In vitro addition of cpd3 to HEK-TRPA1 cells induced an intracellular calcium wave that could be inhibited by the TRPA1-antagonist A-967079. In Trpa1 KO mice, however, the increase in scratch activity after cpd3 administration was not reduced. Conclusions: Cpd3 has in vivo antinociceptive effects but induces scratch activity in mice, probably by activation of multiple pruriceptors, including TRPA1. These results urge screening of antinociceptive candidate drugs for activity with pruriceptors.

Small-Molecule Lysophosphatidic Acid Receptor 5 (LPAR5) Antagonists: Versatile Pharmacological Tools to Regulate Inflammatory Signaling in BV-2 Microglia Cells.

Lysophosphatidic acid (LPA) species in the extracellular environment induce downstream signaling via six different G protein-coupled receptors (LPAR1-6). These signaling cascades are essential for normal brain development and function of the nervous system. However, in response to acute or chronic central nervous system (CNS) damage, LPA levels increase and aberrant signaling events can counteract brain function. Under neuro-inflammatory conditions signaling along the LPA/LPAR5 axis induces a potentially neurotoxic microglia phenotype indicating the need for new pharmacological intervention strategies. Therefore, we compared the effects of two novel small-molecule LPAR5 antagonists on LPA-induced polarization parameters of the BV-2 microglia cell line. AS2717638 is a selective piperidine-based LPAR5 antagonist (IC50 0.038 µM) while compound 3 is a diphenylpyrazole derivative with an IC50 concentration of 0.7 µM in BV-2 cells. Both antagonists compromised cell viability, however, at concentrations above their IC50 concentrations. Both inhibitors blunted LPA-induced phosphorylation of STAT1 and STAT3, p65, and c-Jun and consequently reduced the secretion of pro-inflammatory cyto-/chemokines (IL-6, TNFα, IL-1ß, CXCL10, CXCL2, and CCL5) at non-toxic concentrations. Both compounds modulated the expression of intracellular (COX-2 and Arg1) and plasma membrane-located (CD40, CD86, and CD206) polarization markers yet only AS2717638 attenuated the neurotoxic potential of LPA-activated BV-2 cell-conditioned medium towards CATH.a neurons. Our findings from the present in vitro study suggest that the two LPAR5 antagonists represent valuable pharmacological tools to interfere with LPA-induced pro-inflammatory signaling cascades in microglia.

Probing 2H-Indazoles as Templates for SGK1, Tie2, and SRC Kinase Inhibitors.

The broader and systematic application of a novel scaffold is often hampered by the unavailability of a short and reliable synthetic access. We investigated a new strategy for the design and synthesis of an array of N2-substituted aza-2H-indazole derivatives as potential kinase inhibitors. Guided by a rational ligand alignment approach to qualify the so-far underrepresented aza-2H-indazole scaffold, indazoles were connected at the N2 position with a phenyl spacer and an arylsulfonamide or amide linkage. Initial profiling against a panel of 30 kinases confirmed the in silico predicted selectivity bias. A synthesized focused library of 52 different aza-2H-indazole derivatives showed good initial selective inhibition against SGK1, Tie2, and SRC kinases, with the best representatives having IC50 values in the range of 500 nm. In a comparative computational study, these data were analyzed and rationalized in light of docking studies.

A General One-Pot Synthesis of 2H-Indazoles Using an Organophosphorus-Silane System.

A simple and direct approach for the regioselective construction of the privileged 2H-indazole scaffold is described. The developed one-pot strategy involves phospholene-mediated N-N bond formation to access 2H-indazoles. The amount of organophosphorus reagent was minimized by recycling the phospholene oxide with organosilane reductants. Starting from functionalized 2-nitrobenzaldehydes and primary amines, a mild reductive cyclization, involving the use of commercially available phospholene oxide and silanes, delivered a wide variety of substituted 2H-indazoles in good to excellent yields.

Organophosphorus-mediated N-N bond formation: facile access to 3-amino-2H-indazoles.

A convenient and efficient strategy has been devised to access 3-amino-2H-indazole derivatives in two steps from readily available starting materials. The conversion of 2-nitrobenzonitriles to substituted benzamidines followed by an organophosphorus-mediated reductive cyclization and a subsequent N-N bond formation afforded 3-amino-2H-indazoles in good to excellent yields.