Phenylalanine-Derived ß-Lactam TRPM8 Modulators. Configuration Effect on the Antagonist Activity.

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a Ca2+ non-selective ion channel implicated in a variety of pathological conditions, including cancer, inflammatory and neuropathic pain. In previous works we identified a family of chiral, highly hydrophobic ß-lactam derivatives, and began to intuit a possible effect of the stereogenic centers on the antagonist activity. To investigate the influence of configuration on the TRPM8 antagonist properties, here we prepare and characterize four possible diastereoisomeric derivatives of 4-benzyl-1-[(3'-phenyl-2'-dibenzylamino)prop-1'-yl]-4-benzyloxycarbonyl-3-methyl-2-oxoazetidine. In microfluorography assays, all isomers were able to reduce the menthol-induced cell Ca2+ entry to larger or lesser extent. Potency follows the order 3R,4R,2'R > 3S,4S,2'R ≅ 3R,4R,2'S > 3S,4S,2'S, with the most potent diastereoisomer showing a half inhibitory concentration (IC50) in the low nanomolar range, confirmed by Patch-Clamp electrophysiology experiments. All four compounds display high receptor selectivity against other members of the TRP family. Furthermore, in primary cultures of rat dorsal root ganglion (DRG) neurons, the most potent diastereoisomers do not produce any alteration in neuronal excitability, indicating their high specificity for TRPM8 channels. Docking studies positioned these ß-lactams at different subsites by the pore zone, suggesting a different mechanism than the known N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist.

Influence of freezing on the bioaccessibility of beetroot (Beta vulgaris) bioactive compounds during in vitro gastric digestion.

BACKGROUND: The effect of freezing (in liquid nitrogen at -196 °C , at -80 °C and at -20 °C) on the food matrix structure and the consequent effect on the bioaccessibility of total polyphenol content (TPC) and antioxidant activity (AA) of beetroot was investigated by an in vitro digestion method. The in vitro digestion involved incubation in simulated saliva followed by incubation in simulated gastric juice. Bioaccessibility of bioactive compounds was evaluated by measuring the TPC and the AA during in vitro digestion in both the beetroot matrix and the gastric juice. RESULTS: Release of TPC and AA of beetroot was higher in frozen samples (55 ± 5% for TPC and 68 ± 5% for AA) than in the fresh beetroot sample (∼46% for TPC and ∼53% for AA). The mass transfer kinetics were described by using a Weibull model with mean relative errors lower than 8.5% contributing to a better understanding of the influence of freezing on the bioaccessibility of bioactive compounds. CONCLUSIONS: From the treatments tested in this study, freezing with liquid nitrogen resulted in the greatest increase in bioaccessibility of the antioxidant and phenolic compounds of beetroots. © 2018 Society of Chemical Industry.