Handling and Analysis of 5-Formyl-, 5,10-Methenyl-, 10-Formyl-, 5-Formimno-, and 5,10-Methylenetetrahydrofolates.

As the principal one-carbon carriers in mammalian biology, tetrahydrofolates are crucial for normal and malignant cells to synthesize and repair DNA and are the target of extensive research, including metabolomics analysis. The susceptibility of tetrahydrofolates to oxidization, as well as the propensity of substituted tetrahydrofolates to chemical degradation, mandates the use of carefully controlled experimental conditions to ensure their integrity. Analytical protocols for LC analysis along with handling and storage conditions for 5-formyl-, 5,10-methenyl-,10-formyl-, 5-formimno-, and 5,10-methylenetetrahydrofolate are described.

Novel Synthesis and Pharmacological Characterization of NOP Receptor Agonist 8-[(1S,3aS)-2,3,3a,4,5,6-Hexahydro-1H-phenalen-1-yl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (Ro 64-6198).

The nociceptin/orphanin FQ opioid peptide (NOP) receptor is a widely expressed GPCR involved in the modulation of pain, anxiety, and motor behaviors. Dissecting the functional properties of this receptor is limited by the lack of systemically active ligands that are brain permeant. The small molecule NOP receptor-selective, full agonist 8-[(1S,3aS)-2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (Ro 64-6198) hydrochloride is an active, brain penetrant ligand, but its difficult and cost-prohibitive synthesis limits its widespread use and availability for animal studies. Here, we detail a more efficient and convenient method of synthesis, and use both in vitro and in vivo pharmacological assays to fully characterize this ligand. Specifically, we characterize the pharmacodynamics of Ro 64-6198 in cAMP and G-protein coupling in vitro and examine, for the first time, the effects of nociceptin/orphanin FQ and Ro 64-6198 in arrestin recruitment assays. Further, we examine the effects of Ro 64-6198 on analgesia, anxiety, and locomotor responses in vivo. This new synthesis and pharmacological characterization provide additional insights into the useful, systemically active, NOP receptor agonist Ro 64-6198.

PVP formulated fullerene (C60) increases Rho-kinase dependent vascular tissue contractility in pregnant Sprague Dawley rats.

Pregnancy is a unique physiological state, in which C60 fullerene is reported to be distributed in both maternal and fetal tissues. Tissue distribution of C60 differs between pregnant and non-pregnant states, presumably due to functional changes in vasculature during pregnancy. We hypothesized that polyvinylpyrrolidone (PVP) formulated C60 (C60/PVP) increases vascular tissue contractility during pregnancy by increasing Rho-kinase activity. C60/PVP was administered intravenously to pregnant and non-pregnant female Sprague Dawley rats. Vascular responses were assessed using wire myography 24h post-exposure. Increased stress generation was observed in uterine artery, thoracic aorta and umbilical vein. Rho-Rho-kinase mediated force maintenance was increased in arterial segments from C60/PVP exposed pregnant rats when compared to PVP exposed rats. Our findings suggest that intravenous exposure to C60/PVP during pregnancy increases vascular tissue contractility of the uterine artery through elements of Rho-Rho-kinase signaling during late stages of pregnancy.

Structure-activity correlations for beta-phenethylamines at human trace amine receptor 1.

A cell line in which RD-HGA16 cells were stably transfected with the hTAAR 1 receptor was created and utilized to carry out a systematic evaluation of a series of beta-phenethylamines. Fair agreement was observed with data obtained for aryl and ethylene chain substituted analogs in an AV12-664 cell line in which hemagglutinin-tagged hTAAR 1 was stably co-expressed with rat G alpha(s). Analogs with multiple substituents as well as analogs with bulky groups were found to be partial agonists. Analogs in which the primary amino group was converted to a secondary or a tertiary amino group by N-methylation were also partial agonists. Comparative Molecular Field Analysis (CoMFA) using the potency data yielded a regression coefficient r(2) of 0.824. The steric field contribution to the model was 61% with the balance (39%) contributed by the electrostatic field. The collective results suggest that increasing steric bulk both at the amino nitrogen, particularly by N-dimethylation, and at the 4-position of the aromatic ring leads to low efficacy ligands.

High specific activity tritium-labeled N-(2-methoxybenzyl)-2,5-dimethoxy-4-iodophenethylamine (INBMeO): a high-affinity 5-HT2A receptor-selective agonist radioligand.

The title compound ([3H]INBMeO) was prepared by an O,O-dimethylation reaction of a t-BOC protected diphenolic precursor using no carrier added tritiated iodomethane in DMF with K(2)CO(3). Removal of the t-BOC protecting group and purification by HPLC afforded an overall yield of 43%, with a radiochemical purity of 99% and specific activity of 164Ci/mmol. The new radioligand was suitable for labeling human 5-HT(2A) receptors in two heterologous cell lines and had about 20-fold higher affinity than [(3)H]ketanserin.