α-[Amino(4-aminophenyl)thio]methylene-2-(trifluoromethyl)benzeneacetonitrile; Configurational equilibria in solution.

Inconsistent results have been reported for the effects of the mitogen-activating extracellular kinase (MEK) inhibitor α-[amino(4-aminophenyl)thio]methylene-2-(trifluoromethyl)benzeneacetonitrile (SL 327) on ethanol-induced conditioned place preference (EtOH-CPP). Since such inconsistencies may be due to the configurational composition of administered SL 327, the interconvertibility of the geometric isomers of this class of compounds has been investigated. This study provides conditions for determination of configurational composition of this class of compounds by HPLC and by 1H NMR and reports details of configurational equilibria as a function of medium and time in solution along with solubility data for SL 327 in aqueous DMSO. The results suggest that the apparently inconsistent results reported for CPP-EtOH may be due to the administration of suspension vs. solutions, as well as to different configurational compositions of SL 327.

Distribution and biomarker of carbon-14 labeled fullerene C60 ([(14) C(U)]C60 ) in pregnant and lactating rats and their offspring after maternal intravenous exposure.

A comprehensive distribution study was conducted in pregnant and lactating rats exposed to a suspension of uniformly carbon-14 labeled C60 ([(14) C(U)]C60 ). Rats were administered [(14) C(U)]C60 (~0.2 mg [(14) C(U)]C60 kg(-1) body weight) or 5% polyvinylpyrrolidone (PVP)-saline vehicle via a single tail vein injection. Pregnant rats were injected on gestation day (GD) 11 (terminated with fetuses after either 24 h or 8 days), GD15 (terminated after 24 h or 4 days), or GD18 (terminated after 24 h). Lactating rats were injected on postnatal day 8 and terminated after 24 h, 3 or 11 days. The distribution of radioactivity in pregnant dams was influenced by both the state of pregnancy and time of termination after exposure. The percentage of recovered radioactivity in pregnant and lactating rats was highest in the liver and lungs. Radioactivity was quantitated in over 20 tissues. Radioactivity was found in the placenta and in fetuses of pregnant dams, and in the milk of lactating rats and in pups. Elimination of radioactivity was < 2% in urine and feces at each time point. Radioactivity remained in blood circulation up to 11 days after [(14) C(U)]C60 exposure. Biomarkers of inflammation, cardiovascular injury and oxidative stress were measured to study the biological impacts of [(14) C(U)]C60 exposure. Oxidative stress was elevated in female pups of exposed dams. Metabolomics analysis of urine showed that [(14) C(U)]C60 exposure to pregnant rats impacted the pathways of vitamin B, regulation of lipid and sugar metabolism and aminoacyl-tRNA biosynthesis. This study demonstrated that [(14) C(U)]C60 crosses the placenta at all stages of pregnancy examined, and is transferred to pups via milk.

Distribution and biomarkers of carbon-14-labeled fullerene C60 ([(14) C(U)]C60 ) in female rats and mice for up to 30 days after intravenous exposure.

A comprehensive distribution study was conducted in female rats and mice exposed to a suspension of uniformly carbon-14-labeled C60 ([(14) C(U)]C60 ). Rodents were administered [(14) C(U)]C60 (~0.9 mg kg(-1) body weight) or 5% polyvinylpyrrolidone-saline vehicle alone via a single tail vein injection. Tissues were collected at 1 h and 1, 7, 14 and 30 days after administration. A separate group of rodents received five daily injections of suspensions of either [(14) C(U)]C60 or vehicle with tissue collection 14 days post exposure. Radioactivity was detected in over 20 tissues at all time points. The highest concentration of radioactivity in rodents at each time point was in liver, lungs and spleen. Elimination of [(14) C(U)]C60 was < 2% in urine and feces at any 24 h time points. [(14) C(U)]C60 and [(14) C(U)]C60 -retinol were detected in liver of rats and together accounted for ~99% and ~56% of the total recovered at 1 and 30 days postexposure, respectively. The blood radioactivity at 1 h after [(14) C(U)]C60 exposure was fourfold higher in rats than in mice; blood radioactivity was still in circulation at 30 days post [(14) C(U)]C60 exposure in both species (<1%). Levels of oxidative stress markers increased by 5 days after exposure and remained elevated, while levels of inflammation markers initially increased and then returned to control values. The level of cardiovascular marker von Willebrand factor, increased in rats, but remained at control levels in mice. This study demonstrates that [(14) C(U)]C60 is retained in female rodents with little elimination by 30 days after i.v. exposure, and leads to systemic oxidative stress.

Isolation of Hemiketal and Hydroxy Ketone Tautomers of Synthetic Intermediates Each Affording 18-Hydroxycortisol-1,6,7-d3.

During the synthesis of deuterated 18-hydroxycortisol, two of the synthetic intermediates have been found to exist in tautomeric forms as the acyclic 18-hydroxy 20-ketone and the cyclic 18,20-hemiketal corresponding to the previously identified less polar (L) and more polar (M) forms of C-18 hydroxylated steroids, respectively. Specifically, p-chloranil oxidation of 18-hydroxycortisol-17,21-acetonide afforded two isomers of the 6,7-dehydro analogue; separate catalytic reduction of each isomer under deuterium gave a single isomer of acetonide-protected 18-hydroxycortisol-1,6,7-d3 for each, with the more polar isomer giving a more polar product and the less polar isomer giving a less polar product. The more polar product (corresponding to M) was characterized as 18,20-hemiketal; 18-hydroxycortisol-17,21-acetonide-18,20-hemiketal-1,6,7-d3: in the deuterochloroform solution, it was found to slowly convert to a substance consistent with the hydroxy ketone structure with features resembling those of the isolated less polar isomer (corresponding to L). Deacetonidization of each gave 18-hydroxycortisol as a single product, which was characterized as the 18,20-hemiketal. The issues associated with the existence of 18-hydroxysteroids as hydroxy ketones and hemiketals, both in solution and as isolable solids, are discussed.

Identification of (+)-erythro-mefloquine as an active enantiomer with greater efficacy than mefloquine against Mycobacterium avium infection in mice.

Infection caused by Mycobacterium avium is common in AIDS patients who do not receive treatment with highly active antiretroviral therapy (HAART) or who develop resistance to anti-HIV therapy. Mefloquine, a racemic mixture used for malaria prophylaxis and treatment, is bactericidal against M. avium in mice. MICs of (+)-erythro-, (-)-erythro-, (+)-threo-, and (-)-threo-mefloquine were 32 µg/ml, 32 µg/ml, 64 µg/ml, and 64 µg/ml, respectively. The postantibiotic effect for (+)-erythro-mefloquine was 36 h (MIC) and 41 h for a concentration of 4× MIC. The mefloquine postantibiotic effect was 25 h (MIC and 4× MIC). After baseline infection was established (7 days), the (+)- and (-)-isomers of the diastereomeric threo- and erythro-α-(2-piperidyl)-2,8-bis(trifluoromethyl)-4-quinolinemethanol were individually used to orally treat C57BL/6 bg(+)/bg(+) beige mice that were infected intravenously with M. avium. Mice were also treated with commercial mefloquine and diluent as controls. After 4 weeks of treatment, the mice were harvested, and the number of bacteria in spleen and liver was determined. Mice receiving (+)- or (-)-threo-mefloquine or (-)-erythro-mefloquine had numbers of bacterial load in tissues similar to those of untreated control mice at 4 weeks. Commercial mefloquine had a bactericidal effect. However, mice given the (+)-erythro-enantiomer for 4 weeks had a significantly greater reduction of bacterial load than those given mefloquine. Thus, (+)-erythro-mefloquine is the active enantiomer of mefloquine against M. avium and perhaps other mycobacteria.

Hydrolytic instability of the important orexin 1 receptor antagonist SB-334867: possible confounding effects on in vivo and in vitro studies.

SB-334867 has been an important ligand for the study of the orexin 1 (OX1) receptor due to its high OX1/OX2 selectivity and bioavailability. This ligand however, contains a 2-methylbenzoxazole ring system which is known to undergo hydrolysis, particularly under acidic or basic conditions. The possibility that SB-334867 would be susceptible to significant hydrolysis was evaluated in various formulations and in the solid state. SB-334867 was found to be unstable under conditions commonly employed to prepare stock solutions for in vitro and in vivo studies. In addition, and most alarmingly, the hydrochloride salt of SB-334867 was found to quantitatively decompose to an OX1-inactive product even in the solid state. These findings combine to suggest that studies using SB-334867 (and any other 2-methylbenzoxazole-containing compound) should be performed with great care to avoid the confounding effects of the rapid hydrolytic decomposition of this susceptible structure.

Trace amine-associated receptor 1 is a stereoselective binding site for compounds in the amphetamine class.

The demonstrated ability of amphetamine to functionally activate the rat trace amine associated receptor 1 (rTAAR1) and the subsequent reports of amphetamine activation of TAAR1 in rhesus monkey mouse, human, and human-rat chimeric TAAR1-expressing cell lines has led to speculation as to the role of this receptor in the central nervous system (CNS) responses associated with amphetamine and its analogs. The agonist potencies of ten pairs of enantiomeric amphetamines, including several with known CNS activity, at primate TAAR1 stably expressed in RD-HGA16 cells, robustly indicate the S-configuration to be associated with higher potency. Moreover, the rank order of potency to activate TAAR1 parallels the stimulant action reported by humans for the specific amphetamines. Taken together, these data suggest that TAAR1 is a stereoselective binding site for amphetamine and that activation of TAAR1 is involved in the modulation of the stimulant properties of amphetamine and its congeners. In addition, the observed parallel between hTAAR1 and rhTAAR1 responses supports the rhesus monkey as a highly translational model for developing novel TAAR1-directed compounds as therapeutics for amphetamine-related addictions.

Designer drugs: a medicinal chemistry perspective (II).

During 2012-2018, the clandestine manufacture of new psychoactive substances (NPS) designed to circumvent substance control regulations increased exponentially worldwide, with concomitant increase in fatalities. This review focuses on three compound classes identified as synthetic opioids, synthetic amphetamines, and synthetic cannabinoids and highlights the medicinal chemistry precedents utilized by clandestine laboratories to develop new NPS with increased brain penetration, longer duration of action, and greater potency. Chemical approaches to illicit drug abuse treatment options, particularly for opioid use disorder, are also discussed.

Distribution of carbon-14 labeled C60 ([14C]C60) in the pregnant and in the lactating dam and the effect of C60 exposure on the biochemical profile of urine.

This study was conducted to determine the distribution of [(14)C]C60 in the pregnant rat and fetuses, and in the lactating rat and offspring. Pregnant rats were dosed on gestation day (gd) 15 and lactating rats were dosed on postnatal day (pnd) 8 via tail vein injection with a suspension of approximately 0.3 mg [(14)C]C60 kg(-1) body weight prepared in polyvinylpyrrolidone (PVP), or with PVP alone. Tissues were collected at 24 and 48 h after dosing. The largest portion of the administered dose was detected in the liver (approximately 43%, pregnant dam; approximately 35%, lactating dam) and lung (approximately 25%, lactating dam). Radioactivity (approximately 6%) was distributed to the reproductive tract, placenta and fetuses of the pregnant dam. Lactating rats had radioactivity distributed to the milk (3140 dpm g(-1) tissue, 24 h; 1620 dpm g(-1) tissue, 48 h), and to the pups' GI tract (2.8%, 24 h; 4.4% 48 h) and liver (<1%). Blood radioactivity was significant at 24 h (14-19%) and at 48 h (7%) after dosing; largely accounted for in the plasma fraction. Less that 4% of the dose was recovered in the maternal spleen, heart, brain, urine or feces. Metabolomics analysis of urine indicated that dams exposed to [(14)C]C60 had decreased metabolites derived from the Krebs cycle and increased metabolites derived from the urea cycle or glycolysis, as well as alterations in the levels of some sulfur-containing amino acids and purine/pyrimidine metabolites. This study demonstrated that [(14)C]C60 crosses the placenta and is transmitted to offspring via the dam's milk and subsequently systemically absorbed.

Design, synthesis and biological evaluation of a bi-specific vaccine against α-pyrrolidinovalerophenone (α-PVP) and 3,4-methylenedioxypyrovalerone (MDPV) in rats.

α-PVP (α-pyrrolidinovalerophenone) and MDPV (3,4-methylenedioxypyrovalerone) are potent abused stimulants that are members of the synthetic cathinone class of drugs. Although these drugs are taken with recreational intent, high doses can lead to unintended adverse effects including agitation, cardiovascular effects, sympathomimetic syndromes, hallucinations, and psychoses. One possible treatment is the use of a vaccine to block or attenuate adverse medical effects. These studies report the preparation of a vaccine that generates high affinity antibodies specific for both drugs and the pharmacological testing of this vaccine in male rats. Alkylation of a hydroxy-α-PVP analog with an appropriate thiol-bearing linker afforded the hapten. When hapten-conjugated carrier protein was mixed with adjuvant, the resulting vaccine stimulated production of antibodies in male Sprague Dawley rats that were found to significantly reduce α-PVP- and MDPV-induced hyperlocomotion as well as to significantly reduce the concentrations of MDPV drugs in critical organs. The novel vaccine produced high affinity antibodies against MDPV, (R)-MDPV, (S)-MDPV, and α-PVP. Cross-reactivity testing against nine structurally similar cathinones showed very limited binding, and no binding to off-target endogenous and exogenous compounds. Antibodies generated by this bi-specific vaccine also significantly shortened the duration of locomotor activity induced by both drugs up to a dose of 5.6 mg/kg in male rats.

Amiodarone and its putative metabolites fail to activate wild type hTAAR1.

The ability of amiodarone and its putative metabolites to activate unmodified human trace amine associated receptor 1 (hTAAR1) stably expressed in CHO cell lines was evaluated. Receptor activation was monitored by measuring the accumulation of cAMP, the putative hTAAR1 native second messenger, or calcium mobilization in cells where the receptor was coupled to the promiscuous Gq, Galpha16. Despite literature reports of activation of rodent TAAR1 by these agents, no response was seen in either cell line although robust activation was obtained with the endogenous ligand beta-phenethylamine. These results indicate that TAAR1 activation by amiodarone and its analogs is species specific.

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.

Synthesis and Characterization of the Selective, Reversible PKCß Inhibitor (9 S)-9-[(Dimethylamino)methyl]-6,7,10,11-tetrahydro-9 H,18 H-5,21:12,17-dimethenodibenzo[ e,k]pyrrolo[3,4- h][1,4,13]oxadiazacyclohexadecine-18,20(19 H)-dione, Ruboxistaurin (LY333531).

The demonstrated role of PKCß in  mediating amphetamine-stimulated dopamine efflux, which regulates amphetamine-induced dopamine transporter trafficking and activity, has promoted the research use of the selective, reversible PKCß inhibitor (9 S)-9-[(dimethylamino)methyl]-6,7,10,11-tetrahydro-9 H,18 H-5,21:12,17-dimethenodibenzo[ e,k]pyrrolo[3,4- h][1,4,13]oxadiazacyclohexadecine-18,20(19 H)-dione, ruboxistaurin. Despite the interest in development of ruboxistaurin as the mesylate monohydrate (Arxxant) for the treatment of diabetic retinopathy, macular edema, and nephoropathy, several crucial details in physicochemical characterization were erroneous or missing. This report describes the synthesis and full characterization of ruboxistaurin free base (as a monohydrate), including X-ray crystallography to confirm the absolute configuration, and of the mesylate salt, isolated as a hydrate containing 1.5 mol of water per mole.

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.

Caged Naloxone: Synthesis, Characterization, and Stability of 3- O-(4,5-Dimethoxy-2-nitrophenyl)carboxymethyl Naloxone (CNV-NLX).

The photolabile analogue of the broad-spectrum opioid antagonist naloxone, 3- O-(4,5-dimethoxy-2-nitrophenyl)carboxymethyl naloxone (also referred to as "caged naloxone", 3- O-(α-carboxy-6-nitroveratryl)naloxone, CNV-NLX), has been found to be a valuable biochemical probe. While the synthesis of CNV-NLX is simple, its characterization is complicated by the fact that it is produced as a mixture of α R,5 R,9 R,13 S,14 S and α S,5 R,9 R,13 S,14 S diastereomers. Using long-range and heteronuclear NMR correlations, the 1H NMR and 13C NMR resonances of both diastereomers have been fully assigned, confirming the structures. Monitoring of solutions of CNV-NLX in saline buffer, in methanol, and in DMSO has shown CNV-NLX to be stable for over a week under fluorescent laboratory lights at room temperature. Exposure of such solutions to λ 365 nm from a hand-held UV lamp led to the formation of naloxone and CNV-related breakdown products.

Synthesis and Physicochemical Characterization of the One-Carbon Carrier 10-Formyltetrahydrofolate; A Reference Standard for Metabolomics.

INTRODUCTION: Metabolomics analysis depends on the identification and validation of specific metabolites. This task is significantly hampered by the absence of well-characterized reference standards. The one-carbon carrier 10-formyltetrahydrofolate acts as a donor of formyl groups in anabolism where it is a substrate in formyltransferase reactions in purine biosynthesis. It has been reported as an unstable substance and is currently unavailable as a reference standard for metabolomics analysis. OBJECTIVES: The current study was undertaken to provide the metabolomics community thoroughly characterized 10-formyltetrahydrofolate along with analytical methodology and guidelines for its storage and handling. METHODS: Anaerobic base treatment of 5,10-methenyltetrahydrofolate chloride in the presence of anti-oxidant was utilized to prepare 10-formyltetrahydrofolate. RESULTS: Pure 10-formyltetrahydrofolate has been prepared and physicochemically characterized. Conditions toward maintaining the stability of a solution of the dipotassium salt of 10-formyltetrahydrofolate in solution have been determined. CONCLUSION: This study describes the facile preparation of pure (>90%) 10-formyltetrahydrofolate, its qualitative physicochemical characterization, as well as conditions to enable its use as a reference standard in physiologic samples.

A rapid functional assay for the human trace amine-associated receptor 1 based on the mobilization of internal calcium.

The molecular targets for trace amines (TAs) such as p-tyramine and beta-phenylethylamine have been recently discovered and have been shown to comprise a family of G-protein-coupled receptors based on DNA sequence homologies. These have been termed trace amine-associated receptors (TAARs) because TAs do not activate all of the identified receptors. Because TA may be involved in modulating a variety of behaviors including mood, cognition, and addiction, it is of interest to discover novel ligands for TAARs to probe the role TAs play in brain function. Pharmacophore development for the G(s)-coupled human TAAR1 (hTAAR1) would be aided by a rapid functional assay amenable to screening libraries of compounds. Accordingly, the authors used RD-HGA16 CHO-1 cells from Molecular Devices, which stably express the promiscuous G(q), G(alpha16), to create a cell line stably expressing hTAAR1, thereby coupling receptor activation to the mobilization of internal calcium. They used this cell line to develop a homogenous fluorometric imaging plate reader-based assay using the Calcium 3 fluorescent dye. The EC50 and Emax data obtained for known TAs are in close agreement with previous work using transient hTAAR1 expression systems or a chimeric receptor. These data indicate that the hTAAR1 retains its reported pharmacological characteristics when coupled to G(alpha16).

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.