The enzyme pseudooxynicotine amine oxidase from Pseudomonas putida S16 is not an oxidase, but a dehydrogenase.

The soil-dwelling bacterium Pseudomonas putida S16 can survive on nicotine as its sole carbon and nitrogen source. The enzymes nicotine oxidoreductase (NicA2) and pseudooxynicotine amine oxidase (Pnao), both members of the flavin-containing amine oxidase family, catalyze the first two steps in the nicotine catabolism pathway. Our laboratory has previously shown that, contrary to other members of its enzyme family, NicA2 is actually a dehydrogenase that uses a cytochrome c protein (CycN) as its electron acceptor. The natural electron acceptor for Pnao is unknown; however, within the P. putida S16 genome, pnao forms an operon with cycN and nicA2, leading us to hypothesize that Pnao may also be a dehydrogenase that uses CycN as its electron acceptor. Here we characterized the kinetic properties of Pnao and show that Pnao is poorly oxidized by O2, but can be rapidly oxidized by CycN, indicating that Pnao indeed acts as a dehydrogenase that uses CycN as its oxidant. Comparing steady-state kinetics with transient kinetic experiments revealed that product release primarily limits turnover by Pnao. We also resolved the crystal structure of Pnao at 2.60 Å, which shows that Pnao has a similar structural fold as NicA2. Furthermore, rigid-body docking of the structure of CycN with Pnao and NicA2 identified a potential conserved binding site for CycN on these two enzymes. Taken together, our results demonstrate that although Pnao and NicA2 show a high degree of similarity to flavin containing amine oxidases that use dioxygen directly, both enzymes are actually dehydrogenases.

Nazartinib for treatment-naive EGFR-mutant non-small cell lung cancer: Results of a phase 2, single-arm, open-label study.

INTRODUCTION: Nazartinib, a novel third-generation EGFR-tyrosine kinase inhibitor, previously demonstrated antitumor activity and manageable safety in patients with EGFR-mutant advanced non-small cell lung cancer (NSCLC) who received ≤ 3 prior lines of systemic therapy. Herein, we report phase 2 efficacy and safety of first-line nazartinib. METHODS: This single-arm, open-label, global study enrolled treatment-naive adult patients with stage IIIB/IV NSCLC harboring EGFR-activating mutations (eg, L858R and/or ex19del). Patients with neurologically stable and controlled brain metastases were also eligible. Patients received oral nazartinib 150 mg once daily. The primary endpoint was Blinded Independent Review Committee (BIRC)-assessed overall response rate (ORR) per RECIST v1.1. RESULTS: Forty-five patients received ≥ 1 dose of nazartinib. The median follow-up time from enrollment to data cutoff (November 1, 2019) was 30 months (range: 25-34). The BIRC-assessed ORR was 69% (95% CI, 53-82). The median progression-free survival (PFS) was 18 months (95% CI, 15-not estimable [NE]). The median overall survival was NE. In patients with baseline brain metastases (n = 18), the ORR and median PFS (95% CIs) were 67% (41-87) and 17 months (11-21). Seventeen of 18 patients had brain metastases as non-target lesions; the CNS lesions were absent/normalized in 9 of 17 (53%). Only 2 of 27 patients without baseline brain metastases developed new brain metastases postbaseline. Most frequent adverse events (≥ 25%, any grade, all-causality) were diarrhea (47%), maculopapular rash (38%), pyrexia (29%), cough, and stomatitis (27% each). CONCLUSIONS: First-line nazartinib demonstrated promising efficacy, including clinically meaningful antitumor activity in the brain, and manageable safety in patients with EGFR-mutant NSCLC. TRIAL REGISTRATION: ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT02108964.

Metal-Mediated Organocatalysis in Water: Serendipitous Discovery of Aldol Reaction Catalyzed by the [Ru(bpy)2(nornicotine)2]2+ Complex.

The [Ru(bpy)2(Nor)2]2+ complex (Nor = nornicotine) is an efficient catalyst for the aldol reaction of acetone with activated benzaldehydes in a buffered aqueous solution. The metal plays the role of an activator for the nornicotine organocatalyst ligands. The resulting catalytic activity is potentiated by a factor of about 4.5 as compared to free nornicotine. Similar rate enhancements can be achieved by using Zn(II) cations as the activator. The observations are rationalized with the reduced basicity of the pyrrolidine N in nornicotine due to the enhanced electron withdrawal of the metal-complexed pyridyl moiety.

A Simple and High-Throughput LC-MS-MS Method for Simultaneous Measurement of Nicotine, Cotinine, 3-OH Cotinine, Nornicotine and Anabasine in Urine and Its Application in the General Korean Population.

Measuring nicotine metabolites is the most objective method for identifying smoke exposure. Liquid chromatography--tandem mass spectrometry (LC-MS-MS) can measure multiple metabolites and is sensitive enough to detect low concentrations of metabolites. Therefore, we developed a simple and high-throughput method for measuring nicotine, cotinine, trans-3'-hydroxycotinine (3-OH cotinine), nornicotine and anabasine for population-based studies using LC-MS-MS. Each 30 µL of urine sample was diluted with 90 µL of acetonitrile containing five deuterated internal standards. Chromatographic separation used a C18 column, and LC-MS-MS analysis was performed with a multiple reaction monitoring mode. The chromatographic run time for each sample was 6.5 min. The method was validated by evaluating selectivity, interference, limit of detection, lower limit of quantification, precision, accuracy, linearity, extraction recovery, matrix effect and carryover according to guidelines. Our methods required a short preparation time (∼20 min) while simultaneously measuring five markers for smoking status. No endogenous or exogenous interference was found. Our method showed excellent precision and accuracy: within-run coefficient of variation (CV) 2.9-9.4%, between-run CV 4.8-8.7% and bias -10.1 to 5.3%. Linear dynamic ranges were 1-10,000 ng/mL for nicotine, nornicotine and anabasine; 2-5,000 ng/mL for cotinine and 5-15,000 ng/mL for 3-OH cotinine. Extraction recovery was consistent (87-109%) across concentrations. No significant matrix effect or carryover was observed. The validated method was applied to 849 urine samples. In samples from the 125 current smokers, nicotine, cotinine, 3-OH cotinine, nornicotine and anabasine were detected in 97.6, 99.2, 98.4, 96.8 and 87.2%, respectively. No markers were detected in 93.9% of 609 nonsmokers. The overlapping detection of multiple markers made it possible to identify the smoking status even in current smokers with a low concentration of cotinine. Our LC-MS-MS method using a simple sample preparation technique is sensitive and effective for screening of smoking status in the general population.

Synthetic Methods for the Preparation of Conformationally Restricted Analogues of Nicotine.

In the context of naturally occurring nitrogen heterocycles, nicotine is a chiral alkaloid present in tobacco plants, which can target and stimulate nicotinic acetylcholine receptors (nAChRs), a class of ligand-gated ion channels commonly located throughout the human brain. Due to its well-known toxicity for humans, there is considerable interest in the development of synthetic analogues; in particular, conformationally restricted analogues of nicotine have emerged as promising drug molecules for selective nAChR-targeting ligands. In the present mini-review, we will describe the synthesis of the conformationally restricted analogues of nicotine involving one or more catalytic processes. In particular, we will follow a systematic approach as a function of the heteroarene structure, considering: (a) 2,3-annulated tricyclic derivatives; (b) 3,4-annulated tricyclic derivatives; (c) tetracyclic derivatives; and (d) other polycyclic derivatives. For each of them we will also consider, when carried out, biological studies on their activity for specific nAChR subunits.

Nicotinic aspects of the discriminative stimulus effects of arecoline.

Despite the evidence that the muscarinic agonist arecoline is a drug of abuse throughout Southeast Asia, its stimulus characteristics have not been well studied. The goal of this work was to understand more about the mediation of discriminative stimulus effects of arecoline. Arecoline (1.0 mg/kg s.c.) was trained as a discriminative stimulus in a group of eight rats. The ability of various cholinergic agonists and antagonists to mimic or antagonize the discriminative stimulus effects of arecoline and to modify its rate-suppressing effects was evaluated. A muscarinic antagonist, but neither of two nicotinic antagonists, was able to modify the discriminative stimulus effects of arecoline, suggesting a predominant muscarinic basis of arecoline's discriminative stimulus effects in this assay. However, both nicotine itself and two nicotine agonists with selective affinity for the α4ß2* receptor (ispronicline and metanicotine) produced full arecoline-like discriminative stimulus effects in these rats. The discriminative stimulus effects of the selective nicotine agonists were blocked by both the general nicotine antagonist mecamylamine and by the selective α4ß2* antagonist, dihydro-beta-erythroidine (DHßE). Surprisingly, only DHßE antagonized the rate-suppressing effects of the selective nicotine agonists. These data indicate a selective α4ß2* nicotine receptor component to the behavioral effects of arecoline. Although the nicotinic aspects of arecoline's behavior effects could suggest that abuse of arecoline-containing material (e.g. betel nut chewing) is mediated through nicotinic rather than muscarinic actions, further research, specifically on the reinforcing effects of arecoline, is necessary before this conclusion can be supported.

Nicotine-like discriminative and aversive effects of two α4ß2-selective nicotine agonists, ispronicline and metanicotine.

An attempt to determine the receptor selective nature of some of nicotine's behavioral effects was undertaken through the evaluation of the ability of two nicotinic α4ß2*-selective receptor agonists to produce nicotine-like effects and modify rates of responding in a discrimination assay and in an aversive stimulus assay. A group of eight rats was trained to discriminate the presence of 1 mg/kg nicotine base. Another group of 4-6 rats was trained to report the aversive effects of nicotine by selecting a lever that produced one food pellet over a second lever that produced two food pellets and an intravenous injection of nicotine. Ispronicline and metanicotine, two α4ß2*-selective receptor agonists, increased selection of the nicotine-appropriate lever in a dose-related manner, up to a maximum of approximately 75%. The α4ß2*-selective receptor antagonist, dihydro-beta-erythroidine blocked both the discriminative stimulus effects and the rate-suppressing effects of ispronicline, metanicotine, and small, but not large doses of nicotine. The nonselective antagonist, mecamylamine, antagonized the discriminative stimulus effects of each of the three nicotine agonists as well as the rate-decreasing effects of nicotine and metanicotine. Mecamylamine did not modify the rate-decreasing effects of ispronicline. Both ispronicline and metanicotine as well as nicotine were avoided in the drug + food vs. food choice situation. The receptor-selective nature of ispronicline and metanicotine was hereby confirmed in a behavioral assay, as were earlier reports that the discriminative stimulus effects of relatively small doses of nicotine are likely mediated by activity at the α4ß2* nicotine receptor.

Simultaneous Evaluation of a Vaccine Component Microheterogeneity and Conformational Integrity Using Native Mass Spectrometry and Limited Charge Reduction.

Analytical characterization of extensively modified proteins (such as haptenated carrier proteins in synthetic vaccines) remains a challenging task due to the high degree of structural heterogeneity. Native mass spectrometry (MS) combined with limited charge reduction allows these obstacles to be overcome and enables meaningful characterization of a heavily haptenated carrier protein CRM197 (inactivated diphtheria toxin conjugated with nicotine), a major component of a smoking cessation vaccine. The extensive conjugation results in a near-continuum distribution of ionic signal in electrospray ionization (ESI) mass spectra of haptenated CRM197 even after size-exclusion chromatographic fractionation. However, supplementing the ESI MS measurements with limited charge reduction of ionic populations selected within narrow m/z windows gives rise to well-resolved charge ladders, from which both masses and charge states of the ionic species can be readily deduced. Application of this technique to a research-grade material of CRM197/H7 conjugate not only reveals its marginal conformational stability (manifested by the appearance of high charge-density ions in ESI MS) but also establishes a role of the extent of haptenation as a major factor driving the loss of the higher order structure integrity. The unique information provided by native MS used in combination with limited charge reduction provides a strong argument for this technique to become a standard/required tool in the analytical arsenal in the field of biotechnology and biopharmaceutical analysis, where protein conjugates are becoming increasingly common.

Clinical efficacy and safety of nazartinib for epidermal growth factor receptor mutated non-small cell lung cancer: Study protocol for a prospective, multicenter, open-label.

BACKGROUND: Nazartinib is considered a new, permanent, and mutant-selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI). It has a demonstrated efficacy to treat patients experiencing EGFR-mutated non-small cell cancer (NSCLC). The present study aims to explore the clinical efficacy and safety of nazartinib in patients experiencing EGFR-mutated NSCLC. MATERIALS AND METHODS: The present study is a prospective, multicentre, open-label experiment seeking to assess the clinical safety as well as efficacy of nazartinib in patients suffering from EGFR-mutated NSCLC. The study will randomly divide 78 patients into experimental and control groups using a ratio of 1:1. Additionally, the study will treat the experimental group with nazartinib, and the control group with other chemotherapeutic agents. Besides, the study will treat both the experimental and control groups with standard treatment for a period of 14 days and will be followed up at least 24 weeks. Overall response rate is the major endpoint. Accordingly, the minor endpoints will include progression-free survival, response time, overall survival, and adverse events. Statistical analysis will be performed by SPSS 25.0 software. DISCUSSION: The study will investigate the clinical safety and efficacy of nazartinib in patients suffering from EGFR-mutated NSCLC. The anticipated results of the study are expected to provide clinical basis for nazartinib to treat patients suffering from EGFR-mutated NSCLC.

Mechanistic study of L-6-hydroxynicotine oxidase by DFT and ONIOM methods.

L-6-Hydroxynicotine oxidase (LHNO) is a member of monoamine oxidase (MAO) family and catalyzes conversion of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during bacterial degradation of nicotine. Recent studies indicated that the enzyme catalyzes oxidation of carbon-nitrogen bond instead of previously proposed carbon-carbon bond. Based on kinetics and mutagenesis studies, Asn166, Tyr311, and Lys287 as well as an active site water molecule have roles in the catalysis of the enzyme. A number of studies including experimental and computational methods support hydride transfer mechanism in MAO family as a common mechanism in which a hydride ion transfer from amine substrate to flavin cofactor is the rate-limiting step. In this study, we formulated computational models to study the hydride transfer mechanism using crystal structure of enzyme-substrate complex. The calculations involved ONIOM and DFT methods, and we evaluated the geometry and energetics of the hydride transfer process while probing the roles of active site residues. Based on the calculations involving hydride, radical, and polar mechanisms, it was concluded that hydride transfer mechanism is the only viable mechanism for LHNO.

Challenges associated with quantification of selected urinary biomarkers of exposure to tobacco products.

Tobacco use, of which cigarette smoking is the most common, is a global health concern and is directly linked to over 7 million premature deaths annually. Measurement of the levels of tobacco-related biomarkers in biological matrices reflects human exposure to the chemicals in tobacco products. Nicotine, nicotine metabolites, anatabine, and anabasine are specific to tobacco and nicotine containing products. However, as nicotine and its metabolites are ubiquitous in the environment, background contamination during sample preparation can occur, making the quantification of target analytes challenging. The main purpose of the present study was to examine quality control measures needed in the determination of urinary nicotine, nicotine metabolites, anatabine, and anabasine. Urine samples (n = 75) and NIST standard reference materials SRM 3671 and SRM 3672 were analysed. A one-step extraction procedure using cold acetone was used in this study, which involved no additional clean up. The blank matrices investigated included synthetic urine prepared with HPLC-grade water, synthetic urine prepared with Milli-Q water, and bovine urine. By adopting strategies for minimizing the background levels, very low detection limits for all the target analytes ranging from 0.025 ng/mL for 3-hydroxycotinine to 0.634 ng/mL for nicotine, were achieved. Recoveries ranged between 67% and 118% with RSD values below 20%. Intra-day and inter-day precisions were in the range of 1.1-11.7% and 4.8-25.2%, respectively. The levels of all target analytes were higher in daily smokers than in non-smokers, with the largest difference observed for 3-hydroxycotinine. No difference was observed in the levels of target analytes between individuals who were former smokers, who never smoked or who were exposed to environmental tobacco smoke (ETS), except for total nicotine equivalents (TNE), which was significantly higher in non-smokers exposed to environmental tobacco smoke compared with study participants who never smoked. The results obtained from SRM 3671 and SRM 3672 could inform a potential certification of additional biomarkers of exposure to tobacco products in those standard reference materials.

Identification and characterization of Nornicotine degrading strain Arthrobacter sp. NOR5.

Nornicotine, the primary nicotine metabolite that is formed through demethylation of nicotine in the genus Nicotiana tabacum L. Nornicotine is not only a precursor of tobacco-specific nitrosamine N-nitrosonornicotine but also have detrimental effects to human health. Till now, information on the biotransformation of nornicotine is limited. Herein, we identified and characterized a bacterium Arthrobacter sp. strain NOR5, utilized nornicotine as the sole of carbon and energy source, and degraded 500 mg/L nornicotine completely within 60 h under the optimum conditions of pH 7.0 and 30 °C. In this study, we not only identified previously reported intermediate metabolites such as 6-OH-nornicotine, 6-OH-mysomine, 6-OH-pseudooxy-nornicotine (6HPONor) but also identified a new intermediate metabolite 2,6-di-OH-pseudooxy-nornicotine (2,6DHPONor) by UV spectroscopy and liquid chromatography coupled with time of flight mass spectrometry. About half of 6HPONor could be transformed into 2,6DHPONor that was identified as a novel catabolic intermediate of nornicotine. By the addition of an electron acceptor 2,6-dichlorophenolindophenol (DCIP), the cell-free extract exhibited inducible 6HPONor dehydrogenase activity at 179 ± 60 mU/mg that could convert 6HPONor to 2,6DHPONor. Our study demonstrated that Arthrobacter sp. strain NOR5 has a high potential to degrade the nornicotine completely.

LC/MS analysis of three-dimensional model cells exposed to cigarette smoke or aerosol from a novel tobacco vapor product.

A novel tobacco vapor product (NTV) contains tobacco leaves and generates nicotine-containing aerosols using heating elements. Subchronic biological effects have been evaluated previously using three-dimensional bronchial epithelial model cells by repeated exposure to cigarette smoke (CS) and the NTV aerosols; however, the intracellular exposure characteristics have not been studied in detail. In this study, cells were initially exposed to an aqueous extract (AqE) of cigarette smoke (CS) at two concentration levels, and the cell lysate underwent untargeted analysis by LC-high resolution mass spectrometry to determine the exogenous compounds present in the cells. Among the thousands of peaks detected, four peaks showed a CS-dependency, which were reproducibly detected. Two of the peaks were nicotine and nicotine N-oxide, and the other two putative compounds were myosmine and norharman. The cells were then exposed to an AqE of CS in various combinations of exposure and post-exposure culture durations. Post-exposure culturing of cells with fresh medium markedly decreased the peak areas of the four compounds. The in-vitro switching study of CS to NTV aerosols was investigated by intermittently exposing cells to an AqE of CS four times, followed by exposure to either an AqE of CS, NTV aerosol or medium another four times. Switching to NTV reduced myosmine and norharman levels, which are known CS constituents. The results indicate that extracellular compounds inside cells reflect the exposure state outside cells. Thus, monitoring functional changes to cells in these exposure experiments is feasible.

Analysis of Nicotine and Non-nicotine Tobacco Constituents in Aqueous Smoke/Aerosol Extracts by UHPLC and Ultraperformance Convergence Chromatography-Tandem Mass Spectrometry.

The non-nicotine constituents of tobacco may alter the reinforcing effects of nicotine, but the quantitative and qualitative profiles of these chemicals in tobacco products such as electronic cigarettes (e-cigarettes), cigars, and waterpipe tobacco are not well characterized. The objective of this work was to develop and validate analytical methods to utilize saline both as an extraction solvent for smoke condensates from cigarettes, little cigars, and waterpipe tobacco and aerosols from e-cigarettes and as a delivery vehicle of nicotine and non-nicotine constitents for nonclinical pharmacological studies. Ultrahigh-performance liquid chromatography was used to analyze nicotine and acetaldehyde, and a novel ultraperformance convergence chromatography-tandem mass spectrometry method was developed to analyze anabasine, anatabine, cotinine, myosmine, nornicotine, harmane, and norharmane. Linearity was confirmed for each standard curve with correlation coefficients (r) ≥ 0.99, and relative errors (RE) for the standards were ≤±10% over the calibration ranges. Method validation was performed by preparing triplicate samples in saline to mimic the composition and concentration of each analyte in the smoke or aerosol condensate and were used to determine method accuracy and precision. Relative standard deviation values were ≤15% and mean RE ≤15% for each analyte at each concentration level. Selectivity of the methods was demonstrated by the absence of peaks in blank vehicle or diluent samples. Storage stability was assessed over ∼45 days. Precision (%RSD ≤ 13) and recovery (percent of day 0 ≥ 80%) indicated that the saline formulations of all four products could be considered stable for up to ∼45 days at 4-8 °C. Therefore, the use of saline both as an extraction solvent and as a delivery vehicle adds versatility and improved performance in the study of the pharmacological effects of constituents from mainstream smoke and aerosols generated from cigarettes, little cigars, waterpipes, and e-cigarettes.

Biocompatible SPME fibers for direct monitoring of nicotine and its metabolites at ultra trace concentration in rabbit plasma following the application of smoking cessation formulations.

The ultra-trace determination of nicotine and its 4 major metabolites (cotinine, nornicotine, norcotinine and anabasine) from rabbit plasma was achieved by a newly developed solid phase microextraction-liquid chromatography-tandem mass spectrometry method. Extraction of the target analytes was performed with hydrophilic/lipophilic balance-polyacrylonitrile SPME fibers. Dual fiber extraction was necessary to guarantee improved recovery at parts-per-trillion levels. Liquid chromatographic analysis was achieved in a 6-min run using a C18 (1.9 µm C18, 50 mm x 2.1 mm) column with a mobile phase flow rate of 0.4 mL/min. Tandem mass spectrometry was used for detection and quantification in positive electrospray ionization (ESI+) mode for all the targeted analytes. Two stable isotope-labeled internal standards were used for signal correction and accurate quantification. The mass spectrometer with laminar flow ion flux transport, guaranteed improved signal stability, minimal contamination of the ion guide and reproducibility into the first quadrupole analyzer. The method was validated in line with the Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The results met the acceptance criteria as proposed by the FDA: accuracy was tested at 0.35, 10 and 75 µg L - 1 and ranged between 98.3-112.2% for nicotine, 94.1-101.9% for cotinine, 94.7-107.0% for nornicotine, 81.1-107.2% for norcotinine and 94.3-115.2% for anabasine, with precision up to 14.2%. Stability tests indicated that all the targeted analytes were stable in the desorption solution for at least 1 week. LOQs ranged from 0.05 to 1 µg L-1. The method was successfully applied to analyze plasma samples obtained from rabbits following transdermal application of a smoking cessation formulation loaded with solid lipid nanoparticles containing a nicotine-stearic acid conjugate.

Chiral determination of nornicotine, anatabine and anabasine in tobacco by achiral gas chromatography with (1S)-(-)-camphanic chloride derivatization: Application to enantiomeric profiling of cultivars and curing processes.

The alkaloid enantiomers are well-known to have different physiological and pharmacological effects, and to play an important role in enantioselectivity metabolism with enzymes catalysis in tobacco plants. Here, we developed an improved method for simultaneous and high-precision determination of the individual enantiomers of nornicotine, anatabine and anabasine in four tobacco matrices, based on an achiral gas chromatography-nitrogen phosphorus detector (GCNPD) with commonly available Rtx-200 column using (1S)-(-)-camphanic chloride derivatization. The method development consists of the optimization of extraction and derivatization, screening of achiral column, analysis of the fragmentation mechanisms and evaluation of matrix effect (ME). Under the optimized experimental conditions, the current method exhibited excellent detection capability for the alkaloid enantiomers, with coefficients of determination (R2) > 0.9989 and normality test of residuals P > 0.05 in linear regression parameters. The ME can be neglected for the camphanic derivatives. The limit of detection (LOD) and limit of quantitation (LOQ) ranged from 0.087 to 0.24 µg g - 1 and 0.29 to 0.81 µg g - 1, respectively. The recoveries and within-laboratory relative standard deviations (RSDR) were 94.3%~104.2% and 0.51%~3.89%, respectively. The developed method was successfully applied to determine the enantiomeric profiling of cultivars and curing processes. Tobacco cultivars had a significant impact on the nornicotine, anatabine, anabasine concentration and enantiomeric fraction (EF) of (R)-nornicotine, whereas the only significant change induced by the curing processes was an increase in the EF of (R)-anabasine.

Dealkenylative Alkenylation: Formal σ-Bond Metathesis of Olefins.

The dealkenylative alkenylation of alkene C(sp3 )-C(sp2 ) bonds has been an unexplored area for C-C bond formation. Herein 64 examples of ß-alkylated styrene derivatives, synthesized through the reactions of readily accessible feedstock olefins with ß-nitrostyrenes by ozone/FeII -mediated radical substitutions, are reported. These reactions proceed with good efficiencies and high stereoselectivities under mild reaction conditions and tolerate an array of functional groups. Also demonstrated is the applicability of the strategy through several synthetic transformations of the products, as well as the syntheses of the natural product iso-moracin and the drug (E)-metanicotine.

Effects of nicotine, nornicotine and cotinine, alone or in combination, on locomotor activity and ultrasonic vocalization emission in adult rats.

RATIONALE: The behavioral effects of the nicotine metabolites nornicotine and cotinine have not been investigated extensively. OBJECTIVES: To evaluate the effects of nicotine, cotinine, and nornicotine, given alone or in combination, on locomotor activity and emission of ultrasonic vocalizations in male adult rats. METHODS: Rats were first given home cage nicotine injections to make them tolerant to the drug's locomotor depressant effects. On subsequent days, locomotor activity (LMA) and ultrasonic vocalizations were recorded in an open field, for 60 min after challenge injection, using repeated measures designs. In single-drug experiments, subjects were tested with nicotine 0.05-0.4 mg/kg, cotinine 0.03-3 mg/kg, or nornicotine 0.1-10 mg/kg. In drug-combination experiments, saline or nicotine 0.2 mg/kg challenge was preceded by cotinine (0, 0.3, 3 mg/kg) or nornicotine (0, 0.1, 0.3, 1, 3 mg/kg) injection. RESULTS: High doses of nornicotine increased LMA and blunted the locomotor stimulant effect of nicotine. Less consistently, nicotine and high doses of nornicotine decreased the 50-kHz call rate, with no clear evidence of a nornicotine × nicotine interaction. Cotinine, given alone or before nicotine injection, altered neither LMA nor the call rate. No drug altered the relative prevalence of flat vs. trill 50-kHz call subtypes, except that the highest dose of nornicotine promoted flat calls over trills. No drug evoked 22-kHz calls. CONCLUSION: Nornicotine can exert an acute anti-nicotine effect in vivo, as previously reported in vitro. The finding that nicotine did not detectably alter the 50-kHz call profile appears consistent with this drug's mild subjective effects in human subjects.

A Methyl Scan of the Pyrrolidinium Ring of Nicotine Reveals Significant Differences in Its Interactions with α7 and α4ß2 Nicotinic Acetylcholine Receptors.

The two major nicotinic acetylcholine receptors (nAChRs) in the brain are the α4ß2 and α7 subtypes. A "methyl scan" of the pyrrolidinium ring was used to detect differences in nicotine's interactions with these two receptors. Each methylnicotine was investigated using voltage-clamp and radioligand binding techniques. Methylation at each ring carbon elicited unique changes in nicotine's receptor interactions. Replacing the 1'-N-methyl with an ethyl group or adding a second 1'-N-methyl group significantly reduced interaction with α4ß2 but not α7 receptors. The 2'-methylation uniquely enhanced binding and agonist potency at α7 receptors. Although 3'- and 5'-trans-methylations were much better tolerated by α7 receptors than α4ß2 receptors, 4'-methylation decreased potency and efficacy at α7 receptors much more than at α4ß2 receptors. Whereas cis-5'-methylnicotine lacked agonist activity and displayed a low affinity at both receptors, trans-5'-methylnicotine retained considerable α7 receptor activity. Differences between the two 5'-methylated analogs of the potent pyridyl oxymethylene-bridged nicotine analog A84543 were consistent with what was found for the 5'-methylnicotines. Computer docking of the methylnicotines to the Lymnaea acetylcholine binding protein crystal structure containing two persistent waters predicted most of the changes in receptor affinity that were observed with methylation, particularly the lower affinities of the cis-methylnicotines. The much smaller effects of 1'-, 3'-, and 5'-methylations and the greater effects of 2'- and 4'-methylations on nicotine α7 nAChR interaction might be exploited for the design of new drugs based on the nicotine scaffold. SIGNIFICANCE STATEMENT: Using a comprehensive "methyl scan" approach, we show that the orthosteric binding sites for acetylcholine and nicotine in the two major brain nicotinic acetylcholine receptors interact differently with the pyrrolidinium ring of nicotine, and we suggest reasons for the higher affinity of nicotine for the heteromeric receptor. Potential sites for nicotine structure modification were identified that may be useful in the design of new drugs targeting these receptors.

Structural and functional computational analysis of nicotine analogs as potential neuroprotective compounds in Parkinson disease.

As the mechanism of interaction between nicotinic receptors with nicotine analogs is not yet fully understood, information at molecular level obtained from computational calculations is needed. In this sense, this work is a computational study of eight nicotine analogs, all with pyrrolidine ring modifications over a nicotine-based backbone optimized with B3LYP-D3/aug-cc-pVDZ. A molecular characterization was performed focusing on geometrical parameters such as pseudo-rotation angles, atomic charges, HOMO and LUMO orbitals, reactivity indexes and intermolecular interactions. Three analogs, A2 (3-(1,3-dimethyl-4,5-dihydro-1h-pirazole-5-yl) pyridine), A3 (3-(3-methyl-4,5-dihydro-1H-pyrazol-5-yl)-pyridine) and A8 (5-methyl-3-(pyridine-3-yl)-4,5-dihydroisoxazole), were filtered suggesting putative neuroprotective activity taking into account different reactivity values, such as their lowest hardness: 2.37 eV (A8), 2.43 eV (A2) and 2.56 eV (A3), compared to the highest hardness value found: 2.71 eV for A5 (3-((2S,4R)-4-(fluoromethyl)-1-methylpyrrolidine-2-il) pyridine), similar to the value of nicotine (2.70 eV). Additionally, molecular docking of all 8 nicotine analogs with the α 7 nicotinic acetylcholine receptor (α 7 nAChR) was performed. High values of interaction between the receptor and the three nicotine analogs were obtained: A3 (-7.1 kcal/mol), A2 (-6.9 kcal/mol) and A8 (-6.8 kcal/mol); whereas the affinity energy of nicotine was -6.4 kcal/mol. Leu116 and Trp145 are key residues in the binding site of α 7 nAChR interacting with nicotine analogs. Therefore, based upon these results, possible application of these nicotine analogs as neuroprotective compounds and potential implication at the design of novel Parkinson's treatments is evidenced.