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Regular practice in tobacco farming recognizes that copper is one of the essential micronutrients in tobacco (Nicotiana tabacum). We report here that copper is present in significant levels in post-harvested, cured tobacco. Transition metals have been shown to catalyze the formation of nitrosamines from amines of which nicotine, the most sought-after component in tobacco, is a tertiary amine. We found, using the nicotine-copper model system, Cu2+ facilitates nitrosamine formation. A mechanistically plausible route for this metal ion-mediated nitrosamine formation from alkaloids is the nicotine-Cu2+ complex formation where Cu2+ preferentially binds to the pyridine nitrogen of nicotine in a pH-dependent fashion. Knowing that tobacco also contains other alkaloids, e.g., anabasine and anatabine, Cu2+ could also enhance the production of nitrosamines from other tobacco-specific alkaloids.

The ability of smoking to reduce body weight serves as motivation for continued smoking. It is unclear to what extent non-nicotine constituents in cigarettes are contributing to the weight-reducing effect of smoking. The purpose of the current study was to examine the effects of nicotine and four minor tobacco alkaloids (nornicotine, cotinine, anatabine, and anabasine) on food intake, one of the key regulators of body weight. In addition, a smokeless tobacco extract (STE) and e-cigarette (EC) refill liquid were used to model the effects of actual tobacco product exposure on food intake. Male Holztman rats were trained to lever press for food pellets during daily 2h sessions in operant chambers. In Experiment 1, the effects of subcutaneous injections of saline, nicotine (0.25 – 1.00 mg/kg), nornicotine (0.50 – 6.00 mg/kg), cotinine (1.00 – 100.00 mg/kg), anatabine (0.25 – 3.00 mg/kg), and anabasine (0.50 – 4.00 mg/kg) were assessed. In Experiment 2, rats from Experiment 1 were used to examine the effects of nicotine, STE, and EC liquid. All alkaloids, except cotinine, produced a dose-dependent reduction in overall food intake. The highest doses of all drugs significantly reduced latency and response rate to obtain the first pellet. At some doses, nicotine, anatabine, and nornicotine reduced food intake within the first 45 minutes without compensatory increases in intake later in the session. STE and EC liquid produced dose dependent decreases in food intake similar to nicotine alone. These data suggest that minor tobacco alkaloids have appetite suppressant effects and warrant further investigation into their effects on body weight, energy intake, and energy expenditure under free-feeding conditions. However, findings with STE and EC liquid suggest that nicotine is the primary constituent in these products to effect food intake, whereas levels of minor alkaloids in these products may be too low to influence food intake.

A rapid high pressure liquid chromatography coupled with tandem mass spectrometry method (LC-MS/MS) was developed for simultaneously determining nicotine (NT) and its derivatives including cotinine (CT), trans-3-hydroxycotinine (HC), nornicotine (NNT), anatabine (AT), anabasine (AB) and amino ketone (4-(methylamino)-1-(3-pyridyl)1-butanone) (AK) as well as two important metabolites isomers 2-hydroxynicotine (2-HN) and 6-hydroxynicotine (6-HN) in tobacco leaves. Different from other methods, 2-HN and 6-HN isomers can be successfully separated and quantified through a combination of amide and C18 columns and optimized multiple reaction monitoring mode of tandem mass spectrometry. The limits of detections (LOD) and quantification (LOQ) of these compounds were 0.02 and 0.06 ng/mL for CT and HC, 0.05 and 0.15 ng/mL for AK and 6-HN, and 0.1 and 0.3 ng/mL for AB, NNT, AT, 2-HN and NT, respectively. Their recoveries ranged from 86.1% for 2-HN to 102.2% for AK by using solvent extraction assisted with ultrasonication. The developed method was validated through determination of the compounds in different regular and genetically modified (for lowering nicotine) tobacco leaves. Conclusively, it is a robust and rapid method for simultaneous quantification of nicotine and its derivatives in tobacco products. The protocol of the method could also be applied in developing analysis method for tracking the toxic substances synthesis and metabolism in tobacco leaves or other herbal plants.

Mitochondria are among the first responders to various stress factors that challenge cell and tissue homeostasis. Various plant alkaloids have been investigated for their capacity to modulate mitochondrial activities. In this study, we used isolated mitochondria from mouse brain and liver tissues to assess nicotine, anatabine and anabasine, three alkaloids found in tobacco plant, for potential modulatory activity on mitochondrial bioenergetics parameters. All alkaloids decreased basal oxygen consumption of mouse brain mitochondria in a dose-dependent manner without any effect on the ADP-stimulated respiration. None of the alkaloids, at 1 nM or 1.25 μM concentrations, influenced the maximal rate of swelling of brain mitochondria. In contrast to brain mitochondria, 1.25 μM anatabine, anabasine and nicotine increased maximal rate of swelling of liver mitochondria suggesting a toxic effect. Only at 1 mM concentration, anatabine slowed down the maximal rate of Ca2+-induced swelling and increased the time needed to reach the maximal rate of swelling. The observed mitochondrial bioenergetic effects are probably mediated through a pathway independent of nicotinic acetylcholine receptors, as quantitative proteomic analysis could not confirm their expression in pure mitochondrial fractions isolated from mouse brain tissue.

Nematode parasites infect ∼2 billion people world-wide. Infections are treated and prevented by anthelmintic drugs, some of which act on nicotinic acetylcholine receptors (nAChRs). There is an unmet need for novel therapeutic agents because of concerns about the development of resistance. We have selected Asu-ACR-16 from a significant nematode parasite genus, Ascaris suum, as a pharmaceutical target and nicotine as our basic moiety (EC50 6.21 ± 0.56 μM, Imax 82.39 ± 2.52%) to facilitate the development of more effective anthelmintics. We expressed Asu-ACR-16 in Xenopus oocytes and used two-electrode voltage clamp electrophysiology to determine agonist concentration-current-response relationships and determine the potencies (EC50s) of the agonists. Here, we describe the synthesis of a novel agonist, (S)-5-ethynyl-anabasine, and show that it is more potent (EC50 0.14 ± 0.01 μM) than other nicotine alkaloids on Asu-ACR-16. Agonists acting on ACR-16 receptors have the potential to circumvent drug resistance to anthelmintics, like levamisole, that do not act on the ACR-16 receptors., Graphical abstract Image 1, Highlights • ACR-16 receptor agonists may overcome resistance to anthelmintics like levamisole. • We modeled the Asu-ACR-16 of Ascaris as a target for novel anthelmintic development. • We synthesized novel nicotinic agonists including (S)-5-ethynyl-anabasine. • We expressed Asu-ACR-16 in Xenopus oocytes to determine agonists potencies. • (S)-5-ethynyl-anabasine is more potent (EC50 0.1 μM) than other nicotinic alkaloids.

Acute ischemic stroke (AIS) causes both central and peripheral inflammation, while activation of α7 nicotinic acetylcholine receptors (nAChRs) provides both central and peripheral anti-inflammatory and anti-apoptotic effects. Here, we provide evidence that 4OH-GTS-21, a selective α7 agonist, produces its therapeutic effects via primarily central sites of action because 4OH-GTS-21 was found equally effective in splenectomized and non-spenectomized rats in the sub-acute phase of ischemic stroke (≤1 week). However, the spleen may boost the therapeutic efficacy of 4OH-GTS-21 in certain behavioral tasks as our data also indicated. In our tests, AIS was modeled by transient middle cerebral artery occlusion (tMCAO). Splenectomy was done 2 weeks before tMCAO. We determined that: 1) Daily 4OH-GTS-21 treatments for 7 days after tMCAO significantly reduced neurological deficits and brain injury in both splenectomized and non-spelenectomized rats demonstrating that the spleen is not required for therapeutic benefits of 4OH-GTS-21; 2) The effects of 4OH-GTS-21 in the adhesive sticker removal test were significantly weaker in splenectomized animals suggesting that the spleen boosts the efficacy of 4OH-GTS-21 in the first week after tMCAO; and 3) Ischemic brain injury was not significantly affected by splenectomy in both vehicle-treated and 4OH-GTS-21-treated animals. These data support the hypothesis that the therapeutic efficacy of sub-chronic (≤1 week) 4OH-GTS-21 primarily originates from central sites of action. These results validate brain availability as a critical factor for developing novel α7 ligands for AIS.

Wastewater-based epidemiology (WBE) has been used to estimate tobacco use in the population. However, the increased use of nicotine replacement therapies and e-cigarettes contributes to the load of nicotine metabolites in wastewater, causing over-estimation of tobacco use if nicotine metabolites were used in WBE back-estimation. This study aims to develop a rapid method for determining the tobacco-specific biomarkers, anabasine and anatabine, in wastewater and to evaluate their in-sewer stability for better estimation of tobacco use by WBE. An enhanced direct injection LC-MS/MS was developed to quantify anabasine and anatabine as well as nicotine biomarkers (nicotine, cotinine and hydroxycotinine). The method was optimal when wastewater was filtered through 0.2 μm RC syringe filters and a pre-conditioned SPE cartridge (Oasis HLB 1 cc, 30 mg) before 50 μL was injected into the LC-MS/MS system. Limits of quantification varied between 2.7 and 54.9 ng/L with recoveries from 76% to 103% for all five compounds. In sewer reactors, anabasine and anatabine were less stable than cotinine and hydroxycotinine. They were more stable in the gravity sewer reactor with

In this study, we determined the pyridine alkaloid content (nicotine, nornicotine, anabasine, anatabine, cotinine, and myosmine) of 58 species and 2 subspecies of the Nicotiana genus by ultra-high-performance liquid chromatography coupled with mass spectrometry. We observed clear correlation between Noctiflorae and Suaveolentes sections and their above average accumulation of anabasine in the genus. In addition, the results demonstrated the presence of not only trace amounts but quantifiable levels of myosmine, an alkaloid previously detected in only minute quantities, in the leaves and roots of 16 species. In this study, analysis of gene expression of 58 species and 2 subspecies from the Nicotiana genus by mRNA sequencing was performed for the first time. Sequencing reads were mapped against annotated genes of a Nicotiana tabacum reference genome and expression values were subsequently calculated. Hierarchical clustering of alkaloid biosynthesis pathway genes and alkaloid content composition revealed patterns clearly segregating Nicotiana sections. Correlation of gene expression with alkaloid accumulation phenotypes was evident, including low putrescine methyltransferase expression for all species in the Suaveolentes section or clear correlation of nicotine demethylase with conversion rates of nicotine to nornicotine in the majority of species. Multiple additional correlations between alkaloid accumulation and gene expression values were identified, which makes this study an important fundament toward future scientific exploration of the Nicotiana genus.

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 (GC NPD) 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.

A reversed-phase HPLC-UV method was developed, optimized, and validated for the separation and quantitation of six target alkaloids from leaves of Nicotiana species (nicotine, nornicotine, anatabine, anabasine, myosmine, and cotinine). A bidentate reversed-phase C18 column was used as stationary phase and an alkaline ammonium formate buffer and acetonitrile as mobile phase. The alkaloids were well separated in a short run time of 13min with mobile phase pH 10.5 and a small gradient of 9-13% acetonitrile, and detected using UV at 260nm. Peak parameters were acceptable for all six closely related alkaloids. The proposed method has enough linearity with correlation coefficient >0.999 within the investigated range for all tested alkaloids. Satisfactory precision was achieved for both intra- and inter-day assay, with RSD less than 2% for all alkaloid standards. Reproducibility was also within the acceptable range of RSD

Anabasine copper(II) and zinc(II) complexes have been studied using experimental methods and quantum-chemical studies. Preferred coordination centers and possible structures of the complexes have been determined. It has been revealed, that the main coordination center for anabasine complexes with both Cu(II) and Zn(II) ions is the piperidine nitrogen atom, while in zinc complexes also the nitrogen atom from the pyridine residue of anabasine is engaged to some small degree. In both systems investigated preferable formation of hydroxo-complexes was observed.

Sustained inhibition of fetal movement in livestock species, induced by several poisonous plants, can result in numerous skeletal-contracture malformations. Lupines are responsible for a condition in cattle referred to as "crooked calf syndrome" that occurs when pregnant cattle graze teratogenic lupines. Similar malformations are also seen in animals poisoned by Conium maculatum (coniine) and Nicotiana glauca (anabasine). A proposed management strategy to limit these types of birth defects includes utilizing an intermittent grazing schedule to allow short durations of grazing lupine-infested areas interrupted by movement to a lupine-free pasture. The objective of this study was to use a goat model to determine if an intermittent schedule of five continuous days on treatment followed by two days off treatment would be sufficient to decrease, or prevent, the incidence of anabasine-induced malformations. The data from this study suggest that, for N. glauca in goats, the intermittent grazing program of five days exposure with two days of non-exposure is insufficient to prevent significant skeletal malformations from occurring. However, this study did demonstrate an inverse relationship between the amount of serum anabasine in the dam and the extent of fetal movement.

The aim of the present study was to determine the vapor pressures of three tobacco alkaloids using the high pressure differential scanning calorimeter based technique, in accordance with a standard test method ASTM E 1782. The measurements were conducted in the pressure range from 15 to 1000 kPa. Temperature ranges for ( l )-nicotine were from 447.7 to 617.8 K, for (±)-anabasine from 478 to 644.3 K and for (−)-cotinine from 537.7 to 669.7 K. Hermetic-type sealable pans with a pierced lid (pinhole diameter 50 μm) and a heating rate of 5 K/min were used. The experimental vapor pressure data were fitted to the Antoine equation. Enthalpies of vaporization and the normal boiling temperatures of the compounds were estimated from the measured data. Comparisons are made with published literature data. In addition, a review of the vapor pressure studies of nicotine and anabasine is given.

Numerous plants are known to be teratogenic in livestock. In addition to causing malformations, several plants can also cause embryonic death. These losses decrease the reproductive efficiency of animals exposed to these plants. The aim of this study was to determine if teratogenic plants such as lupines or tobaccos cause embryonic losses. A goat model using the plant Nicotiana glauca was used in this study, as this model has been used to characterize the mechanism of Lupinus, Conium, and Nicotiana-induced terata. Four groups of goats were dosed from gestational day 1–10, 11–20, 21–30, and 31–40. Goats were evaluated via ultrasound imaging for pregnancy after completion of the dosing regimen and kids were evaluated for malformations at the time of parturition. Overall, there was no evidence from this study that N. glauca (anabasine) at this dose (2 g/kg/day) would cause embryonic losses in goats. However, the dose of N. glauca used in this study was at the lower threshold that would be expected to produce terata. Therefore it is possible that higher doses of anabasine could cause embryonic loss. Further work is also needed to characterize the kinetic profile of anabasine, and other teratogenic alkaloids, in the fetal compartments.

Tobacco smoking has been shown to be quite addictive in people. However, nicotine itself is a weak reinforcer compared to other commonly abused drugs, leading speculation that other factors contribute to the high prevalence of tobacco addiction in the human population. In addition to nicotine, there are over 5000 chemical compounds that have been identified in tobacco smoke, and more work is needed to ascertain their potential contributions to tobacco’s highly addictive properties, or as potential candidates for smoking cessation treatment. In this study, we examined seven non-nicotine tobacco constituent compounds (anabasine, anatabine, nornicotine, myosmine, harmane, norharmane, and tyramine) for their effects on nicotine self-administration behavior in rats. Young adult female Sprague-Dawley rats were allowed to self-administer nicotine (0.03 mg/kg/ 50 μl infusion) under a fixed ratio-1 schedule of reinforcement. Each self-administration session lasted 45 min. Doses of each tobacco constituent compound were administered subcutaneously 10 min prior to the start of each session in a repeated measures, counterbalanced order two times. Anabasine displayed a biphasic dose-effect function. Pretreatment with 0.02 mg/kg anabasine resulted in a 25% increase in nicotine self-administration, while 2.0 mg/kg of anabasine reduced nicotine infusions per session by over 50%. Pretreatment with 2.0 mg/kg anatabine also significantly reduced nicotine self-administration by nearly half. These results suggest that some non-nicotine tobacco constituents may enhance or reduce nicotine’s reinforcing properties. Also, depending upon the appropriate dose, some of these compounds may also serve as potential smoking cessation agents.

Within the traditional pharmacopeia, tobacco (Nicotiana spp.) is often cited as an efficient pesticide. This activity is generally attributed to nicotine, but tobacco plants contain other alkaloids that could potentially contribute to this effect. In this study, we tested methanolic extracts of N. glutinosa, N. glauca, N. debneyi, and N. tabacum (putrescine N-methyltransferase line, burley TN90 and Stella, Virginia ITB 683 and K326), selected according to alkaloid content. Their antiparasitic activity was evaluated in bioassays against adult fleas (Ctenocephalides felis), blowfly (Lucilia cuprina) larvae, nematodes (Caenorhabditis elegans), and ticks (Rhipicephalus sanguineus larvae and adults, Ixodes ricinus nymphs). None of the extracts killed fleas and blowfly larvae effectively at the concentrations tested. Only N. tabacum K326 and N. glutinosa exhibited moderate anthelmintic activity. All extracts significantly repelled R. sanguineus ticks, but not I. ricinus, and the nicotine-rich extracts rapidly knocked down all tick species and stages at high concentrations. The link between nicotine and tick knockdown was confirmed by successfully testing the pure alkaloid at concentrations found in the tobacco extracts. In contrast, repellent activity could not be correlated to the individually tested alkaloids (nicotine, nornicotine, anabasine, anatabine), although anatabine and nornicotine were active in the tick bioassay at high concentrations.

Plants of the root holoparasite Orobanche ramosa L. and four of its potential host species, Nicotiana glauca Graham, Nicotiana rustica L., Nicotiana sylvestris Speg. & S.Comes, and Nicotiana tabacum L., grown in the greenhouse in Kiel/Germany, were analyzed for their contents of pyridine alkaloids anabasine ( 1 ) and nicotine ( 2 ). All investigated samples contained both alkaloids in different amounts. The distribution of the alkaloids in the various plant organs of Nicotiana differed significantly between the species. The alkaloid contents of the Orobanche samples relative to the alkaloid contents of the roots of the respective host plants varied between 3.47 ± 1.08 and 28.8 ± 37.5%. Orobanche plants drain water and crucial nutrients from their hosts; also, some examples for the sequestration of specialized natural products have been reported. O. ramosa is not able to synthesize pyridine alkaloids anabasine ( 1 ) and nicotine ( 2 ) itself; therefore, the present study proves the sequestration of pyridine alkaloids by O. ramosa from the four investigated Nicotiana host species.

Alkaloids represent an extensive group of nitrogen-containing secondary metabolites that are widely distributed throughout the plant kingdom. The pyridine alkaloids of tobacco (Nicotiana tabacum L.) have been the subject of particularly intensive investigation, driven largely due to the widespread use of tobacco products by society and the role that nicotine (16) (see Fig. 1) plays as the primary compound responsible for making the consumption of these products both pleasurable and addictive. In a typical commercial tobacco plant, nicotine (16) comprises about 90% of the total alkaloid pool, with the alkaloids nornicotine (17) (a demethylated derivative of nicotine), anatabine (15) and anabasine (5) making up most of the remainder. Advances in molecular biology have led to the characterization of the majority of the genes encoding the enzymes directly responsible the biosynthesis of nicotine (16) and nornicotine (17), while notable gaps remain within the anatabine (15) and anabasine (5) biosynthetic pathways. Several of the genes involved in the transcriptional regulation and transport of nicotine (16) have also been elucidated. Investigations of the molecular genetics of tobacco alkaloids have not only provided plant biologists with insights into the mechanisms underlying the synthesis and accumulation of this important class of plant alkaloids, they have also yielded tools and strategies for modifying the tobacco alkaloid composition in a manner that can result in changing the levels of nicotine (16) within the leaf, or reducing the levels of a potent carcinogenic tobacco-specific nitrosamine (TSNA). This review summarizes recent advances in our understanding of the molecular genetics of alkaloid biosynthesis in tobacco, and discusses the potential for applying information accrued from these studies toward efforts designed to help mitigate some of the negative health consequences associated with the use of tobacco products.

Root-zone temperature affects several physiological processes, but its influence on alkaloid biosynthesis and accumulation in plants has not yet been evaluated. Here we studied the effect of different root-zone temperatures on Catharanthus roseus and Nicotiana tabacum , which produce a variety of alkaloids. C. roseus produces different alkaloids in different organs and tissues. N. tabacum produces its main alkaloid, nicotine, only in the roots. Plants were maintained for two days at root-zone temperatures of 12 °C, 25 °C (control temperature), and 30 °C; the alkaloid content of the roots and shoots of the plants was then analysed, and the expression level of genes involved in alkaloid biosynthesis pathways was determined. In C. roseus , the lowest vinblastine content was detected in the leaves of plants with a root-zone temperature of 30 °C. Incubation at a root-zone temperature of 12 °C greatly enhanced the root ajmalicine content. In general, the expression of genes involved in alkaloid biosynthesis was highest in C. roseus plants with a root-zone temperature at 12 °C. In N. tabacum plants, a root-zone temperature of 30 °C reduced the root nicotine content by 50% compared to controls, while the anabasine content was reduced by approximately 30% in plants with a root-zone temperature of 12 °C or 30 °C compared to control plants. Genes involved in the nicotine pathway were more highly expressed in plants with a root-zone temperature of 12 °C. Our results demonstrate that the root-zone temperature alters the biosynthesis and accumulation of alkaloids in plant tissues. Our results also suggest that cross-talk between the roots and shoots mediates the transcription of genes involved in alkaloid biosynthesis in different parts of the plant.

Unlike most Nicotiana species, leaf tissues of the globally significant weed Nicotiana glauca Grah. (Argentinian tree tobacco) contains anabasine as the main component of its alkaloid pool, with concentrations typically increasing several fold in response to wounding of plants. The Δ 1 -piperidinium ring of anabasine is synthesised from cadaverine, via the decarboxylation of lysine, however the identity of the protein catalysing this reaction remains unknown. Recent studies indicate that ornithine decarboxylase (ODC), an enzyme involved in the synthesis of the diamine putrescine, may also possess LDC activity. Previously we found that ODC transcript is markedly up-regulated in leaves of N. glauca in response to wounding. In order to examine the role of ODC in the synthesis of anabasine in N. glauca , transcript levels were constitutively down-regulated in hairy root cultures and transgenic plants via the introduction of a CaMV35S driven ODC -RNAi construct. In addition to the anticipated marked reduction in nicotine concentrations, demonstrating that the ODC -RNAi construct was functioning in vivo , we observed that N. glauca ODC -RNAi hairy root cultures had a significantly diminished capacity to elevate anabasine synthesis in response to treatment with the wound-associated hormone methyl jasmonate, when compared to vector-only controls. We observed also that ODC -RNAi transgenic plants had significantly reduced ability to increase anabasine concentrations following removal of the plant apex. We conclude that ODC does have an important role in enabling N. glauca to elevate levels of anabasine in response to wound-associated stress.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels found throughout the body, and serve to mediate diverse physiological functions. Muscle-type nAChRs located in the motor endplate region of muscle fibers play an integral role in muscle contraction and thus motor function. The toxicity and teratogenicity of many plants (which results in millions of dollars in losses annually to the livestock industry) are due to various toxins that bind to nAChRs including deltaline and methyllycaconitine (MLA) from larkspur (Delphinium) species, and nicotine and anabasine from tobacco (Nicotiana) species. The primary result of the actions of these alkaloids at nAChRs is neuromuscular paralysis and respiratory failure. The objective of this study was to further characterize the motor coordination deficiencies that occur upon exposure to a non-lethal dose of nAChR antagonists MLA and deltaline as well as nAChR agonists nicotine and anabasine. We evaluated the effect of nAChR agonists and antagonists on the motor function and coordination in mice using a balance beam, grip strength meter, rotarod, open field analysis and tremor monitor. These analyses demonstrated that within seconds after treatment the mice had significant loss of motor function and coordination that lasted up to 1 min, followed by a short period of quiescence. Recovery to normal muscle coordination was rapid, typically within approximately 10 min post-dosing. However, mice treated with the nAChR agonist nicotine and anabasine required a slightly longer time to recover some aspects of normal muscle function in comparison to mice treated with the nAChR antagonist MLA or deltaline.

In this work, urinary nicotine was determined in the presence of the metabolite cotinine and the alkaloid anabasine using surface enhanced Raman spectroscopy and colloidal gold as substrate. Spectra were decomposed using the multivariate curve resolution-alternating least squares method, and pure contributions were recovered. The standard addition method was applied by spiking urine samples with known amounts of the analyte and relative responses from curve resolution were employed to build the analytical curves. The use of multivariate curve resolution in conjunction with standard addition method showed to be an effective strategy that minimized the need for reagent and time-consuming procedures. The determination of the alkaloid nicotine was successfully accomplished at concentrations 0.10, 0.20 and 0.30 μg mL(-1) and total error values less than 10% were obtained.

A series of arylidene anabaseines were synthesized to probe the functional impact of hydrogen bonding on human α7 nicotinic acetylcholine receptor (nAChR) activation and desensitization. The aryl groups were either hydrogen bond acceptors (furans), donors (pyrroles), or neither (thiophenes). These compounds were tested against a series of point mutants of the ligand-binding domain residue Gln-57, a residue hypothesized to be proximate to the aryl group of the bound agonist and a putative hydrogen bonding partner. Q57K, Q57D, Q57E, and Q57L were chosen to remove the dual hydrogen bonding donor/acceptor ability of Gln-57 and replace it with hydrogen bond donating, hydrogen bond accepting, or nonhydrogen bonding ability. Activation of the receptor was compromised with hydrogen bonding mismatches, for example, pairing a pyrrole with Q57K or Q57L, or a furan anabaseine with Q57D or Q57E. Ligand co-applications with the positive allosteric modulator PNU-120596 produced significantly enhanced currents whose degree of enhancement was greater for 2-furans or -pyrroles than for their 3-substituted isomers, whereas the nonhydrogen bonding thiophenes failed to show this correlation. Interestingly, the PNU-120596 agonist co-application data revealed that for wild-type α7 nAChR, the 3-furan desensitized state was relatively stabilized compared with that of 2-furan, a reversal of the relationship observed with respect to the barrier for entry into the desensitized state. These data highlight the importance of hydrogen bonding on the receptor-ligand state, and suggest that it may be possible to fine-tune features of agonists that mediate state selection in the nAChR.

Based on pharmacophore elucidation and docking studies on interactions of benzylidene anabaseine analogs with AChBPs and α7 nAChR, novel spirodiazepine and spiroimidazoline quinuclidine series have been designed. Binding studies revealed that some of hydrogen-bond donor containing compounds exhibit improved affinity and selectivity for the α7 nAChR subtype in comparison with most potent metabolite of GTS-21, 3-(4-hydroxy-2-methoxybenzylidene)-anabaseine. Hydrophobicity and rigidity of the ligand also contribute into its binding affinity. We also describe alternative pharmacophoric features equidistant from the carbonyl oxygen atom of the conserved Trp-148 of the principal face, which may be exploited to further design diverse focused libraries targeting the α7 nAChR.

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of nicotine (NIC), cotinine (COT), nornicotine (NNIC), norcotinine (NCOT), nicotine-N-β-D-glucuronide (NIC GLUC), cotinine-N-β-D-glucuronide (COT GLUC), nicotine-1′-oxide (NNO), cotinine-N-oxide (CNO), trans-3′-hydroxycotinine (3-HC), anabasine (AB) and anatabine (AT) was modified and validated for quantification of these selected analytes in rat brain tissue. This analytical method provides support for preclinical NIC pharmacokinetic and toxicological studies after controlled dosing protocols. After brain homogenization and solid-phase extraction, target analytes and corresponding deuterated internal standards were chromatographically separated on a Discovery® HS F5 HPLC column with gradient elution and analyzed by LC-MS/MS in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. Method linearity was assessed and calibration curves were determined over the following ranges: 0.1 – 7.5 ng/mg for NIC, COT GLUC and AB; and 0.025 – 7.5 ng/mg for COT, NNIC, NCOT, NIC GLUC, NNO, CNO, 3-HC and AT (R2 ≥ 0.99 for all analytes). Extraction recoveries ranged from 64% - 115%, LC-MS/MS matrix effects were ≤ 21%, and overall process efficiency ranged from 57% - 93% at low and high quality control concentrations. Intra- and inter-assay imprecision and accuracy for all analytes were ≤ 12.9% and ≥ 86%, respectively. The method was successfully applied to quantification of NIC and metabolites in the brain of post-natal day 90 rats that were sacrificed 2-h after a single 0.8 mg/kg s.c. administration of (−) NIC. In these tissues, striatal concentrations were 204.8±49.4, 138.2±14.2 and 36.1±6.1 pg/mg of NIC, COT and NNIC, respectively. Concentrations of NIC, COT and NNIC in the remaining whole brain (RWhB) were 183.3±68.0, 130.0±14.1 and 46.7±10.3 pg/mg, respectively. Quantification of these same analytes in plasma was also performed by a previously validated method. NIC, COT, NNIC, NCOT, NNO and CNO were detected in plasma with concentrations comparable to those reported in previous studies. However, and in contrast to brain tissues, COT concentrations in plasma were significantly higher than were those of NIC (194.6±18.6 ng/mL versus 52.7±12.9 ng/mL). Taken together, these results demonstrate that a sensitive and selective method has been developed for the determination of NIC biomarkers in rat brain.

ZnX2 (X = Br, Cl) complexes with N-methylanabasine have been synthesized and characterized by elemental analysis and spectroscopic techniques. The complexes are formulated as [(C11H16N2)2ZnBr2] (1) and [(C11H16N2)2ZnCl2] (2) where C11H16N2 = N-methylanabasine. The X-ray structure of compound 1 is presented. It constitutes the first report on the crystal structure of the metal complex with anabasine or its derivative acting as a ligand. It shows that two N-methylanabasine units and two bromide anions form a tetrahedral coordination around the Zn2+ cation.

A three-component diastereoselective vinylogous Mannich-type reaction (VMR) with the silyl ketene acetal (1) was developed. Adducts from the reactions provided valuable intermediates for construction of a variety of chiral 6-alkylated piperidinone and 2-piperidine compounds. The strategy was applied in a concise synthesis (S)-anabasine.

The synthesis, spectral characteristics and structures of N-1-methyl-6-(pyridin-3-yl)piperidine-2-thione (1) (thioanalogue of N-1-methylanabasine) and N-1-methyl-(5-pyridin-3-yl)pyrrolidine-2-thione (2) (thioanalogue of nicotine) are reported. Both compounds were obtained using Lawesson’s reagent. The structures of compounds 1 and 2 are confirmed by NMR, IR, UV and mass spectroscopy, as well as, by X-ray diffraction analysis. Pyridine ring of compound 1 adopts a pseudo-axial orientation in solution, as well as in a solid state. A substantial lengthening of the C S bond in the crystals of 1 is interpreted as a sign of an enhanced electron delocalization within the thiolactam group due to the presence of several C H groups in the nearest vicinity of the sulfur atom. In the crystals of 2, which differ from 1 in that the relatively puckered piperidine-2-thione moiety is replaced by the flat pyrrolidine-2-thione ring, no short CH⋯S( C) contacts are observed. Instead, the packing is governed by stacking interactions between pyridine rings. The pyrrolidine and pyridine rings in 2 are nearly perpendicular to each other and the pyrrolidine moiety adopts a flattened half-chair conformation.

We have demonstrated that the Ruppert–Prakash reagent is able to react with a number of cyclic imines under acidic condition to afford the corresponding α-trifluoromethyl derivatives of nitrogen heterocycles. 5–7-Membered cyclic imines bearing various alkyl, aryl or heterocyclic group were successfully involved in this transformation. Novel trifluoromethylated analogues of nicotine, anabasine, and homoanabasine alkaloids were synthesized.

Nicotinic acetylcholine receptors (nAChRs) have become targets for drug development in recent years. 3-(2,4-dimethoxybenzylidene)-anabaseine (DMXBA), which selectively stimulates the alpha7 nAChR, has been shown to alleviate some cognitive deficits associated with schizophrenia. In this paper we report an analysis of the interactions between 47 arylidene-anabaseines (including 45 benzylidene-anabaseines) and rat brain alpha7 and alpha4beta2 nicotinic acetylcholine receptors, using three different modeling techniques, namely 2D-QSAR, 3D-QSAR and molecular docking to the Aplysia californica acetylcholine binding protein (AChBP), a water soluble, homomeric nAChR surrogate receptor with a known crystal structure. Our investigation indicates the importance of: (1) the nitrogen atom of the tetrahydropyridyl (THP) ring for hydrogen bond formation; (2) pi-pi interactions between the aromatic rings of the ligands and the nAChBP binding site; (3) molecular surface recognition expressed in terms of steric complimentarity. On the basis of the 3D-QSAR results, bulky substituents at positions 2 (and due to the rotational freedom also at position 6) and 4 of the benzylidene moiety, with highly electronegative atoms projecting approximately 3-3.5A away from the benzylidene ring at position 4 seem optimal for enhancing binding affinity to the alpha7 nAChR.

A novel series of bis-aromatic ring neonicotinoid analogues (1a–1l, 2a–2c), were designed and prepared by introducing a new substituted aromatic ring into nitenpyram and forming a tetrahydropyrimidine ring, with the cis-configuration confirmed by X-ray diffraction. Preliminary bioassays showed most analogues exhibited good insecticidal activities at 100 mg/L, and compound 1d and 2a were highly potent even at 10 mg/L. Modeling the ligand–receptor complexes by molecular docking study explained the structure–activity relationships observed in vitro, which may provide some useful information for future design of new insecticides.

Secondary phosphines, elemental selenium, and the natural alkaloids, lupinine and anabasine, interact in a three-component-type reaction under mild conditions (70 °C, 1–1.5 h, EtOH) without the formation of any by-products to give diselenophosphinates of the above-mentioned alkaloids (in almost quantitative yields) with the retention of their intrinsic optical activity. Thus, the developed synthesis represents an atom-economic route to new selenophosphorus derivatives of alkaloids.

Cut seedlings of Mercurialis annua L. were supplied with solutions containing [1-13C1]glucose or [U-13C4,15N1]aspartate. After 5–7 days, the pyridinone-type chromogen, hermidin, was isolated and analyzed by NMR spectroscopy. In the experiment with [1-13C1]glucose, five single-labelled isotopomers of hermidin were detected at high abundances (2.7–1.8 mol%). In the experiment with [U-13C4,15N1]aspartate, contiguous labelling was observed for carbon atoms 2 and 3 and the nitrogen atom in hermidin. The labelling patterns of hermidin and of amino acids from the same experiments rule out predominant formation of the pyridinone by pathways resembling the biosyntheses of vitamin-B6, anabasine, or polyketides, but suggest a pathway by condensation of aspartate and dihydroxyacetone phosphate affording nicotinate as a precursor of hermidin.

This work describes an on-line molecularly imprinted solid-phase extraction (MISPE) method for spectrophotometric determination of nicotine in urine samples of smokers. This method is based on manganese (VII) to manganese (VI) reduction in an alkaline medium, promoted by nicotine. Two wash solutions (1:4 (v/v) acetonitrile:sodium hydroxide--pH 11.4, and nitric acid--pH 2.5) were employed to circumvent interferences. Aqueous solutions containing nicotine plus different possible concomitants (cotinine, anabasine, norcotinine and caffeine) were tested individually. The analytical calibration curve was prepared in urine samples collected from non-smokers and spiked with nicotine standard from 1.1 to 60 micromol L(-1) (r(2)>0.998). The limit of quantification and the analytical frequency were 1.1 micromol L(-1) and 11 h(-1), respectively. The precision, evaluated using 3, 10 and 30 micromol L(-1) nicotine in urine, was 10, 10 and 4% (intra-day precision) and 12, 13 and 5% (inter-day precision), respectively. Accuracy was checked through high performance liquid chromatography and the results did not present significant differences at the 95% confidence level according to the Student's t-test.

A simple, rapid and sensitive method for the determination of nicotine, cotinine, nornicotine, anabasine, and anatabine in human urine and saliva was developed. These compounds were analyzed by on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS). Nicotine, cotinine and related alkaloids were separated within 7 min by high performance liquid chromatography (HPLC) using a Synergi 4u POLAR-RP 80A column and 5 mM ammonium formate/methanol (55/45, v/v) as a mobile phase at a flow-rate of 0.8 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for MS detection of these compounds. The optimum in-tube SPME conditions were 25 draw/eject cycles with a sample size of 40 microL using a CP-Pora PLOT amine capillary column as the extraction device. The extracted compounds could be desorbed easily from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS method, the calibration curves were linear in the concentration range of 0.5-20 ng/mL of nicotine, cotinine and related compounds in urine and saliva, and the detection limits (S/N=3) were 15-40 pg/mL. The method described here showed 20-46-fold higher sensitivity than the direct injection method (5 microL injection). The within-run and between-day precision (relative standard deviations) were below 4.7% and 11.3% (n=5), respectively. This method was applied successfully to analysis of urine and saliva samples without interference peaks. The recoveries of nicotine, cotinine and related compounds spiked into urine and saliva samples were above 83%, and the relative standard deviations were below 7.1%. This method was used to analyze urinary and salivary levels of these compounds in nicotine intake and smoking.

A fully automated multi-dimensional gas chromatography (MDGC) system with a megabore precolumn and cyclodextrin-based analytical column was developed to analyze the enantiomeric compositions of anatabine, nornicotine and anabasine in commercial tobacco. The enantiomer abundances of anatabine and nornicotine varied among different tobacco. S-(-)-anatabine, as a proportion of total anatabine, was 86.6% for flue-cured, 86.0% for burley and 77.5% for oriental tobacco. S-(-)-nornicotine, as a proportion of total nornicotine, was 90.8% in oriental tobacco and higher than in burley (69.4%) and flue-cured (58.7%) tobacco. S-(-)-anabasine, as a proportion of total anabasine, was relatively constant for flue-cured (60.1%), burley (65.1%) and oriental (61.7%) tobacco. A simple solvent extraction with dichloromethane followed by derivatisation with trifluoroacetic anhydride gave relative standard deviations of less than 1.5% for the determination of the S-(-)-isomers of all three alkaloids. The study also indicated that, a higher proportion of S-(-)-nornicotine is related to the more active nicotine demethylation in the leaf.

Homomeric alpha7 and heteromeric alpha4beta2 nicotinic acetylcholine receptors (nAChR) can be distinguished by their pharmacological properties, including agonist specificity. We introduced point mutations of conserved amino acids within the C loop, a region of the receptor critical for agonist binding, and we examined the expression of the mutant receptors in Xenopus oocytes. Mutation of either a conserved C loop tyrosine (188) to phenylalanine or a nearby conserved aspartate (197) to alanine resulted in alpha7 receptors for which the alpha7-selective agonist 3-(4-hydroxy, 2-methoxybenzylidene) anabaseine (4OH-GTS-21) had roughly the same potency as for wild-type receptors, whereas the physiologic agonist acetylcholine (ACh) showed drastically reduced potency for these mutant receptors. Corresponding mutations in alpha4 receptors co-expressed with beta2 resulted in alpha4beta2 receptors for which ACh potency was relatively unchanged, although the efficacy of the alpha7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in alpha7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both alpha7 and alpha4 resulted in receptors that were largely unresponsive to both ACh and 4OH-GTS-21. Our results suggest that initiation of gating depends both on specific interactions between residues in the C loop domain and, depending on receptor subtype, the physiochemical properties of the agonist, so that in the altered environment of the alpha4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.

Anabasine occurring in wild tree tobacco (Nicotiana glauca) and anabaseine occurring in certain animal venoms are nicotinic receptor agonist toxins. Anabasine lacks the imine double bond of anabaseine; the two possible enantiomers of anabasine occur in N. glauca. A comparision of the relative potencies of S- and R-anabasine has not been previously reported. We separated the enantiomers of anabasine by reaction of the racemic N. glauca natural product with 9-fluorenylmethoxycarbonyl-L-alanine (Fmoc-L-Ala-OH) to give diastereomers, which were separated by preparative reversed phase HPLC. The S- and R-anabasine enantiomer fractions were then obtained by Edman degradation. A mouse bioassay was used to determine the relative lethalities of S- and R-enriched anabasine enantiomers. The intravenous LD50 of the (+)-R-anabasine rich fraction was 11± 1.0 mg/kg and that of the (−)-S-anabasine-rich fraction was 16±1.0 mg/kg. The LD50 of anabaseine was 0.58±0.05 mg/kg. Anabaseine was significantly more toxic in the mouse bioassay than S-anabasine (27-fold) and R-anabasine (18-fold). The relative agonistic potencies of these three alkaloids on human fetal nicotinic neuromuscular receptors were of the same rank order: anabaseine ≫R-anabasineNS-anabasine. © 2006 Published by Elsevier Inc.

The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates. The kinetics of metabolism of (R,S)-N-methylanabasine differ significantly from those of nicotine in that no further degradation of the initial demethylation product occurs. (R,S)-N-Methylanatabine, however, shows kinetics closer to those of nicotine, with loss of alkaloid from the system. Further more, (R,S)-N-methylanabasine does not diminish (S)-nicotine demethylation, indicating a lack of competition. However, the metabolism of (S)-nicotine is affected by the presence of (R,S)-N-methylanabasine. Hence, the demethylation of the piperidine homologues of nicotine is seen to be similar but not identical to that of the pyridine analogues. The implications of these different metabolic profiles in relation to the demethylation activity are discussed.

Reaction of 3-(1-alkylamino)pyridines with electron deficient acetylenes in the presence of acids yields 1,2-dihydro[2,7]naphthyridine-3,4-dialkyldicarboxylates 4 in 35–72% yield. Compounds 4 unsubstituted in position 1 can be easily oxidized with potassium permanganate into the respective naphthyridine-1-ones derivatives 5 in good yields.

Liquid chromatography columns containing stationary phases based upon immobilized nicotinic acetylcholine receptors (nAChRs) were used to screen a series of conformationally constrained nicotine and anabasine derivatives for agonist activity. The alpha3beta4 nAChR and alpha4beta2 nAChR subtypes were used to prepare the chromatographic columns and [(3)H] epibatidine dihydrochloride ([(3)H] EB) was used as the marker ligand. Single displacement experiments were conducted with the test ligands and with nicotine and carbachol. Nicotine was used as an internal control for compounds with agonist activity and carbachol was used as an internal control for compounds with very weak agonistic activity (K(d) > 4700 nM for alpha3beta4). The displacement of [(3)H] EB by each of the test compounds and internal controls was calculated and expressed as Deltaml. Functional studies were then conducted using a stably transfected cell line that expresses the alpha3beta4 nAChR and EC(50) values were determined for the test compounds and the internal controls. A comparison of the Deltaml and EC(50) values indicated that 9/11 compounds had been correctly identified as agonists or non-agonists of the alpha3beta4 nAChR. A similar comparison could not be made for the alpha4beta2 nAChR, since the intact cell line was not available for testing. The results of the study suggest that the immobilized nAChR columns can be used for the rapid on-line screening of compounds for their relative affinities for the immobilized receptor and as an initial determination of qualitative functional activities.

Importance of atom-level topological descriptors like electrotopological state atom (E-state) index in QSAR study is increasing. These descriptors help to relate structure and activity at atomic/fragmental level. In view of the earlier success of E-state index on some azidopyridinyl neonicotinoid insecticides, a relatively new atom-level topological descriptor; refractotopological state atom (R-state) index was used in this work. This was used to identify the important atoms/fragments related to dispersive/van der Waals interactions of neonicotinoids with the nicotinic acetylcholine receptor (nAChR). This study showed the structural requirements for the mammal alpha(4)beta(2) and Drosophila nAChR agonistic activity. It also revealed that substituted imine, nitromethylene at X-position were selective to the insecticidal activity. Azido substitution at pyridine ring of neonicotinoids disfavored the binding with the receptors. This study confirmed the validity of the R-state index as a new tool for quantitative structure-activity relationships. It has the ability to find out the required structural features as well as to predict the activity of the neonicotinoids.

The expression of the alpha7-nicotinic acetylcholine receptor is diminished in selected brain areas of patients with schizophrenia. This diminished expression may account for the pathophysiological deficits of sensory inhibition and smooth pursuit eye movement performance in these patients. Furthermore, the deficits in sensory inhibition and smooth pursuit eye movement performance in schizophrenia appear to be inherited in an autosomal dominant fashion; thus, the "alpha7-nicotinic acetylcholine receptor-deficiency" may be a necessary condition for expression of schizophrenia. This deficit has encouraged speculation about the possible therapeutic benefit of selective alpha7-nicotinic acetylcholine receptor agonist interventions in this disorder. In view of this, we sought to examine the effect of anabasine, a selective alpha7-nicotinic acetylcholine receptor agonist, on popping behavior in mice elicited by MK-801. MK-801, a high affinity analogue of phencyclidine (PCP), is a noncompetitive N-methyl-D-aspartic acid (NMDA) receptor antagonist that binds to the hydrophobic domain of this ligand-gated channel. PCP is known to precipitate a schizophreniform psychosis in susceptible individuals, causing productive (e.g. hallucinations) deficit (e.g. affective blunting, amotivation, and social withdrawal), cognitive and motor symptoms similar to those seen in naturally-occurring schizophrenia. Behaviors elicited by MK-801 in mice reflect a pharmacologically-induced state of NMDA receptor hypofunction (NRH), which has been proposed to exist in schizophrenia. Compounds that attenuate MK-801-elicited behaviors, which are identified in this animal model, may have the potential to treat schizophrenia, including deficit and cognitive symptoms. In the current study, anabasine attenuated MK-801-elicited popping at a dose that did not cause clonic seizures. The development of alpha7-nicotinic acetylcholine receptor agonist interventions for schizophrenia must consider their potential liability to elicit seizure activity.

S-(-)-Nicotine and 13 of the most prevalent nicotine-related alkaloids and metabolites (i.e., S-(-)-nornicotine, myosmine, beta-nicotyrine, S-cotinine, S-norcotinine, S-(-)-nicotine N-1'-oxide, S-(-)-nicotine Delta1'-5'-iminium ion, S-(-)-anabasine, S-(-)-N-methylanabasine, anabaseine, S-(-)-anatabine, nicotelline, and 2,3'-bipyridyl) were evaluated as inhibitors of human cDNA-expressed cytochrome P-450 2A6 (CYP2A6) mediated coumarin 7-hydroxylation. Tobacco alkaloids myosmine, S-(-)-nornicotine, S-cotinine, S-norcotinine, S-(-)-nicotine N-1'-oxide, S-(-)-nicotine Delta1'-5'-iminium ion, S-(-)-N-methylanabasine, anabaseine, and nicotelline had Ki values for inhibition of coumarin 7-hydroxylation ranging from 20 microM to more than 300 microM whereas nicotine and S-(-)-anatabine were much more potent (i.e. 4.4 and 3.8 microM, respectively). The tobacco alkaloids 2,3'-bipyridyl (7.7 microM) and S-(-)-anabasine (5.4 microM), were somewhat less potent compared with S-(-)-nicotine or S-(-)-anatabine in inhibition of human CYP2A6. beta-Nicotyrine, in which the N-methylpyrrolidino moiety of nicotine was replaced by the aromatic N-methylpyrrole ring, was shown to inhibit human CYP2A6 with much greater potency (Ki=0.37 microM) compared with S-(-)-nicotine. Among the compounds examined, only nicotine and beta-nicotyrine were mechanism-based inhibitors of human CYP2A6. The potency of the mechanism-based CYP2A6 inhibitors suggests that, for smokers, modulation of CYP2A6 may be greater than that predicted on the basis of serum concentration of these alkaloids. Our results indicate that the prominent nicotine-related alkaloid beta-nicotyrine present after smoking potently inhibits human CYP2A6.

Tuberomammillary histamine neurons (TM) of the posterior hypothalamus exclusively express alpha-bungarotoxin (alphaBgt) sensitive nicotinic receptors, providing a unique model system for studying physiological properties of native alpha7-like receptors. Here the properties of alphaBgt-sensitive receptors were investigated using the patch-clamp technique and rapid application of acetylcholine (ACh) or the alpha7-selective agonists, 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21), and choline. Alpha-Bgt-sensitive receptor responses to rapid application of high agonist concentrations were characterized by a transient current which rapidly decayed in a voltage-independent concentration-dependent manner to a relatively sustained slow current. Upon agonist removal, current persisted for several milliseconds (or longer) and increased above the level of the slow current (rebound). Lower agonist concentrations did not produce a rebound. Our analysis suggests that the current rebound represents a recovery phase from a low potency inhibition. This inhibition was voltage-dependent for ACh and choline but voltage-independent for 4OH-GTS-21. A slow form of desensitization was present which was relatively agonist-independent and was faster than the rate of 4OH-GTS-21 unbinding. Kinetic analysis revealed that the concentration dependence of the transient response amplitudes was compromised by solution exchange; net charge measurements over the late response phases were chosen as an alternative measure of concentration/response function. Our data suggest that low agonist concentrations can evoke a prolonged or tonic-like receptor activation. Functioning in this modality, receptors would regulate calcium homeostasis over a narrow, but therapeutically important, range of intracellular calcium concentrations. This could then provide the basis for cytoprotective effects of 4OH-GTS-21 and other nicotinic agonists, mediating trophic and neuromodulatory functions.

A novel multistep synthesis of anabasine analogues bearing a bioisosteric pyridazine moiety instead of a pyridine nucleus in 2-position of the piperidine ring of the alkaloid is described. Starting materials are racemic 2-hydroxymethyl-piperidine, racemic pipecolic acid or (S)-(−)-piperidine-1,2-dicarboxylic-acid-1-tert-butyl ester. Key step of this synthetic approach is a Diels–Alder cycloaddition process with inverse electron demand utilizing diverse 1,2,4,5-tetrazines as electron-deficient dienes and the new racemic or enantiopure 2-(2′-methoxyethenyl)-piperidine as electron-rich dienophile. In this [4+2]-cycloadditions 1,2,4,5-tetrazines serve as synthons for introducing the pyridazine ring in the 2-position of the piperidine moiety.

A range of endemic Nicotiana species are chewed as a smokeless tobacco by several Aboriginal populations of Australia. In tobacco research, nicotine to nornicotine conversion is important because nornicotine lowers tobacco quality and is detrimental to health. A diverse group of cytochrome P450 genes with different transcriptional regulations are involved in this conversion. The primary aims of this study were to quantify the pyridine alkaloids and investigate nicotine to nornicotine conversion in laboratory-grown Australian Nicotiana spp. Nicotine, nornicotine, anatabine, anabasine, myosmine and cotinine were quantified in fresh leaves of 24 out of the 26 recognised Australian Nicotiana taxa. Conserved regions of CYP82E related genes were PCR amplified in all studied taxa. The conversion process in fresh leaves was compared with that in leaves that underwent a simulated curing process for species that we identified as being high converters (N. cavicola, N. goodspeedii, N. velutina) and low converters (N. benthamiana, N. excelsior, N. gossei). Agarose gel electrophoretic analysis of CYP82E related genes obtained from the PCR amplification of the cDNA in fresh versus leaves with simulated curing showed about a 3-fold increase in transcript accumulation levels in cured leaves of the high converter species, while the transcript accumulation in N. gossei and N. excelsior maintained a steady basal level and increased by a small amount in N. benthamiana. This suggests the presence of functional loci that are triggered by curing in only high converter species and indicates a potential risk for chewers of high converter species.

A short and straightforward synthesis of the racemic tobacco alkaloids anatabine and anabasine in five and six steps, respectively, from 3-pyridinecarboxaldehyde utilizing Barbier-type Zn-mediated allylation and ring-closing olefin metathesis, as the key steps, is reported. Additionally, a complete NMR spectroscopic analysis of the final products is carried out and full assignment of the NMR spectra of anatabine and anabasine with accurate coupling constants is accomplished and reported here for the first time.

A short synthesis of methyl (SR)-8,10-dichloro-1,2,3,4,5,6-hexahydro-6-hydroxy-1-methyl-1,9-phenanthroline-6-carboxylate (14) has been achieved en route to conformationally restricted analogues of nicotine and anabasine. The key feature of this process involves an efficient intramolecular Hamaguchi–Ibata reaction of 11.

VAN HAAREN, F., K. G. ANDERSON, S. C. HAWORTH AND W. R. KEM. GTS-21, a mixed nicotinic receptor agonist/antagonist, does not affect the nicotine cue. PHARMACOL BIOCHEM BEHAV 64(2) 439–444, 1999.—Identification of nicotinic receptor subtypes involved in nicotine dependence is required for guiding the design of more selective antagonists capable of blocking the nicotine cue and nicotine self-administration. Due to the multiplicity of nicotinic receptors in the mammalian brain, selective agonists and antagonists are needed to assess the functional involvement of a particular subtype in vivo. Only recently have a few nicotinic receptor subtype-selective antagonists and agonists been identified. GTS-21 (also known as DMBX-anabaseine) is the only agent so far reported that selectively stimulates the α7 nicotinic receptor. Here GTS-21 was used to assess the possible mediation of the nicotine cue by this receptor subtype. Long–Evans rats were trained to discriminate between presession administration of 0.10 or 0.40 mg/kg (−)-nicotine bitartrate and its vehicle. GTS-21 did not substitute for nicotine, as all subjects consistently chose the vehicle lever after GTS-21 substitution. In another experiment, different doses of GTS-21 were administered prior to nicotine administration to investigate whether GTS-21 would antagonize the nicotine cue. Such was not the case. The lack of effect of GTS-21 upon the nicotine cue is consistent with the notion that the cue is mediated by nicotinic receptors other than the α7 receptor.