Further Quinolizidine Derivatives as Antiarrhythmic Agents- 3.

Fourteen quinolizidine derivatives, structurally related to the alkaloids lupinine and cytisine and previously studied for other pharmacological purposes, were presently tested for antiarrhythmic, and other cardiovascular effects on isolated guinea pig heart tissues in comparison to well-established reference drugs. According to their structures, the tested compounds are assembled into three subsets: (a) N-(quinolizidinyl-alkyl)-benzamides; (b) 2-(benzotriazol-2-yl)methyl-1-(quinolizidinyl)alkyl-benzimidazoles; (c) N-substituted cytisines. All compounds but two displayed antiarrhythmic activity that was potent for compounds 4, 1, 6, and 5 (in ascending order). The last compound (N-(3,4,5-trimethoxybenzoyl)aminohomolupinane) was outstanding, exhibiting a nanomolar potency (EC50 = 0.017 µM) for the increase in the threshold of ac-arrhythmia. The tested compounds shared strong negative inotropic activity; however, this does not compromise the value of their antiarrhythmic action. On the other hand, only moderate or modest negative chronotropic and vasorelaxant activities were commonly observed. Compound 5, which has high antiarrhythmic potency, a favorable cardiovascular profile, and is devoid of antihypertensive activity in spontaneously hypertensive rats, represents a lead worthy of further investigation.

Five New Alkaloids from the Roots of Sophora flavescens.

Five new quinolizidine alkaloids, including three sparteine-type alkaloids (1 - 3) and two cytisine-type alkaloids (4 and 5), along with four known ones, were isolated from the roots of Sophora flavescens. Their structures were determined by extensive spectroscopic techniques including IR, UV, NMR, and HR-ESI-MS. All the compounds were evaluated for their antibacterial activities against Staphylococcus aureus and Escherichia coli.

The Use of (-)-Sparteine/Organolithium Reagents for the Enantioselective Lithiation of 7,8-Dipropyltetrathia[7]helicene: Single and Double Kinetic Resolution Procedures.

The effect of organolithium reagent (RLi: R=nBu, iPr, sBu, tBu), solvent (diethyl ether, diethyl ether/THF and MTBE), and stoichiometry on the (-)-sparteine-mediated silylation of 7,8-dipropyltetrathia[7]helicene shows that, unusually, substantially more than 0.5 equivalent of RLi (R=iPr, sBu, tBu) and a large excess of (-)-sparteine (R=nBu, sBu) is often needed to achieve substantial conversions and good ee values. With nBuLi, however, just one equivalent of the organolithium reagent is sufficient to obtain high conversions. Our best results were obtained using the convenient tBuLi/(-)-sparteine adduct with which the need for a high (-)-sparteine/RLi ratio can be avoided. Single- and double-kinetic resolution (KR) procedures give enantiopure samples of 2-trimethylsilyl- and 2,13-di(trimethylsilyl)-7,8-dipropyltetrathia[7]helicene and two-step double-KR combining (-)-sparteine/sBuLi and chiral formamides affords the synthetically valuable 2-formyl-7,8-dipropyltetrathia[7]helicene. This is the first use of (-)-sparteine for the enantioselective lithiation of helicenes and the first report of tBuLi outperforming sBuLi in a (-)-sparteine-mediated procedure.

The anticonvulsant effect of sparteine on pentylenetetrazole-induced seizures in rats: a behavioral, electroencephalographic, morphological and molecular study.

Abnormal synchronous activity in neurons generates epileptic seizures. Antiepileptic drugs (AEDs) are effective in 70% of patients, but this percentage is drastically lower in developing countries. Sparteine is a quinolizidine alkaloid synthesized from most Lupine species and has a probable anticonvulsive effect. For this reason, the objective of the present work was to study the anticonvulsant effect of sparteine using a dose-effect curve and to determine its effectiveness against seizures using behavioral, electroencephalographic, morphological and molecular data. Wistar rats were grouped into control [saline solution (0.9%), pentylenetetrazole (90 mg/kg), and sparteine (13, 20 and 30 mg/kg), intraperitoneal (i.p.)] and experimental (sparteine + pentylenetetrazole) groups. The rats were implanted with surface electrodes to register electrical activity, and convulsive behavior was evaluated according to Velisek's scale. The rats were perfused to obtain brain slices for cresyl violet staining and cellular density quantification as well as for immunohistochemistry for NeuN and GFAP. Other animals were used to determine the hippocampal mRNA expression of the M2 and M4 acetylcholine receptors by qPCR. Sparteine exhibited a better anticonvulsant effect at a dose of 30 mg/kg (i.p.) than at the other doses used. This anticonvulsant effect was characterized by a decrease in the severity of convulsive behavior, 100% survival, an inhibitory effect on epileptiform activity 75 min after pentylenetetrazole administration, and the conservation of the cellular layers of CA1, CA3 and the dentate gyrus (DG); however, astrogliosis was observed after 30 mg/kg sparteine treatment. In addition, sparteine treatment increased the mRNA expression of the M4 receptor three hours after administration. According to our findings, the effective dose of sparteine as an anticonvulsant agent by i.p. injection is 30 mg/kg. The astrogliosis that was observed after sparteine administration may be a compensatory mechanism to diminish excitability and maintain neuronal homeostasis, possibly through redistributing potassium and glutamate. The increase in the mRNA expression of the M4 receptor may suggest the participation of the M4 receptor in the anticonvulsive effect of sparteine, as the activation of this receptor may inhibit acetylcholine release and facilitate the subsequent release of GABA. However, the precise mechanisms by which sparteine produces these effects are not known, and therefore, further experiments are necessary.

Selection of an optimal method for screening the collection of narrow-leaved lupine held by the Vavilov Institute for the qualitative and quantitative composition of seed alkaloids.

Narrow-leaved lupine (Lupinus аngustifolius L.) is a widely cultivated leguminous forage and green manure crop with a potential for human nutrition. However, the presence of secondary metabolites - alkaloids - in lupine seeds considerably affects the quality of raw produce, reducing its nutritive value; in addition, high concentrations of alkaloids are toxic to humans and animals. Therefore, plant breeders working with lupine need to gain knowledge about the variability of alkaloid content in seeds of different genotypes and search for the sources of their low concentrations in the crop's gene pool. The collection of narrow-leaved lupine genetic resources held by the N.I. Vavilov Institute of Plant Genetic Resources (VIR) offers wide opportunities for such search by means of mass screening. For its part, largescale gene pool screening requires the selection of an optimal technique to measure alkaloid content in seeds, so that it would be easily reproducible and as little labor-, time- and fund-consuming as possible. The results of the search for such method are presented. Qualitative and quantitative indices were compared when target compounds had been extracted with multicomponent mixtures and individual reagents (chloroform, methanol, etc.) and the extracts analyzed using gas chromatography-mass spectrometry. High-performance liquid chromatography combined with mass spectrometry was also employed. Five major alkaloids were found to be present in all types of extracts: lupanine, 13-hydroxylupanine (dominant ones), angustifoline, sparteine, and isolupanine. The fullest extraction of alkaloids was observed when the extractant with an added alkaline agent was used (425 mg/100 g). The lowest level of extraction was registered with chloroform (216 mg/100 g). The significance of the differences was confirmed statistically.

17 Oxo Sparteine and Lupanine, Obtained from Cytisus scoparius, Exert a Neuroprotection against Soluble Oligomers of Amyloid-ß Toxicity by Nicotinic Acetylcholine Receptors.

Alzheimer's disease (AD) is a neurodegenerative pathology, which is characterized by progressive and irreversible cognitive impairment. Most of the neuronal perturbations described in AD can be associated with soluble amyloid- ß oligomers (SO-Aß). There is a large amount of evidence demonstrating the neuroprotective effect of Nicotine neurotransmission in AD, mainly through nicotinic acetylcholine receptor (nAChR) activation and antiapoptotic PI3K/Akt/Bcl-2 pathway signaling. Using HPLC and GC/MS, we isolated and characterized two alkaloids obtained from C. scoparius, Lupanine (Lup), and 17- oxo-sparteine (17- ox), and examined their neuroprotective properties in a cellular model of SO-Aß toxicity. Our results showed that Lup and 17- ox (both at 0.03µM) prevented SO-Aß-induced toxicity in PC12 cells (Lup: 64±7%; 17- ox: 57±6%). Similar results were seen in hippocampal neurons where these alkaloids prevented SO-Aß neurotoxicity (Lup: 57±2%; 17- ox: 52±3%) and increased the frequency of spontaneous calcium transients (Lup: 60±4%; 17- Ox: 40±3%), suggesting an enhancing effect on neural network activity and synaptic activity potentiation. All of the neuroprotective effects elicited by both alkaloids were completely blocked by α-bungarotoxin. Additionally, we observed that the presence of both Lup and 17- ox increased Akt phosphorylation levels (52±4% and 35±7%, respectively) in cells treated with SO-Aß (3 h). Taken together, our results suggest that the activation of nAChR by Lup and 17- ox induces neuroprotection in different cellular models, and appears to be an interesting target for the development of new pharmacological tools and strategies against AD.

A modified QuEChERS method coupled with liquid chromatography-tandem mass spectrometry for the simultaneous detection and quantification of scopolamine, L-hyoscyamine, and sparteine residues in animal-derived food products.

We developed a modified Quick, Easy, Cheap, Effective, Rugged, and Safe (CEN QuEChERS) extraction method coupled with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS) to identify and quantify residues of three botanical alkaloids, namely, scopolamine, L-hyoscyamine, and sparteine, in animal-derived foods, including porcine muscle, egg, and milk. A combination of ethylenediaminetetraacetic acid disodium buffer and acetonitrile acidified with 0.5% trifluoroacetic acid was used as an extraction solvent, whereas QuEChERS (CEN, 15662) kits and sorbents were applied for cleanup procedures. The proposed method was validated by determining the limits of quantification (LOQs), with values of 1-5 µg/kg achieved for the target analytes in various matrices. Linearity was estimated from matrix-matched calibration curves constructed using six concentration levels ranging from 1- to 6-fold increases in the LOQs of each analyte, and the correlation coefficients (R2 ) were ≥0.9869. Recoveries (at three concentration levels of 1-, 2-, and 3-fold increases in the LOQ) of 73-104% were achieved with relative standard deviations (RSDs) ≤7.7% (intra-day and inter-day precision). Ten types of each matrix procured from large markets were evaluated, and all tested samples showed negative results. The current protocol is simple and versatile and can be used for routine detection of plant alkaloids in animal food products.

Scientific opinion on the risks for animal and human health related to the presence of quinolizidine alkaloids in feed and food, in particular in lupins and lupin-derived products.

The European Commission asked EFSA for a scientific opinion on the risks for animal and human health related to the presence of quinolizidine alkaloids (QAs) in feed and food. This risk assessment is limited to QAs occurring in Lupinus species/varieties relevant for animal and human consumption in Europe (i.e. Lupinus albus L., Lupinus angustifolius L., Lupinus luteus L. and Lupinus mutabilis Sweet). Information on the toxicity of QAs in animals and humans is limited. Following acute exposure to sparteine (reference compound), anticholinergic effects and changes in cardiac electric conductivity are considered to be critical for human hazard characterisation. The CONTAM Panel used a margin of exposure (MOE) approach identifying a lowest single oral effective dose of 0.16 mg sparteine/kg body weight as reference point to characterise the risk following acute exposure. No reference point could be identified to characterise the risk of chronic exposure. Because of similar modes of action for QAs, the CONTAM Panel used a group approach assuming dose additivity. For food, the highest mean concentration of Total QAs (TotQAs) (i.e. the 6 most abundant QAs) was found in lupin seed samples classified as 'Lupins (dry) and similar-'. Due to the limited data on occurrence and consumption, dietary exposure was calculated for some specific scenarios and no full human health risk characterisation was possible. The calculated margin of exposures (MOEs) may indicate a risk for some consumers. For example, when lupin seeds are consumed without a debittering step, or as debittered lupin seeds high in QA content and when 'lupin-based meat imitates' are consumed. For horses, companion and farm animals, other than salmonids, the available database on adverse effects was too limited to identify no-observed-adverse-effect levels and/or lowest-observed-adverse-effect levels and no risk characterisation was possible. For salmonids, the CONTAM Panel considers the risk for adverse effects to be low.

Identification of brain areas sensitive to the toxic effects of sparteine.

Sparteine is one of the most toxic quinolizidine alkaloids found in leguminous plants. Several studies have demonstrated that sparteine affects the nervous system, blocking the nervous ganglion, producing antimuscarinic effects, depressing the central nervous system and causing neuronal necrosis. However, there are no reports identifying the areas of the brain that are sensitive to the toxic effects of this alkaloid. 32 adult Wistar rats were on study, sixteen were implanted with an intracerebral stainless steel cannula and randomly assigned to a control or experimental group (n=8). Animals, control and experimental, received daily intraventricular (ICV) injections of a sparteine or a sterile water solution for five consecutive days. Additionally, two groups of animals (8 rats each) received daily intraperotineal injections (IP) of a sparteine or sterile water solution for five consecutive days. 72h after the last dose, the animals were sacrificed, their brains removed, fixed and embedded in paraffin to obtain 10µm tissue slices. Brain slices were stained with H&E and evaluated under a light microscope. The main brain structures sensitive to sparteine were the cerebral cortex (frontal, fronto-parietal and striate) olfactory and amygdaloid areas, the ventromedial hypothalamic nucleus, the Purkinje cells in the cerebellum, and the CA1, CA3 and dentate gyrus regions of the hippocampus. Administration of sparteine, via ICV or IP, caused neuronal necrosis in brain structures, mainly related with cholinergic pathways.

Anagyrine desensitization of peripheral nicotinic acetylcholine receptors. A potential biomarker of quinolizidine alkaloid teratogenesis in cattle.

Anagyrine, a teratogenic quinolizidine alkaloid found in Lupinus spp., is proposed to undergo metabolism by pregnant cattle to a piperidine alkaloid which inhibits fetal movement, the putative mechanism behind crooked calf syndrome. The objective of this study was to test the hypothesis that anagyrine but not lupanine or sparteine can directly, without metabolism, desensitize nicotinic acetylcholine receptors (nAChR) in a cell culture model. SH-SY5Y cells expressing autonomic nAChR, and TE-671 cells expressing fetal muscle-type nAChR were exposed to lupine alkaloids or Dimethylphenylpiperazinium (DMPP) in log10 molar increments from 10nM to 100µM and then to a fixed concentration of acetylcholine (ACh) (10µM for SH-SY5Y cells and 1µM for TE-671 cells) and the responses measured with a membrane potential sensing dye to assess nAChR activation and desensitization. The selective ganglionic nAChR agonist DMPP used as a positive control, was a potent activator and desensitizer of nAChR expressed by SH-SY5Y cells. Lupanine was a weak agonist and desensitizer in SH-SY5Y cells and sparteine was without effect. Anagyrine acted as a partial agonist in both cell lines with EC50 values of 4.2 and 231µM in SH-SY5Y and TE-671 cells, respectively. Anagyrine was a desensitizer of nAChR with DC50 values of 6.9 and 139µM in SH-SY5Y and TE-671 cells, respectively. These results confirm the hypothesis that anagyrine is a potent and effective desensitizer of nAChR, and that anagyrine can directly, without metabolism, desensitize nAChR. Moreover, serum anagyrine concentrations may be a potential biomarker for lupine teratogenicity in cattle.

C2-Modified Sparteine Derivatives Are a New Class of Potentially Long-Acting Sodium Channel Blockers.

The lupin alkaloid sparteine is a well-known chiral diamine with a range of applications in asymmetric synthesis, as well as a blocker of voltage-gated sodium channels (VGSCs). However, there is only scarce information on the VGSC-blocking activity of sparteine derivatives where the structure of the parent alkaloid is retained. Building on the recent renewed availability of sparteine and derivatives we report herein how modification of sparteine at position 2 produces irreversible blockers of VGSCs. These compounds could be clinically envisaged as long-lasting local anesthetics.

Evaluación de la susceptibilidad in vitro a esparteína, en cuatro cepas de Mycobacterium tuberculosis / Evaluation of in vitro susceptibility to sparteine in four strains of Mycobacterium tuberculosis

RESUMEN La esparteína es un alcaloide con actividad bacteriostática sobre el género Mycobacterium. El objetivo de este trabajo fue evaluar la acción antimicrobiana de la esparteína en el crecimiento de cuatro cepas ATCC de Mycobacterium tuberculosis (susceptible, resistente a isoniazida, resistente a rifampicina y multidrogorresistente) in vitro. La evaluación de la actividad bactericida del sulfato de esparteína se realizó a través de una adaptación del método de ensayo de cultivo y susceptibilidad a medicamentos antituberculosos mediante observación microscópica (MODS, por sus siglas en inglés), según el protocolo descrito en el manual técnico elaborado por el Instituto Nacional de Salud. Los resultados demuestran que a concentraciones de 25; 50 y 100 mM de sulfato de esparteína, no se desarrollan unidades formadoras de colonia en las cuatro cepas evaluadas de Mycobacterium tuberculosis. Los resultados demuestran el potencial efecto antimicrobiano in vitro de la esparteína en la tuberculosis multidrogorresistente.

ABSTRACT Sparteine is an alkaloid with bacteriostatic activity on the genus Mycobacterium. The aim of this study was to evaluate the antimicrobial activity of sparteine on the growth of 4 ATCC strains of Mycobacterium tuberculosis (susceptible, resistant to isoniazid, resistant to rifampicin and multidrug-resistant) in vitro. Validation of bactericidal activity of sparteine sulfate was carried out through an adaptation of the Microscopic-Observation Drug-Susceptibility (MODS) method according to the guidelines of the Peruvian National Health Institute. The results demonstrate that at concentrations of 25; 50 and 100 Mm of sparteine sulfate, there is no development of colony-forming units in any of the 4 evaluated strains. Our results demonstrate the potential in vitro antimicrobial effect of sparteine on multidrug-resistant tuberculosis.

Sparteine as an anticonvulsant drug: Evidence and possible mechanism of action.

Sparteine is a quinolizidine alkaloid extracted from Lupinus that has numerous pharmacological properties both in humans and animal models. In the central nervous system, sparteine reduces locomotor activity, has light analgesic effects, also has no effects on short-term memory or spatial learning and does not induce changes in behavior or electroencephalographic (EEG) activity. However, the anticonvulsant profile of sparteine is not fully characterized in experimental animals and there are no data in humans. Therefore, the present review focuses on the experimental evidence supporting the anticonvulsant action of sparteine in models of acute seizures and status epilepticus (SE), as well as its possible mechanisms of action. The evidence that supports the anticonvulsant effect of (-)-Sparteine sulfate includes the inhibition of seizures induced by maximal electro-stimulation, a delay in the onset of convulsive behavior and the prolongation of survival time in mice treated with pentylenetetrazole (PTZ). Additionally, sparteine delays the onset of convulsive behavior and decreases the severity and mortality of rats treated with PTZ and pilocarpine. Sparteine decreases amplitude and frequency or blocks the epileptiform activity induced by PTZ, pilocarpine and kainic acid. Sparteine may decrease hyperexcitability through the activation of the M2 and M4 subtypes of mAChRs, which is a probable mechanism of action that together with its systemic effects may favor its anticonvulsant effects against seizures and SE.

Effect of sparteine on status epilepticus induced in rats by pentylenetetrazole, pilocarpine and kainic acid.

The long-term effects of status epilepticus (SE) include severe clinical conditions that result in disorders of various organs and systems as well as neurological damage that could lead to death. Sparteine is a quinolizidine alkaloid synthesized from most Lupine species, and its anticonvulsive effect was evaluated in the pentylenetetrazole model of SE. However, efforts to clearly determine the anticonvulsive effect of sparteine have not been made previously. For this reason, we consider it important to study the anticonvulsant effects of sparteine at the level of behavior and EEG activity in three different SE models. The animals of the control groups, which received intraperitoneal pentylenetetrazole (90 mg/kg), kainic acid (9 mg/kg) or pilocarpine (370 mg/kg), exhibited convulsive behavior and epileptiform activity. After sparteine pretreatment (13 mg/kg, administered 30 min before the convulsive drug), the animals administered pentylenetetrazole and pilocarpine exhibited reduced mortality rates compared with the corresponding control groups, while the animals administered kainic acid exhibited a delayed onset of convulsive behavior and decreased seizure duration compared with the corresponding control group. In the three models of SE, a significant reduction in the amplitude and frequency of discharge trains was observed. These results support the anticonvulsant effect of low doses of sparteine and allow us to direct our efforts to other new anticonvulsant strategies for seizure treatment. However, it is necessary to perform more experiments to determine the precise mechanism through which sparteine produces an anticonvulsant effect at this concentration.

Solid-State Structural Investigation of an Organolithium (-)-Sparteine Complex: η3 -N-Boc-N-(p-methoxyphenyl)-3-phenylallyllithium⋠(-)-Sparteine.

A η3 monomeric alkyllithium⋅(-)-sparteine complex has been isolated and characterized in the solid state (see picture). Determination of the absolute configuration of this key intermediate in asymmetric metalation/substitution sequences of N-tert-butoxycarbonyl-N-(p-methoxyphenyl)cinnamylamine allows definitive assignment of the stereochemical course of its electrophile-dependent substitution reactions.