Pseudolycorine N-oxide, a new N-oxide from Narcissus tazetta.

A new N-oxide, Pseudolycorine N-oxide (1) was characterised along with eleven known alkaloids homolycorine (2), O-methylmaritidine (3), 8-O-demethylhomolycorine (4), homolycorine N-oxide (5), lycorine (6), narciclasine (7), pseudolycorine (8), ungeremine (9), 8-O-demethylmaritidine (10), zefbetaine (11) and lycorine N-oxide (12), from Narcissus tazetta. Their structures were established on the basis of spectroscopic data analysis. The extract, fractions and isolated compounds were screened for in vitro cytotoxicity against two human cancer cell lines, human cervical cancer (SiHa) and human epidermoid carcinoma (KB) cells. The study demonstrated the cytotoxic potential of extract and its chloroform and n-butanol fractions. Further, the results revealed the bioactive potential of narciclasine, pseudolycorine and homolycorine alkaloids. However, new N-oxide (1) was not active against these cell lines.

Quantitative analysis of drug distribution by ambient mass spectrometry imaging method with signal extinction normalization strategy and inkjet-printing technology.

Quantitative mass spectrometry imaging (MSI) is a robust approach that provides both quantitative and spatial information for drug candidates' research. However, because of complicated signal suppression and interference, acquiring accurate quantitative information from MSI data remains a challenge, especially for whole-body tissue sample. Ambient MSI techniques using spray-based ionization appear to be ideal for pharmaceutical quantitative MSI analysis. However, it is more challenging, as it involves almost no sample preparation and is more susceptible to ion suppression/enhancement. Herein, based on our developed air flow-assisted desorption electrospray ionization (AFADESI)-MSI technology, an ambient quantitative MSI method was introduced by integrating inkjet-printing technology with normalization of the signal extinction coefficient (SEC) using the target compound itself. The method utilized a single calibration curve to quantify multiple tissue types. Basic blue 7 and an antitumor drug candidate (S-(+)-deoxytylophorinidine, CAT) were chosen to initially validate the feasibility and reliability of the quantitative MSI method. Rat tissue sections (heart, kidney, and brain) administered with CAT was then analyzed. The quantitative MSI analysis results were cross-validated by LC-MS/MS analysis data of the same tissues. The consistency suggests that the approach is able to fast obtain the quantitative MSI data without introducing interference into the in-situ environment of the tissue sample, and is potential to provide a high-throughput, economical and reliable approach for drug discovery and development.

Further studies on pumiliotoxin 251D and hydroquinone content of the skin secretion of Melanophryniscus species (Anura, Bufonidae) from Uruguay.

In whole animal ethanolic extracts from adult specimens of Melanophryniscus atroluteus (27 specimens) and M. devincenzii (16 specimens) as well as of two egg clutches and four tadpole samples from the latter species, the major alkaloid pumiliotoxin (PTX) 251D and hydroquinone were assayed quantitatively by gas chromatography/mass spectrometry. All toad extracts contained high concentrations of PTX 251D and hydroquinone and exhibited considerable variation in the content of these compounds among individual specimens. The extracts of the eggs and tadpoles were entirely free of alkaloids as well as hydroquinone, pointing to a dietary origin of these compounds.

Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: Changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes.

We examined the effects of genetic transformation by Agrobacterium rhizogenes on the production of tylophorine, a phenanthroindolizidine alkaloid, in the Indian medicinal plant, Tylophora indica. Transformed roots induced by the bacterium grew in axenic culture and produced shoots or embryogenic calli in the absence of hormone treatments. However, hormonal treatment was required to regenerate shoots in root explants of wild type control plants. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes, which include, short internodes, small and wrinkled leaves, more branches and numerous plagiotropic roots. Plants regenerated from transformed roots showed increased biomass accumulation (350-510% in the roots and 200-320% in the whole plants) and augmented tylophorine content (20-60%) in the shoots, resulting in a 160-280% increase in tylophorine production in different clones grown in vitro.

On-line identification of phenanthroindolizidine alkaloids in a crude extract from Tylophora atrofolliculata by liquid chromatography combined with tandem mass spectrometry.

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with sequential mass spectrometry (LC/MS(n)) were applied to identify trace-level phenanthroindolizidine alkaloids in crude extracts from Tylophora atrofolliculata. Based on the relationship between the characteristic fragmentation reactions and the structural features of related compounds of known structure from this plant, the bioactive crude extract was analyzed in detail by positive and negative ion ESI-MS(n), LC/UV-MS and LC/MS(n) techniques. A total of nine constituents in the crude extract were identified rapidly, including several isomers; seven of these constituents are new and two are known compounds. The structures of four of these constituents were subsequently confirmed by nuclear magnetic resonance (NMR) and accurate mass measurements using high-resolution fast-atom bombardment mass spectrometry (FAB-HRMS).

Preparative isolation of swainsonine from locoweed: extraction and purification procedures.

The trihydroxy indolizidine alkaloid swainsonine, a plant toxin with potent alpha-mannosidase-inhibitory activity and chemotherapeutic potential, was isolated in gram quantities from locoweed (Astragalus lentiginosus). The key isolation and purification step was a continuous liquid/liquid extraction procedure using dichloromethane to extract a basified aqueous methanol solution obtained after isolation of the polar base fraction by ion-exchange. The concentration of swainsonine was increased from ca. 7% in the polar base material to 68% using the liquid/liquid extraction procedure. Pure swainsonine was then obtained by recrystallisation from ammonia-saturated chloroform or by sublimation. Small samples of swainsonine were also purified by formation of the chloroform-soluble methylboronate derivative, from which the alkaloid could be regenerated easily by hydrolysis.

A flexible approach toward trisubstituted piperidines and indolizidines: synthesis of 6-epi-indolizidine 223A.

2,5,6-Trisubstituted piperidines are readily prepared by a combination of an aza-Achmatowicz oxidation of a furyl-substituted benzenesulfonamide followed by a conjugate addition to the resulting 2H-pyridone and subsequent addition of various nucleophiles to a transient N-sulfonyliminium ion. The stereochemistry of the conjugate addition product is the result of axial attack from the face opposite the diaxial substituents at C(2) and C(6). This can be attributed to steric hindrance between the pseudoaxially oriented 2,6-substituents and the equatorially approaching nucleophile, thereby leading to the exclusive formation of the kinetically favored axial 1,4-adduct. Indolizidine alkaloid 223A was isolated from a skin extract of a Panamanian population of the dendrobatid Dendrobates pumilio Schmidt (Dendrobatidae). Synthesis of the originally proposed structure of this alkaloid was achieved in 13 steps in 13.1% overall yield by using an aza-Achmatowicz oxidative rearrangement and a diastereoselective 1,4-conjugate addition as the key steps. The structure of the natural 223A alkaloid (5b) differs from that of the epi-isomer 5a synthesized in this study in the configuration at the 6-position of the indolizidine ring.

Evaluation of the excretion and metabolism of the new analgesic agent RWJ-22757 in male and female CR Wistar rats.

The excretion and metabolism of (+/-)-trans-3-(2-bromophenyl)octahydroindolizine hydrochloride (RWJ-22757) have been investigated in male and female CR Wistar rats. Radiolabeled [14C] RWJ-22757 was administered orally to each of the rats as a single 60 mg/kg suspension dose. Plasma (0-48 h), urine (0-168 h) and fecal (0-168 h) samples were collected and analyzed. There were no significant gender differences observed in the data. The estimated elimination half-life of the total radioactivity from plasma was 19 h while the estimated elimination half-life of RWJ-22757 was 15 h. Recoveries of total radioactivity in urine and feces were 58.4+/-5.8 and 42.4+/-6.3%, respectively. RWJ-22757 and a total of 11 metabolites were isolated in rat plasma, urine, and fecal extracts. The structures of four of these metabolites were tentatively identified. Unchanged RWJ-22757 accounted for < 4% of the dose in plasma and urine and 28% in feces; thus, indicating the drug was extensively metabolized and either not absorbed well or biliary excreted. Identified metabolites accounted for > 80% of the total radioactivity contained in the samples. The following pathways were used to describe the formation of the metabolites identified in rats: octahydroindolizine ring oxidation, phenyl hydroxylation, octahydroindolizine ring oxidation followed by ring opening to a carboxylic acid function and octahydroindolizine ring oxidation followed by ring opening and N-methylation.

Sequestration of phenanthroindolizidine alkaloids by an Asclepiadaceae-feeding danaid butterfly, Ideopsis similis.

3-Demethyl-14alpha-hydroxyisotylocrebrine N-oxide was isolated along with three homologous isotylocrebrine type alkaloids from the imagines and pupae of Ideopsis similis reared on the host plant, Tylophora tanakae.

Development and evaluation of an immunological approach for the identification of novel acetyl coenzyme-A carboxylase inhibitors: assay optimization and pilot screen results.

Cyclohexanediones, aryloxyphenoxypropionates, indolizidinediones, and triazinediones are four known structural classes of herbicides that inhibit acetyl coenzyme-A carboxylase (ACCase; EC 6.4.1.2). An immunological study to determine the potential of ACCase inhibitor-specific monoclonal antibodies as screening tools to identify novel lead chemistry was undertaken. Using two cyclohexanedione-specific monoclonal antibodies (mAb A and mAb B; Webb, S. R.; Hall, J. C. J. Agric. Food Chem. 2000, 48, 1210-1218) and three different cyclohexanedione hapten coating conjugates, competitive indirect enzyme-linked immunosorbent assays (ciELISA) were developed. Cross-reactivity of the monoclonal antibodies with four structural classes of ACCase inhibitors revealed that the ciELISA using mAb A and a modified cyclohexanedione hapten coating conjugate detected analogues from all four known classes of ACCase inhibitors. A pilot screen using this ciELISA format identified two novel ACCase inhibitors, demonstrating the potential for antibodies as rapid and cost-effective screening tools for identifying novel lead chemistry in pesticide discovery programs.

A novel (5E,9Z)-dialkylindolizidine from the ant Monomorium smithii.

The alkaloidal venom of Monomorium smithii was found to contain (5E,9Z)-3-butyl-5-(4-penten-1-yl)indolizidine [1b], a novel indolizidine, its monocyclic analogue trans-2-butyl-5-(8-nonen-1-yl)pyrrolidine [2], (5E,8Z)-3,5-di(5-hexen-1-yl)pyrrolizidine [3], and trans-2-(5-hexen-1-yl)-5-(8-nonen-1-yl)pyrrolidine [4]. The structure of 1b was based on the results of two independent syntheses. Reductive amination of the appropriate triketone confirmed the carbon-nitrogen skeleton of 1b and suggested its stereochemistry, which was verified by the results of a stereoselective synthesis based on pyrrole hydrogenation. The chemotaxonomic implications of this first report of the concomitance of a 3,5-dialkylindolizidine and a 3,5-dialkylpyrrolizidine in an ant venom are discussed.

Alkaloids from dendrobatid frogs: structures of two omega-hydroxy congeners of 3-butyl-5-propylindolizidine and occurrence of 2,5-disubstituted pyrrolidines and a 2,6-disubstituted piperidine.

Forty alkaloids were detected and characterized from skin extracts of high- and low-elevation populations of the poison frog Dendrobates histrionicus from northwestern Colombia. Combined gc/ms with NH3 or ND3 in a chemical ionization mode detected protonated parent ions and determined the number of exchangeable NH and OH hydrogens. Six previously unknown dendrobatid alkaloids were characterized. Two were 2,5-disubstituted pyrrolidines, which included pyrrolidine 197B, a trans-2-butyl-5-pentylpyrrolidine, while a third was a 2,6-dipentylpiperidine. Indolizidines 239AB and 239CD had the same relative configuration as the parent alkaloid 223AB [(5E,9E)3-butyl-5-propylindolizidine] and contained, respectively, a omega-hydroxy group in the propyl or butyl side chain. The profiles of alkaloids in the new northern populations of D. histrionicus are typical of the species in containing a set of about eight histrionicotoxins, in marked contrast to a related species, Dendrobates lehmanni, which does not contain histrionicotoxins.