Purification of five azaspiracids from mussel samples contaminated with DSP toxins and azaspiracids.

Human intoxications during toxic episodes in shellfish are a very important concern for public health, as well as for economic interests of producer regions. Although initially each toxin appeared in a determined geographical zone, nowadays many of them are found in multiple places worldwide. In addition, more toxic compounds (new toxins or new analogs of known toxins) are being isolated and identified, which bring about new risks for public health. An example of this situation is the group of azaspiracids (AZAs). Initially these toxins were concentrated in Irish coasts but today appear in many different geographic locations; in the first toxic episode only three analogs were isolated, but now it is known that the group is comprised of at least eleven identified compounds. A substantial problem associated with all these new toxins is the extreme difficulty associated with the study of their toxic effects and mechanisms of action due to the very small quantities of purified toxin available. Therefore, the study of procedures to isolate them from contaminated shellfish or to synthesize them is of tremendous importance. In this paper we design a complete procedure to obtain AZAs analogs from mussels contaminated with DSP toxins and azaspiracids by means of three consecutive steps: an extraction procedure to remove toxins from shellfish, a solid phase extraction (SPE) to clean the samples and separate DSP toxins and AZAs, and a preparative HPLC to isolate each analog. In all the steps LC/MS is used to detect and quantify the toxins. Large amounts of AZA1, AZA2, AZA3, AZA4 and AZA5 were obtained by use of this procedure, which can be utilized in future studies relating to the toxins such as the production of certified materials and standards.

Discovery of a new class of potent, selective, and orally bioavailable CRTH2 (DP2) receptor antagonists for the treatment of allergic inflammatory diseases.

A novel chemical class of potent chemoattractant receptor-homologous expressed on Th2 lymphocytes (CRTH2 or DP2) antagonists is reported. An initial and moderately potent spiro-indolinone compound ( 5) was found during a high-throughput screening campaign. Structure-activity relationship (SAR) investigation around the carboxylic acid group revealed that changes in this part of the molecule could lead to a reversal of functional activity, yielding weakly potent agonists. SAR investigation of the succinimide functional group led to the discovery of several single-digit nanomolar antagonists. The potency of these compounds was confirmed in a human eosinophil chemotaxis assay. Moreover, compounds ( R)- 58 and ( R)- 71 were shown to possess pharmacokinetic properties suitable for development as an orally bioavailable drug.

Discovery of N-(2-aryl-cyclohexyl) substituted spiropiperidines as a novel class of GlyT1 inhibitors.

Screening of the Roche compound library led to the identification of cis-N-(2-phenyl-cyclohexyl)-spiropiperidine 1 as structurally novel GlyT1 inhibitor. The SAR, which was developed in this series, resulted in the discovery of highly potent compounds displaying excellent selectivity against the GlyT2 isoform.

Discovery of N-(2-hydroxy-2-aryl-cyclohexyl) substituted spiropiperidines as GlyT1 antagonists with improved pharmacological profile.

During SAR exploration of N-(2-aryl-cyclohexyl) substituted spiropiperidine as GlyT1 inhibitors, it was found that introduction of an hydroxy group in position 2 of the cyclohexyl residue considerably improves the pharmacological profile. In particular, reduction of the binding affinity at the nociceptin/orphanin FQ peptide and the mu opioid receptors was achieved.

Aspirochlorine class compounds from Aspergillus flavus inhibit azole-resistant Candida albicans.

Dereplication of the antifungal extracts of Aspergillus flavus indicated that the primary antifungal compound present was the known aspirochlorine (1). Preparative isolation work resulted in the identification of the new compounds tetrathioaspirochlorine (2) and cyclo(D-N-methyl-Leu-L-Trp) (3).

Liquid chromatography--multiple tandem mass spectrometry for the determination of ten azaspiracids, including hydroxyl analogues in shellfish.

Azaspiracids (AZAs) are a group of polyether toxins that cause food poisoning in humans. These toxins, produced by marine dinoflagellates, accumulate in filter-feeding shellfish, especially mussels. Sensitive liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS(n)) methods have been developed for the determination of the major AZAs and their hydroxyl analogues. These methods, utilising both chromatographic and mass resolution, were applied for the determination of 10 AZAs in mussels (Mytilus edulis). An optimised isocratic reversed phase method (3 microm Luna-2 C18 column) separated 10 azaspiracids using acetonitrile/water (46:54, v/v) containing 0.05% trifluoroacetic acid (TFA) and 0.004% ammonium acetate in 55 min. Analyte determination using MS3 involved trapping and fragmentation of the [M + H]+ and [M + H - H2O]+ ions with detection of the [M + H - 2H2O]+ ion for each AZA. Linear calibrations were obtained for AZA1, using spiked shellfish extracts, in the range 0.05-1.00 microg/ml (r2 = 0.997) with a detection limit of 5 pg (signal : noise = 3). The major fragmentation pathways in hydroxylated azaspiracids were elucidated using hydrogen/deuterium (H/D) exchange experiments. An LC-MS3 method was developed using unique parent ions and product ions, [M + H - H2O - CgH10O2R1R3]+, that involved fragmentation of the A-ring. This facilitated the discrimination between 10 azapiracids, AZA1-10. Thus, this rapid LC-MS3 method did not require complete chromatographic resolution and the run-time of 7 min had detection limits better than 20 pg for each toxin.

Detection of five new hydroxyl analogues of azaspiracids in shellfish using multiple tandem mass spectrometry.

The polyether dinoflagellate toxins, azaspiracids, are responsible for azaspiracid poisoning (AZP), a new human toxic syndrome arising from the consumption of shellfish. To date, five azaspiracids have been isolated and fully structurally elucidated, including, AZA1, its 8-methyl and 22-demethyl analogues, AZA2 and AZA3, respectively, and two hydroxyl derivatives of AZA3, named AZA4 and AZA5. Using a recently developed method involving liquid chromatography with multiple tandem mass spectrometry (LC-MS(n)), five new azaspiracids, AZA7-AZA11, have been found in mussels (Mytilus edulis). AZA6 is a positional isomer of AZA1 and four of the new compounds are isomers with a mass of 857.5 amu. AZA7 and AZA8 are hydroxyl analogues of AZA1 while AZA9 and AZA10 are hydroxyl analogues of AZA6. AZA11 is a hydroxyl analogue of AZA2. The separation of all 11 azaspiracids was achieved using isocratic reversed phase liquid chromatography using a combination of eluent additives, trifluoroacetic acid and ammonium acetate. The ion-trap MS experiments, with electrospray ionisation, involved the fragmentation of the protonated molecule [M+H](+), trapping and fragmenting the product ions due to the loss of a water molecule [M+H-H(2)O](+), together with mass spectral data analysis that included the characteristic A-ring fragmentation for each compound.