Charged Tags for the Identification of Oxidative Drug Metabolites Based on Electrochemistry and Mass Spectrometry.

Most of the active pharmaceutical ingredients like Metoprolol are oxidatively metabolized by liver enzymes, such as Cytochrome P450 monooxygenases into oxygenates and therefore hydrophilic products. It is of utmost importance to identify the metabolites and to gain knowledge on their toxic impacts. By using electrochemistry, it is possible to mimic enzymatic transformations and to identify metabolic hot spots. By introducing charged-tags into the intermediate, it is possible to detect and isolate metabolic products. The identification and synthesis of initially oxidized metabolites are important to understand possible toxic activities. The gained knowledge about the metabolism will simplify interpretation and predictions of metabolitic pathways. The oxidized products were analyzed with high performance liquid chromatography-mass spectrometry using electrospray ionization (HPLC-ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. For proof-of-principle, we present a synthesis of one pyridinated main oxidation product of Metoprolol.

Novel Synthesis of Substituted 2-Trifluoromethyl and 2-Perfluoroalkyl N-Arylpyridinium Compounds-Mechanistic Insights.

We report a new one-pot synthesis of 2-trifluoromethylated/2-perfluoroalkylated N-aryl-substituted pyridiniums, 5,6,7,8-tetrahydroquinoliniums and 6,7,8,9-tetrahydro-5H-cyclohepta[b]-pyridinium compounds starting from an activated ß-dicarbonyl analogue (here a perfluoro-alkylated gem-iodoacetoxy derivative), an aromatic amine and a (cyclic or acyclic) ketone. The key step of this multicomponent reaction, involves the formation of a 3-perfluoroalkyl-N,N'-diaryl-1,5-diazapentadiene intermediate, various examples of which were isolated and characterized for the first time, together with investigation of their reactivity. We propose a mechanism involving a concurrent inverse electron demand Diels-Alder or Aza-Robinson cascade cyclisation, followed by a bis-de-anilino-elimination. Noteworthy, a meta-methoxy substituent on the aniline directs the reaction towards a 2-perfluoroalkyl-7-methoxyquinoline, resulting from the direct cyclization of the diazapentadiene intermediate, instead of pyridinium formation. This is the first evidence of synthesis of pyridinium derivatives from activated ß-dicarbonyls, ketones, and an aromatic amine, the structures of which (both reactants and products) being analogous to species involved in biological systems, especially upon neurodegenerative diseases such as Parkinson's. Beyond suggesting chemical/biochemical analogies, we thus hope to outline new research directions for understanding the mechanism of in vivo formation of pyridiniums, hence possible pharmaceutical strategies to better monitor, control or prevent it.

Identification of a 3-Alkylpyridinium Compound from the Red Sea Sponge Amphimedon chloros with In Vitro Inhibitory Activity against the West Nile Virus NS3 Protease.

Viruses are underrepresented as targets in pharmacological screening efforts, given the difficulties of devising suitable cell-based and biochemical assays. In this study we found that a pre-fractionated organic extract of the Red Sea sponge Amphimedon chloros was able to inhibit the West Nile Virus NS3 protease (WNV NS3). Using liquid chromatography⁻mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the identity of the bioactive compound was determined as a 3-alkylpyridinium with m/z = 190.16. Diffusion Ordered Spectroscopy (DOSY) NMR and NMR relaxation rate analysis suggest that the bioactive compound forms oligomers of up to 35 kDa. We observed that at 9.4 µg/mL there was up to 40⁻70% inhibitory activity on WNV NS3 protease in orthogonal biochemical assays for solid phase extracts (SPE) of A. chloros. However, the LC-MS purified fragment was effective at inhibiting the protease up to 95% at an approximate amount of 2 µg/mL with negligible cytotoxicity to HeLa cells based on a High-Content Screening (HCS) cytological profiling strategy. To date, 3-alkylpyridinium type natural products have not been reported to show antiviral activity since the first characterization of halitoxin, or 3-alkylpyridinium, in 1978. This study provides the first account of a 3-alkylpyridinium complex that exhibits a proposed antiviral activity by inhibiting the NS3 protease. We suggest that the here-described compound can be further modified to increase its stability and tested in a cell-based assay to explore its full potential as a potential novel antiviral capable of inhibiting WNV replication.

Anti-mycobacterial alkaloids, cyclic 3-alkyl pyridinium dimers, from the Indonesian marine sponge Haliclona sp.

Three new dimeric 3-alkyl pyridinium alkaloids, named haliclocyclamines A-C (1-3), were isolated together with five known congeners, cyclostellettamines A (4), B (5), C (6), E (7), and F (8), from the Indonesian marine sponge Haliclona sp. The structures of 1-3 were assigned based on their spectroscopic data (1D and 2D NMR, HRFABMS, ESIMS/MS, UV, and IR). Compounds 1-8 exhibited antimicrobial activities against Mycobacterium smegmatis with inhibition zones of 17, 10, 13, 14, 8, 8, 12, and 12mm, respectively, at 10µg/disc. Compounds 3 and 8 also modestly inhibited the activity of vaccinia H-1-related phosphatase (VHR), a dual-specificity phosphatase, at 17-18µM.

Identification of Pyridinium with Three Indole Moieties as an Antimicrobial Agent.

A novel pyridinium with three indole moieties, tricepyridinium, was obtained from the culture of an Escherichia coli clone incorporating metagenomic libraries from the marine sponge Discodermia calyx. For the important structural elements of tricepyridinium to be investigated for antibacterial activity, tricepyridinium and its analogues were chemically synthesized. Tricepyridinium had antimicrobial activity, but not against E. coli, and cytotoxicity against P388 cells. Additional bioassays with its synthetic analogues revealed that the intriguing combination of the indole moieties, most likely derived from three tryptamines, as well as the pyridinium moiety were chiefly responsible for its potent biological activities.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry quantitative method for the cellular analysis of varying structures of gemini surfactants designed as nanomaterial drug carriers.

Diquaternary gemini surfactants have successfully been used to form lipid-based nanoparticles that are able to compact, protect, and deliver genetic materials into cells. However, what happens to the gemini surfactants after they have released their therapeutic cargo is unknown. Such knowledge is critical to assess the quality, safety, and efficacy of gemini surfactant nanoparticles. We have developed a simple and rapid liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantitative determination of various structures of gemini surfactants in cells. Hydrophilic interaction liquid chromatography (HILIC) was employed allowing for a short simple isocratic run of only 4min. The lower limit of detection (LLOD) was 3ng/mL. The method was valid to 18 structures of gemini surfactants belonging to two different structural families. A full method validation was performed for two lead compounds according to USFDA guidelines. The HILIC-MS/MS method was compatible with the physicochemical properties of gemini surfactants that bear a permanent positive charge with both hydrophilic and hydrophobic elements within their molecular structure. In addition, an effective liquid-liquid extraction method (98% recovery) was employed surpassing previously used extraction methods. The analysis of nanoparticle-treated cells showed an initial rise in the analyte intracellular concentration followed by a maximum and a somewhat more gradual decrease of the intracellular concentration. The observed intracellular depletion of the gemini surfactants may be attributable to their bio-transformation into metabolites and exocytosis from the host cells. Obtained cellular data showed a pattern that grants additional investigations, evaluating metabolite formation and assessing the subcellular distribution of tested compounds.

Antifouling activity of synthetic alkylpyridinium polymers using the barnacle model.

Polymeric alkylpyridinium salts (poly-APS) isolated from the Mediterranean marine sponge, Haliclona (Rhizoniera) sarai, effectively inhibit barnacle larva settlement and natural marine biofilm formation through a non-toxic and reversible mechanism. Potential use of poly-APS-like compounds as antifouling agents led to the chemical synthesis of monomeric and oligomeric 3-alkylpyridinium analogues. However, these are less efficient in settlement assays and have greater toxicity than the natural polymers. Recently, a new chemical synthesis method enabled the production of poly-APS analogues with antibacterial, antifungal and anti-acetylcholinesterase activities. The present study examines the antifouling properties and toxicity of six of these synthetic poly-APS using the barnacle (Amphibalanus amphitrite) as a model (cyprids and II stage nauplii larvae) in settlement, acute and sub-acute toxicity assays. Two compounds, APS8 and APS12-3, show antifouling effects very similar to natural poly-APS, with an anti-settlement effective concentration that inhibits 50% of the cyprid population settlement (EC50) after 24 h of 0.32 mg/L and 0.89 mg/L, respectively. The toxicity of APS8 is negligible, while APS12-3 is three-fold more toxic (24-h LC50: nauplii, 11.60 mg/L; cyprids, 61.13 mg/L) than natural poly-APS. This toxicity of APS12-3 towards nauplii is, however, 60-fold and 1200-fold lower than that of the common co-biocides, Zn- and Cu-pyrithione, respectively. Additionally, exposure to APS12-3 for 24 and 48 h inhibits the naupliar swimming ability with respective IC50 of 4.83 and 1.86 mg/L.

Retinal metabolism in humans induces the formation of an unprecedented lipofuscin fluorophore 'pdA2E'.

Toxic lipofuscin in the RPE (retinal pigment epithelium) is implicated in blindness in AMD (age-related macular degeneration) or recessive Stargardt's disease patients. In the present study, we identified a novel fluorescent lipofuscin component in human and bovine RPEs. Using 1D and 2D NMR and MS, we confirmed the structure of this pigment and called it pdA2E. It exhibits absorbance maxima at 492 and 342 nm, and is susceptible to photocatalytic isomerization and oxidation. This fluorophore was also detected in the eyecup extracts of Abca4(-/-)Rdh8(-/-) (Abca4 encodes ATP-binding cassette transporter 4 and Rdh8 encodes retinol dehydrogenase 8) mice, an AMD/recessive Stargardt's disease model. Excess amassing of pdA2E within RPE cells caused significant cell viability loss and membrane damage. The formation of pdA2E occurred when atRAL (all-trans-retinal) reacted with excess ethanolamine in the absence of acetic acid, and the process is likely to involve the participation of three atRAL molecules. Our findings suggest that endogenous pdA2E may serve as a sensitizer for yielding singlet oxygen and a singlet oxygen quencher, as well as a by-product of retinal metabolism, and its complete characterization facilitates the understanding of biosynthetic pathways by which adverse RPE lipofuscin constituents form.

Spectrophotometric method for the determination of total thiols in human urine.

Thiols have been of enduring interest for many years because of their role in biological and pharmacological processes. Monitoring of total thiols content is very important in order to understand their function in living organisms. This paper describes the spectrophotometric method for the determination of total thiols concentration in urine. The method is based on derivatization with 1-benzyl-2-chloropyridinium bromide and ultraviolet detection of S-pyridinium derivatives at 316 nm. The analytical recovery and RSD values for precision within the calibration range were from 95.7 to 102.9% and from 2.1 to 8.4%, respectively. The concentration of total thiols normalized against creatinine for 38 apparently healthy subjects (19 women and 19 men) occurred in the range 17.2-73.7 and 25.7-83.6 mmol/mol creatinine, respectively. There was no difference in the urinary excretion of thiols in men and women, but there was a significant statistical correlation between urine total thiols and age in the studied group.

Cyclic 3-alkyl pyridinium alkaloid monomers from a New Zealand Haliclona sp. marine sponge.

Bioassay and NMR approaches have been used to guide the isolation of one known and two new cyclic 3-alkyl pyridinium alkaloid (3-APA) monomers from the New Zealand marine sponge Haliclona sp. The new compounds, dehydrohaliclocyclins C (3) and F (4), are the first reported examples of cyclic 3-APA monomers with unsaturation in the alkyl chain. The known compound haliclocyclin C (2) was also isolated from a mixture with 4. The structures of compounds 2-4 were elucidated using NMR spectroscopy, mass spectrometry, and chemical degradation.

Toxicity of the synthetic polymeric 3-alkylpyridinium salt (APS3) is due to specific block of nicotinic acetylcholine receptors.

The in vivo and in vitro toxic effects of the synthetic polymeric 3-alkylpyridinium salt (APS3), from the Mediterranean marine sponge Reniera sarai, were evaluated on mammals, with emphasis to determine its mode of action. The median lethal doses of APS3 were 7.25 and higher that 20mg/kg in mouse and rat, respectively. Intravenous administration of 7.25 and 20mg/kg APS3 to rat caused a significant fall followed by an increase in mean arterial blood pressure accompanied by tachycardia. In addition, cumulative doses of APS3 (up to 60 mg/kg) inhibited rat nerve-evoked skeletal muscle contraction in vivo, with a median inhibitory dose (ID(50)) of 37.25mg/kg. When administrated locally by intramuscular injection to mouse, APS3 decreased the compound muscle action potential recorded in response to in vivo nerve stimulation, with an ID(50) of 0.5mg/kg. In vitro experiments confirmed the inhibitory effect of APS3 on mouse hemidiaphragm nerve-evoked muscle contraction with a median inhibitory concentration (IC(50)) of 20.3 µM, without affecting directly elicited muscle contraction. The compound inhibited also miniature endplate potentials and nerve-evoked endplate potentials with an IC(50) of 7.28 µM in mouse hemidiaphragm. Finally, APS3 efficiently blocked acetylcholine-activated membrane inward currents flowing through Torpedo nicotinic acetylcholine receptors (nAChRs) incorporated to Xenopus oocytes, with an IC(50) of 0.19 µM. In conclusion, our results strongly suggest that APS3 blocks muscle-type nAChRs, and show for the first time that in vivo toxicity of APS3 is likely to occur through an antagonist action of the compound on these receptors.

Rapid purification method for vitamin A-derived aging pigments A2E and iso-A2E using cation exchange resin.

A2E, known to be involved in the pathogenesis of age-related macular degeneration (AMD), is one of the major compounds that accumulate as fluorescent pigments in retinal pigment epithelial (RPE) cells with age and in some retinal disorders. While the biomimetic synthesis of A2E and its cis-isomer, iso-A2E is as simple as 'one-pot' reaction, the purification of these amphiphillic compounds has been a bottleneck for the mass production of these pathophysiologically important eye pigments. In order to provide a new method of rapid purification of A2E and iso-A2E, we employed a cation exchange resin for the separation of these pigments from crude reaction mixture. The reaction mixture was loaded on a weak acid resin and was eluted with 80% methanol with sodium hydroxide (pH 12), 100% methanol, and 100% methanol with 0.1% trifluoroacetic acid (TFA) in sequence. A2E and isoA2E were eluted only with 100% methanol solution containing TFA. Most of unreacted starting materials and intermediates were removed with 80% methanol containing sodium hydroxide. The new method can be used as a relatively simple and economic way to purify A2E and iso-A2E compared to conventional HPLC technique.

Viscosalines†B(1,2) and E(1,2): challenging new 3-alkyl pyridinium alkaloids from the marine sponge Haliclona viscosa.

Four new 3-alkyl pyridinium alkaloids, the viscosalines B(1) (1 a), B(2) (1 b), E(1) (2 a), and E(2) (2 b), were isolated from the Arctic sponge Haliclona viscosa. The structure elucidation of these isomeric compounds was challenging due to ambiguous fragments that derive during "standard" mass spectrometric fragmentation experiments. The final structure elucidation relied on the use of a combination of synthesis, liquid chromatography, and mass spectrometry. Three different mass spectrometers were used to differentiate between the synthetic structural isomers: a time-of-flight (TOF) mass spectrometer and two ion-trap mass spectrometers with different ion-transfer technologies (i.e., skimmer versus funnel optics). Although at first none of the spectrometers returned spectra that permitted structure elucidation, all three mass spectrometers provided analysis that successfully differentiated between the isomers after thorough method optimization. The use of in-source collision-induced dissociation (CID) with the ion trap and TOF instrument returned the most interesting results. The mode of fragmentation of the viscosalines under different experimental conditions is described herein. After successful optimization of the mass spectrometric method applied, the chromatographic method was improved to distinguish the previously inseparable isomers. Finally, both the liquid chromatography and mass spectrometric methods were applied to the natural products and the results compared to those from the synthetic compounds.

Novel Pyridinium compound from marine actinomycete, Amycolatopsis alba var. nov. DVR D4 showing antimicrobial and cytotoxic activities in vitro.

Marine sediment samples from Visakhapatnam coast of Bay of Bengal, India, were investigated as a source of actinomycetes to screen for the production of antibiotics and cytotoxic compounds. Actinomycete strain DVR D4 with interesting bioactivity profile was isolated during our systematic study of marine actinomycetes. Based on biochemical properties and 16S rDNA analysis the isolate DVR D4 was identified as a strain of Amycolatopsis alba. A solvent extraction followed by a chromatographic purification helped to isolate a cytotoxic compound, which was identified as 1(10-aminodecyl) Pyridinium salt antibiotic, on the basis of spectral data. The compound showed potent cytotoxic activity against cancer cell lines of cervix (HeLa), breast (MCF-7) and brain (U87MG) in vitro and also exhibited antibacterial activities against Gram-positive and Gram-negative bacteria.

Partition of bispyridinium oximes (trimedoxime and K074) administered in therapeutic doses into different parts of the rat brain.

The penetration of acetylcholinesterase reactivators (oximes) into the central nervous system is typically restricted by the blood-brain barrier. Although oximes are highly hydrophilic compounds, some contradictory results confirming permeation into the brain exist. The aim of this study is to verify the penetration of oximes through the blood-brain barrier and to detect their levels achieved in different brain regions 60 min after the administration. It was confirmed that oximes are able to penetrate into the brain after injection of therapeutic doses corresponding with 5% of LD(50). The level in whole brain was 0.58% for trimedoxime and 0.85% for the experimental drug oxime K074 as the percentage of their plasma concentration. The highest concentration was found in frontal cortex (trimedoxime 2.27%; oxime K074 0.95%) and lowest in basal ganglia (trimedoxime 0.86%; oxime K074 0.42%). Entry of oximes into the brain is minimal, but some low reactivation effect should be expected. The reactivation potency of oximes might be higher or lower, depending on the real oxime concentration in a given area.

Evaluation of the antifouling properties of 3-alyklpyridine compounds.

One of the most promising alternative technologies to antifouling (AF) biocides based on toxic heavy metals lies in the development of natural eco-friendly biocides. The present study evaluates the AF potential of structurally different compounds containing a 3-alkylpyridine moiety. The products, namely poly 3-alkylpyridinium salts, saraine, and haminols, were either extracted or derived from natural sources (the sponges Haliclona sp. and Reniera sarai and the mollusc Haminoea fusari), or obtained by chemical synthesis. All the molecules tested showed generally good anti-settlement activity against larvae of the barnacle Amphibalanus (=Balanus) amphitrite (EC(50) values between 0.19 and 3.61 µg ml(-1) and low toxicity (LC(50) values ranging from 2.04 to over 100 µg ml(-1)) with non-target organisms. For the first time, the AF potential of a synthetic monomeric 3-alkylpyridine was demonstrated, suggesting that chemical synthesis is as a realistic way to produce large amounts of these compounds for future research and development of environmentally-friendly AF biocides.

Polyaxibetaine, an amino acid derivative from the marine sponge Axinella polypoides.

A new pyridinium derivative, polyaxibetaine (3), has been isolated from the marine sponge Axinella polypoides, together with two known modified amino acids, 1 and 2. The planar structure of compound 3 has been elucidated by spectroscopic methods; definition of the absolute configuration of compounds 1-3 has been carried out through ECD studies.

3-Alkylpyridinium alkaloids from the Pacific sponge Haliclona sp.

The analysis of the polar extracts of the Pacific sponge Haliclona sp. yielded new dimeric (1), trimeric (2), and polymeric 3-alkylpyridinium alkaloids. Their isolation and structural elucidation, based on NMR and MS data, are discussed in detail, along with their cytotoxic activity.

Adsorption kinetics of maxilon yellow 4GL and maxilon red GRL dyes on kaolinite.

Kaolinite, a low-costly material, is the most abundant phyllosilicate mineral in highly weathered soils. In this work, the adsorption kinetics of maxilon yellow 4GL (MY 4GL) and maxilon red GRL (MR GRL) dyes on kaolinite from aqueous solutions was investigated using the parameters such as contact time, stirring speed, initial dye concentration, initial pH, ionic strength, acid-activation, calcination and solution temperature. The equilibrium time was 150 min for both dyes. The results showed that alkaline pH was favorable for the adsorption of MY 4GL and MR GRL dyes and physisorption seemed to play a major role in the adsorption process. It was found that the rate of adsorption decreases with increasing temperature and the process is exothermic. The adsorption kinetics followed the pseudo-second-order equation for both dyes investigated in this work with the k(2) values lying in the region of 1.79 x 10(4) to 107.87 x 10(4)g/mol min for MY 4GL and 3.44 x 10(4) to 72.09 x 10(4)g/mol min for MR GRL. The diffusion coefficient values calculated for the dyes were in the range of 3.76 x 10(-9) to 62.50 x 10(-9)cm(2)/s for MY 4GL and 1.98 x 10(-9) to 44.00 x 10(-9)cm(2)/s for MR GRL, and are compatible with other studies reported in the literature. The thermodynamic activation parameters such as the enthalpy, entropy and free energy were determined. The obtained results confirmed the applicability of this clay as an efficient adsorbent for cationic dyes.

Simplex-optimized chromatographic resolution of selected ionic liquid cations utilizing a polar reversed-phase system.

This study reports on optimization of the RP-HPLC separation of imidazolium and pyridinium ionic liquid cations using a variable-size simplex algorithm. Under the optimized conditions, all critical pairs of ionic liquids were successfully separated in a single chromatographic run. The mobile phase at the point corresponding to the optimum consisted of 10% MeOH and 90% 15 mM KH(2)PO(4)/H(3)PO(4) with pH 3.43. The coefficients of asymmetry for all of the compounds analyzed at the simplex algorithm optimum ranged from 0.83 to 2.91.