Curare alkaloids from Matis Dart Poison: Comparison with d-tubocurarine in interactions with nicotinic, 5-HT3 serotonin and GABAA receptors.

Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated α-bungarotoxin ([125I]-αBgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-αBgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 µM) than d-TC (IC50 = 0.39 µM), while with α7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 µM and 7.77 µM, respectively), but BBIQA2 was similar (IC50 = 5.52 µM). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult α1ß1εδ nAChR or human α7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 µM). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle α1ß1εδ nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal α3ß2, α4ß2, and α9α10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With α3ß2γ2 GABAAR currents were almost completely inhibited by d-TC at a high (100 µM) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-α-cobratoxin for binding to α1ß3γ2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.

Interspecies-cooperations of abutilon theophrasti with root colonizing microorganisms disarm BOA-OH allelochemicals.

A facultative, microbial micro-community colonizing roots of Abutilon theophrasti Medik. supports the plant in detoxifying hydroxylated benzoxazolinones. The root micro-community is composed of several fungi and bacteria with Actinomucor elegans as a dominant species. The yeast Papiliotrema baii and the bacterium Pantoea ananatis are actively involved in the detoxification of hydroxylated benzoxazolinones by generating H2O2. At the root surface, laccases, peroxidases and polyphenol oxidases cooperate for initiating polymerization reactions, whereby enzyme combinations seem to differ depending on the hydroxylation position of BOA-OHs. A glucosyltransferase, able to glucosylate the natural benzoxazolinone detoxification intermediates BOA-5- and BOA-6-OH, is thought to reduce oxidative overshoots by damping BOA-OH induced H2O2 generation. Due to this detoxification network, growth of Abutilon theophrasti seedlings is not suppressed by BOA-OHs. Polymer coats have no negative influence. Alternatively, quickly degradable 6-hydroxy-5-nitrobenzo[d]oxazol-2(3H)-one can be produced by the micro-community member Pantoea ananatis at the root surfaces. The results indicate that Abutilon theophrasti has evolved an efficient strategy by recruiting soil microorganisms with special abilities for different detoxification reactions which are variable and may be triggered by the allelochemical´s structure and by environmental conditions.

Constituents from the bark resin of Schinus molle

ABSTRACT A total of five terpenes was isolated from the bark resin of Schinus molle L., Anacardiaceae, and their structures were determined by spectroscopic techniques. Among these compounds the sesquiterpene hydrocarbon terebinthene showed significant growth inhibitory activity against human colon carcinoma HCT-116 cells. Furthermore, terebinthene and pinicolic acid (5) also showed antibacterial activity against Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633.

Coumarins of Loricaria ferruginea

ABSTRACT In the presented research we isolated and characterized compounds from Loricaria ferruginea (Ruiz & Pav.) Wedd., Asteraceae. To the best of our knowledge no data on any compounds from L. ferruginea have been published to this day. As main compounds of the hexane extract we found four known coumarins: 5,7-dimethoxycoumarin; 5,7,8-trimethoxycoumarin; 5-hydroxyobliquine and 5-methoxyobliquine. All the structures were determined by spectroscopic and spectrometric methods.

Corrigendum to "Synthesis and Characterization of New Palladium(II) Thiosemicarbazone Complexes and Their Cytotoxic Activity against Various Human Tumor Cell Lines".

[This corrects the article DOI: 10.1155/2013/524701.].

Constituents of Corynaea crassa "Peruvian Viagra"

Abstract A phytochemical investigation of methanol and n-hexane extracts of tuber/roots of Corynaea crassa Hook. f., Balanophoraceae, led to the isolation and characterization of β-sitosterol, lupenone, β-amyrone, lupeol, and β-amyrine. Unusual complex 1:1 mixtures of lupenone/β-amyrone and lupeol/β-amyrine obtained from the extracts were identified by NMR and HR-MS experiments. The structure of the 1:1 lupenone/β-amyrone mixture was confirmed by X-ray analysis. These triterpene ketone derivatives, only distinguished either by 5- or 6-membered E ring, co-crystallize in one common unit cell in the solid state.

Synthesis and Characterization of New Palladium(II) Thiosemicarbazone Complexes and Their Cytotoxic Activity against Various Human Tumor Cell Lines.

The palladium(II) bis-chelate complexes of the type [Pd(TSC(1-5))2] (6-10), with their corresponding ligands 4-phenyl-1-(acetone)-thiosemicarbazone, HTSC(1) (1), 4-phenyl-1-(2'-chloro-benzaldehyde)-thiosemicarbazone, HTSC(2) (2), 4-phenyl-1-(3'-hydroxy-benzaldehyde)-thiosemicarbazone, HTSC(3) (3), 4-phenyl-1-(2'-naphthaldehyde)-thiosemicarbazone, HTSC(4) (4), and 4-phenyl-1-(1'-nitro-2'-naphthaldehyde)-thiosemicarbazone, HTSC(5) (5), were synthesized and characterized by elemental analysis and spectroscopic techniques (IR and (1)H- and (13)C-NMR). The molecular structure of HTSC(3), HTSC(4), and [Pd(TSC(1))2] (6) have been determined by single crystal X-ray crystallography. Complex 6 shows a square planar geometry with two deprotonated ligands coordinated to Pd(II) through the azomethine nitrogen and thione sulfur atoms in a cis arrangement. The in vitro cytotoxic activity measurements indicate that the palladium(II) complexes (IC50 = 0.01-9.87 µM) exhibited higher antiproliferative activity than their free ligands (IC50 = 23.48-70.86 and >250 µM) against different types of human tumor cell lines. Among all the studied palladium(II) complexes, the [Pd(TSC(3))2] (8) complex exhibited high antitumor activity on the DU145 prostate carcinoma and K562 chronic myelogenous leukemia cells, with low values of the inhibitory concentration (0.01 and 0.02 µM, resp.).

Allium ursinum L.: bioassay-guided isolation and identification of a galactolipid and a phytosterol exerting antiaggregatory effects.

AIMS: We wanted to investigate the possible antithrom botic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adenosine diphosphate(ADP)-induced aggregation of human platelets by wild garlic, Allium ursinum L. METHODS: For this purpose, a bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. For the bioassay, blood was taken from healthy human volunteers and platelet-rich plasma was prepared for turbidimetric platelet aggregation tests. Platelet-rich plasma, stimulated with 20 µ mol/l of ADP, was treated with extracts of different polarities, fractions and isolated single compounds from A. ursinum. The extracts were investigated by thin-layer chromatography(TLC), HPLC, mass spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and 1/2-dimensional (1)H/(13) C-nuclear magnetic resonance (NMR) spectroscopic techniques. RESULTS: Fresh A. ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited ADP-induced aggregation of human platelets. Thisethanolic extract was subjected to liquid-liquid partition. Whilst the aqueous phase, containing the moiety of cysteine sulphoxide and thiosulphinate derivatives, showed only weak activity on platelet aggregation, the ethyl-acetate and particularly the chloroform partitions showed the high estaggregation-inhibiting potency. Thus, in our bioassay, the effects of alliins/allicins could be neglected. The chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient-elution open column chromatography on silica gel. The most active fractions 11­17 were separated again, yielding 10 subfractions. This afforded 1,2-di-O-α-linolenoyl-3-O-ß-D-galactopyranosyl-sn-glycerol and ß-sitosterol-3-O-ß-D-glucopyranoside, the structures of which were determined by ESI-MS and 1/2-dimensional (1)H/(13) CNMR spectroscopic techniques. Furthermore, the minute amounts of volatile oil of A. ursinum leaves obtained by steam distillation according to Ph. Eur. could be evaluated asa third aggregation-inhibiting principle. CONCLUSION: In our study, for the first time, 2 active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol,could be identified exhibiting inhibitory action on ADP-induced aggregation in human blood platelets. As a major constituent, the galactolipid, 1,2-di-O-α-linolenoyl-3-O-ß-D-galactopyranosyl-sn-glycerol, not yet found in Allium sp., appears as a new, highly useful marker substance for A. ursinum drugs, or their pharmaceutical or food preparations,as shown by our orientating TLC analyses.

Synthesis of peptides containing 5-hydroxytryptophan, oxindolylalanine, N-formylkynurenine and kynurenine.

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5-HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the α-amino group of both amino acids, and the commercially available 5-HTP, with Fmoc-succinimide. High yields of Fmoc-Kyn were obtained by acid hydrolysis of Fmoc-NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M-type), and ß-enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5-HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion-pair reagent on a C18-phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan.

Identification and quantitation of the N-acetyl-L-cysteine S-conjugates of bendamustine and its sulfoxides in human bile after administration of bendamustine hydrochloride.

We recently reported the detection of mercapturic acid pathway metabolites of bendamustine, namely, cysteine S-conjugates in human bile, which are supposed to subsequently undergo further metabolism. In this study, we describe the identification and quantitation of consecutive bendamustine metabolites occurring in human bile using authentic reference standards and the synthesis and structural confirmation of these compounds. Mass spectrometry data along with high-performance liquid chromatography retention data (fluorescence detection) of the synthetic reference standards were consistent with those of the metabolites found in human bile after administration of bendamustine hydrochloride to cancer patients. Analysis of the purified synthetic reference compounds showed a purity of at least 95%. Structural confirmation was achieved by one- and two-dimensional proton as well as carbon-13 NMR spectroscopy and mass spectrometry. A total of 16 bendamustine-related compounds were detected in the bile of patients, 11 of them were recovered as conjugates. Eight conjugates have been structurally confirmed as novel mercapturic acids and sulfoxides. Biliary excretion of the sulfoxides was twice that of the mercapturate precursors. Glutathione S-conjugates of bendamustine have not been detected in bile samples, indicating rapid enzymatic cleavage in humans. Both the lack of glutathione (GSH) conjugates and occurrence of diastereomeric sulfoxides emphasize species-related differences in the GSH conjugation of bendamustine between humans and rats. The total amount recovered in the bile as the sum of all conjugates over the period of 24 h after dosing averaged 5.2% of the administered dose. The question of whether the novel metabolites contribute to urinary excretion should be a target of future investigations.

Characterization of two phase I metabolites of bendamustine in human liver microsomes and in cancer patients treated with bendamustine hydrochloride.

PURPOSE: The metabolism of bendamustine (BM) hydrochloride, a bifunctional alkylator containing a heterocyclic ring, was investigated in vitro and in vivo for identification of cytochromes P450 (CYP) involved in the formation of two phase I metabolites, structural confirmation of these previously unidentified metabolites and assessment of their cytotoxic effect in relation to the parent compound. METHODS: Potential metabolites of BM were synthesized and structurally characterized by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis. In vitro metabolism of BM hydrochloride in human hepatic microsomes was conducted to identify the CYP450 isoenzymes involved in the oxidative metabolism of BM. Samples from cancer patients after treatment with BM hydrochloride and microsomal preparations were analyzed by LC-MS and HPLC with fluorescence detection. The cytotoxic effect of the metabolites was analyzed in several lymphoma cell lines and peripheral blood lymphocytes and compared with that of the parent compound using an MTT assay. RESULTS: LC-MS as well as HPLC with fluorescence detection revealed hydroxylation of the methylene carbon at the C-4 position of the butanoic acid side chain and N-demethylation of the benzimidazole skeleton as the main metabolic pathways in human liver microsomes. Isoform-specific chemical inhibitors and correlation analysis pointed to CYP1A2 as the prominent enzyme in BM oxidation. The rate of formation for both metabolites correlated (r=0.931 and 0.933) with the activity of CYP1A2 and there were no other notable correlations with any of the other CYPs. In addition, both metabolites were identified in plasma, urine, and bile samples from cancer patients under BM hydrochloride therapy as shown by comparison with chromatograms obtained from the authentic reference standards. Cytotoxic activity observed for gamma-hydroxy BM was approximately equivalent to that obtained for the parental compound BM. N-demethyl BM displays five to tenfold less cytotoxic activity than BM. CONCLUSION: The results indicate that CYP1A2-catalyzed N-dealkylation and gamma hydroxylation are the major routes for BM phase I metabolism producing two metabolites less or similarly toxic than the parent compound. In contrast to the metabolic pathways of the structurally related chlorambucil, no beta-oxidation of the butanoic acid side chain leading to enhanced toxicity was detected for BM.

Synthesis and characterization of some new phase II metabolites of the alkylator bendamustine and their identification in human bile, urine, and plasma from patients with cholangiocarcinoma.

The alkylating agent bendamustine is currently in phase III clinical trials for the treatment of hematological malignancies and breast, lung, and gastrointestinal tumors. Renal elimination mainly as the parent compound is thought to be the primary route of excretion. Because polar biliary conjugates were expected metabolites of bendamustine, three cysteine S-conjugates were synthesized, purified by quantitative high-performance liquid chromatography (HPLC), and characterized by NMR spectroscopy and mass spectrometry (MS). HPLC assays with MS, as well as fluorescence detection of bile, urine, and plasma after single-dose intravenous infusion of 140 mg/m(2) bendamustine in five subjects with cholangiocarcinoma, indicated the existence of these phase II metabolites, which were identified as cysteine S-conjugates by comparison with the previously characterized synthetic reference standards. The sum of the three cysteine S-conjugates of bendamustine was determined in human bile and urine to be 95.8 and 26.0%, respectively, expressed as mean percentage of the sum of the parent compound and identified metabolites. The percentage of administered dose recovered in urine as cysteine S-conjugates ranged from 0.9 to 4.1%, whereas the total percentage of the administered dose excreted in urine as the parent drug and seven metabolites ranged from 3.8 to 16.3%. The identification of cysteine S-conjugates provide evidence that a major route of bendamustine metabolism in humans involves conjugation with glutathione. Results indicate the importance of phase II conjugation in the elimination of bendamustine, besides phase I metabolism and hydrolytic degradation, and require further investigation.