The effects of conformational constraints in the polyamine moiety of philanthotoxins on AMPAR inhibition.

Philanthotoxin-433 (PhTX-433) is a known potent inhibitor of ionotropic glutamate receptors, and analogues have been synthesised to identify more potent and selective antagonists. Herein we report the synthesis of four PhTXs with a cyclopropane moiety introduced into their polyamine chain, and their inhibition of an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype by using two-electrode voltage-clamp assays on Xenopus oocytes expressing the GluA1flop subunit. All analogues were found to be more potent than PhTX-343, with trans-cyclopropyl-PhTX-343 being the most potent (∼28-fold) and cis-cyclopropyl-PhTX-343 least potent (∼4-fold). Both cis- and trans-cyclopropyl-PhTX-444 had intermediate potency (both∼12-fold). Molecular modelling indicates that a cyclopropane moiety confers a favourable steric constraint to the polyamine part, but this is compromised by a cis conformation due to enhanced intramolecular folding. Elongated PhTX-444 analogues alleviate this to some extent, but optimal positioning of the amines is not permitted.

Some transformations of tacrolimus, an immunosuppressive drug.

Transformations of the macrocyclic lactone tacrolimus (1), an important immunosuppressive drug produced by Streptomyces species, are described. These transformation products are primarily of interest as reference substances for drug impurity analyses. Upon action of acid (p-toluenesulfonic acid in toluene), tacrolimus is dehydrated by loss of water from the ß-hydroxyketone moiety with partial inversion of configuration at C-8, resulting in formation of 5-deoxy-Δ(5,6)-tacrolimus and 5-deoxy-Δ(5,6)-8-epitacrolimus. The structure of the latter was determined by single-crystal X-ray crystallography. The same products are formed upon action of free radicals (iodine in boiling toluene), along with formation of 8-epitacrolimus. The latter is converted by p-toluenesulfonic acid to 5-deoxy-Δ(5,6)-8-epitacrolimus. Treatment of tacrolimus with weak base (1,5-diazabicyclo[4.3.0]nonene) gives, in addition to 8-epitacrolimus, the open-chain acid corresponding to 5-deoxy-Δ(5,6)-tacrolimus, a rare non-cyclic derivative of tacrolimus. Strong base (t-butoxide) causes pronounced degradation of the molecule. Thermolysis of tacrolimus leads to ring expansion by an apparent [3,3]-sigmatropic rearrangement of the allylic ester moiety with subsequent loss of water from the ß-hydroxyketone moiety. ¹H and ¹³C NMR spectra of the obtained compounds, complicated by the presence of amide bond rotamers and ketal moiety tautomers, were assigned by extensive use of 2D NMR techniques.

Alkaloid analysis by high-performance liquid chromatography-solid phase extraction-nuclear magnetic resonance: new strategies going beyond the standard.

The hyphenated technique HPLC-SPE-NMR is an important tool for rapid dereplication of complex mixtures of in particular small molecules and has been successfully employed in natural product research. However, positively charged alkaloids at low pH are often poorly trapped on the generally used SPE cartridge limiting the general application of the procedure. In this work, two new approaches for efficient SPE trapping of alkaloids and elution efficiencies were evaluated using 24 model alkaloids. Use of a 0.1 M NaOH solution as the post-column dilution greatly enhanced trapping of alkaloids on the commonly used cartridge containing divinylbenzene polymer (GP resin). This procedure, however, was unsuitable for trapping phenolic alkaloids. Severe line broadening and immiscibility with water made chloroform-d(1) unsuited as eluent. None of these problems occurred when methanol-d(4) was used as eluent. Previously, mixed mode cation exchange sorbents have not been used in HPLC-SPE-NMR analysis of natural products. In contrast to GP resin this material showed good retention and elution characteristics for retention and elution of alkaloids. As well the use of methanol-d(4) containing 1% aqueous NaOD (40%) as methanol-d(4) containing 5% aqueous NH(4)OH (30%) as eluents were successful, even though elution of alkaloids with pK(a) of the corresponding acid above 10 proved difficult. Alkaloid extracts of Huperzia selago containing complex aliphatic alkaloids and Triclisia patens containing bisbenzylisoquinoline alkaloids were used for validation of the protocols for analysis of a diverse collection of alkaloids. Mixed mode cation exchange sorbent was efficient for trapping and elution of both types of alkaloids as evidenced by acquisition of 2D NMR data for all trapped compounds. In contrast, GP resin proved only viable for all the H. selago alkaloids whereas trapping and elution of bisbenzylisoquinoline alkaloids were dubious.

Extraction of alkaloids for NMR-based profiling: exploratory analysis of an archaic Cinchona bark collection.

A museum collection of Cinchonae cortex samples (n = 117), from the period 1850-1950, was extracted with a mixture of chloroform-d1, methanol-d4, water-d2, and perchloric acid in the ratios 5 : 5 : 1 : 1. The extracts were directly analyzed using 1H NMR spectroscopy (600 MHz) and the spectra evaluated using principal component analysis (PCA) and total statistical correlation spectroscopy (STOCSY). A new method called STOCSY-CA, where CA stands for component analysis, is described, and an analysis using this method is presented. It was found that the samples had a rather homogenous content of the well-known cinchona alkaloids quinine, cinchonine, and cinchonidine without any apparent clustering. Signals from analogues were detected but not in substantial amounts. The main variation was related to the absolute amounts of extracted alkaloids, which was attributed to the evolution of the Cinchona tree cultivation during the period in which the samples were collected.

Direct (13)C NMR detection in HPLC hyphenation mode: analysis of Ganoderma lucidum terpenoids.

Solid phase extraction (SPE) was introduced as a crucial step in the HPLC-SPE-NMR technique to enable online analyte enrichment from which proton-detected NMR experiments on submicrogram amounts from complex mixtures were possible. However, the significance of direct-detected (13)C NMR experiments is indubitable in simplifying structural elucidations. In the current study, we demonstrated direct (13)C NMR detection of triterpenoids from a Ganoderma lucidum extract in hyphenation mode. The combined advantage of a cryogenically cooled probe, miniaturization, and multiple trapping enabled the first reported application of HPLC-SPE-NMR analysis using direct-detected (13)C NMR spectra. HPLC column loading, accumulative SPE trappings, and the effect of different elution solvents were evaluated and optimized. A column loading of approximately 600 µg of a prefractionated triterpenoid mixture, six trappings, and an acquisition time of 13 h resulted in spectra with adequate signal-to-noise ratios to detect all C-13 signals.

From retrospective assessment to prospective decisions in natural product isolation: HPLC-SPE-NMR analysis of Carthamus oxyacantha.

An extract of Carthamus oxyacantha (wild safflower) was investigated using two approaches: a traditional, nontarget fractionation by VLC and HPLC, and the hyphenated technique HPLC-PDA-HRMS-SPE-NMR followed by targeted isolation of selected constituents for inclusion in a screening library of pure natural products. While the nontarget fractionation involved considerable time spent on pursuing fractions containing well-known or undesired compounds, the hyphenated analysis was considerably faster and required less solvent and other consumables. The results were used to design and execute an optimized, HPLC-HRMS-guided, targeted isolation scheme aiming exclusively at a series of identified spiro compounds. Thus, HPLC-PDA-HRMS-SPE-NMR is a dereplication technique of choice, allowing economical acquisition of comprehensive data about compounds in crude extracts, which can be used for rational, prospective decisions about further isolation efforts. A total of 15 compounds were identified in the extract. Six spiro compounds, of which four have not previously been characterized, and tracheloside (a lignin glucoside) are presented with assigned 1H and 13C chemical shifts.

Production of unusual dispiro metabolites in Pestalotiopsis virgatula endophyte cultures: HPLC-SPE-NMR, electronic circular dichroism, and time-dependent density-functional computation study.

The endophytic fungus Pestalotiopsis virgatula, derived from the plant Terminalia chebula and previously found to produce a large excess of a single metabolite when grown in the minimal M1D medium, was induced to produce a variety of unusual metabolites by growing in potato dextrose broth medium. Analysis of the fermentation medium extract was performed using an HPLC-PDA-MS-SPE-NMR hyphenated system, which led to the identification of a total of eight metabolites (1-8), six of which are new. Most of the metabolites are structurally related and are derivatives of benzo[c]oxepin, rare among natural products. This includes dispiro derivatives 7 and 8 (pestalospiranes A and B), having a novel 1,9,11,18-tetraoxadispiro[6.2.6.2]octadecane skeleton. Relative and absolute configurations of the latter were determined by a combination of NOESY spectroscopy and electronic circular dichroism spectroscopy supported by time-dependent density-functional theory calculations (B3LYP/TZVP level). This work demonstrates that a largely complete structure elucidation of numerous metabolites present in a raw fermentation medium extract can be performed by the HPLC-SPE-NMR technique using only a small amount of the extract, even with unstable metabolites that are difficult to isolate by traditional methods.

Metabolic profiling based on two-dimensional J-resolved 1H NMR data and parallel factor analysis.

Metabolic profiling of natural products is used to map correlated concentration variances of known and unknown secondary metabolites in extracts. NMR-spectroscopy is in this respect regarded as a convenient and reproducible technique with the ability to detect a wide range of small organic compounds. Two-dimensional J-resolved NMR-spectra are used in this context to resolve overlapping signals by separating the effect of J-coupling from the effect of chemical shifts. Often one-dimensional projections of these data are used as input for standard multivariate statistical methods, and only the intensity variances along the chemical shift axis are taken into account. Here, we describe the use of parallel factor analysis (PARAFAC) as a tool to preprocess a set of two-dimensional J-resolved spectra with the aim of keeping the J-coupling information intact. PARAFAC is a mathematical decomposition method that fits three-way experimental data to a model whose parameters in this case reflect concentrations and individual component spectra along the chemical shift axis and corresponding profiles along the J-coupling axis. A set of saffron samples, directly extracted with methanol-d(4), were used as a model system to evaluate the feasibility and merits of the method. To successfully use PARAFAC, the two-dimensional spectra (n = 96) had to be aligned and processed in narrow windows (0.04 ppm wide) along the chemical shift axis. Selection of windows and number of components for each PARAFAC-model was done automatically by evaluating amount of explained variance and core consistency values. Score plots showing the distribution of objects in relation to each other, and loading plots in the form of two-dimensional pseudospectra with the same appearance as the original J-resolved spectra but with positive and negative contributions are presented. Loadings are interpreted not only in terms of signals with different chemical shifts but also the associated J-coupling profiles.

Metabolic profiling of Rhodiola rosea rhizomes by ¹H NMR spectroscopy.

INTRODUCTION: Rhodiola rosea is a broadly used medicinal plant with largely unexplored natural variability in secondary metabolite levels. OBJECTIVE: The aim of this work was to develop a non-target procedure for ¹H NMR spectroscopic fingerprinting of rhizome extracts for pattern recognition analysis and identification of secondary metabolites responsible for differences in sample composition. To achieve this, plants from three different geographic areas (Swiss Alps, Finland, and Altai region in Siberia) were investigated. RESULTS: A sample preparation procedure was developed in order to remove polymeric polyphenols as the ¹H NMR analysis of low-molecular-weight metabolites was hampered by the presence of tannins. Principal component analysis disclosed tight clustering of samples according to population. PCA models based on the aromatic region of the spectra showed that the first two components reflected changes in the content of salidroside and rosavin, respectively, the rosavin content being negatively correlated to that of rhodiocyanoside A and minor aromatics. Score plots and non-parametric variance tests demonstrated population-dependent changes according to harvest time. Data consistency was assessed using score plots and box-and-whisker graphs. In addition, a procedure for presenting loadings of PCA models based on bucketed data as high-resolution plots, which are reminiscent of real ¹H NMR spectra and help to identify latent biomarkers, is presented. CONCLUSION: This study demonstrated the usefulness of the established procedure for multivariate non-target ¹H NMR metabolic profiling of Rhodiola rosea.

Assessment of structurally diverse philanthotoxin analogues for inhibitory activity on ionotropic glutamate receptor subtypes: discovery of nanomolar, nonselective, and use-dependent antagonists.

An array of analogues of the wasp toxin philanthotoxin-433, in which the asymmetric polyamine moiety was exchanged for spermine and the headgroup replaced with a variety of structurally diverse moieties, was prepared using parallel solid-phase synthesis approaches. In three analogues, the spermine moiety was extended with an amino acid tail, six compounds contained an N-acylated cyclohexylalanine, and four analogues were based on a novel diamino acid design with systematically changed spacer length between N-cyclohexylcarbonyl and N-phenylacetyl substituents. The analogues were studied using two-electrode voltage-clamp electrophysiology employing Xenopus laevis oocytes expressing GluA1(i) AMPA or GluN1/2A NMDA receptors. Several of the analogues showed significantly increased inhibition of the GluN1/2A NMDA receptor. Thus, an analogue containing N-(1-naphtyl)acetyl group showed an IC(50) value of 47 nM. For the diamino acid-based analogues, the optimal spacer length between two N-acyl groups was determined, resulting in an analogue with an IC(50) value of 106 nM.

1H NMR spectroscopy-based interventional metabolic phenotyping: a cohort study of rheumatoid arthritis patients.

1H NMR spectroscopy-based metabolic phenotyping was used to identify biomarkers in the plasma of patients with rheumatoid arthritis (RA). Forty-seven patients with RA (23 with active disease at baseline and 24 in remission) and 51 healthy subjects were evaluated during a one-year follow-up with assessments of disease activity (DAS-28) and 1H NMR spectroscopy of plasma samples. Discriminant analysis provided evidence that the metabolic profiles predicted disease severity. Cholesterol, lactate, acetylated glycoprotein, and lipid signatures were found to be candidate biomarkers for disease severity. The results also supported the link between RA and coronary artery disease. Repeated assessment using mixed linear models showed that the predictors obtained from metabolic profiles of plasma at baseline from patients with active RA were significantly different from those of patients in remission (P=0.0007). However, after 31 days of optimized therapy, the two patient groups were not significantly different (P=0.91). The metabolic profiles of both groups of RA patients were different from the healthy subjects. 1H NMR-based metabolic phenotyping of plasma samples in patients with RA is well suited for discovery of biomarkers and may be a potential approach for disease monitoring and personalized medication for RA therapy.

Ring opening of a resin-bound chiral aziridine with phenol nucleophiles.

An efficient and versatile solid-phase route for the preparation of aryl-alkyl ethers is described. Regioselective ring opening of a resin-bound chiral aziridine with phenolic nucleophiles constitutes the key feature of the present protocol that allows incorporation of fluorescent moieties and subsequent on-resin protecting group interconversion. Initial experiments demonstrated that a competing oligomerization may occur by concomitant attacks of transient nosylamide anions on neighboring aziridines, resulting in formation of dimeric and trimeric byproduct. Expectedly, the significance of this alternative reaction pathway was strongly dependent on resin loading, and a low loading (<0.4 mmol g(-1)) was required for obtaining high yields of the desired aryl-alkyl ethers. The developed methodology allowed preparation of novel N-Fmoc-protected coumaryl amino acid building blocks, which were incorporated into peptides by solid-phase peptide synthesis.

H NMR-based metabolomics combined with HPLC-PDA-MS-SPE-NMR for investigation of standardized Ginkgo biloba preparations.

Commercial preparations of Ginkgo biloba are very complex mixtures prepared from raw leaf extracts by a series of extraction and prepurification steps. The pharmacological activity is attributed to a number of flavonoid glycosides and unique terpene trilactones (TTLs), with largely uncharacterized pharmacological profiles on targets involved in neurological disorders. It is therefore important to complement existing targeted analytical methods for analysis of Ginkgo biloba preparations with alternative technology platforms for their comprehensive and global characterization. In this work, (1)H NMR-based metabolomics and hyphenation of high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) were used for investigation of 16 commercially available preparations of Ginkgo biloba. The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that (1)H NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone-d(6). Unexpected or unwanted extract constituents were also easily identified in the (1)H NMR spectra, which contrasts traditional methods that depend on UV absorption or MS ionizability and usually require availability of reference standards. Automated integration of (1)H NMR spectral segments (buckets or bins of 0.02 ppm width) provides relative distribution plots of TTLs based on their H-12 resonances. The present study shows that (1)H NMR-based metabolomics is an attractive method for non-selective and comprehensive analysis of Ginkgo extracts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-009-0195-x) contains supplementary material, which is available to authorized users.

Synthesis and characterization of an epimer of tacrolimus, an immunosuppressive drug.

8-Epitacrolimus (2), a new l-pipecolic acid macrolide lactone, was obtained by base-catalyzed epimerization of tacrolimus (FK-506, 1), an important immunosuppressive drug, and its structure determined by a single-crystal X-ray diffraction method. The compound was fully characterized by spectroscopic techniques. The epimer is of importance due to its potential biological effects as well as because of its possible formation during formulation, handling, and use of tacrolimus products.

Brain nonoxidative carbohydrate consumption is not explained by export of an unknown carbon source: evaluation of the arterial and jugular venous metabolome.

Brain activation provokes nonoxidative carbohydrate consumption and during exercise it is dominated by the cerebral uptake of lactate resulting in that up to approximately 1 mmol/ 100 g of glucose equivalents cannot be accounted for by cerebral oxygen uptake. The fate of this 'extra' carbohydrate uptake is unknown, but it may be that brain metabolism is balanced by a yet-unidentified substance(s). This study used a nuclear magnetic resonance-based metabolomics approach to plasma samples obtained from the brachial artery and the right internal jugular vein in 16 healthy young males to identify carbon species going to and from the brain. We observed a carbohydrate accumulation of 255+/-37 micromol/100 g glucose equivalents at exhaustion not accounted for by the oxygen uptake. Although the cumulated uptake was lower than earlier observed, the results show that glucose and lactate are responsible for the majority of the carbon exchange across the brain. Even during intense exercise associated with the largest nonoxidative carbohydrate consumption, the brain did not show significant release of any other metabolite. We conclude that during exercise, the surplus carbohydrate uptake by the brain cannot be accounted for by changes in the NMR-derived plasma metabolome across the brain.

Acetylcholinesterase inhibitory activity of lycopodane-type alkaloids from the Icelandic Lycopodium annotinum ssp. alpestre.

The aim of this study was to investigate structures and acetylcholinesterase inhibitory activities of lycopodane-type alkaloids isolated from an Icelandic collection of Lycopodium annotinum ssp. alpestre. Ten alkaloids were isolated, including annotinine, annotine, lycodoline, lycoposerramine M, anhydrolycodoline, gnidioidine, lycofoline, lannotinidine D, and acrifoline, as well as a previously unknown N-oxide of annotine. 1H and 13C NMR data of several of the alkaloids were provided for the first time. Solvent-dependent equilibrium constants between ketone and hemiketal form of acrifoline were determined. Conformation of acrifoline was characterized using NOESY spectroscopy and molecular modelling. The isolated alkaloids were evaluated for their in vitro inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Ligand docking studies based on mutated 3D structure of Torpedo californica acetylcholinesterase provided rationale for low inhibitory activity of the isolated alkaloids as compared to huperzine A or B, which are potent acetylcholinesterase inhibitors belonging to the lycodine class. Based on the modelling studies the lycopodane-type alkaloids seem to fit well into the active site gorge of the enzyme but the position of their functional groups is not compatible with establishing strong hydrogen bonding interactions with the amino acid residues that line the binding site. The docking studies indicate possibilities of additional functionalization of the lycopodane skeleton to render potentially more active analogues.

HPLC-SPE-NMR identification of a novel metabolite containing the benzo[c]oxepin skeleton from the endophytic fungus Pestalotiopsis virgatula culture.

HPLC-SPE-NMR analysis of a crude extract of fermentation broth of cultured PESTALOTIOPSIS VIRGATULA isolate TC-320 from TERMINALIA CHEBULA Retz. (Combretaceae) disclosed the presence of a simple but unprecedented low-molecular-weight metabolite, 9-hydroxybenzo[ C]oxepin-3[1 H]-one, subsequently isolated by a targeted purification procedure.

Accelerated dereplication of crude extracts using HPLC-PDA-MS-SPE-NMR: quinolinone alkaloids of Haplophyllum acutifolium.

Direct hyphenation of analytical-scale high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) has been used for accelerated dereplication of crude extract of Haplophyllum acutifolium (syn. Haplophyllum perforatum). This technique allowed fast on-line identification of six quinolinone alkaloids, named haplacutine A-F, as well as of acutine, haplamine, eudesmine, and 2-nonylquinolin-4(1H)-one. Acutine and haplacutine E, isolated by preparative-scale HPLC, showed moderate antiplasmodial activity with IC(50) values of 2.17+/-0.22 microM and 3.79+/-0.24 microM, respectively (chloroquine-sensitive Plasmodium falciparum 3D7 strain).

Expedite protocol for construction of chiral regioselectively N-protected monosubstituted piperazine, 1,4-diazepane, and 1,4-diazocane building blocks.

This paper describes the first study of solution-phase synthesis of chiral monosubstituted piperazine building blocks from nosylamide-activated aziridines. The protocol, involving aminolysis of the starting aziridines with omega-amino alcohols and subsequent Fukuyama-Mitsunobu cyclization, offers the advantage of mild conditions as well as short reaction times, and it leads to optically pure N-Boc- or N-Ns-protected piperazines. This four-step sequence, requiring only a single final chromatographic purification, was extended to include novel diazepane and diazocane derivatives.