Subthalamic nucleus neurones in slices from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mice show irregular, dopamine-reversible firing pattern changes, but without synchronous activity

The loss of dopamine in idiopathic or animal models of Parkinson's disease induces synchronized low-frequency oscillatory burst-firing in subthalamic nucleus neurones. We sought to establish whether these firing patterns observed in vivo were preserved in slices taken from dopamine-depleted animals, thus establishing a role for the isolated subthalamic-globus pallidus complex in generating the pathological activity. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) showed significant reductions of over 90% in levels of dopamine as measured in striatum by high pressure liquid chromatography. Likewise, significant reductions in tyrosine hydroxylase immunostaining within the striatum (>90%) and tyrosine hydroxylase positive cell numbers (65%) in substantia nigra were observed. Compared with slices from intact mice, neurones in slices from MPTP-lesioned mice fired significantly more slowly (mean rate of 4.2 Hz, cf. 7.2 Hz in control) and more irregularly (mean coefficient of variation of inter-spike interval of 94.4%, cf. 37.9% in control). Application of ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-phosphonopentanoic acid (AP5) and the GABAA receptor antagonist picrotoxin caused no change in firing pattern. Bath application of dopamine significantly increased cell firing rate and regularized the pattern of activity in cells from slices from both MPTP-treated and control animals. Although the absolute change was more modest in control slices, the maximum dopamine effect in the two groups was comparable. Indeed, when taking into account the basal firing rate, no differences in the sensitivity to dopamine were observed between these two cohorts. Furthermore, pairs of subthalamic nucleus cells showed no correlated activity in slices from either control (21 pairs) or MPTP-treated animals (20 pairs). These results indicate that the isolated but interconnected subthalamic-globus pallidus network is not itself sufficient to generate the aberrant firing patterns in dopamine-depleted animals. More likely, inputs from other regions, such as the cortex, are needed to generate pathological oscillatory activity. © 2006 IBRO.

Hypervalent iodine in synthesis 92. A facile synthesis of 3-substituted-5,6-dihydroimidazo[2,1-b]thiazoles by cyclocondensation of alkynyl(phenyl)iodonium salts and imidazolidine-2-thione

A simple method for the synthesis of 3-substituted 5,6-dihydroimidazo[2,1-b]thiazoles is achieved by cyclocondensation of alkynyl(phenyl)iodonium salts with imidazolidine-2-thione.

Synthesis of substituted 3-anilino-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepine-2-ones and their evaluation as cholecystokinin-ligands

3-Amino-1,4-benzodiazepines as well as chemically related diverse amines were prepared from oxazepam and subsequently screened on the cholecystokinin receptor in a radiolabel binding assay. Oxazepam 2 was activated via its 3-chloro-1,4-benzodiazepine intermediate 3 and was reacted with a large series of aliphatic and aromatic amines. The substituted 3-anilino-1,4-benzodiazepine structure was identified as lead structure in a diverse series of 3-amino-1,4-benzodiazepines 4-38 and the full SAR (structure-activity relationship) optimisation provided 3-anilinobenzodiazepines 16-38 with CCK 1 receptor selectivity to CCK 2. The compounds 18, 24, 28 and 33 have shown affinities at the CCK 1 receptor of 11, 10, 11 and 9 nM, respectively. These equipotent CCK 1 ligands were fully evaluated in behaviour pharmacological essays. An antidepressant effect was identified in the tail suspension- and the Porsolt swimming-test. The ED 50 values for 24 and 28 were determined in these assays as 0.46 and 0.49 mg/kg. The mixed antagonist 37 showed in addition to the antidepressant effects anxiolytic properties. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

Studies on the neurotoxicity of 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) in SH-SY5Y cells

We have studied the hypothesis that 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) is neurotoxic. Salsolinol induced a significant time and dose related inhibition of 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazoyl blue (MTT) reduction, and increased lactate dehydrogenase release (LDH) release from human SH-SY5Y neuroblastoma cells, at concentrations within the range of 1-methyl-4-phenylpyridinium (MPP+) cytotoxicity, in vitro. Cytotoxicity was not inhibited by the addition of antioxidants, monoamine oxidase inhibitors or imipramine. In confluent monolayers, salsolinol stimulated catecholamine uptake with EC50 values of 17 muM and 11 muM, for noradrenaline and dopamine, respectively. Conversely, at concentrations above 100 muM, salsolinol inhibited the uptake of noradrenaline and dopamine, with IC50 values of 411 muM and 379 muM, respectively. The inhibition of catecholamine uptake corresponded to the increase displacement of [3H]nisoxetine from the uptake 1 site by salsolinol, as the Ki (353 muM) for displacement was similar to the IC50 (411 and 379 muM) for uptake. Salsolinol stimulated catecholamine uptake does not involve the uptake recognition site, or elevation of cAMP, cGMP, or inhibition of protein kinase C. Salsolinol also inhibited both carbachol (1 mM) and K+ (100 mM, Na+ adjusted) evoked released of noradrenaline from SH-SY5Y cells, with IC50 values of 500 muM and 120 muM, respectively. In conclusion, salsolinol appears to be cytotoxic to SH-SY5Y cells, via a mechanism that does not require uptake 1, bioactivation by monoamine oxidase, or membrane based free radical damage. The effects of salsolinol on catecholamine uptake, and the mechanism of toxicity require further investigation.

Organic reactions in ionic liquids; Ionic liquid-accelerated three-component reaction: a rapid one-pot synthesis of 3-alkyl-5-[(Z)-arylmethylidene]-1,3-thiazolidine-2,4-diones

A rapid one-pot synthesis of 3-alkyl-5-[(Z)-arylme­thylidene]-1,3-thiazolidine-2,4-dionesis described that occurs in recyclable ionic liquid [bmim]PF6 (1-butyl-3-methylimidazolium hexafluorophosphate).Significant rate enhancement and good selectivity have been observed.

Synthesis and iron binding studies of myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate, and iron binding studies of all myo-inositol tetrakisphosphates

The first syntheses of the natural products myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate are described. The protected key intermediates 4,5,6-tri-O-benzoyl-myo-inositol and (+/-)-3,4,5,6-tetra-O-benzyl-myo-inositol were phosphorylated with dibenzyl N,N-di-isopropylphosphoramidite in the presence of 1H-tetrazole and subsequent oxidation of the phosphite. The crystal structures of the synthetic intermediates (+/-)-1-O-(tert-butyldiphenylsilyl)-2,3,O-cyclohexylidene-myo-inos itol and (+/-)-4,5,6-tri-O-benzoyl-1-O-(tert-butyldiphenylsilyl)-2,3-O-cycl ohexylidene- myo-inositol are reported. myo-Inositol 1,2,3-trisphosphate (+/-)-myo-inositol 1,2-bisphosphate, and all isomeric myo-inositol tetrakisphosphates were evaluated for their ability to alter HO. production in the iron-catalysed Haber-Weiss reaction. The results demonstrated that a 1,2,3-grouping of phosphates in myo-inositol was necessary for inhibition also that (+/-)-myo-inositol 1,2-bisphosphate potentiated HO. production. myo-Inositol 1,2,3-trisphosphate resembled myo-inositol hexakisphosphate (phytic acid) in its ability to act as a siderophore by promoting iron-uptake into Pseudomonas aeruginosa.

A combinatorial approach to the chemical synthesis and biological evaluation of 3,4,5-trisubstiituted furan-2(5H)-ones

Many important natural products contain the furan-2(5H)-one structure. The structure of this molecule lends itself to manipulation using combinatorial techniques due to the presence of more than one site for the attachment of different suhstituents. By developing different reaction schemes at the three sites available for attachment on the furan-2(5H)-one scaffold, combinatorial chemistry techniques can be employed to assemble libraries of novel furan 2(5H)-ones. These libraries can then be entered into various biological screening programmes. This approach will enable a vast diversity or compounds to be examined, in the hope or finding new biologically active Iead structures. The work in this thesis has investigated the potential that combinatorial chemistry has in the quest for new biologically active lead structures based on the furan-2(5H)-one structure. Different reactions were investigated with respect to their suitability for inclusion in a library. Once sets of reactions at the various sites had been established, the viability of these reactions in the assembly of combinatorial libraries was investigated. Purification methods were developed, and the purified products entered into suitable biological screening tests. Results from some of these tests were optimised using structure activity relationships, and the resulting products re-screened. The screening tests performed were for anticancer and antimicrobial activity, cholecystokinin (CCK-B) antagonism and anti-inflammatory activity (in the quest for novel cyclo-oxygenase (COX-2) selective non-steroidal anti-inflammatory drugs). It has been shown that many reactions undergone by the furan-2(5H)-one structure are suitable for the assembly of a combinatorial library. Investigation into the assembly of different libraries has been carried out with initial screening results included. From this work, further investigation into combinatorial library assembly and structure activity relationships of screened reaction products can be undertaken.

Conformational analysis of the natural iron chelator myo-inositol 1,2,3-trisphosphate using a pyrene-based fluorescent mimic

myo-Inositol phosphates possessing the 1,2,3-trisphosphate motif share the remarkable ability to completely inhibit iron-catalysed hydroxyl radical formation. The simplest derivative, myo-inositol 1,2,3-trisphosphate [Ins(1,2,3)P3], has been proposed as an intracellular iron chelator involved in iron transport. The binding conformation of Ins(1,2,3)P3 is considered to be important to complex Fe3+ in a 'safe' manner. Here, a pyrene-based fluorescent probe, 4,6-bispyrenoyl-myo-inositol 1,2,3,5-tetrakisphosphate [4,6-bispyrenoyl Ins(1,2,3,5)P4], has been synthesised and used to monitor the conformation of the 1,2,3-trisphosphate motif using excimer fluorescence emission. Ring-flip of the cyclohexane chair to the penta-axial conformation occurs upon association with Fe3+, evident from excimer fluorescence induced by π-π stacking of the pyrene reporter groups, accompanied by excimer formation by excitation at 351 nm. This effect is unique amongst biologically relevant metal cations, except for Ca 2+ cations exceeding a 1:1 molar ratio. In addition, the thermodynamic constants for the interaction of the fluorescent probe with Fe3+ have been determined. The complexes formed between Fe 3+ and 4,6-bispyrenoyl Ins(1,2,3,5)P4 display similar stability to those formed with Ins(1,2,3)P3, indicating that the fluorescent probe acts as a good model for the 1,2,3-trisphosphate motif. This is further supported by the antioxidant properties of 4,6-bispyrenoyl Ins(1,2,3,5)P4, which closely resemble those obtained for Ins(1,2,3)P3. The data presented confirms that Fe3+ binds tightly to the unstable penta-axial conformation of myo-inositol phosphates possessing the 1,2,3-trisphosphate motif. © 2010 The Royal Society of Chemistry.

The isolation and identification of dermatologically active constituents of coal tar

The production and uses of coal tar are reviewed as are the uses of steroids and cytotoxic agents in the treatment of psoriasis with a review of the condition also. An attempt was made to improve the efficaciousness and cosmetic acceptability of a low temperature tar, by screening fractions of this tar, derived from a variety of separation procedures. The most efficacious fraction was the highest boiling acid fraction, which is believed to consist mainly of mono- and di-hydric phenols. A time and concentration study showed that the optimum regime was the application of a 10% concentration in 5% wool fat in soft, yellow paraffin daily for 21 days. The mouse tail skin was selected as an experimental model, to ascertain the efficaciousness of fractions, because of the similarities between this skin and the psoriatic lesion. The activity of a fraction was monitored by the inducement of a granular layer in the mouse tail epidermis. Because coal tar is not an easy medium to work with, and the active fractions showed no increase in cosmetic acceptability over the parent coal tar, likely coal tar constituents were selected for screening on the basis of phenolic character, and the molecular weight range elucidated by mass spectroscopy. 32 potential anti-psoriatic agents were screened on mouse tail. Two catechols, 3,5-di-t-butyl and 4-t-butyl catechols were active. Other structures showed little or no activity. 24 catechols were screened and two extremely active catechols were discovered, 3-methyl-5-t-octyl and 5-methyl-3-t-octyl catechols. The screening of catechol-rich coal tar fractions and a coal tar fraction which had had the catechols removed by oxidation, showed that some anti-psoriatic activity was contained in the catechol fraction of coal tar. Attempts to elucidate the mode of action of these two compounds met with little success, but two modes of action are suggested.

Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced aCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced aCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of aCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on aCGRP binding and cAMP production; they are likely to indirectly influence the binding site for aCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired aCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.

Fluorescent probe:complexation of Fe3+ with the myo-inositol 1,2,3-trisphosphate motif

Natural myo-inositol phosphate antioxidants containing the 1,2,3-trisphosphate motif bind Fe3+ in the unstable penta-axial conformation.

The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport

The yeast gene fab1 and its mammalian orthologue Pip5k3 encode the phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinases Fab1p and PIKfyve, respectively, enzymes that generates phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. A shared feature of fab1Delta yeast cells and mammalian cells overexpressing a kinase-dead PIKfyve mutant is the formation of a swollen vacuolar phenotype: a phenotype that is suggestive of a conserved function for these enzymes and their product, PtdIns(3,5)P(2), in the regulation of endomembrane homeostasis. In the current study, fixed and live cell imaging has established that, when overexpressed at low levels in HeLa cells, PIKfyve is predominantly associated with dynamic tubular and vesicular elements of the early endosomal compartment. Moreover, through the use of small interfering RNA, it has been shown that suppression of PIKfyve induces the formation of swollen endosomal structures that maintain their early and late endosomal identity. Although internalisation, recycling and degradative sorting of receptors for epidermal growth factor and transferrin was unperturbed in PIKfyve suppressed cells, a clear defect in endosome to trans-Golgi-network (TGN) retrograde traffic was observed. These data argue that PIKfyve is predominantly associated with the early endosome, from where it regulates retrograde membrane trafficking to the TGN. It follows that the swollen endosomal phenotype observed in PIKfyve-suppressed cells results primarily from a reduction in retrograde membrane fission rather than a defect in multivesicular body biogenesis.

Facile synthesis and proposed mechanism of α,ω‐oxetanyl-telechelic poly(3-nitratomethyl-3-methyl oxetane) by an SN2(i) nitrato displacement method in basic media

The synthesis of a novel heterocyclic–telechelic polymer, α,ω-oxetanyl-telechelic poly(3-nitratomethyl-3-methyl oxetane), is described. Infrared spectroscopy (IR), gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR) spectroscopy have been used to confirm the successful synthesis, demonstrating the presence of the telechelic-oxetanyl moieties. Synthesis of the terminal functionalities has been achieved via displacement of nitrato groups, in a manner similar to that employed with other leaving groups such as azido, bromo, and nitro, initiated by nucleophiles. In the present case, displacement occurs on the ends of a nitrato-functionalized polymer driven by the formation of sodium nitrate, which is supported by the polar aprotic solvent N,N-dimethyl formamide. The formation of an alkoxide at the polymer chain ends is favored and allows internal back-biting to the nearest carbon bearing the nitrato group, intrinsically in an SN2(i) reaction, leading to α,ω-oxetanyl functionalization. The telechelic-oxetanyl moieties have the potential to be cross-linked by chemical (e.g., acidic) or radiative (e.g., ultraviolet) curing methods without the use of high temperatures, usually below 100°C. This type of material was designed for future use as a contraband simulant, whereby it would form the predominant constituent of elastomeric composites comprising rubbery polymer with small quantities of solids, typically crystals of contraband substances, such as explosives or narcotics. This method also provides an alternative approach to ring closure and synthesis of heterocycles.