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The titanium-mediated cyclopropanation reaction using Ti((OPr)-Pr-i)(3)Me/EtMgBr/BF3 center dot OEt2 has been applied to various 2-cyanopyrrolidines for the synthesis of functionalized 2-(1-aminocyclopropyl)pyrrolidine-3,4-diol derivatives (dideoxyiminoalditols). Under the same experimental conditions the trans-5-azidomethyl-2-cyanopyrrolidine derivative was not cyclopropanated but reduced into the corresponding 5-amino-2-cyano derivative. After polyol deprotection 2-(1-aminocyclopropyl)pyrrolidine-3,4-diols were obtained and their inhibitory activity towards 13 glycosidases has been evaluated. (2S,3S,4R,5S)-2-(1-Aminocyclopropyl)-5-methylpyrrolidine-3,4-diol (38), which has the same absolute configuration as L-fucose, is a moderate (IC50=44 mu M), but selective, inhibitor of alpha-L-fucosidase from human placenta. (C) 2011 Elsevier Ltd. All rights reserved.

Roscovitine and purvalanol are purine derivative cyclin-dependent kinase (CDK) inhibitors that induce apoptosis in various types of cancer cells. However, their impact on the apoptotic cell death mechanism requires further elucidation. Natural polyamines putrescine, spermidine and spermine play essential roles in the regulation of cell growth and proliferation. Increased levels of polyamines in cells are considered to be involved in cancer progression. Intracellular polyamine levels are under the control of several catabolic enzymes, such as spermidine/spermine-N-acetyl transferase (SSAT), acetylpolyamine oxidase (APAO) and spermine oxidase (SMO), which could be altered by several therapeutic drugs. However, the possible role of polyamines in drug-induced apoptosis has yet to be clarified. In the present study, our aim was to determine the modulation of the polyamine catabolic pathway related to CDK inhibitor-induced apoptosis in Caco-2 cells. We found that roscovitine and purvalanol (each 20 mu M) induced apoptosis by activating caspase-9 and -3, and inhibiting the mitochondrial membrane potential in Caco-2 cells. CDK inhibitors decreased the intracellular putrescine and spermine levels without affecting spermidine levels. Although both roscovitine and purvalanol induced SSAT expression, they did not exert a significant effect on the APAO expression profile. SSAT transient silencing prevented roscovitine-induced apoptosis compared to parental cells. Thus, we concluded that roscovitine and purvalanol significantly induce apoptosis in Caco-2 cells by modulating the polyamine catabolism, and that SSAT could be an important target in evaluating the potential role of polyamines in apoptotic cell death.

Ser/Thr protein kinase CK2 is involved in several fundamental processes that regulate the cell life, such as cell cycle progression, gene expression, cell growth, and differentiation and embryogenesis. In various cancers, CK2 shows a markedly elevated activity that has been associated with conditions that favor the onset of the tumor phenotype. This prompts to numerous studies aimed at the identification of compounds that are able to inhibit the catalytic activity of this oncogenic kinase, in particular, of ATP-competitive inhibitors. The many available crystal structures indicate that this enzyme owns some regions of remarkable flexibility which were associated to important functional properties. Of particular relevance is the flexibility, unique among protein kinases, of the hinge region and the following helix αD. This study attempts to unveil the structural bases of this characteristic of CK2. We also analyze some controversial issues concerning the functional interpretation of structural data on maize and human CK2 and try to recognize what is reasonably established and what is still unclear about this enzyme. This analysis can be useful also to outline some principles at the basis of the development of effective ATP-competitive CK2 inhibitors.

NOTICE: this is the author’s version of a work that was accepted for publication in Toixcon. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Toxicon, [Volume 57, Issue 6, May 2011] doi:10.1016/j.toxicon.2011.03.014 http://www.sciencedirect.com/science/article/pii/S0041010111001000, peer-reviewed, For many years, the presence of yessotoxins (YTXs) in shellfish has contributed to the outcome of the traditional mouse bioassay and has on many occasions caused closure of shellfisheries. Since YTXs do not appear to cause diarrhoea in man and exert low oral toxicity in animal experiments, it has been suggested that they should be removed from regulation. Before doing so, it is important to determine whether the oral toxicity of YTXs is enhanced when present together with shellfish toxins known to cause damage to the gastrointestinal tract. Consequently, mice were given high doses of YTX, at 1 or 5 mg/kg body weight, either alone or together with azaspiracid-1 (AZA1) at 200 μg/kg. The latter has been shown to induce damage to the small intestine at this level. The combined exposure caused no clinical effects, and no pathological changes were observed in internal organs. These results correspond well with the very low levels of YTX detected in internal organs by means of LCMS/MS and ELISA after dosing. Indeed, the very low absorption of YTX when given alone remained largely unchanged when YTX was administered in combination with AZA1. Thus, the oral toxicity of YTX is not enhanced in the presence of sub-lethal levels of AZA1.

The search for novel compounds of relevance to the treatment of diseases caused by trypanosomatid protozoan parasites continues. Screening of a large library of known bioactive compounds has led to several drug-like starting points for further optimisation. In this study, novel analogues of the monoamine uptake inhibitor indatraline were prepared and assessed both as inhibitors of trypanothione reductase (TryR) and against the parasite Trypanosoma brucei. Although it proved difficult to significantly increase the potency of the original compound as an inhibitor of TryR, some insight into the preferred substituent on the amine group and in the two aromatic rings of the parent indatraline was deduced. In addition, detailed mode of action studies indicated that two of the inhibitors exhibit a mixed mode of inhibition.

The seven-step synthesis of a novel structural isomer of VPA-985, N-[3-chloro-4-(5H-pyrrolo[1,2-c][1.3] benzodiazepin-6(11H)-ylcarbonyl)phenyl]-5-fluoro-2-methylbenzamide, is described. (2-Aminophenyl)(1H-pyrrol-2-yl)methanone was converted with thiophosgene into (2-isothiocyanatophenyl)(1H-pyrrol-2-yl) methanone which was cyclised in the presence of base to 5-thioxo-5,6-dihydro-11H-pyrrolo[1,2-c][1.3]benzodiazepin-11-one. The latter underwent desulfurisation with Raney nickel followed by reduction with lithium aluminium hydride in the presence of aluminium trichloride and the resulting 6,11-dihydro-5H-pyrrolo[1,2-c][13]-benzodiazepine acylated with 2-chloro-4-nitrobenzoyl chloride to afford 6-(2-chloro-4-nitrobenzoyl)-6,11-dihydro-5H-pyrrolo[1,2-c][1.3]benzodiazepine. The nitro group in the latter compound was reduced with zinc and ammonium chloride to give the corresponding aniline derivative which was then acylated with 2-methyl-5-fluorobenzoyl chloride to provide the final product. (C) 2011 Elsevier Ltd. All rights reserved. Tetrahedron

The racemic triflate derivatives 5-8 of the 5-, 6-, 7-, and 8-hydroxylated 2-(di-n-propylamino)-tetralins 1-4 were shown to possess similar pharmacological profiles to their phenolic counterparts in in vitro binding and in vivo biochemical and behavioral assays in rats. Consequently, subcutaneous administration of the 5-, 6-, and 7-triflates displayed essentially dopaminergic agonist properties, while the 8-triflate was shown to be a selective 5-HT1A receptor agonist. With respect to their agonist activities, the triflates were less potent than their phenolic analogs. The absolute oral bioavailability of compound 8 (8-triflate) was 4-5 times greater than the corresponding hydroxylated compound. Interestingly, in the in vivo biochemical assay compound 8 was found to be more potent after oral than after subcutaneous administration, indicating formation of one or more active metabolites. Following a study of the metabolism of compound 8 in rat hepatocytes, the monopropyl analog 9 was identified as the major metabolite and was suprisingly found to be more potent than compound 8. Oral administration of compound 5 (5-triflate) resulted in behavioral and biochemical effects indicative of mixed DA/5-HT1A agonist properties not seen after subcutaneous administration. These results may also be indicative of the formation of active metabolites.

A method is described for the rapid production and purification of new potential dopamine agonists. Via thermal heating (refluxing in an oil bath) and microwave exposure 2-[ N -n-3-fluoropropyl- N -(4-methylphenyl)ethylamino]-5-hydroxytetralin ( 12 ), 2-[ N -n-3-fluoropropyl- N -(4-fluorophenyl)ethylamino]-5-hydroxytetralin ( 13 ), and their isotopic fluorine-18 derivatives were synthesized, respectively. The fluorine-18 label was introduced via N -fluoroalkylation with no-carrier-added (n.c.a.) 18 FCH 2 CH 2 CH 2 I. In 115 min, radiochemical yields of 11% (corrected for decay) were achieved for both compounds. The specific activity ranged from 15–75 GBq/μmol. After i.v. injection in rats, the fluorine-18 labeled compounds were evaluated for their in vivo binding to the D 2 -receptors. The radioactivity levels in the striatum, nucleus accumbens and tuberculum olfactorius were not significantly higher than in the cerebellum and frontal cortex at 15, 30 and 60 min after administration of the tetralin derivatives. Dopamine depletion with reserpine did not affect the uptake in the dopamine D 2 -receptor rich area. Remarkable high uptakes were found in the adrenal for both compounds.

The pharmacokinetics of the dopamine D2 agonist S(-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (1, N-0923) after intravenous, intraperitoneal, and oral administration was studied in freely moving male and female albino Wistar rats. In all cases, the dose was 1 0 mumol . kg-1. Levels of 1 in plasma were monitored up to 6 h after dosing by high-performance liquid chromatography. A biphasic disappearance pattern with a rapid distribution phase was observed in the curve of concentration in plasma versus time. Pharmacokinetic analysis based on a two-compartment model with elimination from the central compartment yielded the following parameters for male rats (mean +/-standard deviation, n = 3): elimination half-life was 108 +/- 7 min, apparent volume of distribution of the central compartment was 397 +/- 44 mL . kg-1, the plasma clearance was 32 +/- 4 mL . min-1 . kg-1, and the apparent volume of distribution at steady state was 2.29 +/- 0.25 L . kg-1. No significant difference (analysis of variance p value > 0.25) was found in the pharmacokinetic data between male and female rats. An extremely fast absorption was found after intraperitoneal administration. Maximal concentrations in plasma were often observed at the first time point 5 min after dosing. The bioavailability (mean +/- standard deviation, n = 3) was 7.9 +/- 2.7% for male rats and 6.5 +/- 2.1% for female rats. An increase of the elimination half-life of at least 40% for male rats and 300% for female rats was observed, indicating dose-dependent kinetics after intraperitoneal administration. After oral dosing, levels of 1 in plasma were extremely low and often below the quantitation limit of the assay (10 pmol . mL-1). This did not allow detailed kinetic analysis. Even in the more favorable cases, the bioavailability did not exceed 0.5%. Although the 5-0-glucuronide and 5-sulfate are the major metabolites of 1, they could not be detected in plasma, indicating efficient excretion into bile and urine.

Based on the hypothesis that simultaneous stimulation of dopamine-D1 and -D2 receptors could be beneficial for the treatment of Parkinson's disease7,13, we prepared 5,6,7,8-tetrahydro-6-[propyl[2-(2-thienyl)ethyl]amino]-1,2-naphthalenediol (PTAT, 4), the diol analogue of N-0437 (3), as a potential mixed D1/D2-receptor agonist. The synthesis of 4 was carried out via a new strategy. The crucial step in this strategy was the conversion of 1-diazo-4-(2,3-dimethoxyphenyl)-2-butanone (15) to the key intermediate 5,6-dimethoxy-2-tetralone (9) via a rhodium(II)-acetate-catalysed cyclisation, followed by transformation under the influence of trifluoroacetic acid. Pharmacological evaluation of the dopaminergic properties of PTAT (4) revealed that this compound displays high affinity for both D1 and D2 receptors and has the same ability as DPAT (1), a mixed D1/D2-receptor agonist, to induce locomotor activity in mice rendered acutely akinetic with reserpine.

Apoptosis or programmed cell death is an inherent part of the development and homeostasis of multicellular organisms. Dysregulation of apoptosis is implicated in the pathogenesis of diseases such as cancer, neurodegenerative diseases and autoimmune disorders. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce apoptosis by binding death receptor (DR)4 (TRAIL-R1) and DR5 (TRAIL-R2), which makes TRAIL an interesting and promising therapeutic target. To identify peptides that specifically interact with DR5, a disulfide-constrained phage display peptide library was screened for binders towards this receptor. Phage-displayed peptides were identified that bind specifically to DR5 and not to DR4, nor any of the decoy receptors. We show that the synthesized peptide, YCKVILTHRCY, in both monomeric and dimeric forms, binds specifically to DR5 in such a way that TRAIL binding to DR5 is inhibited. Surface plasmon resonance studies showed higher affinity towards DR5 for the dimeric form then the monomeric form of the peptide, with apparent K(d) values of 40 nm versus 272 nm, respectively. Binding studied on cell lines by flow cytometry analyses showed concentration-dependent binding. Upon co-incubation with increasing concentrations of TRAIL, the peptide binding was reduced. Moreover, both the monomeric and dimeric forms of the peptide reduced TRAIL-induced cell death in Colo205 colon carcinoma cells. The peptide, YCKVILTHRCY, or its derivates, may be a useful investigative tool for dissecting signalling via DR5 relative to DR4 or could act as a lead peptide for the development of therapeutic agents in diseases with dysregulated TRAIL-signalling.

A number of dioxolane, dioxane, and dioxepine quinazoline derivatives have been synthetized and evaluated as EGFR inhibitors. Their cytotoxic activity has been tested against two cell lines overexpressing and not expressing EGFR. Most derivatives were able to counteract EGF-induced EGFR phosphorylation, and their potency was comparable to the reference compound PD153035. The size of the fused dioxygenated ring was crucial for the biological activity, the dioxane derivatives being the most promising class of this series.

414UR Times Cited:0 Cited References Count:43; The 1,5-benzothiazepine-4-one scaffold was earlier shown to provide efficient protease inhibitors. In this contribution, we describe its use in the design of factor VIIa/tissue factor inhibitors. A series containing a scaffold non-substituted on its aryl part led to compound 20 with an IC50 of 2.16 mu M. Following molecular modelling studies of this compound, a second series was prepared, which necessitated the synthesis of protected 7- or 8-substituted 1,5-benzothiazepine-4-one derivatives. (C) 2009 Elsevier Ltd. All rights reserved.

1 - Article; A new synthesis of unsymmetrical phosphinic acids R'P(O)OHR '' has been evaluated. The first P-C bond was formed by base-promoted H-phosphinate alkylation of a protected H-phosphinate, which is easier and safer to handle. A one-pot methodology was developed for the second P-C bond formation reaction that involves the sila-Arbuzov reaction. This methodology was then extended to the synthesis of a dialkylphosphinic acid with an amino functionality. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

We assessed the renal hemodynamic changes occurring acutely after glomerular mesangial cell immune injury and the effects of thromboxane receptor antagonism on these changes. A single intravenous proteinuric dose of a monoclonal antibody raised against the rat thymocyte antigen Thy 1.1 (ER4), which is also expressed in rat mesangial cells, induced acute decrements in glomerular filtration rate and in renal blood flow in male Munich-Wistar rats. One hour after administration of 4 to 6 mg/kg of ER4 antibody, glomerular filtration rate and renal blood flow decreased by 80 and 36%, respectively. These decrements were associated with significant increments in basal thromboxane B2 synthesis in isolated glomeruli and no changes in glomerular prostaglandin E2 synthesis. Pretreatment of animals with the thromboxane receptor antagonist SQ-29, 548 (2 mg/kg) significantly ameliorated decrements in glomerular filtration rate and renal blood flow. Pretreatment with a structurally dissimilar thromboxane receptor antagonist, L-670,596 (3 mg/kg) had no effect. Both antagonists at the doses employed abolished the decrements in renal blood flow induced by systemic administration of the thromboxane mimetic U-46619. Whereas the SQ-29,548 antagonist had no effect on glomerular leukotriene B4 and 12-hydroxyeicosate-traenoic acid synthesis, the L-670,596 thromboxane receptor antagonist significantly inhibited glomerular synthesis of these eicosanoids in immunologically injured glomeruli. These observations indicate that in mesangial cell immune injury the protective effect of thromboxane A2 receptor antagonism on glomerular filtration rate and renal blood flow is not solely due to inhibition of the vasoconstrictor effects of thromboxane A2. An effect on the synthesis of arachidonate lipoxygenation products may also play a role.

Tolerance to otherwise lethal cerebral ischemia in vivo or to oxygen-glucose deprivation (OGD) in vitro can be induced by prior transient exposure to N-methyl-d-aspartic acid (NMDA): preconditioning in this manner activates extrasynaptic and synaptic NMDA receptors and can require bringing neurons to the “brink of death.” We considered if this stressful requirement could be minimized by the stimulation of primarily synaptic NMDA receptors. Subjecting cultured cortical neurons to prolonged elevations in electrical activity induced tolerance to OGD. Specifically, exposing cultures to a K+-channel blocker, 4-aminopyridine (20–2500 μm), and a GABAA receptor antagonist, bicuculline (50 μm) (4-AP/bic), for 1–2 days resulted in potent tolerance to normally lethal OGD applied up to 3 days later. Preconditioning induced phosphorylation of ERK1/2 and CREB which, along with Ca2+ spiking and OGD tolerance, was eliminated by tetrodotoxin. Antagonists of NMDA receptors or l-type voltage-gated Ca2+ channels (L-VGCCs) applied during preconditioning decreased Ca2+ spiking, phosphorylation of ERK1/2 and CREB, and OGD tolerance more effectively when combined, particularly at the lowest 4-AP concentration. Inhibiting ERK1/2 or Ca2+/calmodulin-dependent protein kinases (CaMKs) also reduced Ca2+ spiking and OGD tolerance. Preconditioning resulted in altered neuronal excitability for up to 3 days following 4-AP/bic washout, based on field potential recordings obtained from neurons cultured on 64-channel multielectrode arrays. Taken together, the data are consistent with action potential-driven co-activation of primarily synaptic NMDA receptors and L-VGCCs, resulting in parallel phosphorylation of ERK1/2 and CREB and involvement of CaMKs, culminating in a potent, prolonged but reversible, OGD-tolerant phenotype.

Some N-(3,5-di-/1,3,5-trimethylpyrazole-4-yl)-4-substitutedbenzamide derivatives were prepared as possible antiociceptive-antimicrobial agents. New amide derivatives (3-12) were synthesized by reacting 4-amino-3,5-di and 1,3,5-trimethylpyrazoles with 4-substitutedbenzoyl chlorides. Hotplate and tail-immersion tests were used for the determination of the antinociceptive activity. Morphine, was used as a standard test drug. All compounds were administered at a dose of 100 mg/kg ip and some of them had significant antinociceptive activity in both tests. Compound 10 (N-(1,3,5-trimethylpyrazole-4-yl)-4-bromobenzamide), was the most active one in both tests among the compounds. The antinociceptive activity of the compounds 10, 11 (N-(1,3,5-trimethylpyrazole-4-yl)-4-chlorobenzamide), and 12 (N-(1,3,5-trimethylpyrazole-4-yl)-4-fluorobenzamide), started at 30 minutes and continued up to 150 minutes in the hotplate test. Also compounds were tested for their in vitro antimicrobial activity, but exhibited weak antibacterial activity.

Subunit vaccines capable of providing protective immunity against the intracellular pathogens and cancers that kill millions of people annually require an adjuvant capable of directing a sufficiently potent cytotoxic T lymphocyte response to purified antigens, without toxicity issues. Archaeosome lipid vesicles, prepared from isoprenoid lipids extracted from archaea, are one such adjuvant in development. Here, the stability of an archaeal core lipid 2,3-di-O-phytanyl-sn-glycerol (archaeol) is used to advantage to synthesize a series of disaccharide archaeols and show that subtle variations in the carbohydrate head group alters the type and potency of immune responses mounted in a mammal. Critically, a glycosylarchaeol was required to elicit high cytotoxic CD8(+) T cell activity, with highest responses to the antigen entrapped in archaeosomes containing disaccharides of glucose in beta- or alpha1-6 linkage (beta-gentiobiose, beta-isomaltose), or of beta-lactose. This first study on synthetic archaeal lipid adjuvants reveals potential for this class of regulatory friendly, easily scalable, inexpensive, and potent glyco-adjuvant

2-(Alkenyloxy)naphthalene-l-carbaldehyde oximes are oxidized with potassium iodide, iodine and sodium bicarbonate directly to novel naphthopyranoisoxazoles. Naphthopyranoisoxazoles are also prepared from 2-(3-chloroallyloxy)napbthalene-1-carbaldehyde oxime or 2-(alkynyloxy)naphthalene-1-carbaldehyde oximes by oxidation with sodium hypochlorite and triethylamine. The oxidation of 2-(alkenyloxy)naphthatene-1-carbaldehyde oximes with sodium hypochlorite and triethylamine afforded novel naphthopyranoisoxazolines. The former reaction is tentatively proposed to occur via activation of the alkene side chain by means of an iodonium intermediate and either 1,3-dipolar interaction with a nitrile oxide side-group or cyclization with a hydroximic acid iodide side-group. Synthesis-Stuttgart

Vaccines capable of eliciting long-term T cell immunity are required for combating many diseases. Live vectors can be unsafe whereas subunit vaccines often lack potency. We previously reported induction of CD8(+) T cells to Ag entrapped in archaeal glycerolipid vesicles (archaeosomes). In this study, we evaluated the priming, phenotype, and functionality of the CD8(+) T cells induced after immunization of mice with OVA-Methanobrevibacter smithii archaeosomes (MS-OVA). A single injection of MS-OVA evoked a profound primary response but the numbers of H-2K(b)OVA(257-264)-specific CD8(+) T cells declined by 14-21 days, and 300 days. CFSE-labeled naive OT-1 (OVA(257-264) TCR transgenic) cells transferred into MS-OVA-immunized recipients cycled profoundly (>90%) within the first week of immunization indicating potent Ag presentation. Moreover, approximately 25% cycling of Ag-specific cells was seen for >50 days, suggesting an Ag depot. In vivo, CD8(+) T cells evoked by MS-OVA killed >80% of specific targets, even at day 180. MS-OVA induced responses similar in magnitude to Listeria monocytogenes-OVA, a potent live vector. Furthermore, protective CD8(+) T cells were induced in TLR2-deficient mice, suggesting nonengagement of TLR2 by archaeal lipids. Thus, an archaeosome adjuvant vaccine represents an alternative to live vectors for inducing CD8(+) T cell memory

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetics tool to silence gene expression in multiple organisms, including plants, nematodes and insects. In this study, DNA vectors capable of promoting the synthesis of long hairpin dsRNAs in vivo from a DNA template to suppress gene expression in insect cells have been successfully constructed. The inhibition of the expression of a gene encoding enhanced green fluorescent protein (eGFP) in insect cells was demonstrated by using plasmid or baculovirus vectors. Both plasmid and baculovirus vectors were able to inhibit eGFP expression in a dose dependent manner. Complete inhibition was obtained when co-transfection ratios of target plasmid to inhibition plasmid were 1:1 and 1:0.1. Eighty percent suppression was still maintained even when the ratio of eGFP plasmid to 'hairpin' plasmid was as high as 1:0.01. When the hairpin dsRNAs were encoded in a baculovirus, the suppression was about 50% when the ratio of 'target' baculovirus to 'inhibition' baculovirus reached 1:10. Therefore, the designed plasmid and baculovirus vectors are useful to induce RNAi in insect cell systems.

A new environment-sensitive fluorophore, 6-N,N-(dimethylamino)-2,3-naphthalimide (6DMN) was introduced in the delta-selective opioid peptide agonist H-Dmt-Tic-Glu-NH(2) and in the mu-selective opioid peptide agonist endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH(2)). Environment-sensitive fluorophores are a special class of chromophores that generally exhibit a low quantum yield in aqueous solution but become highly fluorescent in nonpolar solvents or when bound to hydrophobic sites in proteins or membranes. New fluorescent delta-selective irreversible antagonists (H-Dmt-Tic-Glu-NH-(CH(2))(5)-CO-Dap(6DMN)-NH(2) (1) and H-Dmt-Tic-Glu-Dap(6DMN)-NH(2) (2)) were identified as potential fluorescent probes showing good properties for use in studies of distribution and internalization of delta receptors by confocal laser scanning microscopy.

''Click chemistry" was explored to synthesize two series of 2-(1,2,3-triazolyl) adenosine derivatives (1-14). Binding affinity at the human A(1), A(2A), and A(3)ARs (adenosine receptors) and relative efficacy at the A(3)AR were determined. Some triazol-1-yl analogues showed A(3)AR affinity in the low nanomolar range, a high ratio of A(3)/A(2A) selectivity, and a moderate-to-high A(3)/A(1) ratio. The 1,2,3-triazol-4-yl regiomers typically showed decreased A(3)AR affinity. Sterically demanding groups at the adenine C2 position tended to reduce relative A(3)AR efficacy. Thus, several 5'-OH derivatives appeared to be selective A(3)AR antagonists, i.e., 10, with 260-fold binding selectivity in comparison to the A(1)AR and displaying a characteristic docking mode in an A(3)AR model. The corresponding 5'-ethyluronamide analogues generally showed increased A(3)AR affinity and behaved as full antagonists, i.e., 17, with 910-fold A(3)/A(1) selectivity. Thus, N-6-substituted 2-( 1,2,3-triazolyl)-adenosine analogues constitute a novel class of highly potent and selective nucleoside-based A(3)AR antagonists, partial agonists, and agonists.

While searching for a PET method to determine the density and occupancy of the dopamine D-3 receptor, we found evidence that suggested that the dopamine D-3 antagonist GR218231 could be a substrate of the P-glycoprotein efflux pump. P-glycoprotein protects the brain against toxic substances and xenobiotics, but it also hampers the delivery of various drugs into the brain. In this study, we aimed to explore whether radiolabeled GR218231 could be applied as a PET tracer for monitoring P-glycoprotein activity in the blood-brain barrier. Such an imaging technique could be useful for the development of new drugs and novel strategies to deliver drugs to the brain and for identification of undesirable drug-drug interactions. Methods: As a potential PET tracer, GR218231 was labeled with C-11 by reaction of the newly synthesized desmethyl precursor with C-11-methyl triflate. The biodistribution of C-11-GR218231 was determined in rats. To assess specific binding to the dopamine D3 receptor, blocking experiments with unlabeled GR218231 (0.2 and 2.5 mg/kg) were performed. To demonstrate the influence of P-glycoprotein on cerebral uptake of C-11-GR218231, the efflux pump was modulated with 50 mg/kg cyclosporine A. The sensitivity of C-11-GR218231 for P-glycoprotein modulation was assessed in dose-response studies, using escalating cyclosporine A dosages. Results: C-11-GR218231 was prepared in 53% +/- 8% decay-corrected radiochemical yield and had a specific activity of 15 +/- 10 GBq/mu mol (mean +/- SD). Biodistribution studies in rats revealed a low and homogeneous uptake in the brain. Pretreatment of the animals with unlabeled GR218231 did not demonstrate any specific binding. Modulation of P-glycoprotein with cyclosporine A caused a 12-fold higher C-11-GR218231 uptake in the brain, indicating that the low cerebral tracer uptake was caused by the P-glycoprotein efflux pump in the blood-brain barrier. Cyclosporine A close-escalation studies showed a dose-dependent sigmoidal increase in C-11-GR218231 uptake in brain and spleen (median effective dose [ED50], 23.3 +/- 0.6 and 38.4 +/- 2.4 mg/kg, respectively), whereas a dose-dependent decrease was observed in the pancreas (ED50, 36.0 +/- 4.4 mg/kg). Conclusion: Although C-11-GR218231 is unsuited for dopamine D3 receptor imaging with PET, it appears to be an attractive PET tracer for visualization and quantification of P-glycoprotein activity in the blood-brain barrier.

2‐Benzylamino‐1‐phenyl‐ethanones 1 were converted to the corresponding isoquinolinium perchlorates 2 in high yields using 70% HClO4‐FeCl3 mixture as a cyclization and oxidation reagent. A mild and high yielding method for the oxidation of perchlorates 2 to isoquinolin‐1‐ones 3 involving the treatment of 2 with KOH and K3[Fe(CN)6] in THF‐H2O two‐phase system at room temperature was developed. Compounds 2a–g were shown to be disproportionate to 3 and the corresponding 1,2‐dihydroisoquinoline 4 in the presence of base, which in turn is oxidized by K3[Fe(CN)6] to 2.

2-Amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic acid (4) (ATPC) is an unnatural amino acid with promise in applications as a building block for the synthesis of peptidomimetics. ATPC was obtained from both 3a and 3b thienopyrimidines by hydrolysis and hydrogenolysis, respectively. The synthesis of eleven ATPC-amino acids and two ATPC-peptides is described. ATPC is incorporated as N-terminal moiety in solution or solid-phase peptide synthesis using Boc or Fmoc methodology and without protection of the ATPC amino group. Synthesis-Stuttgart

A set of 1-(arylalkyl)quinolizidines, isosteric thioanalogues, and variously functionalized congeners were synthesized (see 1–25) and tested for affinity to sigma 1 and sigma 2 receptor subtypes, by displacing [3H]- (+)-pentazocine and [3H]DTG from guinea pig brain and rat brain preparations, respectively. All compounds exhibited a good affinity for the σ1 subtype, with subnanomolar Ki values for the best of them, while only modest or poor affinity for the σ2 subtype was observed (Tables 1 and 2). Some structureactivity relationships were put forward.

The pharmacokinetics and metabolic fate of the intrinsically active (anti-HIV) drug carrier succinylated human serum albumin (Suc-HSA) was studied in rats. Suc-HSA was prepared by derivatizing HSA with 1,4-[C-14]-succinic anhydride, a modification by which all available epsilonNH2-groups in HSA were converted into carboxylic groups.After iv injections of 0.3, 1.0, 3.0 and 10.0 mg/kg in freely moving rats, Suc-HSA showed a dose dependent elimination pattern, indicating a saturable elimination pathway. The Michaelis-Menten parameters V-max and K-m were 98.7 mug.min(-1).kg(-1) and 8.5 mug.ml(-1) respectively. The kinetics of Suc-HSA was influenced by anaesthesia. In anaesthetised animals, V-max and K-m were found to be 26.9 mug.min(-1).kg(-1) and 0.26 mug.ml(-1), respectively. This implies an intrinsic clearance of 100 ml.min(-1).kg(-1), which is about 10-fold higher as compared to 12 ml.min(-1).kg(-1) in freely moving animals.Intravenous administration of a sub-saturable dose of 3.0 mg.kg(-1)1,4-[C-14]-Suc-HSA to freely moving rats resulted in a biphasic elimination with an initial t(1/2) of 20 min and a terminal t(1/2) of 40 hrs. Excretion of metabolites in urine and faeces lasted for at least 48 hours. About 70% of the radioactive dose was excreted in urine, whereas maximally 2% was detected in faeces. Suc-HSA was degraded to its individual amino acids including succinylated lysine (the only radioactive product formed). Succinylated lysine was not further metabolised and mainly excreted via the urine. Immunohistochemical staining showed that even after 48 hrs Suc-HSA could be detected in livers. Together with the urinary excretion patterns, this points to a gradual degradation of Suc-HSA.

[Carbonyl-C-11]WAY-100635 ([C-11]WAY) is an established radioligand for the study of brain serotonin(1A) (5-HT1A) receptors in living animals and humans with positron emission tomography (PET). There is a recognised need to develop halogenated ligands for 5-HT1A receptors, either for labelling with longer lived fluorine-18 for more widespread application with PET or with iodine-123 for application with single photon emission tomography (SPET). Here we used autoradiography and PET to assess two new halogenated anlogues of WAY, namely 6BPWAY and 6FPWAY [N-(2-(1-(4-(2-methoxyphenyl)-piperazinyl) ethyl))-N-(2-(6-bromo-/fluoro-pyridinyl))cyclohexanecarboxamide] as prospective radioligands, initially using carbon-11 as the radiolabel. Labelling of 6BPWAY and 6FPWAY with carbon-11 was accomplished by acylation of the corresponding secondary amine precursors with [carbonyl-C-11]cyclohexanecarbonyl chloride. After incubation of human brain crysections with [C-11]6BPWAY or [C-11]6FPWAY, the highest accumulation of radioactivity was observed in cortical areas and the hippocampal formation. Both radioligands had high nonspecific binding. There was a rapid accumulation of radioactivity in the monkey brain after intravenous injection of [C-11]6BPWAY and [C-11]6FPWAY. High accumulation of radioactivity was observed in the frontal and temporal cortex and the raphe nuclei, areas known to contain a high density of 5-HT1A receptors. The ratios of radioactivity in receptor-rich temporal cortex to that in receptor poor cerebellum at peak equilibrium were 1.9 (at 10 min) and 3.0 at (at 20 min) for [C-11]6BPWAY and [C-11]6FPWAY, respectively. In pretreatment experiments with high doses of unlabelled WAY, the level of radioactivity in the frontal and temporal cortex and the raphe nuclei was reduced to the same level as in the cerebellum. Radioactive metabolites of [C-11]6FPWAY appeared at a rate similar to those for [C-11]WAY, with 17% of the radioactivity in plasma represented by unchanged radioligand after 40 min. Radioactive metabolites of [C-11]6BPWAY appeared much more slowly. At 40 min after injection 45% of the radioactivity in plasma still represented unchanged radioligand. The results indicate that 6-pyridinyl radiohalogented analogues of WAY are new leads to radioligands for PET or SPET. (C) 2001 Elsevier Science Inc. All rights reserved.

1 Peroxynitrite (ONOO-) the highly reactive coupling product of nitric oxide and superoxide, has been implicated in the pathngenesis of an increasing number of (inflammatory) diseases. At present, however. selective peroxynitrite antagonizing agents with therapeutic potential are not available. Therefore, the NADPH-oxidase inhibitor apocynin (3-hydroxy-3-methoxy-acetophenone) was tested for its ability to inhibit peroxynitrite formation in vitro. 2 The murine macrophage cell-line J774A.1, stimulated with IFN gamma/LPS, was used as a model. Conversion of 123-dihydrorhodamine (123-DHR) to its oxidation product 123-rhodamine was used to measure peroxynitrite production. 3 Stimulated peroxynitrite formation could be completely inhibited by apocynin, by the superoxide scavenger TEMPO as well as by the nitric oxide synthase inhibitor aminoguanidine. Apocynin and aminoguanidine specifically inhibited superoxide and nitric oxide formation respectively as confirmed by measuring lucigenin enhanced chemiluminescence and nitrite accumulation. 4 It is concluded that J774A.1 macrophages produce significant amounts of peroxynitrite, which is associated with nitric oxide production and NADPH-oxidase dependent superoxide formation. The NADPH-oxidase inhibitor apocynin proved to be a potent inhibitor of both superoxide and peroxynitrite formation by macrophages, which may be of future therapeutic significance in a wide range of inflammatory disorders.

The effect of ritanserin, a potent 5HT(2A/2C) receptor antagonist, used as an add-on medication to neuroleptic treatmentin patients with schizophrenia, was compared with that of placebo, in an international, double-blind, parallel-group study. Previously established neuroleptic therapy was maintained, and ritanserin 10 mg or placebo was given once daily for 8 weeks. Psychopathology was assessed with the Positive and Negative Syndrome Scale (PANSS) and the Clinical Global Impression (CGI) cale. Safety assessments included the Extrapyramidal Symptom Rating Scale (ESRS), and the requirement for antiparkinsonian medication was monitored. About 70 per cent of patients completed the treatment. There was no difference between the two groups in the numbers of patients with clinical improvement at endpoint on the PANSS negative subscale and total PANSS. The CGIs of overall severity of schizophrenia were better under placebo. The overall prevalence of side effects and the requirements for antiparkinsonism medication were comparable in the two groups. Copyright (C) 2000 John Wiley & Sons, Ltd.

Angiotensin-converting enzyme (ACE) inhibition reduces proteinuria in established adriamycin nephrosis. To investigate whether the reduction in proteinuria is due to decreased generation of angiotensin II (AngII) or to decreased degradation of bradykinin, four series of experiments in established adriamycin nephrosis were performed. In the first series, 2 mg/kg lisinopril reduced BP from 117 +/- 4 to 67 +/- 2 mmHg and proteinuria from 335 +/- 66 to 57 +/- 10 mg/24 h after 2 wk of treatment. Subsequent continuous intraperitoneal infusion of AngII: (250 ng/kg per min) for 2 wk partially restored proteinuria to 180 +/- 42 mg/24 h, whereas BP increased to 97 +/- 3 mmHg. Subsequent withdrawal of AngII restored the antiproteinuric effects of lisinopril, whereas subsequent withdrawal of lisinopril restored proteinuria to pretreatment values. In the second series, AT1 receptor blockade induced a fall in BP and proteinuria similar to that by lisinopril. In the third series, lisinopril reduced BP from 121 +/- 5 to 68 +/- 2 mmHg and proteinuria from 355 +/- 90 to 101 +/- 10 mg/24 h. Subsequent intraperitoneal infusion of bradykinin antagonist (HOE 140; 1 mg/kg per 24 h) for 2 wk did not affect BP (72 +/- 2 mmHg) or proteinuria (92 +/- 15 mg/24 h). In the fourth series, bradykinin (3 mg/kg per 24 h) was infused for 2 wk to mimic decreased bradykinin breakdown. This did not affect proteinuria, but induced a fall in BP from 114 +/- 3 to 93 +/- 4 mmHg. The BP-lowering effect of exogenous bradykinin was completely reversed by 1 wk infusion of HOE 140 (93 +/- 4 to 113 +/- 4 mmHg), while proteinuria remained unchanged. In conclusion, the antiproteinuric effect of ACE inhibition appears to be independent of bradykinin in this model, supporting a main role for reduction of AngII in the antiproteinuric action of ACE inhibition.

Negatively charged albumins (NCAs) have been identified as potent inhibitors of HIV-1 replication in vitro. Time of addition studies suggest that succinylated and aconitylated human serum albumin (Suc-HSA and Aco-HSA) act at an early stage of the virus life cycle, and surface plasmon resonance (BIAcore) experiments have confirmed a direct interaction of NCAs with HIV-1 gp120, Resistance to Suc-HSA and Aco-HSA was analyzed by characterizing HIV-1 variants that were selected in cell culture after serial passage of the NL4-3 strain in the presence of the compounds. After 24 passages (126 days) we isolated variants that were resistant to Suc-HSA (>27-fold) and Aco-HSA (37-fold), as compared with the wild-type NL4-3 virus. The binding of the NCA-resistant HIV strains to CD4(+) MT-4 cells could no longer be inhibited by either Suc- or Aco-HSA, The emergence of mutations in the envelope gp120 of the resistant virus paralleled the emergence of the resistant phenotype, The Suc-HSA-resistant strain was 100-fold cross-resistant to the G quartet-containing oligonucleotide AR177 (Zintevir, an HIV-binding inhibitor), and partially cross-resistant to dextran sulfate, but remained sensitive to the bicyclam AMD3100 and the chemokine SDF-1 alpha, which block HIV replication by interaction with the chemokine receptor CXCR4, Furthermore, neither Suc-HSA nor Aco-HSA inhibited the binding of monoclonal antibodies 12G5 and 2D7 (directed to CXCR4 and CCR5, respectively) in SUPT-1 cells or THP-1 cells. These results confirm that NCAs bind primarily to gp120 and do not interact directly with the HIV chemokine receptor but block the binding of the virus particles (through gp120) with CD4(+) cells.

In the present study, we described the interaction of succinylated human serum albumin (Suc-HSA), a negatively charged anti-HIV-1 active protein, with HIV-1 gp120 and in detail with the third variable domain of gp120 (V3 loop). To this end, different assay formats were tested in which gp120- and V3-related peptides were presented in various configurations in order to investigate the effect of the conformational structure of the V3 loop on the interaction with negatively charged albumins. When gp120 presented via a lectin was used, it was observed that Suc-HSA bound to native gp120. The binding site appeared to be located at or near the thrombin digestion site (GPGRAF sequence) in the V3 loop of gp120, since the cleavage of the loop resulted in decreased binding of Suc-HSA. In addition, Suc-HSA was able to protect the V3 region of gp120 from cleaving with thrombin. In contrast, significant binding of Suc-HSA to V3 loop or gp120 peptides was not observed when both were presented in a fluid phase system, suggesting the involvement of a monovalent low affinity binding of Suc-HSA. Using overlapping peptides delineating the whole V3 loop immobilized to CNBr-Sepharose, we noticed that the interaction of the V3 loop with Suc-HSA was predominantly induced by electrostatic interactions between positively charged linearized peptide fragments and Suc-HSA and was positively influenced by the presence of hydrophobic amino in the V3 loop fragments as well. Moreover, the highest affinity site was located at sites near the GPGRAF sequence These observations add to the evidence, collected earlier, that Suc-HSA interferes at the level of virus entry, independent of interaction with the CD4 receptor. Since the recently discovered chemokine receptors are negatively charged, we can hypothesize that Suc-HSA is able to prevent the positively charged V3 loop from interacting with these types of receptors, thereby inhibiting virus entry. BIOCHEM PHARMACOL 57;8:889-898, 1999. (C) 1999 Elsevier Science Inc.

Evidence is provided that dromedary heavy-chain antibodies, in vivo-matured in the absence of light chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of heavy-chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector. By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.

Background & Aims: During endotoxemia, expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in the liver is increased, NO has been suggested to have a hepatoprotective function. The aim of this study was to investigate the distribution of iNOS and the effect of different NO synthase inhibitors on liver damage and hemodynamics during endotoxemia. Methods: Rats were injected with lipopolysaccharide (LPS) and received the NOS-inhibitor S-methylisothiourea (SMT) or NG-nitro-L-arginine methyl ester (L-NAME). iNOS induction was assessed by Western blot, immunohistochemistry, and measurement of NO metabolites in plasma and bile. Liver damage was determined by aspartate aminotransferase and alanine aminotransferase and by histology. The effects of both inhibitors on systemic and portal pressure were measured in normal and LPS-treated rats. Results: LPS treatment strongly induced iNOS in inflammatory cells, macrophages, bile duct epithelium, and hepatocytes, especially at the canalicular membrane, LPS-induced liver damage strongly increased after L-NAME. SMT caused a similar reduction of NO production without enhancing liver damage. In LPS-treated rats, SMT increased the systemic and portal pressure significantly more than L-NAME. Conclusions: During endotoxemia, administration of the NOS-inhibitor L-NAME aggravates liver damage. This liver damage does not seem to be caused by hemodynamic changes, In contrast, SMT caused significant hemodynamic changes but did not increase LPS-induced liver damage.

Anionic charge-modified human serum albumin (HSA) has previously been shown to exert potent in vitro activity against human immunodeficiency virus type 1 (HIV-1). In these studies, introduction of the additional negative charges was performed by derivatizing the epsilon-amino groups of lysine residues with succinic (Suc-HSA) or cis-aconitic anhydride (Aco-HSA), by which primary amino groups are replaced with carboxylic acids. The anti-HIV-1 activity was related to inhibition of gp41-mediated membrane fusion. Here, we investigated the activity of aconitylated and succinylated proteins on influenza Virus membrane fusion, which is mediated by the viral membrane glycoprotein hemagglutinin (HA). Aco-HSA and Suc-HSA markedly inhibited the rates and extents of fusion of fluorescently labeled virosomes bearing influenza HA, with target membranes derived from erythrocytes. The inhibitory activity was dependent on the overall negative-charge density; HSA modified with 36 or less extra negative charges failed to inhibit fusion. The inhibition of fusion showed a certain degree of specificity for the protein carrying the negative charges: polyanionic HSA and beta-lactoglobulin A derivatives had fusion-inhibitory activity, whereas succinylated BSA, lactalbumin, lactoferrin, lysozyme, and transferrin were inactive. Aco(60)-HSA and Aco-beta-lactoglobulin A inhibited influenza virus membrane fusion in a concentration-dependent manner, IC50 values being about 4 and 10 mu g/mL, respectively. HA-mediated membrane fusion is pH dependent. Aco(60)-HSA did not induce a shift in the pH threshold or in the pH optimum. Fusion with liposomes of another low pH-dependent virus, Semliki Forest virus, was not specifically affected by any of the compounds reported here. In view of some structural and functional similarities between influenza HA and the HIV-1 gp120/gp41 complex, it is tempting to postulate that the current results might have some implications for the anti-HIV-1 mechanism of polyanionic proteins. (C) 1997 Elsevier Science Inc.

Anionic charge-modified human serum albumin (HSA) has previously been shown to exert potent in vitro activity against human immunodeficiency virus type 1 (HIV-1). In these studies, introduction of the additional negative charges was performed by derivatizing the epsilon-amino groups of lysine residues with succinic (Suc-HSA) or cis-aconitic anhydride (Aco-HSA), by which primary amino groups are replaced with carboxylic acids. The anti-HIV-1 activity was related to inhibition of gp41-mediated membrane fusion. Here, we investigated the activity of aconitylated and succinylated proteins on influenza Virus membrane fusion, which is mediated by the viral membrane glycoprotein hemagglutinin (HA). Aco-HSA and Suc-HSA markedly inhibited the rates and extents of fusion of fluorescently labeled virosomes bearing influenza HA, with target membranes derived from erythrocytes. The inhibitory activity was dependent on the overall negative-charge density; HSA modified with 36 or less extra negative charges failed to inhibit fusion. The inhibition of fusion showed a certain degree of specificity for the protein carrying the negative charges: polyanionic HSA and beta-lactoglobulin A derivatives had fusion-inhibitory activity, whereas succinylated BSA, lactalbumin, lactoferrin, lysozyme, and transferrin were inactive. Aco(60)-HSA and Aco-beta-lactoglobulin A inhibited influenza virus membrane fusion in a concentration-dependent manner, IC50 values being about 4 and 10 mu g/mL, respectively. HA-mediated membrane fusion is pH dependent. Aco(60)-HSA did not induce a shift in the pH threshold or in the pH optimum. Fusion with liposomes of another low pH-dependent virus, Semliki Forest virus, was not specifically affected by any of the compounds reported here. In view of some structural and functional similarities between influenza HA and the HIV-1 gp120/gp41 complex, it is tempting to postulate that the current results might have some implications for the anti-HIV-1 mechanism of polyanionic proteins. (C) 1997 Elsevier Science Inc.

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.