Synergistic effects of a cremophor EL drug delivery system and its U0126 cargo in an ex vivo model.

Neuroprotection has proven clinically unsuccessful in subarachnoid hemorrhage. We believe that this is because the major component in the early damage pathway, the vascular wall, has not been given the necessary focus. U0126 is a potent inhibitor of vascular phenotypical changes, exemplified by functional endothelin B (ETB) receptor upregulation. The current study aimed to determine the optimal dose of U0126 ex vivo and test the toxicology of this dose in vivo. To find the optimal dose and test a suitable in vivo delivery system, we applied an ex vivo model of blood flow cessation and investigated functional ETB receptor upregulation (using a specific agonist) as the primary endpoint. The secondary endpoint was depolarization-induced contractility assessed by 60 mM K+ stimuli. Furthermore, an in vivo toxicology study was performed on the optimal selected doses. U0126 (10 µM) had a strong effect on the prevention of functional ETB receptor contractility, combined with minimal effect on the depolarization-induced contractility. When cremophor EL was chosen for drug delivery, it had an inhibitory and additive effect (combined with U0126) on the ETB receptor contractility. Hence, 10 µM U0126 in 0.5% cremophor EL seems to be a dose that will be close to the maximal inhibition observed ex vivo on basilar arteries, without exhibiting side effects in the toxicology studies. U0126 and cremophor EL are well tolerated at doses that have effect on ETB receptor upregulation. Cremophor EL has an additional positive effect, preventing functional ETB receptor upregulation, making it suitable as a drug delivery system.

5-hydroxytryptamine2A receptor inverse agonists as antipsychotics.

We have used a cell-based functional assay to define the pharmacological profiles of a wide range of central nervous system active compounds as agonists, competitive antagonists, and inverse agonists at almost all known monoaminergic G-protein-coupled receptor (GPCR) subtypes. Detailed profiling of 40 antipsychotics confirmed that as expected, most of these agents are potent competitive antagonists of the dopamine D2 receptor. Surprisingly, this analysis also revealed that most are potent and fully efficacious 5-hydroxytryptamine (5-HT)2A receptor inverse agonists. No other molecular property was shared as universally by this class of compounds. Furthermore, comparisons of receptor potencies revealed that antipsychotics with the highest extrapyramidal side effects (EPS) liability are significantly more potent at D2 receptors, the EPS-sparing atypical agents had relatively higher potencies at 5-HT2A receptors, while three were significantly more potent at 5-HT2A receptors. Functional high-throughput screening of a diverse chemical library identified 530 ligands with inverse agonist activity at 5-HT2A receptors, including several series of compounds related to known antipsychotics, as well as a number of novel chemistries. An analog of one of the novel chemical series, AC-90179, was pharmacologically profiled against the remaining monoaminergic GPCRs and found to be a highly selective 5-HT2A receptor inverse agonist. The behavioral pharmacology of AC-90179 is characteristic of an atypical antipsychotic agent.

Multistep solution-phase parallel synthesis of spiperone analogues.

A flexible, multistep parallel synthesis of spiperone analogues is described. A library of 4-substituted piperidines, assembled utilizing reductive amination and acylation protocols, was alkylated either homogeneously or heterogeneously, exploiting a product release only concept, to afford an oxa-series of spiperone analogues. Screening of the products at 5-HT2 and D2 receptors revealed 5-HT2A antagonists with improved selectivity compared to spiperone and AMI-193.

An organoiron approach to the benzophenone appendage of the protein kinase C inhibitor balanol.

[formula: see text] A new synthetic route to the benzophenone appendage of balanol, based on sequential iron-assisted nucleophilic aromatic substitution and ring-opening as well as regioselective oxidative cyanation, is described.

Higher order iminodiacetic acid libraries for probing protein-protein interactions.

Full details of the preparation of iminodiacetic acid diamide dimer (2040 compounds), trimer (560 compounds), and tetramer (1596 compounds) libraries by multistep convergent solution-phase synthesis for studying protein-protein interactions are provided. The libraries were assembled in a format providing small 8-10 compound mixtures and the deconvolution of many of the small mixtures to identify screening leads by resynthesis of the individual components have been conducted for 320 of the individual compounds to date. A representative example of the subsequent exploration of the structure-activity relationships for an identified receptor binding antagonist (200 additional individual compounds) and steps taken for potential elaboration to a receptor dimerization agonist are defined with preparation of representative linked dimers (70 compounds).

Antioxidative properties of organotellurium compounds in cell systems.

The protective/antioxidative properties of diaryl tellurides were demonstrated in cellular systems of increasing complexity. In the presence of glutathione, bis(4-hydroxyphenyl) telluride (1a), bis(4-aminophenyl) telluride (1d) and bis(2-carboxyphenyl) telluride (1h) reduced by more than 50% t-butyl hydroperoxide-induced cell death in lung fibroblast cultures at concentrations below 2 microM. Bis(2,6-dimethyl-4-hydroxyphenyl) telluride (2b) reduced by more than 50% leukocyte-mediated and phorbol-12-myristate-13-acetate-stimulated damage to Caco-2 cells at 0.1 microM concentration. As judged by their abilities to reduce formation of thiobarbituric acid reactive substances at concentrations close to 1 microM, diaryl tellurides 1a, 1d and 2b protected rat kidney tissue against oxidative damage caused by anoxia and reoxygenation. The organotellurium compounds also offered protection after systemic administration. In the presence of diaryl telluride 2b (0.1-1 microM), the ischemia/reperfusion-induced vascular permeability increase in the hamster cheek pouch was significantly reduced as compared with the control. Some of the most active organotellurium cell protectants were evaluated for their ability to inhibit formation of the inflammatory mediators leukotriene B4 and interleukin-1beta. An inhibitory effect on the secretion of these species was seen for compounds 1a and 2b at or above 10 microM concentrations. The protective effects of diaryl tellurides against t-butyl hydroperoxide-induced cell injury can be ascribed mainly to the peroxide-decomposing, glutathione peroxidase-like capacity of the compounds. The chain-breaking, electron- or hydrogen atom-donating ability of diaryl tellurides seems to be the main reason for their protection against leukocyte-mediated cell damage in Caco-2 cells and in the oxidatively challenged rat kidney and hamster cheek pouch.

Organotellurium compounds as efficient retarders of lipid peroxidation in methanol.

Diaryl tellurides were found efficiently to retard azo-initiated lipid peroxidation of linoleic acid in methanol. The most efficient compounds, 31 and 39, contained one and two hydroxyl groups, respectively, in the para positions and methyl groups in all four positions ortho to tellurium. As determined by the values of n.kinh, these materials were as effective retarders of lipid peroxidation as vitamin E. Contrary to the conventional antioxidants examined, diaryl tellurides were found to inhibit peroxidation for long times, seemingly with an autocatalytic mechanism. Diaryl tellurides were found to be partially oxidized during the peroxidation. The reduction of tellurium (IV) compounds to the divalent state during the conditions of the experiment is discussed. The reactivity of some diaryl chalcogenides toward 2,2-diphenyl-1-picrylhydrazyl (DPPH) in methanol was studied. All compounds investigated were less reactive than vitamin E. One of the most active organotellurium compounds, bis(4-aminophenyl) telluride, reacted considerably faster with DPPH than the corresponding selenide or sulfide. It was concluded that mechanisms involving both hydrogen atom transfer and electron transfer were operative in the reaction of organotellurides with DPPH.

Synthesis, antioxidant properties, biological activity and molecular modelling of a series of chalcogen analogues of the 5-lipoxygenase inhibitor DuP 654.

2-Phenylsulfenyl- (1b), 2-phenylselenenyl- (1c) and 2-phenyltellurenyl-1-naphthol (1d) were prepared and their antioxidative properties evaluated in comparison with 2-benzyl-1-naphthol (1a; DuP 654). 2-Phenyltellurenyl-1-naphthol had a significantly lower (1.00 V versus SCE) oxidation potential than the other three compounds (1.24, 1.27 and 1.25 V, respectively, versus SCE for compounds 1a, 1b and 1c) as determined by cyclic voltammetry. In contrast to the other materials, compound 1d was able to catalyze the reduction of hydrogen peroxide in the presence of thiols as stoichiometric reducing agents. The organotellurium compound was also the most efficient inhibitor of azo-initiated peroxidation of linoleic acid in a two-phase model system. Ab initio geometry optimization at the 3-21G(*) level revealed infinitesimal changes in the molecular conformations of the carbon, sulfur, selenium and tellurium analogues. As judged by their ability to inhibit stimulated LTB4 biosynthesis in human neutrophils, compounds 1a-1d all turned out to be highly potent 5-lipoxygenase inhibitors with IC50-values ranging from 0.40 microM for 2-benzyl-1-naphthol (1a) to 0.063 microM for 2-phenyltellurenyl-1-naphthol (1d).

Diaryl tellurides as inhibitors of lipid peroxidation in biological and chemical systems.

Diaryl tellurides carrying electron-donating substituents in the para positions were found to efficiently inhibit peroxidation of rat hepatocytes, rat liver microsomes and a chlorobenzene solution of phosphatidylcholine. The most active compound in the microsomal assay, bis(4-dimethylaminophenyl) telluride, showed an IC50-value of 30 nM. This compound also caused a dose-dependent delay of the onset of the linear phase of microsomal peroxidation stimulated by iron/ADP/ascorbate. The peak oxidation potentials of the diaryl tellurides (0.50-1.14 V in MeCN) correlated linearly with the IC50-values in this assay, with a point of inflection around 0.85 V. In the hepatocyte system, all compounds showed similar protective activity. It is proposed that diaryl tellurides exert an antioxidative effect by deactivating both peroxides and peroxyl radicals under the formation of telluroxides. These oxides may regenerate the active divalent organotellurides upon exposure to a suitable reducing agent.

Antioxidant activity of some diarylselenides in biological systems.

The selenoorganic compounds di(4-aminophenyl)selenide (10) and 4-nitro-4'-amino-diphenylselenide (36) were shown to inhibit lipid peroxidation in ADP/Fe2+/ascorbate-treated microsomes and tert-butylhydroperoxide-treated hepatocytes with IC50s of 3 and 10 microM, and 14 and 10 microM, respectively. In the former system, these inhibition constants compare favourably with those of Ebselen and classical antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). In the cell system, these selenium compounds were equipotent with BHA but more potent than Ebselen and its analogues. The diamino compound (10) was also an effective inhibitor of lipid peroxidation initiated by diquat redox cycling in hepatocytes, again being equipotent with BHA but more potent than Ebselen and its analogues, which actually stimulated lipid peroxidation in this test system. Manipulation of the amino functions of (10) and (36) by alkylation or acylation altered the antioxidant capacity. Optimal activity in this series was achieved by N-ethylation or N-isobutylation of (10). This produced antioxidants having IC50s below 1 microM in the microsome system, 3-13 microM in the tert-butylhydroperoxide system, and being 100% effective in the diquat model at 50 microM. On the other hand, acylation or alkylation of the amino groups with long chain acyl or alkyl groups reduced the efficacy of the structures below that of the parent diamine. As with other antioxidant compounds, several of the chalcogenides were relatively selective inhibitors of monocyte 5'-lipoxygenase-dependent secretion of LTB4 as compared to their effect on cyclooxygenase-dependent secretion of PGE2 (for example compound 42 had IC50s of 0.6 microM and 10 microM, respectively). No correlation was observed between the redox-properties of the chalcogenides and their respective abilities to inhibit these enzymes.

Alpha-(phenylselenenyl)acetophenone derivatives with glutathione peroxidase-like activity. A comparison with ebselen.

Here we describe a new class of organoselenium compounds possessing glutathione peroxidase-like activity. The parent compound, alpha-(phenylselenenyl)acetophenone (PSAP), increased the rate of reaction of glutathione with H2O2, tert-butylhydroperoxide, cumene hydroperoxide, linoleic acid hydroperoxide and dilinoleyl lecithin hydroperoxide by 7.0, 25.1, 34.1, 19.1 and 8.4-fold, respectively, as assessed by the oxidized glutathione (GSSG) reductase enzyme assay. Direct assay of the removal of hydrogen peroxide and glutathione from reaction mixtures confirmed the peroxidase-like activities of these selenoorganic compounds, but indicate that the conventional coupled GSSG reductase assay may be unsuitable for the assessment of the catalytic capacity of PSAP and Ebselen. One possible mechanism of catalysis by PSAP involves an initial oxidation at selenium. Thiol may then react with the selenoxide to yield a selenium (II) compound, H2O and a disulfide. Compounds derived from PSAP may provide potential selenium-based anti-inflammatory agents.