Chiral resolution, pharmacological characterization, and receptor docking of the noncompetitive mGlu1 receptor antagonist (+/-)-2-hydroxyimino- 1a, 2-dihydro-1H-7-oxacyclopropa[b]naphthalene-7a-carboxylic acid ethyl ester.

Racemic CPCCOEt ((1aRS,7aRS)-2-hydroxyimino-1a, 2-dihydro-1H-7-oxacyclopropa[b]naphthalene-7a-carboxylic acid ethyl ester, (+/-)-1) derivatives have been shown to be subtype-selective metabotropic glutamate (mGlu) 1 receptor antagonists (Annoura et al. Bioorg. Med. Chem. Lett. 1996, 6, 763-766). The optical isomers of (+/-)-1 have been separated by chromatography on a chiral stationary phase. The absolute configuration at the C-1a and C-7a positions was determined using X-ray crystallography of an amide derivative with the methyl ester of L-phenylalanine (L-PheOMe) ((+)-6). In a phosphoinositol (PI) turnover assay at the cloned human mGlu1b receptor, (-)-1 and the new amide derivatives (-)-5 and (-)-6, all of which have (1aS,7aS)-stereochemistry on the chromane ring system, showed IC(50) values of 1.5, 0.43, and 0.93 microM, respectively. In contrast, (+)-1 and the new amide derivatives (+)-5 and (+)-6were found to be inactive up to a concentration of 30 microM indicating a selectivity for the (-)-enantiomers of at least 70-fold. In a previous study (Litschig et al. Mol. Pharmacol. 1999, 55, 453-461) we demonstrated using site-directed mutagenesis that the interaction site of (+/-)-1 is located in the transmembrane (TM) domain of hmGlu1b. To suggest a plausible binding mode of (-)-1, we have built a molecular mechanics model of the putative seven TM domain of hmGlu1 based on the alpha-carbon template of the TM helices of rhodopsin. A receptor docking hypothesis suggests that the OH of T815 (TMVII) comes in close contact with the oxime OH of (-)-1 and (-)-5, whereas no such close interactions could be demonstrated by docking of (+)-1.

(+)-4-phosphonophenylglycine (PPG) a new group III selective metabotropic glutamate receptor agonist.

A new synthesis of (R,S)-PPG (4-phosphonophenylglycine) and the separation of the protected enantiomers leading after deprotection to (+)- and (-)-PPG are described. Pharmacological characterization at the group III metabotropic glutamate receptors hmGluR4a and hmGluR7b revealed (+)-PPG as the active enantiomer.

Role of hydroxyl radicals and singlet oxygen in the formation of primary radicals in unsaturated lipids: a solid state electron paramagnetic resonance study.

Primary radicals were generated by UV photolysis of samples of trilinolein, at 77 K and under a controlled atmosphere. The resulting EPR spectra clearly show that the amount of radicals is dependent on the purity of the lipid, the exposure to visible light in the presence of a photosensitizer and oxygen, and, finally, the presence of an antioxidant. These solid state EPR experiments indicate that if all of the elements for the production of singlet oxygen (Rose Bengal, molecular oxygen, and visible light) are not present, primary radicals are practically not generated. They also point out the various steps of the oxidation mechanism: formation of singlet oxygen, which reacts with the lipid to form a hydroperoxide; and photolytic formation of the hydroxyl radical, which reacts with the frozen lipid to generate primary lipidic radicals. This constitutes a new method for investigating lipid oxidation and studying the influence of photosensitizers and molecules that are likely to react with singlet oxygen.

Simulated moving bed chromatographic resolution of a chiral antitussive.

The behavior of a laboratory simulated moving bed (SMB) unit for continuous chromatographic separation of enantiomers has been considered. This was applied to the resolution of a chiral antitussive agent, guaifenesin, on Chiralcel OD, during an experimental campaign involving nineteen runs. The application of recently developed criteria for the design and optimization of SMB units allows us to understand and rationalize the experimental results, as well as to indicate how to optimize the separation performances. A three-step procedure to determine the adsorption isotherms needed to apply these criteria is proposed; it is reliable and may be applied also where pure components are not available.

Development and validation of chiral high-performance liquid chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester.

A stereospecific HPLC method for the quantitation of CGP 49309 in samples of its corresponding enantiomer valsartan has been developed and validated. The enantiomeric separation was achieved on a 5 micron silica-bonded, alpha 1-acid glycoprotein column (Chiral AGP) with a phosphate buffer, pH 7, containing 2% (v/v) 2-propanol as a mobile phase. The linearity was established in the range 0.1-4% (r > 0.999). The limit of quantitation was 0.1% and the limit of detection was 0.04%. The accuracy of the method was found to be 96.7% (average). For the precision (repeatability), a relative standard deviation value of 2.4% was found. Similarly, a stereoselective HPLC method was also developed and validated for the quantitation of the enantiomer of the starting material used for the synthesis of valsartan, namely (R)-valinebenzyl ester tosylate. Baseline resolution of the enantiomers of valinebenzyl ester tosylate could be achieved on the chiral crown ether column Crownpak CR (Daicel) at 50 degrees C using water-methanol-trifluoroacetic acid (850:150:1, v/v) as a mobile phase. The linearity was established in the range 0.5-5% (r > 0.999). The accuracy of the method was found to be 100.5% (average). For the precision (repeatability), a relative standard deviation value of 3.4% was found. Both methods were found to be suitable for the analysis of the respective analytes.

Radical intermediates and antioxidants: an ESR study of radicals formed on carnosic acid in the presence of oxidized lipids.

Carnosic acid, an antioxidant extracted from rosemary, is shown to produce radicals when in contact with oxidized methyl oleate in the absence of air above 50 degrees C. Two radical species are formed: the first one, stable up to approximately 110 degrees C, is an hydroxy-phenoxy radical whose ESR spectrum was analyzed by studying its temperature dependence and its sensitivity to deuterium/proton exchange. The second species was observed above 110 degrees C, its ESR spectrum was identical to the spectrum obtained when carnosol, another antioxidant extracted from rosemary, was heated at the same temperature in the presence of oxidized lipid. This observation is probably due to the transformation of carnosic acid into carnosol; the analysis of the corresponding ESR spectrum suggests the formation of a keto phenoxy radical exhibiting a great delocalization of the unpaired electron.

Effect of aromatase inhibitors on estrogen 2-hydroxylase in rat liver.

The effect of aromatase inhibitors, 4-hydroxyandrostenedione, CGS 16949A and aminoglutethimide on the inhibition of estrogen 2-hydroxylase activity in rat liver microsomes in vitro and on its induction in vivo has been examined. Estrogen 2-hydroxylase was found to have over twice the affinity for estradiol compared to estrone. Using high pressure liquid chromatography and employing estradiol as a substrate, the IC50 values were 2.2, 98, 110 and 908 microM for the reference compound ketoconazole and the aromatase inhibitors, 4-hydroxyandrostenedione, CGS 16949A and aminoglutethimide, respectively. Similar IC50 values were obtained using estrone as a substrate and by a tritiated water method employing estradiol as a substrate. The Km value for estrogen 2-hydroxylase with estradiol as a substrate using a tritiated water method was 4.3 microM with a Vmax of 11.89 nmol/h/mg. Ketoconazole, CGS 16949A and aminoglutethimide exhibited non-competitive inhibition whereas 4-hydroxyandrostenedione appeared to be a competitive inhibitor of estrogen 2-hydroxylase. The Ki values were 2.6, 72, 114 and 958 microM for ketoconazole, 4-hydroxyandrostenedione, CGS 16949A and aminoglutethimide, respectively. All three aromatase inhibitors were weak inhibitors of estrogen 2-hydroxylase as compared to the reference drug, ketoconazole. Following treatment of rats with aminoglutethimide (40 mg/kg/day; i.p.; for 3 days), estrogen 2-hydroxylase activity was increased by 28 and 30% using estradiol and estrone as substrates, respectively. Following treatment of rats with CGS 16949A (2 mg/kg/day; p.o.; for 3 days), the corresponding increase in estrogen 2-hydroxylase activity was 48 and 44%. The results of this study indicate that the aromatase inhibitors, aminoglutethimide and CGS 16949A are only weak inhibitors of estrogen 2-hydroxylase activity in vitro and show no evidence of inhibition in vivo. On the contrary, there was some evidence to suggest that both aminoglutethimide and CGS 16949A induce estrogen metabolism following repeated administration. Therefore, aminoglutethimide and CGS 16949A may lower estrogen levels not only by primarily inhibiting their synthesis but also by inducing the metabolism of estrogens.

Pharmacokinetics and tissue distribution of the renin inhibitor N-(2-(R)-benzyl-3-tert-butyl-sulfonyl-propionyl)-His-ChacVal-n-butylami ne in marmosets.

The fate of CGP 38 560 [N-(2-(R)-benzyl-3-tert-butyl-sulfonyl-propionyl)-His-ChacVal-n-bu tylamine], a potent renin inhibitor, has been studied in marmosets. [3H]CGP 38 560 is rapidly cleared from the plasma. The elimination process is biphasic, with a t1/2 of 4.8 +/- 1.0 min (mean +/- SD) in the first phase and 26.6 +/- 8.4 min (mean +/- SD) in the second. The kinetics of elimination from plasma are similar when measured both in a radio-inhibitor binding assay and radiometrically using 3H-labeled substance. The drug is mainly eliminated in the bile, almost 73.8% of the i.v. administered dose being excreted within the first 60 min. It is detectable in bile in both unchanged (8.6%) and metabolized form. HPLC analysis of bile extracts showed at least five tritiated peaks representing constituents capable of binding human renin (A, B, C, D, and E). These fractions were isolated, purified, and analyzed by mass spectrometry. Peak E corresponded to unchanged CGP 38 560. Metabolites A, B, C, and D are more polar than the parent compound, as indicated by their retention times upon HPLC analysis. The metabolic pathways inferable from the respective molecular weights are hydroxylation, oxygenation, and, in one case, cleavage of the n-butylamino group located at the COOH-terminal. From comparisons of the pharmacokinetic parameters after iv (0.1 mg/kg) and oral (10 mg/kg) administration, it can be estimated that the bioavailability of CGP 38 560 in the marmoset is 0.3%.(ABSTRACT TRUNCATED AT 250 WORDS)