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Interest in the 5-, 6-, and 7-trifluoromethyl-substituted 2,3-bis(4-methoxyphenyl)indoles as potent, orally active anti-inflammatory agents required a method for their determination in serum, urine, and feces to permit studies of their absorption, metabolism, and excretion. A simple, rapid, sensitive, and specific procedure was developed based on an ethyl acetate extraction of alkaline specimens and subsequent fluorometric analysis of ethanolic solutions of the extract residues. The method is sensitive to 0.1 microgram/ml, 0.5 microgram/ml, and 0.7 microgram/100 mg of these compounds in serum, urine, and feces, respectively. Overall mean recoveries and standard deviations of the 5-, 6-, and 7-trifluoromethyl-substituted compounds from biological samples were 102.2 +/- 3.0, 102.2 +/- 5.4, and 100.5 +/- 5.7%, respectively. The procedure was applied successfully to absorption studies with 2,3-bis(4-methoxyphenyl)-7-(trifluoromethyl)indole in the dog.

Interest in 2,3-bis(p-methoxyphenyl)imidazo[1,2-a]-pyrimidine as a potent, orally active, anti-inflammatory agent required a method for its determination in serum, urine, and feces to permit studies of its absorption, metabolism, and excretion. A simple, rapid, sensitive, and specific procedure was developed based on an ethyl acetate extraction of alkaline specimens and subsequent fluorometric analysis of ethanolic solutions of the extract residues. The method is sensitive to 0.2 mug/ml, 0.3 mug/ml, and 1.2 mug/100 mg of drug in serum, urine, and feces, respectively. The overall mean recovery and the standard deviation from biological samples are 99.8 +/- 5.1%. The procedure has been successfully applied to absorption studies in the dog.

Small molecules containing the N-nitroso group, such as the bacterial natural product streptozotocin, are prominent carcinogens1,2 and important cancer chemotherapeutics3,4. Despite the considerable importance of this functional group to human health, enzymes dedicated to the assembly of the N-nitroso unit have not been identified. Here we show that SznF, a metalloenzyme from the biosynthesis of streptozotocin, catalyses an oxidative rearrangement of the guanidine group of Nω-methyl-L-arginine to generate an N-nitrosourea product. Structural characterization and mutagenesis of SznF reveal two separate active sites that promote distinct steps in this transformation using different iron-containing metallocofactors. This biosynthetic reaction, which has little precedent in enzymology or organic synthesis, expands the catalytic capabilities of non-haem-iron-dependent enzymes to include N-N bond formation. We find that biosynthetic gene clusters that encode SznF homologues are widely distributed among bacteria—including environmental organisms, plant symbionts and human pathogens—which suggests an unexpectedly diverse and uncharacterized microbial reservoir of bioactive N-nitroso metabolites. The metalloenzyme SznF catalyses the formation of an N-N bond in the biosynthesis of streptozotocin, providing insights into the enzymatic assembly of an N-nitroso group. [ABSTRACT FROM AUTHOR]

We report the synthesis and biological evaluation of a new series of 3- or 4-(substituted)phenylisoxazolones as HNE inhibitors. Due to tautomerism of the isoxazolone nucleus, two isomers were obtained as final compounds (2-NCO and 5-OCO) and the 2-NCO derivatives were the most potent with IC50 values in the nanomolar range (20-70 nM). Kinetic experiments indicated that 2-NCO 7d and 5-OCO 8d are both competitive HNE inhibitors. Molecular modelling on 7d and 8d suggests for the latter a more crowded region about the site of the nucleophilic attack, which could explain its lowered activity. In addition molecular dynamics (MD) simulations showed that the isomer 8d appears more prone to form H-bond interactions which, however, keep the reactive sites quite distant for the attack by Ser195. By contrast the amide 7d appears more mobile within the active pocket, since it makes single H-bond interactions affording a favourable orientation for the nucleophilic attack. [ABSTRACT FROM AUTHOR]

Human neutrophil elastase (HNE) is an important target for the development of novel and selective inhibitors to treat inflammatory diseases, especially pulmonary pathologies. Here, we report the synthesis, structure–activity relationship analysis, and biological evaluation of a new series of HNE inhibitors with an isoxazol-5(2H)-one scaffold. The most potent compound (2o) had a good balance between HNE inhibitory activity (IC50value =20 nM) and chemical stability in aqueous buffer (t1/2=8.9 h). Analysis of reaction kinetics revealed that the most potent isoxazolone derivatives were reversible competitive inhibitors of HNE. Furthermore, since compounds2oand2scontain two carbonyl groups (2-N-CO and 5-CO) as possible points of attack for Ser195, the amino acid of the active site responsible for the nucleophilic attack, docking studies allowed us to clarify the different roles played by these groups. [ABSTRACT FROM PUBLISHER]

Compounds that can effectively inhibit the proteolytic activity of human neutrophil elastase (HNE) represent promising therapeutics for treatment of inflammatory diseases. We present here the synthesis, structure–activity relationship analysis, and biological evaluation of a new series of HNE inhibitors with a cinnoline scaffold. These compounds exhibited HNE inhibitory activity but had lower potency compared toN-benzoylindazoles previously reported by us. On the other hand, they exhibited increased stability in aqueous solution. The most potent compound,18a, had a good balance between HNE inhibitory activity (IC50value = 56 nM) and chemical stability (t1/2 = 114 min). Analysis of reaction kinetics revealed that these cinnoline derivatives were reversible competitive inhibitors of HNE. Furthermore, molecular docking studies of the active products into the HNE binding site revealed two types of HNE inhibitors: molecules with cinnolin-4(1H)-one scaffold, which were attacked by the HNE Ser195 hydroxyl group at the amido moiety, and cinnoline derivatives containing an ester function at C-4, which is the point of attack of Ser195. [ABSTRACT FROM AUTHOR]

This work describes the regioselective synthesis of two new series of 1,1′-oxalylbis[3-(alkyl/aryl/heteroaryl)-4,5-dihydro-5-hydroxy-5-(trihalomethyl)-1 H-pyrazoles], where the 3-substituents are H, Me, CH, 4-FCH, 4-ClCH, 4-BrCH, 4-NOCH, 4,4′-BiPh, and 2-furyl, in a one-pot methodology with ethanol as solvent, from the reaction of 4-alkoxy-4-(alkyl/aryl/heteroaryl)-1,1,1-trihaloalk-3-en-2-ones with oxalyldihydrazide (51-89%). Complementarily, the dehydration reactions of five examples of the described oxalylbispyrazolines are also reported, which furnished the respective 1,1′-oxalylbis[3-(alkyl/aryl/heteroaryl)-5-(trihalomethyl)-1 H-pyrazoles] in 53-78% yields without the two C(O)-N bond cleavages. Graphical abstract: [Figure not available: see fulltext.]. [ABSTRACT FROM AUTHOR]

The oxidation of l-phenylalanine by hexacyanoferrate(III) (abbreviated as HCF) catalyzed by Ir(III) has been studied spectrophotometrically at 35 °C and at a constant ionic strength of 0.50 mol dm−3. The main oxidation product was identified as phenylpyruvic acid by physico-chemical and spectroscopic methods. The stoichiometry was found to be 2:1, i.e. 2 mol of hexacyanoferrate(III) reacted with 1 mol of phenylalanine. The reaction was first order with respect to both HCF and alkali concentration. The order with respect to [Phe] changed from first to zero as the concentration was increased. The effect of ionic strength was also investigated. Thermodynamic parameters were evaluated by studying the reaction at four different temperatures between 35 and 50 °C. Based on the experimental results, a suitable mechanism involving complex formation has been proposed. [ABSTRACT FROM AUTHOR]

Administration of cadmium (Cd) at a dose of 1.23 mg/kg (2.0 mg/kg as CdCl2) markedly decreased the activity of an enzyme (acetohexamide reductase) catalysing the ketone-reduction of acetohexamide, an oral antidiabetic drug, in liver microsomes of male rats. However, the decreased enzyme activity was increased by repeated treatment with testosterone propionate (TP). When male rats were castrated and TP was given to the castrated ones, a similar decrease and increase, as described above, were observed in the microsomal enzyme activity. Cd exposure to male rats induced haemorrhage and atrophy of the testes and significantly diminished serum testosterone levels. There was no possibility that Cd accumulated in liver microsomes of male rats causing direct inhibition of the microsomal enzyme activity. We conclude that Cd exposure decreases androgen-dependent metabolism of acetohexamide in liver microsomes of male rats through its testicular toxicity. Cd exposure had no effect on acetohexamide reductase activity in liver cytosol of male rats. [ABSTRACT FROM AUTHOR]

SUMMARY Objective To study the effects and serum levels of glibenclamide (Gb) and its active metabolites in patients on chronic Gb medication on different daily doses. Material and methods Fifty patients with type 2 diabetes on regular Gb therapy (1.75–14.0 mg daily). Blood samples were taken immediately before and 90 min after regular Gb intake. A standardized breakfast was served 30 min after drug intake. Serum insulin and proinsulin levels were determined by ELISA methods without cross-reactivities. Serum drug levels were determined by HPLC. Fischer's R to Z-test (correlation coefficients) and paired Student t-tests were used when comparing values within the entire group and unpaired non-parametric Mann–Whitney tests were used when comparing high and low dose levels. A p-value < 0.05 was considered significant. Results There were significant correlations between daily Gb dose, on the one hand, and, on the other, HbAlc (r = 0.55), Δ-insulin (r = - 0.59) and Δ-proinsulin (r = - 0.52) levels. Significant correlations between Gb therapy duration and insulin (r = - 0.40) and proinsulin (r = - 0.34) secretion and between Gb dose and ratio proinsulin/insulin (RPI) at both time points (r = 0.32 and 0.30) were also found. The RPI was lower after Gb intake. In patients on ≥ 10.5 mg steady state serum metabolite levels (Ml and Ml + M2) were higher (29(0–120) and 33 (0–120) ng/ml) than those of Gb itself (18(0–64) ng/ml). A great inter-subject variability in Gb levels at both time points was seen. Conclusions Our results indicate that, in patients on chronic medication, Gb is capable of stimulating both insulin and proinsulin secretion; the effect on insulin release is relatively greater. The effect was more pronounced in patients on a low Gb dose, either because of less impaired β-cells in those receiving low doses, or due to reduced sulphonylurea sensitivity in those on high dosage (down-regulation). In patients on a daily dose of 10.5 mg or more, serum metabolite levels of clinical relevance were demonstrated; the metabolites may contribute to hypoglycaemic events. [ABSTRACT FROM AUTHOR]

Aims The main purpose of this paper is to describe the relationship between serum concentrations of glibenclamide and its main metabolites and the effects on blood glucose levels, the clinically most relevant parameter to assess in diabetes. Methods Serum concentrations and blood glucose lowering effects (expressed as percent blood glucose reduction vs placebo) of glibenclamide (Gb) and its active metabolites, 4- trans-hydroxy-(M1) and 3- cis-hydroxy-glibenclamide (M2), were analysed in eight healthy subjects participating in a placebo-controlled, randomized, single-blind crossover study, using intravenous administration of each compound as well as oral administration of Gb. Results Plots of % blood glucose reduction vs log serum concentration demonstrated counter-clockwise hysteresis for parent drug and its metabolites. An effect compartment was linked to appropriate pharmacokinetic models and pharmacokinetic and pharmacodynamic modelling was used to fit the pharmacokinetics of Gb by both routes and the metabolites for each individual. Based on the individual concentration-time profiles a PK/PD-model was applied to all effect data simultaneously. An increase in the steady-state serum concentration when the effect is 50% of maximal, CEss50,, was found in the sequence M1 (23 ng ml−1 ), M2 (37 ng ml−1 ) and Gb (108 ng ml−1 ). Corresponding interindividual variabilities expressed as CV% were 25%, 47% and 26%. The elimination rate constants from the effect site ( kE0 ) were estimated and increased in the order M1 (0.178 h−1, CV 13%), M2 (0.479 h−1, CV 8.5%) and Gb (1.59 h−1, CV 36%). Corresponding equilibration half-lives for the effect site ( kE0-HL ) were 3.9 h, 1.4 h and 0.44 h. Estimated Emax-values obtained for M1, M2 and Gb were 40% (CV 30%), 27% (CV 56%) and 56% (CV 14%), respectively. Conclusions It is concluded that the two major metabolites of Gb are hypoglycaemic in man, that they may have higher activity at low concentrations and that they may have a longer effect duration than the parent drug. [ABSTRACT FROM AUTHOR]