Antiinflammatory beta-arylamidoacrylic acids.

A series of 34 beta-arylamidoacrylic acids was prepared and examined for antiinflammatory activity. These compounds are vinylogous carbamic acids, and several displayed activity equal to phenylbutazone in the rat pleural effusion model. Highest activity was associated with structures bearing halogen and cyano substituents. Amides were inactive.

Antimicrobial 3-methyleneflavanones.

The antimicrobial activity previously attributed to flavanone Mannich bases was found to be due to their breakdown products, 3-methyleneflavanones. Among the latter compounds, highest potency was observed when the flavanone phenyl ring contained bromine or chlorine substituents. 3-Methylene-2-phenylflavanone (8) was synthesized and shown to be equal to hexachlorophene in tests against representative Gram-positive microorganisms.

Synthesis and antiinflammatory activity of some 1,2,3- and 1,2,4-triazolepropionic acids.

All possible "nonadjacent" phenyltriazolepropionic acids were synthesized and tested for antiinflammatory activity. Two of the isomers displayed activity approximately equal to phenylbutazone: the 4-phenyl-1,2,3,-triazole-2-propionic acid (7) and its reverse counterpart, 2-phenyl-1,2,3-triazole-4-propionic acid (6). The other five isomers were inactive. Since these seven acids are geometrically congruent and possess similar lipophilic characters, antiinflammatory activity must depend on some property that is a function of how the carbon and nitrogen atoms are arranged in the triazole ring.

Substrate-labeled fluorescent immunoassay for phenobarbital.

An assay for the anticonvulsant drug phenobarbital (PB) has been developed that is based on the principles of the substrate-labeled fluorescent immunoassay. A fluorogenic enzyme substrate, galactosyl umbelliferone, was covalently linked to a derivative of PB. The labeled drug, galactosyl umbelliferone-PB (GUPB), is nonfluorescent under conditions of the assay; however, hydrolysis of the galactosyl moiety by bacterial beta-galactosidase yields a fluorescent product. When GUPB is bound by antibody to PB, it is not a substrate for enzymatic hydrolysis. Thus, only GUPB not bound to antibody is hydrolyzed. In competitive binding reactions, using a fixed concentration of GUPB and a limiting amount of antibody, the PB in serum and the GUPB compete for antibody-binding sites. The fluorescence produced upon enzymatic hydrolysis of unbound GUPB is directly proportional to the concentration of PB. Unknown serum levels of PB are determined from a standard curve of fluorescent intensity versus standard PB concentrations. The assay is specific, sensitive, and easy to perform. It is carried out by adding the equivalent of 2 microliters of serum standard or unknown directly to a cuvette containing 3 ml of a buffered solution of antibody and enzyme. One-hundred microliters of GUPB is added, and the fluorescence intensity is measured after a fixed time (any time from 5 to 90 min). Using clinical specimens, our assay correlated well with a commercial enzyme immunoassay (correlation coefficient = 0.97) and had an interassay precision of less than 7%.

Simultaneous determination of phenytoin and phenobarbital in serum or plasma by substrate-labeled fluorescent immunoassay.

This assay system for simultaneously determining phenytoin and phenobarbital in serum and plasma is based on the substrate-labeled fluorescent immunoassay technique. A beta-galactosylcoumarin derivative of phenobarbital and a 4-methylcoumarin phosphodiester derivative of phenytoin are used as substrate labels for Escherichia coli beta-galactosidase and Crotalus atrox phosphodiesterase I, respectively. The smallest measurable concentrations are about 1.6 mg/L for phenytoin, 2.7 mg/L for phenobarbital. Within-run coefficients of variation are about 5% for phenytoin and 2% for phenobarbital, about 6% for both between-runs. Results for phenytoin and phenobarbital in serum and plasma correlate well with those determined by the Ames TDA (r = 0.944 and 0.986, respectively) and Syva's EMIT (r = 0.977 and 0.969, respectively) assays.

Homogeneous substrate-labeled fluorescent immunoassay for theophylline in serum.

A substrate-labeled fluorescent immunoassay for theophylline in serum is described. 8-(3-Aminopropyl)-theophylline is covalently attached to a fluorogenic enzyme substrate, 7-beta-galactosylcoumarin-3-carboxylic acid. Hydrolysis of this theophylline-labeled substrate by beta-galactosidase yields a fluorescent product. When antibody to theophylline interacts with this substrate, the resulting complex is inactive as an enzyme substrate. For measuring theophylline, competitive protein-binding reactions are set up, with the theophylline in the sample competing with the substrate for the antibody-binding sites. The substrate not bound to antibody is hydrolyzed by beta-galactosidase, producing fluorescence that is proportional to the theophylline concentration. Results for theophylline determined by this method in clinical samples of serum correlated well (r > 0.96) with results obtained by gas-chromatographic or enzyme immunoassay procedures. The within-run CV for three control samples ranged from 1.1 to 2.8%, the between-run CB from 2.3 to 4.5%.

Substrate-labeled fluorescent immunoassay for phenytoin in human serum.

A homogeneous substrate-labeled fluorescent immunoassay has been applied to the measurement of phenytoin concentrations in human serum. We coupled a fluorogenic enzyme substrate, galactosyl-umbelliferone, covalently to a derivative of phenytoin. Under assay conditions, this drug-substrate conjugate was nonfluorescent but became fluorescent upon hydrolysis catalyzed by bacterial beta-galactosidase. When antibody to phenytoin is bound to the drug-substrate conjugate, it is inactive as an enzyme substrate. Addition of phenytoin to competitive-binding reactions relieves the inactivation, and the resulting fluorescence is proportional to the phenytoin concentration. We validated the fluorescent immunoassay by comparing values for phenytoin obtained with this technique to those obtained by gas chromatography and by enzyme immunoassay (EMIT). All three methods correlated well. The major metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, and other drugs at concentrations expected in serum had no effect on the assay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microL of serum sample per test.