Antioxidative property of T-0970, a new ureidophenol derivative.

We investigated the antioxidative property of T-0970, a newly synthesized ureidophenol derivative. The inhibitory effect of T-0970 on spontaneous lipid peroxidation in rat brain was 10 times greater than those of well-known antioxidants such as butylhydroxytoluene (BHT), probucol and alpha-tocopherol. T-0970 also showed dose-dependent free radical scavenging activities in vitro for both superoxide anions and hydroxyl radicals. The radical-scavenging potencies of T-0970 were about 10-30 times stronger than those of BHT. We evaluated the in vivo antioxidative ability of T-0970 in the animal model of acute oxidative tissue injury in rats. Intraperitoneal injection of ferric nitrilotriacetate (Fe/NTA) caused an acute and remarkable increase in the level of thiobarbituric acid-reactive substances (TBARS) in both plasma and the liver, and also resulted in a considerable elevation of the plasma levels of GOT and GPT indicative of hepatic injury. Both oral and intravenous administration of T-0970 dose-dependently depressed these diagnostic parameters. These results indicate that T-0970 may have a therapeutic potential in various diseases associated with oxidative tissue injury.

Quantitative structure-activity analyses of novel hydroxyphenylurea derivatives as antioxidants.

A series of substituted hydroxyphenylureas was synthesized, the chemical structure of which was designed based on structures of natural antioxidants, vitamin E (alpha-tocopherol) and uric acid. They exhibited high inhibitory activity against lipid peroxidation. In order to gain an insight into the mechanism of the inhibition reaction, we analyzed their structure-activity relationships quantitatively. Electronic and steric effects of substituents on the phenolic hydroxyl group were shown to be of importance in governing the inhibitory potency. An increase in the electron donating property of substituents toward the phenolic hydroxyl group enhanced the antioxidative activity by the stabilization of an electron-deficient radical-type transition state. The steric shielding by ortho-substituents stabilized the phenoxy radicals formed following the transition state. Derivatives having the carboxyl group were only weakly active presumably because of an intermolecular ion-dipole interaction of the phenolic hydroxyl group with the carboxylate anion which could retard the formation of the transition state.

4-acylaminophenol derivatives as novel lipoxygenase inhibitors: synthesis and inhibitory effect on 5-lipoxygenase and leukotriene B4 production.

Structure-activity relationships in the inhibitory effects of 4-acylaminophenol derivatives on the 5-lipoxygenase (5-LOX) from RBL-1 cells and leukotriene B4 (LTB4) production by guinea pig neutrophils were studied. When the N-acyl group was n-octanoyl or 2-thiophenecarbonyl and the size of the two ortho substituents of phenol was varied, the substituents bulkier than isopropyl, i.e., 2,6-di-tert-butyl and 2,6-dicyclohexyl, substantially weakened the inhibitory activity in both enzymatic and cellular systems. Among the 2,6-dimethyl derivatives with an acyl group of various carbon-chain lengths (C1-13), those with a n-alkyl chain of C5 to C12 showed similarly potent inhibitory activities toward 5-LOX with an IC50 ranging from 0.27 to 0.66 microM; in contrast, maximal inhibitory activities toward LTB4 production were observed in a narrower range of the serial compounds: i.e., those with a n-hexyl, n-heptyl, or n-octyl chain on the carbonyl carbon formed by far the most inhibitory group of the series and the inhibitory activity sharply decreased on either side of the chain length. Nearly all the active compounds also inhibited cyclooxygenase (COX), but the IC50 values for COX inhibition were more than ten times higher than the corresponding IC50 values for 5-LOX inhibition in most cases, indicating that the acylaminophenols are relatively selective 5-LOX inhibitors.

Mechanism of phosphate adsorption to a three-dimensional structure of boehmite in the presence of bovine serum albumin.

A new microcrystalline boehmite (tentatively named PT-A) was synthesized as an efficient phosphate adsorbent to replace aluminum hydroxide gel. The characteristic structure of PT-A was examined by nitrogen adsorption/desorption, X-ray diffraction, deviation microscopy, and scanning electron microscopy to establish a pore structural model of PT-A. With this model structure, the details of the mechanism of interaction between PT-A and phosphate in the presence of bovine serum albumin (BSA) are discussed. PT-A is a spherical particle with a diameter of approximately 100 microns and a porous surface structure, and its inside is packed with boehmite microcrystals (crystallite size, 2 nm). PT-A has three types of pores in its structure: a micropore with a narrow size-distribution, a mesopore with a broad size-distribution, and a macropore (radii of pores are 0.7, 1-20, and approximately 300 nm, respectively). When phosphate was incubated with PT-A in human gastric and intestinal juices or in an aqueous solution containing BSA, the amounts of phosphate adsorbed by PT-A were not affected by the presence of proteins. The nitrogen adsorption/desorption isotherms and energy dispersive X-ray analyses demonstrated that phosphate could diffuse to the smaller tunnels freely even if the external surface of PT-A was covered with BSA. It was also demonstrated that the main site of adsorption for phosphate was in micropores of PT-A, whereas BSA was adsorbed only to the external surface and none entered inside smaller tunnels consisting of micro- and mesopores.

Interaction of bovine serum albumin with the surface of a microcrystalline aluminum oxide hydroxide compound: a possible new type of phosphate adsorbent.

Aluminum hydroxide gel (ALG) has been effective for ameliorating acidosis associated with phosphatemia caused by hemodialysis. However, aluminum accumulation in the body causes severe side effects. As substitute for ALG, a new type of aluminum oxide hydroxide (tentatively named PT-A) was prepared with the hope of future clinical use. PT-A has a microcrystalline structure with a high resistance to pH change and has more phosphate-binding efficacy than ALG. It was tested for possible interaction with protein by adsorption test, zeta-potential analysis, X-ray diffraction, and scanning electron microscopy. Bovine serum albumin (BSA) was chosen as a model protein. The interaction of BSA with PT-A depended on the amount of adsorbent. Protein adsorption occurred rapidly and reached the maximal level at near neutral pHs. Phosphate adsorption was not affected by the presence of BSA, but the interaction of BSA with PT-A was significantly reduced by the presence of phosphate. Zeta-potential changes on the surface of PT-A indicated that the positively charged surface of PT-A was covered with negatively charged phosphate ions that repelled negatively charged BSA molecules. X-ray diffraction patterns indicated no observable structural alteration caused by adsorption of BSA or phosphate, and scanning electron microscopy revealed that BSA covered the outer surface of PT-A but did not cover small pores, where phosphate can freely penetrate.

Phosphate and pepsin adsorptions by a new boehmite compound and aluminum hydroxide.

A new microcrystalline compound of aluminum oxide hydroxide (tentatively named PT-A) was synthesized in the hope of providing a better phosphate adsorbent for future clinical use than the currently marketed aluminum hydroxide gels (ALG). An X-ray diffraction study demonstrated a boehmite structure in PT-A but an amorphous structure in ALG. PT-A was more stable in pH change than ALG; in elution tests in artificial gastric and intestinal solutions, aluminum ion eluted from PT-A was maximally 10% of the amount from ALG at pH 1.2; and was undetectable at pH 6.8, at which point ALG still showed some aluminum elution. Phosphate-adsorbing efficacy of PT-A and ALG in vitro was about the same at pH 1.2; however, it was four times greater in PT-A than in ALG at pH 6.8, indicating that PT-A will be effective in the intestine. PT-A also adsorbed pepsin but the amount was at most the same or much less than that adsorbed by ALG, which depended on pH in solution.

In vitro metabolism of the new cardiotonic agent denopamine (TA-064) by rat and rabbit liver preparations. Oxidation, methylation, and glucuronidation.

The metabolic pathways of the cardiotonic agent denopamine, (-)-(R)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol, were studied in vitro with rat and rabbit liver preparations. 4'-O-Demethylated (M-1), 3'-O-demethylated (iso-M-1), and 3-hydroxylated (M-4) metabolites of denopamine were formed by incubation of denopamine with the rat liver microsomal fraction containing the NADPH-generating system. The ratio of M-1 to iso-M-1 formed in this system was 33:1. 3-Methoxydenopamine (M-2) and 3-hydroxy-4-O-methyldenopamine (iso-M-2) were formed via the catechol intermediate M-4, when denopamine was incubated with the rat liver 9000g supernatant fraction in the presence of the NADPH-generating system and S-adenosyl-L-methionine. The ratio of M-2 to iso-M-2 in this system was 7:1. Conversion of iso-M-2 to M-2, i.e. 4-O-demethylation followed by 3-O-methylation, but not vice versa, took place in this system. M-2 was demethylated at 4' to form M-3 by the above microsomal system. M-1 was not ring-hydroxylated by this system, excluding the metabolic route to M-3 via M-1. Denopamine, M-1, M-2, and M-3 were glucuronidated in vitro by the rabbit liver microsomal fraction. The glucuronides of denopamine and M-2 were conjugated at the 4-phenolic hydroxy group, and the glucuronides of M-1 and M-3, which possess two phenolic hydroxy groups, were preferentially conjugated at the 4'-hydroxy group. The order of the rates of in vitro glucuronidation was M-3 greater than M-1 greater than M-2 greater than denopamine.

Metabolism of a new cardiotonic agent, (-)-alpha-(3,4-dimethoxyphenethylaminomethyl)-4-hydroxybenzyl alcohol (TA-064), in man. O-demethylation and ring hydroxylation.

A method is described for the determination of plasma concentration of (-)-alpha-(3,4-dimethoxyphenethylaminomethyl)-4-hydroxybenzyl alcohol (TA-064), a new, selectively inotropic cardiotonic agent, based on selected ion monitoring gas chromatography-mass spectrometry. The plasma TA-064 concentration rose rapidly and reached a peak within 60 min after oral administration. The mean peak value of five volunteers was 14.4 ng/ml. About 30-40% of the dose was excreted as free and conjugated TA-064 and conjugates of five metabolites in the human 24-hr urine. The five urinary metabolites were characterized by mass spectrometry after gas- or high performance liquid chromatographic separation: they were 4'-demethyl-, 3-methoxy-, and 4'-demethyl-3-methoxy-TA-064 as the major and 3'-demethyl- and 3-hydroxy-4-methoxy-TA-064 as the minor metabolites. Therefore, the metabolic reactions involved are demethylation at either one of the two adjacent methoxy functions and hydroxylation at the ortho position to the phenolic hydroxy group followed by methylation of either one of the two vicinal hydroxy groups. The ratio of 4'- and 3'-demethyl-TA-064, which of the dimethoxy functions was the one demethylated, was 17:1, and that of 3-methoxy-4-hydroxy- and 3-hydroxy-4-methoxy-TA-064, the phenolic moiety which was vicinally hydroxylated followed by methylation of one of catecholic dihydroxy function, was 6:1.