Mechanism of horseradish peroxidase inactivation by benzhydrazide: a critical evaluation of arylhydrazides as peroxidase inhibitors

Many compounds are oxidized by haem enzymes, such as peroxidases and cytochromes P450, to highly reactive intermediates that function as enzyme inactivators. To evaluate the potential of arylhydrazides as selective metabolically activated peroxidase inhibitors, the mechanism of HRPC (horseradish peroxidase isoenzyme C) inhibition by BZH (benzhydrazide) was investigated in detail. No oxygen consumption was detected in BZH solutions at pH 7.0–12.0, but addition of HRPC resulted in significant O2 uptake above pH 8.0, indicating that the enzyme catalyses BZH oxidation. Addition of H2O2 to HRPC plus BZH activates the latter as an inhibitor. This involves the three-electron oxidation of BZH in one-electron steps by the peroxidase catalytic intermediates, Compounds I and II, to produce a benzoyl radical that covalently alters the active site and inhibits peroxidase activity. Alternatively, the benzoyl radical could be produced by di-imide (NH=NH) elimination from the BZH radical. Production of Compound III (oxyperoxidase) followed by p-670 (m/z=583, biliverdin-like derivative) was observed for HRPC incubated with excess H2O2, and the addition of BZH resulted in an increase in the rate of p-670 production. BZH is an inefficient inhibitor of HRPC with a KI of 80 μM, an apparent inactivation rate constant (kinact) of 0.035 min−1, and an IC50 of 1.0 mM. This prompted the investigation of HRPC inactivation by a series of related arylhydrazides with known binding affinities for HRPC. The hydrazide with the highest affinity (2-naphthoichydrazide; Kd=5.2 μM) was also found to be the most effective inhibitor with KI, kinact and IC50 values of 14 μM, 0.14 min−1 and 35 μM, respectively.

Thermokinetic study of the inactivation reaction of 1-methyl phthalazinium ylids

The present paper consists in a thermokinetic study on the dimerization reactions of 1-methylphthalazinium ylids with ?NO2 (ylid 1) and respectively, ?O?CH3 (ylid 2) substituents in the p position of the benzoyl radical bound to the ylidic carbanion. From experimental data, the reaction order and rate constants have been calculated. The reaction order n = 2 confirmed the ylids? dimerization reactions, while the values of the rate constants, k2 = 3.093x10-2L/mol s and, respectively, 2.16 x?10-1 L/mol s for the dimerization of ylids 1 and 2 made evident the higher reactivity of ylid 2 versus ylid 1. The same conclusion is also supported by the results of the thermodynamic study based on the chemical affinity of the two reactions, when Ao dim,1 < Aodim,2.

Synthesis of 5-phenyl-10-methyl-7H- pyrimido[4,5-f][1,2,4]triazolo[4,3-a][1,4]diazepine and its evaluation as an anticonvulsant agent

The homolytic benzoylation (benzoyl radical) of 5-tert-butylcarbonylaminopyrimidine (6, 1 equiv.) in the presence of benzaldehyde (3 equiv.), water, sulfuric acid, and acetic acid, upon treatment with FeSO4·7H2O (3 equiv.) and 70% t-BuOOH (3 equiv.) at 5-10°C for 10 min afforded a mixture of 4-benzoyl-5-tert-butylcarbonylaminopyrimidine (7, 23%), 4,6-dibenzoyl-5-tert-butylcarbonylaminopyrimidine (8, 44%), and 4-benzoyl-5-tert-butylcarbonylamino-6- methyl pyrimidine (9, 10%). When a similar reaction was performed using 1 equiv. each of PhCHO, FeSO4·7H2O, and t-BuOOH, to prevent formation of the 4,6-dibenzoyl product 8, the monobenzoyl (7, 38%) and 4-benzoyl-6-methyl (9, 6%) products were obtained. A similar homolytic benzoylation of 5-bromopyrimidine (10) using 1.5 equiv. of reagents to generate the benzoyl radical afforded 4-benzoyl-5-bromopyrimidine (11, 61%) as the predominant product. Elaboration of 11 via a six-step reaction sequence afforded 2-hydrazino-5-phenyl-3H-pyrimido[5,4-e][1,4]diazepine (17) in 5.2% overall yield. The acid-catalyzed reaction of 17 with triethyl orthoacetate gave the title compound 5-phenyl-10-methyl-7H-pyrimido[4,5-f][1,2,4]triazolo[4,3-a][1,4]diazepine (18, 62%). The triazolo compound 18 was more potent than valproic acid (Depakene(r)) in both the subcutaneous metrazol (scMet) and maximal electroshock (MES) anticonvulsant screens, and more potent than clonazepam in the MES anticonvulsant screen but less potent than clonazepam in the scMet anticonvulsant screen.Key words: homolytic benzoylation, pyrimidines, pyrimidodiazepines, anticonvulsants.