Mechanism of Antibacterial and Degradation Behavior of a Chlorinated Isoxazolylnaphthoquinone Patricia M. Bogdanov, Viviana G. Dabbene, Ine ´s Albesa, Marı ´a M. de Bertorello, and Margarita C. Brin ˜o ´n 1 Departamento de Farmacia, Facultad de Ciencias Quı ´micas, Universidad Nacional de Co ´rdoba, Ciudad Universitaria, 5000 Co ´rdoba, Argentina Received July 20, 1999 The chemical stability of 3-chloro-2-hydroxy-(3,4-di- methyl-5-isoxazolyl)-1,4-naphthoquinon-4-imine (ClQ 1 ), a new potential antimicrobial agent was analyzed at different pH values by first-derivative spectroscopy. The degradation of ClQ 1 followed a pseudo-first-order kinetics in aqueous media at different pH values. The interaction of antibiotics with respiratory chain of Staphylococcus aureus generates superoxide anion, an oxygen radical capable of producing damage to the bacteria. The performed assays have demonstrated that ClQ 1 presents higher activity and toxic oxidant generation at pH 5.0 than at pH 7.5. In addition, the antibacterial activity of other halogenated isoxazolyl- naphthoquinones was also studied in different collec- tion and clinical strains which presented the following decreasing activity, ClQ 1 > BrQ 1 > DClQ 1 whereas DBrQ 1 did not show inhibition properties. The anti- bacterial and stability properties evidenced by ClQ 1 are so important that must be taken into account when new alternative treatments against -lactamase- positive S. aureus strains are investigated. © 1999 Academic Press Key Words: Staphylococcus aureus; superoxide anion; antibacterial activity; stability studies; chlori- nated naphthoquinonimine. Isoxazolylnaphthoquinones are investigational syn- thetic drugs which have proven to be effective against Staphylococcus aureus (1, 2) and Trypanosoma cruzi (3, 4). As part of our research on this family of com- pounds, we have synthesized the halogenated deriva- tives, DBrQ 1 , DClQ 1 , BrQ 1 and ClQ 1 (Scheme 1) (5), as well as studied the chemical stability of DBrQ 1 , DClQ 1 and BrQ 1 , in ethanolic solutions (6 –9). In this way, DBrQ 1 and DClQ 1 yielded BrQ 1 and ClQ 1 respectively, as principal degradation products, and BrQ 1 decomposed to Q 1 , the prototype of the series under the same degrading conditions. Previous results from our laboratory showed Q1 is an effective agent against S. aureus, detecting redox-reactions between Q 1 and NADH in presence of diaphorase (10). The interaction of Q 1 with the respiratory chain of S. aureus generates O 2 - which can lead to the release of other oxygen radicals capable of promoting further mi- crobial injury. A number of pharmacological agents undergo rapid redox cycling under aerobic conditions, resulting in an exogenous cause of reactive oxygen species (ROS) increase. These compounds are reduced but in presence of O 2 they are reoxydized, transferring the electrons to oxygen, forming O 2 - and H 2 O 2 via a superoxide dismutase like nitrofurantoin (11–13). Other antimicrobial agents used in the treatment of clinical infections, in addition to conventional mecha- nisms of action, generate reactive oxygen intermedi- ates capable of damaging the cell like the oxidation of DNA produced by penicillin’s and cephalosporin’s due to [ • OH] mediation (11). Taking into account the above results, and the im- portance of learning the stability behavior of poten- tially active compounds, the goal of this paper was to study the hydrolysis of 3-chloro-2-hydroxy-N-(3,4-di- methyl-5-isoxazolyl)-1,4-naphthoquinon-4-imine (ClQ 1 ) in aqueous media, as well as its antibacterial activity against collection bacteria and clinical strains. Kinetic studies were carried out using a zero crossing first- order derivative spectrophotometry as analytical method. The validation of the procedures was also presented. The antibiotic effects of BrQ 1 , DClQ 1 and DBrQ 1 were Abbreviations used: DBrQ 1 , 3-bromo-N-bromo-N-(3,4-dimethyl-5- isoxazolyl)-4-amine-1,2-naphthoquinone; DClQ 1 , 3-chloro-N-chloro-N- (3,4-dimethyl-5-isoxazolyl)-4-amine-1,2-naphthoquinone; BrQ 1 , 3-bromo- 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinon-4-imine; ClQ 1 , 3-chloro-2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naphtho- quinon-4-imine; Q 1 , 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naphtho- quinone-4-imine. 1 To whom correspondence should be addressed. Fax: 54-351- 4334127. E-mail: macribri@dqo.uncor.edu. Biochemical and Biophysical Research Communications 263, 301–307 (1999) Article ID bbrc.1999.1360, available online at http://www.idealibrary.com on 301 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.