Inactivation of anthracyclines by serum heme proteins

We have previously shown that the anticancer agent doxorubicin undergoes oxidation and inactivation when exposed to myeloperoxidase-containing human leukemia HL-60 cells, or to isolated myeloperoxidase, in the presence of hydrogen peroxide and nitrite. In the current study we report that commercial fetal bovine serum (FBS) alone oxidizes doxorubicin in the presence of hydrogen peroxide and that nitrite accelerates this oxidation. The efficacy of inactivation was dependent on the concentration of serum present; no reaction was observed when hydrogen peroxide or serum was omitted. Peroxidase activity assays, based on oxidation of 3,3',5,5'-tetramethylbenzidine, confirmed the presence of a peroxidase in the sera from several suppliers. The peroxidative activity was contained in the >10000 MW fraction. We also found that hemoglobin, a heme protein likely to be present in commercial FBS, is capable of oxidizing doxorubicin in the presence of hydrogen peroxide and that nitrite further stimulates the reaction. In contrast to intact doxorubicin, the serum + hydrogen peroxide + nitrite treated drug appeared to be nontoxic for PC3 human prostate cancer cells. Together, this study shows that (pseudo)peroxidases present in sera catalyze oxidation of doxorubicin by hydrogen peroxide and that this diminishes the tumoricidal activity of the anthracycline, at least in in vitro settings. Finally, this study also points out that addition of H2O2 to media containing FBS will stimulate peroxidase-type of reactions, which may affect cytotoxic properties of studied compounds.     

pH control of nucleophilic/electrophilic oxidation

Finding formulations that prevent degradation of the active pharmaceutical ingredient is an essential part of drug development. One of the major mechanisms of degradation is oxidation. Oxidative degradation is complex, and can occur via different mechanisms, such as autoxidation, nucleophilic/electrophilic addition, and electron transfer reactions. This paper uses three model compounds and determines the mechanisms of oxidation and strategies to reduce degradation. The mechanism of oxidation was established by comparing the results of different forced degradation experiments (radical initiation and peroxide addition), computational chemistry to those of formulated drug product stability. The model compounds chosen contained both oxidizable amine and sulfide functional groups. Although, both oxidative forced degradation conditions showed different impurity profiles the peroxide results mirrored those of the actual stability results of the drug product. The major degradation pathway of all compounds tested was nucleophilic/electrophilic oxidation of the amine to form N-oxide. Strategies to prevent this oxidation were explored by performing forced degradation experiments of the active pharmaceutical ingredient (API) in solution, in slurries containing standard excipient mixtures, and in solid formulation blends prepared by wet granulation. The reaction was significantly influenced by pH in solvent and excipient slurries, with 100% degradation occurring at basic pH values (>pH 8) and no degradation occurring at pH 2 upon exposure to 0.3% peroxide. Wet granulated blends were also stabilized by lowering the pH during granulation through the addition of citric acid prior to the solution of peroxide, resulting in little (0.02% maximum) or no degradation for the four different blends after 6 week storage at 40 °C/75%RH.     

Oxidation of 2-t-butyl-4-methoxyphenol (BHA) by horseradish and mammalian peroxidase systems

Di-BHA, 2,2'-dihydroxy-3,3'-di-t-butyl-5,5'-dimethoxy-diphenyl, was isolated as the product of the reaction of either commercial horseradish peroxidase or partially purified rat intestine peroxidase (Donor-H₂O₂ oxidoreductase, EC 1.11.1.7.) and hydrogen peroxide with 2-t-butyl-4-methoxyphenol (BHA). BHA, Di-BHA and other cyclic compounds possessing a hydroxyl group in the ring were found to be competitive inhibitors with respect to guaiacol, and non-competitive inhibitors with respect to hydrogen peroxide in a system containing guaiacol, hydrogen peroxide and peroxidase. A free radical intermediate generated during peroxidatic oxidation of BHA was detected and identified by means of EPR spectroscopy. It was estimated that during one hour incubation the peroxidase activity present in the rat ileum mucosa is able to oxidise 12μmoles BHA at a saturating concentration. It is suggested that peroxidative oxidation at the intestinal wall may represent a contribution to the inactivation of some phenol derivatives potentially toxic to mammals.     

A prediction system of oxidation reaction as a solid-state stress condition: Applied to a pyrrole-containing pharmaceutical compound

Stress conditions for predicting oxidative degradation products in solid-state pharmaceutical compounds were investigated. 4-Methyl-2-(3,4-dimethylphenyl)-1-(4-sulfamoylphenyl)pyrrole, Compound A, was used as the model compound for this study and its four main degradation products were due to oxidation, as identified by LC–MS and LC-¹H NMR. In order to develop a prediction system for the oxidation reaction, solid-state Compound A was stored under moisture-saturated conditions. Hydrogen peroxide was added to the solution used to saturate the headspace with moisture and oxygen was substituted for the headspace air, in order to stimulate the oxidation reaction. After optimizing the conditions, a similar degradation product profile to that actually observed in the stability studies was obtained in only 3 days under conditions using 3% hydrogen peroxide at 40 °C. The prediction of the oxidative degradation products in a solid-state pharmaceutical compound was successfully achieved in a short term utilizing this newly developed prediction system.     

Mechanistic aspects of the oxidation of phenothiazine derivatives by methemoglobin in the presence of hydrogen peroxide

Mechanistic aspects of the reaction of hydrogen peroxide with methemoglobin with respect to phenothiazine oxidation have been studied. Three phenothiazines, methoxy- (MoPZ), chlor- (CPZ) and methoxycarbonylpromazine (MaPZ), have been used. These phenothiazines differ only in substitution at the 2-position, which contributes substantially to the electron-donating properties of these compounds. Reaction with hydrogen peroxide oxidizes methemoglobin to ferrylhemoglobin, which contains iron(IV)-oxo porphyrin moiety and a protein radical. The phenothiazines are oxidized by ferrylhemoglobin in the presence of H2O2 mainly to their sulfoxides, with a radical cation as intermediate. The conversion rates (MoPZ greater than CPZ greater than MaPZ) decrease with the electron-withdrawing ability of the 2-substituent, as indicated by Hammett sigma para values. Hydrogen peroxide consumption during the reaction is similar for the three phenothiazines. Denaturation reactions that occur upon exposure of methemoglobin to hydrogen peroxide have been investigated. For this heme-protein cross-linking was studied by means of heme retention by the protein after methyl ethyl ketone extraction. Furthermore, oxygen consumption during the reaction was assayed, which indicates formation of protein-peroxy radicals. The extent of both heme-protein cross-linking and oxygen consumption is decreased by phenothiazines in the same order as the phenothiazine conversion rate. CPZ sulfoxide is not converted by methemoglobin in the presence of hydrogen peroxide, and CPZ sulfoxide shows no effect on heme-protein cross-linking and oxygen consumption. The results are explained by electron transfer from phenothiazine to the protein radical. Stronger electron donors (MoPZ greater than CPZ greater than MaPZ) are converted faster and by reducing the protein radical they better protect hemoglobin against denaturation. A catalytic cycle, that takes into account our observation and the existing knowledge of hemoglobin oxidation states, is presented.     

Identification of oxidative degradates of the thrombin inhibitor, 3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methyleneca rboxamidomethylpyridinyl)pyrazinone, using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

Liquid chromatography/mass spectrometry was used to identify reaction products from a solution of 3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methyleneca rboxamidomethylpyridinyl)pyrazinone (L-375,378) and hydrogen peroxide, a system that generates high levels of the oxidative degradates which form in the tablets and intravenous (i.v.) solutions of L-375,378. Two major hydrogen peroxide reaction products of L-375,378 (m/z 407) with m/z values of 369 and 370 were separated and identified. Both compounds were products of ring opening with elimination of three carbon atoms from the center pyrazinone ring. The structural assignments for these two products were alpha-amidinoamide and alpha-diamide compounds, respectively. In addition, five products (m/z 423) with a molecular weight 16 Da greater than that for L-375,378 were separated. Further liquid chromatography/tandem mass spectrometry experiments indicated that three of these M + 16 products were phenolic derivatives of L-375,378. Among them, the para-hydroxy compound has been verified using an authentic standard. The other two phenolic compounds were believed to be the meta- and ortho-hydroxy derivatives of L-375,378. The fourth M + 16 product was derived from hydroxylation of the methyl group on the center pyrazinone ring. The fifth M + 16 product was derived from oxidation on the aminopyridine moiety, most likely N-oxide of the pyridine ring. Other minor hydrogen peroxide reaction products were not studied in detail because they did not appear in tablets or i.v. formulations.     

Hydroxyl radical mediated demethylenation of (methylenedioxy)phenyl compounds.

The oxidative demethylenation reactions of (methylendioxy)phenyl compounds (MDPs), (methylenedioxy)benzene (MDB), (methylenedioxy)amphetamine (MDA), and (methylenedioxy)methamphetamine (MDMA), were evaluated by using two hydroxyl radical generating systems, the autoxidation of ascorbate in the presence of iron-EDTA and the iron-catalyzed Haber-Weiss reaction conducted by xanthine/xanthine oxidase with iron-EDTA. Reaction products generated when MDB, MDA, and MDMA were incubated with the ascorbate or xanthine oxidase system were catechol, dihydroxyamphetamine (DHA), and dihydroxymethamphetamine (DHMA), respectively. The reaction required the presence of either ascorbic acid or xanthine oxidase. Levels of each catechol increased in proportion to ferric ion concentration and were suppressed by desferrioxamine B methanesulfonate (desferal). Catalase (CAT) inhibited the oxidation by the ascorbate system whereas superoxide dismutase (SOD) had little effect. The addition of hydrogen peroxide to the reaction mixture stimulated the oxidation, but the reaction was not initiated by hydrogen peroxide alone, suggesting that hydrogen peroxide acts as a precursor of hydroxyl radical. SOD and CAT suppressed the demethylenation reactions in the xanthine oxidase system. Hydroxyl radical scavenging agents such as ethanol, benzoate, DMSO, and thiourea effectively inhibited the oxidation by both systems. Urea, which has little effect on hydroxyl radical, was without any effect. These results indicated that hydroxyl radical can effect the cleavage of methylenedioxy group on MDPs.     

Oxidative ring-coupling of tyrosine and its derivatives by purified rat intestinal peroxidase.

Intestinal peroxidase was shown to catalyse the oxidative ring-coupling of tyrosine, alpha-methyltyrosine, tyramine and morphine whereas amphetamine was not oxidized to any detectable extent. The oxidative ring-coupling reaction can be monitored by changes in absorbance spectra and the dimers formed in this way with morphine and alpha-methyltyrosine were identified by mass spectrometry. Intestinal peroxidase also catalysed the peroxidatic oxidation of L-DOPA and alpha-methyl-L-DOPA, but in this case the reaction would be expected to be more complicated and to yield a variety of possible products. The kinetic parameters for the oxidation of each of these substrates were determined. Since the products of the oxidative ring-coupling reactions may have different pharmacological properties to those of the parent compounds, these studies suggest that, in the presence of an adequate supply of metabolically produced hydrogen peroxide, the action of intestinal peroxidase may affect the behaviour and pharmacokinetics of these compounds after oral administration.     

2-Alkylpyrrole formation from 4,5-epoxy-2-alkenals

N-Substituted 2-pentylpyrrole formation has been related to the etiology or the consequences of several diseases in which lipid oxidation is involved. This study describes the formation of N-substituted 2-alkylpyrroles in the reaction of 4,5-epoxy-2-alkenals with amino compounds and suggests an alternative pathway for the formation of these compounds that are nowadays commonly accepted to be produced by reaction of the lipid oxidation product 4-hydroxy-2-nonenal with primary amino compounds. The described reaction constitutes a new route for pyrrole production in the lipid peroxidation pathway when it takes place in the presence of amino compounds and implies the loss of one carbon in the 4,5-epoxy-2-alkenal during the formation of the heterocyclic ring, which is proposed to be released as formaldehyde. This reaction also confirms the high reactivity of 4,5-epoxy-2-alkenals, which are usually found in smaller amounts than other lipid oxidation products. Their importance in vivo may be underappreciated in part as a consequence of this high reactivity that brings about their rapid disappearance.     

Indirect determination of paracetamol in pharmaceutical formulations by inhibition of the system luminol-H2O2-Fe(CN)3-(6) chemiluminescence

After a large drug scanning, the system Luminol-H2O2-Fe(CN)6(3-) is proposed for first time for the indirect determination of paracetamol. The method is based on the oxidation of paracetamol by hexacyanoferrate (III) and the subsequent inhibitory effect on the reaction between luminol and hydrogen peroxide. The procedure resulted in a linear calibration graph over the range 2.5-12.5 microg ml(-1) of paracetamol with a sample throughput of 87 samples h(-1). The influence of foreign compounds was studied and, the method was applied to determination of the drug in three different pharmaceutical formulations.     

Degradation of microcystin-RR by UV radiation in the presence of hydrogen peroxide.

Experiments were conducted to investigate the degradation of microcystin-RR in order to assess the effectiveness and feasibility of the combined UV/H(2)O(2) catalytic system for purification of water polluted by microcystins. The operating parameters such as hydrogen peroxide dosage, pH value, UV light intensity, initial concentration of microcystin-RR and reaction time were evaluated, respectively. The degradation efficiency increased nonlinearly with increasing UV light intensity and hydrogen peroxide dosage, respectively. There existed an optimal hydrogen peroxide dosage, beyond which the reagent exhibited an inhibitory effect, for degrading microcystin-RR. The degradation process could be fitted by both of the pseudo-first-order and second-order kinetics well and primarily followed a mechanism of both direct photolysis and hydroxyl radical oxidation. Compared with the treatment using UV radiation and hydrogen peroxide individually, the combined UV/H(2)O(2) system could significantly enhance the degradation efficiency due to the synergetic effect between UV radiation and hydrogen peroxide oxidation. The observed rate constants decreased and the corresponding half-lives prolonged as the concentrations of microcystin-RR increased. The combined UV/H(2)O(2) process provides an effective technology for the removal of microcystins from drinking water supplies.     

Molecular basis of the mechanism of thiol oxidation by hydrogen peroxide in aqueous solution: challenging the SN2 paradigm

The oxidation of cellular thiol-containing compounds, such as glutathione and protein Cys residues, is considered to play an important role in many biological processes. Among possible oxidants, hydrogen peroxide (H(2)O(2)) is known to be produced in many cell types as a response to a variety of extracellular stimuli and could work as an intracellular messenger. This reaction has been reported to proceed through a S(N)2 mechanism, but despite its importance, the reaction is not completely understood at the atomic level. In this work, we elucidate the reaction mechanism of thiol oxidation by H(2)O(2) for a model methanethiolate system using state of the art hybrid quantum-classical (QM-MM) molecular dynamics simulations. Our results show that the solvent plays a key role in positioning the reactants, that there is a significant charge redistribution in the first stages of the reaction, and that there is a hydrogen transfer process between H(2)O(2) oxygen atoms that occurs after reaching the transition state. These observations challenge the S(N)2 mechanism hypothesis for this reaction. Specifically, our results indicate that the reaction is driven by a tendency of the slightly charged peroxidatic oxygen to become even more negative in the product via an electrophilic attack on the negative sulfur atom. This is inconsistent with the S(N)2 mechanism, which predicts a protonated sulfenic acid and hydroxyl anion as stable intermediates. These intermediates are not found. Instead, the reaction proceeds directly to unprotonated sulfenic acid and water.     

Competitive oxidation of 6-hydroxydopamine by oxygen and hydrogen peroxide.

Simultaneous oxygen electrode and conventional polarographic measurements show the net concentration of hydrogen peroxide produced by air oxidation of 6-hydroxydopamine is considerably less than that predicted from the known stoichiometry of the reaction. This is due to competitive oxidation of 6-hydroxydopamine by the generated hydrogen peroxide. The presence of ascorbic acid in this reaction also results in significant decreases of hydrogen peroxide under most conditions. The implications of these results to the molecular mechanism of 6-hydroxydopamine neurotoxicity are discussed.     

Electrochemistry in the mimicry of oxidative drug metabolism by cytochrome P450s

Prediction of oxidative drug metabolism at the early stages of drug discovery and development requires fast and accurate analytical techniques to mimic the in vivo oxidation reactions by cytochrome P450s (CYP). Direct electrochemical oxidation combined with mass spectrometry, although limited to the oxidation reactions initiated by charge transfer, has shown promise in the mimicry of certain CYP-mediated metabolic reactions. The electrochemical approach may further be utilized in an automated manner in microfluidics devices facilitating fast screening of oxidative drug metabolism. A wide range of in vivo oxidation reactions, particularly those initiated by hydrogen atom transfer, can be imitated through the electrochemically-assisted Fenton reaction. This reaction is based on O-O bond activation in hydrogen peroxide and oxidation by hydroxyl radicals, wherein electrochemistry is used for the reduction of molecular oxygen to hydrogen peroxide, as well as the reduction of Fe(3+) to Fe(2+). Metalloporphyrins, as surrogates for the prosthetic group in CYP, utilizing metallo-oxo reactive species, can also be used in combination with electrochemistry. Electrochemical reduction of metalloporphyrins in solution or immobilized on the electrode surface activates molecular oxygen in a manner analogous to the catalytical cycle of CYP and different metalloporphyrins can mimic selective oxidation reactions. Chemoselective, stereoselective, and regioselective oxidation reactions may be mimicked using electrodes that have been modified with immobilized enzymes, especially CYP itself. This review summarizes the recent attempts in utilizing electrochemistry as a versatile analytical and preparative technique in the mimicry of oxidative drug metabolism by CYP.     

Myeloperoxidase-catalyzed oxidation of chloroacetonitrile to cyanide

Chloroacetonitrile (CAN) is a disinfection by-product of chlorination of drinking water. Epidemiological studies indicate that it might present a potential hazard to human health. The present work provides an evidence for CAN activation to cyanide (CN-) by myeloperoxidase (MPO)/hydrogen peroxide (H2O2)/chloride (Cl-) system in vitro. Optimum conditions for the oxidation of CAN to CN- were characterized with respect to pH, temperature and time of incubation as well as CAN, MPO, H2O2 and KCl concentrations in incubation mixtures. The kinetic parameters governing the reaction; maximum velocity (Vmax) and Michaelis-Menten constant (Km) were assessed. Oxidation of CAN to CN- by NaOCl alone was shown. Addition of the MPO inhibitors; sodium azide (NaN3), 4-amino benzoic acid hydrazine (ABAH) or indomethacin to the reaction mixtures resulted in a significant decrease in the rate of CAN oxidation. Inclusion of the antioxidant enzyme catalase (CAT) in the incubation mixtures resulted in a significant decrease in the rate of CAN oxidation and CN- formation. Addition of the sulfhydryl compounds; glutathione (GSH), N-acetyl-L-cysteine (NAC), L-cysteine or D-penicillamine significantly enhanced the rate of CN- release. In conclusion, MPO/H2O2/Cl- system has the ability of oxidizing CAN to CN-. The present results represent a novel pathway for CAN activation and might be important in explaining CAN-induced toxicity.     

Studies with primaquine in vitro: superoxide radical formation and oxidation of haemoglobin.

1. The production of superoxide radicals from primaquine diphosphate in aqueous solution has been demonstrated, using as indicator the reduction of cytochrome C with inhibition of the reaction by superoxide dismutase. 2. Primaquine-mediated oxidation of haemoglobin to methaemoglobin was reduced by the addition of catalase and increased by superoxide dismutase. Mannitol, a hydroxyl radical scavenger, abolished the increase in methaemoglobin observed in the presence of superoxide dismutase. EDTA reduced the oxidation of haemoglobin with and without superoxide dismutase. 3. Although the oxidation of haemoglobin in the presence of primaquine includes the effects of hydrogen peroxide, superoxide and hydroxyl radicals and metal ions, the results indicate that hydrogen peroxide, rather than the superoxide radical, is the main oxidizing species. The increase in haemoglobin oxidation occurring with superoxide dismutase may result from the augmented rate of hydrogen peroxide formation from superoxide radicals.     

N-(2-巯基吡啶-3-甲酰)-N-烃基甘氨酸及其双硫化合物的合成

肾素—血管紧张素系统在调节人体血压时有重要作用。当此系统机能亢进时,会引起血压上升。血管紧张素转化酶(ACE)抑制剂既能对该系统有调节作用,又能抑制有降压作用的缓激肽分解,而起到降血压的效应。此外,它对充血性心力衰竭也有良好疗效。新近报道,ACE抑制剂尚有抗心绞痛作用。     

Mechanism of acetaminophen-stimulated NADPH oxidation catalyzed by the peroxidase-H2O2 system.

The oxidation of NADPH catalyzed by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) is markedly increased by the presence of acetaminophen in a concentration-dependent manner. The oxidation follows pseudo-first order kinetics with respect to acetaminophen concentration. The product of the oxidation is enzymatically active NADP+. The stoichiometry of the reaction shows that 1.4 mol of NADPH are oxidized per mole of H2O2 added, and the addition of superoxide dismutase to the reaction mixture increases the ratio of NADPH oxidized:H2O2 consumed, which suggests formation of superoxide as a product. Monitoring cytochrome c reduction in the presence and absence of superoxide dismutase further suggests formation of superoxide. These results indicate that the HRP-H2O2 system oxidizes acetaminophen to the phenoxyl radical, N-acetyl-p-benzosemiquinone imime, which undergoes a rapid electron transfer reaction with NADPH. The NADP thus formed reacts with molecular oxygen to produce superoxide.     

Development of Estimation Methods of Skin Oxidation and Evaluation of Anti-Oxidative Effects of Genistein in Topical Formulations

The objective of the present study was to establish the method of measurement of hydrogen peroxide and to estimate the anti-oxidative effect of genistein in the skin. UVB induced skin oxidation and anti-oxidative effect of genistein formulations were evaluated by determining levels of hydrogen peroxide. The mechanism involved in the determination of hydrogen peroxide is based on a color reaction between ferric ion (Fe³⁺) and xylenol orange, often called FOX assay and subsequent monitoring of absorbance values of the reactant at 540 nm. The reaction was to some extent pH-dependent and detection sensitivity was greatest at pH 1.75. Genistein liposomal gel demonstrated better anti-oxidative effect with regard to lowering hydrogen peroxide levels elevated by UVB irradiation compared to genistein-suspended gel. A linear relationship has been observed between anti-oxidative effect of genistein and drug deposition in the skin tissue. Genistein liposomal gel resulting in the localization of the drug in the deeper skin led to improved anti-oxidative effect compared to genistein gel. The suggested method for evaluation of oxidation of the skin can be used as a tool to screen effective anti-oxidative agents and their delivery systems acting on the skin.     

Mechanism of the Solution Oxidation of Rofecoxib Under Alkaline Conditions

Purpose The rapid oxidation of rofecoxib under alkaline conditions has been previously reported. The oxidation was reported to involve γ-lactone ring opening to an alcohol, which further oxidized to a dicarboxyclic acid. The oxidation was suspected to be mediated by peroxy radicals. This work further investigates the mechanism of oxidation under the alkaline solution conditions. Methods The pH dependence of the oxidation reaction was determined in 50% acetonitrile/50% aqueous phosphate buffer (pH 9–12). The oxidation reaction products were also examined at early timepoints (from 40 s to several minutes) with only 5% water content. The evolution of hydrogen peroxide by the oxidation reaction was quantitatively followed by reaction with triphenylphosphine (TPP) and high-pressure liquid chromatography determination of the resultant triphenylphosphine oxideformed. Rofecoxib was exposed to the alkaline pH conditions in the presence of formaldehyde, and the primary reaction product was isolated and characterized by liquid chromatography-mass spectrometry and proton 1D, heteronuclear multiple quantum coherence (HMQC), gradient heteronuclear multiple bond correlation (gHMBC), and carbon 1D nuclear magnetic resonance techniques. Transient reaction products were examined for hydroperoxide groups by reaction with TPP. Results The oxidation reaction occurs only near pH 11 and above. In the presence of excess formaldehyde, oxidation products are no longer observed but a new product is observed in which two formaldehyde molecules have added to the methylene carbon atom of the γ-lactone ring. The evolution of hydrogen peroxide corresponds quantitatively to the molar amount of the (minor) aldehyde oxidation product formed. It is demonstrated that the rofecoxib anhydride species is actually the primary product of the oxidation reaction. The existence of a transient hydroperoxide species is shown by reaction with TPP and concomitant conversion to a previously identified alcohol. Conclusions The oxidation of rofecoxib under these high pH conditions is mediated by rofecoxib enolate ion formation. The enolate ion reacts with either formaldehyde or dissolved oxygen at the C₅ position. In the case of oxygen, a transient hydroperoxide species is formed. The major and minor products of the oxidation derive from competitive routes of decomposition of this hydroperoxide. The major route involves a second enolate ion formation, which decomposes with heterolytic cleavage of the RO–OH bond to give the rofeocoxib anhydride and hydroxide ion. The anhydride is rapidly hydrolyzed under the alkaline conditions to give the observed rofecoxib dicarboxylate product. The minor hydroxy-furanone product is formed from hydroxide ion attack on the hydroperoxide intermediate.