Pyridinones Are Not Antioxidants As Shown by Kinetics of Free

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Pyridinones Are Not Antioxidants As Shown by Kinetics of Free Radical Autoxidation, but They Prevent Radical Oxidations Catalyzed by Toxic Heavy Metals Sharwatie Ramsaywack,† Christopher M. Vogels,† S. Laurie Ricker,‡ Stephen A. Westcott,† and L. Ross C. Barclay*,† Departments of †Chemistry and Biochemistry and ‡Mathematics and Computer Science, Mount Allison University, Sackville, New Brunswick, Canada E4L 1G8 S Supporting Information *

ABSTRACT: Three 2-methyl-3-hydroxypyridinones, 1-methyl-, 1; 1-(4methoxy)phenyl-, 2; and 1-(4-dimethylamino)phenyl-, 3, were discovered not to possess strong antioxidant properties contrary to literature reports. These pyridinones were not active chain-breaking antioxidants toward peroxyl radicals generated from styrene or methyl oleate initiated by azobis-2-methylpropylnitrile (AIBN) in solution compared to known phenolic antioxidants, 2,2,5,7,8pentamethyl-6-hydroxychroman (PMHC) or 2,6-di-tert-butyl-4-methoxyphenyl (DBHA). Pyridinone 2 exhibited weak antioxidant activity in cumene, kinh = 1.3 × 103 M−1 s−1, compared to 2,6-di-tert-butyl-4-methylphenol (BHT), kinh = 4.3 × 103 M−1 s−1. The pyridinones were not active antioxidants during lipid peroxidation initiated by azobis-2-amidinopropane·2HCl (ABAP) in aqueous− lipid dispersions of 0.50 M sodium dodecyl sulfate (SDS) micelles where 2 did not inhibit peroxidation of methyl oleate at pH 7.0 or 4.0, while BHT exhibited effective suppression of oxygen uptake. In addition, 2 did not exhibit any cooperative antioxidant effect in combination with Trolox during inhibited peroxidation of linoleic acid in micelles. Pyridinones were effective preventative antioxidants in aqueous−lipid dispersions against reactions initiated by heavy metal ions, notably copper; for example, 2 blocked peroxidation of linoleic acid initiated by Cu ions in SDS micelles. In particular, both 2 and 3 were active in preventing the rapid pro-oxidation effects, “spikes”, of very rapid oxygen uptake when phenolic antioxidants PMHC or Trolox were added to peroxidations initiated by Cu2+. A proposal is given to account for such pro-oxidant effects.

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INTRODUCTION The oxidation of organic compounds by molecular oxygen is generally a free radical chain reaction initiated by peroxyl radicals, for example, by O−O•− or •O−O−H, formed in the respiratory chain. Reactions of such reactive oxygen species (ROS) with biological systems are implicated in a variety of degenerative diseases often associated with aging of humans.1,2 Consequently, there is a continuing interest in the discovery and antioxidant mechanisms of natural and synthetic antioxidants that could deactivate ROS species effectively and help prevent damage to sensitive human tissue. Phenolic antioxidants, like those of the chromanol class (e.g., vitamin E), are common antioxidants where the phenolic −OH group deactivates peroxyl radicals by rapid H-atom transfer (HAT) or proton transfer coupled to electron transfer (PCET) to the •O−O−R group. These mechanisms are given in detail in several reviews.3−6 Various nonphenolic compounds are also known to possess antioxidant properties, including dihydroacridane,7 pyrroles related to blood pigments,8 synthetic aminopyridinols,9 a stilbazulenylbisnitrone by single electron transfer (SET) to peroxyl radicals,10 and the novel designed carbon-centered radicals that trap peroxyl radicals very efficiently.11 The N-substituted pyridinones, readily synthesized © 2013 American Chemical Society

from the natural product, maltol, are known for their metal-ionchelating ability. Our laboratory has employed this property to chelate metal ions for treatment of specific diseases,12,13 and recent reviews14−16 indicate increased activity in this field. Derivatives of the pyridinones that have electron-rich groups at the nitrogen position might be expected to possess strong reducing properties toward peroxyl radicals, making them effective antioxidants. Such properties combined with their heavy metal ion-complexing ability could greatly enhance their beneficial medicinal effects. Indeed, recent reports claimed “good antioxidant properties” of several substituted pyridinones even compared to α-tocopherol (vitamin E) by using their direct reaction (e.g., without substrate) with the ABTS•+ radical.17,18 This prompted us to submit our current results on the antioxidant properties of some pyridinones using quantitative chemical kinetics of autoxidation. The general reactions of autoxidation and its inhibition by an antioxidant are outlined in Scheme 1. These well-known chemical kinetic methods are widely used by various independent groups.1 For a list, see ref 2 given in the current ref 9. These and other Received: December 8, 2012 Published: February 13, 2013 399

dx.doi.org/10.1021/tx300486r | Chem. Res. Toxicol. 2013, 26, 399−409

Chemical Research in Toxicology

Article

kp

Scheme 1. General Reactions of Autoxidation and Inhibition by an Antioxidant

1/2

(2k t)

=

−d[O2 ]/dt [R sH ] × R i1/2

(9)

indicates the susceptibility of the substrate toward oxidation. For inhibited oxidations, chain termination occurs by the antioxidant in reactions 5 and 6. So a different steady state approximation applies between the rate of initiation and the rate of termination where the kinh replaces self-termination of peroxyl radicals. Now the general expression for the inhibited oxygen uptake is given by eq 10. {−d[O2 ]/dt }inh = k p/k inh × [R sH ] × R i /n[ArOH]

(10)

Integration of eq 10 gives the linear eq 11. The kinh of the ratedetermining −Δ[O2 ]t = k p[R sH ]/k inh × ln(1 − t /τ )

step for the antioxidant (its activity, reaction 5) can be determined using eq 10 by measurement of the initial stage of oxygen uptake and determination of the rate of free radical initiation, Ri, using a known antioxidant and eq 7. Alternately the kinh may be determined by eq 11 by a plot of −Δ[O2]t versus ln(1 − t/τ) during the inhibition period. By definition, an antioxidant is “any substance, when present at low concentration compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate”.2 Consequently, when considering the free radical reactions involved, certain requirements must be met to determine quantitative antioxidant properties: (1) There must be a substrate of known susceptibility toward defined reactive oxygen species; (2) the “activity” of the antioxidant must be much greater than the susceptibility of the substrate toward the attacking radical, specifically the ratio of rate constants, kinh/kp ≥ 103; (3) the rate of free radical initiation, Ri, must be known and controlled. The quantitative kinetic method provides significant information on antioxidants for both of their antioxidant activity, through their inhibition rate constants, kinh, and their capacity to trap ROS by determination of the number of radicals trapped, n, per molecule of antioxidant. The initial objective was to apply these quantitative methods to determine the antioxidant properties of pyridinones, 3-hydroxy1,2-dimethylpyridin-4(1H)-one, 1; 3-hydroxyl-(4-methoxy)phenyl-2-methylpyridin-4(1H)-one, 2; and 4-(dimethylamino)phenyl-3-hydroxyl-2-methylpyridin-4(1H)-one, 3; see Chart 1. In the current work, where the compound could not provide a measurable inhibition period of suppressed oxidation, the profiles of reduced oxygen uptake are used, compared to a known antioxidant, to show the ability or lack of the compound’s ability to reduce oxidation of a substrate. Some experiments were also carried out to examine the preventative antioxidant effects of pyridinones 2 or 3 on oxidations catalyzed by heavy metal ions in the presence of phenolic antioxidants since pro-oxidant properties of phenols are becoming more significant in the use of natural and synthetic antioxidants and dietary supplements (see Discussion, part 2). The results obtained help clarify the pathway of pro-oxidant effects.

methods to determine antioxidant properties were reviewed recently for lipid peroxidation.19 In general azo-initiators are used to produce peroxyl radicals at known and controlled rates, Ri (reaction 1), which are readily measured (vide infra). Peroxyl radicals can abstract hydrogen atoms from relatively weak C−H bonds of a substrate, Rs−H, or produce carbon radicals by addition to a double bond (see Results) in a chain propagation reaction, and this releases a substrate radical which in turn rapidly produces peroxyl radicals starting a chain reaction (reactions 2 and 3). The process terminates when two radicals (i.e., peroxyls) recombine (reaction 4). An antioxidant, such as a substituted phenol, may break the chain process, if it is sufficiently reactive, with a large kinh compared to the rate constant of propagation, kp . So these are chain-breaking antioxidants. The aryloxyl radical, ArO•, is normally not sufficiently reactive to start H-atom abstraction but combines with another peroxyl radical. So each phenolic antioxidant traps two peroxyl radicals, providing a stoichiometric factor, n, of 2 (reactions 5 and 6). Oxidation of the substrate is suppressed for a length of time, τ, during an inhibition (induction) period. The n factor for an unknown antioxidant is usually determined by measuring the time of suppressed oxygen uptake and determination of the Ri by a known antioxidant, such as a phenolic antioxidant where n is generally 26 (eq 7). R i = n[antioxidant]/τ

(7)

For uninhibited oxidations (reactions 1−4), the application of a steady state approximation, where the rate of initiation is set equal to the rate of chain termination, is used to derive the general expression (eq 8), which generally applies for uninhibited reactions. 1 kp −d[O2 ] = × [R sH ] × R i1/2 1/2 dt (2k t)

(11)

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MATERIALS AND METHODS

Solvents and Chemicals. Commercial chlorobenzene of highest purity for kinetic experiments was dried over molecular sieves before use. Other solvents were HPLC grade. Styrene was passed through supplied inhibitor remover just before use, and cumene was passed through a column of silica gel to remove traces of hydroperoxides

(8)

The relationship kp/(2kt) (eq 9), called the oxidizability, is characteristic of the substrate and 400



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Chart 1. Compounds Used

before use. Methyl oleate and linoleic acid were obtained in sealed glass vials under nitrogen, stored frozen, and opened and checked for traces of hydroperoxides just before use (see Supporting Information). Sodium dodecyl sulfate, electrophoresis quality, was dissolved in deionized, distilled water containing 1 × 10−4 diethylenetriaminepentaacetic acid, sodium salt (DETAPAC), to trap traces of metal ions. The DETAPAC was not used for experiments on the effects of metal ions. The phenolic antioxidants, 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMHC), 2,6-di-tert-butyl-4-methoxyphenyl (DBHA), and 2,6-di-tert-butyl-4-methylphenol (BHT) were recrystallized from methanol and solutions stored refrigerated before use. Commercial Trolox gave the expected spectral properties (1H and 13C NMR) and was used as received. Azobis-2-methylpropylnitrile (AIBN) was recrystallized from methanol, and the melting point, 101−102 °C (dec) was determined by rapid heating on a preheated hot stage at 80 °C. Azobis-2-amidinopropane·2HCl (ABAP) was used as received. Maltol and 1,2-dimethyl-3-hydroxypyridinone were used as received. 1-(4′-Methoxyphenyl)-2-methyl-3-hydroxy- and 1-(4′-dimethylaminophenyl)-2-methyl-3-hydroxypyridinone were synthesized by modifica-

tions of known methods by making use of microwave heating. Their isolation and identification are described in the Supporting Information. Autoxidation/Inhibition Procedures. Kinetic experiments were carried out under 760 Torr of air in a calibrated dual-channel oxygen uptake apparatus equipped with a sensitive pressure transducer and automatic recorder described previously.51 Runs in solution of chlorobenzene were carried out at 30 and 37 °C in aqueous sodium dodecyl sulfate (SDS)/lipid dispersions. The general method used for experiments in SDS is given in ref 25. The apparatus was evacuated for several hours between changes of solutions used or the use of aqueous mixtures. This was followed by conditioning overnight with any change of solvent used. The sensitivity was checked just before starting a kinetic run by removing known small amounts of air with an airtight syringe. Runs with lipids containing small amounts of hydroperoxides at the start were generally controlled by starting with samples containing a controlled amount of preoxidized linoleic acid. Details of preparing these for kinetic studies in SDS containing ionic copper or iron are given in the Supporting Information. 401



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RESULTS The structures of the main substrates, thermal initiators, compounds tested, and known antioxidants used are given in Chart 1. The substrates were selected for known ranges of reactivity with peroxyl radicals in solution at 30 °C: styrene, kp = 41 M−1 s−1, methyl oleate, kp = 0.22 M−1 s−1 per allylic C− H,20 and isopropyl benzene (cumene), kp = 0.18 M−1 s−1.20 Since the purpose is to determine the antioxidant properties of compounds with varying structural features, it is desirable to employ substrates of various reactivity. 1. Antioxidant Properties in Homogeneous Solution. Significance of the Substrate in Quantitative Studies of Antioxidants. (a). Styrene. The combination of styrene and the azo-initiator azobisisobutyronitrile (AIBN) has key advantages for examining new compounds for antioxidant activity. The homolysis rate constant in solution for AIBN, ki at 30 °C = 9.30 × 10−8 s−1,21 provides a half-life of at least 2 × 103 h. This means that the Ri, required in calculations, will be reasonably constant throughout the period of our experiments. In addition, the rate constant for the chain propagation step of styrene with peroxyl radicals (reaction 12) is sufficiently large, 41 M−1 s−1, to be useful in calculations for even the most reactive antioxidants.

The inhibited oxygen uptake profile of 2 is compared to that of PMHC in Figure 1. PMHC is known to possess an antioxidant activity and stoichiometric factor comparable to α-tocopherol in the styrene/AIBN combination.6 In contrast, 2 reduced oxygen uptake slightly (Figure 1, curve b) but did not give measurable induction periods. Under these conditions in the styrene/AIBN system, pyridinone 3 gave a similar slight reduction of oxygen uptake (not shown) but no inhibition period. A measurable induction period obtained for the phenolic antioxidant, DBHA, curve c, showed a linear plot using eq 11 (R2 = 0.99) and gave a kinh = 11.4 × 104 M−1 s−1, in agreement with the literature value (kinh = 11 × 104).6 The kinh value for α-tocopherol is 320 × 104 M−1 s−1 under these conditions.6 Obviously, it does not seem possible for these pyridinones to possess antioxidant properties comparable to that of α-tocopherol. In an effort to obtain some reliable values for the antioxidant properties for the pyridinones, we decided to use less reactive substrates than styrene for kinetic measurements. (b). Methyl Oleate. Methyl oleate possesses a propagation rate constant with peroxyl radicals of only 2.1 × 10−2 compared to that by styrene so that weak antioxidants might be expected to suppress oxygen uptake under our experimental conditions. By replacing styrene with methyl oleate as substrate with the AIBN initiator, we obtained a more defined profile for suppressed oxygen uptake; for example, the suppressed oxygen uptake (not shown) for DBHA provided a kinh for DBHA of 7.94 × 103 M−1 s−1 under these conditions. The pyridinones 2 and 3 showed only slight reductions of oxygen uptake using this substrate. Unfortunately, they did not provide suitable inhibition periods for measurements, demonstrating again their very weak antioxidant activities. Methyl oleate was also used as a substrate for some experiments in aqueous/lipid dispersions (see section 2b). (c). Cumene. Literature data available for cumene as substrate for such kinetic studies with peroxyl radicals showed that even weak antioxidants give well-defined inhibition periods.23 The suppressed oxygen uptake of both pyridinones 2 and 3 is compared with that of BHT in cumene in Figure 2.

C6H5−CHCH 2 + R−O−O• → C6H5CH•CH 2OOR (12)

So styrene/AIBN have been used to provide reliable kinh values for many antioxidant rate constants with values spanning a range from 0.31 × 104 to nearly 600 × 104 M−1 s−1.6 Our experiments started with maltol since it has been reported to possess antioxidant activity in plant extracts containing maltol, for example, by suppression of the formation of malondialdehyde.22 However, we could not detect any antioxidant effect by maltol to reduce oxygen uptake for diluted styrene in chlorobenzene nor did it act cooperatively with a known phenolic antioxidant (not shown). So this compound was not studied further for possible antioxidant activity. We anticipated in our initial experiments that the pyridinones 1, 2, and 3 would exhibit very strong “antioxidant properties” in the styrene/AIBN combination since previous reports,17,18 supported by a recent review,15 showed them to be comparable in antioxidant properties to that of α-tocopherol. Our actual results do NOT meet any such expectations. Indeed the pyridinones 1−3 in the styrene/AIBN/chlorobenzene system do not possess sufficient antioxidant activity to provide a measurable inhibition period, which is defined by the point where the slope of suppressed oxygen uptake intercepts the line where the uninhibited rate continues (see Figure 1,curve d).

Figure 2. Profiles of oxygen uptake for the autoxidation of cumene, 1.73 M, in chlorobenzene initiated by AIBN, 2.15 × 10−2 at 30 °C: curve a, uninhibited; curve b, effect of pyridinone 3, 8.61 μM; curve c, inhibited by pyridinone 2, 8.95 μM; curve d, inhibited by BHT, 7.69 μM.

As shown in Figure 2, curve d, the relatively weak antioxidant BHT gives a typical induction period from which the kinh and stoichiometric factor, n, are obtained. The profile with pyridinone 2 exhibited much stronger oxygen uptake but a measurable induction period, curve c, in dilute cumene. Compounds 2, BHT, and DBHA were examined in repeated

Figure 1. Profiles of oxygen uptake for the autoxidation of styrene, 0.94 M, in chlorobenzene initiated by AIBN, 2.15 × 10−2 M at 30 °C: curve a, uninhibited; curve b, effect of pyridinone 2, 3.16 μM; curve c, inhibited by DBHA, 3.60 μM; curve d, inhibited by PMHC, 6.45 μM. 402



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these compounds have been carried out in aqueous mixtures, for example, in water/alcohol mixtures, where solvent polarity has remarkable effects on kinetic reactions of oxygen-centered radicals with antioxidants.3 Since the observation that either water-soluble or lipid-soluble initiators can be used in quantitative kinetic measurements of antioxidants in lipid− aqueous dispersions,24 such systems have been widely employed, for example, in cooperative and synergistic effects between antioxidants in aqueous−lipid dispersions,25 and micelles were used as a medium in rapid screening for antioxidant potencies.26 We employed dispersions of unsaturated lipids in sodium dodecyl sulfate (SDS) micelles as a system for kinetic experiments with selected pyridinones 2 and 3. Water-insoluble antioxidants such as PMHC and BHT can be prepared in known concentrations in SDS micelles and their antioxidant activities determined as for homogeneous solutions.27 This section describes results of experimentation on (a) a search for cooperative antioxidant effects of a pyridinone with a phenolic antioxidant, (b) the effect of pH on the antioxidant property of 2, and (c) the effects of 2 or 3 on reactions catalyzed by heavy metal ions in the presence of phenolic antioxidants, that is, the effects of pyridinones on prooxidant effects of phenols. (a). Test for Cooperative Antioxidant Effects. Cooperative antioxidant effects between antioxidants are well-documented in the literature.5 Vitamin C is a relatively weak chain-breaking antioxidant alone in heterogeneous lipid/aqueous phases but is known to act synergistically with vitamin E by regenerating the latter from the tocopheryloxy radical in the lipid phase of lipid membranes.5 Similar synergistic effects were found between vitamin C and vitamin E or Trolox in SDS micelles during peroxidation of linoleic acid.27 From analogy with such results, the pyridinones, 2 or 3, which appear from our results not to be active chain-breaking antioxidants, might act cooperatively with the antioxidant Trolox which distributes between both phases of aqueous SDS.28 Such experiments are usually carried out by determining the effect of a compound in question on the induction period of a known antioxidant. We failed to find any evidence of such cooperative effects when a pyridinone was added together with Trolox. A typical example is shown in Figure 3, which shows an induction period of Trolox, A, curve c, contrasted with the effect of 2, which failed to reduce oxygen uptake, curve b. The induction period of the same amount of Trolox with 2 was not changed when Trolox was added with a similar amount or even with excess 2 (Figure 3B). (b). Search for an Effect of pH on the Antioxidant Property of a Pyridinone. In aqueous acidic media, the pyridinones form a pyridinium structure usually isolated as crystalline salts where the N-ring shows aromatic delocalization.29−32 These appear as vicinal diol or catechol-type structures (Figure 4A). Catechols are known to possess strong antioxidant activities from the HAT or PCET reaction with peroxyl radicals. In catechols, the activities are attributed to stabilization of phenoxyl radicals by strong hydrogen bonding with an adjacent −OH group (Figure 4B).5,25 The pyridinone 2 did not exhibit any antioxidant activity in the aqueous SDS system at pH 7.4 (Figure 3). Since the formation of 4A appears to require an acidic medium,32 the experiment was also carried out at pH 4.0. Methyl oleate in SDS micelles was used as the substrate since this relatively unreactive substrate allows defined induction periods even with weak antioxidants. This is the case for BHT, as shown in Figure 5, curve c, which shows a typical profile of activity in this system. In contrast, the pyridinone 2,

experiments in order to compare their antioxidant activities and n values. The results are summarized in Table 1. Surprisingly, pyridinone 3 (curve b) was not as effective as 2 to inhibit oxidation, and a reliable calculation was not possible for 3. Table 1. Comparison of Antioxidant Activities (kinh) and Stoichiometric Factors (n) of a Pyridinone with Those of Known Phenolic Antioxidants during Inhibited Peroxidation of Cumene (1.73 M) in Chlorobenzene Initiated by AIBN (21 mM) at 30 °C under Air kinh (M−1 s−1 × 10−3)a method compound (μ M)

eq 10

eq 11

2 (7.6−8.2) BHT (6.0−9.0) DBHA (3.6−4.2)

d

d

1.29 3.94 10.6

1.20 4.64 12.3

factor, nb

kcl, at 1/2 τc

1.60 1.95 (2.0)

50 6 4

a

The kinh values by eq 10 method were determined by measuring the initial rate of oxygen uptake {−d[O2]/dt}inh using the Ri determined under the same condition by injecting known amounts of either PMHC or DBHA and calculated using Ri = 2 [ArOH]/τ, where τ is the inhibition period of these phenols. Determinations by eq 11 method were made by plots of −Δ[O2] versus ln(1 − t/τ) and calculated from the linear slopes. bStoichiometric factors were calculated from the Ri measurements by use of the relationship n = Ri × τ/[antioxidant]. The n was assumed to be 2.0 for DBHA. a,bError limits were less than 15% for the determinations. cKinetic chain length determined using the −{d[O2]/dt}inh/Ri at the half point of the inhibition. The uninhibited kcl was approximately 110 under these conditions. dA reviewer reminds us that the resulting high selftermination of peroxyl radicals results in significant errors in calculation of kinh by eq 10 or 11. Corrections can be made for this by correcting for the 2kt by a relationship relating the ratios of initial rates of oxidation of the substrate both in the absence and in the presence of antioxidant by the relationship nkinh[AH]o/(Ri2kt)1/2. However, 2kt values vary with the structure of the radical, being markedly lower with tertiary peroxyls.4 Using a value in cumene4 of 3.5 × 104 M−1 s−1 results in an estimated kinh ≈ 2 × 103 M−1 s−1 for 2 but does not change the relative antioxidant activities in Table 1. Also see ref 12 in ref 8 herein, where we noted that such corrections for retarders in cumene are quite minor.

The results for pyridinone 2 in cumene (Table 1) show that the known weak antioxidants, such as BHT and DBHA, are much more active as antioxidants by 3- and 9-fold, respectively, than the pyridinone from the rate constants with peroxyl radicals. This contrasts with the report showing that the pyridinones (including 2) are much more effective in the ABTS•+ test as antioxidants than BHT.17,18 The stoichiometric factor for BHT, 2.0, is as expected for a phenolic antioxidant.6 The reason for the lower value for 2 is probably due to reaction termination by self-termination of peroxyl radicals in competition with peroxyl trapping by 2 considering the rather high kinetic chain length during inhibition of oxygen uptake by 2 (Table 1). In addition, this number is difficult to measure precisely because the termination of the inhibition period is not sharp since the final rate of oxygen uptake does not return to the uninhibited rate. It is possible that the reaction mechanism of pyridinones with peroxyl radicals is quite different from that of phenols (vide infra). 2. Antioxidant Properties in Aqueous Lipid Dispersions: Micelles. Our kinetic data show clearly that the pyridinones do not act as strong chain-breaking antioxidants in an organic solvent. However, much of the literature reports on 403



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rather than acting as chain-breaking antioxidants. Heavy metal ions as catalysts of oxidation of biological molecules continue to receive special attention because of their implication in degenerative diseases.33−36 A recent design of molecules with both free radical scavenging as well as metal-chelating properties37 is an interesting alternative approach to this continuing problem. However, the pyridinones are generally readily available so we decided to (i) determine if pyridinones, such as 2 and 3 block lipid peroxidation of linoleic acid in SDS micelles when oxidation is initiated by metal ions, Cu2+ or Fe2+; (ii) determine the pro-oxidant effects of typical active phenolic antioxidants, such as water-soluble Trolox and lipid-soluble PMHC, on reactions initiated by these heavy metals; (iii) determine if the pyridinones can block these pro-oxidant effects of antioxidants; and (iv) determine the fate of the pro-oxidant effect on the antioxidant (e.g., Trolox), thereby clarifying the pathway of pro-oxidant effects. In view of a recent highly critical article of the use of copper in vitamin/mineral supplements,36 our experiments focused on copper. The rates of oxygen uptake for reactions catalyzed by heavy metal ions in the presence of antioxidants are affected by several factors: (i) the metal ion and its valence state; (ii) the amount and type of antioxidant; and (iii) the presence of hydroperoxide, and more of the latter may be formed (or lost) during the measurement. Consequently, as explained earlier,38 oxygen uptake rates on the effect of these metal ions are not readily reproducible unlike the profiles of reactions controlled by thermal initiators shown herein. This is because reactions of the metal ions are autocatalytic, resulting in changes in the valence state of the metal ion (e.g., reactions 13 and 14). So we show the effects by calculations of the oxygen uptake rates, especially the changes at crucial points such as on addition of the metal ion, followed by a pyridinone, or followed by an antioxidant, and especially the case of adding a pyridinone followed by an antioxidant to determine if the pyridinones can block the pro-oxidant effects of an antioxidant during the metalion-catalyzed oxidation.

Figure 3. Profiles of oxygen uptake for the autoxidation of linoleic acid, 1.29 × 10−4 m in 0.50 M SDS micelles, phosphate buffer, pH 7.4, initiated by ABAP, 37.4 μm at 37 °C: (A) curve a, uninhibited; curve b, effect of pyridinone 2, 2.00 × 10−8 m; curve c, inhibited by Trolox, 1.99 × 10−8 m. (B) Inhibited by a mixture of pyridinone 2, 2.00 × 10−8 m, and Trolox, 1.99 × 10−8 m, or inhibited by a mixture of pyridinone 2, 2.00 × 10−7 m, and Trolox, 1.99 × 10−8 m.

Figure 4. (A) Vicinal diol structure of a pyridinone formed in acidic aqueous media. (B) Proposed stabilization by hydrogen bonding of the oxygen radical derived from B.

R−O−OH + Cu 2 + → Cu+ + R−O−O• + H+

(13)

R−O−OH + Cu+ → Cu 2 + + RO• + OH−

(14)

(i). Effect of a Pyridinone on a Metal-Ion-Catalyzed Peroxidation. The Cu2+-initiated oxygen uptake profile of linoleic acid in aqueous SDS micelles and its control by the pyridinone 2 are shown in Figure 6. The first part of the figure resembles that reported earlier38 under similar conditions. The oxidation here is expected to be affected both by the autocatalytic cycle Cu2+/Cu+ of reactions 13 and 14 and the consumption of ROOH. The oxidation started by copper Cu2+/ Cu+ is effectively blocked when the ratio of pyridinone/copper ≥ unity. An experiment started with the metal ion in the more reactive lower valence state, for example, by adding Fe2+ to linoleic acid containing a trace of ROOH, in SDS phosphate buffer, pH 7.0, caused an immediate “spike” in oxygen uptake similar to that reported earlier.38 (ii). Pro-oxidation Effect of Phenolic Antioxidants, Trolox, Water-Soluble, and PMHC, Lipid-Soluble. We find that an added phenolic antioxidant has a dramatic effect on oxygen uptake at the instant of injection to a reaction initiated by Cu2+. There results a very rapid surge in oxygen uptake which then decreases followed by a decrease in rate which results in a sharp spike. A similar spike appears immediately on addition of another sample of antioxidant. A typical example is shown in

Figure 5. Profiles of oxygen uptake for the autoxidation of methyl oleate, 1.33 × 10−4 m in 0.50 M SDS micelles, potassium biphthalate, pH 4.0, initiated by ABAP, 34.7 μm at 37 °C: curve a, uninhibited; curve b, effect of pyridinone 2, 1.69 × 10−8 m; curve c, inhibited by BHT, 1.49 × 10−8 m.

also delivered in SDS micelles at comparable amounts, failed even to reduce the oxygen uptake (Figure 5, curve b), compared to the uninhibited reaction (Figure 5, curve a). A 10fold excess of 2 gave some reduction of oxygen uptake, but there was no inhibition period typical of an antioxidant. (c). Pyridinones as Preventative Antioxidants. Lipid Peroxidation Catalyzed by Heavy Metal Ions. We next turned our attention to the possibility that pyridinones might act as preventative antioxidants by blocking processes that form ROS 404



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Figure 6. Plot of the rates of oxygen uptake by linoleic acid, 1.29 × 10−4 m in 0.50 M SDS, phosphate buffer, pH 7.0 at 37 °C, catalyzed by the injection of Cu2+, 1.53 × 10−6 m at 40 min. The effect of injecting pyridinone 2, 1.33 × 10−7 m (in SDS) at 80 min, caused a partial reduction in rate, and a second injection of pyridinone 2, 1.67 × 10−6 m at 100 min, reduced the rate to the initial low rate. Experiments shown in Figures 5−7 used linoleic acid containing traces of hydroperoxide.

Figure 7. These spikes in rates of oxidation were observed for addition of either a water-soluble (e.g., Trolox, Figure 7) or a

Figure 8. Plots of the rates of oxygen uptake by linoleic acid, 1.29 × 10−4 m in 0.50 M SDS, pH 7.0, catalyzed by Cu2+, 1.53 × 10−6 m. (A) Injecting PMHC (in SDS), 4.18 × 10−7 m at 30 min and again at 80 min, gave spikes in rapid oxygen uptake. (B) Experiment was repeated under similar conditions, and PMHC was injected as before at 30 min, resulting in a spike of oxygen uptake, then pyridinone 2, 16.7 × 10−6 m was injected at 85 min, followed by injection of PMHC at 100 min, which did not result in a spike of oxygen uptake.

pyridinones 2 and 3 appear to be effective at preventing the pro-oxidant effect of antioxidants. (iv). Fate of the Pro-oxidant Effect on an Antioxidant, Trolox. The pro-oxidant effect of an antioxidant requires three components: an antioxidant, a trace of hydroperoxide, and heavy metal ions. In order to clarify the nature of pro-oxidation that occurs, it was desirable to determine the role of these components on the fate of the antioxidant during the enhanced oxidation. This determination was focused on Trolox since the main product of its reaction with ROS, Trolox quinone, was characterized earlier.39,40 It would appear that Trolox or PMHC must undergo a rapid reaction during the kinetic experiments where strong oxidation spikes of short duration are observed (Figures 7 and 8) since further experiments indicated that the height of the spike depends on the amount of antioxidant injected in a given experiment (not shown). In order to obtain some measure of the “fate” of Trolox during an oxidation spike, we used two related methods. First, the conversion of Trolox to Trolox quinone was followed by rapid mixing at 37 °C in a stopped flow kinetic apparatus of the same reagents used in the oxygen uptake apparatus, and the absorbance was monitored at 370 nm, near the maximum absorbance for the quinone.39 Formation of the quinone was very rapid by this method (Supporting Information, Figure 7). Second, a more specific confirmation of the quinone formation was followed by UV/vis scans at 37 °C of a mixture of only Trolox and Cu2+, which showed a developing increase in absorbance accompanied by shifts to lower wavelength indicative of Trolox quinone formation, as shown in Figure 9I, traces 1−4. This effect was accelerated by the addition of hydrogen peroxide, traces A−B, to mimic the effect of linoleate hydroperoxide used in kinetic experiments, and the absorbance increased rapidly to go off scale. The control experiment showed that Trolox alone (Figure 9II) showed no change after several hours, although it

Figure 7. Plot of the rates of oxygen uptake by linoleic acid, 1.29 × 10−4 m in 0.50 M SDS, pH 7.0, catalyzed by Cu2+, 1.53 × 106 m. Injecting Trolox, 4.56 × 10−7 m each, at 40 min and again at 80 min gave spikes in rapid oxygen uptake. The experiment was continued by the injection of pyridinone 3, 1.70 × 10−7 m at 130 min, followed by the injection of Trolox which did not cause a spike in oxygen uptake.

lipid-soluble antioxidant, on addition of samples of PMHC (Figure 8). These spikes have a remarkable similarity to those found earlier for the reaction on linoleic acid in SDS micelles catalyzed by initial injection of Fe2+ as catalyst and attributed to a rapid increase in rate due to the more reactive metal in the lower valence state followed by a sharp decrease as the redox couple, Fe2+/Fe3+, is established.38 For comparison purposes, this reaction catalyzed by initial addition of Fe2+ is repeated in the Supporting Information (Figure 5), so this comparison can be made directly. (iii). Pyridinones as Blockers of Pro-oxidant Effects. It was of special interest to determine if the pyridinones prevent such pro-oxidant effects on addition of an antioxidant like those shown in (ii) above. The experiment shown in Figure 7 was actually extended by adding the pyridinone 3 after the second spike followed by injection of a third Trolox sample. This Trolox sample did not cause a third spike, indicating that the pyridinone does act in a preventative manner (Supporting Information, Figure 6). The effect of a pyridinone was tested again (Figure 8A,B) by repeating the experiment by adding the pyridinone 3 before the second PMHC injection B, which resulted in blocking the second pro-oxidant effect. So the 405



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kinetic effects of retarders in contrast to chain-breaking antioxidants are discussed in ref 38. To emphasize the importance of the radical chain reaction again, the kinetic chain lengths at one-half of the inhibition period are compared in Table 1 for two common phenolic antioxidants with that of the pyridinone 2. Compared to the kcl ≈ 110 for the uninhibited reaction under these conditions, the two antioxidants DBHA, kcl = 4, and BHT, kcl = 6, “break” the chain reaction almost completely, whereas the kcl value for 2 of approximately 50 indicates that a high rate of chain reaction continues in its presence. Such results are in marked contrast with the reports based on the ABTS•+ monitor showing the pyridinones to exceed BHT in antioxidant capacity.17,18 Overall, the pyridinones do not possess active centers to trap peroxyl radicals. In nonprotic solvents, the enolic −OH of the pyridinones would not provide hydrogen atom transfer to peroxyl radicals because hydrogen bonding to the adjacent carbonyl group is strong. For example, in maltol, it is approximately 7 kcal/mol41 and the HAT reaction would require breaking of this bond simultaneously with breaking the O−H bond. The possibility of either the HAT or proton transfer coupled with electron transfer is very slight for the alternative diol structure (Figure 4) due to an unfavorable positive charge. Also, the intramolecular H-bonding required for stabilization of the phenoxyl radical from a catechol would be retarded by intermolecular hydrogen bonding with water. This latter effect is known to reduce the antioxidant activity of catechols in aqueous micelles to 2% of the value in a nonprotic solvent;25 consequently, the pyridinones are not effective antioxidants in organic solvents or aqueous media by any known antioxidant mechanism. The apparent lower reactivity of 3 compared to that of 2, as shown in oxygen uptake profiles (Figure 2), is surprising because the former bears a (CH3)2− N− on the phenyl while 2 has the CH3O− group. The former group is known to increase the activity of phenolic antioxidants more than the latter.42 This fact plus other behavior of the pyridinones, such as lack of any cooperative effects with a phenolic antioxidant such as Trolox, part (a) above, may be accounted for by a different mechanism to explain the weak antioxidant effects of these pyridinones. Peroxyl radicals react with alkenes by H-atom abstraction from allylic hydrogens, here the CH3 group, or addition to the double bond.4 The latter reaction could take the form of a polar reaction, involving a Michael-type addition43 to the CC−CO chromophore since peroxyl radicals are highly polarized, bearing significant dipole moments (Figure 10).44 Neither of these possibilities is expected to provide significant antioxidant activity.

Figure 9. UV/vis traces of a the effect of Cu2+, 9.56 × 10−6 M, on Trolox, 3.56 × 10−6 M in pH 7.0 buffer at 37 °C. (I) Traces 1−4 at 15 min intervals followed by injection of 100 μL of 30% hydrogen peroxide for traces A and B determined immediately. (II) Control traces on the same concentration of Trolox without Cu2+ in buffer, (A) initial trace and (B) after 3 h.

was reported earlier that Trolox undergoes very slow oxidation in emulsions, especially SDS.39 Sufficient Trolox quinone was prepared by this method to confirm its identity (see Supporting Information).

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DISCUSSION Substituted pyridinones are typically synthesized in several steps from maltol by conversion of the latter to the benzyl derivative followed by reaction with a primary amine and removal of the protecting group. Our method, given in the Supporting Information, demonstrates the advantage of microwave heating, whereby the synthesis is conveniently done in one step directly on a mixture of maltol with the amines since this provided the products in reasonable yields, 33−54%, after purification. 1. Pyridinones Are Not Effective Chain-Breaking Antioxidants. In stark contrast to literature reports on the “antioxidant properties” of these pyridinones, our quantitative kinetic results employing substrates of varying reactivity (styrene, methyl oleate, cumene) to peroxyl radicals show very little inhibition activity until cumene was used. With the latter, pyridinone 2 gave an activity, kinh, only 1/3 of that found for BHT (Table 1), which is one of the weakest phenolic antioxidants.6 The low stoichiometric factor for 2 is probably due to self-termination of peroxyl radicals (reaction 4), a consequence of its low chain-breaking antioxidant activity. At excess concentrations compared to the phenolic antioxidants used, the pyridinones exhibited some reduction in the rates of oxidation of substrates but no distinct inhibition periods. This behavior is typical of the effects often observed by a wide variety of organic compounds referred to as “retarders”. The

Figure 10. Resonance formulas of a peroxyl radical. The unpaired spin shown on the inner oxygen of structure B is associated with the known significant polarity of peroxyl radicals.44

A critical review more than a decade ago referred to “the present chaos in the methodologies used to evaluate antioxidants”45 is still relevant. Antioxidants generally operate by a chain-breaking process. Their properties include the antioxidant activity, which means determination of a rate or rate constant with attacking radicals and the capacity of the antioxidant, the number of radicals trapped per antioxidant molecule. By using substrates of various reactivity, these 406



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Scheme 2. Proposed Mechanism for the Pro-oxidant Effect by a Phenolic Antioxidant during Metal-Ion-Catalyzed Oxidation

to direct reaction of the metal ion with the antioxidant as observed separately. This could result from rapid π-complex formation between the aromatic ring of the antioxidant and Cu2+, which facilitates electron transfer, producing Cu+ and the aroyloxy radical leading to rapid loss of the antioxidant as proposed in Scheme 2. This is accompanied by a surge in oxygen uptake as more ROS are formed. Such π-complexes were reported earlier between Cu+ and aromatic molecules including phenols with theoretical interpretations and determinations of dissociation energies where the metal ion “interacts with the entire cloud” of the aromatic ring.50 Overall, the pyridinones act as preventers of metal-ioncatalyzed oxidation in two ways. First, by forming stable complexes with toxic metal ions such as copper,17 the bound copper is not “free” to react with a hydroperoxide (reaction 13) and start a chain reaction (Figure 6). Second, the complexed metal ion cannot start the very rapid pro-oxidant effect in the presence of a common antioxidant because the π-complex of Cu2+ with the phenolic ring, required for reduction of Cu2+ to the more active Cu+ by electron transfer (Scheme 2), is not available and the pro-oxidant effect is prevented.

properties can be determined even for weak antioxidants. In the present work, the profiles of oxygen uptake compared to active phenolic antioxidants provided useful insights into the limitations of pyridinones as antioxidants. The color quenching of colored long-lived organic radicals such as 2,2-diphenyl-1picrylhydrazyl (DPPH•) or ABTS•+, in some cases relative to a known phenolic antioxidant, have been popular methods to test antioxidants. The DPPH• radical was widely used to determine the hydrogen atom transfer activity of antioxidants. However, it was discovered that, in the alcohol solvents frequently used, the ArOH/DPPH• reactions actually occurred by prior ionization of the phenolic antioxidant and electron transfer to DPPH•, the sequential proton-loss electron transfer, SPLET mechanism.3 The oxygen-centered 2,6-di-tert-butyl-4-(4′-methoxyphenyl)phenoxyl radical can be used to avoid this complication and was recommended for determination of HAT activities of phenolic antioxidants.8 However, none of these methods actually determine chain-breaking antioxidant activities since a substrate is not used to generate a chain reaction. 2. Pyridinones 2 and 3 Are Effective Preventative Antioxidants. Preventative antioxidants generally block the formation of initiating radicals so that damaging chain reactions are not started. Heavy metal ions may initiate radical chain oxidation in the presence of traces of hydroperoxides (reactions 13 and 14). The pyridinones are known metal chelators,12−16 so they might be expected to block the metal-initiated radical oxidation in the metal-complexed state. The aqueous/lipid dispersion of linoleate in SDS micelles was a convenient system to contain the metal ions, the pyridinones, and antioxidants for these experiments. It is evident from Figure 6 that an oxidation already initiated by Cu2+ can be blocked from continuing by adding a pyridinone as a complexing agent. How do antioxidants become converted into pro-oxidants? This is not a trivial question if one considers the potential damaging effects of certain vitamin supplements containing copper and the relationship to degenerative diseases especially of the elderly.36 Several earlier reports indicate that the reduction of metal ions from their higher valence state to more reactive low valence state by antioxidants is a principal step causing prooxidant activity.33,46−49 For polyhydroxy phenols, such as the flavonoids, the antioxidant/pro-oxidant effects are more complex due to possible chelation of metal ions by adjacent hydroxyl groups.46,47 So the antioxidant/pro-oxidant effect depends on the class of flavonoid48 and depends on the method of generating the radicals in the system.49 The kinetic oxygen uptake method does provide more specific insights into the process of the pro-oxidant effect of phenolic antioxidants. In particular, the phenolic antioxidant causes a very rapid increase in oxygen uptake which then subsides, giving spikes in the oxygen uptake profile. This is much like the spikes shown when one starts with a reactive form of the metal ion. So the increase (pro-oxidant effect) is attributed to formation of the reduced lower valence state of the metal ion. The remarkable speed of the oxygen uptake and loss of the antioxidant is apparently due

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CONCLUSION The application of classical quantitative kinetics of autoxidation and its inhibition still provides a superior method to investigate antioxidant activities, limited only by substrates of known (or measurable) rate constants. The method reached a limit with the pyridinones which are so inactive to be classed as retarders, like very many organic compounds. The popular use of colorimetric methods involving direct reaction between the potential antioxidant and a persistent radical (i.e., DPPH• or ABTS•+) in the absence of a substrate may produce some new and interesting chemistry on hydrogen transfer reactions (HAT or PCET) or even reveal a new mechanism, such as the SPLET reaction of phenols and DPPH• in alcohol media. However, the radical chain-breaking antioxidant properties are not evaluated by these methods, and it is misleading to report such results as antioxidant properties. In keeping with the desirability of having more clarity in this field,45 it is urged that results that provide evidence of reactivities and mechanisms on organic compounds due to their hydrogen atom transfer and/or electron transfer properties be reported as HAT, SET, PCET, etc. properties, whereas antioxidant properties should be reported when radical chainbreaking activities such as rate constants and the number of radicals trapped are determined. Aqueous micelles provide a useful medium to follow the prooxidant effects of the combination of toxic heavy metal ions with phenolic antioxidants and determine the prevention of these effects by the pyridinones. It is also desirable to determine if these pyridinones are effective at blocking pro-oxidant effects in model phospholipid membranes at known “free” metal ion concentrations in biological systems under normal or stressed conditions. 407



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ASSOCIATED CONTENT

S Supporting Information *

Details are given on the synthesis and characterization of pyridinones, Fe2+-catalyzed peroxidation of linoleate in 0.50 M SDS micelles, Cu 2+-catalyzed peroxidation of linoleate enhanced by an antioxidant and blocked by a pyridinone, preparation of partially oxidized linoleic acid, and the progress of Cu2+-catalyzed oxidation of Trolox to Trolox quinone. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (506) 364-2455. Fax: (506) 364-2313. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support by the Natural Sciences and Engineering Research Council provided to S.W. through a Canada Research Chair, Tier 1, is acknowledged. We thank Philip Cormier, Daniel Durant, and Cheryl-Anne Lee for technical assistance.

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ABBREVIATIONS ABAP, azobis-2-amidinopropane·2HCl; AIBN, azobis-2-methylpropylnitrile; BHT, 2,6-di-tert-butyl-4-methylphenol; DBHA, 2,6-di-tert-butyl-4-methoxyphenol; Detapac, diethylenetriaminepentaacetic acid; kcl, kinetic chain length; HAT, hydrogen atom transfer; PCET, proton coupled to electron transfer; PMHC, 2,2,5,7,8-pentamethyl-6-hydroxyphenol; ROS, reactive oxygen species; SET, single electron transfer; SDS, sodium dodecyl sulfate; SPLET, sequential proton loss electron transfer

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Pyridinones Are Not Antioxidants As Shown by Kinetics of Free

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