ARCHIVES

OF BIOCHEMISTRY

AND

Vol. 282, No. 1, October,

BIOPHYSICS

pp. 18-25,

1990

Reactivity of Ebselen and Related Selenoorganic Compounds with 1,2-Dichloroethane Radical Cations and Halogenated Peroxyl Radicals Christian

SchGneich,”

Vasanthy

Narayanaswami,?

Klaus-Dieter

Asmus,*,’

and Helmut

*Hahn-Meitner Institut Berlin, Bereich S, Abteilung Strahlenchemie, Postfach 390128, D-1000 Berlin Federal Republic of Germany; and tlnstitut fiir Physiologische Chemie I, Universittit Diisseldorf, Moorenstrasse 5, D-4000 Diisseldorf, Federal Republic of Germany

Received

February

23,199O

The reactivity of ebselen, 2-phenyl1,2-benzisoselenazol-3(2H)one, and structurally related analogues was studied by pulse radiolysis. The rate constant for the reaction of ebselen with trichloromethylperoxyl radicals was determined to be 2.9 X 10’ M-l s-l, while its sulfur analogue, 2-phenyl-1,2-benzisothiazol3(2H)one, was oxidized at much lower rates, k i 10’ Mm’ s- I. Among several derivatives studied, the only other compound that exhibited a high rate constant was 2(methylseleno)-benzoic acid-N-phenylamide. Oxidation of ebselen by other halogenated peroxyl radicals was also carried out and revealed a direct relationship between rate constant and the degree of halogenation of the oxidant. The transient radicals generated during oxidation of ebselen and the analogues were characterized by optical absorption and conductivity measurements and were attributed to one-electron-oxidized radical cations. The oxidation potentials were determined by cyclic voltammetry. Comparative evaluation of the in vitro behavior during microsomal lipid peroxidation revealed ebselen to be the most potent antioxidant of the compounds investigated. 2-(Methylseleno)benzoic acid-N-phenylamide, despite its high rate constant for oxidation by halogenated peroxyl radicals, was found to be a poor antioxidant. The rate constant of oxidation of ebselen by trichloromethylperoxyl radicals is comparable to that of a-tocopherol under similar conditions, underscoring the potential pharmacological interest of ebselen as an antioxidant. cc) isso Academic

The has

significance become clear

1 To whom

18

SiesjJ

39,

of selenium in recent

correspondence

should

in years

biology (1).

be addressed.

and

The

medicine selenoor-

ganic compound ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)one, 1, exhibits glutathione peroxidase (2, 3), antiinflammatory (4), and antioxidant activity (2). This heterocycle has gained considerable attention due to its potential

pharmacological

applications.

In

contrast,

the

sulfur analogue is devoid of peroxidase-like activity and is a poor antioxidant. For this report, the susceptibilities of ebselen and various structurally related derivatives (Fig. 1) to oxidation by radical cations from 1,2-dichloroethane and several halogenated peroxyl radicals were studied by pulse radiolysis, and their electrochemical oxidation potentials are compared with their in vitro behavior during nonenzymatically induced lipid peroxidation in rat liver microsomes which was monitored as low-level chemiluminescence (5). This study aids in further understanding of the biochemistry and biophysics of ebselen. EXPERIMENTAL Pulse radiolysis. Pulse radiolysis experiments were carried out using 1.55MeV electrons supplied by a Van-de-Graaf accelerator. Pulses between 0.3 and 1 ps were usually applied with doses on the order of 225 Gy per pulse (1 Gy = 1 J/kg). Dosimetry was based on the oxidation of thiocyanate by OH’ radicals in nitrous-oxide-saturated aqueous solutions using the molar extinction coefficient of (SCN); = 7200 M -’ cm-’ (optical absorption), Ail = -360 Scm’val ~’ (conductivity) and G = 5.5 (G denotes the yield of radiation chemically generated or altered species per 100 eV absorbed energy; G = 1 corresponds to ca. 0.1 pmol J-i). A detailed description of the pulse radiolysis technique and the evaluation of data is available (6). Owing to the pronounced hydrophobicity of the compounds under investigation studies were performed either in nitrogen-saturated solutions of the Se- and S-derivatives in pure 1,2-dichloroethane, abbreviated as DCE,’ (Baker, distilled prior to use) or in air-saturated mixtures of 35% water (Millipore Q quality) and 65% tert-butanol (Merck, p.a.) (v/v), the

’ Abbreviations used: DCE, monium hexafluorophosphate.

dichloroethane;

All

TBAFs,

Copyright 0 1990 rights of reproduction

tetrabutylam-

0003.9861/90 $3.00 by Academic Press, Inc. in any form reserved.

PULSE

RADIOLYSIS

0

1

OF

oxidation by free radicals. Pulse radiolysis of nitrogensaturated pure 1,2-dichloroethane leads to the formation of radical cations and P-chloroethyl radicals via reactions 1 and 2 (7):

f&a

2~pknyl-1.2-benz~soselenozol-3l2Hlone PZ 51. Ebsekn 1-Ethyl~2-(2’.phenyl-corbomoyl phenyllthioselane

(DCE) + (DCE): + e&, e,, + Cl-CH,CX-Cl

2-phenyl-1.2.benzlsothlozol-3l2Hlone PZ 25

2-lbenzylseknol-benzoc

acid-N-l2-pyridyll-amloe

+ Cl- + Cl-CH,CH2.

111 [al

Dichloroethane radical cations, (DCE)!, are the only primary oxidizing species in such systems; they disappear via various reactions including deprotonation and hydrogen transfer (8), and act as one-electron oxidants on addition of different solutes, including antioxidants (7-11). The yields of the general reaction (3):

I 2-lmethylthlol-benrolc N-phenylomlde

aud

0

2-(benzylseknol-benzolc

19

EBSELEN

(DCE):

+ substrate + (DCE) + oxidized substrate

octd-N-phenylamjde

[3]

0 2-phenyl-1.2.benzlsoselenazol 3IZHloneselenox,de

2-imethylselenoi-benzolc

FIG. 1. Chemical pounds used.

acid-N-phenylamlde

structures

and names

of the selenoorganic

com-

hydrocarbons and at latter containing 5 X 10 ’ M of t,he halogenated various concentrations of the Se- and S-derivatives. The dosimetry values obtained with aqueous systems were corrected for specific gravity and electron density due to lack of specific dosimeters in such solvent systems. The halogenated hydrocarbons used for the generation of halogenated peroxyl radicals in waterltert-butanol mixtures were obtained from Merck (carbon tetrachloride, Ccl,; chloroform, CHCI?), Aldrich (pentachloroethane, CHCl,CC13; freon, CCl,FCClF,), Schuchardt (hexachloroethane, CCl,,CCl,), and Abbott (enflurane, HCF&CF2 CHFCl; methoxyflurane, CH,-O-CF&HCI,). Although of the highest commercially available purity, the solvents were redistilled or sublimated (CCIz1CClz3) twice prior to use. The selenium and sulfur derivatives were kindly supplied by A. Nattermann and Cie, Rhhne-Poulenc (Kiiln) and used without further purification. Cq’clic voltammetr~~. Cyclic voltammetry experiments were performed with a Princeton Applied Research Model 362 using a standard three-electrode system consisting of a 1.47.cm2 Pt flag working electrode, a Pt counter electrode and a Ag/AgCl (saturated LiCl in ethanol) reference electrode. Data were collected on a Kipp and Zonen BD 90 X-Y recorder. The voltammograms were recorded from argon-saturated solutions of the Se-/S- derivatives (0.5-1.0 X IO-” M) in dry acetonitrile (freshly distilled over phosphorous pentoxide) containing 0.1 M tetrabutylammonium hexafluorophosphate (TBAF,) as the supporting electrolyte. Ferrocene was added as internal standard in controls (I?,$+, = +0.54 V vs. Ag/AgCI). All pulse radiolytic and electrochemical experiments were carried out at room temperature.

RESULTS

AND

DISCUSSION

Pulse radiolysis of ebselen in 1,2-dichloroethane. Experiments were carried out in dichloroethane to study the susceptibilities of the Se- and S-containing drugs to

depend on the chemical nature and concentration of the solute (8). G-values between 0.18 and 2.8 (7-9) have been reported. The P-chloroethyl radicals that are also produced are practically inert with respect to redox reactions and are inactivated mostly by dimerization. Figure 2 shows the transient absorption spectrum obtained on pulse irradiation of 0.1 mM ebselen, 1, in nitrogen-saturated DCE, 50 ps after a ca. 1-1~spulse (curve a), and the control absorption spectrum of 0.1 mM ebselen (unoxidized) in 1,2-dichloroethane (curve b). As the computing unit of the pulse radiolysis equipment normalizes the actual extinction before the pulse as reference baseline, any change recorded immediately after the pulse represents a radical-induced change of the solvent or solute molecule. Neither the dichloroethane radical cation nor the P-chloroethyl radicals exhibit any appreciable absorption during the time scale of observation (>l 11s)(7). The transient absorption monitored with the

Wovelength

FIG. 2. Optical absorption spectra of ebselen 1, in 1,2-dichloroethane. (a) Transient absorption spectrum obtained on pulse radiolysis of 0.1 mM ebselen in nitrogen-saturated dichloroethane, 50 IJS after the pulse; (b) control spectrum of the unoxidized ebselen in the same solvent; (c) spectrum of transient species obtained taking into account that an equivalent yield of the unoxidized donor is destroyed, and taking a reasonable average value of G = 1 for the generation of the oxidized species.

20

SCHoNEICH

UA

260

,

r

3ul Wavelength

L20 (nm)

FIG. 3. Transient spectra obtained after pulse radiolysis ofO.l mM 2-phenyl-1,2-benzisothiazol-3(2H)one (2) (after 50 us) (0) and 0.1 mM 2-(methylseleno)benzoic acid-Wphenylamide, (5) (after 80 ps) (0) in nitrogen-saturated dichloroethane after incorporation of necessary corrections for destruction of donors; compare Fig. 2, curve c, for 1.

ebselen containing solutions is therefore attributed to the ebselen radical cation, generated in the one-electron oxidation as: (DCE)?

+ (-Se-)

+ DCE + (-Se-):

141

This conclusion is based on analogous observations for the oxidation of organic selenides and sulfides in other solvents, particularly aqueous systems (Asmus, K. D. et al., unpublished results). The optical absorption shown in Fig. 2, curve a, is composed of the spectrum of the ebselen radical cation and the bleaching of the ground state absorption of the unoxidized ebselen. The exact determination of the radiation chemical yield G for the underlying reaction, [4], is not possible since the extinction coefficient of the ebselen radical cation is not known. Taking a reasonable average value of G = 1 for Reaction [4], and making corrections for the equivalent destruction of the unoxidized donor on this basis, spectrum c was obtained. This probably represents a more accurate spectrum of the ebselen radical cation. Pulse radiolysis of ebselen analogues in dichloroethane. The above semiquantitative approach was used for the evaluation of the transient absorption spectra of the various (-Se-): and (-S-j? species studied. The series of compounds studied included 2-phenyl-1,2benzisothiazol-3(2H)one (2, PZ 25), 2-(benzylseleno) benzoic acid-N-(2-pyridyl)-amide, (3), 2-(benzylseleno)benzoic acid-N-phenylamide (4), 2-(methylseleno)benzoic acid-N-phenylamide (5) and 2-(3-pyridyl) 1,2-benzisoselenazol-3(2H)one (7). Two examples concerning the oxidation of 2 and 5 are shown in Fig. 3. The spectrum of the transient species (2’) (corrected for

ET

AL.

destruction of unoxidized PZ 25) differs significantly from that of the ebselen radical cation, shown in Fig. 2, curve c. The selenoanisole derivative 5 generates a species 5: with at least two additional peaks besides the strong uv absorption. Similar observations have been made for the oxidation of the sulfur analogue, thioanisole, in DCE and in aqueous solutions (Asmus, K. D. et al., unpublished data). Generally, the transient absorption spectra of the seleno radical cations resemble many characteristic features of the corresponding sulfur compounds (12). Ring substitution also has strong effects. For example, substitution of the N-phenyl group (as in 4) by a N-pyridyl group yielding 3, leads to a considerable red-shift of the absorption; 4 and 3 exhibit maxima at 360 and 425 nm, respectively. Oxidation reactions in irradiated DCE generally occur via one-electron transfer. This has been established with many organic substrates like aniline (8) and halogenated biphenyls (7). The experiments in DCE have verified the oxidizability of the selenium and sulfur derivatives and have provided qualitative information on their corresponding radical cations, which in this solvent are the unambiguous one-electron-oxidized transients. Owing to the uncertainties in the yields of the transients such experiments are, however, less informative from the quantitative point of view. This information is better obtained from investigations in more polar solvents, particularly aqueous systems or water/alcohol mixtures (see below). Oxidation of ebselen in waterltert-butanol mixtures. Pulse radiolysis of air-saturated mixtures containing 35% water and 65% tert-butanol (v/v) (pH 5.5) and 0.05 M carbon tetrachloride leads to the radicals generated in the following well-documented reactions, (R’ is mainly the ‘CH,C(CH:J, OH radical): Hz0 + e& + OH’ + H’ + H,‘, t-butanol

+ e$ + R’, H

OH’ + t-butanol Ccl,

+ R’ + Hz0

+ e& + Cl- + Ccl;

O2 + ep + 0; R’+O,+ROO ccl;

+ 0, + cc1300

[51

[61 [71

if31

[91 WI WI

The primary oxidizing species generated on irradiation of aqueous solutions is the ‘OH radical. Although it has a very positive redox potential it does not usually act as a one-electron oxidant but is engaged in H-atom abstraction and addition reactions, the latter due to its electrophilic character. Organic sulfur and selenium compounds are particularly prone to such addition reactions since they provide free electron pairs at the hetero

PULSE

RADIOLYSIS

atom. In a subsequent step these adducts may then be converted into radical cations (13). This mechanistic complication is avoided by employing other radical oxidants such as halogenated peroxyl radicals; they are useful since they act as one-electron transfer agents (1417). A convenient species of this kind is the trichloromethylperoxyl radical (CCl,Oo’), which is generated as shown in the above reactions. Three other species were also employed for comparison, using the peroxyl radicals derived from freon 113, hexachloroethane, and pentachloroethane. Reactions [lo] and [ll] serve here as an example for the production of peroxyl radicals from the employed halogenated compounds. None of the above reactions gives rise to significant transient optical absorptions; only a weak, uncharacteristic uv absorption is seen. The trichloromethylperoxyl radical produced in a diffusion-controlled reaction between ‘CC& and 02, h = 3.3 X 10’ Mm1 s1 (14), is the actual oxidizing species involved. Oxidation of 0.1 mM ebselen by trichloromethylperoxyl radicals gives rise to transient species, the absorption spectrum of which is shown in Fig. 4A (corrected for the disappearance of ebselen’s control absorption as described above). Some absorption bands at wavelengths higher than 330 nm are also observed, which are only slightly apparent in the spectrum in DCE (Fig. 2), probably due to low yields in the DCE system. However, the ebselen-derived transient intermediates show similar characteristics in both solvents and are thus attributed to the same type of species. Further, the kinetic characteristics are the same at all wavelengths, indicating that the entire transient spectrum is indeed due to the same species. It is, therefore, concluded that ebselen is oxidizable in waterltert-butanol according to Reaction [la]: CCl,OO’

+ ebselen -+ CCl,OO-

+ (ebselen):

[12]

The absorption-time trace at 287 nm shows an absorption being present directly after the pulse followed by a slower buildup (Fig. 4B). The absorption observed directly after the pulse (G X t = 2500 Mm’ cm-‘) is not dependent on ebselen concentration, decreases toward higher wavelengths, and is observed also in the absence of ebselen. The rate of the slow consecutive increase is first-order and is proportional to the concentration of ebselen ranging from 0.02 to 0.12 mM. On the basis of this linear relationship, the bimolecular rate constant for the oxidation of ebselen by the trichloromethylperoxyl radical as shown in Reaction [la] was derived, h,, = 2.9 f 0.5 X 10’ M-l s ‘. The possibility that the transient obtained is a twoelectron oxidation product is eliminated as the control absorption spectrum of 2-phenyl-l&benzisoselenazol3(2H)one-selenoxide, 9, is characterized by only one

OF

21

EBSELEN

A

10,000 h

i t ;

5.000;; ”

01- 300

380

160

Wavelength

8,OCC-

‘;

Inml

’

5

c8

‘i L.OOO-I: w 0

: 0

r 0

I LO

1 80

I 120

I 160

Time lpsecl

a OQM!? 4 I

0

I

I

1

1

LO

80

120

160

Time (pseci

FIG. 4. Oxidation of (A) Transient spectrum selen in water/k&butanol ride, air-saturated. (B) sient species at 287 nm.

ebselen by trichloromethylperoxyl radicals: obtained after pulse radiolysis of 0.1 mM eb(35:65 v/v) with 0.05 M carbon tetrachloAbsorption time-trace of formation of tran(C) Buildup of conductivity signal.

maximum at 275 nm in the tert-butanollwater system (not shown); the spectrum of the species produced upon pulse radiolysis of ebselen is not identical with that of the selenoxide. Electrochemically, it has been demonstrated that the oxidation of sulfides to sulfoxides proceeds in tandem, via two one-electron steps (18). Conductivity measurements carried out simultaneously with optical measurements in the waterltert-butanol system show the production of a conducting species (Fig. 4C). The initial rapid rise in conductivity is also present in the blank and is attributed to the H+/Cll ion pair formed in Reactions [5]-[ 111. The second slower rise is attributed to the (ebselen)+‘/CCl:,OO ion pair from Reaction [ 121, and the kinetics of this buildup are similar to those for the corresponding step in the absorp-

22

SCHoNEICH

Pentachloroethane

ot

v 0

I

005 ConcentratlonlmMl

FIG. 5. Oxidation of ebselen by different ditions see Experimental section.

01

015

peroxyl

radicals.

For con-

ET

AL.

F,OO’/CF&lCClF00’) derived from freon 113, (Ccl&C1200’) derived from hexachloroethane, and (CHCl&C1200’) derived from pentachloroethane. Qualitatively all these radicals yielded similar transient spectra. Kinetic analysis of the formation of the radical cation (at 287 nm) showed a linear relationship between kobsand the concentration of ebselen (Fig. 5). The bimolecular rate constants for oxidation of ebselen by different peroxyl radicals as derived from the respective slopes are shown in Table I. The rate constants increase with increasing degree of halogenation of the peroxyl radicals and also on substitution of fluorine for chlorine. Comparable results have been reported for halogenated peroxyl radical-induced oxidation of various other antioxidants as well (16). In particular, the degree of halogenation at the a-carbon atom has a pronounced effect on the rate of ebselen oxidation. Peroxyl radicals from chloroform (CHCl,OU), enflurane (HCF2-0-CF&HFOO’), and methoxyflurane (CH3-O-CF2CHC100’) did not show any significant reactivity at the concentration of ebselen employed, the

TABLE

tion signal (Fig. 4B). Evaluation of the kinetic data results in a rate constant k = 3.0 X 10’ M-r Sol, similar to that calculated from the absorption data. While the result of the conductivity experiments unambiguously demonstrates the formation of charged species, it does not provide equally conclusive information on the exact identity of the conducting species (6). In particular, the long lifetime of the conductivity signal does not mean that the hydroperoxide anion, CC1300m, is a long-lived species; in terms of conductivity this anion could not be distinguished from a chloride ion, Cll, possibly liberated therefrom. The stability of the conductivity signal would, in fact, suggest such a subsequent process to occur since a persistent hydroperoxide anion would probably be protonated, associated with a corresponding decay of the conductivity signal. Quantitative evaluation of the conductivity data must take into consideration the lower equivalent conductivity of water/alcohol mixtures compared to that of pure aqueous solutions (19, 20). Taking the conductivity change observed in the slow step, G X AA = 300fi21 cm2, and assuming a value of ca. 5OV’ cm2 each for the specific conductivities of the radical cation and the anion formed in Reaction [ 121, respectively, a yield of G = 3 was estimated, which is marginally higher than the yield of trichloromethyl radicals (G = 2.4) initially available for oxidation in our system. The conductivity results clearly prove the formation of ions, and this together with the optical and kinetic data leaves no ambiguity for the formation of radical cations. These considerations also seem to apply to oxidation of ebselen by other peroxyl radicals, such as (CCl,FC-

I

Rate Constants for Oxidation by CC1300’ Radicals, Oxidation Potential by Cyclic Voltammetry and Lag Doubling Concentrations during Microsomal Lipid Peroxidation of Ebselen and Structurally Related Derivatives

k Compound

(10’

M-’

1

2.9

2 3

(4.0)” (2.7)” (1.0)’ 0.1 0.9

4 5 6

1.2 3.9 n.d.

7

n.d.

8

n.d.

Oxidation SK’)

potential W)

1.59 n.d. n. d . n. d . 1.69 1.67 (1.48, 1.85) 1.53 1.46 1.46

Lag doubling concentrationd (NM)

0.13

2.2 23 46 150 0.7

(1.6) 1.43 (1.83) 1.68

0.5 55

Note. Numbers of compounds correspond to chemical names in Fig. 1. Conditions of pulse irradiation as described in the text. Cyclic voltammetry experiments were carried out in dry acetonitrile as described under Methods. Oxidation potentials in parenthesis indicate shoulders in the voltammograms. nd., not determined. Rate constant, k, calculated for oxidation of the different derivatives by trichloromethylperoxyl radicals (except a CCl,FCF,OO’/CF,ClCClFOO’ from freon 113, b CCl&Cl,Oo from hexachloroethane and’ CHC1&C1200’ from pentachloroethane) at 280 nm for 1 and 4, 290 nm for 3, and 425 nm for 5 by optical and conductivity measurements. d From Ref. (51 on microsomal lipid peroxidation experiments. Values are representative of 3-4 independent experiments. Conditions of incubation and other d e t ai‘1s o f c h emiluminescence measurements as described in Ref. (5).

PULSE

rate constants being smaller lower limits of measurability conditions. Oxidation terltert-butanol

of other

seleno mixtures.

RADIOLYSIS

OF

23

ERSELEN

than 5 X 10’ M-’ s-l, the under the experimental

and sulfur compounds in wa-

The sulfur analogue of ebselen, 2, in waterltert-butanol, showed neither a transient absorption nor a conductivity signal upon pulse radiolysis. This demonstrates that the oxidation of 2 by trichloromethylperoxyl radicals is very slow, k < lo7 M-’ sl. This result suggests that the point of oxidative attack is the selenium atom and not the remaining aromatic residue. That the selenium compound is more easily oxidized than the sulfur analogue may be substantiated on the basis of ionization potentials and stereoelectronic configuration. It is known, for example, that in benzoyl selenol esters the overlap of the selenium lone electron pair with orbitals of the carbonyl and aromatic groups is weaker than in the sulfur analogues (21). Assuming that this equally applies to the interaction between Se and the aromatic residues, the selenium lone pairs in ebselen would be more prone to oxidation than the sulfur lone pairs in 2. Similar considerations should also apply for the oxidation of the other compounds investigated. All the open chain selenoether derivatives, for example, compounds 3, 4, and 5 are readily oxidized by trichloromethylperoxyl radicals to their respective radical cations. The spectra of 3t, 4:, and 51 are shown in Figs. 6A and 6B. Qualitatively, similar spectral characteristics were found for the radical cations 3: and 4t, derived from the two benzyl-substituted selenides. At longer wavelengths (>300 nm) their absorptions are considerably weaker compared to 5f, which has distinct absorption maxima at 425 and 360 nm. The radical cation 3f thus exhibits only a minor peak around 350 nm, while 4: shows a shoulder at this wavelength besides two small peaks at 425 and 475 nm. The long wavelength peak around 475 nm has been attributed to a dimer radical cation generated by association of the molecular radical cations and a second unoxidized molecule. Similar three electron-bonded species are well known for organic selenides and, in particular, sulfides (22,23). The radical cation 5 f has distinct absorption maxima at 425 and 360 nm besides the strong band below 300 nm. The rate constant for the buildup of the absorption for 5f at 425 nm was calculated to be k = 3.9 X 10RM ~’ s-’ Table I. The bimolecular rate constants measured for the buildup at 290 and 360 nm are 3.3 and 2.8 X 10’ M ’ s l, respectively. Generally, the transient absorption spectra of the compounds measured in the DCE and waterltert-butano1 systems are similar enough to be assigned to the same kind of species. However, compound 7, the pyridyl derivative of ebselen, was an exception. In DCE 0.05 mM

01

J260

310

L20

500

FIG. 6. Spectra obtained after pulse radiolysis of: (A) 0.2 mM 2.(3 pyridyl))1,2-benzisoselenazol-3(2H)one, (7) (after 180 as) (A); 0.2 mM 2-(methylseleno)benzoic acid-A-phenylamide, (5) (after 60 ps) (0); (B) 0.18 mM 2-(benzylseleno)benzoic acid-N-(2.pyridyl)amide, (3) (after 130 as) (0); and 0.18 mM 2-(benzylseleno)benzoic acid-iV-phenylamide, (4) (after 80 FLS) (H), in water/&-t-butanol with 0.05 mM carbon tetrachloride, air-saturated, after appropriate corrections.

7 yields a spectrum with a maximum at 360 nm (G X c = 1.7 X lo4 M-I cm-‘) while in waterltert-butanol, there is no peak at this wavelength and the absorption amounts to only 1000 Mm1 cm-’ at four times the concentration (Fig. 6A). The entire spectrum in waterltert-butanol exhibits a more or less steady rise toward the uv and resembles to a large extent that of 3:. This behavior is probably connected with the possible protonation of the pyridyl nitrogen in the more protic solvent. It is also noted that the electrochemical properties of 7 seem to be special as will be discussed in the next section. The bimolecular rate constants for the formation of the radical cations, analogous to the oxidation Reaction [ 121 have been evaluated from optical and conductivity measurements and are listed in Table I. Cyclic uoltammetry. Cyclic voltammetry experiments were done in order to study the oxidation potentials of the different derivatives and thereby to relate to their respective rates of oxidation or their antioxidant capacities. All measurements were carried out in dry acetonitrile using 0.1 M TBAFG as the supporting electrolyte. The oxidation (peak) potentials obtained are listed in Table I. Considering the compounds ebselen, its sulfur analogue, and the Se-methyl derivative of ebselen, 5, it was seen that the measured potentials correlated fairly well with the trend of rate constants obtained for oxidation by trichloromethylperoxyl radicals; the benzyl derivatives, however, behaved differently. Both were oxidized by trichloromethylperoxyl radicals with similar rate constants but showed different oxidation potentials (f1.53 V for 4 and 11.67 V for 3). A shoulder at +1.85 V for 3 is probably due to the pyridyl substituent, since the other pyridyl derivative 7 also shows a shoulder at comparable potentials ($1.83 V). The latter compound shows a particularly interesting behavior. The +1.83-V peak potential on the forward scan is complemented by a +1.74-V peak on the reverse scan, indicating a reversible

24

SCHoNEICH

redox process, a feature which has not been observed either for the other derivatives or for any of a large number of organic sulfur compounds (23). The difference in peak potentials between the forward and the reverse scans amounts to 90 mV for 7, and does not correspond to the expected value for a reversible unhindered two-electron (118 mV) process or a one-electron step (59 mV). All the other derivatives exhibited only irreversible oxidation waves and possibly two-electron processes as is known for many organic sulfides (18, 23). This excludes an exact correlation between the rate constants for oxidation and the one-electron redox potentials. Comparison to antioxidant capacity observed in microThe antioxidant activities of somal lipid peroxidation. the different derivatives of ebselen were recently evaluated during nonenzymatically induced microsomal lipid peroxidation where chemiluminescence and measurement of thiobarbituric acid-reactive substances were taken as indices of lipid peroxidation (5). The ability of the compound to prolong the lag phase before the onset of active peroxidation was taken as a measure of the antioxidant capacity (see Ref. (2)). The concentrations of the compounds required to double the lag time (lag doubling concentration) compared to those in controls with no added antioxidant are represented in the right-hand column in Table I. Ebselen had the highest antioxidant capacity as it provides protection at very low concentrations, the lag doubling concentration being 0.13 yM. In contrast, the sulfur analogue 2 was required at a 20.fold higher concentration, 2.2 PM (see Ref. (2)). The pyridyl analogue of ebselen, 7, had a potency comparable to that of ebselen, while the benzyl derivatives 3 and 4 afforded protection at 200- 400-fold higher concentration compared to ebselen. This concurs with the results of pulse radiolysis where 3 and 4 were oxidized at similar rates by the trichloromethylperoxyl radical, which, however, were lower compared to that of ebselen. Interestingly, the Se-methylated derivative 5 was practically inactive in preventing lipid peroxidation, in contrast to the pulse radiolytic experiments, where high rate constants were observed for the oxidation of 5 by trichloromethylperoxyl radicals. CONCLUSIONS

The antioxidant nature of selenium-containing drugs like ebselen (2,24) and some related derivatives (5) have been recognized. They have been shown to afford protection by prolonging the lag phase before the onset of lipid peroxidation in microsomal membranes. In the present study the antioxidant efficacies of these drugs were investigated by pulse radiolysis on the basis of their susceptibilities to oxidation by 1,2-dichloroethane radical cations and halogenated peroxyl radicals. Ebselen is oxidized by organic radicals such as the trichloromethylperoxyl radical, with high rate constants, 2.9 X 10’ M-l s-i,

ET

AL. TABLE

II

Comparisonof Rate Constants for the Reaction of Antioxidants with Trichloromethylperoxyl Radicals k Antioxidant

Ref.

(~@M-‘s-~)

15 5.0 2.9 2.0”, 1.1 b 1.6 1.2 0.2

p-carotene n-Tocopherol Ebselen Ascorbate Trolox Urate Methionine

(27)

(25) this paper

(=A (28) (28) (28) (29)

Note. Oxidation of the different antioxidants by trichloromethylperoxyl radicals generated by pulse radiolysis. Details of experimental conditions as described in text for ebselen and in Refs. (25-27) and (29) for the others. Trolox indicates the water soluble form of cu-tocopherol. ’ From Ref. (25). ’ From Ref. (28).

comparable to those exhibited by other well-established antioxidants such as a-tocopherol and ascorbate, (25) (Table II). On the other hand, the sulfur analogue, 2, was oxidized at much lower rates, k < lo7 M-’ s-r, by these organic radicals, indicating the superiority of ebselen as an antioxidant over the latter. However, ebselen and its derivatives may be expected to react with nonhalogenated peroxyl radicals with lower absolute rate constants as compared to the halogenated peroxyl radicals. The transients generated upon oxidation of ebselen and the different derivatives were characterized by optical absorption and conductivity measurements. The results indicate the generation of radical cations which are one-electron-oxidized species, upon reaction of ebselen with halogenated peroxyl radicals. The measured rate constants generally correlate well with the electrochemitally determined oxidation potentials, and with the evaluation of antioxidant efficacy in membrane peroxidation studies (Table I). These results highlight the free radical scavenging efficiency of ebselen and some of its analogues, and when juxtaposed with its glutathione peroxidase (2, 3) and antiinflammatory (4) activity, ebselen appears to be an attractive candidate of pharmacological interest during disturbances in the prooxidant/antioxidant ratios called oxidative stress (26). ACKNOWLEDGMENTS We thank A. Aced for the cyclic voltammetry experiments. V.N. is a Stipendiatin of the Alexander van Humboldt-Stiftung. We thank Dr. Graf, Nattermann/Rhone-Poulenc, for the generous gift of the selenium and sulfur compounds.

REFERENCES 1. Wendel, A. Springer-Verlag,

(Ed.)

(1989) Berlin.

Selenium

in

Biology

and

Medicine,

PULSE 2. Miiller, A., Cadenas, E., Graf, Pharmacol. 33,3235-3239. 3. Wendel, Riochem.

A., Fausel, Pharmacol.

4. Parnham, 324773250.

P., and

Sies,

M., Safayhi, H., Tiegs, 33,3241-3245.

M. J., and Kindt,

S. (1984)

RADIOLYSIS

H. (1984)

G., and Otter,

Rio&em.

R. (1984) 33,

5. Narayanaswami, V., and Sies, H. (1990) Free Radical Res. Commun., in press. 6. Asmus, K.-D. (1984) in Methods in Enzymology (Packer, L., Ed.) Vol. 105, pp 1677178, Academic Press, New York. 7. Miinig, ,J., Asmus, K.-D., Robertson, L. W., and Oesch, J. C’hcm. Sot. Perkins Trans. II, 891-896. 8. Sumiyoshi, (1986) Bull. 9. Wang, 83(15),

T., Sugita, N., Watanabe, K., Chem. SW. Jpn. 6 1, 3055-3059.

Y., Tria, J. J., and Dorfmann, 1946-1951.

and

L. M. (1979)

F. (1986)

Katayama, J. Phys.

M. Chem.

10. Anklam, E., Asmus, K.-D., and Robertson, SW. l’erkin Trans. II, 156991572.

I,. W. (1989)

J. Chem.

11. Anklam, E., Asmus, K.-D., and Robertson, Sot. Perkin Trans. II, 1573-1576.

L. W. (1989)

J. Chem.

12. Fujihara,

H.. Schiineich,

13. Asmus,

K.-D.

(1979)

14. Miinig, Intrract.

J., Bahnemann, 47, 1527.

C., and Asmus, Act.

Chum.

K.-D.,

to he submitted.

Res. 12, 436-442.

D., and Asmus,

K.-D.

(1983)

Chem.

Riol.

15. Packer, J. E., Willson, R. L., Bahnemann, D., and Asmus, (1980) J. Chem. Sot. Perkin Trans. II, 296-299.

K.-D.

16. Lal, M., Schb;neich, C., Miinig, Rndiat. Biol. 54, 773-785.

Int.

,J., and Asmus,

K.-D.

(1988)

J.

25

EBSELEN

17. Neta, P., Huie, Maruthamuthu, 4099-4104.

Biochem.

Pharmacol.

OF

R. E., Mosseri, S., Shastri, P., and Steenken, S. (1989)

L. V., Mittal, J. P., J. Phys. Chem. 93,

18. Glass, R. S., Hojjatie, M., Petsom, A., Wilson, G. S., G6h1, M., Mahling, S., and Asmus, K.-D. (1985) Phosphorous Sulfur 23, 143-168. 19. Asmus, K.-D., Chaudhri, S. A., Nazhat, N. B., and Schmidt, W. F., (1971) Trans. Faraday Sot. 67,2607?2612. 20. Chaudhri, S. A. and Asmus, K.-D. (1972) J. C’hem. Sot. Faraday Trans. I, 68,385-392. 21. Torchinsky, Y. M. (1981) in Sulfur in Proteins, P. 10, Pergamon Press. 22. Nishikada, K., and Williams, Ff. (1975) Chem. Phys. L&t. 34, 302-306. 23. Coleman, B. R., Glass, R. S., Setzer, W. N., Prahhu, U. D. G., and Wilson, G. S. (1982) Adu. Chem. Ser. 201,417-441. 24. Hayashi, M., and Slater, T. F. (1986) Free Rad. Res. Commun. 2, 179-185. 25. Willson, R. L. (1985) in Oxidative Stress, (Sies, H., Ed.), pp. 4172, Academic Press, London. 26. Sies, H. (1985) in Oxidative Stress, (Sies, H., Ed.), pp. l-8, Academic Press, London. 27. Packer, J. E., Mahood, J. S., Mora-Arellano, V. O., Slater, T. F., Willson, R. L., and Wolfenden, B. S. (1981) B&hem. Biophys. Res. Commun. 98,901-906. 28. Neta, P., Huie, R. E., Maruthamuthu, P., and Steenken, S. (1989) J. Phys. Chem. 93,7654-7659. 29. M&rig, J., G6h1, M., and Asmus, K.-D. (1985) J. Chem. Sot. Perkin Trans. II, 647-651.