Chem. Res. Toxicol. 1990, 3, 199-203
199
Microsomal Metabolism of 2 4 Methylseleno)benzanilide Nancy J. John,? Rolf Terlinden,* Hartmut Fischer,* Michel Evers,t and Helmut Sies*pt Institut fur Physiologische Chemie I , Universitat Dusseldorf, Moorenstrasse 5, 0-4000 Dusseldorf, FRG, and Rhone-PoulenclNattermann,Cologne Research Center, Nattermannallee 1, 0-5000 Cologne 30, FRG Received September 12, 1989
2-(Methylseleno)benzanilide,a metabolite of ebselen, was oxidatively demethylated by rat liver microsomes to regenerate the parent compound, ebselen. The rate of this novel reaction exceeds that of ring hydroxylation, and it was inhibitable with metyrapone. The sulfur analogue, 2-(methylthio)benzanilide, was oxidized to the corresponding sulfoxide. T h e regeneration of ebselen from 2-(methylse1eno)benzanilide is suggested to result from an intermediate selenoxide and hydrolysis with formation of methanol, since only very small amounts of labeled formaldehyde were detected in incubations using 2- [(14C)methylseleno]benzanilide.In an analogous reaction, 2-(benzylse1eno)benzaldehydeyielded ebselen upon oxidation with hydrogen peroxide and, as detected by gas-liquid chromatography, benzyl alcohol.
Introduction Ebselen [ 2-phenyl-1,2-benzisoselenazol-3(2H)-one, PZ 511, a biologically active selenoorganic compound, exhibits glutathione peroxidase (GSH-Px)like activity by catalyzing the degradation of H,Oz and organic hydroperoxides (1-3). In the presence of GSH as a substrate, ebselen has been shown to inhibit ADP-Fe-induced lipid peroxidation in isolated hepatocytes ( 4 ) , and in vivo antiinflammatory activity has been demonstrated (see ref 5). Recent biotransformation studies showed ebselen to be metabolized by rat liver cells into a number of Se compounds, all of which shared the common characteristic that the selenazole ring was opened by cleavage of the Se-N bond. We found (6,7)that after ring opening the selenium moiety was either methylated to form 2-(methylse1eno)benzanilide or glucuronidated to form a novel Se glucuronide, 2- (glucuronylseleno)benzanilide.Para hydroxylation of 2-(methylseleno)benzanilideat the phenyl ring resulted in formation of N-(4-hydroxyphenyl)-2-(methylse1eno)benzamide. This reaction was suggested to be catalyzed by cytochrome P-450. In order to further study the cytochrome P-450 catalyzed metabolism of ebselen, we have incubated 2-(methylse1eno)benzanilide with rat liver microsomes and analyzed the resultant metabolic products. Surprisingly, our in vitro results show cytochrome P-450 to catalyze significant oxidative demethylation of this compound, resulting in the regeneration of ebselen. Under the conditions employed, much less para hydroxylation occurs in vitro. Materials and Methods Preparation and Incubation of Microsomes. Phenobarbital-induced and uninduced (control) liver microsomes were prepared from male Wistar rats (180-220 g body weight) as described (8). Microsomes (1 mg/mL) were incubated in 25-mL conical flasks containing an incubation medium (5 mL total volume) at 37 "C. Final concentrations were as follows: sucrose, 125 mM; 2-(methylseleno)benzanilide,dissolved in DMSO, 100 pM; isocitrate, 8.1 mM; MgClz and nicotinamide, 50 mM; and isocitrate dehydrogenase, 0.4 units/mL (in 25 mM Tris-50 mM *Address correspondence to this author. Universitat Diisseldorf. t Cologne Research Center.
0893-228x/90/2703-0199$02.50/0
KCl buffer, pH 7.4). The reaction was started upon addition of NADP' (0.5 mM). At the indicated times, the incubation mix was collected (500 pL) and reactions were stopped with the addition of 6% metaphosphoric acid (100 pL). The entire sample was used for subsequent extraction and HPLC analysis. Extraction and HPLC Analysis. The organoselenium compounds were extracted three times each with ethyl acetate and dichloromethane, followed by evaporation of the solvents. The residue was dissolved in DMSO (0.5 mL) and after centrifugation stored for HPLC analysis. Recovery was 90% with this extraction procedure. Samples of 50 pL were injected to the reverse-phase column (RP18, 100 X 8 mm, LiChrosorb 10 pm; Waters Associates). Separation was performed by using a 0.1% H,PO,/ acetonitrile gradient, at a flow rate of 1.5 mL/min. The gradient was from 70/30 to 3/97 in four 10- and 5-min intervals (6, 7). The metabolites were measured a t 254 nm. (14C)Formaldehyde Production from 2-[ (W)Methylselenolbenzanilide. 2- [ ('*C)Methylseleno] benzanilide, 9.7 mCi/mmol (Nattermann, Cologne), was diluted with unlabeled 2-(methylse1eno)benzanilide and used as a substrate a t a final concentration of 100 pM with a specific activity of 0.06 mCi/mmol. Phenobarbital-induced microsomes (1mg/mL) were incubated in 100 x 16 mm (15-mL) test tubes containing an incubation medium (400 or 500 p L total volume) a t 37 OC a t a shaking frequency of 120 min-'. Throughout the incubation, the test tubes were capped with rubber stoppers containing center wells (Kontes, Vineland, NJ) with 200 pL of phenylethylamine on folded filter papers. In addition to identical final concentrations of the compounds in the microsomal incubation medium described above, the incubation medium contained the following: formaldehyde dehydrogenase, 0.5 units/mL; formate dehydrogenase, 0.2 units/mL; and NAD+, 5 mM (in 25 mM Hepes, 25 mM Tris-50 mM KCI, pH 7.4). To inhibit cytochrome P-450, 0.2 mM metyrapone was added to the incubation mix. (%)Formaldehyde, 15.0 mCi/mmol, was used as a substrate a t a final concentration of 0.75 pM for control experiments. (dimethylamino-14C)Aminopyrinewas diluted with unlabeled aminopyrine and used as a substrate at a final concentration of 1 mM with a specific activity of 0.67 mCi/mmol. Reactions were started upon addition of NADP' (0.5 mM). At the indicated times, reactions were stopped by the injection of either 10% trichloroacetic acid (400 pL for 400 pL incubation volume) or 6% metaphosphoric acid (100pL for 500 p L incubation volume). COPwas allowed to diffuse for 20 h with gentle shaking a t 40 "C. The 14C02evolved was trapped in center wells with phenylethylamine. The center wells were added to 10 mL of Quickzint 212 scintillation fluid, and the 14C was counted in a
0 1990 American Chemical Society
John et al.
200 Chem. Res. Toxicol.,Vol. 3, No. 3, 1990 Beckman scintillation counter (Model LS 1801) with a counting efficiency of 94%. Data were corrected for efficiency of (14C)formaldehyde conversion to 14C02(34%). Samples were used for subsequent extraction and HPLC analysis as described above. Chemicals a n d Biochemicals. Chemicals and biochemicals were obtained from Merck (Darmstadt, FRG) and Boehringer (Mannheim, FRG). Formaldehyde dehydrogenase from Pseudomonas putida (EC 1.2.1.46) and formate dehydrogenase from Pseudomonas oxalaticus (EC 1.2.1.2) were obtained from Sigma (Munich, FRG). 2-(Methylse1eno)benzanilideand analogues were from Nattermann (Cologne, FRG); the purity of the starting materials was checked and confirmed by HPLC and mass spectrometry (6, 7). (14C)Formaldehyde (specific activity 15.0 mCi/mmol) and (dimethylamino-14C)aminopyrine(specific activity 116.0 mCi/mmol) were obtained from Amersham (Braunschweig, FRG). S y n t h e s i s of 2-(Methylseleninyl)benzanilide. A mixture of 2-(methylse1eno)benzanilide(9) (6.9 mmol) and aqueous hydrogen peroxide (20 mL of 30% solution) was stirred for 19 h a t room temperature. The solid material present in the reaction mixture was filtered and washed with water (2 X 20 mL) and dried. The solid was washed in hexane (2 X 20 mL) and dichloromethane (30 mL) and then dried in vacuo to give 2-(methylseleniny1)benzanilide in 100% yield: mp 127 "C. IR (KBr) v = 1646 (CONH), 786 or 800 cm-' (Se=O); 'H NMR (DMSO-d,/TMS) 6 2.62 (s, 3 H, SeCH,), 7.1-8.4 (m, 9 H, H-aromatic); m/e 307 (%e) 14%), 275 (80 Se) (M" - CH,OH, (M', 7 % ) , 291 (%e) (M - o", 63%), 195 (M - CH30H - Se, 100%). Synthesis of 2-(Benzylse1eno)benzoic Acid. Solid sodium hydroxide (0.95 mol) was added to a stirred suspension of diselenosalicylic acid (0.125 mol) in 380 mL of water (IO);the temperature reached 45 "C. Sodium carbonate (0.94 mol) and sodium dithionite (0.33 mol) were added to the yellow solution. The solution was then refluxed for 2 h and then allowed to cool to room temperature. Benzyl bromide (0.4 mol) was added dropwise within 10 min. The mixture became difficult to stir, and 100 mL of water was added. Stirring was continued over 14 h at rmm temperature. Aqueous hydrochloric acid (355 mL, 32%) was added dropwise with stirring. The precipitated crude 2(benzylse1eno)benzoic acid was filtered, washed with water, and recrystallized from 2-propanol (1600 mL) to give pure 2-(benzylse1eno)benzoicacid with 67% yield: mp 209-210 "C; IR (KBr) u = 1668 cm-' (C=O); 'H NMR (DMSO-$/TMS) S 4.16 (s, 2 H, SeCH,), 7.18-7.68 (m, 8 H, H-aromatic), 7.96 (d, 1 H, H3); m / e 292 (BOSe) (M', 12%), 201 (soSe) (M - CH2C6H5, 8 % ) , 91 (CHzC6H5,100%). Anal. Calcd for C14H1202Se( M , 291.2): C, 57.74; H, 4.15; 0, 10.99;Se, 27.11. Found: C, 57.8; H, 4.4; 0, 10.5; Se, 28. S y n t h e s i s of 2-(Benzylseleno)benzanilide.A suspension of 2-(benzylseleno)benzoic acid (20.6 mmol) in dichloromethane was cooled to -10 "C. Dimethylchloroformiminium chloride (Vilsmeier reagent) (20.6 mmol) was added in one portion under vigorous stirring. After an additional stirring over 30 min at 0 "C, the clear solution was allowed to warm to room temperature, and then a solution of aniline (40 mmol) and triethylamine (41.6 mmol) in dichloromethane (60 mL) was added dropwise within 15 min. The reaction mixture was left 48 h at ambient temperature and then successively washed with 1 N aqueous hydrochloric acid (100 mL), distilled water (100 mL), 1 N aqueous sodium hydroxide (100 mL), and distilled water (100 mL). The organic layer was dried over sodium sulfate. The solvent was evaporated under reduced pressure. The crude solid obtained was recrystallized from 2-propanol (200 mL) to give 2-(benzylse1eno)benzanilide in 72.9% yield: mp 132-133 "C; IR (KBr) v = 1641 cm-' (CONH); 'H NMR (DMSO-$/TMS) 6 4.19 (s, 2 H, SeCH,), 7.05-7.50 (m, 10 H , H-aromatic), 7.60-7.70 (m, 4 H, H-aromatic), 10.35 (s, 1 H, CONH); m / e 367 (80Se)(M+, 17%), 276 (@%e) (M - CH2C6H5+,loo%),91 (CH2C6H6+,48%). Anal. Calcd for C2,Hl,NOSe ( M , 366.2): C, 65.58; H, 4.68; N, 3.82; 0, 4.37; Se, 21.55. Found: C, 65.8; H , 4.5; N, 3.8; 0, 4.6; Se, 21.6.
Results Microsomal Metabolism of 2-(Methylse1eno)benzanilide. Incubation of 2-(methylse1eno)benzanilidew i t h u n t r e a t e d r a t liver microsomes resulted i n oxidative de-
A
I T
I
-5:
3
-e C
0
L
C
300
0 0
10 20 Incubation Timelmin)
30
B
I-=+
0 0.
L'-Hydroxy-2-Methylselenobnzmilid~
10
20
30
Incubation Time h"
F i g u r e 1. Time course of microsomal metabolism of 2-(methylse1eno)benzanilide and product formation of ebselen and 4'-hydroxy-2-(methylseleno) benzanilide. (A) Liver microsomes from control rats; (B) liver microsomes from phenobarbitalpretreated rats. The curves represent three or four independent experiments (means & SEM). m e t h y l a t i o n to produce significant amounts of ebselen, while significantly smaller a m o u n t s of 4'-hydroxy-2-(methylse1eno)benzanilide were p r o d u c e d (Figure 1A). The production of ebselen [ 1.0 nmol/ (mimmg of protein)] rose proportionally to the metabolism of 2-(methylseleno)benzanilide [1.1 nmol/(min.mg of protein)] over the 30-min t i m e course (Figure 1A). M e t a b o l i s m of 2-(methylse1eno)benzanilide b y liver microsomes f r o m rats pretreated w i t h p h e n o b a r b i t a l res u l t e d i n a %fold increase in the metabolic conversion of s u b s t r a t e to ebselen over the linear portion of the curve (from 2 1 to 44 pM ebselen produced in 15 m i n ) (Figure 1B). Over t h i s 15-min t i m e period, the production of ebselen [2.0 n m o l / (min-mg of protein)] r e m a i n e d propor-
Microsomal Metabolism of 2-(Methylseleno)benzanilide Table I. Production of 14COzfrom 2-[(14C)Methylseleno]benzanilideand (14C)Formaldehydein Liver Microsomesa “COZ production, dpm 2 4 (14C)methylseleno]- (14C)formincubation conditions benzanilide aldehyde complete 929 4023 minus NADP+ 240 ndb minus formaldehyde 368 418 dehydrogenase plus metyrapone 349 6266
Chem. Res. Toxicol., Vol. 3, No. 3, 1990 201
1.5
Liver microsomes (1mg/mL) from phenobarbital-induced rats were incubated at 37 “C for 30 min in the incubation medium (400 or 500 pL total volume; Materials and Methods) with the indicated benzmodifications. The concentrations of 2 4 (14C)methylseleno] anilide (632000 dpm) and (“C)formaldehyde (11700 dpm) were 100 pM. Reactions were started upon the addition of NADP+ (0.5 mM) or buffer (25 mM Tris-50 mM KCl, pH 7.4) and stopped by the injection of either 10% TCA or 6% metaphosphoric acid. Where indicated, metyrapone (0.2 mM) was present at time zero. Data are means from two or three independent experiments. nd = not determined. Timelminl
tional to the metabolism of 2-(methylse1eno)benzanilide F i g u r e 2. Time course of the production of 14C02from 2[2.1 nmol/(min.mg of protein)]. Although the reaction [ (14C)methylseleno]benzanilidein liver microsomal incubations rates decreased after 15 min (Figure lB), the net rates for from phenobarbital-pretreated rats. [14C ]Formaldehyde was ebselen production and 2-(methylse1eno)benzanilide meenzymatically converted to 14C02as indicated under Materials tabolism remained proportional over the 30-min incubation and Methods. Data are means f SEM (n = 6). period. The production of 4’-hydroxy-2-(methylseleno)was increased to 1 mM, the rate of aminopyrine metabobenzanilide remained low. lism was 2.11 nmol/(min.mg of protein). Isolated hepaThe metabolism of 2-(methylse1eno)benzanilide and tocytes were found to metabolize 1 mM aminopyrine a t product formation of ebselen for both control and phea rate of about 0.36 pmol/(min.g wet weight) (12);with 1 nobarbital-pretreated liver microsomes were dependent on g wet weight equal to lo* cells and 2 mg of protein/106 the presence of NADP+; in the absence of NADP+, there cells, this rate equals approximately 1.8 nmol/(min.mg of was no loss of 2-(methylse1eno)benzanilide. Addition of protein). metyrapone (0.2 mM), an inhibitor of cytochrome P-450 The bulk of one-carbon unit products, therefore, is dependent reactions, inhibited metabolism (not shown). concluded to be released as methanol. (14C)Formaldehyde Production from 2-[ (14C)The rate of 2- [ (14C)methylseleno]benzanilidemetaboMethylseleno]benzanilide by Rat Liver Microsomes. lism, as determined by HPLC analysis, was significantly The one-carbon unit formaldehyde is liberated by cytohigher than that detected by I4CO2recovery. Over a 30chrome P-450 catalyzed oxidative demethylation from a min time course, results showed 2-[ (14C)methylseleno]variety of drug substrates ( 2 1 ) . As shown in Table I, the benzanilide to be metabolized at a rate of 0.74 nmol/ amount of demethylated product recovered as 14C02from (minemg of protein) and ebselen to be produced a t a rate the labeled 2-(methylse1eno)benzanilideis dependent on of 0.23 nmol/(min-mg of protein). the presence of NADP+ and formaldehyde dehydrogenase. Microsomal Metabolism of 2-(Methylthio)benzAddition of metyrapone inhibits 14C02product formation. anilide. The sulfur analogue of 2-(methylse1eno)benzThe amount of demethylation product recovered as anilide was metabolized by rat liver microsomes (Figure 14C02from (14C)formaldehyde (0.75 pM) in the presence of NAD+ was 34%. As expected, 14C02was not recovered 3). The product was the corresponding sulfoxide. There was no evidence for demethylation and subsequent ring in the absence of formaldehyde dehydrogenase. Metaclosure to 2-phenyl-1,2-benzisothiazol-3(2H)-one (PZ 25), pyrone did not inhibit 14C02production from (14C)formthe sulfur analogue of ebselen. aldehyde (Table I). Thermal Cyclization of 2-(Methylseleninyl)benzUnder our incubation conditions (400-500 pL total reanilide into Ebselen. The reaction of regeneration of action volume), isolated microsomes from phenoebselen was mimicked chemically by thermal cyclization barbital-induced rat liver metabolized the drug substrate, of 2-(methylseleniny1)benzanilide into ebselen. For this, 2-(methylseleno)benzanilide, by oxidative demethylation a suspension of 2-(methylseleniny1)benzanilide(6.85 mmol) at a rate of about 0.04 nmol/(min.mg of protein). Thus, in acetonitrile (25 mL) was heated at reflux. After 1.5 h, the net amount of demethylated product recovered as the clear solution obtained was allowed to cool to room 14C02from the labeled 2-(methylse1eno)benzanilidewas temperature. The solid material was collected by filtration much lower than expected from the results shown in Figure and washed with n-hexane (20 mL) to yield ebselen in 46% 1 (0.2%), but the production was linear over a 60-min time yield. ‘H NMR and infrared spectroscopy and comparison course (Figure 2). When aminopyrine, a drug substrate with an authentic sample fully confirmed the nature of the known to undergo P-450-catalyzed oxidative demethylproduct obtained. ation, was incubated under similar conditions (100 pM Reaction of 2-(Benzylseleno)benzanilidewith Hysubstrate concentration), 2.7 % of demethylated product drogen Peroxide. There was no suitable method for the was recovered as 14C02from (dimethyl~mino-~~C)aminodetection of the low amounts of methanol to be expected pyrine. A t this concentration, the rate of substrate mein incubations such as those shown in Figure 1. Therefore, tabolism was 0.36 nmol/ (min-mg of protein), likewise we synthesized the benzyl analogue (see Materials and considerably lower than observed in larger incubation Methods) and oxidized with hydrogen peroxide. Indeed, vessels (12). However, when the substrate concentration
John et al.
202 Chem. Res. Toricol., Vol. 3, No. 3, 1990
It\
601
Demethylation
Ebselen, PZ 51
2-Methy Isulfobenzanihde
-f C
0 .c
e
4-
% !
30c
0 0
I
2-Methylselenobenzonilide
vSe-CH3
f2-Methylsulfoxidobenlonilide
L‘- Hydrox y -2- methy lsekno benzanilide
Figure 4. Scheme of microsomal reactions with 2-(methylseleno)benzanilide. 0
0
10 20 Incubation Time (min)
1
30
Figure 3. Time course of microsomal metabolism of 2-(methy1thio)benzanilide. Incubations were analogous to those shown for (methylse1eno)benzanilide in Figure 1B.
the corresponding products, benzyl alcohol and ebselen, were obtained. Detection of Benzyl Alcohol. To a solution of 2(benzylse1eno)benzanilide(0.1 mmol) in dichloromethane (0.5 mL), maintained at 40 “C, an aqueous hydrogen peroxide solution was added (0.025 mL of a 30% solution). A white solid was formed after 1 min which disappeared very rapidly. After 10 min, the mixture was allowed to cool to room temperature. The oily material that separated was discarded. The gas-liquid chromatography analysis of the supernatant dichloromethane layer showed the presence of benzyl alcohol together with small amounts ( € 5 % ) of benzaldehyde and benzoic acid. Detection of Ebselen and Its Se Oxide. The same mixture as described above was maintained at 25 “C. Under these conditions, it was possible to isolate the white transient solid formed, which was collected by filtration and immediately dissolved in ethanol (0.2 mL). The thin-layer chromatography analysis of the ethanolic solution and the comparison with authentic samples unequivocally showed the presence of ebselen and a small amount of its Se oxide (13)together with benzyl alcohol and traces of benzaldehyde. Preparative Synthesis of Ebselen Starting from 2-(Benzylseleno)benzanilide. In order to investigate the preparative potential of this reaction, a large-scale synthesis was run. To a solution of 2-(benzylseleno)benzanilide (2.7 mmol) in dichloromethane (30 mL) cooled to 0 “C, an aqueous hydrogen peroxide solution (0.31 mL of a 30% solution) was added in 10 min; a white solid was formed. The suspension was warmed to room temperature overnight. The clear yellow solution obtained was washed with water (2 X 50 mL) and dried over sodium sulfate and the solvent evaporated under reduced pressure. The oily residue obtained was dissolved in 2-propanol (20 mL) and warmed 2 h a t reflux (to reduce to ebselen the small amount of ebselen Se oxide formed). The clear solution obtained was allowed to cool to room temperature. The solid material that appeared was collected by filtration and washed with diethyl ether (10 mL) to yield ebselen in 80% yield. ‘HNMR and infrared spectroscopy and comparison with an authentic sample fully confirmed
0
*Se-CH3
I
1
Gonooxygenation,
Figure 5. Pathway of regeneration of ebselen from 2-(methylse1eno)benzanilide. For discussion, see text.
the nature of the product obtained.
Discussion The results demonstrate that 2-(methylseleno)benzanilide is metabolized by rat liver microsomes in vitro (Figure 4). Interestingly, the major metabolite found is ebselen; only comparatively low amounts of 4’-hydroxy2-(methylse1eno)benzanilide are produced. Therefore, there is significant oxidative demethylation of the substrate while very little para hydroxylation occurs (Figure 1). The one-carbon unit that is released from 2-(methy1seleno)benzanilide is concluded to be methanol, since very low amounts of (14C)formaldehydewere found to be produced (Figure 2; Table I). The mechanism by which methanol could be produced is shown in Figure 5. Rearrangements of selenoxides, R’Se(O)R’’, to selenenic esters have already been postulated (14, 15). It is likely that the selenenic acid methyl ester undergoes ring closure with concomitant release of methanol very rapidly (bottom step in Figure 5 ) due to the stability of the benzisoselenazolone ring system. The mechanism as suggested in Figure 5 was supported by chemical evidence in two ways: (a) the thermal cyclization
Microsomal Metabolism of 2-(Methylse1eno)benzanilide 0
Figure 6. Oxidation of 2-(benzylseleno)benzanilideto ebselen and benzyl alcohol.
Figure 7. Oxidation of 2-(methylthio)benzanilide to %(methylsulfinyl)benzanilide.
of 2-(methylseleniny1)benzanilide into ebselen and (b) the detection of benzyl alcohol by gas-liquid chromatography from the oxidation of 2-(benzylseleno)benzanilide,in analogy to methanol generated from the oxidation of 2(methylse1eno)benzaldehyde(Figure 6). Although little is known about selenium demethylation, it has been reported to occur. Rats injected with either a high dose (2 mg of Se/kg) or a low dose (0.064mg of Se/kg) of 75Se-labeledtrimethylselenonium exhale 75Selabeled dimethyl selenide (16, 17). Also, exhalation of volatile %e following a dose of (75Se)trimethylselenonium was observed (18). Further, using a doubly labeled selenobetaine methyl ester, Foster et al. (16) have indicated that labeled trimethylselenonium can be demethylated, and then remethylated, causing a drop in the 14C/75Seratio in trimethylselenonium compared to dimethyl selenide. The sulfur analogue may not isomerize to the analogous methoxy derivative, so that the stable sulfoxide is detected (Figure 7). This is similar to the finding that the antiinflammatory drug 2-acetamido-4-(chloromethyl)thiazole is reported to be excreted as a stable methyl sulfoxide (19).
Acknowledgment. Skillful technical assistance was provided by Bettina Aufmbruch and Marion Feige. Stimulating discussion with Dr. N. Dereu is gratefully acknowledged. Registry No. 2-(Methylseleninyl)benzanilide,126543-39-3; diselenosalicylic acid, 2-(benzylse1eno)benzoic acid, 126543-40-6; 126543-41-7; 2-(benzylseleno)benzanilide,126543-42-8; 2-(methylthio)benzanilide, 22978-26-3;2-phenyl-1,2-benzisothiazol-3(2H)-one, 79054-68-5;2-(methylseleno)benzanilide,60940-24-1; ebselen, 60940-34-3.
References (1) Sies, H. (1989)Metabolism and disposition of ebselen. In Selenium in Biology and Medicine (Wendel, A., Ed.) pp 153-162, Springer Verlag, Heidelberg.
Chem. Res. Toxicol., Vol. 3, No. 3, 1990 203 (2) Muller, A., Cadenas, E., Graf, P., and Sies, H. (1984)A novel biologically active seleno-organic compound. I. Glutathione peroxidase-like activity in vitro and anti-oxidant capacity of PZ 51 (ebselen). Biochem. Pharmacol. 33, 3235-3239. (3) Wendel, A., Fausel, M., Safayhi, H., and Otter, R. (1984)A novel biologically active seleno-organic compound. 11. Activity of PZ 51 in relation to glutathione peroxidase. Biochem. Pharmacol. 33, 3241-3246. (4) Muller, A., Gabriel, H., and Sies, H. (1985)A novel biologically active seleno-organic compound. IV. Protective glutathione-dependent effect of PZ 51 (ebselen) against ADP-Fe induced lipid peroxidation in isolated hepatocytes. Biochem. Pharmacol. 34, 1185-1189. (5) Parnham, M. J., and Graf,E. (1987)Seleno-organic compounds and the therapy of hydroperoxide linked pathological conditions. Biochem. Pharmacol. 36,3095-3102. (6) Muller, A., Gabriel, H., Sies, H. Terlinden, R., Fischer, H., and Romer, A. (1988)A novel biologically active selenoorganic compound. VII. Biotransformation of ebselen in perfused rat liver. Biochem. Pharmacol. 37, 1103-1109. (7) Fischer, H., Terlinden, R., Lohr, J. P., and Romer, A. (1988)A novel biologically active selenoorganic compound. VIII. Biotransformation of ebselen. Xenobiotica 18, 1347-1359. (8)Kuhn-Velten, N., and Sies, H. (1989)Optical spectral studies of ebselen interaction with cytochrome P-450of rat liver microsomes. Biochem. Pharmacol. 38,619-625. (9) Weber, R., and Renson, M. (1976)Les chlorures de chloro-3 benziso-s616nozolium-l,2:synth8se, hydrolyse, thiolation, aminolyse. Bull. SOC.Chem. Fr., 1124-1126. (10) Lesser, R., and Weiss, R. (1912)"Selenindigo" (Bisselenonaphthene indigo) and aromatic compounds containing selenium I. Chem. Ber. 45, 1835-1841. (11) Tephly, T.R., Watkins, W. D., and Goodman, J. 1. (1974)The biochemical toxicology of methanol. Essays Toxicol. 5,149-177. (12) Waydhas, C., Weigl, K., and Sies, H. (1978)The disposition of formaldehyde and formate arising from drug N-demethylations dependent on cytochrome P-450in hepatocytes and in perfused rat liver. Eur. J.Biochem. 89,143-150. (13) Kamigate, N., Iizuka, H., Izuoka, A,, and Kobayashi, M. (1986) Photochemical reaction of 2-aryl-1,2-benzisoselenazol-3(2H)-ones. Bull. Chem. SOC.Jpn. 59,2179-2183. (14) Entwistle, I. D., Johnstone, R. A. W., and Varley, J. H. (1976) Rearrangement of aryl alkyl selenoxides to aldehydes. J. Chem. SOC.,Chem. Commun. 61-62. (15) Reich, H. J., Shah, S. K., Gold, P. M., and Olson, R. E. (1981) Selenium-stabilized anions. Preparation of a,@-unsaturatedcarbonyl compounds using propargyl selenides. Synthesis of (&)-7hydroxymyoporone. J. Am. Chem. SOC.103,3112-3120. (16) Foster, S.J., Kraus, R. J., and Ganther, H. E. (1986)Formation of dimethyl selenide and trimethylselenonium from selenobetaine in the rat. Arch. Biochem. Biophys. 247, 12-19. (17) Foster, S. J., Kraus, R. J., and Ganther, H. E. (1986)The metabolism of selenomethionine, Se-methylselenocysteine,their selenonium derivatives, and trimethylselenonium in the rat. Arch. Biochem. Biophys. 251,77-86. (18) Obermeyer, B.D., Palmer, I. S., Olson, D. E., and Halverson, A. W. (1971)Toxicity of trimethylselenonium chloride in the rat with and without arsenite. Toxicol. Appl. Pharmacol. 20, 135-146. (19) Chatfield, D.H.,and Hunter, W. H. (1973)The Metabolism of acetamidothiazoles in the rat. 2-Acetamido-4-chloromethylthiazole. Biochem. J. 134, 879-884.
