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Free radicals as metabolites of drugs derived from phenothiazine
VOL. 2 9 ( I 9 5 8 )
BIOCHIMICA ET BIOPHYSICA ACTA
Preliminary
44I
Notes
Free radicals as metabolites of drugs derived from phenothiazine* I n a n i n v e s t i g a t i o n of t h e m e t a b o l i s m of c h l o r p r o m a z i n e a n d s o m e o t h e r d r u g s derived f r o m p h e n o t h i a z i n e b o t h in vivo a n d in vitro, i n t e r m e d i a t e s were f o u n d t h a t heretofore h a d n o t been described. T h e i n t e r m e d i a r y m e t a b o l i t e of c h l o r p r o m a z i n e , i o - ( 3 - d i m e t h y l a m i n o p r o p y l ) - 2 - c h l o r o p h e n o t h i a z i n e , as t h e p r o t o t y p e of t h i s class of drugs, h a s n o w been f o u n d to c o n t a i n free radicals as d e t e r m i n e d b y e l e c t r o n - s p i n resonance. T h e o x i d a t i o n level of t h i s n e w m e t a b o l i t e 1 Was b e t w e e n t h a t of t h e c h e m i c a l l y u n c h a n g e d d r u g (I) a n d t h a t of its physiological m e t a b o l i t e 2, t h e sulfoxide (V). T h e sulfone (VI), t h e c o m p o u n d c o r r e s p o n d i n g to t h e h i g h e s t o x i d a t i o n level, h a s so far n o t been e n c o u n t e r e d in o u r s t u d i e s on t h e e x c r e t i o n of c h l o r p r o m a z i n e m e t a b o l i t e s . T h e n e w m e t a b o l i t e w a s t e n t a t i v e l y considered a t h i o n i u m h y d r o x i d e (II) w i t h t h e O H g r o u p or g r o u p s directly a t t a c h e d to t h e sulfur, since t h i o n o l t y p e c o m p o u n d s ( I V ) - - a s a s s u m e d e.g. b y SMILES 3 a n d o t h e r a u t h o r s 4 w h o h a v e s t u d i e d p h e n o t h i a z i n e m e t a b o l i s m - - w i t h one or t w o O H g r o u p s in different positions, m i g h t be e x p e c t e d to yield I
I~ / / N /!l~ / / "I,~ zJCl
II
[-%. fl\ N , / ~II/ / " ~1
I ~CH3 CH,CH~CH~-N<~cH3 [OH]
OH\/OH
CH~CHICH _ N ~ C H * C1-13
III
IV OH~%./S\/~./OH
t
H II
ICl
II /
II /Cl
I ,CH3 CH2CH2CH2-N~cH3 O II
~\(%/~ II II
I
~/\NI~.+~
V
IC1
-
I /CH3 CH2CH2CH2--N X..CH 3
[ /CH3 CH2CH~CH2--N~cH a O~,~O .~'-../S\~x~.,.
~\ II II ~/'..NI~/.~
gI
Icl
I
CH2CH2CH2_N~CH3 CH3
c h a r a c t e r i s t i c u.v. a b s o r p t i o n spectra. T h e s e s h o u l d be d i s t i n c t f r o m t h e chemically u n c h a n g e d d r u g s a n d t h e i r sulfoxides, owing to c h a n g e s in t h e heterocyclic ring s t r u c t u r e a n d position of t h e double bonds. T h e a b s o r p t i o n d a t a for t h e m e t a b o l i t e s h o w e d no d i s t i n c t m a x i m a or m i n i m a , a n d t h e a b s o r p t i o n v a l u e s in t h e u l t r a v i o l e t from 23o to 34 ° m # , as well as in t h e visible w a v e l e n g t h s were f o u n d to lie b e t w e e n c h l o r p r o m a z i n e a n d its sulfoxide. T h e s u b s t a n c e yielded a n u m b e r of i n t e n s e a n d v a r i e d color r e a c t i o n s 5 w i t h ferric chloride, v a r i o u s c o n c e n t r a t e d acids a n d s o d i u m n i t r i t e (kindly s u g g e s t e d b y Prof. 13. K l s c a of Y e s h i v a University), all d i s t i n c t f r o m t h e corres p o n d i n g r e a c t i o n s of c h l o r p r o m a z i n e or o t h e r d r u g s derived f r o m p h e n o t h i a z i n e , a n d t h e i r sulfoxides. Thege n e w m e t a b o l i t e s are e x c r e t e d in considerable a m o u n t s in t h e urine of p a t i e n t s after i n g e s t i o n of t h e c o r r e s p o n d i n g p h e n o t h i a z i n e d r u g s e a n d m a y be b o t h r e d u c e d to t h e chemically u n c h a n g e d d r u g s , or oxidized to t h e sulfoxides. T h e c h l o r p r o m a z i n e m e t a b o l i t e h a s also been p r e p a r e d b y u.v. i r r a d i a t i o n of a dilute a q u e o u s c h l o r p r o m a z i n e solution w i t h a General Electric R S 275 W s u n l a m p . (IOOO ml, c o n t a i n i n g z m g c h l o r p r o m a z i n e / m l , were i r r a d i a t e d in a n o p e n vessel for 3 h at 5o-60°). T h e i r r a d i a t e d solution * A p a r t of t h i s c o m m u n i c a t i o n h a s been p r e s e n t e d a t t h e I33rd M e e t i n g of t h e A m e r i c a n C h e m i c a l Society, S a n Francisco, Calif., April i 3 t h - 1 8 t h 1958.
442
PRELIMINARY NOTES
VOL. 29 (I958)
gave the s a m e color reactions as the n a t u r a l metabolite, e.g. a characteristic deep-blue color d e v e l o p m e n t with conc. HC1, with which chlorpromazine itself yields a pale-pink, and its sulfoxide an orchid color reaction. I n this regard the u.v. irradiation can duplicate the physiological oxidation of phenotbiazine drugs to a level preceding t h a t of the sulfoxides, and it seems t h a t the chlorpromazine metabolite constitutes an active oxido-reduction s y s t e m 1 in the form of a free radical. This conclusion also finds s u p p o r t in earlier w o r k 7 in which it was proved by p o t e n t i o m e t r i c titration t h a t the interm e d i a r y oxidation p r o d u c t s of dyes derived from phenothiazine are free radicals. Hence it seems likely t h a t free radicals are n o r m a l metabolites of chlorpromazine and other phenothiazine drugs. Stored in the dark and u n d e r refrigeration, the u r i n a r y metabolite as well as the one p r e p a r e d by u.v. irradiation, is stable for m a n y m o n t h s in the original acid medium. The r e m a r k a b l e stability of phenothiazine-derived radicals seems to be characteristic of this class of compoundsL F o r identification of the free radical by electron-spin resonance m e a s u r e m e n t , t w o solid crystalline derivatives* of the i n t e r m e d i a r y c h l o r p r o m a z i n e metabolite were prepared in f o l m of a deep-blue nitrite c o m p o u n d and a dark-purple 2,4-dinitrophenylhydrazine derivative, obtained by precipitating a dilute a q u e o u s c h l o r p r o m a z i n e solution after u.v. irradiation with an excess of freshly prepared N a N O 2 solution and a z,4-dinitrophenylhydrazine solution in 2 N HC1 respectively. D e t e r m i n a t i o n of electron-spin resonance was carried o u t on 5-mg samples of the t w o polycrystalline derivatives by Prof. P. A. MILES, Dept. of Electrical Engineering, Massachusetts I n s t i t u t e of Technology, to w h o m we are greatly indebted for generous help. A resonance of 16. 5 Gauss line width and a g-value of 2.004(5) were detected in the nitrite compound, while a Gauss line width of 23 and a g-value of 2.OO6(5 ) w e r e found for the 2,4-dinitrophenylhydrazine derivative. I n b o t h cases, a noticeable a s y m m e t r y of the line suggested an unresolved resonance structure, and t h e resonance intensity indicated t h a t a b o u t o 5 % of the molecular groups present were in freeradical form. These crystalline derivatives of t h e chlorpromazine metabolite had been o b t a i n e d in relatively low yields of a p p r o x i m a t e l y to %, calculated for chlorpromazine applied before u.v. irradiation. This m i g h t be due to inactivation b y polymerization of a large p o r t i o n of the free radical during irradiation, immediately following its formation. This does n o t necessarily reflect physiological conditions since the free radical resulting from biological oxidation m i g h t react with cell cons t i t u a n t s instead of polymerizing. The tentati,,e s t r u c t u r e of the free radical is s h o w n (III). XVe believe t h a t these results are significant for an eventual i n t e r p r e t a t i o n of the metabolism and m e c h a n i s m of action of the phenothiazine-derived drugs. Several aspects of this p r o b l e m are now under investigation, specifically the enzymic formation of the free radicals and the effect of ring s u b s t i t u e n t s and side-chains on their formation. \Ve wish to t h a n k Smith, Kline and French Laboratories, Philadelphia, for a generous s u p p l y of clllorpromazine, its sulfoxide and sulfone.
5:. 3 . Hospital, Brockton, .'~Iass. Dept. o[ Biochemistry, Columbia College o/Physicians and Surgeons, New York, and Department of Experimental Psychiatry, New York, State Psychiatric Institute, New York, N . Y . (U.S.A.)
IRENE S. FORREST FRED M. FORREST MAGDALENA BERGER
1 F. M. FORREST AND 5. S. FORREST, Am. J. Psychiat., 113 (1957) 931. 2 z~. P. SALZMAN, N. C. MORAN AND B. B. BRODIE, Nature, 176 (1955) 1122. 3 E. DE BARRY BARNETT AND S. SMILES, J. Chem. Soc., 95 (19o9) 1253; ibid., 97 (lOlO) 186. 4 F. DE EDS, R. H. \VILSON AND J. O. THOMAS, .f. -//m. Med. Assoc., 114 (194 o) 2095; G. H. BENrtAM, Can. J. Research, 23, Sec. E (I945) 71. 5 35. BERGER AND I. S. EORREST, in p r e p a r a t i o n . 6 F. M. FORREST, I. S..FORREST AND A. S. MASON, .4m. J. Psychiat., I i 4 (1958) 931. 7 L. MICHAELIS, S. GRANICK AND N. P. SCntmERT, J. Am. Chem. Soc., 63 (1941) 351 ; L. MICNAELm AND S. GRANICK, J. Am. Chem. Soc., 63 (1941) 1636; L. MICHAELIS, J. Am. Chem. Soc., 63 (1941) 2446; L. MICNAELm, in J. B. SUMNER ANn K. MYRBT,CK, The Enzymes, Vol. I I , p a r t I, Academic Press, Inc., N e w York, 1951, pp. 1-54. Received April 2nd, 1958
* M e a s u r e m e n t of electron-spin resonance h a d to be carried o u t on solid derivatives since the free-radical concentrations in the urine of p a t i e n t s as well as in the irradiated solutions were a p p a r e n t l y too low to be detected b y the available E S R a p p a r a t u s . The s t r u c t u r e of b o t h crystalline derivatives is c u r r e n t l y f u r t h e r investigated.
BIOCHIMICA ET BIOPHYSICA ACTA
Preliminary
44I
Notes
Free radicals as metabolites of drugs derived from phenothiazine* I n a n i n v e s t i g a t i o n of t h e m e t a b o l i s m of c h l o r p r o m a z i n e a n d s o m e o t h e r d r u g s derived f r o m p h e n o t h i a z i n e b o t h in vivo a n d in vitro, i n t e r m e d i a t e s were f o u n d t h a t heretofore h a d n o t been described. T h e i n t e r m e d i a r y m e t a b o l i t e of c h l o r p r o m a z i n e , i o - ( 3 - d i m e t h y l a m i n o p r o p y l ) - 2 - c h l o r o p h e n o t h i a z i n e , as t h e p r o t o t y p e of t h i s class of drugs, h a s n o w been f o u n d to c o n t a i n free radicals as d e t e r m i n e d b y e l e c t r o n - s p i n resonance. T h e o x i d a t i o n level of t h i s n e w m e t a b o l i t e 1 Was b e t w e e n t h a t of t h e c h e m i c a l l y u n c h a n g e d d r u g (I) a n d t h a t of its physiological m e t a b o l i t e 2, t h e sulfoxide (V). T h e sulfone (VI), t h e c o m p o u n d c o r r e s p o n d i n g to t h e h i g h e s t o x i d a t i o n level, h a s so far n o t been e n c o u n t e r e d in o u r s t u d i e s on t h e e x c r e t i o n of c h l o r p r o m a z i n e m e t a b o l i t e s . T h e n e w m e t a b o l i t e w a s t e n t a t i v e l y considered a t h i o n i u m h y d r o x i d e (II) w i t h t h e O H g r o u p or g r o u p s directly a t t a c h e d to t h e sulfur, since t h i o n o l t y p e c o m p o u n d s ( I V ) - - a s a s s u m e d e.g. b y SMILES 3 a n d o t h e r a u t h o r s 4 w h o h a v e s t u d i e d p h e n o t h i a z i n e m e t a b o l i s m - - w i t h one or t w o O H g r o u p s in different positions, m i g h t be e x p e c t e d to yield I
I~ / / N /!l~ / / "I,~ zJCl
II
[-%. fl\ N , / ~II/ / " ~1
I ~CH3 CH,CH~CH~-N<~cH3 [OH]
OH\/OH
CH~CHICH _ N ~ C H * C1-13
III
IV OH~%./S\/~./OH
t
H II
ICl
II /
II /Cl
I ,CH3 CH2CH2CH2-N~cH3 O II
~\(%/~ II II
I
~/\NI~.+~
V
IC1
-
I /CH3 CH2CH2CH2--N X..CH 3
[ /CH3 CH2CH~CH2--N~cH a O~,~O .~'-../S\~x~.,.
~\ II II ~/'..NI~/.~
gI
Icl
I
CH2CH2CH2_N~CH3 CH3
c h a r a c t e r i s t i c u.v. a b s o r p t i o n spectra. T h e s e s h o u l d be d i s t i n c t f r o m t h e chemically u n c h a n g e d d r u g s a n d t h e i r sulfoxides, owing to c h a n g e s in t h e heterocyclic ring s t r u c t u r e a n d position of t h e double bonds. T h e a b s o r p t i o n d a t a for t h e m e t a b o l i t e s h o w e d no d i s t i n c t m a x i m a or m i n i m a , a n d t h e a b s o r p t i o n v a l u e s in t h e u l t r a v i o l e t from 23o to 34 ° m # , as well as in t h e visible w a v e l e n g t h s were f o u n d to lie b e t w e e n c h l o r p r o m a z i n e a n d its sulfoxide. T h e s u b s t a n c e yielded a n u m b e r of i n t e n s e a n d v a r i e d color r e a c t i o n s 5 w i t h ferric chloride, v a r i o u s c o n c e n t r a t e d acids a n d s o d i u m n i t r i t e (kindly s u g g e s t e d b y Prof. 13. K l s c a of Y e s h i v a University), all d i s t i n c t f r o m t h e corres p o n d i n g r e a c t i o n s of c h l o r p r o m a z i n e or o t h e r d r u g s derived f r o m p h e n o t h i a z i n e , a n d t h e i r sulfoxides. Thege n e w m e t a b o l i t e s are e x c r e t e d in considerable a m o u n t s in t h e urine of p a t i e n t s after i n g e s t i o n of t h e c o r r e s p o n d i n g p h e n o t h i a z i n e d r u g s e a n d m a y be b o t h r e d u c e d to t h e chemically u n c h a n g e d d r u g s , or oxidized to t h e sulfoxides. T h e c h l o r p r o m a z i n e m e t a b o l i t e h a s also been p r e p a r e d b y u.v. i r r a d i a t i o n of a dilute a q u e o u s c h l o r p r o m a z i n e solution w i t h a General Electric R S 275 W s u n l a m p . (IOOO ml, c o n t a i n i n g z m g c h l o r p r o m a z i n e / m l , were i r r a d i a t e d in a n o p e n vessel for 3 h at 5o-60°). T h e i r r a d i a t e d solution * A p a r t of t h i s c o m m u n i c a t i o n h a s been p r e s e n t e d a t t h e I33rd M e e t i n g of t h e A m e r i c a n C h e m i c a l Society, S a n Francisco, Calif., April i 3 t h - 1 8 t h 1958.
442
PRELIMINARY NOTES
VOL. 29 (I958)
gave the s a m e color reactions as the n a t u r a l metabolite, e.g. a characteristic deep-blue color d e v e l o p m e n t with conc. HC1, with which chlorpromazine itself yields a pale-pink, and its sulfoxide an orchid color reaction. I n this regard the u.v. irradiation can duplicate the physiological oxidation of phenotbiazine drugs to a level preceding t h a t of the sulfoxides, and it seems t h a t the chlorpromazine metabolite constitutes an active oxido-reduction s y s t e m 1 in the form of a free radical. This conclusion also finds s u p p o r t in earlier w o r k 7 in which it was proved by p o t e n t i o m e t r i c titration t h a t the interm e d i a r y oxidation p r o d u c t s of dyes derived from phenothiazine are free radicals. Hence it seems likely t h a t free radicals are n o r m a l metabolites of chlorpromazine and other phenothiazine drugs. Stored in the dark and u n d e r refrigeration, the u r i n a r y metabolite as well as the one p r e p a r e d by u.v. irradiation, is stable for m a n y m o n t h s in the original acid medium. The r e m a r k a b l e stability of phenothiazine-derived radicals seems to be characteristic of this class of compoundsL F o r identification of the free radical by electron-spin resonance m e a s u r e m e n t , t w o solid crystalline derivatives* of the i n t e r m e d i a r y c h l o r p r o m a z i n e metabolite were prepared in f o l m of a deep-blue nitrite c o m p o u n d and a dark-purple 2,4-dinitrophenylhydrazine derivative, obtained by precipitating a dilute a q u e o u s c h l o r p r o m a z i n e solution after u.v. irradiation with an excess of freshly prepared N a N O 2 solution and a z,4-dinitrophenylhydrazine solution in 2 N HC1 respectively. D e t e r m i n a t i o n of electron-spin resonance was carried o u t on 5-mg samples of the t w o polycrystalline derivatives by Prof. P. A. MILES, Dept. of Electrical Engineering, Massachusetts I n s t i t u t e of Technology, to w h o m we are greatly indebted for generous help. A resonance of 16. 5 Gauss line width and a g-value of 2.004(5) were detected in the nitrite compound, while a Gauss line width of 23 and a g-value of 2.OO6(5 ) w e r e found for the 2,4-dinitrophenylhydrazine derivative. I n b o t h cases, a noticeable a s y m m e t r y of the line suggested an unresolved resonance structure, and t h e resonance intensity indicated t h a t a b o u t o 5 % of the molecular groups present were in freeradical form. These crystalline derivatives of t h e chlorpromazine metabolite had been o b t a i n e d in relatively low yields of a p p r o x i m a t e l y to %, calculated for chlorpromazine applied before u.v. irradiation. This m i g h t be due to inactivation b y polymerization of a large p o r t i o n of the free radical during irradiation, immediately following its formation. This does n o t necessarily reflect physiological conditions since the free radical resulting from biological oxidation m i g h t react with cell cons t i t u a n t s instead of polymerizing. The tentati,,e s t r u c t u r e of the free radical is s h o w n (III). XVe believe t h a t these results are significant for an eventual i n t e r p r e t a t i o n of the metabolism and m e c h a n i s m of action of the phenothiazine-derived drugs. Several aspects of this p r o b l e m are now under investigation, specifically the enzymic formation of the free radicals and the effect of ring s u b s t i t u e n t s and side-chains on their formation. \Ve wish to t h a n k Smith, Kline and French Laboratories, Philadelphia, for a generous s u p p l y of clllorpromazine, its sulfoxide and sulfone.
5:. 3 . Hospital, Brockton, .'~Iass. Dept. o[ Biochemistry, Columbia College o/Physicians and Surgeons, New York, and Department of Experimental Psychiatry, New York, State Psychiatric Institute, New York, N . Y . (U.S.A.)
IRENE S. FORREST FRED M. FORREST MAGDALENA BERGER
1 F. M. FORREST AND 5. S. FORREST, Am. J. Psychiat., 113 (1957) 931. 2 z~. P. SALZMAN, N. C. MORAN AND B. B. BRODIE, Nature, 176 (1955) 1122. 3 E. DE BARRY BARNETT AND S. SMILES, J. Chem. Soc., 95 (19o9) 1253; ibid., 97 (lOlO) 186. 4 F. DE EDS, R. H. \VILSON AND J. O. THOMAS, .f. -//m. Med. Assoc., 114 (194 o) 2095; G. H. BENrtAM, Can. J. Research, 23, Sec. E (I945) 71. 5 35. BERGER AND I. S. EORREST, in p r e p a r a t i o n . 6 F. M. FORREST, I. S..FORREST AND A. S. MASON, .4m. J. Psychiat., I i 4 (1958) 931. 7 L. MICHAELIS, S. GRANICK AND N. P. SCntmERT, J. Am. Chem. Soc., 63 (1941) 351 ; L. MICNAELm AND S. GRANICK, J. Am. Chem. Soc., 63 (1941) 1636; L. MICHAELIS, J. Am. Chem. Soc., 63 (1941) 2446; L. MICNAELm, in J. B. SUMNER ANn K. MYRBT,CK, The Enzymes, Vol. I I , p a r t I, Academic Press, Inc., N e w York, 1951, pp. 1-54. Received April 2nd, 1958
* M e a s u r e m e n t of electron-spin resonance h a d to be carried o u t on solid derivatives since the free-radical concentrations in the urine of p a t i e n t s as well as in the irradiated solutions were a p p a r e n t l y too low to be detected b y the available E S R a p p a r a t u s . The s t r u c t u r e of b o t h crystalline derivatives is c u r r e n t l y f u r t h e r investigated.