- Email: [email protected]
Synthesis of a potential estrogen metabolite with disrupted ring A
136
PRELIMINARY NOTES
cartilage and 2.85 for epiphyseal plate-metaphyseal iunction). This experiment shows that KC1 will extract some calcium together with a roughly equivalent amount of phosphate from a completely calcified tissue containing a very small quantity of chondroitin sulfate. The Ca/P ratios show that some of the KCl-soluble calcium from the epiphyseal tissue must be in a different form from that in teeth. In the cold, water dissolves only a small amount of collagen from cartilage 1°. The present results indicate that a large fraction of cartilage calcium is bound to a fraction of the chondroitin sulfate associated with collagen insoluble in cold water. I t seems likely t h a t this accumulation of calcium is connected, as a preliminary stage, with the deposition of solid hydroxyapatite on the collagen fibre, which has been demonstrated b y electron diffraction ~. Other means of calcium accumulation which m a y operate in addition to chondroitin sulfate, particularly in the epiphyseal tissues, are, however, not excluded by these preliminary experiments. I wish to t h a n k Mr. K. ARULCHOTI-IYof the Animal Infirmary, Singapore, and Dr. A. Mom~:I)I)IN for their help in obtaining the animal material. I am grateful to Mr. CHIA CHIAP MONG for technical assistance.
Department of Biochemistry, University of Singapore, Singapore (Malaya)
J. M. BOWNESS
I. GROSS, M. B. MATHEWS AND A. DORFMAN, J. Biol. Chem., 235 (196o) 2891. M. BOWNESS, Biochim. Biophys. Acta, 5 ° (1961) 41o. EICHELBERGER AND M. ROMA, Am. J. Physiol., 178 (1954) 296. M. BOWNESS, Brit. J. Nutrition, II (1957) 152. N. BACHRA, A. DAOER AND A. E. SOBEL, Clin. Chem., 4 (1959) lO7. 6 L . ERNSTER, R . ZETTERSTROM AND O. LINDBERG, A c t a Chem. Scand., 4 (195o) 9 4 2 . j. J. L. 4 j. B. z
7 E . J . K I N G AND I. D . P . WOOTTON, i n
"Micro-Analysis i n M e di c al Biochemistry", J. & A.
Churchill, L o n d o n , 1956, p. 78. s j . M. BowNEss, Biochem. J., 67 (1957) 295. 9 j . 1R. DtlNSTONE, Biochem. J., 77 (196o) 164. 10 j . SHATTON AND M. SCHOBERT, J. Biol. Chem., 211 (1954) 565. 11 S. FITTON JACKSON, Proc. Roy. Soc. (London), B 146 (1957) 27o.
Received J a n u a r y 9th, 1962 Biochim. Biophys. Acta, 58 (1962) 134-136
Synthesis of a potential estrogen metabolite with disrupted ring A Recent studies in vitro and in vivo have established that estrogen hormones are metabolized in part b y ortho-hydroxylation of C-2 of the benzene ring to catechol-type steroidsJ, 2. These catechol-like steroids m a y be further metabolized b y cleavage of the benzene ring. JELLINCK obtained water-soluble, radioactive intermediates which he extracted with ether from an acidic aqueous phase prepared from an incubation mixture consisting of rat-liver slices and [I6-14Clestrone3, 4. He suggested that these intermediates resulted from enzymic cleavage of the benzene ring of estrone after its initial ortho-hydroxylation. Other examples of the splitting of benzene rings in biological systems m a y be cited. BHARGAVAet al. ~ showed that, after the c~rcinogen 1,2,5,6-dibenzanthracene was applied to mouse skin, it was metabolized, b y rupture of one of ~ts benzene rings, to a-phenylphenanthrene-3,2'-dicarboxylic acid (a muconic acid derivative). The enzymes pyrocatechase 6 and metapyrocatechase ~, found in Biochim. Biophys. Acta, 58 (1962) 136-138
137
PRELIMINARY NOTES
various strains of Pseudomonas, convert catechol to cis-cis-muconic acid and ahydroxymuconic semialdehyde, respectively. We have synthesized an estrogen derivative with a ruptured benzene ring in order to facilitate identification of potential estrogen metabolites with this novel configuration. Successful scission of the benzene ring of estrone and the isolation of an analogue (I) of a potential acidic estrogen metabolite were accomplished b y the following reaction sequence : O II
OZ\( HO~/~/
wo= , HO~/~
Kishner Redn.
HN0a HAc
J'--,l/~
O~N\~J~/I
z-.V\ ]
H,S04
o=c o>( ( _t-l/ "-H
I
Estrone was converted to i7-deoxoestrone by a Wolff-Kishner reduction. This was nitrated b y the procedure previously described for estrone s. The resulting 2- and 4-nitro-I7-deoxoestrones were separated b y chromatography on aluminum oxide. 2-Nitro-I7-deoxoestrone was eluted first and crystallized from absolute methanol; yellow plates, m.p. 123.5-124.5 °. (Calc. for C18HzaOaN: C, 71.49; H, 8.00. Found: C, 71.59; H, 7.83). Light-absorption max. in ethanol: 295-296 m ~ (e 8400) and 363-364 (e 38o0). 4-Nitro-i7-deoxoestrone was then eluted from the aluminum oxide column and was crystallized from a petroleum e t h e r - m e t h a n o l mixture; yellow needles, m.p. 159-16o °. (Calc. for CjsH2303N: C, 71.49; H, 8.00. Found: C, 71.63; H, 7.71, light-absorption max. in ethanol: 276-277 m/~ (e 17oo ). The infrared absorption spectra of these two nitro compounds have already been reported 9. The assignment of the nitro groups in the nitrodeoxoestrones is based on their order of elution from the aluminum oxide column and their absorption spectra, both ultraviolet and infraredS,L The 2-nitro-i7-deoxoestrone was heated at 95-1oo ° for 3o min in conc. H2SO 4. The reaction mixture was cooled, poured into water, and extracted with ether. The ether extract was washed with water, dried, and evaporated. The oily residue was crystallized from aqueous ethanol, and the colorless crystals obtained sublimed at about 155 ° and melted at 17o-175°; [~]2De = - - 9 4 . 6 ° (chloroform). (Calc. for CzsH~404: C, 71.o2; H, 7.95. Found: C, 71.5o; H, 8.39. ) The material showed end absorption in the ultraviolet region with max. in ethanol 220 m/~ (e 137). A strong carbonyl band appeared at 173o cm -z in KBr. The material was neutral, and was recovered unchanged after sublimation or chromatography on aluminum oxide. Tests for unsaturation (bromine and potassium permanganate) were negative. Similar degradation of para-substituted nitrophenols has resulted in the formation of unsaturated lactonic acids or nitrogenous acidsl°, zz. I t occurred to us that the product expected from the benzene-ring cleavage of 2-nitro-I7-deoxoestrone, II, might have cyclized to the y,7'-dilactone (I). Biochim. Biophys. Acta, 58 (1962) I36-138
138
PRELIMINARY NOTES
O,~.C\o. q "] Ho~C\ C~'.../' H/
\H
II
T h e 7,~/-dilactone s t r u c t u r e of I, which can be d e s i g n a t e d d e c a h y d r o - 5 ~ - m e t h y l 3~,IO~-(epoxyethano)-3flH-cyclopenta [5,6]naphtho [2,I-r] f u r a n - 2 , I 2 (3H)-dione, was confirmed b y its n u c l e a r m a g n e t i c resonance s p e c t r u m . A d o u b l e t a p p e a r e d (z 7.1 a n d 7.2) which can be a s c r i b e d t o t h e m e t h y l e n e p r o t o n s in t h e ~ j - d i l a c t o n e moiety. Since I is n o t s y m m e t r i c a l , t h e p r o t o n s a p p e a r as a d o u b l e t . I n t e g r a t i o n of t h e a r e a s u n d e r t h e curve showed t h a t t h e d o u b l e t a c c o u n t e d for four p r o t o n s , a finding in accord w i t h t h e p r o p o s e d s t r u c t u r e of I. T h e s p e c t r u m confirmed t h e absence of d o u b l e bonds. I s h o u l d p r o v e useful in a s c e r t a i n i n g w h e t h e r e n z y m e s are p r e s e n t in liver t h a t a r e c a p a b l e of r u p t u r i n g t h e benzene ring of estrogen hormones, t h e r e b y c o n v e r t i n g t h e m to muconic a c i d d e r i v a t i v e s . If I 7 - d e o x o e s t r o n e were p a r t i a l l y m e t a b o l i z e d in this way, t h e r e s u l t i n g muconic acid would yield I a f t e r acidic t r e a t m e n t 12. I is of a d d i t i o n a l i n t e r e s t because t h e four carbon a t o m s in its ~,,7'-dilactone rings are d e r i v e d from t h e benzene ring of estrone. B y d e g r a d a t i v e chemical procedures it s h o u l d be possible to isolate these c a r b o n a t o m s i n d i v i d u a l l y . T h e developm e n t of such d e g r a d a t i v e procedures w o u l d a d v a n c e our knowledge of t h e bios y n t h e t i c p a t h w a y s of estrogen f o r m a t i o n from precursors such as cholesterol a n d a c e t i c acidlZ, 14. A liberal gift of estrone from Dr. PRESTON PERLMAN of Schering Corporation m a d e t h i s s t u d y possible. W e are i n d e b t e d to Dr. KEITH FREEMAN" of D o n n e r L a b o r a t o r y for t h e i n f r a r e d s p e c t r u m a n d to Dr. J. N. SHOOLERY a n d Dr. L. F. JOHNSON of V a r i a n Associates for t h e nuclear m a g n e t i c resonance s p e c t r a a n d helpful discussions on t h e i r i n t e r p r e t a t i o n . T h e n o m e n c l a t u r e for I was r e c o m m e n d e d b y Dr. DONALD F. WALKER, Jr., of t h e Chemical A b s t r a c t s Service. This w o r k was s u p p o r t e d b y a g r a n t from t h e U.S. P u b l i c H e a l t h Service. Department of Physiology, f.niversity of California, Berkeley, Call/. (U.S.A.)
HAROLD WERBIN M . R . IMADA I . L . CHAIKOFF
1 j. FISHMAN,R. I. COX AND T. F. GALLAGHER,Arch. Biochem. Biophys., 90 (196o) 318. 2 R. J. B. KING, Biochem. J., 79 (1961) 355z p. H. JELLINCK, Biochem. J., 71 (1959) 665. 4 p. H. JELLINCK, Biochim. Biophys. Acta, 41 (196o) 37. 5 p. IV[.BHARGAVA,H. I. HADLER AND C. HEIDELBERGER,J. Am. Chem. Soc., 77 (1955) 2877e O. HAYAISHIAND Z. HASHIMOTO,J. Biochem. (Tokyo), 37 (195o) 3717 N. ITADAAND O. HAYAISHI,J. Biol. Chem., 236 (1961) 2223. s H. WERBIN AND C. HOLOWAY,J. Biol. Chem., 223 (1956) 651. 9 1R. A. PICKERINGAND H. WERBIN, J. Am. Chem. Soc., 80 (1958) 68o. 10 H. PAULY, R. GILMOURAND G. WILL, Ann. Chem., Liebigs, 403 (1914) 119. 11 O. NEUNHOEFFER AND H. KOLBEL, Ber. deut. chem. Ges., 68 (1935) 255. 12 J . A. ELVIDGE, R. P. LINDSTEAD,B. A. ORKIN, P. SIMS, H. BEAR AND D. B. PATTISON,J. Chem. Soc., (195o) 2228. lZ K. J. RYAN, J. Biol. Chem., 236 (1961) 705 . 14 H. WERBIN, J. PLOTZ, G. V. LEROY AND E. M. DAVIS,J. Am. Chem. Sot., 79 (1957) lO12. R e c e i v e d J a n u a r y I o t h , 1962 Biochim. Biophys. Acta, 58 (1962) 136-138
PRELIMINARY NOTES
cartilage and 2.85 for epiphyseal plate-metaphyseal iunction). This experiment shows that KC1 will extract some calcium together with a roughly equivalent amount of phosphate from a completely calcified tissue containing a very small quantity of chondroitin sulfate. The Ca/P ratios show that some of the KCl-soluble calcium from the epiphyseal tissue must be in a different form from that in teeth. In the cold, water dissolves only a small amount of collagen from cartilage 1°. The present results indicate that a large fraction of cartilage calcium is bound to a fraction of the chondroitin sulfate associated with collagen insoluble in cold water. I t seems likely t h a t this accumulation of calcium is connected, as a preliminary stage, with the deposition of solid hydroxyapatite on the collagen fibre, which has been demonstrated b y electron diffraction ~. Other means of calcium accumulation which m a y operate in addition to chondroitin sulfate, particularly in the epiphyseal tissues, are, however, not excluded by these preliminary experiments. I wish to t h a n k Mr. K. ARULCHOTI-IYof the Animal Infirmary, Singapore, and Dr. A. Mom~:I)I)IN for their help in obtaining the animal material. I am grateful to Mr. CHIA CHIAP MONG for technical assistance.
Department of Biochemistry, University of Singapore, Singapore (Malaya)
J. M. BOWNESS
I. GROSS, M. B. MATHEWS AND A. DORFMAN, J. Biol. Chem., 235 (196o) 2891. M. BOWNESS, Biochim. Biophys. Acta, 5 ° (1961) 41o. EICHELBERGER AND M. ROMA, Am. J. Physiol., 178 (1954) 296. M. BOWNESS, Brit. J. Nutrition, II (1957) 152. N. BACHRA, A. DAOER AND A. E. SOBEL, Clin. Chem., 4 (1959) lO7. 6 L . ERNSTER, R . ZETTERSTROM AND O. LINDBERG, A c t a Chem. Scand., 4 (195o) 9 4 2 . j. J. L. 4 j. B. z
7 E . J . K I N G AND I. D . P . WOOTTON, i n
"Micro-Analysis i n M e di c al Biochemistry", J. & A.
Churchill, L o n d o n , 1956, p. 78. s j . M. BowNEss, Biochem. J., 67 (1957) 295. 9 j . 1R. DtlNSTONE, Biochem. J., 77 (196o) 164. 10 j . SHATTON AND M. SCHOBERT, J. Biol. Chem., 211 (1954) 565. 11 S. FITTON JACKSON, Proc. Roy. Soc. (London), B 146 (1957) 27o.
Received J a n u a r y 9th, 1962 Biochim. Biophys. Acta, 58 (1962) 134-136
Synthesis of a potential estrogen metabolite with disrupted ring A Recent studies in vitro and in vivo have established that estrogen hormones are metabolized in part b y ortho-hydroxylation of C-2 of the benzene ring to catechol-type steroidsJ, 2. These catechol-like steroids m a y be further metabolized b y cleavage of the benzene ring. JELLINCK obtained water-soluble, radioactive intermediates which he extracted with ether from an acidic aqueous phase prepared from an incubation mixture consisting of rat-liver slices and [I6-14Clestrone3, 4. He suggested that these intermediates resulted from enzymic cleavage of the benzene ring of estrone after its initial ortho-hydroxylation. Other examples of the splitting of benzene rings in biological systems m a y be cited. BHARGAVAet al. ~ showed that, after the c~rcinogen 1,2,5,6-dibenzanthracene was applied to mouse skin, it was metabolized, b y rupture of one of ~ts benzene rings, to a-phenylphenanthrene-3,2'-dicarboxylic acid (a muconic acid derivative). The enzymes pyrocatechase 6 and metapyrocatechase ~, found in Biochim. Biophys. Acta, 58 (1962) 136-138
137
PRELIMINARY NOTES
various strains of Pseudomonas, convert catechol to cis-cis-muconic acid and ahydroxymuconic semialdehyde, respectively. We have synthesized an estrogen derivative with a ruptured benzene ring in order to facilitate identification of potential estrogen metabolites with this novel configuration. Successful scission of the benzene ring of estrone and the isolation of an analogue (I) of a potential acidic estrogen metabolite were accomplished b y the following reaction sequence : O II
OZ\( HO~/~/
wo= , HO~/~
Kishner Redn.
HN0a HAc
J'--,l/~
O~N\~J~/I
z-.V\ ]
H,S04
o=c o>( ( _t-l/ "-H
I
Estrone was converted to i7-deoxoestrone by a Wolff-Kishner reduction. This was nitrated b y the procedure previously described for estrone s. The resulting 2- and 4-nitro-I7-deoxoestrones were separated b y chromatography on aluminum oxide. 2-Nitro-I7-deoxoestrone was eluted first and crystallized from absolute methanol; yellow plates, m.p. 123.5-124.5 °. (Calc. for C18HzaOaN: C, 71.49; H, 8.00. Found: C, 71.59; H, 7.83). Light-absorption max. in ethanol: 295-296 m ~ (e 8400) and 363-364 (e 38o0). 4-Nitro-i7-deoxoestrone was then eluted from the aluminum oxide column and was crystallized from a petroleum e t h e r - m e t h a n o l mixture; yellow needles, m.p. 159-16o °. (Calc. for CjsH2303N: C, 71.49; H, 8.00. Found: C, 71.63; H, 7.71, light-absorption max. in ethanol: 276-277 m/~ (e 17oo ). The infrared absorption spectra of these two nitro compounds have already been reported 9. The assignment of the nitro groups in the nitrodeoxoestrones is based on their order of elution from the aluminum oxide column and their absorption spectra, both ultraviolet and infraredS,L The 2-nitro-i7-deoxoestrone was heated at 95-1oo ° for 3o min in conc. H2SO 4. The reaction mixture was cooled, poured into water, and extracted with ether. The ether extract was washed with water, dried, and evaporated. The oily residue was crystallized from aqueous ethanol, and the colorless crystals obtained sublimed at about 155 ° and melted at 17o-175°; [~]2De = - - 9 4 . 6 ° (chloroform). (Calc. for CzsH~404: C, 71.o2; H, 7.95. Found: C, 71.5o; H, 8.39. ) The material showed end absorption in the ultraviolet region with max. in ethanol 220 m/~ (e 137). A strong carbonyl band appeared at 173o cm -z in KBr. The material was neutral, and was recovered unchanged after sublimation or chromatography on aluminum oxide. Tests for unsaturation (bromine and potassium permanganate) were negative. Similar degradation of para-substituted nitrophenols has resulted in the formation of unsaturated lactonic acids or nitrogenous acidsl°, zz. I t occurred to us that the product expected from the benzene-ring cleavage of 2-nitro-I7-deoxoestrone, II, might have cyclized to the y,7'-dilactone (I). Biochim. Biophys. Acta, 58 (1962) I36-138
138
PRELIMINARY NOTES
O,~.C\o. q "] Ho~C\ C~'.../' H/
\H
II
T h e 7,~/-dilactone s t r u c t u r e of I, which can be d e s i g n a t e d d e c a h y d r o - 5 ~ - m e t h y l 3~,IO~-(epoxyethano)-3flH-cyclopenta [5,6]naphtho [2,I-r] f u r a n - 2 , I 2 (3H)-dione, was confirmed b y its n u c l e a r m a g n e t i c resonance s p e c t r u m . A d o u b l e t a p p e a r e d (z 7.1 a n d 7.2) which can be a s c r i b e d t o t h e m e t h y l e n e p r o t o n s in t h e ~ j - d i l a c t o n e moiety. Since I is n o t s y m m e t r i c a l , t h e p r o t o n s a p p e a r as a d o u b l e t . I n t e g r a t i o n of t h e a r e a s u n d e r t h e curve showed t h a t t h e d o u b l e t a c c o u n t e d for four p r o t o n s , a finding in accord w i t h t h e p r o p o s e d s t r u c t u r e of I. T h e s p e c t r u m confirmed t h e absence of d o u b l e bonds. I s h o u l d p r o v e useful in a s c e r t a i n i n g w h e t h e r e n z y m e s are p r e s e n t in liver t h a t a r e c a p a b l e of r u p t u r i n g t h e benzene ring of estrogen hormones, t h e r e b y c o n v e r t i n g t h e m to muconic a c i d d e r i v a t i v e s . If I 7 - d e o x o e s t r o n e were p a r t i a l l y m e t a b o l i z e d in this way, t h e r e s u l t i n g muconic acid would yield I a f t e r acidic t r e a t m e n t 12. I is of a d d i t i o n a l i n t e r e s t because t h e four carbon a t o m s in its ~,,7'-dilactone rings are d e r i v e d from t h e benzene ring of estrone. B y d e g r a d a t i v e chemical procedures it s h o u l d be possible to isolate these c a r b o n a t o m s i n d i v i d u a l l y . T h e developm e n t of such d e g r a d a t i v e procedures w o u l d a d v a n c e our knowledge of t h e bios y n t h e t i c p a t h w a y s of estrogen f o r m a t i o n from precursors such as cholesterol a n d a c e t i c acidlZ, 14. A liberal gift of estrone from Dr. PRESTON PERLMAN of Schering Corporation m a d e t h i s s t u d y possible. W e are i n d e b t e d to Dr. KEITH FREEMAN" of D o n n e r L a b o r a t o r y for t h e i n f r a r e d s p e c t r u m a n d to Dr. J. N. SHOOLERY a n d Dr. L. F. JOHNSON of V a r i a n Associates for t h e nuclear m a g n e t i c resonance s p e c t r a a n d helpful discussions on t h e i r i n t e r p r e t a t i o n . T h e n o m e n c l a t u r e for I was r e c o m m e n d e d b y Dr. DONALD F. WALKER, Jr., of t h e Chemical A b s t r a c t s Service. This w o r k was s u p p o r t e d b y a g r a n t from t h e U.S. P u b l i c H e a l t h Service. Department of Physiology, f.niversity of California, Berkeley, Call/. (U.S.A.)
HAROLD WERBIN M . R . IMADA I . L . CHAIKOFF
1 j. FISHMAN,R. I. COX AND T. F. GALLAGHER,Arch. Biochem. Biophys., 90 (196o) 318. 2 R. J. B. KING, Biochem. J., 79 (1961) 355z p. H. JELLINCK, Biochem. J., 71 (1959) 665. 4 p. H. JELLINCK, Biochim. Biophys. Acta, 41 (196o) 37. 5 p. IV[.BHARGAVA,H. I. HADLER AND C. HEIDELBERGER,J. Am. Chem. Soc., 77 (1955) 2877e O. HAYAISHIAND Z. HASHIMOTO,J. Biochem. (Tokyo), 37 (195o) 3717 N. ITADAAND O. HAYAISHI,J. Biol. Chem., 236 (1961) 2223. s H. WERBIN AND C. HOLOWAY,J. Biol. Chem., 223 (1956) 651. 9 1R. A. PICKERINGAND H. WERBIN, J. Am. Chem. Soc., 80 (1958) 68o. 10 H. PAULY, R. GILMOURAND G. WILL, Ann. Chem., Liebigs, 403 (1914) 119. 11 O. NEUNHOEFFER AND H. KOLBEL, Ber. deut. chem. Ges., 68 (1935) 255. 12 J . A. ELVIDGE, R. P. LINDSTEAD,B. A. ORKIN, P. SIMS, H. BEAR AND D. B. PATTISON,J. Chem. Soc., (195o) 2228. lZ K. J. RYAN, J. Biol. Chem., 236 (1961) 705 . 14 H. WERBIN, J. PLOTZ, G. V. LEROY AND E. M. DAVIS,J. Am. Chem. Sot., 79 (1957) lO12. R e c e i v e d J a n u a r y I o t h , 1962 Biochim. Biophys. Acta, 58 (1962) 136-138