July 20, 1956
COMMUNICATIONS TO THE EDITOR
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Most of the described compounds showed re-62' (CHC13) [Calcd. for C21H~004c, 72.80; H, 8.73. Found C, 72.74; H, 8.88 and C, 72.86; markable anabolic activity and low androgenic H, 8.741. The assignment of configuration was effect. The following anabolic androgenic ratios, based on rotatory power, taking as reference 4P,5- determined according to Hershberger, Shipley and oxido coprostane-3-one, whose structure has been Meyer,4a t 500 y daily doses, were obtained: demonstrated. Testosterone propionate 0.28 4-Chlorotestosterone acetate .88 Rearrangement of the p- or a-epoxide with boron 4-Hydroxytestosterone acetate .61 trifluoride in benzene yielded A4-androstene-4,17p4-Fluorotestosterone acetate .35 diol-3-one, m.p. 222 -223', strong ferric chloride .72 19-Nortestosterone cyclopentylpropionate test, while acetic acid containing 2% of H2S04 gave 4-Chloro-19-nortestosterone cyclopentylpropionate 1 .82 4-Chloro-19-nortestosterone acetate 1.60 A4-androstene-4,17-P-diol-3-one 17-acetate (4-hydroxytestosterone 17-acetate) m.p. 194- 196'., We are indebted t o Dr. Sala and G. Baldratti of ( a ) +~8 3 O , Xmax 277 mp, E = 12,100 [Calcd. for our Department of Pharmacology for the data on CalHaoO4:C, 72.80; H, 8.73; Found: C, 72.40; anabolic and androgenic potency and to Dr. F. H, 8.801. Canal for the microanalyses. The latter compound formed with O-phenyleneL . G. Hershberger, E. G. Shipley and R. K. Meyer, Proc. SOC. diamine a quinoxaline derivative and with acetic E x(4) P . B i d . M e d . , 83, 176 (1953). anhydride in pyridine a 4,17-diacetate, m.p. LABORATORI RICERCHE FARMITALIA 170-172', ( a ) +l05', ~ Xmax 246 mp, E = 15,500 SEZIONE CHIMICA B. CAMERINO [Calcd. for C23H3205:C, 71.10; H, 8.30. Found: VIA DEI GRACCHI B. PATELLI 35 MILASO,ITALY A. VERCELLONE C, 71.03; H, 8.251. RECEIVED MAY29, 1956 The 4~,5-oxidoetiocholane-17~-ol-3-one in acetone containing dil. rearranged to 2ahydroxytestosterone, m.p. 160-162', ( a ) ~ 166' A NEW PANCREATIC CARBOXYPEPTIDASE (reported2: m.p. 161-162', (LY)D +120'), while Sir: acetate gave the 4a,5-oxidoandrostane-l7~-ol-3-one A hitherto unreported carboxypeptidase has the normal product of epoxide fission androstanebeen found in the euglobulin fraction obtained from 4/3,50,17P-triol-3-one 17-acetate, m.p. 213-215' [Calcd. for C21H3205: C, 69.20; H, 8.85; Found: bovine pancreas after autolysis. It manifests a rapid activity in releasing lysine and arginine from C, 69.07; H, 9.181. Treatment of the 4/3,5-oxidoetiocholane-l7j% the carboxyl terminal position of the synthetic subol-3-one acetate with the requisite hydrogen halide strates, a-N-benzoylglycyl-L-lysine (BGL) and in acetic acid formed the 4-bromotestosterone ace- benzoylglycyl-L-arginine (BGA), which is not tate, m.p. 196-197', Xmax 261 mp, E = 11,600 ascribable to the known carboxypeptidase (CP). [Calcd. for C21H2903Br: C, 61.61; H, 7.14. The new enzyme is tentatively designated basic Found: C, 61.66; H, 7.181, the 4-chlorotestosterone carboxypeptidase (BCP) because of its apparent acetate (3), m.p. 228-230°, (a)D + 1 1 8 O , Xmax 255 requirement for a basic carboxyl terminal amino Like C P i t exists in fresh frozen pancreas as mp, E = 13,300 [Calcd. for C21H2803C1: C, 69.11; aacid. proenzyme which is liberated in active form upon H, 8.01; Found: C, 69.37; H, 8.221 and the 4fluorotestosterone acetate, m.p. 178-180', Amax 241 treatment of pancreas extract with trypsin. In Table I are shown the proteolytic coefficients mp, E = 11,670 [Calcd. for C21H2903F;C, 72.38; of some of the fractions obtained during a typical H , 8.38. Found: C, 72.50; H, 8.611. The reaction of the 4a,5-oxidoandrostane-l7~TABLE I ol-3-one acetate with hydrogen chloride in acetic PROTEOLYTIC COEFFICIENTS~ OF CARBOXYPEPTIDASE PREacid produced the 4P-chloroandrostane-5a, 17PPARATIVE FRACTIOXS WITH DIFFERENT SUESTRATES diol-3-one-17-acetate, m.p. 202-204' [Calcd. for Substrateb CGPC BGLd BGAO CZIH~IO~CI: C, 65.86; H, 8.15. Found: C, 65.68; 1.12 0.85 0.64 H, 8.321 with on prolonged heating eliminated Crude pancreas extract water to give 4-chlorotestosterone acetate. 3 26 2.55 1.90 Crude euglobulin precipitate In the same way the a-epoxide acetate on reac- Barium hydroxide extract 3.62 3.00 2 . 8 5 tion with hydrogen bromide in acetic acid yielded Once crystallized carboxypeptidase 18.00 2.85 2.10 Three times crystallized carboxypepdirectly 4-bromotestosterone acetate. tidase 19.40 The p- and a- epoxides reverted t o testosterone on treatment with aqueous hydrogen iodide in Six times crystallized carboxypepti0 dase 19.10 chloroform or by heating with potassium iodide in acetic acid. The corresponding derivatives were a First order proteolytic coefficient defined as the first obtained also from 116-hydroxytestosterone, 17a- order velocity constant, Kt = log (a/a - x ) , per mg. proN/ml. The extent of hydrolysis was measured on 0.2methyltestosterone, 19-nortestosterone, progester- tein ml. samples by the alcoholic KOH titration procedure of one, 1lp-hydroxyprogesterone, desoxycorticoster- Grassman and Heyde, Z . physiol. Chem., 183, 32 (1929). Substrates employed a t 0.02d M concentration with 0.025 one, cortisone and other steroids and will be deM "tris" buffer PH 7.65 a t 25 . c Hofmann and Bergmann, scribed in forthcoming papers. J . BioZ. Chem., 134,225 (1940). Hofmann and Bergmann,
+
'
(1) P1. A . Plattner, H. Heusser and A. B. Rulkarni. Helo. Chim. Acto, 31, 1822 (1948). (2) F. Sondheimer, St. Kaufmann, J. Romo, H. Martinez and G. Rosenkranz, THISJOURNAL, 76, 4712 (1953).
(3) 4-Chlorotestosterone acetate was prepared also by direct chlorination of testosterone acetate.
ibid., 134, 225 (1940); Folk, Arch. Biochem. Biophys., in
press. Folk, unpublished data. f Five t o 10% hydrolysis in 24 hours at a substrate-enzyme molar ratio of 300 to 1. 0 Very little hydrolysis, only detectable by paper chromatographv after 24 hours a t a substrate-enzyme moldr ratio of 30 tu 1.
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1-d.7s
COMMUNIC.\TIONS TO THE EDITOR
preparation of CP' from frozen pancreas. It is demonstrated that purification of C P through recrystallization practically deletes the BGL and BGA splitting activity. Graded extraction of the euglobulin precipitate from pH 6.3 to pH 8.5 with barium hydroxide, shows decreasing ratios of BGL and BGA splitting activity relative to carbobenzyloxyglycyl-L-phenylalanine (CGPj splitting activity. Action of BCP as a carboxypeptidase is demonstrated in its failure to hydrolyze benzoylglycyl-Llysine amide. The existence of BCP as an enzyme distinct from C P is further demonstrated by the results obtained with crude euglobulin precipitate employing various competitive inhibitors (Table 11). A C P in-
THE ACONITE ALKALOIDS. XXXII. THE STRUCTURE OF DELPHININE
Sir: Recent developments, particularly infrared studies, lead us to propose for delphinine, C33H4jNOq, a hexacyclic modified diterpenoid structure (I) which accommodates the following data. Oxidation furnishes a-oxodelphinine, containing an N-formyl group, and P-oxodelphinine, most likely a d-lactam (uNujol 1645 cm.-l). n'ith the nitrogen contained in a six-membered ring,4 the data suggest nitrogen bridging between the gemmethyl a t C-16 and the angular methyl a t C-17 as in some atisine alkaloids.5-s In p-oxadelphinine the CO would be a t (2-16. The formation of the
TABLE I1 EFFECT OF COMPETITIVE ISHIBITORS O N THE HYDROLYSIS BY PANCREATIC EUGLOBCLIS O F ~7ARIOLWSUBSTRA4TES" Substrate Inhibitor
CGP
BGL
BGA
None 30.0 25.5 2 4 . 5 3-Iodolepropionate," 0.0025 X 11.5 25.7 2 5 . 0 eA%minocaprate,'0.0025 Ji 30.0 0 7.5 6-Amino-n-valerated 0.0025 x 29.0 6 . 0 17.0 a Values reported as per cent. hydrolysis in 20 minutes. mg. of protein The enzyme concentration was 2.2 X N/ml. Conditions for hydrolysis as reported in Table I. * Eastman Kodak Company, Rochester, Iiew York, obtained as the free acid. California Foundation for Biochemical Research, Los Angeles, California, obtained as the free acid. California Foundation obtained as the acid mono HC1.
- OB2
dicyclopentanobenzene hydrocarbon, C17HZ4, from hexahydrobenzoyloxodelphonine acetate9 is explicable in terms of a contraction of ring A during dehydrogenation. l o I n order of lability'l the first methoxyl group is tertiary and placed a t C-11. Subsequent cleavage hibitor, 3-indolepropionate, does not inhibit the of the methoxyl a t C-15 exposes a primary hydroxyl hydrolysis of BGL or BGA. e-Xminocaproate group which can be oxidized via an aldehyde to an N O ~ methyl , ester is resistant to and 8-aminovalerate, inhibit the hydrolysis of BGL acid, C ~ S H ~ ~ whose and BGA and are without effect upon the hydrolysis hydrolysis.'* The proximity of the methoxyls at of CGP. The inhibition of BCP by e-aminocap- C-11 and C-15 is shown by formation of oxido deroate and 8-aminovalerate suggests a similarity in rivatives" accompanying demethylation of isopyro-a-oxodelphinine. The third methoxyl is termechanism of action of the two carboxypeptidases. The p H optimum for partially purified BCP is tiary and placed provisionally a t (2-13. Hydroly7.6 to 7.7. Its action is unaffected by natural oc- sis of the fourth methoxyl at C-4 exposes a secondcurring trypsin inhibitors and diisopropyl phos- ary hydroxyl yielding desmethylanhydroisopyrophorofluoridate. There is no observed hydrolysis a-oxodelphinine. This can be oxidized cia a sixof the following compounds after 24 hours with 0.3 membered ketone, C?7H27N07(yKBr 1712 cm.-l), to mg. crude enzyme N iml. : benzoyl-glycylnitro-L- the dicarboxylic acid, C2iHsiNOlo, whose monoarginine, a: - benzoylglycyl - E - carbobenzyloxy - L- methyl ester (C-4) resists hydrolysis. Hydrolysis of the acetoxy group placed a t C-14 lysine, carbobenzyloxyglycyl-L-proline, carbobenzyloxyglycyl-L-glutamic acid and carbobenzyloxy(1) T o emphasize similarity t o t h e diterpenes. our numbering syqtem P-glycyl-L-histidine. Details of purification, sub- is based on t h a t proposed for abietane by \V, Klyne 1.7. Ciienz. .?or , (1953)l. Our skeleton is formally derived frum abietane 1 , ~ ' strate specificity and inhibition of BCP as well as 3072 cleavage between C-6 and C-7 and closure between C-C, a n d C - 8 w i i h the synthesis of BGA will be reported subsequently. bridging of C-9 and (2-17 b y C-19. The existence of a second type carboxypeptidase (2) W. A. Jacobs and S. W. Pelletier, C h e m . aiid I i a d , , 948 (1V.i.i). (3) T h e erroneous conclusion of W. Schneider [ A n i ! . , 590, 153 has been independently observed by Dr. J. Gladner (1954)] t h a t a-oxodelphinine is a &lactam wa.; based on infrared and K. Laki. They find that upon incubation of sorption a t 1668 cm.-'. This absorption is due t o its K f o r m y l DIP-trypsin (among other protein substrates) group.% (4) W.Schneider [ A r c h , P h a v m . , 2 8 3 ,281 (1950)l obtained piperidine with C P (3X cryst. DFP-treated 50-fold molar excess, S:E = 2 3 ) , one equivalent of lysine is from delphinine with zinc dust. (5) K. Wiesner, R. Armstrong, M. P. Bartlett a n d J . A . I l d w a r d i . rapidly r e l e a ~ e d . ~ Chem. a n d Itid., 132 (195-1). 311-
(1) ?VI.L. Anson, J . Geiz. Physiol., 2 0 , 663 (1937). (2) K . Hofmann and M. Bergman, J . B i d . Chem., 130, 81 (1939). (3) E. Elkins-Kaufman and H. Keurath, ibid.,178, 645 (1945). (4) Personal communication, J. Gladner and K. Laki, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, manuscript in preparation.
RESEARCH ASSOCIATE,AMERICANDENTALASSOCIATIONAT THE XATIOSAL IXSTITUTE OF DENTAL RESEARCH. SATIOSAL ISSTITUETS OF HEALTH, PUBLIC HEALTH SERVICE, u.s.DEPT. O F HEALTH,EDUCATION AND WELFARE BETHESDA, h l D . J. E. FOLK RECEIVED MAY3 , 1936
(6) S. W. Pelletier and W. A . Jacobs, THISJ O I ' R N A L , 76, 44!3ti (1951). (7) S . W. Pelletier and W. A. Jacobs, Chein niiii t i i d . , 138.7 (8) K. Wiesner and J . A. Edwards, E x p e i i r n t i u . 11, 255 (19 (9) W. A. Jacobs and C. P. Huebner, J . H i o l C h ~ i i i, 170, 20:) (1947). (10) Precedent for such a contraction is availahle in t h e cevine [W.4. Jacobs and S. W. Pelletier, J Oyg. Cifvrii , 18, 7(i3 ( 1 THISJOURNAL. 78, 191-1 (1950)l (11) W.4.Jacobs and Y . Sato J B i d . Chuiia., 180, 133 (113.74) (12) W. A. Jacobs and S . W. Pelletier, Tins JOUKNAI., 76, l l i l (lQ5-1).
