Nov. 20, 1958
COMMUNICATIONS TO THE EDITOR
6147
+)2-TROPINONE
tropanol. This is a case wherein metal-alcohol reduction of an unhindered ketone affords preWe wish to report the synthesis of ~(+)2-tropin- dominantly if not exclusively the axial alcohol. one, a key degradation product of the alkaloid STERLING-WINTHROP RESEARCH INSTITUTE M. R. BELL dioscorine.' L-Cocaine hydrochloride2 was hydro- RENSSELAER, KEWYORK S. ARCHER RECEIVED OCTOBER 4, 1958 lyzed to ecgonine, which in turn was treated with phosphorus oxychloride to produce the unisolated acid chloride of anhydroecgonine. Cold aqueous HYDANTOINJ-PROPIONIC AClD : A NEW URINARY ammonia converted the chloride to L( -)-anhydroMETABOLITE OF UROCANIC ACID ecgonine amide, m.p. 142.5-145', [ffIz5D -51.2' Sir : (1% in HzO). Some unidentified acidic metabolites have been (Anal. Calcd. for C9H14N20: C, 65.03; H, 8.49; N, 16.86. Found: C, 65.27; H, 8.35; N, found in urine after administration of radioactive 16.66).4 Treatment of the amide with sodium histidine and urocanic acid (I) to mammals.' hypochlorite in aqueous methanol and then acid They could not be accounted for as intermediates in hydrolysis furnished L( +)2-tropinone, b.p. 60-61 ' the known catabolic pathway of I which proceeds (0.55 mm.), m.p. cu. 27', [ c x ] ~ O D +23.0' (1.6% through breakage of the ring to formiminoglutamic in H20) (anal. Calcd. for CsH13NO: C, 69.03; H, acid (11) and finally glutamic acid.* Alternate 9.41; N, 10.06. Found: C, 69.36; H, 9.46; N, pathways have been demonstrated in bacteria3 and have been induced in vitro by the addition of 9.89). The infrared spectrum showed carbonyl absorp- oxidants (dichlorophenolindophenol (111) or ferrition a t 5.82 p (CHC13 solution and liquid film). cyanide) to I in the presence of purified liver uroIt was suggested to us by Dr. B. Witkop The methiodide, m.p. >330', ( a n d . Calcd. for ~ a n a s e . ~ C9H1dNO: I, 45.14. Found: I, 45.28) absorbed that the hydantoin structure could account for the a t 5.76 p (Nujol). The hydrobromide, m.p. 266- marked acidity of these unknown compounds. Ring labelled (2-CI.l) I was made enzymatically 266.5', reconvertible to the parent base, crystallized from methanol as a methanolate which was devoid from 1.-histidine (Nuclear-Chicago), 6 and 4.8 pc. of carbonyl absorption (KBr disc) and was therefore (9.3 mg.) were injected intraperitoneally into a 200formulated as the hemiketal (anal. Calcd. for Cs- g. white male rat. Urine was collected under H13N0.HBrCH40: C, 42.86; H, 7.19; Br, 31.69; toluene; 10% of the injected radioactivity was CHBO, 12.31. Found: C, 42.78; H, 6.84; Br, excreted during the first 12 hours. Urine was chromatographed directly on Dowex32.20; CH30, 11.78).5 Apparently the proximity of the positively 1X8-acetate (100-200 mesh, 50 drops/tube). charged nitrogen in the 2-tropinone salts to the Samples were collected automatically, dried, and trigonal carbon is responsible for both the shift of counted on a Packard Tri-Carb liquid scintillation ketonic absorption to lower wave lengths and the counter after the addition of hyamine and phosphor.6 Figure 1 shows the elution pattern. Unready solvation of the carbonyl group. Reduction of 2-tropinone with lithium aluminum hydride gave mainly 2a-tropano1, m.p. 73-76' (anal. Calcd. for CEH~LNO: C, 68.04; H, 10.71; N, 9.92. Found: C, 67.61; H, 10.63; N, 9.88), (hydrochloride, m.p. 268-269.5' (dec.) ( a n d . Calcd. for C E H ~ & ~ N O N,: 7.88. Found: N, 7.81), and a small amount of the liquid 20-tropanol isolated as the hydrochloride, imp. 326-328' (dec.) ( a n d . Found: N, 7.70). Reduction of 2tropinone by a slight modification of Dev'ss sodium-propanol-2 method afforded in about 5SY0 yield, 2P-tropanol substantially free of the alpha isomer (vapor phase chromatography and infrared spectrum). Since the intensity of the broad hydroxyl band in the spectrum of the solid isomer C 50 I CC was concentration dependent and the intensity of TUBE NJWBEE the sharp hydroxyl band of the liquid isomer did +Gwdie-st 0 5h Acetic A c i d - Z C O r . H2C 9 e s e r r o r - c 6 h Acetics Atid not change on dilution, the latter alcohol shows intramolecular hydrogen bonding and must be 2P- Fig. 1.-Urinary metabolites of 2-C" urocanic acid in the rat. L(
Sir:
(1) D. E. Ayer, G. Biichi, P. Reynolds Warnhoff and Dwain M. White, THISJOURNAL, 80, 6146 (1958). (2) E. Hardegger and H. Ott, Heiv. Chim. Acta, 38, 312 (1955). (3) A. Einhorn, Ber., 20, 1221 (1887). ( 4 ) Analyses and spectral determinations were carried out under the supervision of M. E. Auerbach and F. C. Nachod, respectively. (5) Earlier, R . E. Lyle, e f nl., J . Oug. Chem., in press, found t h a t I-methyl-3-piperidone behaved similarly, ;.e., t h e base absorbed at 5.82 p (liquid film) and the corresponding hydrochloride formed a stable hydrate t h a t was transparent in t h e carbonyl region in the infrared. We wish to thank Dr. Lyle for supplying this information in advance of publication. (6) S. Dev, J. I d . Chem. Soc., 3 3 , 7G9 (1933).
(1) M. Kraml and L. P. Bouthillier, Canad. J. Biochem. Physiol., 34, 783 (1956); G. 'Wolf, P. L. Wu and W. W. Heck, J. B i d . Chem., 222, 159 (1956). ( 2 ) H. Tabor, Pharmacal. Rev., 6 , 299 (1954). (3) K. ichihara, Y. Sakamoto, H. Satani, N. Okada, S. Kakiuchi, T. Koizumi and S. Ota, J . Biochem. ( J a p a n ) , 43, 797 (1956). (4) A. Miller and H. Waelsch, J . B i d . Chem., 228, 365 (1957); R. H. Felnberg and D. M. Greenberg, Nature, 181, 897 (1958). (5) A. H. Mehler, H. Tabor and 0. Hayaishi, Biochem. Preps., 4, 50 (10.55). ( 6 ) J. M. Passmann, N. S. Radin and J. A. D. Cooper, Anal. C h e m . , 28, 484 (195G).
614s
1-01,so
COMMUNICATIONS TO THE EDITOR
changed I (peak A) was identified by recrystallization with carrier from water to constant specific activity. The radioactivity in peak B could be volatilized by treatment with ammonia and acidification with formic acid, identifying it indirectly as II.’ Peak D was shown to be hydantoin-5propionic acid (IV) by recrystallization with carrier to constant specific activity from both ethanol-benzene and water and co-chromatography in six solvents with the synthetic compound.8 Synthetic IV (Found: C, 41.69; H, 4.GO; N, 16.29) prepared from L-glutamic acidg was eluted in the identical position as peak D. The dotted line in Fig. 1 is synthetic radioactive IV. Peaks A-D have 72, 1.1, 1.5 and 8.5% of the urinary radioactivity, respectively. Incubation of I with rat liver slices forms small amounts of IV in the presence or absence of 111; radioactive IV is present in monkey and human urine after intravenous C14 histidine. The biochemical steps from I to IV have not yet been elucidated, Acknowledgment.-The authors are grateful to Dr. Herbert Tabor for his helpful criticism. ( 7 ) U. A. Borek and H. Waelsch, J . Bioi. Chem., 205, 459 (1953). ( 5 ) Synthetic I V sprayed with 0.1 X I AgKOs: 0.1 M NHIOH (1: 1) is white against a brown background. Radioautograph spots matched the outline and position of stained spots exactly. RF values for benzene: 1-butano1:methanol:HzO (1:l:Z:I ) , 2-hutano1:formic acid:HzO (lQ:Z:G),acetic acid:l-butano1:ethyl acetate:H20 ( l : l : l : l )ethanol: , etber:Hs0:7.4 N NHa (4:5:1:0.1), 1-propano1:l N acetic acid (3:1), 2-propanol:NHa:H?O ( 8 : l : l ) were 0.64, 0.07, 0.68, 0.13, O.GS, 0.07, respectively. 19) €1. D. Dakin. Biorhem. J . , 1 3 , 396 (1919). LABORATORY OF CLINICAL SCIENCE NATIONAL INSTITUTE O F MENTAL DONALD D. BROWN
IIEAL~H MARIANW. KIES INSTITUTES O F HEALTH BETHESDA, MARYLAND RECEIVED JULY31, 105s NATIONAL
MICROBIOLOGICAL TRANSFORMATIONS. III.’ THE HYDROXYLATION OF STEROIDS AT C-9 Sir:
when treated with pyridine and acetic anhydride. Fermentation of I with a species of Arthobacter (B 20-17s) that converts 4-androstene-3,l’i-dionc to 1,4-androstadiene-3,17-dionein excellent yield, gave 9,lO-seco-3-hydroxy-1,3,5(10)-androstatriene9,17-dione. The latter compound was purified as its acetate, n1.p. 143.5-146’, which proved to be identical in all respects (m.p., mixed m.p., and 10)infrared) with the 9,10-seco-3-acetoxy-l,3,5( androstatriene-9,17-dione reported previously.’ Thus, the pOSitiOJlS of the three oxygen atonis in 1 were established. The Sa-configuration was assigned to the new hydroxyl group because of its molecular rotatory contribution ( A M D ~ ~ I = - l S ) 4 and because of the recent evidence that microbiologically introduced hydroxyl groups have the same configuration as the hydrogens replaced.5 I n the aromatization-degradation of 4-androstene-3,17-dione i t seems probable that this species of Nocardia6 first hydroxylates a t C-9 then introduces the AI-double bond. This is just the opposite sequence originally found with Psezidonzonns. A paper chromatographic study of the fermentawith tion of 9~u-hydroxy-4-androstene-X,l7-dione Pseiidomonas showed the formation of, a t most, only trace quantities of phenolic material. Tl-itli Pseudomonas the sequence in which the reactions occur seems to be limited. ( 4 ) T h e molecular rotatory contribution o f the Da-hydroxyl groui) A. S. IIallsworth and €1. 1%. in 38-acetoxyergostau-~a-ol was -31. Henbest, J . Chem. SOL., 4604 (19;7). T h e nlolecular rotatory COIItribution o f the new ( 8 or 9) hydroxyl group in the steroids h y J r o x y l ated with Nrlicosljliiwa piv:;ic>vntr, S f x i o r parasilictcs, Xi‘u~ov c!’i.vrflcyaiiiis and A’eurospova S I ’ U S S I ~ (Ref. 2) indicates t h e prohaliility of Sa, rather t h a n 8 p , hydruxylation. See: S. H . Eppstein, P. D. hleister, H. C. Murray and D. H. Peterson, mones,” Vol. S I V , 388 (1956), Academic Press, Inc., iiew T o r k , li,Y. However, t h e specific rotation of the previously described 88 (or 9a)-hydroxy-4-androstene-3,17-dione,la m.p. ?14-217‘o, [a][> 1 G A O (CHCIa), obtained via the hydroxylation of 11-deoxycrirtiwl wit11 11. Pirifoume, does not agree with ours. ( 5 ) (a) M. H a y a n o , &I. G u t , R. I. Dorfman, 0. K. Sebek aud D . H. Peterson, Trixs J O U K N A L . 80, 2376 (1955); (b) E. J . Corey. G. A. Gregoriou and D. €1. Peterson, ibid.,8 0 , 2338 (1928). (0) n’e have isolated another strain of Nucardia (A20 0 ) which apparently follows the alternnt e w l i i e n c e .
+
We wish to report the microbiological prepara- G. D. SEARLE ASD COMPANY tion and proof of structure of 9a-hydroxy-4-andro- P. 0. Box ,5110 11. hI. Donsox I
