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May 20, 19.54
matographic determination of hippuric acid, which emerged a t about 245 ml. from the Dowex 2 column. Normal adult males may excrete 1.0 to 2.5 g. of hippuric acid per day, a value which is higher than others previously reported and accounts for 65 to 75Oj, of the observed conjugated glycine in 24 hour urines2 Hippuric acid and PAG together account for about half of the 2 g. of bound amino acids excreted daily. The quantities of PAG and hippuric acid observed in the urines from fasting individuals, were similar to those reported above, indicating that both compounds probably are normal metabolic products, and do not arise only as a result of the ''detoxication" of dietary precursors. I n phenylpyruvic oligophrenia, PAG excretion was 2.4 g. per day (cf. 7) whereas the quantity of hippuric acid was diminished to about 0.3 g. I n Wilson's disease, urinary hippuric acid was similarly reduced in amount but PAG excretion was normal. We wish to acknowledge the technical assistance of Miss Joyce F. Scheer.
PSa and PSm are not identical as claimed by Marker and co-workers.6 On acetylation a t room temperature in pyridine-acetic anhydride, 20-iSa and 20-iSm both form monoacetates; 20-iSa acetate (m.p. 167O, [(Y]25D +30°; Calcd. for C29H4604: C, 75.95; H, 10.11. Found: C, 75.94; H, 9.94); Calcd. 20-iSm acetate (m.p. 160°, [cx['~D -49'; for C29H4604:C, 75.95; H, 10.11. Found: C, 75.77; H, 10.05). Infrared spectra of both compounds showed a peak a t 1732-1735 kr.7of strength corresponding to a monoacetate. Treatment of 20-iSa and 20-iSm with acetic anhydride a t re$ux or a t 200' in a sealed tube resulted in smooth formation of PSa and PSm (after hydrolysis of the acetates).
I
= Natural Sapogenins Rz = CH, R1 = H,
LABORATORIES OF THE WILLAIMH. STEIN ROCKEFELLER INSTITUTE ALEJANDRO C. PALADIN+ FOR MEDICAL RESEARCH C. H. W. HIRS NEWYORK21, N. Y. STANFORD MOORE RECEIVED APRIL9, 1954 Fig. 1.-Configuration (7) L. I. Woolf, Biochem. J., 49, ix (1951). (8) Rockefeller Foundation Fellow, 1951-1953.
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I1 = 20-Isosapogenins R1 = CHI, Rp H
of natural and 20-isosapogenins at c20.
Both 20-iSa and 20-ism have complex infrared spectra in the region 650-1400kr. associated with the STEROIDAL SAPOGENINS. XIX. STEREOCHEMIS- spiroketal linkage a t C22.82QThe spectra of these TRY OF SAPOGENINS AND CHOLESTEROL AT CAR- two steroids are completely different from each BON 201 other and also from Sa and Sm; among others 20Sir : iSa has strong bands a t 985, 965, 951, 917 and 905 The configuration of the methyl and hydrogen kr.; 20-iSm a t 974,964,920 and 897 kr. groups attached to the asymmetric CZOof steroid Catalytic hydrogenationlo of 20-iSa and 20-iSm sapogenins has never been determined. We have resulted in formation of D20-iSa (m.p. 167', [(rIzsD established that naturally occurring sapogenins of Calcd. for C27H48O3: C, 77.46; H, 11.08. both the 22b- and 22a-spirostane series have struc- -8'; Found: C, 77.57; H, 10.98; diacetate, m.p. 96O, ture I a t C2,,. We have also prepared for the first Calcd. for C2gH&,: 74.06 H, time a new series of 20-isosapogenins with structure [cY]"D -3'; H, 10.17) and D2010.025. Found: C, 74.08; 11. The evidence for these formulations follows. Found: C, ism (m.p. 161°, [ ( Y ] ~ ~ D+3O; Calcd. for c 2 7 PSa2t3 (m.p. 169-170°, [(rIz6D +12'. H4603: C, 77.46; H, 11.08. Found: C, 77.73; +20°) H, 77.79; H, 10.63) and PSm (m.p. 161°, [cY]*~D 11.02; diacetate, m.p. 96', [ a I z 5 D -4'; Calcd. on brief treatment a t room temperature with alcofor C2QH6005: C, 74.06; H, 10.025. Found: C, holic hydrochloric acid or 24 hours in ethanol10.09). As with DSa and DSm,8,g 74.36; acetic acid form two new compounds which we have D20-iSa H, and D20-iSm do not have complex infradesignated as 20-iSa and 20-iSm, respectively. The red spectra in the region 65G1400 kr. and have esnew compounds are isomeric with Sa and Sm; sentially identical spectra, which differ from that of Calcd. 20-iSa (m.p. 176-177", [(r]"D +31.9'; for C27H4403: C, 77.83; H, 10.65. Found: C, DSa." However, their respective X-ray diffrac77.70; H, 10.62); 20-iSm (m.p. 185O, [ C Y ] ~ ~ Dtion powder patterns are completely different. ( 6 ) R. E. Marker, et al., ibid., 61,3592 (1939); 62, 648 (1940). -60'; Calcd. for C27H4403: C, 77.83; H, 10.65. For the spectroscopic symbolism, cf. J. Optical SOC.Am., 43, Found: C, 77.92; H, 10.74). Refluxing 20-iSa 410(7) (1953). and 20-iSm in alcoholic hydrochloric acid gave, (8) M. E. Wall, C. R. Eddy, M . L. McClennan and M. E.Klumpp, respectively, Sa and Sm. Thus as we previously Anal. Chem., 24, 1337 (1952). (9) R . N. Jones, E. Katzenellenbogen and K. Dobriner, THIS indicated4 and later was shown by another group,s JOURNAL, 75, 158 (1953).
c,
(1) Paper XVIII. 76, in press (1954).
M.M. Krider and M. E. Wall, THISJOURNAL,
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(2) Abbreviations used in this paper: Sa = sarsapogenin; Sm = smilagenin; P = pseudo; D = dihydro; 20-i 20-iso. T h u s PDSa = pseudodihydrosarsasapogenin. (3) All melting points obtained with Kofler micro hot stage. Rotations in chloroform with exception of PSa and PSm which were in dioxane. Infrared spectra were obtained with CSn solvent. (4) M. E. Wall, C. R . Eddy, S. Serota and R. F. Mininger, Tars JOURNAL, 75, 4437 (1963). (6) I. Scheer, R . B. Kostic and E. Mosettig, ibid., 76, 4871 (1953).
(10) R. E. Marker and E. Rohrmann, ibid., 61, 846 (1939). (11) We have found that compounds isomeric at carbon 25 cannot be distinguished by infrared spectra which are essentially identical. However, their X-ray diffraction patterns are markedly different. Compounds differing both a t Cns and Czr can be distinguished by infrared spectra. Thus in the case of the infrareh spectra of PSa, PSm; DSa, DSm; DaO-iSa, DPO-ism: each pair has essentially identical spectra characteristically different from every other pair. Each individual compound has a characteristically different X-ray diffraction pattern. Full details of these findings will be presented in a detailed paper which will be submitted to THISJOURNAL.
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Catalytic hydrogenation of PSa and PSm diace- which completely substantiatesKlyne’s formulation tates followed by alkaline hydrolysis yielded the for cholesterol. Marker and Turnerlg converted known DPSa and DPSml2 which were found to be diosgenin to cholesterol by a route which could not identical to D20-iSa and D20-iSm, respectively. (I) found in affect the acid stable C ~ configuration O Mild oxidation of 20-iSa and 20-ism with Cr03- all natural steroidal sapogenins. Marker and copyridinels gave the respective 3 keto derivatives; workersZ0s2lalso showed that diosgenin, tigogenin 3 keto-20-iSa (m.p. 15lo, [ a I z 5+20°, ~ strong ke- and smilagenin all have the same side chain, a fact tonic band a t 1714 kr.; Calcd. for C27H4203: C, also confirmed by infrared s t u d i e ~ . ~Conse.~ 78.21; H, 10.21. Found: C, 78.24; H, 10.04); quently the side chain configurations of cholesterol 3 keto-20-ism (m.p. 162”, [ a I z 5-55’; ~ ketonic and smilagenin a t CZOare identical. We have shown band a t 1714 kr.; Calcd. for C27H4203: C, 78.21; that the CZO configuration of smilagenin is 20-alpha. H, 10.21. Found: C, 78.14; H, 10.18). Reflux Hence cholesterol and most other natural sterols with alcoholic HC1 resulted in formation of the and bile acids which have been related to it have known 3 keto-Sa (sarsasapogenone), map. 223’ and the 20-alpha configuration with respect to the rest 3 keto-Sm (smilagenone), m.p. 188’ identical with of the molecule. the products of CrO3-pyridine oxidation of Sa and These findings confum by an independent route Sm. the previous conclusions of Wieland and Miescher.22 Mild oxidation of 20-iSa and 20-ism with Cr03- These workers showed that A5-3P-acetoxy-bisnoracetic acid yielded amorphous acids which on treat- cholenic acid could be converted to A6-pregnen-3P,ment with KOH in t-butyl alcohol were smoothly 20a-diol as a result of the action of perbenzoic acid. cleaved to the known 16-pregnen-3,20-dione, (m.p. Turnerza later showed that this type of reaction 20&201O, [ a I z 5 D +69.3’, Am,, 239 mp, log E 3.98). proceeds with retention of configuration. Hence Similar treatment of DBO-iSa and D20-ism also the bisnor-cholenic acid and the longer chain bile resulted in formation of 16-pregnen-3,20-dione. acids from which it can be derived have the 20Under similar oxidative conditions the linkage be- alpha configuration. tween C ~ and O C22 in Sa, Sm, DSa, DSrn is not af(19) R. E. Marker and D. L. Turner, THISJ O U R N A L , 63, 767 11941) fected. (20) R . E. Marker, T. Tsukamotu and D. L. Turner, rbid., 62, 2525 The data presented permit a reasonably certain (1940). (21) R. E. Marker, E. Rohrmann and E. M. J o n e s , i b i d . , 62, 1162 assignment of configuration of steroidal sapogenins a t (220. Molecular models constructed for the two (1 940). (22) P. Wieland and K . Miescher, Hclc. Chim. Acfa, 32, 1922 (1949). possible geometrical isomers show that I is under (23) R. B . Turner, THISJ O U R N A L , 72, 878 (1950). relatively little strain whereas in I1 the methyl UNITEDSTATESDEPARTMENT OF AGRICULTURE groups attached to carbons 13 and 20 put a tre- AGRICULTURAL RESEARCH SERVICE MONROE E. WALL EDDY UTILIZATION RESEARCH BRANCHC. ROLAND mendous strain on ring E. The configuration I1 is EASTERN 18, PENNSYLVANIA SAMUEL SEROTA assigned to 20-isosapogenins. It is in accord with PHILADELPHIA RECEIVED MARCH13, 1954 the facile oxidative cleavage of such compounds and their dihydro analogs, and with the formation of pseudosapogenins on refluxing with acetic anhy- STEROIDAL SAPOGENINS. XX. CONFIGURATION dride. Configuration I is assigned to the more staOF SPIROKETAL SIDE CHAIN AT CARBON 22’ ble naturally occurring steroidal sapogenins. For- Sir : mation of 20-isosapogenins is not confined to sarsasaIn a recent communication Scheer, Kostic and pogenin and smilagenin but has been observed with Mosettigz state that Saa and Sm are not isomeric a t diosgenin, tigogenin and hecogenin indicating it is a both C22 and C Zas ~ previously believed4 but differ general reaction. only a t Czs. We feel this view is incorrect. Not The configuration of cholesterol and related ster- only is there excellent evidence available to show ols and bile acids a t CZOis still unsettled. Fieser that Sa and Sm are isomeric a t (222, but in view of and Fieser assigned the non-relative designations the establishment of the configuration of steroidal 20-a or 20-b to differentiate the side chains of such sapogenins a t Cz01 it is now possible for the first ~ t e r 0 i d s . l ~Based largely on optical rotation dif- time to designate the actual configuration of Sa and ferences, they later assigned (in terms of their C20 Sm at CZZ. convention) the relative configuration 20-beta to The evidence that Sa and Sm are isomeric at CZZ the side chains of cholesterol and bile acids.16*16 is convincing: (a) Sa and Sm have different infraKlyne” deduced from the X-ray studies of Carlisle red spectra in the region 850-1350 K.6p6 These are and Crowfootls that the cholesterol side chain has believed to be due to the vibrations of the -C-0-Cthe 20-alpha configuration. 0-C- spiroketal system constrained by the two E There is now available direct chemical evidence and F rings. When this system is disrupted, as in (12) R. E. Marker and E . Rohrmann, THISJOURNAL, 62, 521 (1 940). (13) G. I. Poos,G.E. Arth, R. E. Beylen and L. H. Sarett, ibid., 76, 422 (1953). (14) L. F . Fieser and M. Fieser, “Natural Products Related to Phenanthrene,”3rd ed., Reinhold Publ. Corp., New York, h‘. Y.. 1949,
(1) Paper XIX. M. E. Wall, C. R. Eddy and S. Serota, THISJOUR7 6 , 2849 (1954). (2) I . Scheer, R. B. Kostic and E. Mosettig, THIS JOURNAL, 76, 4871 (1953). (3) Abbreviations used in this paper: Sa = sarsasapogenin, Sm similagenin, P pseudo, D = dihydro, 20-i = 20-iso. Thus D20-
pp. vi-viii.
iSa
(15) L. F. Fieser and M. Fieser, ref. 14, pp. 412-419. (16) L. F . Fieser and M. Fieser, Expc~ienria,4, 285 (1948). (17) W. Klyne, C h c m i s l ~ yand Induslry, 426 (1951). (18) C. H. Carlule and D. Crowfoot, Prec. Roy. Soc. (London), 184A, 64 (1946).
(4) R. E . Marker and E. Rohrmann, THISJOURNAL, 61, 846 (1939). (6) M. E. Wall, C. R. Eddy, M . L. McClennan and M. E. Klumpp, Anal. Chcm., 94, 1337 (1952).
NAL,
-
-
-
dihydro 20-isosarsasapogenin.
(6) R . N. Jones, E. Katzenellenbogen and K. Dobriner, THIS 7% 168 (1953).
JOUR”.,
