Jan., 1949 [CONTRIBUTION FROM
INFRARED SPECTROMETRY OF
11- AND 18-OXYGENATED STEROIDS
24 1
THESLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH AND THEDIVISIONOF CHEMISTRY OF T H E NATIONAL RESEARCH COUNClL OF CANADA']
Studies in Steroid Metabolism. VI. The Characterization of 1.1- and &Oxygenated Steroids by Infrared Spectrometry BY R. NORMAN JONES, P. HUMPHRIESAND KONRAD DOBRINER The use of infrared spectrometry for the detection and identification of steroid metabolites in human urine has been reported in a previous publication in this series.2 It has also been demonstrated that the presence of certain functional groups in the steroid molecule can be recognized by infrared spectrometryJa and a distinction between ketosteroids in which the carbonyl group is at the 3, 17 or 20 positions can be made from a determination of the location of the carbon-oxygen stretching vibration in the region between 1660 and 1780 cm.-'. It seemed reasonable to anticipate that a carbonyl group a t positions 11 and 12 might also absorb at characteristic frequencies and a systematic investigation of the infrared spectra of a number of steroids containing ketonic and hydroxyl groups a t these positions has been carried out. The recognition of steroids oxygenated at position 11 is of special importance in connection with steroid metabolism, since carbonyl or hydroxyl groups a t this position are indicative of steroids originating in the cortex of the adrenal gland. Among the twenty-eight steroids isolated from the adrenal gland4 fifteen contain a ketone or hydroxyl group a t this position. It has already been demonstrated5J' that metabolites of 11-oxygenated adrenocortical hormones are present in human urine. Androstanediol-3cu,1lp-one-17, etiocholanol-3a-dione-l1,17, and androstanol-3a-dione-11,17occur in the urine of both normal and diseased person^^^^^^^ while etiocholanediol-3aJ110-one-17, its transformation product Ag~11-etiocholenol-3 a-one-17 and pregnanol-3a-dione-11, 20 have been observed frequently in the urine of diseased persons and rarely in the urine of healthy individualsa8 In addition to these completely characterized compounds, a variety of additional steroids containing a hydroxyl or a carbonyl group a t position 11 may be found in human urine, and some of (1) Published as Contribution No. 1667 from the Laboratories of The National Research Council of Canada. (2) Dobriner, Lieberman, Rhoads, Jones, Williams and Barnes, J. B i d . Chcm., l T S , 297 (1948). (3) Jones, Williams, Whalen and Dobriner, THISJOURNAL, TO, 2024 (1948). (4) T. Reichstein and C. W. Shoppee, "The Hormones of the Adrenal Cortex," Vitamins and Hormones, Vol. I, Academic Press, Inc., New York, N. Y., 1943, pp. 845-413. (5) Mason, J . B i d . Chcm., 168, 719 (1945). (6) Lieberman, Fukushima and Dobriner, Federation of American Societies for Experimental Biology, Proceedings, Vol. VII, 168 (1948). See also Dobriner, Lieberman, Hariton, Sarett and Rhoads, J. B i d Chem., 169, 221 (1947). (7) Lieberman and Dobriner, J . B i d . Chem.. 166, 773 (1946).
R CH3
I
I
H-C-OH
I
I1
CHa I
I11
H-C-CH=C
CE"
!
IV
1
VI1 (n = 0) VI11 (n = 1) IX (n = 2)
X
-H
XI
-H
H-C-OH
XI1
-H
OH
XI11
---COCHs
0-COCH,
XI\'
-H
COOCHI
CHs
I I
I
TABLE I CAKDON-OXYGEN STRETCHING VIBRATION OF
11-KETOSTEROIDS Position of max. cm.-l (CS)
NO,
structure
Compound
Sourcea
.4. 11-Monoketosteroids 3a,Scu-Epoxypregnanol-~~-~rie-l1 Azo,22-3a,9a-Epoxy-22,22'-diphenylbisnorcholenone-l 1 A22-3~,9~-Epo~y-23,23'-diphenyl~iorel~olenone-1 1 A2~-3~,9a-Epoxy-24.24'-diphenylcholenone-l 3 Xa,Ya-Epoxy-23,23'-diphenylnorrholanol-23-onr-l1 3cu,9cu-Epoxy-24,23.'-diphenylcholanol-24-one-1L Azo,22-22,22'-Diphenylbisnoreliolanol-3cu-one-l I Pregnanediol-3a,20(epi)-one-11' Etiocholanedio1-3a,17a-anc-l 1
1713 1713 1713 1713 1713 1713 1713 1710, 1742d 1716"
1
I1 IIr I\
v
VI X XI XI1 B. 11-Ketosteroid Esters 3a,9a-Epoxy-ll-ketobisnorcholanicacid methyl ester VI1 3a,Sa-Epoxy-ll-ketonorcholanic acid methyl ester VI11 3a,Sa-Epoxy-ll-ketocholanic acid methyl ester IX Etiocholanediol-3ru,l7a-one-11-diacetate XI11 3a-Hydroxy-11-ketoetiocholanic acid methyl ester XIV Bar-Hydroxy-11-ketobisnorcholanic acid methyl ester XY 3a-Hydroxy-11-ketonorcholanicacid methyl ester XVI 3a-Acetoxy-11-ketonorcholanicacid methyl ester XVII 3a-Hydroxy-1 I-ketocholanic acid methyl ester XT'III 3a-Acetoxy-1I-ketocholanic acid methyl ester XIX Pregnanediol-3a-20-one-ll-diacetate xx Pregnanediol -3a-20(epi)-one-11-diacetateb lyXl
12 12 12 12 1% I:! 12 9
9
1742,1713 12 1742,1713 12 1745,1713 12 1742,1713 9 1742,1713 12 1742,1710 12 1742,1710 12 1739,1710 12 1742,1710 12 1742,1710 12 1739,1710 9 1739,1710 9 C. 3,l I-, 11,20- and 3,11,20-Ketosteroids and Related Esters Pregnano1-3a-dione-11,20 XXII 1713 8, 9,lZ Pregnanol-3a-dione-l1,20-acetate XXIII 1739,1710 12 Uranedione-3,11, XXI v 1713 5 Pregnanetrione-3,11,20 xxv 1713 1,12 ill. 11,17-*3,ll,l7-Ketosteroids and Related Esters Androstanol-3cudione-11,17 . . 1751,1713 6 Etiocholanol-3a-dione-11,17 ".. 1754,1716 9 Androstanol-3a-dime-1 IJ7-acetate XXVI 1745". 1713 6 Etiocholanol-3a-dione-11,17-acetate . . 1748,1739,1713 9 Androstanetrione-3,11,17 1751,1716" 4 Etiocholanetrione-3,11,17 XXVII 1751,1716" 4,9 A4-Androstcnetrione-3,11,17 (adrenosterone) XXVIII 1751, 1719, 1674 2 E. 11,2O-Diketo-21-acetoxysteroids Pregnanediol-3a-21-dione-l1,2O-acetate-21 XXIX 1758,1732,1713 9 Pregnanol-21-trione-3,ll,2O-acetate WIX 17.58', 1732, 1713 9 .laPregncnol-21-trione-3,11,20-acetate(1 1 -dchvdroI*1758,1732 9 corticosterone acetate) 1716,1674 (1) T. F.Gallagher, Sloan-Kettering Inst., Kew York, N. Y. (2) E. C. Kendall, Mayo Clinic, Rochester, Minn. (3) A. Lardon, University of Basel, Basel, Switzerland. (4) S. Lieberman, Sloan-Kettering Inst., New York, N. Y. (5) R. E. Marker, Pennsylvania State College, State College, Penna. (6) Compound prepared or isolated at Memorial Hospital, Sew York, S. IT. (7) V. Prelog, Eidg, Tech. Hochschule, Zurich, Switzerland. (8) T. Reichstein, University of Basel. Basel, Switzerland. (9) L. H. Sarett, M a c k and eo.,Rahway, N. J. (IO) C. R. Scholz, Ciba Pharmaceutical Products, Inc., Summit, i S.J. (11) E. Schwenk, The Schering Corp.. Bloomfield, E.J. (12) R. Turner, Harvard University., Cambridge, Mass. (13) A. L. Wilds, University of Wisconsin, Madison, Wis. In these compounds the configuration a t position 20 is uncertain and the prefix "epi" is used arbitrarily for convenient reference. c Solvent tetrachlorethane. d Weak band attributed to an impurity. e Broad maximum. Suspension of crystalline material in saturated solution in carbon disulfide. I
.
'
these may be included among compounds which have already been isolated, but which have not, as yet, been fully characterized? 1 1-Ketosteroids.-The recognition of an absorption band associated with the 11-ketone ( 8 ) Lieberman. Dobiiner, ITill Firser and Khoads, J . B i d Chcm.,
173,263 (1948).
group could be achieved unequivocally fronl measurements on 11-monocarbony1 compounds in which there could be no interference from carbony1 groups located elsewhere in the molecule. Unfortunately, the number of such compounds is limited, but through the kind cooperation of Dr. R. Turner and Dr. L. R. Sarett we have obtained
nine 11-ketosteroids which satisfy this criterion. The infrared absorption spectra of carbon disulfide solutions of eight of these substances (I-VI, X-XI) exhibit one maximum only in the carbonyl region a t 1710-1713 cm.-’. The ninth compound (XII) gave a maximum a t 1716 cm.-l in solution in tetrachlorethane (Table IA). Similar data are also listed in Table IB for twelve 11-ketosteroid esters (VII-IX, XIII-XXI), all of which possess two maxima in the carbonyl region a t 1710-1713 and 1739-1742 cm.-’, respectively.
CHJ -c0CH3
I
H-C-OCOCHS
I
CHa
XXI
-‘OCHs
CHIIOCO-C!-H
I
It has been shown previously3 that both the 3- and 17-acyl esters of steroid alcohols as well as the methyl esters of cholanic acid derivatives possess a maximum a t 1739-1742 cm.-’. Consequently in these compounds i t may be inferred that the absorption band a t 1710-1713 ern.-' is associated with the carbonyl group at position 11, and that no interactions occur between the vibrations associated with the ketone group a t this position and ester groups a t positions 3, 17 or on the side chain. 11,2O-Diketones.-In the non-conjugated 20ketosteroids the carbonyl absorption maximum is a t 1706-1710 cm.-‘ and because of the proximity of the 11-ketone and 20-ketone band maxima a simple spectrometric differentiation of these two important structures is uncertain (vide p. 246). Two compounds containing 11,20-diketone grouping have been examined. I n XXII only a single band at 1713 ern.-' occurs, while X X I I I has two bands a t 1710 and 1739 ern.-', the latter due to the ester group at position 3. CHs
I
co
RO’
CHa
CH3
CH?
co
I
I
O
y
y
AI
R2
Ri
xx
The only compound of this kind so far examined is uranedione-3,ll (XXIV) which gives a single maximum a t 1713 cm.-’. Measurements on compounds XXV and XXVII support the evidence from XXIV in indicating that no anomalous displacements of the band positions are associated with the 3,ll-diketone structure.
XXII XXIII
(R = -H) (R = -CO-CHs)
3,11-Diketones.-A similar situation arises in the identification of 11-ketosteroids which contain a second (unconjugated) ketone group a t position 3, as the non-conjugated 3-ketone group absorbs a t 1715-1719 cm.-l which is also very close to the frequency of the 11-ketone band.
In 11-ketosteroids containing a conjugated ketone group a t position 3, the band a t 1674-1677 ern.-' characteristic of this conjugated group occurs a t the expected position. 11,1’7-Diketones.-In 17-ketosteroids in which ring D is saturated, a n absorption maximum occurs a t 1742-1745 cm.-’, but in 11,17-diketones this band is displaced to 1748-1754; the band normal to the 11-ketone a t 1710-1716 cm.-’ is present 0
0
0
Interaction Eff ects.-These measurements indicate that interaction occurs between carbonyl groups a t positions 11 and 17 which displace the bands from their normal positions. A similar interaction effect has been noted previously between a ketone group at position 20 and an acetoxy group a t position 21 in the acetylated derivatives of steroids containing the keto1 side chain.a Compounds XXIX-XXXI ccntain both the 11-ketone and the 20-keto-21-acet~xygroupings and are of particular interest as they are derivatives of adrenocortical stero‘d hormones. The bands a t 1732 and a t 1756-1758 cm.-l, (9) In compound XXVI which contains an acetate group at position 3 in addition to the 11,117-diketone group, the band maxima are at 1745 and 1713 cm.-l. It is presumed that the former band is an unresolved doublet, due to the acetate band at 1739-1742 and a band at 1748-1754 cm.-1. .In the corresponding compound in the etiocholane stereochemical series this band is resolved (see Table ID).
Vol. 73 TABLE IT CARBON-OXYGEN STRETCHING L'IBRATIoN OF
Compound
12-KETOSTEROIDS
Striicture
Pregnanone- 12 .. 3~~-Acetoxy-l2-ketocholanic acid methyl ester XXXII .14-Pregnenol-21-trione-3, 12,aO-acetate XXXIII ~3~~,7~-Dihydroxy-12-ketoetiocholanic acid methyl ester XXXII' 3cu-7a-Didcetoxy-12-ketoetiocholanic acid methyl ester XXXV 3~~-Succinoxy-7~-~icetoxy12-ketoetiocholanic acid methyl ester XXXVI A95 '-3 u-Acetoxy-l2-ketocholenic acid ~~~~1-3~-Acetoxy-12-ketocholenic acid methyl ester ll,l2-Diketonorcholanic acid methyl ester ~3,11,12-Triketonorcholanic acid methyl estel 3,12-Diketocholanic acid methyl ester . .(. See footnote to Table 1. See footnote to Table I. Inflection only
.
characteristic of the 20-keto-21-acetoxy group in addition to the 11-ketosteroid maximum a t 17'10-1715 mi.- occur in all three compounds.I0
Position of malt ( c m solvent CSa
-1)
1710 1742,1710 1758,1732,1716,1677 1739, 1723b 1742,1723 1742,1723 1742,1713,1684 1742,1680 1742,1726 1739', 1726 1743,1716
Source0
S
10,E 10 8 8 8 10 I1 3
12 12
12-Ketosteroids.-Eleven compounds containing a carbonyl group a t position 12 have been examined (see Table 11). I n pregnanone-12 and XXXII the band a t 1710 cm.-' can be assigned to the unconjugated 12-ketone grouping. The spectrum of XXXIII, which contains the acetvlated keto1 side chain and the conjugated ketone group a t position 3 exhibits the maxima characteristic of all four carbonyl groups (cf. Fig. 1). I n compounds XXXIV-XXXVI which contain the 12-keto-17-carbomethoxy grouping, the 12ketone band is displaced to 1723 ern.-' (vide p . 2-26). CIS
CHCH~CH~COOCHi
0 I
/A\,+,.'I '?
CH20COCHz i
0
It may be concluded that a band a t 17.541760 em. is suggestive of the presence of either the ll,l7-diketone or the 20-keto-21-acetoxy grouping. The former structure also produces at 1710-1716 cm.-l whereas he second band is near 1730 cm. -l. The distmction between these two g r ~ u p ings can be confirmed from measurements in the 1200-1250 cm.-l region since a strong band chaxacteristi acetaxy group and lacking in the carbonyl ccurs near 1230 cm. '.
COOCHI
~
(IO) I n XXX the non-conjugated 3-ketone group cuntributes also to the maxlmuin at 1713 c m
-1
no
doubt
In the Ay-12 ketones the maximum is a t 1680This is significantly higher than the 1684 cm:-l.
Jan., 1949
INFRARED SPECTJXOMETRY OF
frequency observed for A4-3-ketosteroids (16741677 cm.-l) and for A16-20-ketosteroids (16661669 cm.-I). The two 11,12-diketones listed in Table I1 possess a maximum a t 1726 cm.-'. The displacement of this band from 1706-1716 to 1726 cm.-l is indicative of interaction in the adiketone structure. Other Ketosteroids.-As these investigations were initiated with the primary object of improving techniques for the identification of steroid hormones and hormone metabolites, attention has been chiefly directed to steroids containing functional groups a t the positions which are of major importance in relation to the hormonal activity. Up to the present time only a few steroids containing carbonyl groups a t other positions have been examined; the results, summarized in Table 111, suggest that the nonconjugated 7-ketone group absorbs near 1719 ern.-', the non-conjugated 16-ketone near 1754 cm.-l, the 3,6-diketosteroid group a t 1723 cm.-' and the A3g5-diene-one-7group a t 1663 cm.-'. One monoketosteroid of uncertain structure kindly supplied by Dr. Heard" and believed to contain a ketone group a t position 15 had an absorption maximum a t 1751 ern.-'. The location of this band is in general agreement with the observation that a carbonyl group in a cyclopentanone ring system absorbs a t a high frequency, and would seem to be consistent with the postulated structure of this compound. 11-Hydroxy Compounds.-The position of the hydrogen-oxygen stretching vibration of the hydroxyl group in a steroid molecule cannot be related t o the position of substitution in the molecule in a manner similar to t h a t for the carbon-oxygen bond of the carbonyl group3 Nevertheless, the presence of an absorption band near 3600 cm.-l in the spectrum of a solution of a steroid is associated with the presence of a hydroxyl group in the molecule.
11- .%ND 12-OXYGENATED STEROIDS
245
1760 1720 1680 Wave number, cm.-I. Fig. 1.-Infrared absorption spectrum of ll-dehydrocorticosterone acetate between 1640 and 1800 cm.- l. Maxima A and B are associated with the acetylated keto1 side chain, maximum C with the ketone group a t position 11, and maximum D with the &unsaturated ketone group a t position 3 (solvent carbon disulfide). 1800
No simple 11-monohydroxy steroids have been available for measurement, but several dihydroxy steroids in which one of the hydroxyl groups is a t position 11have been examined. I n these spectra the normal hydroxyl absorption band near 3600 cm.-l is observed and no behavior specifically relatable to the 11 position is to be noted. I n a compound such as androstanediol-3a, 1lp-one17-acetate-3, where the hydroxyl group a t position 3 is esterified, the absorption band a t 3616 cm.-' can be attributed to the 11-hydroxyl group. Discussion
TABLE I11 The relationships which have been observed CARBON-OXYGENSTRETCHING VIBRATION OF KETObetween the frequencies of the carbonyl band STEROIDS WITH CARBONYL GROUPSAT P o s ~ r ~ o xOTHER s maxima, and the structure for all types of steroids THAN 3, 11, 12, 17 AND 20 Position
of
max. (cm.-1) solvent Compound
Sitostanedione-3,6 AZ2-Sitostenedione-3,6 3a,l2a-Diacetoxy-7-ketocholanic acid methyl ester
cs1 1723 1723 1742 1719
3a-Succinoxy-12~-acetoxy-7-ketoetio-1742 cholanic acid methyl ester 1719 3o-12~~-Dihydroxy-7-ketocholanic acid A~-S-Cholestadiene-one-7 111 3.h 'O>"*-Estrapentaene-one-16 a See footnote to Table I. ( 1 1) t 1946).
1719 1663 1754
Sourcw
5 5 1
8 1 7 13
Heard and McKay, J. B i d . Chem., 181, 371 (1939); 166,677
are summarized in Table IV. I n evaluating these data, i t must be noted that they apply to solutions in carbon disulfide only. The significance which is to be attached to them is dependent on the number of compounds of each type examined, and this is indicated in the third column of the table. The recognition of the 11-ketone group in a steroid by infrared spectrometry is only partially successful, Such identification is possible provided certain other ketone groupings are known to be absent, but non-conjugated ketone groups a t other positions on the hexacyclic rings or in the side chain may absorb quite close to the same frequency. This is certainly true of 3-ketones and 20-ketones, and probably also of ketone groups a t positions 6, 7 and 12. With the spec-
TABLE I1 as also is the observation that the introduction SUMMARY OF POSITIOSS OF CARBO?IYL DA\ I) MAXIMA of the 11-ketone group does not disturb the posiCarbon tlisulfitii. ~ o l u t i o r i ~ tion of these bands. An example is provided by
the spectrum of dehydrocorticosterone acetate (XXXI) in which all of the four carbonyl maxima Carbonyl type c il's lie, ' f
