V O L U M E 2 7 , NO. 5, M A Y 1 9 5 5 cases where y activity of t h e matrix interferes in the -, spectrometric analysis of t h e sample, and where t h e half life is not short enough to permit rapid decay of the interference, chemical separation can be employed. The method possesses a sensitivity of from 0.001 to 1 y for the majority of t h e elements and exhibit8 good accuracy and precipion in this trace range. .4CKNOW-LEDGMENT
=\clino\vledgment ip gratefully given to E:. R. Bowerman fov hip niany valuable suggestions and critirisnis. The authors aldo wish to thank P. H. Keck of the Squier Signal Corps Lahoi.atory for furnishing the single crystal of silicaon grown hy t h e floatiiig zone technique.
813 LITERATURE CITED
(1) Boyd, G. E., ~ N A L CHEM., . 21, 335 (1949). (2) Connally, R. E., and Leboeuf, 31. B., Ibid., 25, 1095 (1953). (3) Hollander, J. M., Perlman, I., and Seaborg, G. T., Reus. M o d . Phys., 25, 469 (1953).
(4) Keck, P. H., Van Horn, IT., Soled, J., and RIacDonald, A , , Rec. Sei.Instr., 25, 331 (1954). (5) Leddicotte, G. W., and Reynolds, S. A , , ,Vucleonics, 8, 63 (1951). ( 6 ) Litton, F. B., and Andersen, H. C., J . Electrochem. Soc., 101, 287 (1954). (7) Lyon, D. W., Olson, C. lI.,arid Lewis, E. D., Ibid., 96, 359 (1949). (8) Lyon. IT,5..a ~ i d~ l a i ~ n i.J.n ~ .J.., Phys. Rev., 93, 501 (1954). (9) Taylor. T.I.. and Hareii., K. K . , .Vrrcleo!iics, 6, 54 (1950). RECEI\LD fur. reyiew Xoi-ember 27, 1Y.54.
Accepted J a n u a r j 21, 1923.
Optical-Analytical Studies on Steroids Reducing Characteristics of Hydroxylated and Ketonic Steroids toward Blue Tetrazolium ANDRE S. MEYER
and
MARJORIE C. LINDBERG
Worcester Foundation for Zxperimental Biology, Shrewsbury, Mass.
The need for implementation of micromethods leading to an assignment of rerognized functional groupings in complex molecules to their correct position instigated an investigation of the reducing characteristics toward Mire tetrazoliuni of a number of A4-3-ketosteroids with a hqdroxjl or a keto grouping in various positions of the molecule. These functions, dependent on their location and configuration, gave rise to varied rates and intensities of color formation. The complex nature of reaction of the .!~‘-3-hetosteroids limited the diagnostic \ alue of those rate nieasurements for structural characterization on a microlew el to certain well-studied examples. The conversion of A4-androstene-3,17-dione to ~*-androsten-6~-0l-3,17-dione and A4-androstene-3,6,17-trione by alkaline blue tetrazolium has been demonstrated.
T
HE increasing importance of tetrazolium salts as hydrogen
acceptors in biological redox reactions with the subsequent formation of deeply colored formazans has stimulated the preparation of new members of this class (for reviews, see 1 , 15-19). Some of these compounds have been introduced recently in steroid chemistry for the qualitative demonstration ( 4 ) and the quantitative estimation ( 5 )8, 10, 13)of corticosteroids. The observation t h a t the non-a-ketolic 6a-hydroxy-A4-androstene-3,17dione ( 2 ) exhibited, when tested on paper (12), a retarded formazan formation with the blue tetrazolium [2,2’-p-(di-o-methoxy)iliphenylene-3,3‘5,5’-tetraphenylditetrazoliumchloride] (16-18) reagent, whereas the analogous 6B-hydroxyl derivative did not, instigated the present investigation of a number of biologically important A4-3-ketosteroids (parent compounds) and their hydroxylated derivatives with respect to their reducing characteristics (Tables I and I1 and Figure 1). This was done in order to determine whether a correlation between the color production and the location of the structural function in the polycyclic iiucleus could be established. This report deals mith the results of such a study. RESULTS
The steroids were exposed t o blue tetrazolium in ethanolic solution a t various alkalinities and temperatures, and the color development was measured a t appropriate intervals of time.
The ohserved absorbance (equal to the negative logarithm of the transmittance) was, for comparison purposes, referred to 0.040 micromole of substance. Steroids with a primary a-keto1 group reduced blue tetrazolium more readily than those formazanproducing rompounds without t,h:it grouping and were thus easily distinguishalile. Representative data for one comImund of each series, investigated u n d ~ ra varirtJ- of conditions, aw listril iu Table 111. Deoxycorticosterone (2) reached maximum intensity of its chromogen in all instances within 45 minutes (6-23 X lo3), whereas the slower forniazan formation of 6a-hydroxy-A4-androstene-3,17-dione (1)was a function of the alkalinity and the temperature. S o t only the rate of color development but also its maximum intensity (reproducibility st50/0) was dependent on these conditions. T h e range of basicity was achieved by the use of three tetraalkylammonium hydroxides. Tetramet,hyl:inimoniuni hydroxide ( T M A H ) had the one disadvantage that the color formed was destroyed to a larger degree, over the estensive reaction period or a t the elevated alkalinity required, than TThen triton B (R1 = benzyl) or choline (RI = p-hydrosj.ethyl) or approximately equimolar solutions of these bases ivith tetramethylammonium hydroxide were used. T h e less reproducible destruction of the formaean (9, 16) did not seriously inipair the react,ion, since the concurrent production of the chroniogen was usually predominant. Although a reaction period of 2 to 3 hours-e.g., conditions 1- and XII-could be considered suitable for general application, it may in certain cases be desirable t o preserve valuable characteristics of the color formation as can be observed under less vigorous conditions in an early stagee.g., I11 and IX. As an expedient, the reaction can he started a t room temperature a t an elevated basicity and, after a few readings, terminated a t a higher temperature, thus keeping the react,ion time within practical limits-e.g., conditions X I . With a more stable formazan compound i t should be possible to carry out the reaction a t higher alkalinities and room temperature in a shorter period of time. The chromogen formation by simple A4-3-ketosteroids with various substituents a t carbon-17 (under conditions IX) is illustrated in Figure 2. Changed conditions influenced the shape of the curves to some extent, although the order of magnitude was in most cases maintained. Similar time curves t o that of compound 3 were recorded for 1la-hydroxy-A4-androstene-3, 17dione ( l l ) , to t h a t of compound 4 for lla-hydroxy (12), 16ahydroxy (13), and 17a-hydroxyprogesterone (14), and to that of
ANALYTICAL CHEMISTRY
814 Table I. NO. 1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20 21
as was observed with 6ahydroxyandrostenedione ( 1) (Figure 3). Furthermore, the 11-keto group in the androstenedione molecule (27) contribiited a notable color (Figure 4). As only a limited number of a n d r o s t e n e d i o n e d e r i v a t i v e s hydroxylated in various positions were known, the study was completed with hydroxylated d e r i v a t i v e s of other A4-3-ketosteroids. T o facilitate a comparison, curves were plotted for the formular absorbance increment of these steroids over their parent compounds determined simultaneously-e.g., the difference in absorbance of compound 1 and of 3 was calculated for each reading (Figure 4). The increment for 7~-hydroxy-A4-cholestenone (23) was high, nearly twice as large as that found for 2a-hydrosytestosterone (24). Removal of the angular methyl group a t carbon-10 (19-norandrostenedione) (26) also gave rise to an intensified color
Compounds Investigated
[Nomenclature Compound Ga-Hydroxy-A4-androstene-3,17-dioneand acetate 11-Deoxycorticosterone Ad-Androstene-3,17-dione Progesterone ( A'-pregnene-3,20-dione) Ad-Androsten-3-one Testosterone (A4-androsten-17pol-3-one) AWholesten-3-one 3-Keto-A'-etienic acid A%~-~4ndrostadiene-3,17-dione A*t'-Androstadiene-3,17-dione 1la-Hydroxy-A~-androstene-3,17-dione 1la-Hydroxyprogesterone 16a-Hydruxyprogesterone 17a-Hydroxyprogesterone lla-Hydrory-3-keto-A~-etienicacid 16a-Hydroxy-A'-androstene-3.17-dione 19-Hydroxy-A~-androstene-3,17-dione 14&Hydroxy-A~-androstene-3,17-dione 12a-Hydroxy-A~-androstene-3,17-dione 11/3-Hydroxy-A4-androstene-3,17. dione G&Hydroxy-A4-androstene-3,17-dione and acetate
according t o ( B ) ] NO. Compound 22 Androstane-3,6,17-trione 23 7/3-Hydroxy-A~-cholesten-3-one 24 2a-Hydroxytestosterone 25 19-Hydroxy-3-keto-A4-etienic acid 26 19-Nor-Ad-androstene-3,17-dione 27 A4-Andros tene-3,11,17-trione 28 Ga-Hydroxyprogesterone 29 6a-Acetoxy-17a-hydroxyprogesterone 30 Ga-Acetoxy-A~-oholesten-3-one 31 Ga-Acetoxy-deoxycorticosterone acetate 32 6a-.~cetoxy-17a-hydroxydeoxycorticosterone acetate 33 19-Norprogesterone 34 19-Hydroxyprogesterone 3.5 2a-Acetoxyprogesterone 36 11-Ketoprogesterone 37 Cortisone (17a-hydroxy-I l-de!iydrocortico>teronel 38 Aldosterone (18-oxocorticosterone) 39 A~-Androstene-3/3,17&diol-16-one 40 Cortisone-21-aldehyde 41 1,4-Cyclohexanedions 42 Hydroquinone (1,4-benzenediolf
Table 11. Compounds with N o Significant Reaction toward Alkaline BlueTetrazolium (under Conditions XII) SO.
43 44 45 46
47 48 49 50
[Somenclature according t o Conipound NO. Androstane-3.17-dione 51 Androstan-3a-o!-17-~ne (androsterone) 52 Androstan-33-01-17-one (epiandrosteronei 53 A~-Androsten-33-01-17-one !drliydroepinndrosteronei 54 A~-Androstnne-3a,l73-di~~l A?-Androstene-3p,17$-diol 56 55 A4-Androstene 57 Ad-Cholestene-3,R-dione 58
A
& &$,&
B
( 5 , 6. 1 4 ) l
Compound Cholestan-3-one enol acetate Estrone Solanidine Isorubijervine Icterogenin Methyl hederagonate 5.5-Dimethyl-l.3-c~cloliexanedione (dimedone) Formaldehyde
& C
C1J3
0
'' 0
D E F Figure 1. Types of structures investigated A . Androstane B. Progesterone (4), R = COCHa 3-Keto-Ad-etienic arid (S), R = COOH C. Corticosterone D. Ad-Cholesten-3-0110 (7) E. Solanidine (53), R = H ; Isorubijervine (54), R F. Methyl hedera onate (55), R = CHs, R' = H Icterogenin (567, R = H, R = O.COGH,
=
formation. T h e increment rates have been found to be similar for various molecules with a particular structural function. A close correspondence vias found for the rates of the increase in color production of the 6a-hydroxyl derivatives of androstenedione (1) and progesterone (28)) and the 6a-acetates of l7a-hydroxyprogesterone (29) and cholestenone (30). I n good agreement xvith these were the analogous curves for the diacetates of Ga-hydroxydeoxycorticosterone (31) and 6a-hydroxy-17a-hydroxydeoxycorticosterone (32) \Then the relatively appreciable destruction of the chromogen of the parent compound was taken into account. The above measurements were made under conditions I11 and I X ; conditions VI1 were less favorable for such a comparison. A correlation was also found with 19-norprogesterone (33) and 19-norandrostenedione (26). Again 19-hydroxypr-o-
OH
compound 8 for lla-hydrox~-3-keto-~4-etiocholenicacid (15). Substances found to produce practically no color are listed in Table 11. ilmong the trsted solvents, purified ethyl alcohol was found to give relatively smallest blank readings (Figure 2). The hydroxylated A'-androstenedione derivatives studied showed remarkably different curves (Figure 3). According to its position and configuration, the hydroxyl group may inhibit the chromogen formation of its parent compound (20 and 21), influence it little (19 and other examples mentioned above), or make an additional contribution to the color production as in compounds 1, 16, 17, and 18. The acetates of compounds 1 and 21, on comparison n-ith the respective free substances, resultcd in the same time curves. Compound 1 obeyed Beer's law a t steroid concentrations from 2 to 8 X 10-6LTf. It is remarkable that androstane-3, 6, 17-trione (22) shoived under conditions I11 approximatply the 2ame rate of formazan formation
4
6
8
I ?
1"
Figure 2. Rate of blue formaaan formation for 0.040 micromole of various A4 -3-ketosteroids under conditions IX Curve numbers correspond to rompound numbers listed in Table I . Absorbance of reaction blank read against ethyl alcohol
V O L U M E 27, N O . 5, M A Y 1 9 5 5
815
It was confirmed that several pure corticosteroids and their acetates produced essentially the same chromogen as did deoxycorticosterone (2). Comparable values were further obtained in 45 minutes of reaction time with 18-oxocorticosterone (aldosterone) (38) under conditions I X and with the cyclic secondary a-ketolsteroid A6-androstene-38, 17,8-diol-16-one (39) under conditions 111. T h e delayed formazan formation of the two other types of secondary m-ketolsteroids examined (16, a 17,16m-ketol and 21, a A4-3,2a-ketol) has been mentioned. T h e pronounced difference in the reducing characteristics of the 16,178-keto1 and the 1 7 , 1 6 ~ k e t oisl noterrorthy. Cortisone aldehyde (A4-pregnen21-oxo-17a-o~-3,1lJ2O-trione) (40) led to a gradual formation of
Figure 3. Rate of blue formazan formation for 0.040 micromole of hydroxylated A4androstene-3,17-dione derivatives under conditions XI1
Table 111. Blue Formazan Formation under Various Conditions
Condition NO.
I
I1 111 IV V VI VI1 VI11
IX X
XIS
SI1 503
Maximum Color Formation, -4bsorbance per 0.040 Micromole Ga-Hydroxyandrostenedione (1) D ~ ~ Nornial- Temp., Absorbafter ity Base C. Hours ance 45 min. TMAH 0,0055 37 0.71 30 0.77 25 0.017 0.61 0.60 TMAH 20 TRlAH 37 0,017 0.60 8 0.71 0.56 TMAH 4 37 0.028 0.68 TMAH 37 0.055 2 ,5 0.41 0.65 Choline 37 0,066 3.5 0.45 0.69 Triton B 0.060 37 3.5 0.48 0.68 Triton B 0.060 45 0.57 2.5 0.82 ThIAH 0.008 37 8 0.62 0.74 Choline ThlAH 50 4 0.60 0.76 Choline TllAH 25 1 .i 0.60 0 74 Choline 50 1.5 TlIAH 37 3 0.53 0.72 Choline
Color developed b y exposure first t o 25. C. for 1.5 hours and then t o
c.
gesterone (34) and 19-hydroxyandrostenedione (17) were comparable in their chromogens, and 19-hydroxy-3-keto-A4-etienic acid (25)developed an increment of the same maximum intensity, however, at a slower rate (Figure 4). Furthermore, under conditions XI1 the increments of 2a-hydroxytestosterone (24) and 2a-acetoxyprogesterone (35) agreed well. Adrenosterone (27) and 11-ketoprogeGterone (36) showed a similar increase in color formation over their parent compounds, whereas that of cortisone ( 3 7 ) appeared to be less. Analogous comparisons should be possible a i t h other polycyclic systems, since the tested steroidal alkaloids (compounds 53 and 54) and triterpenes (compounds 55 and 56) without conjugated ketonic grouping did not react with alkaline blue tetrazolium (Table 11).
~
~
~
$
~
-
Figure 4. Increment of blue formazan formation rate for 0.040 micromole of oxygenated or 19-nor-A4-3-ketosteroids under conditions IX Color formation of parent compounds, see Figure 1
the formazan which eventually amounted to one third that of the standard deoxycorticosterone. 1,4Cyclohexanedione (41)) hoxever, gave after 45 minutes a chromogen of the magnitude of the standard (under conditions I, a someiThat higher and under conditions X I I , a somewhat lower value), and hydroquinone (42) showed (in both instances) half the intensity of the latter, whereas dimedone (57) did not reart. METHOD
Commercially available steroids were purified by direct crystallization or after chromatography, while other compounds were prepared according t o published procedures. The 95% ethyl alcohol (c.P.) was tested for its reducibility of blue tetrazolium by running a reaction blank. Some lots of distilled ethyl alcohol could be used as such; other batches produced too high blank readings and had to be refluxed over alkaline blue tetrazolium, and redistilled. In cases where the reaction blanks were still high, a prior refluxing over finely ground hydrated ferrous sulfate for 21 hours iva? found advantageous. T h e purified ethyl alcohol \vas stored under nitrogen in a dark bottle. The allcaline ethyl alrohol reagent was freshly prepared as follovcs. For conditions I11 (Table I I I ) , 6 ml. of 10% (-1.liV)
ANALYTICAL CHEMISTRY
816 aqueous tetramethylammonium hydroxide were mixed with 94 nil. of the purified ethyl alcohol and, if necessary, filtered under a nitrogen atmosphere; t h e reagent lvas thus 0.066N in the hydroxide. Because, in the course of the reaction, i t was diluted to four times its volume, the final alkalinity (indicated in Table 111)was 0.0165LV, Choline (-2.61V) and triton B (-2.ON) were employed as 30% aqueous solutions. The final ethyl alcohol content of the niedium ranged thus b e t w e n (32 to %yo,varying for t,he conditions employed. .4n aliquot of 0.50 nil. containing a known quantity betiyeen 10 t,o 16 y of steroid (-0.045 micromole) in purified ethyl alcohol was pipetted into a glass-stoppered 10 X 75 mm. Coleman cuvette, and 0.25 ml. of a 0.1% (weight/volume) ethanolic solution * of recrystallized blue tetrazolium (-0.35 micromole) was added. Finally, 0.25 ml. of the alkaline reagent was added and the solution was immediately mixed by inverting the stoppered cuvette twice. Each steroid was determined in duplicate. I n each series of measurement deoxycorticosterone was run as standard of reference. The rack holding the series of up to 30 cuvettes was then placed in a light-free, thermostatically controlled cabinet a t the desired temperature. The absorbance was read at, appropria t e time intervals against a reaction blank in a Coleman Junior spectrophotometer a t 530 nik a t room temperature (25" e.), requiring 5 to 10 minutes for a series. The duplicates were generally found to differ by not more than 0.02 in absorbance. The mean value of the duplirates was recorded, refrrred to 0.040 micromole and plotted with respect to time. DISCUSSION
This investigation can perhaps be best related to an earlier st,udy of Heard and Sobel ( 7 ) in which the reducing power of various steroids was measured by means of the color devekpment when the substances were heated a t 100" C. for 20 minutes to 3 hours with a phosphomolybdic acid reagent in an acetic acid niedium. Many similarities in the reducing charactc)ristics of the various steroid classes t,o\\-ard both reagents were discerned. T h e principal distinction consisted in the fact that steroids reacted with blue tetrazolium under more gentle conditions, and those with a primary a-keto1 grouping formed formazan cvhroniogens of comparable intensities (21j. I n contrast, the molybdenum blue development by various side chain ketolic steroids diff ered considerably-e.g., cortisone produced a chromogen of only i o % of that of deoxycorticosterone. The data on the saturated steroids with a cyclic secondary a-keto1 grouping a t carbon-1 1,12, carhon-l2,ll(a and p), and carl~on-3,2(aand p), of which these xorkrrs studied one rxample of each type, indicated that the molybdenum blue reached an absorptivity of the magnitude of 45% of deoxycorticosterone. The blue tetrazolium reagent has found extensive application for the quantitative estimation of corticosteroids folloxving methods described by Chen and coworkers ( 5 ) and Xader and Buck (10). Under the proper conditions the presence of moderate amounts of simple A'-3-ketosteroids does not interfere with the measurement of the corticosteroids ( 5 , 10). The gathered data revealed t h a t at minimum bmicity-e.g., 0.0025N in tetramethylammonium hydroxide or conditions I-relative short reaction time (45 and 15 minutes, respectively), and a temperature of about 40" C., the specificity of blue tetrazolium toward primary a-ketols is the greatest. The corticosteroids in this investigation were used for reference only. T h a t certain hydroxyl and ketone groups in the steroid molecule demonstrate considerable reducing power toward blue tetrazolium is noteworthy (Figures 3 and 4). (In paper chromatography, many of these compounds developed the purple color when the strip, after being sprayed with alkaline blue tetrazolium, was exposed to a stream of warm air.) According to their position and configuration in the molecule, remarkable differences in the reducing characteristics exist which may be used as a diagnostic means for the location of these functions in unknown compounds. Using conventional instrumentation, only 20 y of the pure substance are required. The specificity of the changes in rate due t o a particular structural grouping could not be established by this investigation of limited scope. Many more compounds would have to be examined and i t is, for the time
being, inipossible to derive from these data alone definite characterization of unknowns. Aloreorer, possible interactions between the grouping under consideration and the parent substance should not he lost sight of when dealing with new structures. However, in specific case8 Lvhe1.e alternate possibilities are in question, the formazan rates may allon- a reasonable choice. Tn-o applications of this method hnve been described (11, 12). Some discrepancies in increment rates for the same function have been noted, which limits the general applicability of this procedure. Two applications (12) of this method with presumably ]I)-droxylated A4-androstene-3,17-dione derivatives may exemplify its usefulness. One of t h r v unknonn compounds, isolated in small quantities, showed n .similar, although somewhat tlecreased formazan formatioii t hail did 6a-hydroxyandrostenedione (1j. Infrared analyses indicated that another hydroxyl group was attached a t C-llp. Since a hydroxyl group at t h a t posit,ion was shown t o inhibit the chromogen formation of the parent A4-3-ketosteroid, the correlation of the curves improved. Fina the characteristic light absorption in alkaline ethanol for a A keto-6a-hydrosyl structure (11) v a s noted, and these data permitted the tentative conclusion that t,his unknown sample \vas the 6a,llp-dihydrosy driivtitire. .\nother isolated compound deinonstrated an unusual rate of chromogen formation which allowed the exclusion of all the .stiuctural p9Fsibilities prerioas1y examined.
Figure 5 .
Transformation products
1-arious isolated carbonyl, hydroxyl (primary and secondai,!- ),
or enol groupings or olefinic linkages, as well as a,8- or 8,y-utisaturated hydroxyl groups did not induce a dehydrogenation in the steroid molecule by the blue tetrazolium reagent (Table I1 and compounds 11 to 15). However, the A'-en-3-one system gave rise to ample formazan formation, in contrast to the limited ability of the A1?'-dien-3-one (10) and A4s6-dien-3-one (9) structures (Figure 2). I n the case of A'-androstene-3,17-dione ( I ) a conversion to 6p-hydroxyandrostenedione (11) and 6-ket,oandrostenedione (111) wft~ ascertained through isolation of these transformation products (Figure 5). This oxidation appeared to be influenced t o a n unexpectedly large extent by functional groupings quite distant from the conjugated ketonic system. For example, the different side chains at carbon-17 affected the color development, and the 118-hydroxyl group (20), whirh is situated in y-position to the conjugated double bond in ring -4, distinctly suppressed the reaction. The lla-hydroxyl group (11, 12, and 15), however, had no influence on the chromogen, n-hrreas the presence of the 11-keto group (27 and 36) enhanced it. The tertiary hydroxyl group a t carbon-14 (18) added significantly to the color. T h a t t h e 68-hydroxy1 group (21) decreased the formazan production is explained b y the demonstrated course of reaction. I t would appear t h a t t h e 6a-hydroxy-A'-3-keto system (1) was isomerized in the alkaline medium to the 3,g-diketo Sa-configuration (22) or a common enolate, as both substances showed approximately the same rate of formazan formation. I n another ydiketone, 1,4-~yclohexanedione(41), a n oxidation to the quinone appeared to occur, as hydroquinone (42) yielded only half the color intensity. The 19-norsteroids (26 and 33) produced considerably more color than their corresponding 10-methylated compounds. T h k was due to the more complex alterations these compounds underwent, as was seen in an experiment on a larger scale with 19-
V O L U M E 2 7 , NO. 5, M A Y 1 9 5 5
817
I I ( I I :mdrostenedione.
Some products showed typical aromatic properties when examined spectrometrically, indicating a convei,sion to estrone-type steroids. Such a course of reaction might possibly also apply t o the 19-hydroxylated A4-3-ketosteroids (17 niid 34), as their transformation in alkaline medium t o 19-nor compounds has been demonstrated ( 1 1 ) and the formazan increiiients reached their maximum in both cases after 1.5 hours (Figure 4). Ultraviolet measurements indicated that the 7phydrosy~'-cholesten-3-one (23) was dehydrated in alkaline medium (in :thence of a tetrazolium salt) to the A4s6-dien-3-onederivative (9 j (unpublished observation). In the presenre of blue tetrazolium ruch dehydration was not favored, as coinpound 9 reacted only very slowlv, whereas with compound 23 an efficient dehydrogenation took place. It has been demonstrated that the secondary aketol 2-hydror~-l-cyclohesanor,cis osidized with triphenyltetrazoliuni chloride to the corresponding dione (20). An analogous reaction can be assumed as having occurred on the ketol moiety of conipounds 16 and 24. Deoxycorticosterone and its lia-hydrosyl analog have been degraded by blue tetrazolium to their corresponding 17-carboxylic acids, as shown by the isolation of :i-keto-A4-etienic acid and 3-keto-17cu-hydroxy-A4-etienicacid ('f !. Formaldehyde (58) xhich conceivably could arise from the Iri,eakdo\vn of this side chain, would not be further oxidized by i ~ l u etetrazolium, as it did not give any color with the reagent. similar course of reaction as v i t h the 20,21-ketols seemed to be indirated for the 16,17~-ketol(39); in the latter case a fission of ring D would be involved. EXPERI-MESTAL
Conversion of A4-Androstene-3,17-dione( I ) to 6pHydroxyandrostenedione (11) and 6-Ketoandrostenedione (111). Chroniatographically pure A'-androstene-3,li-dione ( I )(26.3 mg.) and Itlue tetrazolium (170 mg.) were dissolved in purified ethyl alcohol (155 ml.), and were made alkaline wit'h a 5070 aqueous choline solution (0.80 ml.) and a 10yo aqueous tetramethylammonium hydroxide solution (3.95 nil.). T h e batch was heated in the dark for 3 hours a t 37' C. After that period, 95% of the mixture was cooled to room temperature and L-ascorbic acid (100 me.) was added for reduction of unreacted tetrazolium salt, The misture was then neutralized with lAj7 hydrochloric acid (7.4 ml.), and evaporated t o complete dryness in vacuo below 40' C. under a nitrogen atmosphere. The residue was shaken with distilled water (500 ml.) a t room temperature for 4 hours, when the undissolved formazan was quantitatively separated by suction through a Celite filter. The filter residue was shaken overnight with another portion (300 ml.) of water. T o the combined aqueous filtrate sodium chloride (60 grams) was added, and the solution estrarted in 60-ml. portions consecutively wit,h three portions of ether (each 400 ml.). The ether extracts were washed with t w o portions of water (15 nil.), dried over anhydrous sodium sulfate, and evaporated in vacuo. T h e residue (23 mg.) was applied in the usual manner (4,1 2 ) on two sheets of Whatman paper S o . 1 (17 cm. wide) and chromatographed with propylene glycol-saturated ligroin at 25" CI. for, 30 hours. A minute quantity of unreacted I had been caught nith the overflow. Scanning the chromatograms with ultraviolet light revealed the presence of two a,p-unsat,urated ketonic products in larger concentrations. The first zone located a t 1 to 2.5 cni. from the origin was eluted and rechromatographed on a 6-cm. sheet in the toluene-propylene glycol system. The material yielded after recrystallization from acetone-ether, 2.5 mg. of slightly impure 6p-hydr0~y-A~nndrostene-3,li-dione ( I I ) , melting point 192-195" C., 2x6 nip. T h e melting point, was not depressed on admixture with a reference compound ( 1 2 ) and the infrared spectra of both saniplrs were dentical. The second zone a t 23 to 20 cm. from the origin produced with 14YG ethyl alcoholic potassium hydroxide a strong yellow color, rhar:tcteristic of the A4-3,6-diketo structure. Less intense coloration was exhihited on the paper in the adjoining zone a t 15 to 23 cm. The eluate of this trailing zone was rechromatographed on a 7-cm. sheet for 3 hours ascendingly in the toluene-propylene glycol system in which the substance moved as a relatively narrow band with a R, 0.5. T h e combined eluates yielded, after crystallization from ethyl acetate and acetone-ether, 8.5 mg. of A4androstene-3,6,17-trione (111), melting point 225-227' C. ; 251 mp (log E 4.04), 259 m p (log E 4.03), 380 nip (log E 3.96) (data to be published); 6 1730 em.-'
AF:z60H
AF2°H-KoH
A;d:
(17-keto), 1686 cm.-' (A4-3,6-diketo), 1612 cm.? (conjugated olefine); the infrared spectrum was identical with that of a reference sample. With the m-dinitrobenzene reagent (12) a small quantity of a reduced 17-ketosteroid, which did not absorb the ulfraviolet light, was located in a zone at 5 to 6 em. from the origin. An aliquot of 5% of the batch was withdrawn after 0.5 hour of reaction time and processed in a manner analogous t o the above. According to evidence by chromatogram, the yields of I1 and 111 were of the same order of magnitude as in the experiment of longer duration, although much unreacted I was still present and the formaaan absorbance amounted to only two fifths the value of the 3-hour run. It thus would appear that especially I11 nu. rather instable in the reaction medium. 1 sample of the above 6p-hydroxyandrostenedione (11) (1.5 mg.) was reacted for 3 hours under conditions analogous to thoqe for the androstenedione (I). T h e absorbance of the formazan formed was determined under the dilution used in the anal) tical measurements t o give a value of 0.28 for 0.040 micromole of 11. Of the recovered material, approvimately one half had been converted to I11 as estimated bv ultraviolet measurements oi the eluates of the paper chromatogram. ACKNOWLEDGMENT
This study was made possible bl- the generous contrihution.4 of those \Tho kindly furnished the required samples. Thanks are given to D. H. R . Barton for compound8 55 and 56: to JIaximilian Ehrenst,ein for all nine specimens oxygenated at rarhon-6 (with the one listed exception) and for compounds 25 and 34; to L. F. Fieser for compounds 30 and 5 0 ; to Josef Fried, Squit,t, Institute for Medical Research, for compounds 13 and 16; to Marcel G u t for compounds 7 , 10,26, 47, and 48; to W. A. Jacobs for compounds 53 and 54; to D. H . Peterson, Upjohn Co., for compounds 11 and 36 and reference ronipound 111; to Tadeua Reichstein for compound 38; to I. T.-. Rollins, Chemical Specialties Co., Inc., for compounds 9 and 33; to Franz Sontlheimer, Syntex S. B., for compounds 24 and 35; to A . F. St. And&, C'itln Pliarmaceutical Products, Inc., for compounds 5 and 18; to Max Tishler, Merck & Co., Inc., for compounds 12 and 40; and to .4lbert Kettstein, Ciba, Ltd., for compound 19. The support of this investigation by grants from the Sational Institutes of Health (Research Grant G-3247) of thr G. S. Public Health Service, and from G. D. Searle and C o . , Chicago, Ill., is gratefully acknowledged. LITERATURE CITED
;Ishley, J. N., Davis, B. H., Xitleha~n.A K., and Sla(
