593
~ ~ R O M L I A T I Z A TOF ~ O10P-HYDROXY-3-KETO N STEROIDS
Septeniber, 1964
T 1
0.9 0.e
- 0.0.at _____ O.D. ai
235 my 240mp
1
0.2
'
0
.
0 210
'
240 ' z j o Wavelength
'
zbo
aio
'
330
in mp
Fig. 2.-Ultraviolet absorption spectra of equimolar quantities (5,466 x 10-6 M) of lop-hydroxy-19-nortestosterone and estradiol. The arrow (245 mp) indicates the wave length a t which the spectrophotometer was set in order to obtain a record of optical density against time.
\\
\
L\/J
zio
1
niethanol in water for 99 transfers in a Craig Countercurrent Distribution instrument. The number of equilibration strokes was set for 20 and the settling time for 10 min. The concentration of solute was obtained by determining the optical density Fig. 1.-Countercurrent distribution of the products of the preparation of 17a-ethynyl-lOp-hydroxy-19-nortestosterone. a t 235 and 240 m p in methanol of equal aliquots from both upper Peak I ( K = 0.37) is pure lop-hydroxy-170-ethynyl-19-nortestos- and lower phases of each tube. In the preparation of both 1013hydroxy-19-nortestosterone and of the 1Ta-ethynyl analog, terone, which absorbs maximally at 235 mp in ethanol. Peak distribution of the crude products gave two peaks with K values I1 ( K = 1.47) is 17a-ethynyl-19-nortestosterone which has xE~OH of 0.37 and 1.47, respectively. In earh case the 10-hydroxy The system was 60c0 ethyl acetate in hexane and mnx 240 nip. derivatives were found in the first peak while the second peak .io! Iniethanol in water, and the number of transfers was 99. contained 19-nortestosterone or 17a-ethynyl-19-nortestosterone. The physical constants of the purified products were as follows: butioii system suitable for the facile purification of lop10P-hydroxy-19-nortestosterwq m.p. 208-210", lit.6 208-210", 235 mp ( e 13,270), lit.6 " : :A 234-236 m p ( E 13,200); 1701hydroxy steroids prepared by the method of Ruelas, et " : :A ethynyl-lOP-hydroxy-19-nortestosterone,m.p. 258 -260", lit.6 ~ l . in, ~which the 3,lO-bond of either estr-5(10)-en-l7p263-264", X?AH 235 mp ( E 13,240), lit.6 " : :A 236 nip ( E 14,5iO).6 01-3-one or its 1Ta-ethynyl analog is epoxidized by These two compcunds were pure as judged by chromatography iiioiioperphthalic acid in chloroforni a t low temperaon thin layer silica gel.10 ture and the epoxide rearranged in niethaiiolic potasKinetic Experiments.-lleasurements of the rates of aroniatization were made in the following manner. A solution containing siuiii hydroxide. Ruelas6 reported a crude product iii approximately 0.7 mg. of steroid in 1 nil. of methanol was added the first reaction of 65%. I n our hands, it v a s not to a rapidly stirred solution (49 ml.) of HCI in methanol or in possible to obtain pure epoxide without a coiisiderable water. The addition was carried out at :37", and the time of decrease in yield aiid it was found expedient to carry addition was taken as zero. A capped cell, previously placed in the therniostated (37") sample compartment of a Cary Model 14 out the two steps of the reaction without attempting a spectrophotometer was filled with this solution, and a record of purification of the epoxide. The final reaction iiiixture optical density 2)s. time was started at an accurately timed interval was then purified by countercurrent distribution. after zero time. Measurements were made at 252 nip for aqueous This procedure offers the advantage of iiicreasiiig the solutions and 245 mp for methanolic solutions. The spectroyield aiid avoiding the risk of dehydration of the photometer was balanced using a solution of reagent grade hydrochloric acid in the appropriate solvent. The various soluproduct 011 chromatography columns. tions of methanolic HC1 were made by dilution of a stock solution of calculated 2 M acid in methanol. .;in accurate duplicate of this solution in water byas standardized and found to be 2.01 Experimental .
10
20
30
40 50 Fractions
80
70
bo
SO
'TI.
Chemistry.-Estr-5(lO)-en-lip-ol-3-one, 17a-ethynylestr-5 (lO)-en-lip-ol-3-one, and a small reference sample of 17aethynylestr-4-ene-l0~,lip-diol-3-one were obtained from G. D. Searle and Co., Chicago, Ill., through the courtesy of Dr. Frank Colton. lop-Hydroxy-19-nortestosterone and 17a-ethynyl-108-hydroxy-19-nortestosterone were prepared as described by Ruelas.6 The two steps of the reaction were carried out without attempting a purification of the epoxide. The crude reaction product was then distributed between 6OC2 ethyl acetate in hexane and 50%
The record was stopped after 6 to 7 half-lives, and t'he spectrum of the solution was examined. For calculation of the halflife (t i / *), A D was first obtained from the spectrophotometer chart by subtraction of the final fixed optical density from the values a t various time intervals. Log A D O was determined by plotting log A D against time, and extrapolating the resulting gave log straight line t o zero. Subtraction of log 2 from log 0, ADtl,%. The half-life (till)was then obtained graphically. (10) T. Golab and D. S. Lrtyne, J . Chromatog., 9, 321 (1962).
391 0.c
I.?
2,
tib = 5.75 minutes -
1.c
0
a
-2.0-
0 0
-
-2 .!
3.5
-
-3.01 2.C
1
1
i
21.5 T. 0 log conc. o f H C I in methanol
i.5
2.0
Time (minuter)
b’ig. Calculatiuii of the half-life ( t i / * ) of l’ia-ethyiiyl-10$h?drosy-l!)-riortestnsterone in 0.008 Jf HC1 iii methanol.
‘Vlie 10-hydroxy steroids were coiiverted to products whicli :il)sorheti at 2% nip in Kater and a t 286 and 278 m p in methanol. ‘l’licw spectra were assumed to be due, respectively, t o the correspoiidiny ring h phenols and their methyl ethers. To coiifirni tlie ideiitificaation the reaction mixture \vas extracted with ether x i i d the extrayt chromatographed 011 thin layer silica gel G 1)latcs in a system of 50cz ethl-1 aretate in cyclohexane, using the :tiit lientic (.ompounds as standards. The developed chromatogram was heated at 100” aiid sprayed with saturated solut ioii of antimciny trichloride in chloroform. Coincidence of the, (.stracted material with the standard and similar color reactions i i i thc visible and ultraviolet regions ivere taken as positive ideiitificAatitin !o
Results and Discussion The results of the couiitercurreiit distribution of tlic crude products of the preparatioii of l7a-ethyiiylIO~-hydroxy-19-nortestosteroiie are showii in Fig. I . curve of similar character was obtained froiii t h e products of the preparation of 10P-hydroxy-19-nortestosterone. Optical densities are plotted a t 235 and 210 iiip aiid demonstrate the presence of the 10P-hydroxy S4-3-ketonc in the first peak arid of the unsubstituted 14-3-ketone in the secoiid. The latter conipouiid iiiidoubtedly resulted froiii isoiiierization of the Aj(ln’cx-:!,-oiie starting inaterial in either the acidic epoxidatioii or in the basic rearraiigenient media. I’edcrsoii, et u Z . , ~ reported the acid-catalyzed delipdratioii of 1OP-hydroxy-19-iiortestosteroiie to estradiol, but did iiot give details of the reaction conditions. liuclas:, et U Z . , ~ obtaiiied estradiol 17-acetate in 80T7 yield from lOp-hydroxy-19-iiortestosteroiie by thc uctioii of hydrogen chloride i i i acetic acid at 3- 10’ ’l’li(. present iiivestigatioii of the aroniatizatioii of’ tlio
lO$-liydroxy coiiipouiids by acid ill aqueous soliitioii iiidicated that the rcwtioii \vas very -low a t acid coliceiitratioiis belo\v 3 -11. Well-defined spectra of i itig plieiiols were iiot observed iii t h e aqueous reactioii t i i c d i u i i i below this coiiccliitratioii of acid iii periods ot up to 12 hi., aiid .idid cjuaiititatioii of thc rcJtlctioti rate \vas iiot possiblc. Iicceiitly, Crardi and l ’ ~ l r a 1 1’ ~fouiid that vai iou. 5,lO-substituted 3-keto l!)-iiorstcroids gave 3-iiwtliosy poiidiiig phrw)ls by tieatiiiciit A l l v a r e ~coiifiriiied Y this oh- ( n a t i o n using lOd-acetoxy derivatives as startiiig caoiiipouiids. 1 hc prescwt 1) (irk >lion-edthat in alcoholic. wlutioii, tlw aroniatizatioti of the ;,lo-substitutcd :iketo steroids procccdctl. rapidly, and it was possiblr i t 1 this iiiediuiii to study tlic ratc of reaction at 37’ aiid t l i c b dfect of acid conceiitrntioii thereon. The sc+xtioii OI 24.i iiip (or 232 iiip for a q w o u - wlutioiisj for ineab~rc>tii(wt of the disappcaraiice of thtl 10P-hydrosy stc.ioiti. i.; based on the rewlt- sliouii in Fig. 2 , i i i which t l i v epcxtra of starting iiiaterial aiid product (in equiiiiol:it quaiitityj are supcriiiiposed. While 213 iiip is olil> 10 iiip reiiiovecl froiit thc3 n a w leiigth at which thc 103hydroxy steroids absorh iiiaxiii~ally( E 12.630), it i,i tlic poiiit a t which the iiivtliyl &her.; of the correspoiidiiig pheiiols absorb iiiiiiiiiially ( E 400). 1Ieasureiiiciitcan thus be iiiadc at thih n-ave length with little> 1 0 s of seiisitivity and with iniiiitiiuiii interference frotii tllo product of tlie rcwtioli. Fig. :!, shoivs that t h e plot 01 the log of coilcentratioti 01
1;ol-t:thyiiyl-10d-li~~i,osy-l!)-iiort~stoster~ii(~ iii (‘1 ab a fuiictioii of‘ time is a straight ’I-, thc reactioii is psmdo first ordm. with
Septeniber, 1964
AROMATIZATIOS O F 10P-HYDROXY-3-KETO STEROIDS
595
nounced effects of l7a-substitution on the rate of isomerization of A5(lo)- and A6c6)-3-ketosteroids to their A4-analogs, but the present results indicate that substitution of an ethynyl group for the 17a-hydrogen Acid atoin does not affect the acid-catalyzed aromatization concn., t’/7, of ring A in lop-hydroxy-A4-3-keto steroids. M min. Compound solvent 108-Hydroxy-19-nortestosAIeOH 1.o 4.35 The concentration of HC1 in the stomach immediately 0.012 terone MeOH 4.25 after a meal is about 0.5 M.12 .4s mentioned above, MeOH 5.75 0.008 accurate measurements of the rate of aroniatizatioii 0.003 MeOH 10.75 of lop-hydroxy-A4-3-keto steroids were not possible a t 66.5 3.0 Water aqueous acid concentrations of this order, but the 1.25 1.0 17~Ethynyl-108-hydroxy-19MeOH results in Table I are sufficient to indicate that the rate 0.100 4.5 nortestosterone MeOH would be very slow. The major conversion of ingested 0,014 MeOH 4.5 norethynodrel to phenols would require prior hydroxy0.012 MeOH 4.5 lation a t C-10, with the 10-hydroxy 3-ketone remaining 5.0 0,010 MeOH in the stomach for several hours. Since the eniptying 0.008 MeOH 5.75 time of the stomach, although highly variable, is from 0.006 7.25 MeOH 0.004 8.75 3 to 5 hr. after a mixed the conversion of MeOH 0.003 11.0 MeOH norethynodrel to ethynylestradiol would be small. 27.25 0.002 MeOH This speculation takes no account of the possible 0.001 41.00 MeOH catalysis of the reaction by other substances in the 5.5.5 3.0 Water stomach, and enzymatic aromatization of lop-hy0.10 26 hr.* MeOH droxy steroids in other parts of the body is possible. a Values expressed as half-life of the starting material. This However, the results support the previous observavalue is of little quantitative significance, but is presented because tions12 based on an examination of the urinary and of its special importance in the discussion. fecal conversion products of norethynodrel, that the respect to steroid since it is also dependent on hydrogen per cent conversion of this compound in viuo to estroion concentration. Figure 4 is a graph of log k’ for the genic phenols is not large. It must, however, be borne reaction of 17a-ethynyl-10B-hydroxy-19-nortestosterone in mind that even a 1% conversion of a 10-mg. dose of against the log of the iiiolarity of HC1 in methanol. norethynodrel to ethynylestradiol would result in a At concentrations of HC1 greater than 0.01 ill in methdose of this estrogen (0.1 mg.) which has physiological anol the measured k‘ values were independent of acid effe~ts.1~The aromatization of the lop-hydroxy concentration. At the two concentrations of acid derivative in the stomach might therefore explain studied below 0.003 X,the half-life increased rapidly the low levels of estrogenicity exhibited by orally adwith the decreasing hydrogen ion concentration and the ministered norethynodrel. relation of the latter to log k‘ was not linear. Theories for the niechanisiii of aroniatization have been adAcknowledgment.-The authors are indebted to ~anced.’-~ A h . E‘rancoise Gospodarowicz for technical assistance. Table I compares the rate of aroniatization of lophydroxy-19-nortestosterone and its 17a-ethynyl analog in methanol and in water for various conceiitratioiis of (12) .4. C a n t a r o a and AI. Truiiiper, “Clinical I3ioche1nistry,” W. 13. acid. S o difference n7as observed in the rates for the Haunders Co., Philadelphia, Pa., 1962, p. 463. (13) C. H. Best and N. 13. Taylor. “The Physiological Basis of Medical two conipounds. Xes, et U L . , ~ ~ have reported proTABLE I
CoNVERSlON OF
10-HYDROXY-3-ICETO STEROIDS BY DIFFERENT COXCENTRATIONS OF HC1“
(111 W.R. N e s , E. Loeser, R. Kirdani, and J. Marsh, Tetrahedron, 19, 299 (1963).
Practice,” 5th Ed., FVilliains and Willianis Co., Baltimore, RId., 1950, p. 568. (14) I).S. Layne, T. Golab, K. Arai, and G. Pincus, Biochem. Pharrnacol., 12, 905 (1963).
