The Ultraviolet Absorption Spectra of Allyl and ... - ACS Publications

The Ultraviolet Absorption Spectra of Allyl and Propenyl Substituted Derivatives of Diethylstilbestrol and Hexestrol. Emil Kaiser, Virgil L. Koenig. J...
0 downloads 0 Views 348KB Size
740

EMILKAISERAND VIRGILL. KOENIG

which agrees with t h a t reported by Ruzicka and Wettstein.¶ ~~-Androstene-3,17-dibenzoate.-The dibenzoate was prepared by heating on the water-bath a mixture of A~-androstene-3,17-diol, benzoyl chloridc and pyridine. The crude dibenzoate was recrystallized from methanol; m. p. 203-205". Anal. Calcd. for C ~ ~ H ~ BC, O +79.5; : H, 7.6. Found: C, 79.3; H, 7.8. Dehydrogenation of A6-Androstene-3,17-diacetatewith Benzoquinone.-A mixture of 0.3 g. of the diacetatc aiid 0.0!14 g. of berizoquiiione was heated i n a sealed evacuated t u l x a t 1:30" for three hours. The product from this rcact i o i l was tlissolvcd i i i ether atid thc solution thoroughly shakeii n i t h a 20yc solution of sodium liydroscilfitc, then estractcd sevcral times with a l0yo sodium hydroxide solutioti, dried, and the ether removed. The slightly tiiscolored rcsidue was purified from nicthanol and a product was obtained the spectrum of which showed a singlc band with a inaxiinilm at 288 nip having a molecular estinctioii coefliciciit of 1116. A6-Aiidrosteiie-3,17-tliacetateesliibitetl no selective absorption in this region. A eompariS O I L of the extinction coelliciciit with that of ergosterol (lO,GOO a t 282 nip) ititlicatctl that the iiiatcrial coiitaitictl the clcsircd dehydrogeriatetl product t o the estciit of about 10%. Irradiation of A5~i-Androstadiene-3,17-diol.-A sainplc OT t Iic iurtially tleliydrogciiiatctl tliacctate was 1iytfrolyzc.ci with iiiciliyl alcohnlic potash arid the iioii-sai,oiiiliable

[CONTRIDUTION FROM T H E

Vol. 68

diol irradiated in ether for four hours using a quartzmercury lamp. T h e product was theii assayed biologically by Professor R. S. Harris who reported no activity at a level of 10,500 U. S. I?. XI vitamin D units per gram. Almost a year after these experiments were performed, Dimroth and PalandB reported similar biological results with this substance made by a different procedure.

The authors wish to express their appreciation for a generous sample of dehydroandrosterone supplied to one of us (N. A. 11.1.) by the Schering Corporation through the courtesy oi Dr. Erwin Schwenk.

Summary 3 . ~5-Aiitlrosteiiol-3(GiS 3 : 10) aritl its acetate antl benzoa.te were prepared in the pure state. 2. l'he partial dehydrogenation (if h5-antlrostenol-3-benzoate antl &5-androstc:tic-R,li-diacetate with benzoquinone has been studied. The irradiated dehydrogenation products were devoid of antirachitic action when tested at levels of 4000 and 10,500 U. S. I-'. XI vitmiiii D units pcr grain, respcctively. CAMBRIDGE:, MASSACHUSETTS h C E I V E D NOVEMHER

30, 1'3-15

ARMOUR LAUORATORIES]

The Ultraviolet Absorption Spectra of Allyl and Propenyl Substituted Derivatives of Diethylstilbestrol and Hexestrol BY EMILKAISERAND VIRGILL. KOENIC In a previous communication' the preparation of 3,3'-allyl and 3,3'-propenyl diethylstilbestrol and hexestrol are described. The 3,S'-allyl substituted derivatives were rearranged by heating in alkaline solution to coinpounds which were ;I&sui 1 ictl to 1)e :$, '-i)ropcnyl tlcrivativcs in ~tiialog~. to rvactioiis rcpurtctl i i i thc litcriiturc.? Obviously, thc reaction described by Ealbiario3 for thr: cliff crentintiori between allyl and propenyl groups cannot bc applied here inasmuch as the large number of double bonds would introduce complications. For this reason it was decided to exatninc the ultraviolet absorption spectra of these coiiipouiids. An important difference in the structure of the 3,3'-allyl and 3,3'-propenyl tlerivativcs tinder discussinti here resides iii the positioii of tlie tlorible bonds ol' the side chains. In the pr(JT)CllYl roinpountts the double bonds are i n a conjugatccf position to the double bonds of the aromatic iiuclei; i n the allyl derivatives the double bonds are not conjugated. Since the presence of the double bonds is shown by the shape of the ultraviolet absorption curves of organic compounds, i t was thought possible to demonstrate the shifting of the double bonds by comparing the f I ) Kaiser aricl Svarz, 'L'iirs fuunsni.. 68, 036 (1046). ( 2 ) "Organic Keactioiir," Vu!. 11, J o l i i l \Vilcy alltl Suns, Inc., New York, N . Y . , 1941, p. 19. (3) l ~ : l l l , , , m < > l h, , . , 48, 3!bt (101.->).

absorption spectra of 3,3'-allyl derivatives of diethylstilbestrol and hexestrol with the absorption spectra of 3,3'-propenyl derivatives of diethylstilbestrol and hexestrol. Acknowledgment.-The authors are indebted to Professor F. C. Koch for his Iielpftil criticism of the 1ii;iiiuscript and for his eiicouragcmeiit during the work.

Experimental The ultraviolet absorption Curves of the followi~~g coinpounds were determined: diethylstilbestrol, diallyl ether of dicthylstilbestrol, 3,3'-allyl diethylstilbe.stroi, 3 3 ' propenyl diethylstilbestrol and hexestrol. hexestrol diallyl ether, 3,3'-allylhexestrol and 3,:3'-propeiiylhexestrol. The diethylstilbestrol and hexestrol w r r e recrystallized froin commercial preparations. The othcr compouncls were prepared accordiiig to a previous coininunicatioti.' Acetotic-free absolute niethauol, tlistillctl froin alkali, was used as solvent. The coiicciitration of the solutions was 0.00570. The tiieasurenients were made on a Bechman spcctrophotonletcr using the hydrogen discharge tribe as the source of light. Transmittance values were determined at 10 millimicron increments from wave length 220 millimicrons t o 400 millimicrons. Extinction values were obtained from the transmission values and, from these, molecular extinctions were calculated and used for plotting the graphs.

Discussion In Fig. 1 the absorption spectra of diethylstilbestrol, clinllyl ether of tliethylstiibestrol, 3J'-

May, 1946

SPECTRA OF DERIVATIVES OF DIECTHYLSTXLBESTROL AND HEXESTROL

741

A maximum of the absorption is observed close to 250 millimicrons, then a drop in the absorption occurs until a wave length of about 280 millimicrons. There instead of an inflection of the curve the light absorption increases again until a maximum is reached a t 310 millimicrons. The presence of conjugated double bonds in the molecule shows up very definitely by this particular peak of the absorption curve. The difference between the light absorption of the allyl and propenyl compounds is even more pronounced in the hexestrol series, Fig. 2. The maxima of absorption of hexestrol, hexestrol diallyl ether and 3,3’-allylhexestrol are all close to 230 millimicrons and 280 millimicrons, in good agreement with the values for hexestrol reported by Elvidge4 and by Goetze and Serf.7 The absorption curve of 3,3’-propenylhexestrol, however, had maxima a t 250 and 310 millimicrons. The maxima are a t the same wave Iengths as the absorption maxima of 3,3’-propenyldiethylstilbestrol and seem to be characteristic for the conjugated double bonds of 3,3’-propenyldiethylstilbestrol and hexestrol. 20,000

220

280 340 Wave length in mp. Fig. 1.--4, diethylstilbestrol; @, diethylallyl ether; 0 , 3,3’-allyldiethylstilbestrol; 9, 3,3’-propenyldiethylstilbestrol.

allyldiethylstilbestrol and 3,3’-propenyldiethylstilbestrol are shown. The diethylstilbestrol absorption curve was very similar to the absorption curves reported for this compound by El~ i d g eKharasch ,~ and Kleiman6and by Solmsson.6 The maximum of the absorption curve was close to 250 millimicrons, a value somewhat different from Elvidge, who found the maximum close to 240 millimicrons. The highly purified diethylstilbestrol of Kharasch and Kleiman5 has a maximum close to 250 millimicrons in accordance with our measurements. The inflection a t 280 millimicrons, pointed out by Solmsson,e is shown in Fig. 1. The absorption curve of the diallyl ether of diethylstilbestrol shows a maximum in the region of 240-250 millimicrons and has no inflection at 280 millimicrons. Another ether of diethylstilbestrol, the dimethyl ether, has an inflection a t 280 millimicrons in the absorption curve.6 The maximum of the curve of the 3,3’allyldiethylstilbestrol was very close to the maximum of the diethylstilbestrol curve and the inclination at 280 millimicrons was very marked. An entirely different picture is presented by the absorption curve of 3,3‘-propenyldiethylstilbestrol. The absorption of light is greater than in the case of diethylstilbestrol and its allyl derivatives. (4) Elvidge, Quart. J . Phorm. Phormacol., 12, 347 (1937). TOURNAL. . 66.. I 1 (1943). ( 5 ) Kharasch and Kleiman. THIS . . (6) Sulmsson, &id., 66, 2370 (1043).

2

15,000

’ i

t

220

280 340 Wave length in mp. Fig. 2.-0, hexestrol; .S, hexestrol allyl ether; 0 , 3,3’allylhexestrol: 9, 3,3’-propenylhexestrol.

Summary Ultraviolet absorption curves are reported for diethylstilbestrol, diethylstilbestrol diallyl ether, 3,3’-allyldiethylstilbestrol, 3,3’-propenyldiethylstilbestrol, hexestrol, hexestrol diallyl ether, 3,3’allylhexestrol and 3,3’-propenylhexestrol. The shifting of the double bonds from the allyl t o the (7) Coetze a n d Serf, J . Ani. Phnrm. A S S O C .S, X S I V , 209 (1045)

HENRYB. BULLAND BYRONT. CURRIE

742

Vol. 68

propenyl grouping is accompanied by a shift of bestrol and of 3,3'-propcnylhexrstrol arc a t identithe absorption maxima in the ultraviolet. The cal wave lengths. absorption maxima of 3,3'-propenyldiethylstil- CHICAGO,ILLINOIS RECEIVED J A N U A R Y 5, 1946

[CONTRIBUTION FROM

THE

DEPARTMENT OF CHEMISTRY, NORTHWESTERN UNIVERSI~ Y MEDICAL SCHOOL]

Osmotic Pressure of @-LactoglobulinSolutions BY HENRYB. BULLAND BYRONT. CURRIE' through t h e courtesy of Dr. C . J. 13. Thor. The moist casing was securely knotted at one end and the other end was slipped over a rubber stopper which had bcen trimmed to size, and attached t o the end of the tube which was to contain t h e protein. T h e stopper had been previously coated with stopcock grease. The end of the sack which had been slipped over t h e rubber stoppcr was wrapped TABLE I tightly with a rubber band t o complete the attachment t o MOLECULAR WEIGHTOF LACTOGLOBULIN AS REPORTED the rubber stopper. T h e sack was thcn filled with pro:ciii solution through the larger glass tube t o which !vas a t BY VARIOUSWORKERS tached a second, larger rubber stopper which was coated Molecular with stopcock grease. The apparatus was then asMethod weight Workers sembled as shown in Fig. 1. T h e larger rubber stopper Ultracentrifuge (equilibrium) 38,000 Pedersen" was held in place by a metal clamp which is n o t shown in and diffusion Fig. 1. Small one-hole rubber stoppers were placed in the Ultracentrifuge (rate seditubes containiiig the outside solution and the protein solumentation) 41,500 Pedersen" tion. Their purpose was to decrease the evaporation of );-Ray (wet crystals) 33,000 Crowfootb these solutions during a n experiment. McMeekin and Wiariier' The wholc apparatus was clamped in a fixed position X - R a y (air dried crystals) 35,000 Crowfootb in a constant temperature bath a t 25". The stopcock McMeekin and Warner' was allowed t o remain open until the apparatus had come Chemical analysis mini42,000 Brand and Kasseld t o temperature. The position of the toluene level in the Chemical analysis mum 42,000 Chibnall' capillary was marked by appropriate means and the stop 42,020 Brand el d' Chemical analysis (m. w , ) cock was closed. The toluene meniscus immediately be38,000 Gutfreundv Osmotic pressure gan t o rise or fall depending on whether the hydrostatic 2 X 17,100 Bullh Film pressure previire was greater or less than the osmotic pressure. 35,000 Iieller and Klevensi Light scattering Protei:i solution was added t o or removed from the protei11 Osmotic pressure 35.020 Bull and Currie solution column t o bring the toluene meniscus t o its original Crowfoot, level. Adjustinent of the protein solution level was made O1 Pedersen, Biochem. J . , 30, 961 (1936). Chew. Rev., 28, 215 (1941). McMeekin and Warner, from tiitie t o time as needed. The toluene column thus THISJOURXAL,64, 2393 (1942). Brand and Kassel, acted essentially as an indicator and in the ideal case there J . Biol. Chem., 145, 365 (1942). ' Chibnall, Proc. Rny. is no net motion of liquid across the sausage casing memSoc. Londm, B131, 136 (1942). f Brand, Saidel, Gold- hrane. Usually, no further adjustment of the protein water, Kassell and Ryan, THISJOURNAL, 67, 1.524 (1945). solution column was necessary after about two hours. I n Bull, THISJOURGutfreund, Nulure, 155, 237 (1945). all cases, however, t h e experiment was allowed to continue NAL, 68,.745 (1946); Heller and Klevens, private com- overnight. A t the end of this tiinc, the positioii of ~ l i c munication. toluene column had usually shifted one or two nim. The drift of the toluene level was mi!!tiq"cJ by the density of The present paper reports the result of 20 toluene and applied as a correctio:: t o the osmotic pressure. difference in lcvcl between the outside solution and osmotic pressure measurements on solutions of P- The the protcin solution \vas measured with a catlictoinetcr. lactoglobulin along with the calculation of the This difference iii lcvcl whcn niultiplicd by the density of molecular weight of this protein. the protein solution gave, after applying the correction d u c to the small excursion of the tolu('!ie colunln, the osmotic pressure of thc protein solutiori 1 1 1 i ! .'iiicters of water. Experimental The P-lactogloliulin Mas prepared by a modification of a T h e osmotic pressure apparatus was a modification of method suggested in a private coiiiniunication by Dr. A. t h a t described by Bull.* This modification is diagrammed H. Palmer. Raw whole niilb was b r o ~ l g i ~t ot 60% saturain Fig. 1. T h e capillary tube had an inner diameter of 0.2 mm. and was furnished by Corning Glass Works. tion with arnmoniuni su1Iatc. Thc solution was filtered About 5 CC.of the outside solutipn (solution whose com- and the filtrate brought t o 807, saturation with aiiiiiioposition was identical with t h a t of the protein solution niuin sulfate. A siiia11 ainouiit of water a l o i ~ g\\itli some toluene was aticlctl t o thc precipitate. 'The precipitate except for the absence of proteiii) was placed in the botwas transferrctl t o .;aus:ipe caGiiig w i r l c i i u l y z d agaiiist Iretom of t h e apparatus. Toluene was added from the top of the clean, dry capillary and forced dowii the capillary quent changes of rlistillcd \rat('r a t ;7' for four or five days. with gentle air pressure. Suction was then applied t o the T h e protcin so1utio:r was th-ii rrniovrd Iroiii the sausage capillary t o remove the trapped air. T h e apparatus was casing and any cstr:riieous snlid iiiatcrial filtered off. The then filled with outside solution. T h c sack which coii- filtrate was adjustcid to PH 5.1 by the cautious addition of tained t h e protein solution was made from Visking sausage dilutc hydrochloric acid. The turbid so!ution was then casing whose flat width was 1.5 cin. and was supplied dialyzed agaiiirt distilicu water and the dialysis contiiiued until 110 furthvr scl>aratiwi of the "oily" phasr tool; place. ( 1 ) On leave ol absence from the Corn Products llefinitiy Coni T h r protcin was thew crystallizvrl as ticscribeti by Pa1111cr.~

A number of values for the molecular weight of P-lactoglobulin from cow's milk have been reported. There is, however, a disconcerting lack of agreement between these values as is shown in Table I.

1

*

.

paw. (2) Bull. J . B i d Ckcm.. 137, 1.13 (1'311)

( 3 ) l ' , ~ I i i i c r ,i b i d . . 104, 3 3 3 ( l ' J 3 4 j