SHORT COMMUNICATIONS 609

Adrenal estrogens |1. Further characterizatlons of isolated urinary 11p-hydroxyesterone and 11p-hydroxyestr~diol

In a previous publication 1, we reported the isolation of I Ifl-hydroxy [4J4C i estrone and x Ifl-hydroxy [4J4C]estradiol from the pooled urine samples of an o6phorectomized and adrenalectomized patient with metastatic mammary carcinoma, after the adminis- tration of a tracer dose of [4-z4Clcortisone acetate. In the present communication, we wish to report results on studies of the monob~nzoate and dibenzoate derivatives of the isolated 11-oxTgenated estrogens. .

Preparation of benzoate derivatives -

Ilfl-Hydloxy[4-14C]estrone monobenzoate and Ilfl-hydroxy[4J4C]estradiol di- benzoate were prepared as described by HUFFMA,'~ AND LOTT 2, with slight modifi- cations (Table I). When the reaction was completed, the mixture was poured into xoo ml of ice water, forming a reddish oil at the bottom. The aqueous mixture was allowed to stand at 3-5 ° for 24 h. The aqueous phase, separated from the oil by de- cantation, contained approx. IO % of the radioactivity, as shown by ether extraction. The oil was dissolved in aq. ethanolic acetone in the presence of a trace of pyridine. After several hours of standing at room temperature, it formed a greasy solid. Th~ greasy solid was recrystaUized once from 95 % ethanol and ,once from ethyl acetate and heptane, giving colorless needle crystals.

Counter-current distributions of the benzoates

The crystalline benzoates were distributed in an all-glass Craig apparatus with 2o transfers in a solvent systerh consisting of 5o% ethyl acetate, 5o% cyclohexane per 7 ° % ethanol, 3o % water. Partition coefficients were calculated as described by CRAIG AND CRAIG 3. The method for the assay of radioactivity is the same as described previouslyL Concentratim,s of the carriers were estimated spectrophotometrically at 230 m# in absolute ethanol with a Beckman Spectrophotometer model DO.

Distribution of 1Tfl-hydroxy[4-14C]estrone monobenzoate (Fig. I) showed that the radioactivity as well as the weight of the carrier was concentrated between tubes tI-19, with a peak at tube I6. A small amount of crystalline material without radio- activity was isolated from tubes 3-7- This material has a melting point of I r8 - I2 I °. The infrared spectrum of this material is identical with that of benzoic acid. A small peak at the ascending limb, as shown by both radioactivity and spectrophotometric estimation, may represent contamination of iifl-hydroxyestradiol dibenzoate, since previous separations did not separate these two compounds completely. Material between tubes I4- I9 was pooled, and was submitted to successive recrystaUizations.

Distribution of II~-hydroxy[4J4C]estradiol dibenzoate (F;.g. 2) showed that the radioactivity and bulk of the carrier was concentrated between tubes 6--i6 with a peak at tube t 2. The skewed contour of the ascending limb is possibly due to contamine.tion by benzoic acid.Tubes 6-x6 were pooled and were submitted to multiple recrystalfization.

Carrier rerrysiallizations

Crystallizations were first carried out in hot ethanol. After several recrystalliza- tions, the carrier was recrystallized from ethyl acetate and beptane. After collecting

Biochim. Biophys. Aaa, 57 (x06.,) 6oo--rzz

TA

BL

E

l O

PR

EPA

RA

TIO

N A

ND

PI/

YSI

CA

L

PRO

PER

TIE

S O

F B

EN

ZO

AT

E D

ER

IVA

TIV

ES

4bso

rptw

n in

eth

anol

In

frar

ed

Car

rie~

B

enzo

yl

Rea

ctio

n

Ben

zoat

e m

.p.

" F

orm

ula

Rad

ioac

tive

met

abol

ite

adde

d ch

lori

de

tim

e yi

dd

~ma.

x ~'

max

E

ster

H

ydro

xyl

(°C

) (r

ag)

(ml)

(h

) (c

ou

nts

/ra

in)

(m/O

(c

m-I

) (c

m-l

)

C a

lid

H A

naly

sis

C%

H

%

Cal

c.

Fou

nd

Ca

lc.

Fou

nd

z zfl

-Hyd

roxy

[4-z

4C] -

z 7

25

estr

on

e(3

32

3co

un

ts/m

in)

28

0. 5

15

z269

23

o 3

32

00

z2

7o

345 °

26

7-26

8 C

~H

z604

76

.89

76.6

7 6.

7z

6.37

t Ifl

-Hyd

roxy

[4-z

~C

] -

7725

es

trad

iol

(475

2cou

nts/

min

i 31

1.

o 25

i6

6o

230

39 o

oo

z27o

35

00

197-

I98

Cai

Haz

05

77.3

9 75

.94

6-49

5.

98

800

m= tO

0

~. ~o

o 800'

6 i 6 :4.

Fig

. z.

Cou

nter

-c,l

rren

t d

istr

ibu

tio

n

of

z i-

hy

dro

xy

estr

on

e m

ono-

• ~

.;

i/*

be

nzoa

te.

0--

0,

rad

ioac

tiv

ity

; ×

- ×

, ab

sorp

tio

n a

t .-3

° m

~u.

4 6

6 ro

,'e

i4

16 !

0

Fig

. 2.

C

ou

nte

r-cu

rren

t d

istr

ibu

tio

n

of

z z-

hy

dro

xy

estr

adio

l di

ben-

TU

BE N

UMBE

R zo

ate.

@

--@

, ra

dio

acti

vit

y;

×--

×,

abso

rpti

on a

t 23

0 ra

p.

8 8

I00

0

u~60

0 =/

'=

4°°lJ

I 2 "

200

I •

•

2 4

6 8

I0

12 1

4 16

IB

~0

TUBE

NUMB

ER

SHORT COMMUNICATIONS 611

the second crop of crystals, the mo the r liquors from the var ious crystal l izat ions were pooled a n d assayed for to ta l radioact iv i ty . The results of the recrystal l izat ions are summar ized in Table II .

S a p o n i f i c a t i o n o f benzoates

After recrystal l izat ion, the crystal l ine mater ia l was pooled and refluxed wi th 3 ml of 4 N methanol ie KOH for 30 rain. W h e n the react ion was complete, the mix ture was poured in to 25 ml of water , a n d the pH was ad jus ted to 7-8 by the addi t ion of dil. H2SO 4. The aqueous mix tu re was then ex t rac ted wi th e ther (4 t imes wi th 50 nil). The combined e ther ex t rac t was washed wi th wat%r (2 t imes wi th 20 ml), and then dried over anhyd . Na,SO, . Evapora t i on of e ther under ni t rogen yielded free 11/3- hydroxyes t rone - : id free x I/3-hydroxyestradiol. The free estrogens were character ized b y u l t ravio le t absorp t ion and de te rmina t ion of par t i t ion coefficients by counter- cur ren t d i s t r ibu t ion in 5 0 % ethyl acetate , 4 o % cyclohexane/33°o aqueous e thanol (solvent sys tem II) as described previouslyL The result2; are summar ized in Table I I I .

T'A BLE II

CARRIER RECRYSTALLIZAT1ONS

Recrystallizations ufl-Hydroxyestrone .'onobenzoate llfl-Hydroxyestradiol dibenzoate

Solvent couats/min/mg Solvent counts, min/mg

ISt Hot ethanol 56 *Hot ethanol I !

2nd Hot ethanol 54 Hot ethanol Io

3rd Hot ethanol 63 Hot ethanol Io

Ethyl acetate Ethyl acetate 4th 5 ° 8

Heptane Heptane

Ethyl acetate Ethyl acetate 5 th 5 t 7 Heptane Heptane

Total radioactivity 660 928 in mother liquor

TABLE 1I[

IDENTIFICATION CF PRODUCTS ISOLATED AFTER SAPONIFICATION OF BENZOATE DERIVATIVES

.SaponiIicatlon product

Benzoate ~max Yield in ethanol K* (counts/rain)

(ml~)

xxfl-Hydroxy[4-14C]estrone 280 2.84 704 monobenzoate

x xfl-Hydroxy [4-a4C]estradiol dibenzoate 280 L27 I42o

• Determined by counter-current distribution with 5 ° transfers in 5 ° % ethyl acetate, 50 % cyelohexane-33 % aq. ethanol.

Biochim. Biopkys . Acta, 57 (I962) 6o9-.-6Iz

6 1 2 SHORT COMMUNICATIONS

In summary, the exper imental results reported in this communicat ion have provided additional evidence concerning the nature and pur i ty ot radioactive I lf l - hydroxyest rone and I l f l -hydroxyestradiol isolated from the urine subsequent to administrat ion of a tracer dose of [4-x4Clcortisone acetate.

We are grateful to Dr. J . A. HOGG, Upjohn Research Laboratories, Kalama,zoo~ Mich. for a generous supply of ! i f l -hydroxyestrone. Also, we wish to acknowledgu the technical assistance of Miss EVA BOKaNY.

This stucty was supported in part by a grant (A-2878) from the National Ins t i tu te of Heal th , U,S. Public Heal th Service.

Roswell Park Memorial Institute Buffalo, N. Y. ( U.S.A .) EDWARD CHANG THoMas L. DAO

x E. Clta.~ A.~D T. L. DAO, J. Clin. Endocrinol. and Metabolism, 2t (I96t) 6z 4. ~- M. N. Ht:FF,~IAr~ A~D l~J. H. LOTT, J. Biol. Chem., 2t 5 (x955) 627. z L. C. CRAIG AND D. CRAIG in WEISABERGER (Ed.), Technique of Organic Chemistry, Vol. 3, Inter-

science, New York, I95o, p. I7I.

Received October 5th, x96x

Bioehim. Biophys. Acta, 57 (1962) 6o9-6xa