A convenient preparation of high specific activity tritiated estrogens

925

A CONVENIENT

PREPARATION

ACTIVITY

M.M.

OF HIGH

TRITIATED

Coombs

a n d H.R.

SPECIFIC

ESTROGENS

Roderick*

Division of Chemistry and Biochemistry, I m p e r i a l C a n c e r R e s e a r c h Fund, Lincoln's Inn Fields, London, W.C.2., England.

Rece£ved January 23, 1968

ABSTRACT 2,4-Dibromo-estriol and - 1 6 - o x o e s t r a d i o l w e r e p r e p a r e d b y b r o m i n a t i o n of the h o r m o n e s w i t h Nbromosuccinimide. Selective dehalogenation by r e d u c t i o n in t r i t i u m o v e r palladitum y i e l d e d 2 , 4 - ~ H ] estriol and 2,4-[3H]-16-oxoestradiol with specific a c t i v i t i e s of 38 a n d 51 c u r i e s / m , mole, r e s p e c t i v e l y .

Steroidal activities catalytic through

with

tritium

tritiation

reductive

derivatives. I allows

estrogens

the

activity forward

estrogen

saturation

o f 50

a need

such

labelled

of c a r r i e r - f r e e

mole

here

arose

tritium with

an

In

o n the m e c h a n i s m

for certain

tritium.

halogen

by straight-

~ 6-derivative.

as 1 6 - o x o e s t r a d i o l with

both by

of amenable

curies/m,

o f the

specific

intermediates, and

of 6 , 7 - ~ H ] - e s t r a d i o l

in progress

action,

to h i g h

prepared

of unsaturated

the u s e

synthesis

of e s t r a d i o l similarly

Thus

work

have been

dehalogenation

in excess

biological

labelled

metabolites

and estriol

Since

of

unsaturated

926

ST ER O ID S

derivatives

of these

inaccessible,

a n d all b u t

transformations activity were

are

on steroids

prepared

from

diiodo-derivative the u s e

with

conservation

oxoestradiol.

of t h i s

This

2,4- ~H]-estrogens under

Aromatic

constants

from

mild

over Raney

the

American

authors,

rotation

were

noted

its d i a c e t a t e .

The

similar

serious

with

(IIb)

their

steroids

the p h y s i c a l

reported

and

structure.

by

in optical and

spectroscopic coupled

2,4-dibromoestradiol on reduction,

of

dibromo-

for 2,4-dibromoestradiol

that

establish

synthesis

its d i a c e t a t e

differences

analytical

in 1 6 -

N-bromosuccinimide

to t h o s e

the f a c t

estradiol

the

and

for our dibromocompounds,

yielded

compatible

function

While

evidence

our

not

of p h e n o l i c

of 2 , 4 - d i b r o m o e s t r o n e b y us w e r e

nickel.

conditions.

temperature. )

prepared

of the 2 , 4 -

corresponding

selective

conveniently

at r o o m

was

describes

and

et al. 2 w h o

o f the = - k e t o l

paper

m a y be

hormones,

catalyst

level

of l a b e l l i n g

by reduction

dibromination

effected

in ethanol

to t h i s

methods

taken by Nunez

in tritium

However,

m a y be

chemical

tritiated

the parent

o f this w a s

compounds

simplest

other

2,4- ~H]-estradiol

the

are relatively

2,4-Halogenated-estrogens

easily

advantage

the

impractical,

studied.

obtained

compounds

11:6

with

readily

appears

to

2,4-Dibromo-estriol

and 2,4-dibromo-16-oxoestradiol

(Ib) w e r e

the

of

June 1968

STEROIDS

also

prepared

without

927

difficulty

by the use

of N-

bromosuccinimide. Hydrogenolysis with

of these

palladium-on-charcoal

potassium hydroxide parent

hormones

tritium

four d i b r o m o e s t r o g e n s

catalyst

smoothly

regenerated

in good yield.

in place

of hydrogen,

by p a p e r

These

chromatography

chemically

were r e d u c e d

compounds

of the radioactivity from 2,4-

(IId)

demonstrated

entered

phenolic (Id)

positions

hydroxy

list

940/0

approximately the

original

hydroxide Estimation reaction

respectively.

to r e a c h

but

on e q u i l i b r a t i o n situated

of the u n d i l u t e d

0H]-16-oxoestradiol (IId),

than those

ortho

to the

2,4- ~ H ] - 1 6 - O x o e s t r a d i o l

t r i t i u m was

of 51 and

of

H]-estriol

since

of the r a d i o a c t i v i t y

steroid

Loss

than i°/0 of the t r i t i u m

on r e b r o m i n a t i o n ,

and by u.v.

activities

other

group.

6°/o

this

less

by

samples.

99%

had

purified

analysis.

investigated

of c a r r i e r - d i l u t e d

that

were

to the

and shown to be radio-

of l a b e l l i n g was

rebromination

pure

2,4-dibromoestriol

pure by c a r r i e r - d i l u t i o n

Specificity

the

By e m p l o y i n g

and 2 , 4 - d i b r o m o - 1 6 - o x o e s t r a d i o l 2,4- D H ] - d e r i v a t i v e s .

in m e t h a n o l i c

was lost

from

with N sodium

at C-16.

estrogens

by the K o b e r

spectroscopy

indicated

38 curies/m,

mole

specific

for the 2,4-

(Id) and 2 , 4 - ~ H J - e s t r i o l The

the t h e o r e t i c a l

failure maximum

of these

activities

for the i n t r o d u c t i o n

928

ST ER O ID S

of two mole)

tritium

atoms

per

probably

stems

from

aqueous

methanolic

dehalogenation protons gas

with

the

end

Attempts

the

but

it w a s

term

at

the

of local

label.

in a q u e o u s

between

3 and

to a r i s e

i0

that

in v i v o

Acid-catalysed rapid; after

exposure

detailed

detritiation

is

involving

of

after

the

the

at

It was,

of

the

may

2 months, be

acid end

this

expected

exchange. relatively

radioactivity

of

stability

2,4-[3H] -

for

to b e

the

short

pH v a l u e s

chemical

appeared

in p r o g r e s s .

the

storage

at

of a c t i v i t y

investigation

in

chemical

temperature

remained

hormones,

with

the

to N h y d r o c h l o r i c

one-third

at

estrogens

application.

buffered

half

tritium

radioactive.

animals

activity

purely

exchange

approximately

only

A more

from

the

recovered

unimportant

vaginal

loss

of

2,4-tritiated

unaffected

no

employing

solvent

highly

connection

at r o o m

the

the n a t u r a l

intact

to be

media

of

curies/m.

unsuccessful.

specific

was

steroid

always

to a s c e r t a i n

The

16-oxoestradiol

in

in

of

solvent

that

from

envisaged

essential

indicating

was

58

in t h e

to d i l u t i o n

were

C-2

necessity solvent

the

recognized

considered

effects

however,

and

since

of r a d i o a c t i v i t y

experiments

of

leading

dioxan

metabolized

loss

as

(about

Exchange

reaction

to u s e

It w a s are

the

reaction.

hydrogen, of

molecule

alkali

occurred,

11:6

for

was

lost

2 weeks,

of 2 m o n t h s . acid-catalysed

June 1968

s T ER O ID S

x

.OR

929

x ~ ~ O

I

H

II a

X

=

H, R = H

b

X

= Br,

R = H

c

X

= Br,

R = Ac

d

X

= 3H, R = H

EXPERIMENTAL The

following

instruments

were

used:-

ultraviolet spectra...Perkin-Elmer 137 U v and U n i c a m sPSO0 spectrophotometers, ethanol solutions; infrared optical

s p e c t r a ...... P e r k i n - E l m e r r o t a t i o n s ..... Z e i s s

237

spectrophotometer;

L E P A1 p o l a r i m e t e r ;

n.m.r, spectra ........ Varian A60 t e t r a m e t h y l s i l a n e as i n t e r n a l

spectrometer, indicator.

Radioactivity measurements were performed by scintillation counting, using a Nuclear Chicago Mark I i n s t r u m e n t ; q u e n c h i n g c o r r e c t i o n s w e r e m a d e u s i n g the automatic external standard. The counting efficiency w a s 9 3 0 / 0 . M e l t i n g - p o i n t s w e r e d e t e r m i n e d on a h o t s t a g e a p p a r a t u s a n d are u n c o r r e c t e d . 50/0 Palladiumon-charcoal catalyst was purchased from Koch-Light L a b o r a t o r i e s and the s a m e b a t c h w a s e m p l o y e d in all the experiments. 16-Oxoestradiol (Ia) w a s p r e p a r e d a c c o r d i n g to the m e t h o d o f H u f f m a n n a n d D a r b y 4, m.p. 2 3 5 - 2 3 8 0 (lit., 5, m.p. 2 3 4 - 2 3 7 0 ) C a l c d . f o r C 1 8 H 2 2 0 ) : C, 75.5; H, 7.75. F o u n d : C, 7 5 . 8 5 ; H, 7.95. The other steroids were obtained from commercial sources. 2,4-Dibromination. - 2,4-Dibromo-16-oxoestradiol ~.4-Dibromq-3,17~-dihydroxyestra-l,~,5 l l O ) - t r i e n - 1 6 - o n e , Ib~ 16-Oxoestradiol (Ia) 1 . 1 7 g.) i n e t h a n o l ( 3 5 0 m l . ) w a s t r e a t e d w i t h Nbromosuccinimide ( 1 . 6 3 g.) at r o o m t e m p e r a t u r e .

9 30

ST ER O ID S

11:6

After 18 hr. the solvent was removed in vacuo at 20 o , and the residue was d i s s o l v e d in chloroform (300 ml.). The solution was washed with two portions (each 30 ml.) of water and dried. After removal of the solvent the residue was r e c r y s t a l l i s e d from methanol y i e l d i n g c h r o m a t o g r a p h i c a l l y homogeneous material (905 mg.). The 2 ~ 4 - d i b r o m o - d e r i v a t i v e was purified by further r e c r y s t a l l i s a t i o n from methanol followed by drying at 600 in high vacuum, granules, m.p. 227-229°; [~] 23 _ 580 (C 0.13 in chloroform-methanol, 2:1); ~ 2~2 (shoulder 287) m~ ( £ 2954); T ( d i m e t h y l - s u l ~ x i d e , (DMSO) 2.60 (C-I proton) and 9.36 (3 protons, 18methyl). Anal.

Calcd. for CIsH20Br202 : C, 48.65; Found: C, 48.8; H, 4.35; Br,

H, 4.55; Br, 36.0.

36.25.

Treatment of (Ib) with acetic anhydridepyridine gave the diacetate (Ic), needles from methanol, m.p. 187-1880; ~ ] 24 _ 740 (c 0.23 in chloroform> ~ 274, 2 8 2 . 5 Dm~ (~ 781 A 747)~n'9~

(cs=) 178o, 1~, 1 7 4 5 , 1 2 2 5 , 119o cm '; v ( ~ T } ~ 2 . 4 7 ({-1 p r o t o n ) , 7 . 5 9 (3 p r o t o n s , 3-OAc), 7 . 7 9 (3 protons, Br,

17-OAc),

9.13

(3

protons,

18-methyl).

Anal. Calcd. for C22H24Br205: C, 50.0; H, 4.6; 30.25. Found: C, 50.05; H, 4.6; Br, 30.8.

2,4-Dibromoestriol ~,4-dibromoestra-l,3.5 (lO)-tricne-3,16=,lT~-triol, IIb] prepared by similar b r o m i n a t i o n of estriol had m.p. 281-282 o ; [ ~ 23 + (shoulder 288) m~ (£ 3066); ~ (DMSO) and 9.36 (3 protons, 18-methyl). H, 4.95;

Anal. Calcd. for C18H22Br203: C, 48.5; Br, 35.8. Found: C, 48.5; H, 5.1; Br,

A c e t y l a t i o n as above afforded a product though homogeneous, could not be crystallised. material had 9 (CS2) 1780, 1740, 1240-1220, --I cm

n)

2.62

35.7. which, This ll90

max .

2,4-Dibromoestradiol [2,4-dibromoestra-l.3.5 (10)-triene-~tlT~-diol q obtained as above from estradiol had m.p. 226-'2280; C~7, 22 + 61.10 (c 0.36 in chloroform-methanol, 2:1) (li~.,) m.p. 218-2190, [ ~ 29 + 1220); ~ 292 (shoulder 287) m~ ( ~ 2900); W (BMSO) 2.62 ( c - W ~ r o t o n ) and 9.35 (3 protons, 18methyl). Anal. Calcd. for C18H22Br202: C, 50.25; H, 5.15; Br, 37.15. Found: C, 50.3; H, 5.3; Br, 37.0.

June 1968

STEROIDS

2,4-Dibromoestradiol 3,17-diacetate prepared by a c e t y l a t i o n of the a b o v e s t e r o i d had m.p. 180-1820; [~]D 23 + 36.10 (c 0.24 in c h l o r o f o r m ) (lit., 3 m.p. 167_1680, [m~ 27 + 106o; ~ 275, 284 m~ (~ 672,

579); ~m-x (Cg2) 1780, 174~x1245, 1190 cm-'; ~(CDC13) 2 . 5 0 (C-~ proton), 7.62 (3 p r o t o n s , 3-0Ac), 7.94 (3 p r o t o n s , 1 7 - 0 A c ) , and 9.17 (3 p r o t o n s , 1 8 - m e t h y l ) .

H,

5.1;

Anal. Calcd. for C22H26Br204: C, 51.4; Br, 31.1. F o u n d : C , 51.3; H, 5.35; Br, 31.0.

B r o m i n a t i o n of e s t r o n e y i e l d e d 2 , 4 - d i b r o m o estrone[2,4-dibromoestra-l,3,5(10)-trien-17-one~, m.p. 231_233~; [~ 22 + 1220 (c O . 2 1 in c h l o r o f o r m - m e t h a n o l , 2 : 1 7 (lit., 3 m.p. 2 2 5 - 2 2 6 0 , [ ~ 2 7 + 1330) w h i c h gave the a c e t a t e m.p. 1 9 1 - 1 9 4 ° ; [ ~ 21 + l l l 0 (c 0.22 in c h l o r o f o r m ) (lit., 3 m.p. 1 8 4 - 1 ~ 5 0 , ~D 27 +

io7 0 )

D e h a l o g e n a t i o n of 2 , 4 - d i b r o m o e s t r o g e n s . 16-oxoestradiol (Ia). - 2 , 4 - D i b r o m o - 1 6 - o x o e s t r a d i o l (Ib) (230 mg.) in m e t h a n o l (i00 ml.) c o n t a i n i n g p o t a s s i u m h y d r o x i d e (i.0 g.) was s t i r r e d w i t h 50/0 p a l l a d i u m - o n - c h a r c o a l (80 mg.) under hydrogen. A f t e r 2 hr. the c a t a l y s t was r e m o v e d and the s o l u t i o n was d i l u t e d w i t h w a t e r (10o ml.). After being acidified with hydrochloric a c i d the s o l u t i o n was e x t r a c t e d w i t h c h l o r o f o r m . F r o m the c h l o r o f o r m e x t r a c t was o b t a i n e d the s t e r o i d (Ia) (150 mg.) w h i c h , a f t e r b e i n g r e c r y s t a l l i s e d f r o m m e t h a n o l , h a d m.p. 2 3 7 - 2 3 9 0 , u n d e p r e s s e d by a d m i x t u r e w i t h the a u t h e n t i c sample, m.p. 2 3 5 - 2 3 8 0 . T h e i n f r a r e d s p e c t r a of the s a m p l e s w e r e i d e n t i c a l . In the same w a y d e h a l o g e n a t i o n of 2 , 4 - d i b r o m o estrone, of 2 , 4 - d i b r o m o e s t r a d i o l and of 2 , 4 - d i b r o m o e s t r i o l (IIb) gave r e s p e c t i v e l y , estrone, e s t r a d i o l and e s t r i o l . The i d e n t i t y of the p r o d u c t in e a c h case was establis]led t h r o u g h co:nparison of the m.p. and i n f r a r e d s p e c t r a w i t h those of a u t h e n t i c s a m p l e s and t h r o u g h m e & s u r e m e n t of the ~nixed m e l t i n g points. T r i t i a t i o n of 2,4-d.ibro:ninated estrp;~ens. T h e v a c u u m m a n i f o l d shown, a m o d i f i c a t i o n of the o r i g i n a l a p p s r a t u s d e s c r i b e d by R o c h e et al. 6, was u s e d e:aployin,5 s t a n d a r d h i g h v a c u u m p r o c e d u r e s . '±'he tritiunl r e s e r v o i r s , A and B, each c o n t a i n i n g uraniulfl t u r n i n g s (2 g.) p a c k e d in glass wool, w e r e h e a t e d ~it 4500 for 15 alia. in v a c u o by m e a n s of an e l e c t r i c f u r n a c e , a f t e r w h i c h tap b_ was c l o s e d and the r e s e r v o i r s w e r e cooled. The evacuated system was i s o l a t e d f r o m the pump by the tap _s, t r i t i u m gas (25 curies, a b o u t l0 ml.) f r o m the a m p o u l e C, o b t a i n e d

931

932

ST ER O I D S

11:6

f r o m the R a d i o c h e m i c a l Centre Amersham, was admitted and was rapidly absorbed by the uranium i n A. When absorption was complete (30 m i n . ) , t a p ~ w a s c l o s e d and t h e s y s t e m w a s a g a i n p u m p e d to r e m o v e a t r a c e of residual gas.

Tritiation Ap_paratus Pirani Gauge

Blo Elo

to pump

I

B10

M

JlO

B10

BIO F A

B

C

W

T h e 20 ml. r e a c t i o n f l a s k R, p r o v i d e d with a v a c u u m t a p ~, c o n t a i n e d 2,4-dibromoestriol (12.0 mg.), 5 0 / 0 P d - C (15 m g . ) , p o t a s s i u m hydroxide (50 m g . ) a n d methanol (5 m l . ) a n d w a s de~,assed~ b y t h e u s u a l procedure of repeated freezing in liquid nitrogen, pumicing a n d t h a w i n g . ~{ith t h e e v a c u a t e d system isolated from the pump, R frozen and taps ~ and o p e n , the a p p a r a t u s w a s f i l l e d w i t h t r i t i u m gas, at a pressure measured on the m a n o m e t e r M, b y h e a t i n g A. The tap E of the filled reaction flask R was closed a n d a f t e r the e x c e s s o f t r i t i u m h a d b e e n r e a d s o r b e d in the c o o l e d r e s e r v o i r A, t h e f l a s k w a s r e m o v e d from the l i n e a n d g e n t l y s h a k e n f o r 2 hr. at r o o m t e m p e r a t u r e . The flask was then returned to t h e line, f r o z e n and the unreacted tritium was adsorbed in t h e s e c o n d uranium reservoir B. After rethawing, the solvent was removed by allowing it to c o n d e n s e in t h e w a s t e v e s s e l W w h i c h w a s c o o l e d in l i q u i d n i t r o g e n . Aqueous acetic a c i d ( 5 0 / 0 v/v, 3 m l . ) , c o n t a i n e d in F a n d p r e v i o u s l y degassed, was condensed into R which was allowed to w a r m to r o o m t e m p . to e f f e c t n e u t r a l i s a t i o n and equilibration, and the water was then removed to ~. The solid contents of R were transferred to a f i l t e r with chloroform-methanol (2:1, t o t a l I0 m l . ) a n d the catalyst was removed. The solution was washed twice

June 1968

s T ER O ID S

933

w i t h water, e v a p o r a t e d to d r y n e s s in a c u r r e n t of n i t r o g e n a n d r e d i s s o l v e d in e t h a n o l (50 ml.). The a q u e o u s m e t h a n o l in W was f o u n d to c o n t a i n 7 . 7 0 c u r i e s of r a d i o a c t i v i t y w h i l e the two w a t e r w a s h e s c o n t a i n e d ll m. c u r i e s each. T h e p r o d u c t (IId) (0.81 curies) was p u r i f i e d by c h r o m a t o g r a p h y on W h a t m a n 3MM p a p e r u s i n g the s y s t e m t o l u e n e (1400 ml.) and ethyl a c e t a t e (600 ml.) I m o b i l e p h a s e ) - m e t h a n o l (I 1. ) and w a t e r (1 1.) ( s t a t i o n a r y phase). T h e e s t r i o l zone, R ~ 0 . 5 0 l o c a t e d by a u t o r a d i o g r a p h y on K o d a k X - r a y film, was e l u t e d w i t h e t h a n o l a n d the s t e r o i d was e s t i m a t e d by u.v. s p e c t r o s c o p y at 280 m~. An aliquot containing 1.37 x l08 d p m was a d d e d to i n a c t i v e e s t r i o l ( 1 . 0 0 6 1 g.) w h i c h was t w i c e r e c r y s t a l l i s e d f r o m chloroform-methanol:let 2nd

c r y s t a l l i s a t i o n . . . . . . . . . . . . 1 . 3 6 x i0 ~ dpm/mg. crystallisation(0.8017 g.) 1.39 x l05 d p m / m g .

showing

that

(IId)

was

radiochemically

pure.

2,4-Dibromo-16-oxoestradiol (Ib) was s i m i l a r l y t r i t i a t e d to give (Id) w h i c h was p u r i f i e d by p a p e r chromatography (R F 0.56) u s i n g the s y s t e m t o l u e n e (100 ml.) ( m o b i l e p h a s e ) - m e t h a n o l (60 ml.) and w a t e r (40 ml.) s t a t i o n a r y phase). R a d i o c h e m i c a l p u r i t y was d e m o n s t r a t e d as a b o v e and the e s t r o g e n was e s t i m a t e d by the K o b e r r e a c t i o n ( ~ 515 m~) u s i n g the e s t r a d i o l r e a g e n t of B a u l S ~ x7 R e b r o m i n a t i o n of t r i t i a t e d e s t r o g e n s . Carrier-diluted (Id) (0.19 p. c u r i e s / m , mole; 332 mg.), treated with N-bromosuccinimide as d e s c r i b e d above, gave the 2 , 4 - d i b r o m o - d e r i v a t i v e (Ib) ( 0 . 0 1 2 p. c u r i e s / m. mole; 2 6 3 . 1 mg.) w i t h m.p., R_ v a l u e and i n f r a r e d spectrum identical with those ofrthe sample described above. T h e m i x e d m.p. of the 2 s a m p l e s s h o w e d no depression. Silnilarly

brominated

(IId)

(18.1

as above yielded

mole; 192 mg.) i d e n t i f i e d s p e c t r u m and R F v a l u e .

~.

(lib)

curies/m,

(0.17

by its m.p.,

mole;

314 mg.

curies/m. infrared

A C K N 0 WLEDG~HDNT

T h e a u t h o r s w i s h to t h a ~ Dr. G.F. M a r r i a n his i n t e r e s t in this work. T h e y are i n d e b t e d to M i s s J.¥. C o m b e n for s k i l f u l t e c h n i c a l a s s i s t a n c e , to Mr. J.F. R i c h a r d s for the m i c r o a n a l y s e s .

for and

93~,

ST ER O ID S

11:6

REFERENCES

I.

Evans, E.A., 'Tritium Butterworths, London,

2.

Nunez, J., Jacquemin, A. and Roche, J., INTERNAT. J. APPL. R A D I A T I O N AND ISOTOPES, i_~, 573, (1962).

.

.

.

Slaunwhite, W.R. 1749, (1962).

and its compounds', 1966, p. 169.

and Neely,

Huffmann, M.N. and Darby, 66, 150, (1944). Huffmann,

M.N.

and Lott,

L.,

J.

ORG.

CIIEM., 27,

H.H. , J. AbbR.

M.H.,

J. AMER.

CHEM.

CHEM.

SOC.,

SOC.,

7~, 719, (1949). .

Roche, J., Nunez, J., Jacquemin, A. and Pommier, J., 'Proceedings of the G o n f e r e n c e on M e t h o d s of p r e p a r i n g and s t o r i n g blarked Molecules', Brussels, Nov. 13th-16th, 1963, Euratom, May 1964, p. 1035.

7.

Bauld,

*

Present

W.S.,

BIOCHEH.

address:

J.,

56,

426,

(1954).

Tobacco Research Council Laboratories, Otley Road,

Harrogate.