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UNITED STATES ATOMIC ENERGY COMMISSION
MICRO-SYNTHESES WITH TRACER ELEMENTS. XVI
THE SYNTHESIS OF HEXESTROL LABELED WITH TRITIUM
D. L. Williams
Anthony R. Ronzio
Los Alamos Scientific Laboratory
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MICRO-SYNTHESES WITH TRACER ELEMENTS. XVI.
THE SYNTHESIS OF HEXESTROL LABELED WITH TRITIUM
D. L. Williams an! Anthony R. Ronzio
information was desired regarding the stability of organic compounds
containing the isotope tritium in place of hydrogen. Since a higher concen-
tration of radioactivity can be imparted to an organic compound by means
of tritium than with almost any other element, this method of labeling should
prove to be ideal for those chemical entities which produce profound biologic
changes in the living system when present in exceedingly small concentrations.
A group of compounds having such an effect are the sex hormones. Minute
amounts of these chemical entities can bring about deep seated physiologic
changes in both the male and female.
One of the female sex hormones, "hexestrol" is synthetic in the sense
that it does not occur naturally in living organisms, but is a very powerful
estrogen. This compound may be easily labeled with tritium and was accord-
ingly chosen for study.
THEORETICAL AND HISTORICAL
Hydrogenation of the unsaturated aliphatic linkage of either "Stilbestrol" (I)
(4, 4'-dihydroxy-a. p-diethylstilbene)
HO < C = C OH (I)
C H5 C H5
or "Dienestrol" (II) (3, 4-di(p-hydroxyphenyl)-2, 4-hexadiene)
HO C C OH (II)
should afford an efficient means of labeling "Hexestrol" (III)
HO < C -C C C- OH (III)
C2H5 C H5
with the radioisotope of hydrogen, tritium (H3). Hexestrol, first isolated
from the demethylation products obtained from anethole by Dodds et al,
displays an extremely high estrogenic activity.
The d-, -, meso-, and racemic forms of hexestrol are possible since
the compound contains two asymmetric carbon atoms. The same workers
isolated an isomer of hexestrol melting at 128C0 (hexestrol, m.p. 184-50C)
which was a much less potent estrogen. Wessely and Welleba(z succeeded
in resolving isohexestrol racemicc form) into d- and -- forms and accordingly
assigned the meso form to hexestrol. An equimolar mixture of the d- and -
forms melted at 129 C, confirming the accuracy of their separation.
Hydrogenation of stilbestrol gave a mixture of isohexestrol and hexestrol;
the proportions of the two compounds depended upon the catalyst used and the
conditions of the hydrogenation.
Wessely and Welleba obtained an 88%6 yield of isohexestrol and a 12%
yield of hexestrol when hydrogenation was carried out in acetic acid at 18 C
using palladium sponge.
Docker and Spielman3 claimed a quantitative yield of isohexestrol by
the hydrogenation of stilbestrol using either palladium, copper chromite, or
Raney nickel catalysts. The high yields of isohexestrol obtained by the hydro-
genation of stilbestrol has been confirmed by others,5 and stilbestrol
apparently does not seem desirable as a starting material for the labeling
Dodds et al reported the successful synthesis of dienestrol and the
hydrogenation of the compound to hexestrol in acetone solution using a
palladium catalyst. A quantitative yield was reported. The exact nature of
the catalyst, e.g. whether supported or not, was not given.
In view of this work, dienestrol was selected for hydrogenation to
hexestrol. The H atoms are located at the positions marked with ap
HO -C C -< OH (IV)
In a series of preliminary experiments, dienestrol (m.p. 231-2 C) was
hydrogenated for the purpose of studying the conditions of hydrogenation and
procedures for purification of the product. A resume of these experiments is
given in Table I.
The apparatus used for carrying out the preliminary experiments is shown
in Figure 1. To a high vacuum manifold was attached a mercury manometer,
a gasometer and a 25 ml. flat-bottomed reaction flask. The flask contained
a short glass enclosed iron rod which served as a magnetic stirrer. Experi-
ments 1, 2, 3, 4, 5, and 9 were carried out in this apparatus.
Since the health hazards from tritium are as yet unknown and since we
were advised that tritium was tenaciously adsorbed on glass surfaces, the
system shown in Figure 2 was designed and used in the tritium hydrogenation.
All contact of tritium with grease was eliminated as much as possible. No
semi-ball joints were used throughout the entire system. The system was so
arranged that the contaminated portion could be isolated and disposed of after
the experiment was completed. A diagram of the apparatus is shown in
The labeling of the dienestrol was carried out according to the following
procedure. H 3 0.15 ml., diluted with H was sealed in "U" tube A which
contained two "breakoffsky" sections y and z B was a 25 ml. flat bottomed
reaction chamber containing catalyst, solvent, dienestrol and a short glass
enclosed iron rod which served as a magnetic stirrer. C, a "U" tube having
a 25 ml. capacity was connected to a gasometer D the outlet of which con-
tained a ground glass valve, E. This was used to prevent the accidental
flow of mercury out of D and into the system. The system contained con-
strictions at points marked F, G and H.
In carrying out the hydrogenation, tube A containing H 3-H2 and 2 steel
balls and reaction chamber B containing the catalyst, solvent, dienestrol and
stirrer were sealed to the system. The contents of B were frozen with liquid
N2 and the entire system was evacuated to 3-5 microns. During this process
the mercury rose in the gasometer until stopped by valve E. After pieing
tested for leaks the reaction system was isolated from the exhaus' manifold
by sealing the constricted points F and G. After the contents in B hrj melted
completely, the "breakoffsky" at Y was broken a id the H -H wa allowed tV
react for 45 min. with the well-stirred reaction mixture. Meanwhile the gas
buret D was filled with H The levelin- bulb K was raised in order to place
a positive pressure on the system, and "breakoffsky" Z was then brok -1. The
steam of H2 served to carry over into the reaction chamber any residue of
H -H remaining in "U" tube A. The course of the reaction was followed
by the decrease in volume of H .
After hydrogenation was complete the mercury reservoir K was lowered
and tube C and the gas bulb of D were both cooled with liquid N2. When the
solvent from A had disappeared entirely, the system was sealed at H and the
reaction flask was removed.
Since the health hazard for tritium is as yet unknown, the glass system
was melted apart at points F, G, and H, the tubing connected to the reservoir
closed with clamps and cut, and the portion of the system thus isolated was
removed for disposal.
Experiments 6, 7 and 8 were carried out in this type of apparatus.
Platinum oxide was used in Experiments 1, 2 and 3. The catalyst was
rr duced in the solvent. The water and solvent was then removed in a high
vacuum and additional solvent with the dienestrol to be reduced, was intro-
duced into the reaction chamber.
At the completion of the experiment the reaction mixture was filtered
thru a medium sintered glass funnel. The solvent was removed under vacuum
and the product was also dried under vacdum.
The Adams platinum oxide catalyst proved not to function well in the
reduction of dienestrol. The rate of hydrogenation diminished rapidly and
the total hydrogenation was not quantitative. The addition of ferrous sulfate,
which sometimes inhibits deactivation of this catalyst, did, in this experiment,
both increase the rate and complete the hydrogenation.
Some material insoluble in benzene, the nature of which was not in-
vestigated, was formed in Experiments 2 and 3. Since Dodds et al used as
catalyst, palladium and since the platinum catalyst gave uncertain results, a
catalyst of 10% palladium on carbon was used for the hydrogenation of dienestrol.
Solubility data of pure hexestrol in benzene(6) at 15-160C indicated that
a ratio of 5 ml. of benzene per 100 mg. of hexestrol should give a 90% yield
of product upon recrystallization. The yield of material melting between
183-185 C obtained in Experiments 5, 6, 7, 8, and 9 was 72 to 77% of theory.
Upon concentration of the mother liquor to 1/5 the original volume an additional
5-7% of product melting at 175-1800C was always obtained.
The second mother liquor obtained in this manner in Experiment 8 was
evaporated to a volume of 1. 5 ml., refluxed gently to dissolve all material
present and cooled. A quantity of substance, 137 mg. (26.7% of theory),
melting at 126-80C, separated in the form of fine, colorless needles. This
compound corresponds to isohexestrol (m.p. 128-129 C(2 4). This compound
was not reported by Dodds and co-workers(5) who hydrogenated dienestrol in
acetone solution with palladium catalyst. These investigators either overlooked
this fraction, or used a catalyst possessing a different composition from the
one used in the present experiments.
Evaporation of the final mother liquor to complete dryness left a colorless
residue which melted at 123-125C (7.1% of theory).
It should be noted that other solvents tried did not give this clean-cut
separation of meso-hexestrol from racemic-hexestrol (iso). Dioxane-H 0 and
carbon tetrachloride solvents gave an 85% yield of material melting 10 C low for
the meso form.
3. SOLVENT OF CRYSTALLIZATION
In all experiments the yield of crude product was in excess of 100%. In
no instance did additional drying, even under vacuum decrease the weight.
In those experiments in which absolute ethanol was used as solvent, the
excess in weight amounted to approximately 23% of the theoretical (Experiments
1, 3, 9). When acetone was used as solvent the excess weight was about 19.0%
of the theoretical. (Experiments 4, 5, 6, 7, 8).
The first assumption was that some inorganic salt was extracted from the
catalyst. However, when a weighed quantity of catalyst was extracted either
with acetone or with alcohol, the weight of catalyst remained unchanged. No
residue remained after evaporation of the solvent. It is significant that the
same percentage excess in the weight occurred for each solvent regardless of
the amount of material hydrogenated or of the catalyst used.
The possibility that a pinacol was formed from the acetone is not tenable
since an increase in weight also appeared with ethanol as solvent. This was
further checked by attempting to hydrogenate acetone with the palladium catalyst
used. There was no hydrogen uptake over a four hour period.
The excess weight of crude product corresponds closely to one mole of
solvent per mole of compound. It then appears likely that a mole of solvent is
held as solvent of crystallization.
4. H3 LABELED HEXESTROL
The reaction chamber containing a mixture of 503 mg. of dienestrol,
54 mg. of 10% palladium on charcoal, and 5 ml. dry acetone was sealed to the
apparatus in the manner described above, Figure 2, and the H 3-H2
hydrogenation completed. After complete hydrogenation the solvent was
removed, as described previously, and the hexestrol was isolated.
The product was redissolved in acetone and the solution was filtered in
a medium sintered glass funnel thru a bed of infusorial earth -- "Norite"
carbon. This was necessary in order to remove the suspended catalyst. The
flask and catalyst were washed with 2 ml. portions of acetone. The total solu-
tion, 50 ml., was evaporated to dryness in a vacuum, and completely dried
in a vacuum desiccator. The crude product weighed 594.6 mg. (116.2% of
The crude product was purified by recrystallation from 30 ml. of benzene.
The crystals were allowed to form at room temperature, and then at 6-80C
(2 hours). The mother liquor was removed and collected in a tared 40 ml.
cone with a filter stick. The colorless product after drying in a desiccator
under vacuum weighed 386.4 mg. (75.8% of theory) and melted at 184-5 C.
This is the meso form commonly called hexestrol and is the isomer of high
Evaporating the mother liquor to dryness left a colorless residue weighing
220. 8 mg. The residue was dissolved in 5 ml. of benzene with gentle refluxing,
then cooled to 6-8 C for 1 hour. The mother liquor was removed with the
filter stick as before. The colorless crystalline residue weighed 34. 9 mg.
(6. 8% of theory) and melted at 160-40C. This undoubtedly is a mixture of
hexestrol and isohexestrol.
The second mother liquor was evaporated, under vacuum to a volume of
1-1/2 ml. and then was cooled to 5-6 C for 1 hour. The mother liquor was
removed as before and the colorless needles were washed with 0. 5 ml. of dry
benzene. This fraction after drying weighed 142. 8 mg. (28% of theory) and
melted at 126-7 C. This fraction is the racemic form called isohexestrol.
The mother liquor when evaporated to dryness left a residue weighing 30.2
mg. and which melted at 124-5 C.
DISCUSSIONr OF RESULTS
It was surprising to observe that the rate of hydrogenation was much
more rapid when the H2 -H2 mixture was used than in the last trial run
using H2, Experiment 8, in which the conditions were identical in every
The two rates of hydrogenation are shown in Figure 3 in which quantities
of hydrogen consumed per unit times are compared.
Solvent, catalyst and hydrogen from the same stocks in the same
apparatus was used throughout both experiments. The hexestrol obtained
in this experiment possessed I mc. of radioactivity per mg.
The estrogen, hexestrol, labeled with the radioactive isotope of hydrogen,
tritium, has been prepared.
Both meso- and racemic forms of hexestrol were obtained by the hydro-
genation of dienestrol with a palladium on carbon catalyst.
The presence of a small amount of tritium exerted a very significant
influence upon the rate of consumption of ordinary hydrogen under conditions
which were otherwise identical.
1. 'ampbell, Dodds and Lawson, Nature, 142, 1121 (1938).
2. Wessely and Welleba, Ber. 74, 777 (1941).
3. Docker and Spielman, J. Am. Chem. Soc., 62, 2163 (1940).
4. Campbell, Dodds and Lawson, Proc. Roy. Soc. (London) B128, 253
5. Dodds, -Goldberg, Lawson, Robinson, Proc. Royal Soc. (London)
B-127, 140 (1939).
6. Cheymol and Carayon Gentil. Bull. Soc. Chim. Biol. 28, 136 (1946)
C.A. 41, 3518e.
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