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Synthesis of estrone-6,7-3H sulfate-35S
VOLUME 1
JANUARY 1963
117
STEROIDS
SYNTHESIS OF ESTRONqS-6,7-3H SULFATE-35S 1 Mortimer Levit z 2 Department of Obstetrics and Gynecology, New York University School of ~dicine, New York, N.Y.
Received November 29, 1962
The view that steroid conjugates serve only as readily excreted end products of steroid metabolism has been modified with the discovery that many conjugates undergo further transformations in vivo. to steroid sulfates°1'2 estrogen,
3
This applies particularly
Although estrone sulfate is a major circulating
very little is excreted unchanged in the urine as such.
A better
understanding of the metabolism of estrone sulfate could be achieved by studying the double-labeled conjugate. Accordingly, a convenient synthesis of estrone-6,7-3H sulfate-35S was sought. Estrone~6,7-3H (estra-l,3,5(10)-triene-3-ol-17-one), easily synthesized by the catalytic reduction of estra-l,3,5(10),6-tetraene-3-ol-17-one with tritium, is available co~ercially.
Reported methods for the sulfurylation
of estrone require either chlorosulfonic acid~ or sulfur trioxide 5 fuming reagents which could constitute a radioactivity hazard to the routine laboratory, if these were labeled with 35S. The synthesis reported here is based on a method described by Sobel, Drekter and Natelson 6 for the sulfurylation of cholesterol.
The starting
35S containing substance is sulfuric acid which is inexpensive and safe to
ST ER O I D S
118
handle.
VOLUME 1
JANUARY 1963
Pyridine sulfate was formed by treating sulfurie acid with pyridine6
In the presence of a 10% molar excess of acetic anhydride, pyridine sulfate reacts v.~th estrone to produce pyridinium estrone sulfate in good yield. Without isolation, the latter is converted to sodium estrone sulfate by the addition of sodium hydroxide.
Apparently the amount of acetic anhydride used
is critical. When the acetic anhydride exceeded the pyridine sulfate twofold, the yield of estrone sulfate was negligible, estrone acetate being the major product° Although the method was designed for the synthesis of radioactive estrone sulfate, it is apparently applicable for the routine preparation of steroid sulfates on any scale,7 EXPERIMENTAL Isotopes: Corporation.
Estrone-6,7-3H was obtained from the New England Nuclear Carrier-free sulfate-35S (0.15 mgo, 20 millicuries) in O.06N HC1
was obtained from the Oak Ridge National Laboratory. Pyridine Sulfate-35S: The sulfate-35S solution was dehydrated at reduced pressure over P205 in a 15 ml. ground glass centrifuge tube. After dilution of the isotope with O.OA ml. (0.73 mmole) of sulfuric acid pyridine sulfate was prepared as described previousl 6
except that the order of addition of
reagents was reversed. One milliliter of dry chloroform and 0.2 ml. (2°5 mmeles) of dry pyridine were added to the sulfuric acid. The white precipitate formed after vigorous stirring was centrifuged and washed 3 times with chloroform°
The yield of pyridine sulfate-35S was quantita~iveo
Sodium Estrone-6~7-3H Sulfate-35S: Because of the short half-life (87 days)
VOLUME 1
JANUARY 1963
ST ER O I D S
119
of 35S, sodium estrone-6,7-3H sulfate and sodium estrone sulfate-35S were prepared separately.
Before conducting a metabolic study, the desired amounts
of the 2 isotopes were mixedo The synthesis was conducted with dry, freshly distilled solvents, and anhydrous reactants.
To 125 rag. (0.~? mmole) of pyridine sulfate in a ground
glass test tube equipped ~ t h a stopper, there was added 1.6 ml. of pyridine and 0.05 ml. (0.53 mmole) of acetic anhydride.
The pyridine sulfate was
quickly pulverized ~ t h a rod and a magnetic stirring bar added. was stirred one-half hour.
The mixture
A solution of 60 mg. (0.22 mmole) of estrone in
0.6 ml. of pyridine was added and stirring was continued overnight.
The
contents of the tube (a fluffy white suspension) was evaporated to dryness at ~0" under vacuum.
The addition of water produced a faint turbidity.
The
solution was adjusted to pH 8 with sodium hydroxide and extracted with ether. The ether extract which would contain unreacted estrone and estrone acetate yielded only 2 rag. upon evaporation.
The pH was elevated to ll-12 with sodium
hydroxide and extracted 5 times with butanol.
After removal of the butanol,
80 mg. of crude sodium estrone sulfate remained. tion from methanol-ether was 50 rag. (60%).
The yield after crystalliza-
The colorless needles, m.p. 229-
231 ° displayed an infrared spectrum identical with authentic estrone sulfate. More than 99% of the radioactivity had the same mobility on paper as sodium estrone sulfate in the solvent system: toluene:n-butanol (lOO: lOO)/ammonium hydroxide:water (20"~).
8
ST ER O I D S
120
VOLUME 1
JANUARY 1963
~O~OT~ 1.
This work was supported by research grants PHS-CY 2071 from the National Cancer Institute, United States Public Health Service and Grant P-206 from the American Cancer Society. A preliminary account of this work was presented at the 46th Annual Meeting of the Federation of American Societies for Experlmnntal Biologists, Atlantic City, April, 1962.
2. Research Career Development Awar~ Ynvestigator, N.I.H., GM-K3-18, ~22.
REFER1~CF_~
i.
Roberts, K.D., VandeWiele, R°L., and Liebermen, S., J. BIOL. CHEM. 236,
2213 (1961). 2.
Twombly, G.H., and Levitz, M., AM. J. OBST. and GYNEC. 80, 889 (1960).
3.
Purdy, R.H., Engel, L.L., and Oncley, J.L., J. BIOL. CHEM. 236
lO&3
(1961). 4. Butenandt, A., ~,d Hofstetter, H., Z. PHYSIOL. CHEM. 259, 222 (1939). 5.
McKenna, J., and Norymberski, J.K., J. CHEM. SOC. 3889 (1957).
6.
Sobel, A.E., Drekter, I.J., and Natelson, S. J. BIOL. CHEM. llS, 381 (1936).
7.
Personal Communication from Dro H. Leon Bradlowo
8.
Schneider, J.J., and Lewbart, M.L., Recent Progr. Hormone Res. 15, 201
JANUARY 1963
117
STEROIDS
SYNTHESIS OF ESTRONqS-6,7-3H SULFATE-35S 1 Mortimer Levit z 2 Department of Obstetrics and Gynecology, New York University School of ~dicine, New York, N.Y.
Received November 29, 1962
The view that steroid conjugates serve only as readily excreted end products of steroid metabolism has been modified with the discovery that many conjugates undergo further transformations in vivo. to steroid sulfates°1'2 estrogen,
3
This applies particularly
Although estrone sulfate is a major circulating
very little is excreted unchanged in the urine as such.
A better
understanding of the metabolism of estrone sulfate could be achieved by studying the double-labeled conjugate. Accordingly, a convenient synthesis of estrone-6,7-3H sulfate-35S was sought. Estrone~6,7-3H (estra-l,3,5(10)-triene-3-ol-17-one), easily synthesized by the catalytic reduction of estra-l,3,5(10),6-tetraene-3-ol-17-one with tritium, is available co~ercially.
Reported methods for the sulfurylation
of estrone require either chlorosulfonic acid~ or sulfur trioxide 5 fuming reagents which could constitute a radioactivity hazard to the routine laboratory, if these were labeled with 35S. The synthesis reported here is based on a method described by Sobel, Drekter and Natelson 6 for the sulfurylation of cholesterol.
The starting
35S containing substance is sulfuric acid which is inexpensive and safe to
ST ER O I D S
118
handle.
VOLUME 1
JANUARY 1963
Pyridine sulfate was formed by treating sulfurie acid with pyridine6
In the presence of a 10% molar excess of acetic anhydride, pyridine sulfate reacts v.~th estrone to produce pyridinium estrone sulfate in good yield. Without isolation, the latter is converted to sodium estrone sulfate by the addition of sodium hydroxide.
Apparently the amount of acetic anhydride used
is critical. When the acetic anhydride exceeded the pyridine sulfate twofold, the yield of estrone sulfate was negligible, estrone acetate being the major product° Although the method was designed for the synthesis of radioactive estrone sulfate, it is apparently applicable for the routine preparation of steroid sulfates on any scale,7 EXPERIMENTAL Isotopes: Corporation.
Estrone-6,7-3H was obtained from the New England Nuclear Carrier-free sulfate-35S (0.15 mgo, 20 millicuries) in O.06N HC1
was obtained from the Oak Ridge National Laboratory. Pyridine Sulfate-35S: The sulfate-35S solution was dehydrated at reduced pressure over P205 in a 15 ml. ground glass centrifuge tube. After dilution of the isotope with O.OA ml. (0.73 mmole) of sulfuric acid pyridine sulfate was prepared as described previousl 6
except that the order of addition of
reagents was reversed. One milliliter of dry chloroform and 0.2 ml. (2°5 mmeles) of dry pyridine were added to the sulfuric acid. The white precipitate formed after vigorous stirring was centrifuged and washed 3 times with chloroform°
The yield of pyridine sulfate-35S was quantita~iveo
Sodium Estrone-6~7-3H Sulfate-35S: Because of the short half-life (87 days)
VOLUME 1
JANUARY 1963
ST ER O I D S
119
of 35S, sodium estrone-6,7-3H sulfate and sodium estrone sulfate-35S were prepared separately.
Before conducting a metabolic study, the desired amounts
of the 2 isotopes were mixedo The synthesis was conducted with dry, freshly distilled solvents, and anhydrous reactants.
To 125 rag. (0.~? mmole) of pyridine sulfate in a ground
glass test tube equipped ~ t h a stopper, there was added 1.6 ml. of pyridine and 0.05 ml. (0.53 mmole) of acetic anhydride.
The pyridine sulfate was
quickly pulverized ~ t h a rod and a magnetic stirring bar added. was stirred one-half hour.
The mixture
A solution of 60 mg. (0.22 mmole) of estrone in
0.6 ml. of pyridine was added and stirring was continued overnight.
The
contents of the tube (a fluffy white suspension) was evaporated to dryness at ~0" under vacuum.
The addition of water produced a faint turbidity.
The
solution was adjusted to pH 8 with sodium hydroxide and extracted with ether. The ether extract which would contain unreacted estrone and estrone acetate yielded only 2 rag. upon evaporation.
The pH was elevated to ll-12 with sodium
hydroxide and extracted 5 times with butanol.
After removal of the butanol,
80 mg. of crude sodium estrone sulfate remained. tion from methanol-ether was 50 rag. (60%).
The yield after crystalliza-
The colorless needles, m.p. 229-
231 ° displayed an infrared spectrum identical with authentic estrone sulfate. More than 99% of the radioactivity had the same mobility on paper as sodium estrone sulfate in the solvent system: toluene:n-butanol (lOO: lOO)/ammonium hydroxide:water (20"~).
8
ST ER O I D S
120
VOLUME 1
JANUARY 1963
~O~OT~ 1.
This work was supported by research grants PHS-CY 2071 from the National Cancer Institute, United States Public Health Service and Grant P-206 from the American Cancer Society. A preliminary account of this work was presented at the 46th Annual Meeting of the Federation of American Societies for Experlmnntal Biologists, Atlantic City, April, 1962.
2. Research Career Development Awar~ Ynvestigator, N.I.H., GM-K3-18, ~22.
REFER1~CF_~
i.
Roberts, K.D., VandeWiele, R°L., and Liebermen, S., J. BIOL. CHEM. 236,
2213 (1961). 2.
Twombly, G.H., and Levitz, M., AM. J. OBST. and GYNEC. 80, 889 (1960).
3.
Purdy, R.H., Engel, L.L., and Oncley, J.L., J. BIOL. CHEM. 236
lO&3
(1961). 4. Butenandt, A., ~,d Hofstetter, H., Z. PHYSIOL. CHEM. 259, 222 (1939). 5.
McKenna, J., and Norymberski, J.K., J. CHEM. SOC. 3889 (1957).
6.
Sobel, A.E., Drekter, I.J., and Natelson, S. J. BIOL. CHEM. llS, 381 (1936).
7.
Personal Communication from Dro H. Leon Bradlowo
8.
Schneider, J.J., and Lewbart, M.L., Recent Progr. Hormone Res. 15, 201