Patented Apr. 21, 1953 2,636,042

UNITED STATES PATENT OFFICE 2,636,042 WATER-SOLUBLE HORMONE COMPOUNDS Ralph Salkin, Jackson Heights, N.Y., assignor to S. B. Penick and Company, New York, N. Y., a corporation of Delaware No Drawing. Application July 8, 1949 Serial No. 103,759 5 Claims. (C. 260-39.4) 1. 2 My invention relates to an improvement in the ether, and the sulfate is then Salted out of the manufacture of water-soluble compounds of the aqueous solution by the addition of a, caustic estrane series, and in particular it is concerned solution under cooling. The liberated hormone With an improvement in the synthesis of alkali sulfate is extracted into a suitable Solvent, for and alkaline-earth metal salts of the sulfates of 5 instance butanol, pyridine being preferred how the estranes. ever. The hormone sulfate solution is exhaus The estranes to which my invention applies are tively extracted with ether to remove the solvent. steroids having a free hydroxyl group in the The resultant semicrystalline product is recrys 3-position and a hydroxy or keto group in the tallized from a dilute monohydric alcohol. Or 17-position of the molecule, such as estrone, O Water to give the pure sterol.ester. equilin, equilenin, estradiol and similar com In order to get pure ester Salts, I have found pounds. it essential that the tertiary amine-sulfur trioxide These products which are commonly known as adduct be absolutely pure when being reacted With conjugated estrogens can be obtained from nat the hormones. Improved yields and more readily ural sources such as the urine of pregnant mares purifiable light colored granular products result, or of stallions. The principal water-soluble if the tertiary amine-Sulfur trioxide adduct is Compound which they contain is. Sodium estrone separately prepared, as above described, for in Sulfate which, however, is difficult to isolate from stance by addition of "Sulfan B' (which is stabi the natural Sources in pure crystalline form. lized SO3), or chlorosulfonic acid to a solution Attempts to synthesize the sulfuric acid ester 20 of the tertiary amine in the inert chlorinated hy of estrone and its salts have been commercially drocarbon Solvent under cooling and filtering and unsatisfactory in that the ester was obtained as Washing the reaction product, and using the ad a reddish brown oil (A. Butenandt and H. Hof duct thus prepared to effect the reaction as Stetter, Zeitschr, physiol. Chemie 259, 222 at 232, described in the attached examples. Traces of 1939). In that form the ester, cannot conven 25 Occluded acids and moisture in an adduct em iently be purified and be converted into its pure ployed without previous isolation as above de Sodium salt in good yields and in a manner Scribed seem to have an adverse effect on the feasible for production. reaction. It is therefore an object of my invention to find The alkali sulfate salts thus obtained are the an improved method by which the ester can 30 basis for the preparation of the alkaline earth readily be obtained in a novel form which Will metal Salts. The latter are prepared, for in lend itself with particular ease to purification stance, by adding a concentrated solution of the and Conversion into the salts. Other objects of alkaline earth acetate to a concentrated aqueous my invention are this novel form of the ester, Solution of the acidified alkali metal sterol ester and the various new salts which can be obtained 35 SaltS. The crude alkaline earth metal salts may therefrom. be further purified by crystallization from water. I have found that these various objects can be With this improved procedure it is now pos accomplished, if the pure hormones or, if desired, Sible for the first time in this art to manufacture Crude mixtures containing them are reacted with Water-soluble compounds of the estrane series an exceSS of a pure tertiary amine-sulfur trioxide 40 conveniently in excellent yields, and in a high adduct in a Suitable solvent mixture, for instance State of purity. an inert chlorinated hydrocarbon and pyridine, The following examples illustrate my inven under anhydrous conditions. tion: I have discovered that the reaction products thus obtained, instead of being oily, are practically 45 Eacample I-Estrone sulfate sodium sait colorless and granular and in that form lend Ten grams of estrone (m. p. 254-256°) are dis themselves with particular ease to purification Solved in 40 cc. of pyridine and diluted with 100 cC. of carbon tetrachloride and chilled in an ice and conversion into the salts. They are presum bath. Forty grams of dry pyridine SO3 are sus ably a mixture of unreacted adduct and tertiary 50 pended in 200 cc. of chloroform and cooled in amine Salts of the Sulfate esters of the estranes. an ice bath. The chilled estrone solution is The granular products can be purified and con added dropWise over a period of twenty minutes verted into a salt by decomposing them with an in an apparatus protected from moisture. The alkali or alkali earth metal carbonate or bicar ice bath is removed and the reaction mixture bonate. The liberated amine is extracted with 5 s heated to boiling and refluxed for 15 minutes. 2,636,042 3 4 The hot solution is filtered and the filter cake Eacample IV.-Estrone sulfate ammonium salt Washed With a fresh portion of carbon tetra chloride. 500 mgs. Sodium salt obtained as described in The granular reaction product is decomposed Example I Were dissolved in 25 cc. Water. The With 150 ml. of Water and 35 gms. NaHCO3. Solution was acidified with three drops of 2.5 N The aqueous Solution containing the reaction HCl and to it was added 15 cc. of a 50% am product is chilled and to the chilled reaction monium acetate Solution. The flocculent pre product is added 20 ml, pyridine and 40 ml. 50% cipitate thus produced was redissolved in the NaOH solution. The mixture is well agitated reaction mixture by warming. Filtering and and allowed to settle. The aqueous phase is chilling the filtrate gave micro-needles of the discarded, and the pyridine solution of the re desired compound in a 85% yield. action product extracted four times with 100 ml. Empirical formula C18H2O5SNH4.2H2O: portions of ether. The Semicrystalline reaction product is taken up in anhydrous ethyl alcohol, Theory Found charcoaled and filtered. The colorless filtrate is 5 adjusted to 90% alcohol concentration by the Moisture content------percent.-- 8.9 9. addition of water and concentrated to incipient Estrone by dinitrophenylhydrazone ---do---- 66.9 67.5 Crystalization and refrigerated. The Snow-white (alpi (0.4% in H2O)------...--- degrees-- --134 ------crystals are filtered off and dried to constant Sulfated ash------None ------Weight in a desiccator over Sulfuric acid. Yield 20 i1.70 gns. Empirical formula C18H2O5SNa.1H2O Eacample V.-Sodium salt of sulfuric acid ester of Found Theory natural estrogens The following initial material was reacted un Estrone by dinitrophenylhydrazone 25 der the conditions described in Example I supra: percent- 68.8 69.2 (0.4% H2O)------degreeS.-- A Semi-purified crystallizate of estrogenic sub cy20 (95%. EtOH).------do---- Stances, imelting point 235 C., and total ketone (ahs. MeOH)------do---- Moisture------percent. Content of 86.3% as determined by dinitro-phen Sulfur as (SO4): by micro Preg--- do---- Kober (chromogens as estrogens)---do--- 30 ylhydraZone, (a)0--147, and a potency of Sulfated ash------do---- 8, 3 8.2 8,600,000 international estrone units per gram. Melting point------after sintering at 200° C. The reaction product thus obtained had the - - - Imelts with decompo sition at about 225°C. following constants: Found Ketones by dinitrophenylhydrazone 56.5% Bioassay of the above compound by Oral admin 35 istration to rats showed a potency of 490,000 in Lal (0.4% 95% EtOH)------105.3° ternational units per gram expressed as estrone. Solubility in H2O at pH 8 (2% soln.) -- complete It Will be understood that the constants of this Eacdnple II.-EStrone Satlfotte potassium salt type of material are widely variable and a func The procedure of Example I Was followed but 40 tion Solely of the constituents of the estrogenic KHCO3 was used to decompose the crude reac Substance used. tion product. 60% KOH solution was employed to Salt out the final product. The compound Eacd/mple VI.-Equilenin sulfate sodium salt was crystallized from dilute alcohol. It had Equilenin Was isolated thru the picrate from the following constants. Empirical formula mother liquors of the production of estrogenic 45 substance. Picrate M. P. 203-205° C., a 20--87° (95% EtOH) (0.4% solution). The picrate was hydrolyzed and the resultant ketone recrystallized Found. Theory from 95% ethanol. It showed 96.4% ketone when Estrone by dinitrophenylhydrazone.---- percent.-- 69.3 69.5 50 aSSayed by the dinitrophenylhydrazone technique. oD2 (0.4%, 95%. EtOH) -degrees. --114 ------This material was converted to the sulfate ester Sulfated ash------percent - 22.7 22.4 Sodium Salt by the procedure outlined in Example, Moisture------do---- 0.2 O I. The Crude product was recrystallized from dilute ethanol and gave a white microcrystalline, Eacample III.-Estrone sulfate calcium salt 55 hygroscopic Solid instantly soluble in water. Ernpirical formula C18H17O5SNa.1H2O. M. W. One gram of estrone Sulfate Sodium Salt is dis 386.4 Solved in 50 cc. of Water and the Solution acidi fied to pH 4 With dilute acetic acid. A slight excess of a 25% solution of calcium acetate solu Found. Theory tion is added and the flocculent precipitate cen 60 trifuged off and Washed With Small portions of Percent Percent, ice cold Water. The Washed calcium Salt is dis Retone Content (by DNPH) ------69.2 9.9 Solved in a minimum quantity of hot distilled Moisture------5.3 4.7 water, filtered and allowed to Crystallize. Sulfated ash------18.9 8.4 The crystals are filtered off and dried to con 65 stant weight over sulfuric acid. Empirical for Eacample VII-Equilin sulfate sodium salt mula (C18H21O5S)2Ca.3H2O: An impure equilin was isolated from estrogenic Substance. With the following constants: rotation. Found Theory 70 in alcohol --245; ketones by dinitrophenylhy drazone 98.8%. The material was sulfated by the Estrone by dinitrophenylhydrazone.----. percent. 68.5 68. Standard procedure and recrystallized from Moisture------do---- 7.1 6.8 Sulfated ash------do---- . 17. 7.1 ethermethanol. The product was a white crys Rotation alpi (ydrate) 0.4%H2O.----- degrees. -120 ------talline hydroscopic solid instantly soluble in water. 2,686,042 5 6 Empirical formula C18H1905SNa.1H2O, M. W. 7.0, 8.0, 9.0 showed no perceptible decomposi 388.4: tion. On Several months' standing at room tem perature in flint glass vials. Solutions in aqueous Found Theory propylene glycol have also shown excellent sta bility. Ketone Content (by DNPH).------percent. 69.4 69.5 What I claim is: Moisture------do---- 4.7 4, 6 Sulfated ash------do. 8.5 8.3 1. The process of making a water-soluble sul ap25 (0.4% in Methanol (25° C))-- --degrees. 187.5 ------fate of the estrane series which comprises mix ing, in a pre-cooled inert chlorinated hydrocar The method described in Example I may also O bon medium under anhydrous conditions and in be applied to the preparation of Sulfate esters the presence of a free tertiary amine, a hormone of estradiol, Such as of the estrane series and an excess of a tertiary amine-Sulfur trioxide adduct free from occluded Estradiol. 3 Sulfate moisture and acids, boiling the mixture until Estradiol. 3,17,disulfate 5 formation of the Solid reaction product is com Estradio 17-sulfate. plete, filtering off the product, decomposing it Each of the above described compounds has a in an alkaline medium, removing the liberated characteristic U. W. absorption curve. amine from said medium, then isolating the free The inert chlorinated hydrocarbon in which the estrane sulfate from the medium and purify reaction occurs may be carbon tetrachloride 20 ing it. which I prefer, or it may be CH2Cl2, C2H4Cl2, 2. The process of claim 1 in which carbon. CHCl3, tri- or tetra, chlorethylene or tetra chlor tetrachloride is the Solvent and pyridine is the ethane, and the tertiary amine of the Sulfur free tertiary amine used. trioxide adduct may be pyridine as shown in EX 3. The process of claim 1 in which estrone is ample I, or picoline, dimethylaniline, N-ethyl 25 the hormone used. morpholine, trimethylamine and the like. 4. The process of claim 1 in which equilin is These new synthetic alkali and alkaline earth the hormone used. metal salts of compounds of the estrane Series 5. The process of claim 1 in which equilenin is have various novel and outstanding characteris the hormone used. tics which distinguish then clearly from the nat 30 RAPH. SAKIN. ural products. They are crystalline, and they are free from odor and taste. They are com References Cited in the file of this patent pletely soluble in water and stable in aqueous Solu UNITED STATES PATENTS tions. They possess oral biological potency, and are easily standardized by U. W. Spectral analy 35 Number Name Date sis. They are readily formulated into compounds 1,810,758 Drescher et al. --- June 16, 1931 Suitable for oral administration and Subcutaneous 1835,841. Beckett et al. ------Dec. 8, 1931 injection; they can be formulated into either tab 1921,497 Barnes et al. ------Aug. 8, 1933 lets, elixirs, or similar types of pharmaceutical 2,035,361 Bauer et al. ------Mar. 26, 1936 preparations in the usual manner. AS Vehicles 40 2,267,258 Ruzicka ------Dec. 23, 1941 for tablet manufacture such substances as mag 2,403,226 Lecher et al. ------July 2, 1946 nesium phosphate, magnesium trisilicate and SO FOREIGN PATENTS dium bicarbonate are Suitable excepients. It is Number Country Date preferred in all formulations to keep the vehicles 251,491 Great Britain ------May 23, 1941 neutral to slightly alkaline to avoid any possible 45 decomposition of the Salts. These properties OTHER REFERENCES make the sgynthetic salts a definite improvement Schachter et al., Chen, Abs, Vol. 33 (1939), Over the natural products and more useful in the page 1005. treatment of menopausal Symptoms, as they can Jensen et al., Chem. Abs, vol. 40 (1946), pages be given orally without discomfort. The stability SO 284-285. of aqueous Solutions is a function of their pH; Butenandt et al., Zietschr fur Physio. Chen, 0.5% solutions in U. S. P. buffer solutions at pH vol. 259 (1939), pages 222-234.