Patent GB-513525-A (GB)
Title: A process for separating corpus luteum hormone and androstendione from sterol degradation products
Filed: 1938-03-24
Abstract: 513,525. Sex hormones. CHINOIN GYOGYSZER ES VEGYESZETI TERMEKEK GYARA, R. T. (DR. KERESZTY & DR. WOLF). March 24, 1938, Nos. 9077 and 9078. Convention dates, March 26, 1937, and Feb. 24, 1938. [Class 2 (iii)] Corpus luteum hormone and androstendione are separated from one another and from the other sterol degradation products obtained by oxidizing sterols which have the sterol skeleton of cholesterol, epi-cholesterol, or allo-cholesterol by treatment of the oxidation mixture by a process of chromatographical selection, after the removal from the oxidation mixture of both the acid constituents and the sterol oxidation products cxidized only at position -3. The latter bodies are separated by subjecting the sterol oxidation mixture to a distribution between water-miscible and water-immiscible solvents such as aqueous methyl alcohol and petroleum ether. The prepurified product thus obtained is then dissolved in a solvent such as benzene and passed through a column using an absorbent such as aluminium oxide. The sterol degradation products containing two carbonyl groups pass through the column unadsorbed. To obtain the hormones in crystalline condition, the benzene solution after passing through the column is evaporated, dissolved in petroleum ether and subjected to a chromatographical separation. A suitable dye for the analysis of the column is Sudan-III. The separation may be repeated if necessary, and if desired the solution may be supplemented by other methods of purification such as fractional crystallizaticn and distillation and the use of ketone reagents. In examples (1) cholesterol is oxidized with chromic acid in glacial acetic acid solution with intermediate protection of the nuclear double bond. The acid fractions are removed by treatment with alkali. and the cholestsrone by crystallization from acetone solution. The mother liquours are evaporated and treated with methyl alcohol to give a residue which shows a strong corpus luteum reaction. This product is subjected to a chromatographical selection by being passed in solution through a tube filled with aluminium oxide according to Brockmann. The tube is washed out with benzene, evaporated, dissolved in petroleum ether and re-chromatographed using Sudan-III as indicator. The upper third part of the tube contains androstendione and in the lower two-thirds there is corpus luteum hormone containing androstendione as impurity. These two parts are separated and extracted with methyl alcohol and cholestandione is precipitated by chilling. (2) Cholesterol is oxidized with potassium permanganate with intermediate protection of the nuclear double bond and the acidic materials and the cholesterone removed as in example (1). The material is then subjected in benzene solution to a chromatographic selection in a column filled with aluminium oxide. (3) The material produced in example (1) at the stage prior to treatment in the column is further purified by distribution between methyl alcohol and petroleum ether, and the methyl alcohol extracts evaporated, taken up in benzene and subjected to chromatographic selection using Sudan-III as indicator. (4) The material from the lower two-thirds of the chromatogram in example (1) is converted into its semi-carbazone to effect a further purification. From the less soluble semi-carbazone fraction there is obtained androstendione and from the mother liquours on treatment with pyruvic acid there is obtained corpus luteum hormone. (5) The product of example 3 is further purified by a distribution between methyl alcohol and petroleum ether. The petroleum ether part is again distributed with a further amount of methyl alcohol, and the process then repeated a third time. The three methyl alcohol extracts are then chromatographed to give a separation of corpus luteum 'hormone and androstendione. (6) The product from the lower two-thirds of the chromatogram obtained in example (3) is purified as in example (4). Specificaticn 449,379 is referred to. The Specification as open to inspection under Sect. 91 includes also the separation of the diketone products of the oxidation of sterols by any convenient method such as by crystallization and distribution between two immiscible solvents. This subject matter does not appear in the Specification as accepted.