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Patented Sept. 3,_ 1946 ,1 ‘ 2,406,885 s’rA'rEs ‘ PATENT OFFICE , v -, v‘ 2,406,885 SAPOGENIN OXIDATION ‘ PRODUCTS ., AND PROCESS FOR PREPARING THE SAME ‘ \ Russell Earl'xMarker, State College, Pa., assignor 1' to/Parke, Davis & Company, Detroit, Mich., a 5 corporation of Michigan" No'Di'awing. Original application August 3, 1940, Serial No. 351,147. Divided and this applicaa . v tionlNovember 22,1943, Serial No. 511,340 y ' isolaim‘s, > (01. 260-3971) Jjj 3‘ .f ‘ The‘ ‘invention relates to 'sapogeninoxidation product's; andmore vparticularly to‘compounds ‘which’ may be described‘ ‘ Czé-keto acids, having v v as in ring D the following formula, ‘ " ' CHa (I? ‘ 10. mann, J. Am. Chem.‘S‘oc.‘61, 846, 1516, 2724, 3479, 15 The steroidal sapogenins'havaingeneral, the formula C27H42-4O3-5, _ of which the genin contain one type of side chain‘ characterited by the fact that a sapogenin of this type is readily CaHmOz is known to be present as a side chain 20 The following sapogenins differ from one an otherlrnainlyuin regardtoathe connections be-_ ' " , ‘ '. HO ‘CH3 'A’ ‘ using alcoholic . hydrochloric acid, and amal tives in which the side chain has 4 more hydrogen atoms than in the sapogenins themselves. Tigo CH3 . ‘ reduced according to the Clemmensen procedure gamated zinc (Marker and ,Rohrmann, J. Am. Chem. Soc. 61, 846 (1939)) to give tetrahydro derivatives, that is to say, new sapogenin deriva tween rings ‘A’ and’ B, 'the degree ,of. saturation of the ring system, and the number of sub stituents attached to these rings. (1939); (i2, 647, 896, 1162) that the side chain of the naturally occurring’ sapogenins exists in 1 two modi?cations. Sarsasapogenin‘and neotigoé portion attached to ring D of the__ steroid skeleton. Sarsasapogenln ‘ __Recently ‘it has been found (Marker and‘ Ruhr where R is a member ofnthe class consisting of —OH and groups hydrolyzable to -OH. This application is a division of my copending application, Serial No. 351,147,“ filed August 3, 19%10. Dlosg'ernln‘ ‘I genin, gitogenin, digitogenin, chlorogenin and diosgenin behave differently from sarsasapo genin and neotigogenin in that they are not re duced byvthe Clemmensen procedure, that is to ‘ " ‘ say, they are recovered unchanged after boiling with ‘__. H: _ ‘ l H Tigogenin Gltogemn .- .C‘Hz , _ 5 others which are cited in the references listed, appear to me to be best explained by assuming 1 ‘ , HO I ’ HO , ‘ - , 40 , ' w "H ‘ Digitogenin HR * 1'11 " and Y amal sarsasapogenin itself yields. Theseyfacts, and O‘Ha‘ ’ acid under the conditions of the Clemmensen reduce tion to the same tetrahydrosarsasapogenin that ~ HO__/\/\_ alcoholic hydrochloric gamated zinc. Isosarsasapogenin is converted H r‘ (:Jhlorog'enin 45 that thetwo types of steroidal sapogenin side chains differ in regard to optical isomerism about C22. Accordingly, when it ‘is necessary to dis tinguish between isomers about C22 I represent the :two types. of side chains ‘byformulae of the following type: 1 . , ‘ I ‘ 2,406,885 4 from the group consisting of compounds of hexa valent chromium and compounds of heptavalent CH: CH; CH; / E /0——CH1 1 C CH~CHa V ‘ $\CH2——C§a H° \/ H Sarsasapogenin (“Sarsasapogenin type" side chain) manganese. The reaction mixture is separated into alkali-soluble and alkali-insoluble fractions, and each of these fractions is further separated into its components by crystallization, partition between immiscible solvents, conversion into in soluble derivatives, high vacuum distillation, chromatographic adsorption or other methods 10 adapted to the properties of the substances to be isolated. My invention also comprehends certain new classes of steroidal sapogenin oxidation products. One of these classes of compounds may be repre-i CH: '* CHI CH: ' (\ H0 sented by the following formula 15 é / 1E /CHr-CE: \/ C\ CH-CH; (L O—-——C§a nA 20 CH: CHI CH: O 4} l Yl_ /Y Y H\/ Y A B 25 Isosarsasapogenin (“Tigogenin type” side chain) where the symbol nA represents 11, carbon-t0 carbon double bonds in the A-B portion of the Chem. Soc. 62, 896 (1940) . Ordinarily where 30 steroid nucleus, n having one of the values 0 and 1, Y1, Y2 and Y: are substituents attached-to isomerism about C22 is not of importance, the methylene carbon atoms in the A—B portion of formula for the sarsasapogenin type of side the steroid nucleusI said substituents being se chain will be used for con?gurations both of sar lected from the class consisting of See especially Marker and Rohrmann, J. Am. sasapogenin and tigogenin type. These two types of sapogenin side chains ap pear to be subject to an equilibrium, the velocity of attainment of which is catalyzed by acidic re agents. The equilibrium is in?uenced by the con?guration of the hydrogen atom at C5, and it groups hydrolyzable to appears that the following rule holds true. For compounds of the allo series (cholestane type) the tigogenin type of side chain is the more sta OH ble, while for compounds of the regular series (coprostane type) at C5 the sarsasapogenin side 45 chain is the more stable. For the purpose of greater clari?cation, the Marker-Rohrmann formulation of the side chain of the steroidal sapogenins will be used in de H and groups hydrolyzable to H scribing the invention. It is to be understood, 50 however, that the processes and products of the present invention may be obtained by the use of is a member of the class consisting of (=0) and the methods herein to be described and these groups hydrolyzable to (=0), and R is a mem ber of the class consisting of —OH and groups processes and products are claimed without any hydrolyzable to -—OH. implications that the reactions and structures By the term “methylene carbon atom” is involved will ultimately be proved to be as rep resented herein. - meant a carbon atom having not more than tWo carbon atoms attached to it. The methylene I have found that the steroidal sapogenins and certain derivatives thereof may be oxidized to 60 carbon atoms in the A--B portion of the steroid nucleus are carbon atoms 1, 2, 3, 4, 6 and '7. give new sapogenin oxidation products which are Groups which are hydrolyzable to (=0) in valuable as intermediates for the preparation of clude groups such as =N-O-H, =N-NH-acyl, steroidal hormones. and =N-NH2. Groups which are hydrolyzable to In practicing my invention I proceed as fol lows: The sapogenin or the derivative thereof halogen OH O-acyl O-aralkyl which is to be oxidized is ?rst treated with re , and agents to protect nuclear hydroxyl groups and/or H H double bonds. For instance, nuclear hydroxyl groups are treated with acylating, aralkylating < include <H or halogenating agents to form the correspond- » Groups hydrolyzable to-OH include —O-alkyl, ing -O-acyl, ~—O-aralkyl or halogen derivative. --NH2, and halogen. Nuclear double bonds are protected by addition of hydrogen halide or halogen. Then the nu My invention may be illustrated by the follow ing partial formulae in which only transforma tions of the side chain attached to ring D are clearly protected sapogenin or derivative thereof is oxidized, preferably with a reagent selected 3 shown; ' ‘ 2,406,885 CH, CH1". 6H : . . ' _ ‘ VCR. CH: CH:—CH: _ CH-CHa (l)\o—-caz D . CHI _ B—-CH|CHa HCHaOE cat. ' hydrog ' ' Sapogenln 2526000. ‘ éH . ’ Dlhydrosapogenln 12sec. C ‘ 01-0; CH: CH: \ CHI n (EH-?r-CHICILCHCOOH 25- 70°C. 0 ' 0 — Sapogenoic acid CH‘ CH: \ /K'JH CH: EH-CHaCHaJJHOOOB a N, Semantic acid CH: 10- \ /(5 . 10913830 ' -——» 100°C. ~ / .= CnKeto acid CH: -_COOH (‘3710: 70-100 OH; 0. H eno-Blllanlc _ acid. E=O 25 --——s 70°C. N & > On Lactone 25- ‘ -—» 10° 0. 7 oxo‘sapogenin . ‘(O-100° C. My invention may be further illustrated by the following examples: nor-cholanic acid, may be‘ represented by the formula: Example 1 Dihydrosarsasapogenin, M. P. 165—166° C., is ' ‘ 4.5 CH: prepared by the method described by Marker and Rohrmann, J. Am. Chem. Soc., 61, 846 (1939). l3 A mixture of 20 g. of dihydrosarsasapogenin, M. P. 165—166° C., and 200 cc. of acetic anhydride 50 is re?uxed for thirty minutes. The acetic‘ an hydride is evaporated in vacuo and the residual sirup dissolved in 400 cc. of acetic acid. The well stirred solution is heated at 90-95‘? C. on a steam-bath while 44 g. of chromic anhydride in H Example ‘2 250 cc. of 80% acetic acid is added over a period 55 of two hours, after which the mixture is heated for an additional two hours. The mixture is con ‘To a solution of ,1 g. of'sarsasapogenin acetate ‘ethereal solution, after thorough washing with water, is washed twice with 3% sodium hydroxide . diluted with water and extracted with ether. The in 100 cc. of aceticacid at 20° C. is added 50 cc. centrated in vacuo to a volume of about 100 cc. of l N aqueous potassium permanganate solu The residual material is diluted with water and tion. The temperature is maintained at 18 to 20° the precipitated solids taken up in ether. The 60 solution to remove the acidic fraction. C. for ?fteen hours, after which the mixture is ethereal extract is washed well with water and then with 3% sodium hydroxide solution. The sodium hydroxide washings containing the 65 ' The sodium hydroxide washings containing the acidic material from the oxidation is heated on acidic fraction from the oxidation is heated on the steam-bath for twenty minutes to complete the steam-bath for ?fteen minutes to ‘complete the hydrolysis. The cooled mixture is acidi?ed the hydrolysis. Acidi?cation of the cooled solu with hydrochloric acid and the precipitated acids ‘ tion with hydrochloric acid yields a white solid taken up in ether. Upon standing, the ethereal solution deposits 600 mg. of small compact white 70 which is taken up in ether and crystallized from ' this solvent to give 225 mg. of white crystals. The crystals which are recrystallized once from meth product is recrystallized from methanol to ‘give anol to givea product, M. P. 285-288°‘C. dec. a product, M. P. 286-288° C. dec. 7 This is the C22 This is the C22 keto acid, M. P. 285-287“ C. dec.‘ This substance,“ . 3-(p)-hydroxy-16-keto-bis acid amplql 7.5 keto identical .with that described'in Ex . . < _ ; . 2,406,885 " 7 rived from sarsasapogenin. It may be represent Example 3 ed by the following formula: Ha Evaporation of the ether solution containing the neutral products gives a sirup which is hydro lyzed with hot ethanolic potassium hydroxide. UT Dilution of the resulting solution with water gives a slight precipitate which is taken up in ether and discarded. The aqueous alkaline solution is acidi?ed with hydrochloric acid and the precipi tated material taken up in ether. After sublima 10 tion in high vacuum at ISO-180° the product is an H crystallized from ether-pentane to give white needles, M. P. 198-200° C. This is the C22 lactone related to sarsasapogenin. It may be represent ed by the formula: It has a melting point of 285-28? C. dec. (gas evolution), and it analyzes for the formula 15 C22H34O2. ' Better yields of this acid may be obtained by CH3 CH3 conducting the oxidation of sarsasapogenin ace | C tate at 80-85° 0., using two parts of chromic an hydride to one part of sarsasapogenin acetate. 20 However, the yield of other oxidation products is less at these higher temperatures. This C22 keto acid is very sparingly soluble in ether, acetone, chloroform and ethyl acetate, but . it is fairly soluble in methanol and ethanol. 25 _ The methyl ester of the above C22 keto acid may be obtained by allowing a suspension of 200 To a solution of 800 mg. of ‘the above men mg. of the keto acid in 20 cc. of methanol-ether tioned lactone in 30 cc. of glacial acetic acid is (1:1) containing an excess of diazomethane to added a solution of 2 g. of chromic anhydride in stand over night. Then the solvent is evaporated 40 cc. of 80% acetic acid. The resulting solu tion is heated on the steam-bath at 90° C. for 30 and the residue crystallized from the ether-pen tane to give clusters of small plates which melt three hours. The mixture is diluted with water ric acid and the solid acid extracted with ether. The ether upon slow evaporation deposits com at 124-126" C., solidify at 127° C. and remelt at 159° C. Other esters of this acid may be prepared, for example, by treatment with an appropriate alco hol and a suitable catalyst. For example, 200 mg. of the C22 keto acid may be refluxed with 100 cc. of 1% ethyl alcoholic hydrogen chloride for three pact white crystals. These, after crystallization hours. from ether-methanol, melted at 285-287° C. dec., and give no depression with a sample of the C22 small volume, diluted with water and extracted with ether. The ethereal layer is separated, washed with dilute sodium hydroxide solution and water, and the ether evaporated. The residue is crystallized from ether~pentane to give white nee dles of the ethyl ester, melting point 163-464o C. Derivatives of the ketone grouping may be ob tained by treating the acid or its esters with ke tone reagents having a reactive NHz grouping. and extracted with ether. The ethereal extract is washed well with water and then extracted with 3% sodium hydroxide solution. The sodium hy droxide extract is heated for twenty minutes on the steam-bath, cooled, acidi?ed with hydrochlo keto acid, M. P. 285-287° C- . Treatment of the methyl ester of the keto acid with boiling acetic anhydride for thirty minutes followed by decomposition of the excess acetic anhydride with water yields a methyl ester ace tate which crystallizes from ether-pentane as compact white crystals, M. P. l98-199.5° C. Treatment of the acid with hydroxylamine hy drochloride under the usual conditions gives an oxime which crystallizes from aqueous methanol as small compact white crystals, M. P. 206-208° C‘. dec. Example 4 To a solution of 20 g. of sarsasapogenin acetate in 500 cc. of glacial acetic acid at 60-70° is added Then the solution is concentrated to a For example, a solution of 100 mg. of the C22 keto acid, 100 mg. of semicarbazide hydrochloride and 150 mg. of sodium acetate in 10 cc. of alcohol and 2 cc. of water is re?uxed on the steam-bath for one hour. The solution is diluted with Water and the white solid collected and crystallized from ether to give a semicarbazone of melting point 204-207° C. dec. Other sapogenins may be treated in accordance slowly over a period of 4 hours, a solution of 12 with the directions of this example to give anal g. of chromic anhydride in 200 cc. of 90% acetic ogous products. Such sapogenins include tigog acid. The mixture is stirred an hour longer, then alcohol is added and the solution evaporated 60 enin, gitogenin and diosgenin. The latter con tains a nuclear double bond which is protected to a sirup. This sirup is dissolved in ether and by addition of bromine prior to oxidation. In the ethereal solution extracted with 3% sodium stead of protecting the hydroxyl groups by acetyl hydroxide solution. The alkaline extract is warmed On a steam bath for a few minutes and then it is cooled and acidi?ed. The precipitated solid is crystallized from dilute acetone to give sarsasapogenoic acid of melting point 187-189° C, ' The mother liquor is evaporated to dryness and the residue dissolved in a small amount of ether. ‘After this concentrated ethereal solution has stood at room temperature for several days, small compact white crystals are deposited. These are collected, washed with ether and recrystal lized from methanol to give the C22 keto acid de ation during the oxidation, other protecting agents may be used. For example, the sapogenin may be benzoylated, benzylated or halogenated, for example, by treatment with phosphorus pen tachloride in carbon disul?de. Example 5 To 10 cc. of fuming nitric acid maintained at 20° C. by an external cooling bath is added 1 g. of sarsasapogenin acetate in small portions. After awhile when the sarsasapogenin acetate has com pletely dissolved, the solution is diluted with water 2,406,885 10 and the gummy precipitate collected. This gummy precipitate is boiled for a short time with alcoholic sodium hydroxide. Then the solution is separating the alkali soluble fraction, thereby oo taining an alkali metal salt of a steroid acid having in ring D the structure, diluted, extracted with ether and the alkaline layer separated. This alkaline extract is acidi?ed ‘ CH; ' (EH. 0 and the precipitated acid taken up in ether. The ethereal layer is separated and most of the ether D removed. On standing for several days, this con 0 centrated ethereal solution deposits crystals of the C22 keto acid derived from sarsasapogenin. 10 where M represents an alkali metal, and hydro This acid ‘has the formula C22H34O4, and it melts lyzing said fraction. at 285-287° C. dec. (gas evolution). 2. Process for preparing 3-e-hydroxy-16-keto It will be apparent that in View of this disclo bis-nor-cholanic acid which comprises subjecting sure my invention is capable of numerous varia tions with regard to conditions of reaction, re 15 sarsasapogenin acetate to oxidation at 40-100° C. by treatment with a strong oxidizing agent, sep agents and sapogenins employed. arating the alkali soluble fraction, hydrolyzing Steroidal sapogenins on which this invention said fraction and crystallizing 3-,8-hydroxy-16 may be practiced include not only the aglycones keto-bis-nor-cholanic acid. of the naturally occurring steroidal sapogenins, but also their nuclear transformation products, 1. e., the substances derived from the aglycones by changes in rings A and/or B which leave the side chain attached to ring D still intact and like that in the aglycones. Thus, this invention may 20 be practiced on steroidal sapogenins‘such as smil 25 agenin, sarsasapogenone, 3-desoxysarsasapogenin, 3. An acid having the formula CH: CH: /\ I the‘ sarsasapogenyl chlorides, and the like. It is apparent that sapogenins having in the side chain either con?guration with regard to C22 may be employed in practicing this invention, 30 that is to say, compounds having a side chain either of the type of sarsasapogenin or of the type tigogenin may be oxidized in the manner set forth in this speci?cation, and regardless of the con ?guration of the side chain of the sapogenin oxi 35 dized the structure of the oxidation products re main the same, for example, sarsasapogenin and isosarsasapogenin give the same oxidation prod ucts. CH: 11-00011 \/=0 (a) H ‘ HO 4. An ester having the formula CHI CH’ OH‘ on coo on '_ /\ ‘_ I , =0 (B) H no What I claim as my invention is: _ 1. Process for obtaining sapogenin oxidation products which comprises subjecting a member of the class consisting of steroidal sapogenins, 40 5. A compound having the formula, . CH: sapogentic acids, dihydrosapogenins, sapogenoic / acids, and C22 lactones, to vigorous oxidation at 45 40-100° C. by treatment with a strong oxidizing agent after previously protecting hydroxyl groups therein with a member of the class consisting of acylating agents, aralkylating agents, and halo genating agents, thereby producing a C22 keto acid having in ring D the structure CH: D CH: CH: 50' H>k/ n-coon I ;=0 (5)31 where R is selected from the class consisting of hydrogen and alkyl radicals and R1 is selected from the class consisting of —0H and groups hydrolyzable to ---OH. RUSSELL EARL MARER.