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2,408,835 Patented Oct. 8, 1946 UNITED STA'l‘E s PATENT. OFFICE - 2,408,835 STEROIDAL COMPOUNDS AND METHODS FOR OBTAINING THE SAME Romeo B. Wagner, State College, Pa., assignor to Parke, Davis & Company, Detroit, Mich., a cor poration of Michigan No Drawing. Application May 15, 1944, Serial No. 535,759 7 Claims. (Cl. 260—-239.5)' 1 I 2 Y , . where Y1 and Y2 are the same members of the The invention relates to new carbon ring satu class consisting of rated steroidal sapogenin compounds having hy droxyl groups and/or groups hydrolyzable to hy OH droxyl at positions 2 and 3 of the steroid nucleus H and also one of these same groups or a ketone group at position 12 of the steroid nucleus. It is known that steroidal compounds, that is, and groups hydrolyzable to on compounds containing a 10,13-dimethyl cyclo pentanopolyhydrophenanthrene nucleus, are present in plant tissues such as Beth root (Tril 10 and Y3 is a member of the class consisting of :0, Zium erectum), Digitalis purpurea, etc. However, the number of known plant sourcesof such ste roidal compounds is relatively quite limited and vII the types of steroidal compounds which have thus far been found in practical quantities in plant tis 15 and groups hydrolyzable to I sues are also quite limited in number.' Only one steroidal compound has been known which occurs in plant tissue and which at the same time has an oxygen atom attached to the OH 11 C12 carbon atom of the steroidal nucleus. That 20 These compounds are new valuable starting materials for the preparation of physiologically compound is digoxigenin having a hydroxyl group active substances such as sex hormones and the (—OH) attached to the C12 carbon atom and be like. They are especially valuable for the prep ing a compound of the cardioactive type having no aration of physiologically active steroidal com practical value as an intermediate for sex hor mone type products. Although digoxigenin is a 25 pounds of thetype derived from the mammalian suprarenal cortex whichlhave an oxygen contain__ steroidal compound, it is not a sapogenin and ing group in ring C of the cyclopentanopolyhy'a its side chain attached to ring D of the steroid drophenanthrene nucleus.I nucleus is of a distinctly different type from the In order to get a sapogenin fraction from sapogenin type side chain present in the new 30 plants, for use in ‘obtaining the new compounds genins of my invention. 7 of the invention, I use one of the methods de I have now discovered that certain plants and scribed in the following general procedure. products derived therefrom can be treated by physical and chemical methods in order to ob GENERAL PROCEDURE FOR OBTAINING A SAPOGENIN tain new valuable Ola-substituted steroidal sapo FRACTION genins. - _ The new compounds of the invention have the general formula, Ya CH3 ' CH3 OH: I CHa-CHa / CH—C\O———CHz/CH--CH3 D g O 2 . 35 In obtaining the new compounds, the plant or part of the same which is to be extracted is cut up, ground or shredded and then extracted either with hot water or a lower aliphatic. alcohol such as ethyl alcohol. ' The plant may bedried before extraction, but I prefer to extract the plant without any prelim inary drying. I also prefer to use an alcohol sol vent such as ethanol or about 95% hot aqueous ethanol. For example, if one has about 25 kilo grams of undried and cut up parts of the plants, 2,408,835 3 such as stems, soft roots, leaves or fruit, these‘ A sample of manogenin is boiled with acetic anhydride, excess anhydride distilled oil and the residue crystallized from methanol. It consists of manogenin diacetate and has M. P. 253-255° C. AnaL: Calc’d for C31H4aO-1t C, 70.2; H, 8.7. can be covered over with about 32 liters of 95% ethanol and heated under a re?ux condenser at moderate steam bath temperature for about 12 hours. The hot extract can then be strained through cheese cloth, the ?lter cake Washed with two 7-liter portions of hot ethanol and then squeezed dry. The extract and wash alcohol can Found: C, 70.2; H, 8.6. Example 2.—(Mea:ogenin from .S'amuela be evaporated to a syrup and the syrup concen trated by passing a current of air over its surface. 10 camerosana (TreZ.)) 272 kgs. of the caudex from young plants of When using hot water as the extracting agent, the evaporation takes place much more slowly. the species Samuela carnerosana Trel. were col liberate the ,sapogenins. This is best accom plished by aqueous or alcoholic strong mineral... acid. For example, the above mentioned con centrate from 25 kg. of plant'materiaLcanw-bes the alcoholic extraction oft-the 2721kgssin accord ance with the general procedure given above is dissolved in 192 liters of ethanol and hydrolyzed with. .43. liters ..of.. concentrated hydrochloric acid lected during the month of March along the Rio After evaporation, the concentrate containing saponins .andelikeccombinationsmf. thensteroidall. Grande River at Black Gap in the Big Bend sapogeninsz; must" be& hydrolyzed ini ordenztoc 15 reg-ioncoiq’I‘exas.‘ The alcoholic concentrate from forr3i‘liours... The reaction mixture is then ex hydrolyzed by re?uxing it for 2 hours withi-3 liters ; trated with 160 liters of butyl alcohol. of 2N ethanoli’c hydrochloric acid. . The reaction. The butanoln extract is: washed several times with mixture is cooled and ?ltered..- Ifanye-considéne water'andithemwitlri 10% caustic solution. The butanol'layerv'visiwvashed free of alkali with water and. evaporatediin vacuo. The solid residue is powdered and thoroughly extracted with 160 liters of acetone. The acetone extract is then able tar is present at this stage, it can be ground ' and digested several times with an equal volume of hot alcohol. The combined alcoholic-?ltrates are diluted with 20 liters of diethyl ether and‘th‘v solution washed successively with watery. 5%». evaporated. sodium hydroxide and water and evaporated. One half of the residue from evaporation of Fatty_.esters.in the residue .thus obtained. can be hydrolyzed by re?uxing‘th‘e residue with‘ 3“vol-l 30I the racetone nextractwis-"hydrolyzed .witlrzanrexcess of‘alcoholicipotashior 30 minutes: Thereaction'a umes of 10% alcoholic potash for 30“minutes. mixture is 'COOlEdi to1359C. and extracted:with-40=i~ The cooled mixture from‘ the alkaline hydrolysis liters 1 of: ether: and: the 1; ether .‘extr acttseparated,‘ is extracted with ether and the ethereal solution washed with water and then evaporated to give washed with: water andsevaporated: 'I’he1resid11ec a sapogenin fraction whichaaissdissolvedjinaace-e -- 35 from theieva‘porati'on' is‘ lsepa-rated‘iinto; an ‘ace-7 tone-soluble fraction (fraction H,“ see crystalliza tone, treated with active. charcoal such as the product known as “Noritef’ and ?ltered. The clari?ed ?ltered acetone’: solution contains the sapogenin fraction of the plant and can be sepa tion" chart) ‘ and" an» acetone-insoluble fraction; B”; The ‘-B'—fr‘action- weighs-148 -‘ grams and ‘ is further crystallized v-from"ether 'to ' give- an ether rated from the acetone by evaporating the latter. 40 Such sapogenin fractions-consist of mixturesof sapogenins. The individual sapogenins are next isolated or separated out ‘of this fraction. The above description :isra: general procedure‘: which I use for obtaining a sapogenin fraction or mixture. It is merely illustrative and is capable of considerabler. variation, as will be understood. by those skilled in the art. Any known ‘method ‘of obtaining asapogenin fraction by ,.' hydrolytic ‘and solvent extraction ‘procedures maybe used, provided solvents 'areusedtwhich’: make'vpossible the separation'and ‘isolation from the-plant; tissue of ‘tlie‘greater part .zotjth'e h'y: droxyl I and; ketone substituted 1 steroidal; sapoe genius. liberated by , theh'ydrolyticlsteps. . solublé fraction; _D,‘ and "an‘ eth'ereinsoluble frac-. tion; C,weig'_l_1ing_46‘grams.;. The ether-.insolubleir'action.(C) . is 'acetylated. (e.; g.-_v with. .acetioanhydride) . and thenzcrystal lized from methanolyand- ?nally .from ether.- The» crystals have-M; and‘ mixed. Mr. P. with samogeniniacetate 0f:;195—?198°o C; Yield: is‘-;3.0. grams: Hydrolysis...’ of: this diacetates.‘ gives' samogenin;~ M.=.P:‘ and-mixed M.fP;;.208-214"- C. AnalJCalcfd forCi-zHdOi: C;-.75.0; H,"'10.'3.' Found 2' 'CI‘ 74.8 f ‘12310.25 Tiles-properties ‘of-'samogenir'r and‘ its"diacetate ' are described in-J. Amer: Chem. Soc. vol.‘ 65; pages '1199 ‘to‘1209..(I943).'. The. sapogenins in..th‘e ethere-solublesfraction... D; areacetylated ..and-.treated .'.with. 40 grams: ‘of. . Girardfs ireagentsT- in . 1 .liter . oi ethanol dew Example 1.- (Man'ogenin- from ‘ Agave -' H u'achizcensis Baker) ' scribed for A. HuachucensisfBakerlunderqExam 8 kilograms (8 kg.) of’: entire .plants“ of: this . ple 4. -. The ketonefraction, E,-.is crystallized from speciesare collected .imseptembereast of Tucson‘; ether-.pentane;rM;.P. .195-'200°5 “CI, and weighs 34 Arizona. . The .sapogenin .fractionntthese plants grams: This material: ‘is recrystallized .from 125' is first obtained by- thengeneral’ procedure de cc." of “ ether-‘to ' give 2a 'small' ethereinsoluble frac-' scribed above. It is then taken up in and crystal tion, M. P. and mixed M. P. with kammogenin' lizedjfromacetone.“ The product has ‘a‘melting diacetate, 258=-261° 'Cfand'weig‘hs1.5‘grams. See point” (M: P.) " of ‘ 234-236x‘C." and‘ weighs ‘10.65 above article in J. A. C. S. 65, 119941209 (1943).‘ grams. It "is "recrystallized‘from' ether‘ ‘and then‘ Analysis for/kammogenin diacetate: Calc’d for C31H4407251C; 70.4; H,‘9.4.r.. Found: C, 70.5; H, 8.7. gives the" dihydroxy'ketone 'manogenin" of ”M.‘ P.‘ 241 to 243° 0. ‘ A‘ solutlon‘of manogenin'in-ethanol‘is ‘treated with; a‘ 2 %' solution ‘of ' ‘digitonir'r invethan'olg' Im mediately: a‘ heavy -' white? precipitate or‘ the digitonide of inanogenin‘isidrmedi‘ I The etheri‘?ltrateHF, after; removal of kam mogenin diacetate, iszdiluted with apentane, con centrated and cooled to'give mexogenindiacetate, M. P. and mixed M. P., 204_206‘?' C.'; 'yield'ris 25 grams. Anal: Calc’d for C31H4607: C, 70:2;,H-,'8.7. 75 Found: C, 70.3; H, 9.0. 2.4085835 6 the March collection (272 kg.) of Samueldcar The Girard’s non-ketone fraction, G, is frac tionally crystallized from methanol to give an additional 7 grams of samogenin diacetate, M. P. and mixed M. P., 195-198?’ 0. nerosana-Trel: l H steps is concentrated.cooled._'and ?ltered to give a dark semi-solid mass.‘ This mass is hydrolyzed with excess 20% alcoholic potash for 30 minutes. - L , ~ 'P._.‘172-180° C., C , acetate .. = . ._ . .' ~ ‘ In this case, however, no‘ >_ B‘ D Smilagenin is acetylated and treated with Girard’s reagent as 10 described previously. ' ll j ‘ The acetone-soluble fraction, H, from the above The product from methanol, :i _ G ~. E -. ., Semogenin ~L , diacetate ketone fraction is obtained. The non-ketone diacetate , F fraction is crystallized from acetone, M. P. and mixed M. P. with smilagenin acetate, 148-150° C., 15 yield being 30 grams. Mexogenin Kammogenln diacetate diacetate Anal: Calc’d for C29H46O41 C, 75.9; H, 10.1. Found: C, 75.9; H, 10.2. A. Crystallization from acetone; B. Crystalliza Hydrolysis of the acetate gives smilagenin, tion from methanol and then ether; C. Acetyla M. P. and mixed M. P., 184-186° C. tion. Crystallization from methanol and then Anal: Calc’d for C27H44O32 C, 77.8; H, 10.7. 20 ether; D. Evaporation. Acetylation. Treatment Found: C, 77.8; H, 10.7. with Girard’s reagent T; E. Crystallization of ketone fraction from ether; F. Dilution of ether Memogem'n mother liquor with pentane. Slow evaporation The above described mexogenin diacetate can N and crystallization; G. Crystallization of non; ketone fraction from methanol; H. Evaporation. be hydrolyzed, e. g. with excess 20% alcoholic Hydrolysis. Crystallization _from methanol. potash, and the hydrolyzed product extracted Acetylation. Treatment with Girard’s reagent T. and crystallized from ether to give mexogenin of (No ketone fraction obtained.) Crystallization M. P. 245-246” C. ofnon-ketonefraction from acetone. " ’ ' Anal: Calc’d for 0271-14205! C, 72.6; H, 9.5. Samogenin : ' I Found: C, 72.9; H, 9.6. ’ " ' Example 3.-—Synthesis of agavogem'n from'mdn ogem'n Boiling acetic anhydride converts it back into its diacetate of M. P. 208° C. Mexogenin is (a) By catalytic hydrogenation: An ‘ethereal isomeric with manogenin. It is saturated to bro solution'of 0.3 g. of manogenin containing sev-‘ mine-acetic acid solution. When added to ‘a 2% 35 eral drops of acetic acid is shaken with hydrogen alcoholic solution of digitonin, it forms an insol and Adams catalyst for three hours at room tem-‘ uble saponide very slowly. perature and three atms. Agavogenin is crystal-' Although mexogenin forms a monosemicarba lized from aqueous methanol, M. P.‘233—240°.' A zone, it is not a?ected by’ the conditions, ‘of a he. mixture with the starting‘ material (240°) gives a‘melting point elevation, 242-247°. ' mild Clemmensen reduction reaction, thereby in Anal: Calc’d for 0271-14405: C, 72.3; dicating the inert position of the carbonyl group. Found: C, 72.2; H, 9.9. > I In this respect, it is like the other l2-keto The triacetate is prepared by re?uxing a solu removes the carbonyl group, giving samog'enin. .3, tion of the triol in acetic anhydride for one hour. The solvent is removed and the residue is crys The reduction product when converted .to its di tallized from methanol as plates, M. P. 226-228?’ acetate, is identical with samogenin diacetate. Hence, the relationship of mexogenin to samo Anal: Calc’d for CssHsoOsZ C, 69.0; H,"8.8. genin is thereby further established. The trans Found: C, 68.5; H, 8.7. I formation to samogenin can be illustrated as fol (b) By sodium-ethanol reduction: To a solu lows. tion of one gram of manogenin diacetate in 300 cc. of absolute ethanol is added 23 g. of sodium steroidal sapogenins. Wol?-Kishnerv reduction C ' ’ ‘ over a period of thirty minutes. 'After the so dium has reacted the reaction mixture is diluted O with water and ether extracted. The ethereal solution is washed free of alkali with 10% my O drochloric acid and‘ evaporated. Wolff-Kishner A —-—> reduction Agavogenin crystallizes from ether as White needles,_M. P. 238-2402 yield, 0.25 g. A mixture with material from (a)_melts 240°. ‘ I _ Anal: Calc’d'for C27H44O5I C, 72.3; H. 9.9. H Mexogenin Found: C, 72.0; H, 9.8. ' ' The triacetate is prepared by re?uxing with C: ,um ‘acetic anhydride for one hour. It is crystallized from methanol as plates, M. P. and mixed M. P. triacetate from (a) , 226-228". ' Anal: _Calc’d for CasHaoOe'Z C, 69.0; H, 8.8. Found: 0, 68.5; H, as. ' s In addition to the above described properties of agavogenin, it also is converted by mild (e. g. 25° C.) chromic anhydride oxidation to hecogenic acid. The latter oxidation product is identical Samogenin with the hecogenic acid from mild oxidation by Crystallization chart for the sapogenins from 75 CrOs of manogenin. Hence, agavogenin is 12 2,413.8;835 7 dihydro-manogenin. These changes can be '11 lustrated as follows. ‘ 7“ -H .Azavozenin where ‘Y1 and Yz-are- the same members of the classconsisting of The examples given are for the purpose of il lustrating the invention and can be varied in a OH good many Ways. For instance, acetic anhydride H is used as the acylating agent for the alcoholic 30 and groups hydrolyzable to hydroxyl groups of the new compounds. How ‘OH ever, one may use any of the known organic agentsfor converting alcoholic OH to an ester or» ether or other group capable of hydrolysis to give ——OH. -Such agents are, for example, or H and Ya, is a member of the class consisting‘of =0, ganic acid halides, acetyl chloride, benzoyl chloride, furoyl chloride, butyric anhydride, forma ‘ 01! tion of the alkali metal alcoholate followed by reaction with an alkyl halide to form an ether, and groups hydrolyzable to use 01' triphenyl, methyl chloride to form the so 40 <03 called trityl ethers, halides such as sulfuryl chlo ride, phosphorus chlorides and the like. Ring dihydroxy compounds, such as mano genin and mexogenin, or ring trihydroxy com ‘ pounds such as agavogenin can be completely ‘1 aoylated or etheri?ed at all of their ring hy droxyls. Completely esteri?ed or acylated agavo genin can be partially hydrolyzed at the number 2 and number 3 carbon atoms of the ring sys H What I clalmis: 1. A ring saturated ?zz-substituted steroidal sapogenin or the fromula tem to obtain derivatives partially esteri?ed or ' etheri?edat Cm but with free hydroxyls at C2 and Cs. ,Similar derivatives can be made by using alkali metal and alcohol, or hydrogen in pres ence of a platinum catalyst, to reduce the C12 keto of completely esteri?ed or etherifled mano genin or mexogenin to get the corresponding compound with a free hydroxyl group at 012. "V where Y1 and Y: are the same-members of the class consisting of When hydrogen and a catalyst are used for re duction, thegroups at C2 and C3 are not. hydro lyzed and the C12 hydroxyl alone can then be 60 esteri?ed or etheri?ed to replace it by the same or by a different ester or ether group from that OH and ester groups hydrolyzable to at carbon atoms 2 and 3. Thus, it isv easy for those skilled-in the art to prepare any of the new compounds of the invention having the gen (55 eral formula, CH: UHF-CH: 7 <03‘ H and Ya is a member of the class consisting 0! =0, on Q~H"‘C,Hl H and'ester groups hydrolyzable to Yx= YFL/ ‘ 75 ' 0H 2,408,835 sapogenin of the formula 10 and ester groups hydrolyzable to 2. A ring saturated Cm-substituted steroidal OH ' <11 4. A 2,3 - dihydroxy - 12 - keto - ring saturated steroidal sapogenin of formula C21H4205. 5. Manogenin of the formula, (7271-14205, the structure of which is 10 O CH; CH3 1 CE! | / CHr-CH: CH—O\ U l O_c§. where Y1 and Y2 are the same members of the class consisting of OH . 15 H and ester groups hydrolyzable to on no HO H-OH! / 1 u H 20 6. Mexogenin of the formula, C2'zH420s, the structure of which is and Y3 1s a member of the class on o ' I on CH; CHI-‘CH2 cH-cm 25 and ester groups hydrolyzable tL OH 30 3. A ring saturated Cur-substituted steroidal 7. Agavogenin of the formula, 0211-14405, the structure of which is HO HO where Y1 and Y2 are the same members of the class consisting of OH H 45 o ROMEO B. WAGNER.