IL23907A - Lincomycin analogues and process for producing them - Google Patents

Description

C O H E N Z E D E K & S P I S B A C H R E G D. PAT E N T A TTO RN EYS 24, LEVONTIN STR., P. O. B. 1169 I E L . A V I V P A T E N T S & D E S I G N S O R D I N A N C E 1345865 SPECIFICATION ■gRSGBSg ^¾¾-GO PqraraS- jPR&DOCKD- gBBRE!B¾ LINCOMYCIN ANALOGUES AND PROCESS . FOR PRODUCING THEM WE, THE UPJOHN COMPANY, a Corporation of the State of Delaware, of 501 Henrietta Street, Kalamazoo, Michigan, U.S.A., HEREBY DECLARE the nature of this invention and in what manner the same is to be performed to be particularly described and ascertained in and by the following statement : This invention relates to a method of producing lincomycin and analogs and isomers thereof and to new compounds produced thereby.

Lincomycin is an antibiotic obtained as an elaboration product of a lincomycln-producing actinomycete . Methods for the . production, recovery, and. purification of lincomycin are described in U. S. Patent 3*086,912. Partly as a result of this invention the structure of lincomycin has ,been elucidated as methyl 6- ( trana- -prop l-1-meth 1-L- prolylamino) -6, 8-dideoxy-l-thio-D-eryth.ro-q-D-galacto- octopyranoside which has the following structural formula: . It has. now been found that lincomycin and isomers and analogs thereof can now be synthesized by acylating with a -substituted L-proline of the formula: wherein Z is a protective hydrocarbyloxycarbonyl group which is removable by hydrogenolysis or trityl( triphenylmethyl) , ■ diphenyl(p-methoxyph.enyl )methyl, bis- (p-methoxyphenyl )phenyl■ 20β2Α f wherein Rx is alkyl of not more than 20 carbon atoms, cyclo alkyl of from three to not more than 8 carbon atoms, or aralkyl of not more than 12 carbon atoms and Y, R2 and 3 are as given above.

Examples of alkyl of not more than 20 carbon atoms are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl , decyl, undecyl , dodecyl, tridecyl, tetradecyl, pentadecy 1 , hexadecyl , heptadecyl , octadecyl, nonadecyl, and eicosyl and the isomeric forms thereof. Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-methyl cycl openty 1 , 2,3-dimethylcyclobutyl , 4-methy 1 cycl obuty 1 , and 3-cyclo-pentyl propyl . Examples of aralkyl are benzyl, phenethyl, a-phenyl propyl , and a-naphthylmethy 1. Examples of hydro-ca byl oxycarbonyl groups are tert i a ry-butoxycarbonyl ; benzyloxycarbonyl groups of the formula: wherein X is hydrogen, nitro, methoxy, chloro, or bromo, for example, carbobenzoxy (benzyloxycarbonyl ), p-ni t roca rbobenzoxy (p- n i t robenzy 1 oxyca rbony 1 ) , p-bromo-, and p-chlorocarbobenzoxy (p-bromo-, and p-chl orobenzyl oxycarbonyl ) ; and phenyloxy-carbonyl groups of the formula: wherein Xx is hydrogen, allyl, or alkyl of not more than 4 carb n a oms such as hen l x carb n - l l ox b l-4 20β2·! R«P(CeH3)3 VIII wherein R* is alkylidene of not more than 12 carbon atoms, cycloalkylidene of from 3 to not more than.8 carbon atoms, and aralkylidene of not more than 12 carbon atoms. Suitable alkyliderie, cycloalkylldene, and aralkylidene groups include methylene, ethylidene, propylidene, butylidene, pentylidene, hexylidene, heptylldene, octylidene, nonylidene, decylidene, undepylldene, and dodecylldene and the isomeric forms thereof, cyclopropylidehe, cyclobutylidene , cyclopentylldene, cyclohexylidene, cycloheptylidene, cyclooctylidene, 2-cyclo-propylethylidene, 3-oyclohexylpropylidene, benzylidene, 2-phenylethylidene, 3-phenylpropylidene, and dx-naphthylmethylene These Wittig reagents are prepared by reacting an alkyl-cycloaikyl, or aralkyltriphenylphosphonium halide with a base such as sodamide, or sodium or potassium hydride, or the sodium or potassium metalate of dimethylsulfoxide and the like, For example, the elimination of hydrogen halide from aikyltriphenylphosphonium halide,: produces alkylidenetriphenyl-phosphorane. [The preparation of phosphoranes: is discussed in detail by Trippett, Quart. Rev. XVII, No. 4 , p. 4o6 ( 1963 ) . ] The reaction is generally carried out in an organic solvent, such as benzene, toluene, ether, dimethylsulfoxide, tetrahydrofuran, or the like, at temperatures between 10°C. and the reflux temperature of the reaction mixture. The thus-obtained product, a alkylidene-, -cyclo-aikylidene-, or -aralkylidene-l-hydrocarbyloxycarbonyl-L-proline (VII), is recovered from the reaction mixture in a conventional manner, generally by extraction from aqueous solutions of the reaction mixture. The crude product can be purified by conventional means, such as recrystallization, or formation and recrystallization of easily formed derivatives such as amine salts of the amino acid, e.g., the dicyclohexylamine salt, and the like, and liberating the amino acids from such compounds The thus-obtained 4-alkylidene-, -cycloalkylidene , or 4-aralkylidene-l-hydrocarbyloxycarbonyl-L-proline (VII) is then hydrogenated in the presence of platinum, which is generally deposited on a carrier for example, carbon or an anion exchange resin like Dowex-l, a cross-linked polystyrene trimethylbenzylammonium resin. A mixture of the cis- and trans-epimers of the l-hydrocarbyloxycarbonyl-4-substituted-L-proline (Ila and lib) is obtained.

Alternatively, the process can be carried out by acylating compound III with compound VII to form a compound of the formula and then hydrogenatlng the alkylidene group with a platinum or palladium catalyst. With a platinum catalyst as given above the Z radical is preserved but with a palladium catalyst, e.g., palladium on carbon, hydrogenolysis Of the Z group concurrently with saturation of the alkylidene group is obtained.

The starting compounds of Formula III can be obtained in a variety of ways. Methyl 6-amino-6,8-dideoxy-l-thio-D- 2062'A methyl α-thioliricosamlnlde or MTL, is obtained by subjecting lincomycin to hydrazinolysis and ethyl 6-amino-6, 8-dideoxy-l-thio-D-erythro-q-D-galacto-octypyranoslde, also known as ethyl a-thiolincosaminide or ETL, is obtained by hydrazinolysis of lincomycin C, an antibiotic obtained when the lincomycin fermentation of U.S. Patent 5,086,912 is carried out in the presence of ethionine.

The hydrazinolysis advantageously is effected by heating the antibiotic under reflux with an excess of hydrazine, say for 20 hours or more. The desired 6-amino-6,8-dideoxy-l-thio-D-erythro-q-D-galacto-octopyranoslde can be isolated by distilling off the excess hydrazine and crystallizing from a polar solvent such as ethanol.

Other .6-amino-6,8-dideoxy-l-thio-D-erythro-a-D-gaiacto-octopyranosides can. be prepared by the following representative sequences: Ac is alkanoyi or ara Ikanoy 1 of not more than 12 carbon and 0 is (^-}~' . atom . Examples are formyl-, acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanoyl, pctanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, and the isomeric forms thereof, and benzoyl, pheny lacety 1 , 3 -pheny 1 prop i ony 1 , -pheny lbutyryl , -pheny valeryl and, the isomeric forms thereof.

The process is effected as fol lows : Methyl a-thiol inco-saminide is N-acylated, the methyl N -a.cy 1 -a-th i ol i ncosami n ide (IX) then O-acylated with thiobenzoyl chloride, and the resulting thiobenzoyl ester (x) is brominated to form compound XI which on treatment with a base is converted to compound XI I . Treatment of compound XI I with mi ld alkal i (aq. carbonate-bicarbonate) gives compound XI I I which on a 1 ky lat i on wi th a alkyl halide yields compound XIV.

Hydrolysis of the benzoyl and thiobenzoyl groups and hydra-zinolysis of the N-acyl group gives compound 1 Mb. In a like manner compounds in which Y is 2 -hydroxyethyl or 2 -a 1 koxyethy 1 can be, prepared by reacting compound XI I I with ethylene chlor-hydrin or a -a 1 koxyethy 1 hal ide. 20β2Α SEQUENCE A-2 Ac can be alkanoyl or aralkanoyl as above or an acyl of the formula: wherein, Rj Ri and Z are as given above. R0 can be alkyl of 2 to not more than 12 carbon atoms, or -CH2CH2OR4 wherein R4 is as given above. The starting glycoside A, which advantageously can be l incomycin, is subjected to mercapta 1 at i on with a mercaptan of formula R0SH to form the dithioacetal ed a d r d rm 20β2Α SEQUENCE Α-3 Ac and R# -a e as given in Sequence A-2. The starting compound, A, is dissolved in water as a soluble salt, e.g., the hydrochloride, and bromine is added with cool ing, advantageous- o ly to between about -10 and 20 C. A satisfactory procedure o is to cool the aqueous solution to about- 0 C. and the bromine is added dropwise. The stoichiometric amount of bromine is 1 mole for each mole of starting compound, though more or less can be used. Advantageously a sl ight excess, say from 5 to 20<£ excess, of bromine is used. The th i ogl ycos i de is thus converted to a sugar, F, in which the pyranose form Fa is in equil ibrium with the -aldose form Fb. This sugar can be isolated if desi red but ordinari ly this is neither necessary nor desirable. In the presence of acid, e.g., hydrochloric acid or other strong non-oxidizing acid such as p- tol uenesu 1 f on i c acid, and sulfonic acid type anion exchange 20β2Α the thiogl ycoside G. Effective conversion is obtained by introducing the mercaptan, oSH, into the aqueous solution resulting from the bromination and treating the resulting system, which wil l ordinari ly be a two phase system because of the insolubil ity of the mercaptan with hydrogen chloride gas or concentrated hydrochloric acid advantageously with o cool ing to a temperature between . about -10 and + 20 C. A satisfactory procedure is to cool the reaction mixture to 0° C. and to bubble into it hydrogen chloride gas. It is advantageous, especial ly with the longer chain mercaptans, to partial ly solubi l ize them by the addition of tetrahydro-furan to the. reaction mixture. Advantageously from 15 to parts of tetrahyd,rof u ran. for each part of water is used.

The temperature of the reaction may be allowed to rise, o advantageously not higher than about 55 C., during the addition of hydrogen chloride gas. After cool ing to about o C, the tet rahyd rof u ran is removed under vacuum. The reaction mixture can be worked up in the usual way which may include solvent extraction at an acidic pH, solvent extract-ion at a basic pH, fractional 1 i qu i d- 1 i qu i d extractions such as counter current distribution or part i t i on . chromatography, crystal l ization, and the l ike. Concomitantly some dithio-acetal .of Formula B (Sequence A-2) may be formed which after separation can be cycl ized as described above to form more of the desired th i ogl ycos i de G.

The β-epimers of the above can be prepared by the fol lowing representative sequence: 2062A Ac and Aci can be alkanoyl or aralkanoyl of not more than 12 carbon atoms as given above .

The process Is effected as follows: treating methyl N- acyl-a-thiollncosaminlde (IX) with mercuric chloride in a warm aqueous solution to obtain a mixture of 6-acylamino- 6, 8-dldeoxy-D-ery hro- -D-galacto-octopyranose or N-acyl-a- lincosamine (XVa) and N-acyl-3-lincosamine (XVB); acyiating this mixture with an acyiating reagent selected from acyl halldes and acid anhydrides to obtain a mixture of N-acyl- l,2,3,4,7-penta-0-acyl-a- and β-lincosamine (XVIa and XVI6); ' treating compound XVIa or compound XVI0 or a mixture of compounds XVIa and XVIB with hydrogen bromide in acetic acid to obtain 6-acylamino-2,;5,4,7-tetra-0-acyl-la-bromo--l,6,8-tri- deoxy-D-erythro-D-galacto-octopyranose or N-acyl-2, 5,4,7- tetra-O-acyl-la-bromo-l-deoxylincosamine (XVII); treating compound XVII successively with thiourea, a mixture of potassium carbonate and sodium bisulfite in water, and an alkyl iodide to obtain alkyl N-acyl-2, 3,4, 7-tetra-0-acyl-B- thiolincosaminide (XVIII ) treating compound XVIII with dry ammonia gas in methanol to obtain alkyl N-acyl-6-thiolinco- saminide (XIX) . Hydrazinolysis of compound XIX or of compound XVIII gives the alkyl 3-thiolincosaminide (IIIc). In a like manner compounds in which Y (formula III) is 2-h.ydroxyethylth.io or 2-alkoxyethylthio can be prepared by reacting compound XVII with ethylenechlorhydrin or a 2- alkoxyethyl halide.

Compounds of Formula III where Y is hydrogen can be obtained by the following representative sequence: 20β2Α: Sequence C The process is carried out as follows: methyl Nracyl- a-thiOlincosaminide (IX) (or methyl N-acyl~3~thiolincosaminide ) is treated with acetone in the presence of sulfuric acid to form methyl N-acyl-5. -0-lsopropylidene-a-thiolincosam- 2062A ..)■ ethanol to 6«acylamino-_5, -0- lsopropylidene -1,6,8-trideoxy-D-eyythro-D-galaoto-octopyranose, or N*acyl-3, -0-lsopropyll-dene-l-deoxylincosamine (XXI) . The desulfurization advantageously Is achieved by heating under reflux in ethanol with loosely packed Raney nickel for a period of 2 to 2 hours.

Thereafter, the catalyst is removed by filtration and the filtrate is evaporated to give a residue which can be purified by conventional means such as recrystallizatlon from organic solvents, The lsopropylidene group can be removed by mild acid hydrolysis, e.g., 80^ aqueous acetic acid or a dilute mineral acid such as hydrochloric acid or sulfuric acid, to give N-acyl-l-deoxylincosamine (XXII) and the N-acyl by hydrazinolysis to give 1-deoxylincosamine (XIII). Alternatively the hydrazinolysis can be effected first to give 3i -0-isopropylidene-l-de0xylincosamine (XXIV) . Advantageously, however, the lsopropylidene group is removed first.

Staptlag D-erythro-D-gaiacto- compounds of Formula EK6 wherein Ra and/or R3 is alkyl of not more than 12 carbon atoms can be obtained by the following representative sequence Sequence D CH3 RBC- can be alkanoyl or aralkanoyl of not more than 12 carbon atoms as given above. Re and R7 can be different alkyls if compounds XXVIIIa and/or XXVIIIb are alkylated with a different alkylating agent from that used to alkylate compound XXVII . Re and Rr lh compound XXVIIIc can thus be different alkyls.

The process is effected as follows: treating a ;, 6-»acylamino-68Tdldeoxy-D-erythro-D-galacto-octppyrano3e compound of formula XXV with dry acetone In the presence of an acid catalyst, for example, sulfuric acid, to give a mixture of compound XXVH and the oxazollne derivative thereof XXVI; hydrolyzin compound XXVI in hot water to convert it to compound XXVIIj methylating compound XXVII with an alkyl hallde, for example alkyl chlorides, alkyl bromides, or alkyl iodides of not more than 12 carbon atoms, in the presence of a base and separating the three products thus obtained (compound XXVIIIa, compound XXVIIIb, and compound XXVIIIc); and hydrolyzing the compounds XXVIII under mild hydrolyzing conditions, for example, with 80 aqueous acetic acid or a dilute mineral acid, such as, hydrochloric acid or sulfuric acid, to eliminate the lsopropylidene mpiety and treating with hydrazine to remove the N-acyl group to give the compounds XXIX. When Y is 2-hydroxyethylthio in the above sequence or when 2-hydroxyethyl 6-amino-6,8-dideoxy- 7»0-methyl»l-thlo»D»erythro-a-D-galacto-octopyranoside, also known as 2-hydroxyethyl a-thiocelestosaminide, obtained by the hydrazinolysis of celesticetin, an antibiotic produced according to U.S. Patent 2,928,844, Is used, the 2-hydroxy group will also be alkylated to give a 2-alkoxyethylthio group In whloh the alkoxy group will contain not more than 12 carbon atoms according to the alk 1 halide used to alkylate. When desired the 2-hydroxythio group can be preserved by protecting it with a trityl group. Thus cpmpounds XXV pr XXVII where Y is 2-hydroxyethylthio can be reacted with trityl chloride (triphenylmethylchlorlde) chlorodiphenyl-(p-methoxyphenyl)methane, or ..chloro-bis- (p-methpxypheny1 )phenylmethane, to cpnvert Y to a 2-trityloxy-ethy thio and the trityl group removed by mild hydrolysis, for example with 80# aqueous acetic acid, after the alkylatipn. The 2-hydrpxy group can be alkylated with a different alkyl group.

In carrying put this prpcess 6-acylaminc-6,8-dideoxy-D-erythro-D-galaoto-octopyranose compound of formula XXV Is suspended in dry acetone. A acid catalyst, such as sulfuric acid, p-tPluenesulfpnic acid, o-toluenesulfonic acid, p-ethylbenzenesulfonic acid, or the like, is added to the suspension, with sulfuric acid preferred. The acetone is generally used in large excess, such as a volume of 20 to 200 times the amount of compound XXV. The acid catalyst is used in a quantity between 0.25 and 5 by weight with respect tp the acetpne . The reaction can be carried out between 10°C. and the reflux temperature of the solution, but is generally carried out at room remperature . The reaction time is between 15 minutes and 6 hours, depending on the temperature, after which the reaction mixture is neutralized to terminate the reaction. The inorganic salts precipitated by the neutralization are removed by filtration, and the filtrate is evaporated to give a mixture containing crystalline solids. This mixture is separated with water into a water-soluble and water-inso uble fraction the water-soluble material is compound XXVII; the insoluble fraction is the oxazoline derivative thereof (XXVI).

The waters-insoluble oxazoline derivative (XXVI) can be" converted to compound XXVII by heating.with water. After this hydrolysis is complete, the product is obtained by evaporating the solution until crystallization occurs.

The. alkylation of compound XXVII is performed with an alkyl halide, preferably an alkyl chloride, bromide or Iodide, in the presence of a. strong base. As alkylating agents, methyl iodide, methyl bromide, ethyl iodide, ethyl bromide, and propyl, butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl chlorides, bromides and iodides and any of the branched-chain alkyl chlorides, bromides or iodides having up to 12 carbon atoms can be used. As a base, aikoxldes, such as sodium methoxide, potassium methoxide, potassium isopropoxide, potassium tert-butoxide, sodium . tert-butoxide, and sodamide and the like can be used. The alkylation can be performed at a temperature between 0° and 50° C. using inert solvents such as benzene, toluene, dioxane, tetrahydrofuran, and the like. After the reaction is terminated (usually between 1/2 hour and > hours) the reaction mixture is filtered to remove solids, such as sodium or potassium chloride, bromide or iodide, and the filtrate is evaporated in vacuo to a syrupy residue. The three products therein, namely compound XXVIIIa, compound XXVIIIb, and compound XXVIIIc, are separated by conventional methods, for example, by chromatography or by counter-current distribution. The removal of the N-acyl and isopropyli-dene groups is effected as described above for sequence C.

It will be understood that 2-0-, and 7-0-, and 2,7-di-0- ■ 2062A hydrazine as described above to form the desired methyl epl-g-thlolincosaminide (methyl 6-amino-6, 8-dideoxy-l-thio-L-threo-ct-D-galacto-octopyranoside ) or the hydraz i nol ys i s can be effected first and the removal of the isopropylidene group last as described for sequence C.

Removal of the protective group Z in compounds of formula IV is effected by hydrogenolysis using a palladium catalyst. The palladium is usually deposited on a carrier, for example, carbon. Any of the conventional methods of hydrogenolysis can be used.

The proline nitrogen in compounds of formula V where Ri is hydrogen can be alkylated with an alkyl halide, for example, an alkyl iodide. Milder conditions than those given above for alkylating the 2-0 and 7-0-positions are used. Thus the N-alkylation can be effected without a strong base and at room temperature or at most with mild heating, say, to not more than 50°C . or so. Advantageously the alkylation is effected by reacting the compound with an oxo (an aldehyde or a ketone) and hydrogenating the resulting adduct. The hydrogenation can be effected with palladium or platinum as the catalyst or any hydrogenating catalyst effective to saturate an olefinic double bond.

Suitable oxo compounds have the formula: 8RgC0 where RgRgC- is alkylidene of not more than 20 carbon atoms, cycloalkylidene of from 3 to not more than 8 carbon atoms, or aralkylidene of not more than 12 carbon atoms. Examples are formaldehyde, acetaldehyde , propionaldehyde , butyraldehyde acetone, isobutyl methyl ketone, benzaldehyde , phenylacetal-dehyde, hydrocinnamaldehyde, acetophenone, propiophenone , butyrophenone , 3-methyl-4-phenyl-2-butanone, 2-methyl-5-phenyl 2062 A 2, 2-dimethylcyclopropylacetaldehyde, 2,2-dimethylcyclopropyl methyl ketone, pyolopentyl methyl ketone, oyclobutyl methyl ketone, cyclobutanone, oyolohexanone, 4-methylcyclohexanone, and the like.

The oia and trans epimere oan be separated by partition or gradient chromatography. The separation can be effected most advantageously at the stage represented by Formulas IV or V. The respective epimere can also be formed by starting with the trans or cis forms of the 4-substltuted-L-prolines (Formulas Ilavand lib.) Various aoid-addition salts of the free base form of the compounds of formula V wherein Ri is hydrogen, alkyl, cyolo-alkyl, or aralkyl as given above, can be made by neutralizing the free base with the appropriate add to below about pH 7.0, and advantageously to about pH 2 to pH 6. Suitable acids for this purpose include hydrochloric, sulfuric, phosphoric, thiocyanlo, fluosilicic, hexafluorodrsenio, hexafluorophosphoric, acetic, succinic, citric lactic, maleic, fumarlc, pamoic, oholic, palmitic, mucic, camphoric, glutario, glycolic, phthalic, tartaric, lauric, stearic, salicylic, 3-phenylsalicyllc, 5^phenylsalicylic, , 5-methylglutaric, orthosulfobenzoic, cyclo-hexanesulfamio, cyclopentaneproplonic, 1,2-cyclohexanedi-carboxylic , 4-cyclohexenecarboxylic, octadecenylsucclnlc, octenylsucclnic , methanesulfonic, benzeneeulfonic, helianthic, Reinecke's, dlmethyldithiocarbamic, cyclohexylsulfamic, hexa-decylsulfamlc , octadecylsulfamic, sorbic, raonoohloroacetlc, undecylenic, · -hydroxyazobenzene-4-sulfonic, octyldecyl-Bulfuric, picric, benzoic, cinnamic, and like acids. .. 2062'' The acid-addition salts can be used for the same purposes as the free base or they can be employed to upgrade the same. For example, the free base can be converted to an insoluble salt, such as the picrate, which. can be subjected to purification procedures, for example, solvent extractions and washings, chromatography, fractional liquid-liquid extractions, and crystallization and then used to regenerate the free base form by treatment with alkali or to make a different salt by metathesis. Or the free base ,can be converted to a water-soluble salt, such as the hydrochloride or sulfate; and the aqueous solution of the salt extracted with various water-immiscible solvents before regenerating the free base form by treatment of the thus-extracted acid solution or converted to another salt by metathesis.

The compounds of formula V where Ri is hydrogen, alkyl, cycloaikyi, or aralkyl as given above, can be used as a buffer or as an antacid. The compounds of formulas IV and V react with isocyanates to form urethanes and can be used to modify polyurethane resins. The long chain compounds, i.'ei, where Ri is alkyl of from 8 carbon atoms up, have surface active properties and can be used as wetting and emulsifying agents. The thiocyanic acid addition salt when condensed with formaldehyde forms resinous materials useful as pickling inhibitors according to U.S. Patents 2,^25,320 and 2,606,155, The free bases also make good vehicles for toxic acids. For example, the fluosillclc acid addition salts are useful as mothproofing agents according to U.S. Patents 1, 15,53 and 2,075,359 and the hexafluoroarsenic acid and hexafluorophosphoric acid addition salt? are useful as parasiticides according to U. S. Patents 2,122,536 and 3,122,552. 2062A The close analogues of 1IncornycIn, I.e., where RH- is trans-alkyl of not more than 6 carbon atoms; Rx is methyl or ethyl; and Y is a-alkylthio of not more than β carbon atoms, have antibacterial properties comparable to lincomycin and can be used for the same purposes as lincomycin. The other analogues and isomers have similar antibacterial properties but to a lesser degree and can be used for the same purposes as lincomycin where larger amounts are not objectionable .

The following examples are illustrative of the process and products of the present invention but are not to be construed as limiting. The parts and percentages are by weight and the solvent ratios are by volume unless otherwise specified.

J 2062A complete reaction . A solution of 26 g. of 4-keto-l-carbo-benzoxy-L-proline in 100 ml. of dimethylsulfoxide was added, and the resulting mixture was heated at 70°C. for 3 hours. The reaction mixture was cooled to 25° C, and 1 liter of 2.5$ aqueous potassium bicarbonate added, This mixture was washed twloe; with 700 ml , portions of ether and the ether was discarded after back extracting with 150 ml. of 2.5# aqueous potassium bicarbonate. The bicarbonate solutions were combined and acidified with 4N hydrochloric' acid. The acidified mixture was extracted with four 5P0-ml. portions of ether. The combined ether extracts were washed successively with 250 ml * of water, three 250-ml . portions of saturated aqueous sodium bisulfite, and 250 ml. of water, and dried over anhydrous sodium sulfate. Evaporation of the solvent under vacuum gave 2 g. of an oily residue which was 4-butylidene-l-carbobenzoxy-L-proline .

This residue was dissolved in 31 ml. of acetonitrile and treated with l8 ml. of dicyclohexylamine and refrigerated. The crystals were collected, washed with acetonitrile and dried In vacuo giving 21 g, (46.8 ) of the crystalline dicyclohexylamine salt melting at 136-l40°C. After two recrystallizations from acetonitrile, an analytical sample was obtained which melted at l42-l44°C, and had a rotation of [o]D - 4° ( 0 = 0.99, CHCI3 ) · Anal . Calcd. for C2eH-_.4 20*: C, 71.86; H, 9.15; N, 5.78 Pound: C, 71.69; H, 9-30; N, .7 .

Ten grams of the dicyclohexylamine salt of 4-butylldene-1-carbobenzoxy-L-proline was shaken with ether and excess 5 aqueous potassium hydroxide until no solid remained. The 2062A layers were separated and each one was backwashed. The aqueous alkal ine layer was combined with the backwash from the ether layer and acidified with 4N hydrochloric acid. The mixture was repeatedly extracted with ether and the ether extracts were combined, dried over sodium sulfate, and evaporated in vacuo to give 6.3 g„ (93$) of 4-butyl idene 1-carbobenzoxy-L-prol i ne as an oil .

A-2. 4-Pentyl idene-l-carbobenzoxy-L-prol i ne and the di cyclohexylami ne salt thereof Fol lowing the procedure of Part A-l substituting the buty 1 t r i phenyl phosphon i urn bromide by pentyl tr i phenyl phosphon i urn . bromi de, 4-pentyl i dene-l-carbobenzoxy-L-prol i ne and the dicyclohexylami ne salt thereof were obtained. The free acid was an oi l but the d i cycl ohexy 1 ami ne salt after re-crystal l ization from acetonitri le had a melting point of o o 124-128 C, an optical rotation of [ ]Q -6 (c, 0.7^2, CHCI3), and the following analysis: Calcd. for C30H46N2O4: C, 72.25; H, 9-30; N, 5.62.

Found: C, 72.38; H, 9.52; N, 5.97- A-3. 4-Hexy i dene-l-ca rbobenzoxy-L-prol i ne and the d i cycl ohexy 1 am i ne salt thereof Following the procedure of Part A-l substituting the buty 1 t r i phenyl phosphon i urn bromide by hexy 1 t r i phenyl phosphon-i urn bromide, -hexyl i dene-l-carbobenzoxy-L-prol i ne and the d i cycl ohexy 1 am i ne salt thereof were obtained. The free acid was an oi l but the d i cycl ohexy 1 am i ne salt after recrystal- o l ization from acetonitrile had a melting point of 109-111 C o an optical rotation of [a]Q -7 (c, 0.941, CHC13), and the fol 1 ow i ng ana 1 ys i s : 20β2Α Α-4. 4-Octyl i dene-l-ca rbobenzoxy-L-prol i ne and the d i cycl ohexyl ami ne salt thereof Fol lowing the procedure of Part A-l substituting the - ■ butyl t r i phenyl phosphon i um brom i de by octyl tri phenyl phosphon- i um bromide, 4-octyl i dene-l-carbobenzoxy-L-prol i ne and the di cycl ohexyl ami ne salt thereof were obtained. The free acid was an oi l but the di cyclohexylami ne salt after recrystal- l ization from acetonitrile had a melting point of 113-118° C. , an optical rotation of [a]Q -11° (c, 1.020, CHC13), and the fol lowi ng anal ysi s: Calcd. for C33H52N2O4: C, 73.29; H, 9.69; N, 5.18.

Found: C, 73.52; H, 10.06; N, 5.28.

B-1. 4- Butyl -1-carbobenzoxy-L-prol i ne The oil from Part A-l was hydrogenated in 200 ml. of methanol over 2.1 g„ of 10 platinum on Dowex-1 catalyst under 40 lbs0 hydrogen pressure. The catalyst was removed by fi ltration and the. filtrate evaporated to yield 6.3 g. of 4-butyl- 1-carbobenzoxy-L-prol i ne as an oil. The product contained about 2 parts cis-4- butyl -1-carbobenzoxy-L-prol i ne to each part of rans-4- butyl -1-carbobenzoxy-L-prol i ne, B-2. 4-Pentyl -1-carbobenzoxy-L-prol i ne Hyd rogenat i on of the oi ly free acid from Part A-2 by the procedure of Part B-1 gave 4-pentyl -1-carbobenzoxy-L- prol i ne as an oi l .

B-3. 4-Hex 1 -1-ca rbobenzoxy-L -prol i ne Hyd rogenat i on of the oi ly free acid from Part A-3 by the procedure of Part B-1 gave 4-hexyl -1-carbobenzoxy-L-prol i ne as an oi 1.

B-4„ 4-0ctyl -1-carbobenzoxy-L-prol i ne - 2062A the procedure of Part B-l gave 4-octyl -1-ca rbobenzoxy-L -prol i ne as an oi l .

C. Methyl 6-amino-6,8-di deoxy-1- th i o-D-erythro- -D- ga lacto-octopyranos i de (Methyl a- th i ol i ncosam i n i de ) A solution of 40 g. of l incomycin (U„ S. Patent 3,086,912) in 20 ml., of hydrazine hydrate (98-100$) was re-fluxed for 21 hours; excess hydrazine hydrate was then removed i n vacuo under nitrogen at steam bath temperature, leaving a residue. The residue, a pasty mass of crystals, was cooled, acetonitri le was added, and the mixture was stirred unti l the crystals were suspended. The crystals were col lected on a filter, washed with acetonitrile and with ether. The yield of whi te, . crystal 1 i ne methyl a-thiol inco-saminide after drying in vacuo at room temperature was 21 g. (8 ). Recrysta 11 i zat i on was accompl ished by dissolving methyl a- th i ol i ncosam i n i de in hot d imethy 1 formam i de and adding an equal volume of ethylene glycol dimethyl ether.

Methyl a- th iol i ncosam i ni de has a melting point of 225-228° Ca i an optical rotation of [a]2Q5 + 27β° (c = .768, water) and a pKa' of 7,45, Calcd. for C9H_9N05S: C, 42.7; H, 7,56; N, 5.53; S, 12.66.

Found: C, 42.6; H, 7.49; N, 5.75; S, 12.38.

D. Methyl N- (4-a 1 ky 1 -1-ca rbobenzoxy-L -p ro 1 yl ) -a- th i o- 1 i ncosam i n i de D-l. Methyl N- (4- butyl -1-ca rbobenzoxy-L -prol yl )- 2Α To a solution of 6.3 g. of 4-butyl -1-carbobenzoxy-L-proline (the oi l from Part A) in.175 ml. of distilled aceton- o . . itrile cooled to 0 was added 3.46 ml. of t r i ethyl am i ne followed by 3.34 ml. of isobutyl chl oroformate. The mixture o o was stirred at 0 C. + 3 ) for 15 min. A solution of 6.2 g. of methyl a- thi ol i ncosami n i de (MTL) from Part B ί n 85 ml. of water was added, and the reaction mixture was stirred at o o 0 C. for 0.5 nr.. and at 25 C. for 1 hr. The reaction product was then filtered and dried yielding 4.57 9· (37.7 ) of methyl N- (4- butyl -1-ca rbobenzoxy-L -prol yl ) -a- thiol i ncosami nide. The mother liquor was concentrated under vacuum and an additional 4.25 g. (35.2$) of product recovered. Recrysta 1 i za-tion from acetoni tri Re produced crystals of methyl N-(4-butyl- 1-ca rbobenzoxy_l -prol yl )- -thiol incosami nide melting at 194- o 196 C. , A second recrysta 11 ί zat i on from aceton i tr i 1 e afforded o o an analytical sample, m.p. 197.5-200 C, [a]D + 111 (c = 0.98, MeOH) .

Calcd. for C2eH4oN20eS: C, 57.75; H, 7.46; N, 5.13; S, 5.93.

Found: C, 57.58; H, 7.l6; N, 5.50; S, 6.07.

D-2 Methyl N- (4-pentyl -1-ca rbobenzoxy-L-prol yl )-a- thiol i ncosami nide Following the procedure of Part D-l substituting the 4-butyl -1-carbobenzoxy-L-prol i ne by 4-penty 1 -1-ca rbobenzoxy-L-prol ine (the oil of Part B-2), there was obtained methyl N- (4-pentyl -1-ca rbobenzoxy-L-prol yl ) -a- th iol i ncosami n i de o having a melting point of 191-193 C, an opt i cal'- rotat i on of [a]D .+ 108° (c = 0.722, MeOH) and the f o l o.wi ng ana 1 ys i s : Calcd. for C H N 0 S: C 58.46 H .6 N 5.05. 20β2Α D-3. Methyl N- (4-hexyl -l-carbobenzoxy-L-prol yl )-a-thio- 1 i ncosam i n i de Following the procedure of Part D-1 substituting the 4-butyl -l-carbobenzoxy-L-prol i ne by 4-hexyl -l-carbobenzoxy-L-prol ine (the oil of Part B-3), there was obtained methyl N- (4-hexyl -l-carbobenzoxy-L-prol yl ) -a- thi ol i ncosami n i de hav i ng o a melting point of 176-I8O C, an optical rotation of o [a] + 103 (c = 0,951 methanol ), and the f ol 1 ow i ng ana 1 ys i s: Calcd. for C28H44N208S: C, 59.13; H, 7..80; H, 4.93; S, 5.64.

Found: C, 59.16; H, 7.46; N, 5.09; S, 5.96.

D-4 Methyl N- (4-octyl -l-carbobenzoxy-L-prol yl -a-thio- 1 i ncosam i n i de Following the procedure of Part D-1 substituting the 4-butyl -l-carbobenzoxy-L-prol i ne by 4-octyl -l-carbobenzoxy-L-prol ine (the oil of Part B-4), there was obtained methyl N- (4-octyl -1-ca rbobenzoxy-L- prol yl -a- thi ol i ncosam i n i de hav i ng o a melting point of 181-202 C, an optical rotation of o + 99 (c = 1.083, methanol ), and the fol lowing analys Calcd. for C3oH48 208S: C, 60.38; H, 8.11; N, 4.70; S, 5.37.

Found: C, 60.35; H, 8.Ο8; N, 4.73.

E. Methyl N- (4-a 1 kyl -L -prol yl ) -a- th i ol i ncosam i n i de hydrochloride 20β2Α E-1, Methyl N- (4- buty 1 -L -p rol y 1 ) -a- th i ol i ncosam i n i de hyd roch lor i de A solution of 7.8 g. of methyl N- (4-butyl -1-carbobenzoxy-L -prol yl ) -a- th i ol i ncosam i n i de from Part D in 200 ml . of methanol was shaken over 2 g. of 10$ pal ladium on carbon under 40 lbs. of hydrogen pressure for 17 hours. The catalyst was removed by fi ltration and the solution concentrated under vacuum. The residue was dissolved in a mixture of 20 ml . of acetone and 20 ml . of water and acidified with 6N hydrochloric acid. Di lution with 4 volumes of acetone precipitated methyl N- (4-butyl -L-prol yl )-a-thi ol i ncosami ni de hydrochloride which was col lected by filtration and dried.. The crystals, dried at • o o 5 C. under vacuum, weighed 4.7 g. and. melted at 188-194 C.

The analytical sample obtained by recrysta 11 i zat i on from acetone melted at 197-199° C. and gave [a] + 150° (water, c = 0.89).

Anal . Calcd. for CieriUtNaOeS. HC1 : C, 48.80; H, 7.96; N, 6.32; S, 7.24.

Found., (corrected for 5.54$ water): C, 48.58; H, 8.19; N, 6.04; S, 7,36.

This material possesses Q$ of the anti acterial activity of l incomycin by _S. 1 utea assay, E-2. Methyl , N- (4-pentyl -L-prol yl ) -a- thi ol i ncosam i n i de hydrochloride Hydrogenol ys i s of methyl N- (4-pentyl -1-carbobenzoxy-L-prplyl )-a-thiol i ncosami nide by the procedure of Part E-1 gave methyl N- (4-pentyl -L-prol yl )- -thi ol i ncosami ni de hydrochloride having a melting point of 212-214° C, an optical rotation of [a] + l4l° (c = Ο.968, H20)^ and the fol lowing 20β2Α Calcd. for CigHayNsOeSCl : C, 9.93; H, 8.l6; N, 6.13; S, 7.02.

Found: C, 50.22; H, 7.96; N, 6,0 ; S, 7.18. (Cor rected for 5Λ34 H20)... .

Er5. Methyl N- (4-hexyl -L-prolyl )- - thiol i ncosam i nide hydrochloride Hydrogenol ys i s of methyl N- (4-hexyl -1-carbobenzoxy-L-prol yl )-a-thiol i ncosami ni de by the procedure of Part E-1 gave methyl N- (4-hexyl -L-prolyl )- - thiol i ncosami ni de hydrochloride having a melting point of 197-209° C. (dec. ) and an optical rotation of [α] + 134 (c = 0,875, H20).

Calcd. for C20H39N2OsSCl : C, 50.99; H, 8.35; N, 5,95.

Found: C, 50.32.; H, 8.07; N, 6.8l (Corrected for .62^ water). .

E-4. Methyl N- (4-octyl -L-prol yl ) - ct- h i o 1 i ncosami ni de hydrochloride Hyd rogenol ys i s of methyl N- (4-o'ctyl -1-carbobenzoxy-L-prol yl )-a-thi ol i ncosami ni de by the procedure of Part E-1 gave methyl N- (4-octyl -L-prol yl )-a-thiol i ncosami ni de hydrochloride o having a melting point of 181-200 C. (dec. ), an optical rotation of [ ]D + 128 (c = 0.850, H20) and the following analysis: Calcd. for C22H42N206S° H l : C, 52.94; H, 8,68; N, 5.6l; S, 6.43.

Found: C, 52.62; H, 8.36; N, 5.6l; S, 6.36.

F. Methyl N- (4-a 1 ky 1 - 1 -methyl - L - pro 1 y 1 ) - a- thi ol i ncosam - inide hydrochloride 20β2Α F-l. Methyl N- (4 -butyl -1 -methyl -L-prolyl )- - thiol inco- saminide hydrochloride A solution of 2,0 g. of methyl N- (4-buty 1 -L-prol y 1 ) -a-th i o I ϊ ncosam Ϊ n i de hydrochloride from Part E and 2,0 ml . of 37$ formal in in 150 ml. of methanol was shaken over 500 mg. of 104, palladium on carbon under 40 lbs. of hydrogen pressure for 3. hrs. Removal of the catalyst by filtration and the solvent by d i st i 11 at i on i n vacuo yielded partially crystal l ine methyl N- (4-butyl -1-methyl -L-prol yl )-a-thiol i ncosami ni de hydrochloride which was found by TLC (thin layer chromatography) on sil ica' gel using a mixture of ethyl acetate, acetone, water (8:4:1) for elution and KMn04 solution for detection to consist chiefly of two materials, the ci s and t rans epimers of methyl N- (4 -butyl -1 -methyl -L-prol yl ) -a- thiol i ncosam i ni de hydrochloride in a ratio of about 3 to 2.

Separation of the c i s and t rans forms by chromatography The methyl N- (4-butyl -1-methyl -L-prol yl )-a-thiol i nco-saminide hydrochloride from Part F-l was dissolved in a mixture of methanol and methylene chloride (1:1) and 1.5 ml. of t r i ethyl ami ne added. To this solution was added 7 g. of si l ica gel and the solvent evaporated under vacuum leaving the antibiotic deposited on the si l ica gel which was sifted on top of a chromatographic column of 200 grams of sil ica gel packed with a solvent mixture consisting of ethyl acetate, acetone, water in a ratio of 8:4:1. The column was developed with the same solvent and 20 ml . portions were collected. TLC of each fraction as described above showed that fractions 31-38, 310 mg., were essential ly pure t rans epimer and that fractions 4 - 4 2 were essen ial l ure ci s e imer ractions 20β2Α 59-48 consisted of a mixture of epimers which could be further separated by repeated chromatography. Each epimer was dissolved in a few drops of dilute hydrochloric acid and the hydrochloride precipitated by addition. of acetone. In this manner, there was obtained 50 mg. of methyl N- ( t rans-4- butyl -1-meth l -L-prol yl ) -a- th i ol i ncosami n i de hydrochloride, m.p. 135-157 °, and about 150 mg. of methyl N- (ci s-4- butyl -1-methyl - o L -prol yl ) -a- thi ol i ncosami n i de hydrochloride softening at 105 • o C. with further melting at 175-185 C.

The trans epimer recrysta 11 i zed from the same solvent melted at 159- 1° C. and had the fol 1 ow i ng .ana 1 ys i s : Calcd. for Ci9H3eN20eS- HC1 C, 49.95; H, 8.l6; N, 6.15; S, 7.02.

Found (Corrected for 4.07$ H20): C, 48.81; . H, 8.54; N, 6,49; S, 6.67.

Simi larly recrysta 11 izat i on of the ci s epimer gave a product, softening at 108° C. and. further melting at about I890 C. (solvated) which had the fol lowing analysis: Found (Corrected for 4.95$ water): C, 50.27; . H, 9.00; N, 6.05; S, 6.65.

The t ra ns epimer was about 2.2 times as active as 1 i neomycin by S . 1 utea assay, about times as active by the broth dilution assay, and 2.5 times as active in mice infected with _S„ aureus.

T he cis epimer was about 1/2 to 1/5 as active as the t rans epimer, being about equal to l i.ncomycin.

F-2. Methyl N- (pentyl -1-methyl -L-prol yl )-a-thiol i nco- saminide hydrochloride Reductive methylation of methyl N- (4-pentyl -L-prol yl )- 20β2Α gave methyl N- (4-pentyl -1-methyl -L-prol yl )-a-thiol i ncosami ni de hydrochloride as a mixture of the ci s and t ran s isomers which on partition chromatography by the procedure of Part F-l gave methyl N- (t rans-4-penty 1 -1-methyl -L-prolyl ) - a- thiol i ncosami n- o ide hydrochloride having a melting point of 188-191 C, and methyl N- (cj_s -4-pentyl -1-methyl-L-prolyl ) - a- thiol i ncosam i ni de o hydrochloride having a melting point of 189-193 C. (sinter o 135 C. ). The trans isomer assayed about 2 to 4 times the antibacterial activity of lincomycinj the c i s isomer about 1 to 2 t imes.

F-3. Methyl N- (4-hexyl -1-methyl -L-prol yl )-a-thi ol i nco- saminid.e hydrochloride Reductive methylation of methyl N- (4-hexyl -L-prol yl )-a-thiol incosami nide hydrochloride and chromatographic separa-tion of the isomers by the procedure of Part F-l gave methyl N- (trans-4-hexyl -L-prolyl ) - a- thiol i ncosam i ni de hyd rochl or i de o having a melting point of 93-104. C. (dec. ) and methyl N- (c i s- 4- hexy 1 -L -p rol y 1 ) -a- th i ol i ncosam i n i de hydrochloride having a o melting point of 92-102 C. (dec ). The trans isomer assayed about 2 to 4 times the antibacterial activity of l incomycin, the ci s isomer about 1 to 2 times.

F-4. Methyl N- (4-octy1 -1-methyl -L-prol yl )-a-thi ol i nco- saminide hydrochloride Reductive methylation of methyl N- (4-octy 1 -L-prol yl ) -a-thiol incosami nide hydrochloride and chromatographic separation of the isomers by the procedure of Part F-l gave methyl N- (trans 4-octy 1 -1-methyl -L-prolyl ) - a- thiol i ncosami n i de hydrochl or i de o having a melting point of 97-100 C. (dec. ) and methyl N-(c i s-4-octy 1 -1-methyl -L-prolyl )- - hiol i ncosami n i de hydro - hl ride 20β2Α G. Methyl N- (4-al kyl -1-ethyl -L-prol yl )-a-thi ol i nco- saminide hydrochloride G-l Methyl N- (4- butyl -1-ethyl -L-prol yl )- - thiol i nco- saminide hydrochloride A mixture of 2.0 g. of methyl N- (4-butyl -L-prol yl )-a-thi ol i ncosam i n i de hydrochloride, 1.5 ml . of aceta 1 dehyde, and 150 mg. of 10K> pal ladium on carbon in 150 ml . of methanol was shaken under 35 lbs. of hydrogen pressure for 5.5 hrs. The catalyst was removed by fi l tration to give a residue consisting chiefly of the ci s and t rans epimers of methyl N- (4-butyl-1-ethyl -L-prol yl )- -thiol i ncosami ni de hydrochloride.

Separation of- epimers As described in Part F-l, the mixture of epimers of Part G-l, (2 g. ) was chromatographed over 200 g. of si l ica gel using for elution a solvent system of ethyl acetate, acetone, water (8:4:1), Fractions 33-42 which by TLC were found to be pure t rans epimer were combined, and fractions 49-64 which were essential ly pure ci s epimer were al so combined.- Fractions 43-48 were a mixture of the epimers which could be pur i f i ed by rechromatog raphy . Each epimer was dis-solved in a few crops of di lute hydrochloric acid and the crystal l ine hydrochloride precipitated on di lution with a large volume of ether..

The crude t rans epimer fraction of 415 mg. gave 340 mg. (15.4 ) of crystal 1 ine methyl N- (t rans-4- butyl -1-ethyl -L- - - - ° 2062A Recrysta 11 i zat ion from di lute acetone raised the m.p. to 148-151° C The c i s epi.mer fraction of 6 5 mg. afforded 300 mg. (14.116) of crystal 1 i ne methyl N- (c_i_s-4- butyl -1-ethyl -L-prol yl )- o a-thiol i ncosaminide hydrochloride, m.p. 135-139 C. When re-crystal l ized from dilute acetone, the melting point was 134-138° C.

The t rans epimer showed about 1-1.2 times the activity of l incomycin by the _S. lutea assay, 2-4 times the activity of l incomycin against Gram-positive organisms, and 8 times or more the activity of l incomycin against Gram-negative organisms. In mice against _S. aureus the t rans epimer was about 2 times as active as l incomycin. The ci s epimer was about 1/2 as active as the t rans epimer.

G-2. Methyl N- (4-pentyl -1-ethyl -L-prol yl )- -thiol i ncosaminide hydrochloride.

Reductive ethylation and separation by the procedure of Part G-1 gave the ci s and trans epimers as the free bases and hydrochlorides. The solvated trans hydrochloride (crystal- . o l ized from aqueous acetone) had a melting point of 90-95 C. (dec. ) .

G-3. Methyl N- (4-hexyl -1-ethyl -L-prol yl )- -thiol i ncosaminide hydrochloride Reductive ethylation and separation by the procedure of Part G-1 gave the ci s and trans epimers as the free bases and the hydrochlorides. The t rans hydrochloride had a melting o point of 102-121 C. (dec. ) and the cis hydrochloride a melt- o ing point of 93-106 C. (dec ).

G-4. Methyl N- (4-octy 1.-1 -ethyl - L-prol yl )- a- thiol inco- 2062A 4 Reductive ethylation and separation by the procedure of Part G-l gave the ci s and trans epimers as the free bases and the hydrochlorides. The hydrochlorides were solids but non-crystal 1 i ne.

By substituting the a 1 kyl t r i phenyl phosphonium bromides of Part A in the above example by other substituted triphenyl-phosphonium bromides where the substituent is methyl, ethyl, propyl, heptyl, nonyl, decyl, undecyl, and dodecyl, and the isomeric forms thereof, as well as the isomeric forms of butyl, pentyl, and hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclo-hexyl, cycloheptyl, cyclooctyl, 2-cycl opropyl ethyl , 3-cycl ohexy 1 -propyl, benzyl, phenethyl , 3-phenyl propyl , and 1-naphthy lmethy 1 , the corresponding methyl N- (ci s- and t rans-4-a 1 kyl - , 4-cyclo-alkyl-, and 4-aral kyl -1-carbobenzoxy-L-prol yl )-a-thiol i nco-saminides, the correspond! ng 'methyl N- (c i s and trans-4-al kyl -, 4-cycl oal kyl -, and 4-aral kyl -L-prol yl )-a-thi ol incosami ni des; the corresponding methyl N- (ci s- and t rans-4-a 1 kyl - , 4-cyclo-alkyl-, and 4-aral kyl -1-methyl -L-propyl )-a-thiol i ncosami nides and the corresponding methyl N- (ci s and trans-4-al kyl -, 4-cycloal kyl and 4-a ral kyl -1-ethyl -L-prol yl ) -a- thi ol i nco-saminides are obtained. For example, when propyl tri phenyl -phosphonium bromide and formalin are used l i neomycin and a 11 ol i ncomyci n (the ci s-epimer ) are obtained both of which are active antibiotics, By substituting the formalin or acetaldehyde by other oxo compounds of the formula RaRgCO, for example, propi onal dehyde, acetone, butyra 1 dehyde, i so-butyl methyl ketone, benza 1 dehyde, pheny 1 aceta 1 dehyde, hydro-ci nnama 1 dehyde, acetophenone, prop i ophenone, but rophenone, 3-methyl -4-phenyl -2-butanone, 2-methyl -5-phenyl -3-pentanone, 20β2Α cycl oheptaneca rboxa 1 dehyde, 2, 2-d imethyl cycl opropaneaceta 1 -dehyde, 2, 2-d imethyl cycl opropy 1 methyl ketone, cyclopentyl methyl ketone, cyclobutyl methyl ketone, cyclobutanone, cycl ohexanone, and 4-methy 1 cycl ohexanone, the corresponding methyl N- (c i s and trans-4-al kyl -, 4-cycloal kyl -, and 4-aral kyl -l-R8R9CH-L-prol yl ) -a- thiol i neosam i nides where R8R9CH- is propyl, isopropyl, butyl, 4-methyl -2-pentyl , benzyl, phenethyl, 3-phenyl propyl , 1-phenyl ethyl , 1-phenyl-propyl, 1-phenyl butyl , 3-methyl -4-phenyl -2-butyl , 2-methyl-5-phenyl -5-pentyl , 3-cycl opentyl propyl , 2-cycl ohexy 1 et hy 1 , eye 1 oheptyl methyl , 2- (2, 2-d imethyl cycl op ropy 1 ) ethyl, 1- (2,2-d imethyl cyclopropyl )ethyl , 1-cycl opentyl ethyl , 1-cycl obutyl -ethyl, cyclobutyl, cyclohexyl, and 4-methy 1 cycl ohexyl are obtained. For example, when ethyl tri phenyl phosphoni urn bromide and acetaldehyde are used, the ant i bi ot i ca 11 y active methyl 6-(t rans-4-ethyl -L-prol ylami no) -6,8-d i deoxy-1- th i o-D -ery thro-a-D-ga 1 acto-octopy ranos i de, methyl 6 - (trans -4- ethyl -1-ethyl-L-prol yl ami no) -6,8-d i deox.y-1- thi o-D -ery thro-a-D-gal acto-octopy-ranoside and the, c i s forms thereof are obtained. .

By substituting the methyl a- th i ol i neosam i n i de by other a- thi ol i ncosami n i des or by β-thiol i ncosami nides or generally by other 6,8-dideoxy-6-ami no-D-erythro- and L- threo-D-ga 1 acto-octopyranose compounds of Formula I I I, examples of which are given hereinafter, the corresponding amides of formulas IV and V are obtained.

Example 2 - Preparation of a 11 ol i ncomyc ί n A. 4-Propyl idene-l-carbobenzoxy-L-prol i ne Sodium hydride (3.8 g. ) was warmed with 75 ml. of di- o methyl sulfoxide at a temperature of 70-75 C. until the o 20β2Α propyl tri phenyl phosphoni urn. bromi de was added, and the resulting red solution was stirred for 30 minutes to insure complete reaction. A solution of 5.2 g. of 4-keto-l-carbobenzoxy-L-prol ine in 15 ml. of di methyl sul foxide was added over a period of 15 minutes, and the resulting mixture was stirred o o for 20 minutes at 26 C. and then at 70 C. for 4 hours. The reaction mixture was cooled, 100 ml. of 5 aqueous potassium bicarbonate and 100 ml. of water added, and filtered. The filtrate was washed twice with 150 ml. portions of ether and the ether was discarded after back extracting with bicarbonate. The bicarbonate solutions were combined, di luted with 200 ml. of water, and acidified with 4N hydrochloric acid. The acidified mixture was' extracted with three 200-ml. portions of ether. The combined ether extracts were washed with three 50-ml. portions of saturated aqueous sodium bisulfite, then with water and. dried over anhydrous sodium sulfate. Evaporation of the solvent gave 5.7 g. of a sol id residue which was 4-propyl idene-l-carbobenzoxy-L-prol i ne.

This residue was dissolved in 18 ml. of acetonitrile and treated with 2.8 ml. of d i cycl ohexy 1 ami ne. The crystal l ine di cyclohexylami ne salt, 5.2 g. (55$ yield), melted at 154- o 157 C. After three recrysta 11 i zat i ons from acetonitri le, o an analytical sample was obtained which melted at Ιβ4-ΐββ C. o and had a rotation of [a] - 8 (c = Ο.3898, CHC13).

D Calcd. for C28H 2N2O4: C, 71.45; H, 9.00; N, 5.95.

Found: C, 71.77; H, 9.59; N, 5.1.

Eight grams (17 mmoles) of the d i eye 1 ohexy 1 am i ne salt of 4-propyl i dene-l-carbobenzoxy-L-prol i ne was shaken with d d and e her un il. 20β2Α solution was complete. The layers were separated and each one was backwashed. The aqueous alkal ine layer was combined with the backwash from the ether layer and acidified with 4N hydrochloric acid. The mixture was extracted with ether and the ether extracts were combined and evaporated to give 4.8 g. (97.8 ) of 4-propyl i dene-l-carbobenzoxy-L-prol i ne as an oil.

B. Methyl N- (4-propyl i dene-l-carbobenzoxy-L-prol yl )- a- th i ol i ncosam i n i de To a solution of 2.25 g. of 4-propyl i dene-l-carbobenzoxy- L-prol ine from Part A and 1.40 ml . of t r i ethyl am i ne in 80 ml . o of distil led acetonitri'le cooled to 0 there was added 1.08 ml . of isobutyl chl oroformate in 1 ml. of aceton i t r i 1 e. The o o mixture was sti rred at 0 (+ 5 ) for 15 min. A solution of 2.92 g. of methyl a- th ί ol i ncosam i n i de (MTL ) in 100 ml . of water was added rapidly. The resulting solution was sti rred o at 0 for 1 hr., the cool ing bath removed and sti rring continued for another hour. The acetonitrile was removed by distil lation under vacuum, leaving a partial ly crystal l ine o residue. The mixture was cooled to 10 and filtered and the o product was dried at 55 under vacuum to yield 2.3 g. of crystall ine methyl N- (4-propyl i dene-l-carbobenzoxy-L-prol yl )- o a-thi ol i ncosami n i de, m.p. I78-I86 C. Two recrysta 11 i zat i ons from moist ethyl acetate afforded ,an analytical sample, m.p. 180-187° C, [a]D + 137° (MeOH, c = Ο.92).

Calcd. for C25H3eN208S: 2 6 2 2 S 6 1 20β2Α C. . Methyl 4- (4 -propyl -L-prolyl )- a- thiol i ncosam i n ide A solution of 100 mg. of methyl N- (4-propyl idene-1-ca rbobenzoxy-L -prol y 1 ) -a- th i ol i ncosami n i de from Part C in 50 ml. of methanol was shaken over 100 mg. of 7K> platinum on Dowex-1 under 40 lbs. of hydrogen pressure for 3 hours.

There was then added 100 mg. of 10 pal ladium on charcoal and the reaction mixture was shaken under 40 lbs. pressure for another J> hours. The catalyst was removed by filtration and the solvent was removed under vacuum. The residue was dissolved in 0.1 ml. of 0.5 N hydrochloric acid. Dilution with 15.0 ml. of acetone precipitated methyl N- (4-propyl -L-p rol yl ) -a- th i ol i ncosam i n i de hydrochloride which was col lected o by filtration. The cry sta 1 s, . d r ί ed at 55 C. under vacuum, o weighed 20 mg. and melted at l8l-l88 C. The product con-tained about 4 parts of the ci s i somer for each part of the trans i somer .

D. Methyl N- (4-propyl -1-methyl -L-prol yl )- -thi ol i nco- saminide hydrochloride A solution of 100 mg. of methyl N- (4-propyl -L-prol yl )-a- d 20β2Α formal in in 50 ml . of methanol was shaken over 100 mg. of 10$ pal ladium on carbon under 0 lbs. of hydrogen pressure for 5 hours. The catalyst was removed by fi ltration and the solvent was removed in vacuo. The residue showed 2 spots on TLC (thin layer chromatography ) , one being l incomycin hydrochloride, the other being a 11 ol i ncomyc i n hydrochloride.

E. Separation of the ci s and trans forms by ; chromatography One gram of methyl N- ( -propyl -1-methyl -L-prol yl )-a-th I ol i ncosam i n i de hydrochloride from Part D is dissolved in 15-20 ml . of methylene chloride containing 0.5 ml . of tri-ethylamine and 2 g. of si l ica gel for chromatography added. The solvent is evaporated under vacuum leaving the antibiotic deposited on the free-flowing si l ica gel. One hundred grams of si l ica gel is slurried with 804, aqueous acetone and poured in a chromatographic column. The solvent is drained down to the level of the gel . The sample of compound deposited on sil ica gel is. dusted on top of the column fol lowed by a layer of sand. The column is eluted with &04, aqueous acetone and fractions of 20 ml . are col lected. A sample of each is evaporated to dryness and assayed by TLC on sil ica gel using 8C aqueous acetone as. the eluting solvent. The antibiotic is detected by spraying with alkal ine permanganate solution.

The fractions containing the desired products are combined, evaporated to dryness, and the antibiotic crystal l ized as the hydrochloride by dissolving in excess dilute hydrochloric aci d and diluting with acetone. The t rans i somer is el uted fi rst fol lowed by a mixture of ci s and trans isomers and finally pure c i s isomer. The mixture may be rechromatographed d 0β2Α fol lowing elemental analysis: Calculated for CieH34N20eS . HC1 : C, 48.80; H, 7.96; N, 6.52.

Found (Corrected for 9-47 H20)s C, 49.15; H, 7.8o; N, 6.39.

By substituting the p ropy 11 r i pheny 1 phosphon i um bromide of Part A in the above example by other substituted triphenyl-phosphonium bromides where the substituent is butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl and the isomeric forms thereof as well as methyl, ethyl, and isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-cycl opropyl ethyl , 3-cycl ohexyl -propyl, benzyl, phenethyl, 3-phenyl propyl , and a-naphthyl-methyl , the corresponding methyl N- (c i s and trans-4-alkyl idene, cycl oa 1 kyl i dene- , and aral kyl i dene-l-carbobenzoxy-L-prol yl )-a- th i ol i ncosam i n i des, the corresponding methyl N- (ci s and t rans-4-a 1 kyl , cycloalkyl-, and aral kyl -L-prol yl )-a- thi ol i nco-saminides, and the corresponding N- (c i s and trans-4-a 1 kyl - , cycloalkyl-, and aral kyl -1-methyl -L-prol yl )-a-thiol i ncosami n-ides are obtained. By substituting the formal in of Part D by other ketaldones of the formula R8 9C0, for example, aceta 1 dehyde, p rop i ona 1 dehyde, acetone, buty ra 1 dehyde, i so-butyl methyl ketone (4-methy 1 -2-pentanone ) , benza 1 dehyde, phenyl aceta 1 dehyde, hyd roc i nnama 1 dehyde, acetophenone, pro-piophenone, buty rophenone, 3-methy 1 -4-pheny 1 -2-butanone, 2-methyl-5-phenyl-3-pentanone, 3- cycl open tanep rop i onal dehyde, cycl ohexaneaceta 1 dehyde, cycl oheptaneca rboxa 1 dehyde, 2,2-d i methyl cycl opropaneaceta 1 dehyde, 2, 2-di methyl cycl opropyl methyl ketone, cyclopentyl methyl ketone, cyclobutyl methyl ketone c cl obutanone c cl ohexanone and 4-meth l c cl o- 2062 A hexanone, the corresponding methyl N- (c i s and trans-4-alkyl-, 4-cycl oai kyl - , and 4-aral kyl -l-R8R9CH-L-prol yl )- -thiol i ncosam i n i des- whe re RaRgCH- is ethyl, propyl, isopropyl, butyl, 4-methyl -2-penty 1 , benzyl, phenethyl, 2-phenyl propyl , . 1-phenyl ethyl , 1 -phenyl propyl , 1-phenyl butyl , 3-methyl-4-phenyl -2-buty 1 , 2-methy 1 -5-phenyl -3-pentyl , 2-cycl openty 1 -propyl, 2-cycl ohexyl ethyl , cycl ohepty Imethyl , 2- (2,2-dimethyl -cycl opropyl )ethyl , 1- (2, 2-d i met hy 1 cycl op ropy 1 )ethy 1 , 1-cyclo-pentylethyl, 1-cycl obutyl ethyl , cyclobutyl, cyclohexyl, and 4-methyl cycl ohexyl are obtained.

By substituting the methyl a- 1 h i ol i ncosam i n i de by other -thiol i ncosami ni des or by β- th i ol i ncosami n i des or general ly by other 6,8- d i deoxy-6-ami no-D-erythro-D-galacto-octopyranose compounds of Formula I I I, examples of which are given herein-after, the corresponding amides of Formulas IV and V are obtained.

Example 3 - Ethyl a- th i ol i ncosam i n i de Lincomycin C hydrochloride (2 g„ ) was dissolved in 50 ml. of water. The pH of the solution was adjusted to 9 by the addition of an anion exchange resin in the hydroxide form. (An anion exchange resin obtained by chl oromethy 1 at i ng by the procedure given on pages 88 and 97 of Kunin, Ion Exchange Resins, 2nd Ed., [1958], John Wiley and Sons, Inc., polystyrene cross- 1 i nked, if desi red, with di vi nyl benzene, prepared by the procedure given on page 84 of Kunin, supra, and quaternizing with tr imethyl ami ne or d imethyl ethanol ami ne by the procedure given on page 97 of Kunin, supra. ) The alkal ine solution was then freeze-dried to a residue which was dissolved in 50 ml . of hydrazine hydrate (98-ΙΟΟΌ and re- . yield 900 mg. A solution of 600 mg. of. the dried insoluble material in ml . of dimethylformamide (heat was used to promote solution) was then clarified by. filtration and . the filtrate was held at room temperature for 4 hours. The crystalline ethyl a-thiolincosaminide which precipitated was isolated by filtration, washed with ether and dried; .'yield 500 mg.

. Ethyl a-thiolincosaminide has the following physical and chemical characteristics; felting .Point: 191-195° C; Optical Rotation: [a] s + 258° (c, Ο.76 in water); and Titration: pKa' e> 7.17, and the following elemental analysis Calculated for CioHaiNOsS: ■ C,. .93i H, 7.92; N, 5,2¾;-S, 115¾9; , 29.92.

Found: C, 44.09; H, 7-91; N, 5.24; S, 11.32. was prepared as follows: ■ FERMENTATION : A soil slant of Streptomyoes llncolnensis var. lincol-nenslsy NRRL 2936, was used to inoculate a series of 500-ml. Erlenmeyer flasks each containing 100 ml. of seed medium con-sisting of the following ingredients: Yeastoiac* 10 g.

Glucose monohydrate 10 g.

N Z-amine B** 5 g. i Tap water q.s. 1 liter *Yeastolac is a protein hydrolysate of yeast cells.

**N-Z-amine B is Sheffield's enzymatic digest casein. The seed medium presterilizatlon pH was 7.3, The seed was grown for 2 days at 28° C, on a (Jump rotary shaker operating at 250 rpm.

A 5 inoculum of the seed described above (5 ml.) was added to each of 30500-ml . Erlenmeyer flasks each containing Glucose monohydrate 15 S - Starch 40 g .

Molasses 20 g- Wilson ' s Peptone Liquor No. 159* 10 g .

Corn Steep Liquor 20 g- Calcium carbonate 8 g .

Lard oil 0.5 ml t Tap water, q.s. 1 liter *Wilson' θ Peptone Liquor No. 159 is a preparation of enzymatically hydrolyzed proteins from animal origin.

At the time of inoculation, DL-ethlonine was added to a final concentration of 2 mg./ml.

The shake flasks were harvested after 4. days of fermentation at 28° C. on a Oump rotary shaker at 250 rpra. They assayed 200 meg./ml. on the S. lutea assay, hereinafter described. The whole beer solids was about 20 gm./liter.

PURIFICATION Whole beer ( 23>5 liters) from a DL-ethionine fermentation was filtered at harvest pH using a filter aid as required. The mycelial cake was washed with water and the cake was then discarded. The filtered beer and water wash ( 275 liters) was stirred for 45 minutes with 12.5 kg. of activated carbon and 2 .5 kg. of diatomaceous earth . The mixture was filtered and the filtrate was discarded, The carbon cake was washed.with 60 liters of water and the w¾ter wash. was discarded. .The cake was washed with 70 liters of 20# aqueous acetone and the 20$ aqueous acetone wash was discarded. The cake was then eluted twice with IQO liter portions of 90$ aqueous acetone. The. eluates "-we're combined ( 215 liters) and the solution was concentrated (l8 liters). This concentrate was adjusted to pH 10.0 with a 50$ aqueous sodium hydroxide solution and extract-ed three times with 20 liter portions of methylene chloride. The methylene chloride extracts were combined ( 60 liters) and then concentrated to give an oily preparation (7.14 g.) containing llncomycln and 1incornycin C in equal -amounts and both in the free base form. This preparation was then dissolved in 200 ml. of methylene chloride. The solution was clarified by filtration and then concentrated to dryness in vacuo. The residue was dissolved in 100 ml . of Γ N methanolic hydrogen chloride. The methanolic solution was then mixed with 3.2 liters of ether under stirring. The resulting precipitated colorless, crude llncomycln hydrochloride and llncomycln C hydrochloride was isolated by filtration and dried; yield 7.1 g. assaying .940 meg./mg. against Sarclna lutea. (The assay against Sarclna lutea Is conducted on agar buffered to pH 6-8 with pH 7.0 phosphate buffer [O.I ], A unit volume [ 0.08 ml.] of solution containing the material to be assayed is placed on a 12.7 ml. assay disc which is then placed on an agar plate seeded with the assay microorganism.) Thin layer chromatography showed the presence of both llncomycln hydrochloride and llncomycln C hydrochloride in approximately equal amounts.

Crude llncomycln C hydrochloride ( 7.0 g.) was dissolved in 20 ml, of water and 20 ml. of butanol, pH adjusted to 4.2 with 1 N HCl, and the solution distributed in a counter current distribution apparatus for 1000 transfers. Analysis by thin-layer chromatography showed. that, the fractions in tubes 13 to 190 contained llncomycln C. These fractions were comb i ned , ' and the solution was concentrated to an aqueous and freeze dried to give 2.44 g. of l incomycin C hydrochloride assaying 1400 mcg./mg. against Sa rci na 1 utea . Five hundred mg. of this preparation was dissolved in 2 ml. of water, 1 ml. of methanol, and 100 ml. of acetone. The solution was clarified by filtration. The filtrate was mixed with ether until crystals appeared. The mixture was allowed to stand at room temperature for 1 hr. Crystal line (cubes) l incomycin C hydrochloride was separated from the supernatant material solution by decantation. These crystals were recrysta 11 i zed from one ml. of water, one ml. of methanol, 80 ml. of acetone and 20 ml. of ether; yield, 250 mg. of crystal l ine (cubes) l incomycin C hydrochloride. The supernatant (obtained as described above) was al lowed to stand at o C for 4 hours. Crystal l ine (needles) l incomycin C hydrochloride which precipitated was filtered and dried; yield, 150 mg. of crystal l ine (needles) l incomycin C hydrochloride, m.p. 151-157° C Following the procedure of Example 1 substituting the methyl a- thiol i ncosami n i de by ethyl a- th i ol i ncosam i n i de, compounds of formulas IV and V wherein R, and Z are as given about and Y is -SCH2CH3 in the a-conf i gurat i on are obtained. For example, when a 1 kyl t r i phenyl phosphon i urn bromide and formal in or acetaldehyde are used ant i b i ot i ca 11 y active ethyl 6-(trans-substituted-|_-proly1amino)-6,8-di deoxy-1-thio-D-erythro-g-D-ga 1 acto-octopy ranos i des and the c i s forms thereof are obtained where the substituents are 4-alkyl, 4-a 1 ky 1 -1-methy 1 , and 4-a 1 ky 1 -1-ethy 1 , where alkyl can be ethyl, propyl, butyl, pentyl, and hexyl, or the isomeric forms thereof.

Immediately. The reaction was continued with occasional- h at- o · ing to 40 C. for a period of 3 days. Thin-layer chromatography then showed the absence of starting material. The precipitate of mercuric chlorid mereaptide (CIHgSMe ) was removed by filtration. The colorless filtrate, together with aqueous washes of the precipitate, was stirred magnetically at room temperature, and the excess of mercuric chloride was removed by the addition of pyridine in small portions until the precipitation of the insoluble mercuric chloride-pyridine complex was complete. After storing the mixture in the o . . ' · ■■ ■ refrigerator at 0 C. for 3 hours, the solid was removed by filtration, the precipitate was washed well with cold water, and the combined filtrate and washings were stirred with a small amount of silver carbonate until the solution was neutral: to pH paper. The solution was thereupon filtered through a "Millipore" filter (Millipore Filter Corporation, Bedford, Massachusetts)* the filter was washed thoroughly with water, and the washings were added to the colorless filtrate. Excess silver ion was precipitated by saturating the solution with hydrogen sulfide and removing the silver sulfide by filtration. The silver sulfide was washed with water, the washings were added to the filtrate, and the solution was . lyophilized to give a colorless amorphous solid, which was admixture of 6-aceta-mldo-6,8-dldeoxy-D-erythro-q- and β-D-galacto-octopyranoses (N-acetyl-a-llncosamine and N-acetyl-&-lincosamine) .

C. N-Acetyl-l,2,3,4,7-penta-0-acetyl-3-lincosamine The crude mixture of N-acetyl-a-lincosamine and N-acetyl-β-lincosamine of part B was slurried in 400 ml. of pyridine and 200 ml. of acetic anhydride and stirred magnetically overnight at room temperature. The resulting colorless solu- o 1 mm, Hg pressure to a pale yellow syrup which was dissolved in a mixture of water and chloroform. The aqueous layer was extracted with chloroform and the chloroform extracts were combined, washed with dilute sulfuric aci (2 N), then twice with wate , with saturated aqueous sodium bicarbonate, with water until neutral, and finally dried over anhydrous sodium sulfate. The resulting chloroform^extract was then evaporated on a rotating evaporator at 35 C. and 15 mm. Hg pressure to give a colorless solid which was dissolved in hot ethyl acetate o this ethyl acetate solution was added Skellysolve B hexanes until a solid began to crystallize. The solid was removed by filtration and twice recrystallized from ethyl acetate-Skellysolve B hexanes to give N-acetyl-1, 2,3,4,7-penta-O-acetyl-0-lincosamine (9.27 g.) of melting point 227- Q . S3 0 230 G. and rotation [a]p + 33 (c, 0,832, chloroform).

Anal. Calcd. for 02οΗ2θΝ0χ2: C, 50.52; H, 6.15; N, 2.95 Pound: C, 50.40; H, 6.42; N, 3,04.

The. ethyl acetate-Skellysolve B hexanes filtrate from the first: crystallization was allowed to stand at room temperature, whereupon colorless prismatic needles of N-acetyl-l,2,3,4,7-penta-0-acetyl-a-lincosamine separated; - o m.p. 169-172 C. The melt resolidified on cooling in the o form of hexagonal platelets which then melted at 237-238 C; after recrystallizatlon from ethyl acetate-Skellysolve B o hexagonal platelets were obtained melting at 240-240.5 C. 23 0 and having a rotation of [a]D + 132 (c, 0.9842, chloroform). Anal, Calcd. for C, 50.52; H, 6.15; N, 2.95 Pound: C, 50.62; H, 6,08; N, 3.02. chloroform layer was separated, the aqueous solution was extracted thoroughly with chloroform,, and the combined ehloro- form extracts were washed with water until the aqueous wash was neutral to Congo red paper, and dried over anhydrous sodium sulfate. The chloroform was thereupon evaporated in a rotatory evaporator at _55°C« and 15 mm. Hg pressure to give an almost colorlees amorphous solid, After three crystallizations of the so id fro . c or - s e s ri at c needles of N-acetyl-2, 3,4,7-tetra-O-acetyl-la-bromo-l- deoxyllncosamine were obtained which had a melting point of 188-I890 C. and a rotation of [a] s + 231° (c, 0.8133, chloroform) .

Anal. Calcd. for Ci8HaeBrNOi0: .

C, 43.56; H, 5.28; N, 2.82; Br, l6.10.

Found: C, 43.68; H, 5·39; N, 2.88; Br, 17.22. E. Methyl N-acetyl-2,3,4,7-tetra-0-acetyl-e-thio- lincosaminide Two grams of N-acetyl-2,3,4,7-tetra-O-acetyl-la-bromo- 1-deoxylincpsamlne of part D was dissolved in 25 ml. of acetone which had been dried previousl over potassium carbonate, and to this solution was added 350 mg. of thiourea. After warming briefly on a steam bath until the solids had dissolved, the colorless reaction mixture was left overnight at room temperature... To it was then added a solution of 680 mg. of potassium carbonate and 86Ο mg. of sodium bisulfite in 10 ml. of water, followed by 900 mg. (0.40 ml.) of methyl iodide. The mixture was kept in a stoppered bottle which was shaken mechanically at room temperature for 3 hours. The reaction mixture was then extracted thoroughly with chloroform, the combined extracts '.were washed twice with water, dried over anhydrous sodium sulfate, and the solvents were removed on a rotating evaporator at 40° C. and 15 mm. Hg pressure.

In this manner 1.3.8 g. of colorless, amorphous solid was obtained, which upon thin-layer chromatography was shown to be a single compound. This solid was dissolved in hot ethyl acetate which was thereupon diluted with Skellysolve B hexanes to give 880 mg. of colorless platelets of melting point 268-272° C. Recrystallization from the same solvents ave methyl N-acet l- 4.

Example .5 6-Amino-l, 6,8rtrideoxy-D-erythro-D-galacto-octo pyr'anose .■ (l-deoxylincosamine ) XXXIX A. Methyl N-acetyl-3,4-0-isopropylidene-e-thlolincosam- inide A suspension of 5.3 g. of finely-powdered methyl N-acetyl- 0-thiolincosaminide was stirred for 30 minutes at room temperature with 500 ml. of acetone and 5 ml. of concentrated ■ sulfuric acid. After 30 minutes another 5 ml. of concentrated sulfuric acid was added, and the solution was stirred for another half hour at room temperature. A suspension of 150 g. of barium carbonate in 100 ml. of water was added, and the > mixture was stirred until neutral. The barium sulfate and excess carbonate were removed by filtration, and the precipi- tate was washed with acetone. : The filtrate and washings were combined and evaporated to dryness in vacuo at 40° C.

The residue was treated with acetone tether (10:1) and insoluble materials were removed by filtration. The filtrate was evaporated to dryness and the residue was dissolved in 100 ml. of ethanol. Removal of the alcohol by heating in vacuo left a gummy material which was dissolved in 20 ml. of warm water containing a small amount of sodium carbonate. - After removal of some insoluble material by filtration, the filtrate was cooled. The crystals which formed were collected after stand- ing in the refrigerator for 4 hours, washed with cold water, and dried in vacuo to give methyl N-acetyl-5, 4-0-isopropyl-idene-0-thiolincosaminide .

B. N-Acetyl-3>4-0-isopropylidene*i-deoxy- lincosamlne Five grams of methyl N-acetyi-3, 4iO-isopropylidene-e-thioiincosaminide was heated under reflux for 7 hours with ?5 ml. of loosely-packed Raney nickel in 150 ml. of ethanol. The mixture was thereupon filtered and the catalyst was washed with a total of 400 ml. jof boiling ethanol. The filtrate and washings were combined and evaporated to dryness, leaving a partially crystalline residue. This residue was purified by counter current distribution in a system of butjanpl:water to give N-acety1-3, 4-0-isopropy1idene-1-deoxy-linoosamine. ■ G. 1-Deoxylinoosamine One gram of N-acetyl-3, -0-isopropylidene-l-deoxylincosamine in 5 ml. of hydrazine hydrate (98-100^) is heated under reflux for 21 hours. The excess hydrazine hydrate is distilled In vacuo . The residue is stirred with acetonitrile and again evaporated. Five ml. of water is added and the pH adjusted to 2 with hydrochloric aoid. After 3-5 hre. at 26° the solution is diluted with 10 ml. of water and excess silver carbonate added. The mixture is then filtered and the filtrate lyophillzed. The resulting residue is crystallized from water to yield 1-deoxylincosamine .

Following formu^e XL wherein X = Rj, or Z as given ■ above and R Is as given above are obtained.

Example 6 2-Hydroxyethyl 6-amino-7-0-methyl-6>8-dideoxy-l thlo-D-erythro- -D>galacto-oetbpyranQslde (2- hydroxye A. 2-Hydroxyethyl a-thiocelestosaminide hydrazine solvate A mixture of 5 g. (0.0094 mole) of celesticetin (Example 3, U S. Patent 2,928,844) and 25 ml. (excess) of hydrazine hydrate was heated under reflux for 21 hours. The excess hydrazine was removed by distillation in vacuo and the residue was crystallized from 55 ml, of absolute ethanol. White crystals (1.2 gv)' of 2-hydroxyethyl a-thiocelestosaminide hydrazine solvate were obtained; which melted at 98-108° C. Recrystallization from., absolute ethanol gave 0.65 g. having an optical rotation of [a]gs = + 245° (c = 0.8, water); an infrared absorption spectrum in Nujol mull at the following frequencies: 3400, 163O, l600, 1460, 1 50 (sh), 1305, 1275, 1260, 1200, 1115, 1085, 1055, 1010>, 978» 95Q. 2 , 10, 873, Soo"¾20, 705, 690, and 680 -1··· cm ; an equivalent weight of 161 with two basic groups having ■ pKas in the region of 7. and : the following elemental analysis : Calculated for Ci HaaNOeS'NeH*: C, 40.11;. H, . 8.26; N, 12.76; S, 9.74. ' ' ■■'.-.·■ '..' : ' ■.· .· ' . '. ·'· . .. ■ t ■ Pound: C, 40.15; H, 8.04; N, il.69; S, 9- .

B. 2-Hydroxyethyl othiocelestosaminide hydrazine '-.■Solvate 0 Desalicetin (Example 1, U. S. Patent 2,851,465) (10 g.) was dissolved in hydrazine hydrate (100 cc.) and heated under reflux in an oil bath at 1650 C. for 18 hours. The almost colorless solution was concentrated to dryness on a steam bath, first at 15 mm. .and finally at less than 1 mm. , giving 5 a solid residue which was triturated with. acetonitrile, filtered, and washed with the same; solvent. Crystallization from ethanol gave colorless needles: (2,64 g,) of 2-hydroxy- ethyl a-thiocelestosaminide hydrazine solvate. Recrystallization from the same solvent gave crystals of 2-hydroxyethyl a-thio- celestosaminide hydrazine solvate which had an equivalent weight of 168 with two basic groups having pKas in the region of 7· 5; an optical rotation of [a]p5 = + 248° (c = 1, 95$ ethanol); and the following elemental analysis: Calculated for CuI^NOeS'NaH*: C, 40.11; H, 8.26; N, 12.76; S. 9-7 Found: C, 40.27 H, 7«95; N, 11.63; ^S. 9.80.

C. 2fHydroxyethyl a-thlocelestosaminide A solution of 2 g. of 2-hydroxyethyl -thiocelestosaminide hydrazine solvate, prepared as in part A or part B, in 30 ml. of dimethylformamide was concentrated to a volume of about A.2-Hydroxyethyl N-acetyl-3>4-0-isopropylidene-a-thio- celestosaminide To a solution of 1 g. (0.047 mole) ; of 2-hydroxyethyl -thiooelestosaminide in 150 ml. of ethanol was added 14 ml. of acetic anhydride. The reaction mixture was stirred fbr 1/2 hours, . refrigerated overnight* and evaporated to dryness In vacuo. The residue, 2-hydroxyethyl N-acetyl-a-thloceiestos-amlnlde, was slurried with ether and dried under reduced pressure. · The thus-obtained dried material was dissolved in 1500 ml . of acetone and 15 mi. of concentrated sulfuric acid was added to the solution with stirring. . he mixture was stirred for about 2 hours and then neutralized by adding dry ammonia. The; mixture was filtered and the filtrate was evaporated to dryness, leaving 2-hydroxyethyl N-acetyl-3,4-.0-isopropylidene-a-thlocelestosamlhide as an oil.

B-l. N-Acetyl-3,4-0-isopropylidene-7-0-methyl-l-deoxy- llncosamine The oil of part A was dissolved in 500 ml. of ethanol, 150 ml. of loosely-packed Raney nickel in ethanol was added thereto, and the mixture was heated under reflux for 10 hours.

Thereafter the mixture was filtered, the catalyst was washed with 1 liter of boiling ethanol and the combined filtrate and washings were evaporated to dryness. An oil resulted which was distributed (500 transfers) in the system 1-butanol :water. A peak fraction, K = 0,82,, obtained by evaporation of pooled tubes 200-250, gave 4,6 g. (33$) of N-acetyl-3, -0-isopropyl-idene-7-0-methyl-l'«deoxylincosamine which crystallized upon drying; melting point, 198-205° C; rotation (a]*s + 71° (c = 1, 50ji ethanol) .

Anal. Calcd. for Ci4H25N0e: C, 55.43; H, 8.51; N, 4.62; Methoxyl, 10.63.

Found: C, 55.03; H, 8.28; N, 4.γθ; Methoxyl, 10.43.

B-2 Potassium metal (l.l6g.) was dissolved in 100 ml. of tert-butyl alcohol. The solvent was removed as completely as possible by disti llation (unless specified all distillations are at atmospheric pressure) and finally by vacuum distillation at 1 mm. Hg pressure. To the dry sol id residue was added 100 ml. of dry benzene and the benzene was removed by disti llation to leave a fine powder. To the thus -obta i ned powdery potassium tert-butoxide was added 200 ml. of dry benzene, and the mixture was stirred magnetically at room temperature unti l an opalescent solution resulted. To this solution was added 5 g. of N -acety 1 -3 ,4 -0- i sopropy 1 i dene -1 -deoxy 1 i ncosam i ne, prepa red accord i ng to part B of Example 5* and the mixture was then stirred overnight at room temperature. To this mixture was added 42.4 g. (18.6 ml.) of methyl iodide and the mixture was stirred at room temperature for 2 hours. The mixture was fi ltered to remove potassium iodide, and the f iltrate was distilled in vacuum at about 35°C . to give a colorless syrupy material which was subjected to counter current distribution in a system of ethyl acetate: ethanol: water in a ratio of 4:1:2. The fractions containing N-acetyl-3,4 -0 - i sopropy 1 i dene -7 -0 -methyl -1 -deoxy 1 i ncosam i ne, as determined by thin-layer chromatography, were combined, evaporated to dryness and the residue was crystallized to give pure N -acety 1 -3,4 -0- i sopropy 1 i dene-7 -0-methy 1 -1 -deoxy -1 incosami ne. 0. 7-0-Methyl-l-deoxylincosamine Following the procedure of Example 5, part C, N- acetyl-5, «0-isopropylidene-7-0-methyl-l-deoxylincosamine is al yl- 20β2Α < 'V can be recrysta 11 i zed from acetone or ethyl acetate by the addition of Skellysolve B (technical hexane).

J3, 2-Hydroxyethyl N-acety 1 -2-0- benzoy 1 -3, 4, 7- 1 r i -0- th i onobenzoy 1 - a- th i ol i ncosam i n i de The tetrathionobenzoate (X) of part A is dissolved in ethanol-free chloroform (to give a 2 to 10$ solution) and treated with a solution of bromine (2 molecular equivalents) in chloroform (a 1-2$ solution, vol, /vol. ) with magnetic stirring at room temperature with the exclusion of atmos-pheric moisture. .After 2 hours the volatile material ÷is o removed as completely as possible by evaporation at 30 C, and 15 mm. Hg pressure, more chloroform (ca_„ 100 ml. per 2 g. of starting material ) is then added and removed as before. To the residue is added acetone (ca. 25 ml . per 2 g. of starting material ) and t r i ethyl am i ne (2 molar equivalents), and the ; resulting solution heated under reflux on a steam bath for 1 hr. with the exclusion of atmospheric moisture. [The initial bromination product (XI ), is thus converted to the cycl ised intermediate (XI I ). ] To the cooled solution is then added potassium carbonate (5 molecular equivalents), sodium bicarbonate (wt. equal to the potassium carbonate used), and water (ca . 10 ml. per g. of potassium carbonate), fol lowed by ethylene iodohydrin (preferably, ca. 6-10 mol ecu 1 a r equivalents), and the stoppered mixture is shaken mechanically for hrs.. at room temperature. The reaction mixture is extracted with chloroform and the chloroform solution is washed with water to remove inorganic salts, and dried over anhydrous sodium sulfate. o Removal of the chloroform at 3 C. i n vacuo yields 2-hydroxy-ethyl N-acetyl -2-0- benzoyl -3, 4,7- 1 r i -0-th i onobenzoy 1 -a-thio- ylene a-thlolincosaminide; melting point, 1 1 -192. ° C; rotation (ct]gs + 126° (c, 0.8508, ethanol).

Anal . Calcd. for Ci Ha3N0sS: C, 52.95; H, 7.30; N, 4,41; £3, 10.10; ',< 0, ,25.20.

Found: C, 52,77; H, 7,3 ; N, 4.40; S, 10,12; 0, 25-11.

The aqueous mother liquors were concentrated in vacuo to give a solid which was recrystallized from acetone it consisted of methyl N-acetyl-3,4-0-isopropyHdene-a-thloHncosaminlde of melting point 178-180° C. and rotation [a]gB + 1890 (c, 0.5137» water) , Β. Conversi on of the oxazoline derived from methyl N- acetyl-3,4-0-isopropylldene a-thiolincosamlnide to methyr N-acetyl-}, 4-0-isopropylidene-a-thiolincos- • aminide A solution was prepared of the oxazoline derived from methyl N-acetyl-J, 4-0-isopropylidene-a-th.iolincosaminide In 20 •■ml. - of hot water and heated under reflux for 2 hours at which time thin-layer chromatography on silica gel revealed the conversion of the starting. material to methyl N-acetyl-3, 4-0- isopropylidene-a-thiolincosaminide . The water was then removed in vacuo at 40° C . , . leaving a colorless crystalline solid which was recrystallized from a small volume of hot water to give colorless needles of methyl N-acetyl-5, 4-0-isopropylidene-a-thio- lincosaminlde of melting point 178-I8O0 C. and rotation [a]^5 + 190° (c, I.223, water).

C_. Methylation of methyl N-acetyl-3, 4-0-isopropylidene- -thiolincosaminide Potassium metal (l.l6 g.) was dissolved in 100 ml. of t-butyl alcohol (previously dried over sodium metal) under stirring and reflux. The solvent was removed as completely as possible by distillation and finally by distillation in a vacuum of 1$ mm. Hg. To the dry, solid residue was added 100 ml. of dry.', benzene, which was removed by distillation to leave a. fine powder. This was treated again with benzene Which was distilled off to Insure complete removal of tert-butyl alcohol.

To the thus-obtained, powdery potassium tert-butoxide was added 200 ml. of dry benzene and the mixture was stirred magnetically at room temperature until an opalescent Buspenelon resulted, .To this was added 5 g. of methyl N-acetyl-3,4-0-ieopropylidene- -thiolincosaminide and the mixture was then stirred overnight at room temperature at the end of which time all of the solid had dissolved.

To this mixture was- added 42.4 g. (18.6 ml.) of methyl iodide and the mixture was stirred at room temperature for .1-1/2 hours; after 1 hour, the mixture gave a neutral reaction with moist pH paper. The reaction mixture was filtered to remove potassium iodide and the potassium iodide was washed with benzene; the benzene was added to. the filtrate. The ' filtrate and washings were distilled in vacuo at 35° C. to give a colorless syrup which was subjected to counter current distribution in the system ethyl; acetate :ethanol : water in the ratio 4:1:2. After 500 transfers the three components, as indicated by thin-layer chromatography, had been completely resolved. The major components were methyl N-acetyl-3,4-0- isopropylidene-7-O-methyl- -thiolincosaminide and methyl N- acetyl-3, 4-0-isopropylidene-2-0-methyl-a-thiolincosaminlde . A minor component was methyl N-acetyl-3, 4-0-isopropylidene- 2,7-di-O-methyl-a-thiolincosaminide .

Removal of solvents from' combined tubes No. 2 0-310 (K=l. JO) inclusive gave a glassy material which crystallized from ethyl acetate tSkellysolve B hexanes to yield methyl N- acetyl-3,4-0-isopropylidene-2-0-methyl-a-thiolincosaminide as short, colorless prisms of melting point 176-177° C. and rotation [ ]J5 + 176° (c, 0.6220, chloroform).

Anal. Calcd. for Ci5H27N0eS: C, 51,57J H, 7.79; N, 4.01; S, 9-17; OMe, 8.88.

Pound? C, 51,82; H, 8.10; N, 4.08; S, 8. ^; OMe, 8.49- Removal of solvents from combined tubes No. 330-384 (K«2.52). inclusive by distillation gave a glassy product which orystalilzed slowly on standing.^ Recrystaiiizatlon from ether gave clusters of minute, colorless needles of methyl N-acetyl- 7-0-methyl-3,4-0-isopropylidene-a-thiolincosaminide .

Similarly,, evaporation of the solvents from combined tubes No. 410-450 (Κ=5. β7 ) gave a colorless glassy product.

Recrystallization from ether gave colorless, chunky needles of methyl N-acetyl-2, 7-di-0-methyl-3, 4-0-isopropylidene-a- thiolincosaminide of melting point 124.5-126° C. and rotation • .'[a] Js + 184° (c, 0.8390, chloroform).

Anal.. Caicd. for deHggNOeS: C, 52.88; H, 8.04; N, 3.85; S, 8.82; 0CH3, 17.08.

Pound: C, 53-02; H, 7 - 95; N, 4 .05 ; S, 8.73; 0CH3, 15 - 92. . p_. Methyl N-acetyi-2r0-methyl-a-thiolincosamlnide A mixture of 2 g. of methyl N-acetyl-2-0-methyl-3, 4-0- isopropylidene- -thiolincosaminide in 50 ml. of 0. 25 hydro- chloric acid was. magnetically stirred at room temperature (about 25° C). The solid starting material dissolved within minutes. After 1-3/4 hours, thin-layer chromatography showed the complete absence of starting material.

The strongly acidic solution was stirred with a polystyrene quaternary ammonium 'anion exchange resin until the colorless supernatant solution gave a neutral reaction with pH paper.

Filtration, washing of the .resin with water, and removal of the water from the combined filtrate and washes in vacuo gave. 1.68 g. ( 95 ) of a colorless crystalline residue which was crystallized from methanol-ether to give long, colorless, felted needles of methyl N-acetyl-2-O-methyl- -thiolincosaminide of melting point 237-238° C.

Anal . Caicd. for Ci2H23N0eS: C, 46.56; Ή, 7.49; N, 4.53; S, 10.36 Pound: C, 46.72; H, 7.44; N, 4.37; S, 10.34.

D. Methyl 2-0-methyl-a-thiolincosaminide The thus-obtained methyl N-acetyl-2-0-methyl.-a-thiolin-cosaminide was heated with 6 ml . of hydrazine hydrate under reflux for a period of 22 hours. The excess hydrazine hydrate was removed by distillation in vacuo and the residue was three times recrystallized from ethanol -water to give methyl 2-0-methyl-a-thiolincosaminide.

E. . Methyl 7-O-methyl-a-thiolincosaminide In the manner given in parts C and D, methyl N-acetyl-7-0-methyl-3, -0-isopropylidene-a-thiolincosamlnide was hydrolyzed and then hydrazinolyzed to give methyl 7-0-methyl- -thiolin-cosaminide.

F. Methyl 2,7-dl-O-methyl-a-thiollncosaminide In the manner given in parts C and D, methyl N-acetyl-2, 7-di-0-methyl-2, -0-lsopropylidene- -thiollncosaminide was hydrolyzed and then hydrazinolyzed to give methyl 2,7-di-0-methyl-a-thiolincosaminide .

By substituting the methyl a-thiolincosaminide by methyl ' β-thiolincosaminide * or by the other aikyl a- or β-thiolincos-aminides given above, the corresponding alkyl 2-0-, 7-0-, and 2,7-di-O-alkyl a- and β-thiolincosaminides are obtained.

Following the procedure of Example 1, compounds of the formula: XLIX alkyl 20β2Α wherein X = Ri or Z as given above, R is as given above, and one of R6 and R7 is alkyl of not more than 12 carbon atoms and the other is hydrogen or alkyl of not more than 12 carbon atoms, are obtained.

By substituting the methyl N-ace tyl-a-thiollncosamlnlde by 2-hydroxyethyl a- and g-thlolincosaminides, the corresponding alkoxyethyl N-acetyl-2-0- , 7-0-, and 2, 7-di-O-alkyl-a-and -thiolincosaminides are obtained. By substituting the methyl N-ace tyl- -thiolincosaminide by 2-trityloxyethyl N-acetyl-a- and β-thiolincosaminides, the corresponding 2-trityloxyethyl N-ace tyl-2-0- , 7-0-, and 2, 7-di-0-alkyl-ct-and -thiolincosaminides are obtained. By removing the trityl group by hydrolyzing with 80 aqueous acetic acid and the ' N-acetyl. by hydrazinolysis, the corresponding 2-hydroxyethyl 2-0-, 7-0-, and 2, 7-di-0-alkyl-a- and 3-thio-linocsaminides are obtained.

F compounds of the fo 0R7 wherein X = R3. or Z as given above, R and R4 are as given above, and at least one of Re and R7 is alkyl of not more than 12 carbon atoms and the other is hydrogen or alkyl of not more than 12 carbon atoms, are obtained. 20β2Α Exampl e 11 Methyl 6 -am i no-6, 8-d i deoxy-l-thio-L-threo-a-D- ga 1 a c to -oc top y ranos i de 3,4-0-1 soprop l i denel i n corny c A solution of .8 9· of l incomycin in 150 ml. of acetone is added to a solution of 9.8 9· of £- to.l uenesu 1 fon i c acid monohydrate in 100 ml. of acetone with good stirring and avoidance of exposure to moisture. The mixture is stirred at ambient temperature for 1 hour, after which 100 ml. of anhydrous ether is added and stirring is continued in an ice-bath for 0.5 hour. The mixture is fi ltered and the sol id is dried o in vacuo at 50 C. ; yield 13.35 g. (85.5$) of 3,4-0- i sopropyl -i denel i ncomyci n _- tol uenesu 1 fonate. An additional 1.15 g. (7.4$) can be recovered from the mother l iquors by adding 350 ml., of anhydrous ether to the mother l iquor from the previous fi ltering operation and chi ll ing the solution for 1 hour. The l4.5 g. so obtained are suspended in 200 ml. of ether and shaken vigorously with 125 ml. of 5$ potassium bicarbonate solu tion. The a ueous la er i s back-ext ra-cted with two 100-ml. 20β2Α portions of ether. The ether extracts are washed with 50 ml. of saturated sodium chloride solution and then filtered through anhydrous sodium sulfate. The ether is evaporated under vacuum, leaving 7.9 9» (73.1 ) of 3*4-0- i sopropyl i dene-l incomycin which is dissolved in 25 ml . of ethyl acetate and concentrated to about 10 to 15 ml . The concentrate is al lowed to stand at room temperature for several hours and then refrigerated overnight. The crystals thus obtained are filtered from the solution and washed sparingly with cold ethyl acetate; yield 4„55 9· (42„2#) of 3,4-0- i sopropyl i denel i nco- o mycin having a melting point of 126-128 C„, and. an optical o rotation of [a]Q + 101-102 (c, 1, methylene chloride). _B. 7-Dehydro-3,4-0-i sopropyl i denel i ncomyci n To a solution of 6 g. (0..0135 moles) of 3, -0- i sopropyl -i denel ί ncomyci n i n 75 ml . of pyridine was added 12 g. (excess) of chromium trioxide (chromic acid). The temperature of o solution rose about 20 C. After one hour the mixture was added to a solution containing 250 ml. each of ethyl ether and ethyl acetate, filtered, and evaporated to 8?4 g.„. of syrupf This syrup was distributed in a 500-transfer counter current distribution using the system, water;ethyl acetate: ethanol : cycl ohexane (l:l:l;l). 7-Dehyd ro-3, -0- i sopropyl -i denel i ncomyci n was isolated from tubes 220-380, K = 2.45.

Anal. Calcd. for C31H36 2O6S; C, 56.72; H, 8.l6; N, 6.3Ο; S, 7.21, Found: C, 56.37; H, 7.62; N, 6.51; S, 6.84.

C 3,4-0- I sopropyl i deneepi 1 i ncomyci n 20β2Α To 1.6 g. of Craig pure 7-dehyd ro-3, 4-0- i sop ropy 1 i dene-l i neomycin in 75 ml. of methanol was added 400 mg. of sodium borohydride. After 1.5 hr. this solution was evaporated to dryness on a rotary evaporator. The residue was added to 25 ml. of water which was extracted three times with 25 ml . portions of methylene chloride. The extract was back-washed with 15 ml. of water, dried over magnesium chloride, and evaporated to dryness. The residue, 1„4 g,,. was distributed in a 500-transfer counter current distribution using the solvent system, watenethyl acetate: ethanol : cycl ohexane (1:1:1:1) and a single peak which fit the theoretical was observed at K = 1.05. The material in pooled tubes 240-280 was isolated as a syrup.

Anal . Calcd. for C21H38N2O6S: C, 56.47; H, 8.58; N, 6.27; S, 7.18.

Found: C, 56.24; H, 8.54; N, 6.13; S, 7.01, Thin layer chromatography (TLC) showed that this material consisted of two substances, one of which was 3,4-0- i sopropyl -i denel i ncomyci n. The other, 3, 4-0- i sop ropy 1 i deneep i 1 i ncomyc i n moved sl ightly slower. p_. Epi 1 i ncomyc i n 20β2Α ml. of 0.25 _N hydrochloric acid and 4θ ml . of ethanol and al lowed to stand at room temperature for 5 hours. The solu- o tion was then kept at 0 C. for 4 days, neutral i zed- wi th sodium bicarbonate, concentrated to 25 ml ., and extracted with chloroform. The extract was washed with a little water and dried over magnesium sulfate, then evaporated to a residue. Thin layer chromatography of the residue showed two substances, both of which were active against S_. 1 utea, The residue was chromatographed on a 14" x 3/4" Florisi l (a synthetic sil icate of the type described in U. S. Patent 2,393,625) column which was eluted gradiently with a total volume of 5,000 ml. of solvent which varied continuously from 100$ Skellysolve B (technical hexane) to 100$ acetone, 40 ml. fractions being col lected. Fractions 53-65 yielded ep i 1 i ncomyc i n, assaying 450 mcg/mg„ Anal . Calcd. for CieH3 N20eS: C, 50.92; H, 8.55; N, β,βθ; S, 7.56.

Found: C, 50.19; H, 7.91; N, 6.05; S, 6.42, Fractions 73-104 yielded l incomycin, assaying 950 mcg,/m9> E_. Methyl ep i - a- thi o1 i ncosami n i de Fol lowing the hydraz i nol ys i s procedure of Example 1, part B, ep i 1 i ncomyci n is converted to methyl jsgj -a- thi ol i nco-saminide.

Lincomycin can be substituted by methyl N-acetyl-a-thi ol i ncosami ni de. The process can also be appl ied to other 6-acylamino-6,8-di deoxy-D-erythro-D-galacto-octopyranose compounds of the formula: where Ac, Y and R3 are as given above to form the correspondin epimers : which can be alkylated by the procedure given above to provide compounds -There can -thus be obtained 6-amino-6, 8-dideoxy-L-threo-D- galacto- ; 6-amino-2-0-alkyl-6, 8-dideoxy-L-th.reo-D-galacto-; 6-amino-7-0-alkyl-6, 8-dideoxy-L-threo-D-galacto- and 6- amino-2, -di-0-alkyl-6, 8-dideoxy-L-threo-D-galacto-octopyranose compounds which in turn can be treated by the procedure of Example 1, part C et seq., to give compounds of the formula: wherein X = Z or Rj. as given above and Y, R, R2, and R3 are as given above .

The intermediates of formula III can be used as a buffer or as an antacid. They react with isocyanates to form urethanes and can be used to modify polyurethane resins. The thlocy.anic acid addition salt when condensed with formaldehyde forms resinous materials useful as pickling inhibitors according to U. S. Patents 2 , 425 , 320 and 2, 606, 155. The free' bases also make good vehicles for toxic acids. For example, the fluosillcic acid addition salts are useful as mothproofing agents according to U. S. Patents 1,915, 33 and 2 , 075, 359 and the hexafluoroarsenic acid and hexafluorophosphoric acid addition salts are useful as parasiticides according to U. S. Patents 3, 122, 536 and 3, 122,552.

Various acid-addition salts of the free base form of the intermediates of formula III can be made by neutralizing the free base with the appropriate acid to below about pH 7 · 0, and advantageously to about pH 2 to pH 6. Suitable acids for this purpose include hydrochloric, sulfuric, phosphoric, thiocyanic, fluosillcic, hexafluoroarsenic, hexafluorophosphoric, acetic, succinic, citric, lactic, maleic, fumaric, pamoic cholic, palmitic, mucic, camphoric, glutaric, glycollc^ phthalic, tartaric, lauric, stearic, salicylic, 3-phenyl-salicylic, 5-phenylsalicylic, 3-methylglutaric, orthosulfo-benzolc, cyclohexanesulfamic,. eyelopentanepropionic, 1,2-cyclohexanedicarboxylic, 4-cyclohexenecarboxylic, octadecenyl-succinic, octenylsuccinlc, methanesulfonic, . benzenesulfonic, helianthlc, Reinecke's, dimethyldithiocarbamic, cyclohexyl-sulfamic, hexadecylsulfamic, octadecylsulfamic, sorbic, monochloroacetic, undecylenlc, ' -hydroxyazobenzene-4-sulfonic, octyldecylsulfuric, picric, benzoic, cinnamic, and like acids .

The acid addition salts can be used for the same purposes as the free base or they can be employed to upgrade the same. For example, the free base can be converted to an Insoluble salt, such, as the picrate, which can be subjected to purification procedures, for example, solvent extractions and washings, chromatography, fractional liquid-liquid extractions, and crystallization and then used to regenerate the free base form by treatment with alkali or to make a different salt by metathesis. Or the free base can be converted to a water-soluble salt, such as the hydrochloride or sulfate and the aqueous solution of the salt extracted with various water-immiscible solvents before regenerating the free base form by treatment of the thus-extracted acid solution or converted to another salt by metathesis. 2062A Typical compounds prepared according to Examples 4, 8, and 9 Include both the cls and trans forms of the following compounds according to each of formulas XXXVII, XXXVIII, XLV, and XLVII .

. TABLE ' I RH Alkyl a) CH3 H CH3 b) CH3 CH3 CH3 c) CH3 C2H5 CH3 d) C2H5 H C2H5 e) C2H5 C2H5 C2H5 f) n-C3H7 H C2H5 g) n-C3H7 CH3 C2H5 h) n-C4H9 H CH3 i) n-C4H9 CH3 CH3 J) n-C4H9 C2H5 C2H5 k) n-C4H9 . H C2H5 1) n-C4H9 CH3 C2H5 m) CH3 H H n) • CH3 CH3 H o) CH3 CH3 CH3 P) H H q) 02¾· C2H5 C2H5 r) n-C3Hy H H s) n-C3H7 CH3 H t) ■ n-C3H7 CH3 CH3 ) n-C4H9 H H v) n-C4H9 CH3 H y) n-C4H9 CH3 CH3 20β2 A Typical compounds prepared according to Examples 5, 6, and 7 include both the cis and trans forms of the following compounds according to formulas XL, XLII, and XLIV.

TABLE II ■ RH a) CH3 H b). CH3 . CH3 c) CH3 C2H5 d) C2H5 H e) ' C2H5 CH3 f) C2H5 C2H5 g)~ n-C3H7 H h) n-C3H7 CH3 i) n-C3H7 C2H5 J) n-C4H9 H k) n-C4Hg CH3 1) n-C4H9 C2H5 Typical compounds according to formulas XLIX and L prepared according to Example 10 include each of the compounds listed above in which the 2-0-hydrogen (R ), and the 7-0-hydrogen (Re), and the 2-0- and 7-0-hydrogens (R7 and R6) are replaced by alkyl, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl and the isomeric forms thereof.

Typical compounds according to formula LVI I include each of the above compounds in the L-th^eo configuration . 20β2Α Example 12 - Conversion of 1 i ncomyc i n hyd rochl or i de to the S-ethyl analog of l incomycin hydrochloride Lincomycin hydrochloride (8.85 g.., ■ 0.02 mole) was o dissolved in 20 ml. of water, cooled to 0 and stirred while adding bromine (5.5 g. , 0.022 mole) drppwise over a one minute period. Ethanethiol (25 ml . ) was added ,and the mix- o ture sti rred at 25 for 2 hours. The clear, colorless, 2-phase' system (ethanethiol is relatively insoluble in water) was cooled in'an ice-bath and hydrogen chloride gas bubbled in for about 5 minutes. The lower aqueous phase turned red. The reaction mixture was then extracted 5 times with 100 ml. portions of Skel lysolve B (technical hexane) and aqueous sodium hydroxide sol ut i on . added to bring the aqueous phase to pH 11. The basic phase was extracted well with chloroform. The chloroform extracts were washed with saturated sodium chloride solution, dried, and evaporated under vacuum to yield 6.2 g. of a white, so l id. Of this so l id, ^.8 g. was adsorbed on 15 g. of sil ica gel in methylene chloride, dried and sifted onto a 2 1/2 inch diameter column of 800 g. of sil ica gel, The column was then eluted with a mixture of methanol and chloroform in the proportions of 1 to 7. After 800 ml. of forerun, eighty 25-ml . fractions were collected. Fractions ½0 through 58 were combined, evaporated to dryness, and the residue recrysta 11 i zed from acetone. About 0.5 g. of 6 ,8-d i deoxy-6- 1 rans-l-methyl propyl -L-2-pyrrol i d i necar-boxami do)-D-erythro-D-galacto-al dehydo-octose diethyl dithio-acetal of the fol lowing formula: 20β2Α was obtained.

Anal. Calcd. for C2i H42N20eS2 : C, 52.25; H, 8.77; N, 5.80; S, 13.29.

. Found: C, 52.16; H, 8.77; N, 5.84; S,| 13.30.

Fractions 65 through 75 were combined-, evaporated to dryness, and the residue converted to the hydrochloride salt and recrysta 11 i zed from aqueous acetone to yield about 0.5 g. of the S-ethyl analog of l incomycin hydrochloride, ethyl 6,8-d i deoxy-6- (trans-l-met'hyl - -propyl -L-2-pyrrol idi necarbox-ami do)-l- th i o-D-ery thro- a-D -gal acto-octopy ranos ide hydrochlor-ide; [ ]2D5 + 147° (c = 1.1206 in H20).

Anal. Calcd. for Ci9H36N20eS. HC1 : C, 9.9 ; H, 8.16; CI, 7.76; N, 6.13; S, 7.01.

Found: C, 49.53; H, 8.23; CI, 7.39; N, 6.04; S, 6.88.

This product had an antibacterial activity about 2 times l incomycin and was identical with the S-ethyl analog of l incomycin (l incomycin C) prepared by carrying out the fermentation according to U. S. Patent 3*086,912 in the presence of added ethionine.

Example 13 - Conversion of l incomycin hydrochloride to the S- isopropyl analog of 1 i ncomyci n hydrochlor ide L i ncom ci n . h drochlori de (35.4 . , 0.08 mole) was 20β2Α dissolved in 80 ml . of water, cooled to 0 C. and stirred while adding bromine (l4.08.g., 0.088 mole) dropwise over a 5 minute period. 2-Propanethi ol (80 ml. ) and ΐ θΟ ml. of tetrahydrofuran were added and hydrogen chloride gas bubbled in vigorously unti l the temperature of the reaction rose to o 3 C At this temperature the two phase system became homogeneous. Hydrogen chloride gas addition was stopped and the reaction solution stirred without external heating or cool ing for two hours. · The tetrahydrofuran was distil led under vacuum and the aqueous residue was worked up as described in Example 12 to yield the S-isopropyl analog of l i neomycin hydrochloride. This compound exhibited about the same antibacterial activity as l incomycin hydrochloride and o had an optical rotation of [a] + 1 7 (c = Ο.6798, H20), and the following elemental analysis: Calcd. for C20H38 N206S . HC1 : C, 50.99; H, 8.35; N, 5.95; S, 6.81; CI, 7.53.

Found: C, 51.23; H, 8.39; N, 5.9≥; S, 7.02; CI, 7.58. Example 14 Fol lowing the procedure of Example 13 substituting the ethanethiol by butanethiol, the S-butyl analog of l incomycin hydrochloride is obtained, .This product had an antibacterial activity of about 2 times that of l incomycin, an optical o rotation of [a] + 130 (c = 0.4070, H20), and the following elemental analysis: Calcd. for C2i rl4oN20eS. HC1 : C, 51.99; H, 8.52; N, 5.78; S, 6.61, Found: C, 50.64; H, 8.33; N, 5.68; S, 6.4l.

Example 15 Fol lowin the rocedure of Exam le 13 substitutin the 20β2Α l incomycin hydrochloride is obtained. This compound had an antibacterial activity about 2 times that of l incomycin, an ° optical rotation of [a] n + 123 (c = 0.99'52, H20), and the fol lowing elemental analysis: Calcd. for CaaF a aOeS. HC1 : C, 54.04· H, 8,48; N, 5.48; S, 6.27; CI, 6.94.

Found: C, 53.57; H, 8.74; N, 5.37; S, 6.25; CI, 6,66.

By substituting the . a 1 kaneth i ol s of Examples 12, 13, and l4 by other al kanethiol s, for example, propane-, pentane-, hexane-, heptane-, octane-, nonane-, decane-, undecane-, and dodecanethiol and the isomeric forms thereof, the correspond-i ng al kyl 6,8-d i deoxy-6- (t rans-l-methyl -4 -propyl -L-2-pyrrol i -d i neca rboxam i do ) -1 - 1 h i o-D-erythro-a-D-ga 1 acto-octanopy rano-sides are obtained.

On hydraz i nol ys i s, or on applying the above procedural steps to the free sugar hydrochloride which bears no acyl group on the amino group in the 6-position, the corresponding al kyl 6- ami no -6,8-d i deoxy-1- th i o-D-erythro- -D-galacto-octo-pyranosides are obtained.

Lincomycin C is also obtained by reacting l incomycin with ethanethiol (ethyl mercaptan) to form a diethyl dithio-acetal and heating the' latter in the presence of p-toluene-sulfonic acid or by heating to fusion. The fol lowing example is i 11 ust rat i ve.

Example l6 · · A. 6,8-di deoxy-6- (t rans-l-methyl -4- propyl -L-2-pyrrol i-d i neca rboxam i do)-D-erythro-D-galacto-a1 dehydo-octose d i ethyl d i th i oaceta 1 20β2Α In a 1-l iter, 3-necked flask were placed concentrated hydrochloric acid (150 cc. ) and ethanethiol (50 cc. , previously o cooled to 0 ), fol lowed by l incomycin hydrochloride (15.0 gm. ) After stirring magnetically at room temperature for 5 hours, the reaction mixture was diluted with an equal volume of ice-water, and the solution extracted thoroughly with Skel lysolve B (technical hexane), these extracts being discarded.

The majority of the acid was neutral ized by the careful addition of sol id potassium hydroxide (100 gm. ), keeping the temperature of the well -stirred reaction mixture between 20 o and 30 C. by cool ing in acetone-Dry Ice. Sol id potassium chloride was removed by fi ltration, and the sol i d washed well with chloroform. Additional chloroform was added to the fi ltrate (ca. 150 cc. ) and the mixture, stirred magnetically, was adjusted to pH 10 by the addition of aqueous sodium hydroxide (2 N). The chloroform layer was separated, the aqueous layer extracted thoroughly with chloroform, the combined extracts washed twice with water, and dried over o anhydrous sodium sulfate. Removal of the solvent at 30 C. i n vacuo gave a semi-sol id residue which on being crystal- - - 1 -methyl -4 -propyl -L-2 -pyrrol i d i neca rboxami do) -D -eryth ro -D -qlacto-a 1 dehydo-octose diethyl dithioacetal as colorless flattened needles, m.p. 130-132°. Concentration of the mother-l iquors gave additional material (1.50 gm.), m.p. 129-131°. (Total yield, 6.91 gm . , 42.4$).

Ana 1 ys i s : Calcd. for C≤ iH42N20eS2 : C, 52.25; H, 8.77; N, 5.80; S, 13.29$.

Found: C, 52.38; H, 8.71; N, 5.93; S, 13.46< .

B. Cycl ization to Lincomycin C (a) One part each of the diethyl dithioacetal of Part A and p -tol uenesu 1 f on i c acid monohydrate were ref luxed in 25 parts of acetonitrile unti l substantial antibacterial activity was obtai ned. The reaction mixture was cooled and evaporated to dryness and ch romatographed on si lica gel using a solvent mixture of ethyl acetate, acetone and water in the ratio of 8:5:1, respectively. Fractions 102 through 131 showed antibacterial activity. Of these, fractions 105 through 125 were pooled, evaporated to dryness, crystall ized from acetone acidif ied with hydrochloric acid, and recrystal l ized by dissolving in water and adding acetone to give crystals of l incomycin C hydrochloride, m.p. 1 9-153°. (b) The diethyl dithioacetal of Part A was heated to 260° for about 3 minutes and the odor of ethyl mercaptan was noted. The product on being ch roma tog raphed as in Part B(a) yielded l incomycin C.

Claims (1)

1. WHAT IS CLAIMED wher protective carbyioxycarbonyl group removable by or diphenyl benzyl or Q designates R wherein and R are alkylldene or of not more than 20 carbon lkylldene or respectively of 3 to hot more than 8 carbon atoms or aralkylidene o respectively of not more than 12 carbon or not more than 12 carbon Y is alkyl of not more than 12 carbon or wherein allcyl of not more than 12 carbon and is alkyl of not more than 20 carbon cyoloalkyl of not more than 8 carbon or aralkyl of not more than 12 carbon the with the exception of Wherein and A process which comprises ting with a of the formula or the formula wherein Z is a protective hydrocarbyloxycarbonyl group removable by trityl diphenyl or and arid respectively not more than 20 carbon and respectively cycloa to not more than 8 carbon and aralkyl or not more than 12 carbon a compound of the formula wherein and are hydrogen or alkyl o not more than 12 carbon Y is of wherein alkyl of not more than 12 carbon and hydrogenating the compound thus formed over a palladium catalyst to remove Z group and to form a compound of the formula wherein and Y are as given 2062 A HAVING NOW the nature of A process which comprises reacting a compound of the formula wherein Z a protective group removable by hydrogenolysis wit a Wittig reagent of the formula wherein R is a member of the group consisting of alkylidene of not more than 20 carbon cycloalkylidene of to not more than 8 carbon and aralkylidene of not more than 12 carbon atoms to form a compound of the formula wherein Z and R are as given hydrogenating the pound of formula C over a platinum catalyst to saturate the alkylidene group to form a compound of the formula wherein Z and are as given with a pound of Formula D a compound of the formula f s a member of the group consisting of of no 40 than 12 carbon and wherein 41 is alkyl of not more than 12 carbon to form 42 pound of the ting emove 20β2Α if desired wherei n R3 and 1 ky 1 at i ng the compound of formula G to give a compound of the formula wherein and Y are as given above and i s a member of the group consi sti ng of kyl of not more than 20 carbon cycloal kyl of not more than 8 carbon and aral kyl of not more than 12 carbon The process of clairri i in which the al kylation i s effected by reacting the compound of Formula G wi th an oxo compound of the formula Rs wherei n i s a member the group consi sting of al kyl i dene of not more than 20 carbon oa ky i dene of from 5 to not more than 8 carbon and aral kyl i dene of not more than 12 carbon atoms and then hydrogenat i ng i n the presence of a catal yst effective to saturate an olef inic double A ch compri ses reacti ng a compound of formula A wi th a i g reagent of formula B to form a compound of formula rogenat i ng the compound of formula C over a i num catal yst to saturate the al kyl idene group to form a pound of formula ng a compound of formula E wi th a c m u d f formula D form a c m d f formu a nd 2062A hyd i ng the compound of formula F over a pal ladium catalyst to remove the Z group and to form a of formula wherein and Z are as given in claim according to Claim 3 A process i ch comprises reacting a compound of formula A with a reagent of formula B to form a compound of formula hyd rogenat i ng the compound of formula C over a platinum catalyst to saturate the idene group to form a compound of formula a compound of formula E with a compound of formula D to form a compound of formula wherein and Z are as given in a im according to Claim A comprises hydrogenat i ng wi th a platinum catal yst a compound of formula C to form a compound of ula a compound of formula E with a compound of formula D to form a compound of formula hyd rogenat i ng the compound of formula F over a pal ladium catalyst to move the Z group and to form a compound of formula and alkylating the compound of formula G to form a compound of formula wherein and Z are as given in claim The process of which the alkylation is effected by reacting the compound of formula G with an oxo compound of the formula wherein is a member of the group consisting of al idene of not more than 20 carbon atomSj cycl oa Iky 1 dene of from to not more than 8 carbon and aralkyl idene of not more than 12 carbon and then hyd rogenat i ng in the presence 2062A 8 of a catalyst effective to saturate an olefinic double 9 according to Claim 3 1 A process wh i ch comprises hyd rogenat i ng over a catalyst a compound of formula C to form a compound of formula a compound of formula E with the 4 pound of formula D to form a compound of formula and 5 hydrogenat ng the compound of formula F over a pal ladium β catalyst to remove the Z group and to form a compound of 7 formula wherein Z are as given in claim 8 according to Claim 1 A process comprises hydrogenat ng over a 2 inum catalyst a compound of formula C to form a compound of formula D and a compound of formula E with 4 a compound of formula D to form a compound of formula 5 and Z are as given in claim according to Claim 3 1 A comprises reacting a compound of 2 formula A with a Wittig reagent of formula B to form a compound formula a compound of formula E 4 with a compound of formula C to form a compound of the 2 20β2Α hyd rogenat i ng the compound of formula I to saturate the olefinic double bond connecting the alkyl idene group and to remove the Z group to form a compound of formula G and al kyl ating the compound of formula G to form a compound of formula Hj wherein and Z are as given in claim as claimed A p wh i ch compr i ses eact i ng a compound of formula A with a Wittig reagent of formula B to form a compound of formula a compound of formula E with compound of formula C to form a compound of formula hydrogenat ng the compound of formula I to saturate the olefinic double bond connecting the alkyl idene group and to remove the Z group to form a compound of the formula wherein Yj Z are as given in claim formula E with a compound of formula C to form a compound of formula I and hyd rogenat i ng the compound of formula I to saturate the olefinic double bond connecting the alkyl idene group and to remove the Z group to form a compound of the formula wherein and Z are as given in claim A compound of the f a 20β2Α r 9 10 wherein R3 and Z are as given in claim 1 and alkyl 11 is of not more than 12 carbon 17 1 A compound of the A 8 wherein and Z are as given in claim 8 wherein R3 and Z are as given in claim 9 wherein and Z are as given in claim 1 and alkyl 10 is of not more than 12 carbon wherein and Z are as given in claim 1 and alkyl is of no more than 12 carbon 21 A compound of the formula wherein and Z are as given in claim 22 A compound of the wherein and Z are as given in claim 1 23 A compound the 20β2Α 9 wherein R R2i and Z are as given in claim 1 free base or 2 acid addition salt forms of a compound of the 0 wherein and R3 are as given in claim 1 and alkyl is 1 of not more than 12 carbon 1 free base or 2 acid addition salt forms of a compound of the 0 wherein and R3 are as given in claim 1 and alkyl 1 is of not more than 12 carbon 26 1 free base 2 acid addition salt forms of a compound of the 2062A 0 wherei n and are as given in claim The 1 free base or 2 acid addi tion sal t forms of a compound of the 9 0 wherein and are as given i n claim 28 1 ffhe free base or 2 acid addi tion sal t forms of a compound of the 0 wherein and R3 are as given i n claim 1 free base or 2 acid addi sal t forms of a compound of the 0 wherei n and R3 are as given in claim 1 and kyl 1 i s of not more than 12 carbon 30 base 9 0 wherein and are as given in claim 1 he free base 2 acid addi tion sal t forms of a compound of the 20β2Α 0 wherein and are as given in claim 1 base 2 acid addition salt forms compound of the 0 wherein and R3 are as given in claim 1 free base 2 acid addition sal t forms of a compound of the 0 wherein R3j and are as given in claim V 20β2Α i A compound of the 9 0 wherein R3j and Z are as given in claim 1 35 1 A compound of the 9 wherein and Z are as given in claim 36 1 free base or 2 acid addition salt forms of a compound of the 20β2Α 1 free base or 2 acid addi tion forms of a compound of the 0 wherei n and Y are as given in claim 38 1 free base 2 acid addi tion sal t forms of a compound of the 0 wherei n al kyl i s of not more than 12 carbon R i s a 1 member of the group consi sting of a 1 kyl of not more than 2 20 carbon cycl oa 1 ky 1 of 3 to not more than 8 3 carbon and of not more than 12 carbon 4 is a member of the group consi sting of 5 al kyl of not more than 20 carbon cycloal kyl of 3 to 6 more than 8 carbon and aral kyl of not more than 7 12 carbon and one of e and i s an al kyl group of 8 not more than 12 carbon atoms and the other i s a member of the 2062A 39 free base acid addition salt forms of a compound of the wherein and are as given in claim and is a member of the group consisting of hydrogen and of not more than 12 carbon 4o free base acid addition salt forms of a compound of the wherein R6j and are as given in claim A process which comprises reacting a compound of the formul a 20β2Α with acetone to form a compound of the oxidizing with chromic acid the compound of Formula form a compound of the converting the obtained compound to a compound of the formula by a borohydride and removing the i sopropyl i dene group by acid and the Ac group by hyd i nol ys i s to form a compound of the wherein is a member of the group consisting of hydrogen and alkyl of not more than 12 carbon atomsf Ac is a member of the group consisting of alkanoyl and aralkanoyl of not more than 12 carbon atoms and acyl of the R is a mem more carbon of to not more than 8 carbon atoms and of not more than 12 carbon atoms arid is a member of the group consisting of alkyl not more than 20 carbon alky1 of to not more than 8 carbon atoms and aralkyl of not more than 12 carbon and Y is a member of the group consisting of of not more than 12 carbon and wherein the alkyl is not more than 12 carbon The free base or acid addition salt forms of a compound of the 20β2Α 1 free base 2 acid addition salt forms of a compound of the e base 2 acid addition salt forms of sopropy i deneep i 1 i ncomyc having the 1 A process which comprises desu 1 f u i i ng with mercuric 2 de a compound of 8 and acylating the resulting product to form a compound of 20β2Α brominating with HBr to a compound of the thioal kylati ng with thiourea and a compound of the formula halogen to form a compound of the hydrolyzing the Ac groups and hydraz i nol yz ng the group to form a compound of the OH wherein Ac and are members of the group consisting of alkanoyl and aralkanoyl of not more than 12 carbon atoms and alkyl is of not more than 12 carbon and 0 i a ber of the group consisting of alkyl of not more than 12 20β2Α A process which comprises acylating a compound of the with thiobenzoyl hal ide to form the tetrathi brominating the tetrathionobenzoate to form a compound of the heating the resulting compound in acetone in the absence of water to form a compound of the 2062A al kylati ng the resul ting compound with a compound of the in the presence of mi base form a compound of the wherein Ac i s a member of the group consi sting of al kanoyl and aral kanoyl of not more than 12 carbon is an integer of 1 to and 0 i s a member of the group consist i ng of al kyl of not more than 12 carbon atoms which i s di ferent from the starting al kyl and wherein R i s al kyl of not more than 12 carbon The process of claim the compound of BF i s subjected to hydrazi nol ysis to form a compound of the wherein i s as given in claim 96 2062A The free base or the acid addition salt forms of of the wherein R is a 1 ky 1 of 4 to not more than 12 carbon j and R3 are as given in claim and alkyl is of not more than 12 ca rbon A member of tho group consisting free base acid addition salt forms of a compound of the wherein is alkyl of 2 to not more than 12 carbon and R3 are as given in claim and alkyl is of not more than 12 carbon of tho group consisting The free base acid addition salt forms of a compound of the 20β2Α 9 wherein and R3 are as given in claim 1 and al kyl i s 0 of more than 12 carbon 57 1 free base 2 acid addi tion sal t forms of a compound of the 9 1 free base or the 2 acid addi tion sal t forms of a compound of the 9 2 acid addi tion sal t forms of a compound of the 1 base or the 2 acid addi tion sal t forms of a compound of the 1 i fhe free base oa th 2 acid addi tion sal t forms of a compound of the 2 acid addi tion sal t forms of a compound of the 20β2Α the free base or th acid addition salt forms of a compound of the acid addition salt forms of a compound of the Tthe free base or t acid addition salt forms of a compound of the 2062A 1 ree base or th 2 acid addition salt forms of a compound of the H3 9 OH The 2 acid addition salt forms of a compound of the 1 free base or th 2 acid addition sal t forms of a compound of the 20β2Α 2 acid add t i on sa t forms of a compound of the 1 fir 4 free base l or 2 acid addition salt forms of a compound of the 1 free base or 2 acid addition salt forms of a compound of the 2062 A 1 free base or the 2 acid addition salt forms of a compound of the 1 The free base or the 2 acid addition salt forms of a compound of the 1 free or the 2 acid addition sal t forms of a compound of the 2062A 1 free base or 2 acid addition salt forms of a compound of the free base or t 2 acid addition salt forms of a compound of the 1 he free base oi th ound of the 20β2Α free base or th acid addition salt forms of a compound of the free base or the acid addition salt forms of a compound of the OH acid addition salt forms of a compound of the OH 20β2Α he free base acid add i on forms of a compound of the base acid addition salt forms of a compound of the as c laimed Ln Claim 3 A preparing a compound of formula H which comprises alkylating a compound of formula G wherein and Y are as given in claim The process of the alkylation is effected by reacting the compound of formula G with an oxo compound of the formula wherein is a member of the group consisting of from to not more than 8 carbon and a ra i dene of not more than 12 carbon and then hyd rogenat ί ng in the presence of a catalyst effective to saturate an olefinic double free base acid addition salt forms of ep i 1 ncomyc n of the he free base d or the acid addition salt forms of a compound of the wherein R3i and Y are as given in claim DATED THIS 6th day of COHEN ZEDE SPISBAOH TEL Attorneys for insufficientOCRQuality