IL25010A - Lincomycin derivatives and process for producing same - 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 , PA T E NT ATT O R N E YS 24, LEVONTIN ST R ., P. O. B. 1169 T E L - AV I V P A T E T S & D E S I G N S O R D I N A N C E SPECIFICATION 14090/66 PROCESS AND COMPOUNDS PRODUCED ' THEREBY We, THE UPJOHN COMPANY, a corporation of the State of Delaware, of 301 Henrietta Street, Kalamazoo, State of Michigan, U.S. A DO 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: more than 12 carbon atoms and Is selected from the group consisting of hydrogen, and a protective group removable by hydro enolysis or alkyli and halo designates chlorine or bromines Examples of alkyl of not more than 20 carbon atoms ( » HR^, and HR2) are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetra-decyl, pentade cyi, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl ar d the isomeric forms thereof. Examples of cyclo-alkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-methylcyclopentyl, 2, 3-dlmethylcy-clobutyl, -m€ thylcyclobutyl, and 3-cyclopentylpropyl . Examples of aralkyl are benzyl, phenethyl, ct-phenylpropyl, and a-naphthyl-methyl. Examples of alkylidene, cycloalkylidene, and aralkyl^ idene groups I Ri and R2) include methylene, ethylidene, propyl-idene, butyli ene, pentylidene, hexylidene, heptylidene, octyl-idene, nonylidene, decylidene, undecylidene , dodecylidene , tri-decylidene, tetradecylidene , pen adecylldene , hexadecylidene , heptadecylidene, octadecylidene, nonadecylidene , eicosylidene , and the isomeric forms thereof, cyclopropylidene, cyclobutyli-dene, cyclopentylidene , cyclohexylidene , cycloheptylidene , cyclooctylidene, 2-cyclopropylethylidene , 3-cyclopentylpropyl -idene, benzylidene, 2-phenylethylidene, 3-phenyipropylidene, and -naphthylmethylene.

The novel compounds of the invention, Formula I, as well as other related compounds, can be prepared by replacing by halogen ct.lQCi.ft», the 7-hydroxy of a compound of the formula The replacement is effected advantageously by mixing the , starting compound of Formula II with Rydon reagent and heating. In the process Ac and R can be any radical which is non-reactive with Rydon reagent ^u for preparing compounds of the invention Ac and R are as given above. For example, when a compound of Formula IIA (Ac in Formula II is that of the acid of Formula A) is used as the starting compound, a novel compound of Formula IA is obtained. When this compound or the starting compound (Formula IIA) is hydrogenated with a catalyst effective to saturate an olefinic double bond, a compound of Formula IB is obtained as a mixture of cis and trans epimers according to the formulas III and IV which, if desired, can be separated by counter current distribution or chromatography.

When R3 in Formulas B, IB, and IIB is hydrogen, it can be replaced by suitable alkylation or like procedure. Advantageously, this replacement is effected by reacting the compound accord-ing to Formula B, IB, or IIB, wherein R3 is hydrogen with, an oxo compound (an aldehyde or a ketone) and hydrogenating the resulting adduct with a catalyst effective to saturate an olefinic double bond. Either platinum or palladium can be used as the catalyst. Suitable oxo compounds have the formula R4R5C0 where the same as R2 given above. Examples of suitable oxo 1980A compounds are formaldehyde, acetaldehyde , propionaldehyde , butyraldehyde, acetone, isobutylmethyl ketone, benzaldehyde, phenylacetaldehyde, hydrocinnamaldehyde, acetophenone, propio-phenone, butyrophenone , 2-methyl-4-phenyl-2-butanone , 2-methyl- 5-phenyl-3-pentanone, 3-cyclopentanepropionaldehyde, cyclohex-aneacetaldehyde, cycloheptanecarboxaldehyde, 2, 2-dimeth.ylcyclo-propylacetaldehyde, 2, 2-dimethylcyclopropyl methyl ketone, cyclopentyl methyl ketone, cyclobutyl methyl ketone, cyclo-butanone, cyclohexanone, -methylcyclohexanone, and the like.

The starting compounds of Formula II are prepared by acylating a compound of the formula wherein R is as given above with a -substltuted-L-2-*pyrrol.i-dinecarboxylic acid of Formula A or B. This acylation and like acylatlons referred to herein can be effected by procedures already well known in the art for acylating amino sugars. The starting acid of Formula A can be prepared by reacting a 4-oxo compound of the formula wherein Z is a protective y rocar y oxycar onyl group which is removable by hydrogenolysis, trityl, i.e., triphenylmethyl, dipheny1 (p-methoxypheny1 ) methyl, bis-(p-methoxypheny1 )phenyl-methyl, benzyl, or p-nitrobenzyl .with a Wittig agent, e.g., an alkylidenetriphenyiphosphorane [see e.g., Wittig et al , , Ber., 87, 13 8' (1954); Trippett, Quarterly Reviews, XVII, No. 4, p. 4θ6 (1963)]. Examples of hydrocarbyloxycarbonyl groups (Z) are tertiary-butoxycarbonyl; benzyloxycarbonyl groups of the formula wherein X Is hydrogen, nltro, methoxy, chloro, or bromo, for example, carbobenzoxy, p-nitrocarbobenzoxy, p-bromo-, and p- chlorocarbobenzoxy; and phenyloxycarbonyl groups of the formula wherein Χχ is hydrogen, allyl, or alkyl of not more than 4 car- bon atoms, such as phenyloxycarbonyl, p-tolyloxycarbonyl, p- ethylphenyloxycarbonyl-, and p-allylphenyloxycarbonyl and the like.

In carrying out this process the 4-oxo-L-2-pyrrolidine- carboxylic acid (Formula C) is added to a freshly prepared Wittig reagent. The Wittig reagents herein used can be generally represented by the following formula: Ri=P( CeHs ) 3 wherein Rx is as given above. These Wittig reagents are prepared by reacting an alkyl, cycloalkyl, or aralkyltriphenylphos-■■ phonium halide with a base such as sodamide, or sodium or potas slum hydride, or the sodium or potassium metalate of dimethyl- sulfoxide and the like. ■ For. example, the elimination of hydrogen halide from alkyltriphenylphosphonium halide, produces alkylidenetriphenylphosphorane . [The preparation of phosphor- anes is discussed in detail by Trippett, Quart. Rev. XVII, No. , p. 6 (1963)].. The reaction is. generally carried out in an organic solvent, such as benzene, toluene, ether, dimethyl-sulfoxide, tetrahydrofuran, or the like, at temperatures be- o tween 10 C. and the reflux temperature of the reaction mixture. The thus-obtained product, a 4-alkylidene-, -cyclo-alkylidene-, or 4--aralkylidene-l-protected-L-proline which has the following formula 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, chromatography, or formation and recrystallization of easily formed derivatives such as amine salts of the amino acid, ce.g., the dicyclohexyl-amine salt, and the like, and liberating the amino acids from such compounds. By hydrogenating an acid of Formula D in the presence of a catalyst, e.g., platinum, which is effective to saturate a double bond, but which is ineffective to effect hydrogenolysis, a compound of the following formula is obtained. Platinum deposited on a carrier, e.g., carbon or an anion exchange resin like Dowex-1, a cro.ss-linked polystyrene trimethyl benzylammonlum resin in the hydroxide cycle is suit- V able. If desired, the starting compounds of Formula /LjL/ can be acylated with acids of Formula C, D, or E to form compounds IIC, IID, and HE, respectively., Compound IIC can then be converted to compound IID by treatment with a Wittlg reagent and compound lib hydrogenated to compound HE by the proced- ures given above . The hydrogenation, both of the acid D and the acylate IID, gives a mixture of cis and trans epimers which, if desired, can be separated by counter current distribution or chromatography. The starting acids of Formula B in which R3 is hydrogen are obtained when an acid of Formula ' D or E is subjected to hydrogenolysls over a palladium catalyst, e.g., palladium on carbon. Likewise, compounds of Formula IID and HE are converted to compounds of Formula IIB in which R3 is hydrogen by the same process. The starting acids of Formula B in which R3 is hydrogen as well as compounds of Formula IIB in which R3 is hydrogen can be converted respectively to compounds of Formulas B- and IIB in which R3 is HR2 by the pro- and formula B cedures given above. The starting acids of. Formula A/are obtained by treating an acid of Formula D or Formula E with, hydrogen bromide in acetic acid to remove the Z group and then replacing the N-hydrogen with an HR2 group by the procedure given above. Compounds of Formula IID and .HE are converted to compounds of Formula IIB by the same process.

Some of the starting compounds of Formula II are obtained biosynthetically . Lincomycin, methyl 6, 8-dideoxy-6- ( trans-1- methyl- -propyl-L-2-pyrrolidinecarboxamldo ) -1-thio-D-erythro- q-D-galacto-octopyranoside, is obtained as an elaboration product of a lincomycin'-producing actinomycete according to U. S. Patent 3 , 086, 912. It has the following structural formula: wherein R and R3 are methyl and RiH is propyl. - Lincomycln B, methyl 6, 8-dideoxy-6- ( trans-l-methyl- -ethyl-L-2-pyrrolidine-carboxamido ) -1-thlo-D-erythro-g-D-galacto-octopyranoside (Formula VI wherein R and R3 are methyl and -RiH is ethyl) also is an elaboration product of the same microorganism when cultured according to the procedure given in U. S. Patent 5, 086, 912 .

( S-ethyl-S-demethy1-1incomycinjj) Lincomycln C, /ethyl 6, 8-dideoxy-6- ( trans-l-meth.yl-4-propyl-L-2-pyrrolidlnecarboxamido )-l-thio-D-erythro-q-D-galacto-octopyrano- . side (Formula VI wherein R is ethyl, -RiH is propyl, and R3 is methyl) is obtained when the process of U.-S. Patent 3, 086, 912 is carried out in the presence of added ethionine. . Lincomycin D, methyl 6, 8-dideoxy-6- ( trans-4-propyl-L-2-pyrrolidinecarbox-amido)-!-thlo-D-erythro-q-D-galacto-octopyranoside (Formula VI wherein R is methyl, -RiH is propyl, and R3 is hydrogen) is obtained when the fermentation of U. S. Patent 3, 086, 912 is 6, 8-dideoxy-6- ( trans-4-ethyl-L-2-pyrrolidinecarboxamido ) -l-thio-D-erythro-q-D-galacto-octopyranoside (Formula VI wherein R is methyl, -RXH is ethyl and R3 is hydrogen) is also produced when a-MTL is added to the fermentation of U. S. Patent I9 O 3,086,912. Similarly, lincomycin K, ethyl 6, 8-dideoxy-6-( trans-jl—propyl-L^-2-pyrrolidinecarboxamido ) -1-thlo-D-erythro-g-D-gaiacto-octopyranoside (Formula VI wherein R is ethyl, -RiH is propyl, and R3 is hydrogen) is produced when the fermentation of U. S. Patent 3,086,912 is carried out in the presence of added a-ETL, ethyl 6-amino-6, 8-dideoxy-D-erythro-q-thio-D-galacto-octopyranoslde, a compound obtained by the hydrazinoly- §^ethyl-S,N-didemethyllincomycint B-sis of lincomycin C/ eEthyl 6, 8-dideoxy-6-( trans- -ethyl-L-2-pyrrolidinecarboxamido)-l-thio-D-erythro-q~D-gaiacto-octopyran-oside (Formula VI wherein R is ethyl, -RiH is ethyl, and R3 is hydrogen) is also obtained when a-ETL is added to the fermentation of U.-S. Patent 3,086,912. The above-described N-desmethyl products which are' obtained when a-MTL and a-ETL are added to the fermentation process of U. S. Patent 3,086,912 are examples of starting compound IIB wherein R3 is hydrogen which, by the procedure described above can have the N-hydrogens replaced when it is desired for R3 to equal HR2, e.g.,, when it is desired to produce methyl 6, 8-dideoxy-6-( trans-l-ethyl-4-propyl-L-2-pyrrolidinecarboxamido) -l-thlo^D-erythro-q-D-galacto-octopyrano-side or ethyl 6, 8-dideoxy-6-( trans-l-methyl-4-ethyl-L-2-pyrrolldinecarboxamido )-l-th.io-D-erythro-a-D-galacto-octopyran-oside or ethyl 6, 8-dideoxy-6-( trans-l-ethyl- -ethyl-L-2-pyrroli-dinecarboxamido ) -1-thlo-D-erythro-q-D-galacto-octopyranoside or methyl 6, 8-dideoxy-6- ( trans-1-ethyl- -ethyl -L-2-pyrr'olidine-· carboxamido) -1-thio-D-eryth.ro-q-D-galacto-octopyranoside .

Lincomycin or any of the starting compounds of Formula II which has the D-erythro configuration can be converted to the oxidizing L-threo configuration by cnn_irFi,r1vi up—the 7-hydroxy group to a reducing the latter 7-oxo group and fjiei^ l able, .agfljjri to a 7-hydroxy group. A suit-able procedure for this purpose is illustrated in the follow For example, llncomycin on treatment with acetone In the presence of p-toluene sulfonic acid is converted to 3>^-0-isopropyl idenelincomycin which on oxidation with chromic oxide gives 7-dfthydro exQ-J^-O-isopropylldenelincomycin (methyl 6, 8-dideoxy-3,4-0-isopropylidene-6- ( tra s-1-methyl- -propyl-L-2-pyrrolldlnecar-boxamido ) -l-thio-D-glycero-q-D-galacto-octanopyranos-7-uloside) which on treatment with sodium borohydrlde 1B converted to 7-epllincomycin (methyl 6, 8-dideoxy-6- ( trans-l-methyl-4-propyl-L-2-pyrrolidinecarboxamldo ) -1-thio-L-threo-q-D-galacto-octopy-ranoslde). Any of the starting compounds of Formula II having a D-eryth o configuration can be converted to the corresponding D-threo configuration by this procedure.

As the biosynthetlc-produced lincomycins, as well as the amino sugars derived therefrom, are either methyl or ethyl thio glycosides, it is sometimes desirable to convert them to higher o!* lower glycosides. It is also sometimes desirable to convert any of the compounds of Formulas1 I, I I , or V to higher or lower glycosides. Th i s. can effect i vel y be accompl ished by reacting the compound to be converted wi th a mercaptan of the Formula ReSH wherein Re is an alkyl group of not more than 20 carbon atoms, but one different from R, for example, compounds of Formula I and I I on reaction with a mercaptan of Formula ReSH produce d i th i oaceta 1 s of the formula wherein X is hydroxy or halogen, which on treatment with acid and/or on heating is recycl ized to give a compound of the following formu The process can be appl ied directly to any of the starting pro ducts of Formula I I, i.e., I I A, MB, I IC, I ID, and ME. The resulting products can be subjected to hyd raz i nol s i s to form compounds of the following formula: which can be N-acylated as described above with acids of Formula A, B, C, D, and E to provide compounds, accord! ng to Formula XI I wherein X is hydroxy. The process can also be appl ied to the starting compounds of Formula V. For example, a-MTL on treatment with ethyl mercaptan fol lowed by recycl i zat i on as described above is converted to a-ETL.

An alternative process for making compounds of Formula XIJL or Formula X I I I is to brominate the starting material and then react it with the mercaptan according to the fol lowing sequence: 9 0 The starting compound, XIV, is dissolved in water as a soluble salt, e,, g,', the hydrochloride and bromine added with cooling advantageously to between about -10 and 20° C. It is sufficient if the aqueous solution is cooled to about 0° C. and the bromine 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 bromine initially replaces the RS- group and the resulting intermediate hydrolyzes to the sugar in which the pyranose form XVa is in equi l ibrium with the aldose form XVb. In the presence of acid, e.g, hydrochloric acid or other strong non- ox i d i z i ng acid such as p- toluene sulfonic acid, and sulfonic acid type anion exchange resins, the mercaptan R6SH reacts with the sugar XV to form the thio-glycoside XVI . Concomitantly some diacetal of Formula XI may be formed which after separation can be cycl ized as described above to form more of the desired th i og 1 ycos i de XVI .

The mechanism by which Rydon reagent effects the substi tution of the 7"hydroxy by halogen is not ful ly understood.

T Lt-4«&-lye4- ve'd' ~ti»®we¥& Rydon reagents are formed by the addition of halogen to tri phenylphosph i ne or t r i pheny 1 phosph i te or addition of an alkyl hal ide to tr i pheny 1 phosph i te and can be represented by the f ormu la : wherein X is halogen, e,g., chlorine, bromine, and iodine. Rydon et al., J. Chem. Soc, 2224 (1953); Ibid, 2281 (1954); Ibid, 3043 (1956). The Rydon reagent can be formed in situ by addition of halogen or methyl halide to a solution of the tri pheny 1 hosph i ne or t r i pheny 1 phosph i te in an inert solvent such as acetonitrile or d imethy 1 formami de, or it can be isolated as a separate entity. In either case the reaction with the l incomycin or related compound is effected by contacting the Rydoi reagent therewith in an inert solvent, e.g., acetonitrile or d imethy 1 formami de, unti l the desired substitution of the 7"hydroxy is obtained. The reaction takes place at ordinary temperature, though gentle heating can be maintained between about 20° C. and about 55 C. The product can be recovered from the reaction mixture by wel l known techniques such as fi ltration, solvent extraction, etc. The reaction mixture advantageousl is treated with methanol to destroy any excess Rydon reagent, fi ltered to remove any sol id such as t r i pheny 1 phosph i ne oxide, formed in the reaction, and then treated to recover the product. The methanol can be added either before or after the fi ltration. Advantageously the treated and fi ltered reaction mixture is evaporated to dryness and purified by solvent extraction and/or chromatography.

The compounds of Formulas IA, IB, I I A, I IB, and V exist either in the protonated or non-protonated forms according to the pH of the environment. When the protonated form is intended the compound is qual ified as an acid-addition salt and when the non-protonated form is intended it is qual ified as the free base. The free bases can be converted to stable acid-addition salts by neutral izing the free base wi th 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, fluosi l icic, hexaf 1 uoroarseni c, hexaf 1 uorophosphor i c, acetic, succinic, citric, lactic, malei c,. f umar i c, pamoic, chol ic, palmitic, mucic, camphoric, glutaric, gl ycol i c, phthal i c, tartaric, lauric, stearic, sal icyl ic, 3-phenyl sal i cycl i c, 5-pheny 1 sal i -cycl i c,- 3-methyl gl utar i c, orthosul fobenzoi c, cycl ohexanesul -famic, cycl opentanep rop i on i c, 1 , 2-cycl ohexaned i carboxyl i c, 4-cycl ohexanecarboxy 1 i c, octadecenyl succi ni c, octenyl succi ni c, methanesul fon i c, benzenesul on i c, hel ianthic, Rei necke' s, dimethyl di thiocarbamic, cycl ohexyl sul fami c, hexadecy 1 sul fami c, octa-decyl sul fami c, sorbic, monochl oroacet i c, undecylenic, 4' -hy-droxyazobenzene-4-sul foni c, octyl decyl sul f ur i c, picric, benzoic, cinnamic, and l i ke acids.

The acid-addition sal ts 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 sal t, such as the picrate, which can be subjected to purification procedures, for example, solvent extractions and washings, chromatography, fractional 1 i qui d- 1 i qu i d extractions, and crystal 1 i zat i on and then used to regenerate the free base form by treatment wi th al kal i 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- immi sci bl e solvents before regenerating the free base form by treatment of the thus-extracted aci d solution or converted to another salt by metathesis. The free bases of Formulas I A, IB, MA, MB, and V can be used as a buffer or as an antacid. The compounds of Formulas 1 , 1 1 , and V react with isocyanates to form urethanes and can be used to modify polyurethane resins. The long chain compounds, i.e., where H 2 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, 425, 320 and 2, 6o6, 1 55. The free bases also make good vehicles for toxic acids. For example, the fluosilicic acid addition salts are useful as mothproofing agents according to U. S. · Patents 1 , 91 5, 33 and 2, 075, 359 and the hexaf 1 uoroarseni c i aci d and hexa 1 uorophosphor i c aci d addi t i on sal ts are useful as parasiticides according to U. S. Patents 3, 1 22, 536 and 3, 1 22, 552. 7-halo-7-deoxy The close analogues of/ 1 i ncomyci n, i . e. , where -RXH is cis or transalkyl of not more than 8 carbon atoms; R3 is methyl or ethyl ; R is alkyl of not more than 8 carbon. atoms have antibacterial properties and some are comparable or superior to 1 i ncomyci n and can be used for the same purposes as li neomycin. » · . " The other analogues and i somers 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.

♦ The corresponding compounds where R«j.is hydrogen have like antibacterial properties, and moreover have improved gram-negative activity.

Example 1 . 7~ B romo-7" deoxy 1 i ncomyc i n and its hydrochloride .

A solution of Rydon reagent was prepared by stirring a dry solution of 52.6 g. (0.2 M) of tr i phhen 1 phosph i ne and 800 ml , of acetonitri le at 30° under nitrogen and 10 ml. (0.19 M) of bromine added over a '20-mi n . period. After stirring for 10 min, more, 8.2 g. of lincomycin was added and the reaction stirred at 30° for 18 hr. A white solid was then present. The reaction was fi ltered and the sol id discarded. Methanol (100 ml, ), was added to the filtrate and the solvents then evaporated under vacuum. The viscous residue was dissolved in 100 ml. methanol, diluted with 1800 ml. of water and extracted six times with 200 ml, portions of ether. The ether extracts were discarded, the aqueous phase made basic (pH 11) with aqueous K0H and then extracted four times with 200 ml, portions of methylene chloride. The extracts were dried and evaporated, leaving 11 g. of a yel low solid which was chromatographed over 1 kg. of si l ica gel us i ng methanol : chl oroform 1:9 (v/v) as the solvent system. After a forerun of 1200 ml ., 22 fractions of 56 ml . were collected. The. last 6 (fractions 17-22) were pooled and evaporated to dryness yielding 2.8 g. of 7* romo-7" deoxy-lincomycin. This was converted to the hydrobromide by dissolving in water, adding HBr to pH 1, filtering, and lyophi l-izing the fi ltrate. The hydrobromide had an Og +114° (c, O.931 , H20) and the following analysis: Calcd. for Ci8H34Br2N205S : C, 39.28; H, 6.23; N, 5.09; S, 5.85; Br, 29.04.

Found: C, 39.64; H, 6.19; N, 5.07; S, 6.04; Br, 28.59- In place of . bromine, there can be substituted other halogens. Chlorine, for example, yields 7~chloro-7_deoxy- l incomycin which is identical with the product obtained by. ch 1 or i nat i ng lincomycin with thionyl chloride. In place of t r i pheny 1 phosph i ne there can be substituted triphenyl phosphite. Also, in that case a methyl hal ide can be used in the place of halogen. In place of the l incomycin, there can be substituted other l incomycins and analogs thereof. Thus, when lincomycin C is substituted for l incomycin 7~b romo-7-deoxyl i ncomyci n C is obtained.

B . Preparation of Lincomycin C Lincomycin C is obtained by reacting lincomycin with ethanthiol (ethyl mercaptan) to form a diethyl dithioacetal fol lowed by heating in the presence of p- tol uenesu 1 f on i c acid or by fusion. The fol lowing procedure is i l lustrative.

Bl. 6,8-d ideoxy-6- (trans- 1- me thy 1 -4- p ropy 1 - L- 2- pyrrol i di necar boxamido)- D-ery thro- D-qa lacto-a 1 dehydo- octose diethyl dithio-aceta 1.

In a 1-1 iter, J-necked flask were placed concentrated hydrochloric acid (150 cc.) and ethanethiol (50 cc, previously cooled to 0°), followed by lincomycin hydrochloride (15.0 gm. ) After stirring magnetical ly at room temperature for hours, the reaction mixture was di luted with an equal volume of ice- 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 -sti rred reaction mixture between 20 and 30° C. by cool ing in acetone-dry ice. Sol id potassium chloride was removed by f i 1 tration, and the sol id washed well with chloroform. Additional chloroform was added to the filtrate (ca. 150 cc. ) and the mixture, stirred magnetical ly, 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 anhy- o drous sodium sulfate. Removal of the solvent at 30 C. i n vacuo gave a semi-sol id residue, which on being crystall ized from acetone, gave.5. 1 gm. of 6,8-dideoxy-6- (trans-1 -methyl -4-p ropy 1 -L-2-pyrrol idi necarboxami do) -D-ery thro-D-gal acto-al de-hydo-octose diethyl dithioacetal as colorless flattened needles, o m.p. 130-132 . Concentration of the mother- 1 i quors gave additional material (1.50 gm. ), m.p. 129-131°. (Total yield, 6.9I gm., 42.4%).

Anal ys? s; Cal cul ated . for C2iH 20e 2S2 : C, 52.25; H, 8.77; N, 5.81 ; S, 13.29%.

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

B2. Cycl ization to Lincomycin C. (a) One part each of the diethyl dithioacetal of Part Bl and p- tol uenesul fon i c acid monohydrate were refluxed in 25 parts of acetonitrile unti 1 substantial antibacterial activity was obtained. The reaction mixture was cooled and evaporated to dryness and chromatographed on sil ica gel using a solvent mixture 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, and crystall ized from acetone acidi f ied wi th hydrochloric acid and recrys-tallized by dissolving in water and adding acetone to give crystals of I I neomycin C hydrochloride, m.p. 149-155°, (b) The diethyl d!thfoacetal of Part Bl was heated to 26o° for about 3 minutes and the odor of ethyl mercaptan was noted. The product on being chromatographed as in Part B2(a) yielded l incomycin C.

C. Preparation of Lincomycin C by fermentation The lincomycin C hydrochloride was prepared as follows: FERMENTATION A soi 1 si ant of Streptomyces 1 ? ncol nensi s var. 1 i ncol -nens i s, NRRL 2956, was used to inoculate a series of 500-ml .

Erlenmeyer flasks each containing 100 ml. of seed medium consisting of the following ingredients: Yeastolac 1 10 g.

Glucose monohydrate 10 g.

, N-Z-amine B 11 5 g.

Tap water q. s. 1 1 i ter 1 Yeastolac is a protein hydrolysate of yeast cells. i:LN-Z-amine B is Sheffield's enzymatic digest casein.

The seed medium prester i 1 I zat ion pH was 7.3. The seed was grown for 2 days at 28° C. on a Gump rotary shaker operating at 250 rpm.

A 5% inoculum of the seed described above (5 ml . ) was added to each of 30 500-ml. Erlenmeyer flasks each containing 100 ml. of the fol 1 owl ng fermentat I on medium: Glucose monohydrate 15 g.

Starch 40 g.

Molasses 20 g.

Wilson' s Peptone Liquor No. 1 9 1 10 g.

Corn Steep Liquor 20 g.

Calcium carbonate 8 g.

Lard oi 1 0.5 ml.

Tap water, q. s. 1 1 i ter 1 Wi 1 son1 s Peptone Liquor No. 159 is a preparation of enzymati-cal ly hydrolyzed proteins from animal origin.

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

The shake flasks were harvested after 4 days of fermen- o tation at 28 C. on a Gump rotary shaker at 250 rpm. They as-sayed 200 meg. /ml . on the,. _S. 1 utea assay, hereinafter described. The whole beer sol ids was about 20 gm./l iter PURIFICATION Whole beer (235 l iters) from a DL-ethionine fermentation was filtered at harvest pH using a filter aid as required. The mycel ial cake was washed with water and the cake was then discarded. The filtered beer, and water wash (275 l iters) was stirred for 5 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 l iters of water and the water was discarded. The cake was washed with 70 l iters of 20% aqueous acetone and the 20% aqueous acetone wash was discarded. The cake was then eluted twice with 100 l iter portions of 90% aqueous acetone. The eluates were combined (215 l iters) and the solution was concentrated (18 l iters). This concentrate was adjusted to pH 10.0 with a 50% aqueous sodium hydroxide solution. and extracted three times with 20 1 i ter portions of methylene chloride. The methylene chloride extracts were combined (60 l iters) and then concentrated to give an oi ly preparation (7.14 g. ) containing l incomycin and l incomycin 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 1 _N methanol ic hydrogen chloride. The methanol ic solution was then mixed with 3.2 l iters of ether under sti rring. The resulting precipitated colorless, crude 1 i ncomyci n hydrochl or i de and l incomycin C hydrochloride was isolated, by f i 1 trat ion .and dried; yield 7.14 g. assaying 9 0 mcg./mg. against Sarcina lutea. (The assay against Sarci na 1 utea is conducted on agar buffered to pH 6-8 with pH 7.0 phosphate buffer [0.1 ]. 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 l incomycin hydrochloride and l incomycin C hydrochloride in approximately equal amounts.

Crude l incomycin C hydrochloride (7.0 g. ) was dissolved in 20 ml . of water and 20 ml. of butanol , pH adjusted to 4.2 with 1 H.C1, and the solution distributed in a counter current di stribut ion apparatus for 1000 transfers. Analysis by thin-layer chromatography showed that the fractions in tubes 135 to I90 conta i ned 1 i ncomyci n C. These fractions were combined, 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 Sarcina lutea. 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. Crystall ine (cubes) 1 ('neomycin .C .hydrochloride was separated from the supernatant material solution by decantation. These crystals were recrystal 1 i zed from one ml. of water, one ml. of methanol, 80 ml. of acetone and.20 ml. of ether; yield, 250 mg. of crystall ine (cubes) l incomycin C hydrochloride. The super-natant (obtained as described above) was al lowed to stand. at 5° C. for 4 hours. Crystall ine (needles) l incomycin C hydrochloride which precipitated was filtered and dried; yield, 150 mg. of crystall ine (needles) l incomycin C hydrochloride, m.p. 151-157° C D . Alternative method for preparation of l incomycin C Lincomycin hydrochloride (8.85 g. - 0.02 moles) was dissolved in 20 ml. of water, cooled at 0° and stirred while adding bromine (3.52 g. - 0.022 moles) dropwise over a 1 minute period. Ethanethiol (25 ml. ) was added and the mixture stirred 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 3 times with 100 ml. portions of Skellysolve B and aqueous sodium hydroxide solution 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 sol id. 4.8 g. of this sol id was chromatographed over 800 g. of sil ica gel by the procedure of : Exam le 2f usi ng methanol -ch 1 orof orm : (l : J , respect i ve 1 y ) as the solvent system. After 800 ml. of forerun, 80 fractions of 25 ml . each were collected. Fractions 40-58 were combined and evaporated to dryness and the residual sol id recrystal 1 i zed from acetone to yield 0.5 g. of material identical with the diethyl dithioacetal of Part Bl . Fractions 65-75 were combined, evaporated to dryness, and dissolved in a mixture of 5 ml. methanol and 400 ml. diethyl ether. Hydrogen chloride gas added and the white sol id which precipitated was col lected. On being recrystal 1 i zed from aqueous acetone, 0.5 g. of l incomycln C hydrochloride, Identical with that of Part C, was obtained.

E. Other alkyl 7-halo-6,7>8-trideoxy-6-(trans-l -methyl -4- L-threo p ropyl -L-2-pyr rol i d i necarboxami do) - 1 - thi oV - D- qa 1 acto-octopyranosi des.

By substituting the ethanethiol in Part Bl and Part D of this example by other alkyl mercaptans, for example, propyl, butyl, pentyl, hexyl , heptyl , octyl , nony.l , decyl, undecyl, dodecyl, tri decyl , tetradecyl , pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl mercaptans and the isomeric forms thereof; by cycloalkyl mercaptans, for example, cyclo-propyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-methy 1 cycl openty 1 , 2, 3-dfmethyl cycl obutyl , 2-methy 1 cycl obuty 1 and J-cycl opentyl propyl mercaptans; or by aral kyl mercaptans, for example, benzyl, phenethyl , 3-phenyl-propyl , and a-naphthylmethyl mercaptans, the corresponding alkyl, cycloalkyl, and aralkyl 6, 8-d i deoxy-6- ( trans- 1 -methyl -4-p rop l - L-2-pyrrol idi necarboxami do) - 1 -thio-D-erythro- -D-galacto-octopy ranos i des are obtained, which on treatment by the procedure of Part A are converted to the corresponding alkyl, cycloalkyl, and aralkyl 7- ha 10-6, 7, 8- 1 r ί deoxy-6- (trans- 1 -methyl - L-threo 6, 8-d i deoxy and the J- ha 1 o- 6,7, 8- t r i deoxy compounds) wherein alkyl is propyl, butyl, pentyl, and hexyl (obtained respectively when propyl, butyl, pentyl, and hexyl mercaptans are used) are especially effective ant i bacter i a 1 s, having the same spectrum as lincomycin and equal or greater activity.

Also, when the lincomycin is substituted by methyl 6,8-d i deoxy- 6- ( t rans- 1-methy 1 - or l-ethyl-4-butyl- L-2-pyrrol id ine-ca rboxam i do) - 1- thf o- D-ery thro- - D- qa 1 acto- octopy ranos i de, the corresponding 7-bromo-7"deoxy compound, methyl 7~ bromo- 6,J, 8-trideoxy-6- (trans- 1-methy 1 and l-ethyl-4-but J-L-2-pyrrolidine- L— tnrea ca rboxam i do)- 1- th i o- -D- a acto- octopy ranos i des are obtained having the same antibacterial spectrum as lincomycin and equal or greater activity.

On substituting the cis epimers, there are obtained methyl 7" b romo- 6, , 8- t r i deoxy- 6- ( c i s- 1-methy 1 and l-ethyl- - L— thr eo bu ty 1 - L- 2- py r rol id i neca rboxami do) - 1- th i o-/ - D-qal acto- octo-pyranosides having the same antibacterial spectrum.

The cis and trans epimers used as starting materials in the above examples were prepared as follows: F. -Buty P dene- 1- carbobenzoxy- L- pro! i ne and the cyclo-hexylamine salt thereof.

Sodium hydride (19 g.) as a 53$ suspension in mineral oi l was warmed with 350 ml. of d ime thy 1 su 1 f ox i de at a temperature of 70-75° C. unti l the reaction was complete (about 30 minutes). After cooling to 32° C, 16.2 g. of butyl triphenyl-phosphonium bromide was added, and the resulting reaction mixture was stirred for 1 hour to insure complete reaction. A solutionof 26 g. of keto- 1- carbobenzoxy- L-prol i ne in 100 ml. of d i methyl su 1 fox i de was added, and the resulting reaction 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 twice wi th 700 ml portions of ether and the ether was discarded after back extracti ng wi th 150 ml. of 2.5$ aqueous potassium bicarbonate The bi carbonate A solutions were combined and acidi f i ed wi th 4N hydrochloric acid. The acidified mixture was extracted wi th four 500-ml . portions of ether. The combined ether extracts were washed success i vel y wi th 250 ml. of water, three 250-ml . portions of saturated aqueous sodi urn bi sul fi te, and 250 ml. of water, and dried over anhydrous sodium sulfate. Evaporation of the solvent under vacuum gave g. of an oily residue which was - butyl i dene- 1 -carbobenzoxy-L-prol i ne.

This residue was dissolved in 21 ml. of acetonitrile and treated with 18 ml. of d i cycl ohexy.1 ami ne and refrigerated. The crystals were col lected, washed with acetonitrile and dr i ed in vacuo giving 21 g. (46.8% ) of the crystalline dicyclo-hexylamine salt melting at 156-1 0° C. After two recrystall i-zations from acetonitrile, an analytical sample was obtained o o which melted at 142-144 C. and had a rotation of [ ]Q - 4 (c : 0.99, CHC ).

Anal. Calcd. for C29H4 N20 : C, 7K86; H, 9.15; N, 5.78.

Found: C, 71.69; H, 9-30; N, 5.74.

Ten grams of the d i cycl ohexyl ami ne salt of 4-butyl i dene-1 -carbobenzoxy-L-prol i ne was shaken with ether and excess 5% aqueous potassium hydroxide until no solid remained. 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 repeatedly extracted with ether and the ether extracts were combined, dried over sodium sulfate, and evaporated in vacuo to give 6.3 g. (93¾ of 4-butyl i dene- 1 -carbobenzoxy-L-prol i ne as an oi 1.

G, . 4- Butyl - 1 -carbobenzoxy-L-prol i ne F The oi l from Part B was hydrogenated in 200 ml. of methanol over 2.1 g. of 10# platinum on Dowex-1 catalyst under 40 lbs. hydrogen pressure. The catalyst was removed by fi ltration and the fi 1 trate evaporated to yield 6.3 g- of 4-butyl-l-carbobenzoxy-L-prol i ne as an oi l . The product contained about 2 parts ci s-4-buty 1 -1-carbobenzoxy-L-prol i ne to each part of trans-4-butyl -1-carbobenzoxy-L-prol i ne .

If desired, the hydrogenati on of the 4-ylidene group can be postponed to any later step, even to the final step, in the process.

By substituting the buty 1 tr i pheny lphosphon i um bromide of F Part & by other substituted tri pheny Iphosphoni um bromide where the ^-substi tuent is methyl, ethyl, propyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl , undecyl, dodecyl, tridecyl, tetra-decy 1 , pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl and the isomeric forms thereof; cyclopropyl, cyclo-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-cyc lopropy lethy 1 , and 3-cyc lopenty 1 p ropy 1 ; benzyl, phenethyl, 3- pheny lpropy 1 , and a-naphthy lmethy 1 , the corresponding 4-a 1 ky 1 i dene- , 4 -eye 1 oa 1 ky 1 i dene- , and 4-a ra 1 ky 1 i dene-1 -ca rbo-benzoxy-L-prol i nes and the corresponding 4-alkyl-, 4-cyclo-alkyl-, and 4-ara 1 ky 1 -1-ca rbobenzoxy-L-prol i nes are obtained. For example, when the buty 1 t ri phen lphosphon i um bromide is substituted by ethyl-, propyl-, isobutyl-, pentyl-, and hexyltri-pheny 1 phosphon i um bromides there are obtained 4-ethy 1 idene-l-carbobenzoxy-L-pro 1 i ne, 4 -propyl i dene -1-ca rbobenz ox y-L- pro 1 i ne, 4- i sobuty 1 i dene -1-ca rbobenzoxy-L-prol i ne, 4 - pen ty 1 i dene -1-ca rbobenzoxy-L-prol i ne, and 4-hexy 1 i dene-l-carbobenzoxy-L-prol i ne, and cis and trans 4-ethy 1 -1-carbobenzoxy-L-prol i ne, 4-propyl-l- 9 0 A -pen ty 1 -1 -ca rbobenzoxy -L -p ro 1 i ne , and 4-hexy 1 -1-carbo-be nz ox y - L - p r o 1 i ne .

H 0. Methyl 6 -ami no-6,8-di deoxy- 1-thio-D-erythro-a-D-gal ac to- octopy ranos i de (a-MTL).

A solution of 40 g. of lincomycin free base (U. S, Patent 5,086,912) in 20 ml. of hydrazine hydrate (98-100%) was re-fluxed for 21 hours; excess hydrazine hydrate was then removed in vacuo under nitrogen at steam bath temperature, leaving a residue. The residue, a pasty mass of crystals, was cooled, acetonitrile was added, and the mixture was stirred until the crystals were suspended. The crystals were collected on a filter, washed with acetonitrile and with ether. The yield of white, crystalline a-MTL free base after drying in vacuo at room temperature was 21 g. (84%). Recrysta 11 i zat i on was accomplished by dissolving a-MTL free base in hot d imethyl formami de and adding an equal volume of ethylene glycol dimethyl ether.

Methyl 6 -ami no-6, 8-d i deoxy- 1 - th? o-D-erythro-a-D-gal acto- I o octopy ranos i de free base has a melting point of 225-228 C, an O optical rotation of [a]g -y 276 (c = .768, water) and a pKa' of 7.45.

Anal, paled, for C9H19NO5S: 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.

By substituting l incomycin by other alkyl or by cyclo-alkyl or aralkyl 6, 8-dideoxy-6- (trans- 1 -methyl -4-propyl -L-2-py r rol i d i necarboxami do) - 1 - th ί o-D-erythro-a-D-gal ac to- octopy rano-sides where alkyl is ethyl propyl , butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl and the isomeric forms thereof; cycloalkyl is cyclo- 19 0 A propyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclo octyl, 2-methyl cycl opentyl , 2, 3-dimethyl cycl obutyl , 2-methyl-cyclobutyl, and 3-cycl opentyl propyl ; and aral kyl Is benzyl, phenethyl, 3-phenyl propyl , and a-naphthylmethyl , the corresponding alkyl, cycloalkyl, and aralkyl 6-aml no-6, 8-d I deoxy- 1 -thio-D-erythro- -D-galacto-octopyranosides are obtained. For example, by substituting the 1 i ncomycl n . by ethyl-, propyl-, butyl-, pentyl -, and hexyl 6, 8-di deoxy-6- ( trans- 1 -methyl -4-propyl -L- 2- pyrrol I d i necarboxaml do) - 1 - thi o-D-ery thro- a-D -gal acto octopy ranos i des , ethyl 6 -ami no-6,8-dideoxy- 1 - thi o-D-erythro-g-D-gal acto-octopy ranos i de, propyl 6-ami no-6, 8-dideoxy-l -th i o-D-erythro-g-D-gal acto-octopyranos ide, butyl 6-ami no-6, 8-di deoxy- 1 - thi o-D-e ry thro- a-D- gal acto-octopyranos i de/ pentyl 6-ami no-6, 8-d i deoxy- 1 - thi o-D-ery thro- a-D- gal acto-octo-pyranoside, and hexyl 6-ami no-6, 8-di deoxy- 1 -thi o-D-ery thro- a-D-qa 1 acto-octop ranos i de are obtained.

If desi red, the procedure of Part A can. be appl ied to H the compounds of Part Causing the hydrochloride or other salt of a strong acid and the resulting 7-halo compounds processed by the ensuing stepsof this example to the final products of the example.

J?. Methy 1 6,8-dideoxy- 6 » ( 1 -carbobenzoxy- -butyl -L-2-pyrrol i di necarboxaml do;- I -thi o-b-erythro-a-D-ga I acto-octopyranos ide tree"" base. " XIX A To a solution of 6.3 g. of 4-butyl - 1 -carbobenzoxy-L- G prol ine (the oil from Part tf) in 175 ml . of distilled acetoni- o r trile cooled to 0 there was added 3.46 ml. of tri ethyl ami ne followed by 3.3 ml . of i sobutyl chl oroformate. The mixture was stirred at 0° C. ( + 3°) for 15 min. A solution of 6.2 g. of a-MTL free base from Part C in 85 ml. of water was added, o and the reaction mixture was- sti r red at 0 C. for 0.5 hr. and o at 25 C. for 1 hr. The reaction product was then filtered and. dried yielding 4.57 g. (57.7%) of methyl 6,8-deoxy-6-( 1 -carbobenzoxy-4-buty 1 -L-2-pyrrol idi necarboxami do) - 1 -thi o-D -erythro-g-D-gal acto-octopyranosi de free base. The mother l iquo was concentrated under vacuum and an additional 4.25 g. (35. $) of product recovered. Recrysta 11 i zat i on from acetoni-trile produced crystals of methyl 6, 8-deoxy-6- ( 1 -carbobenzoxy-4-butyl -L-2-pyrrol idi necarboxami do)- 1 - thi o-D-erythro- -D-gal -acto-octopyranos i de free base melting at 19 -196° C. A second recrystal 1 i zat ion from aceton i tr il e afforded the analytical sample, m.p. 195.5-200° C, [a] +111° (c = 0.98, MeOH) .

Anal. Calcd. for C, 57.75; H, 7.46; N, 5.13; S, 5.95.

Found: C, 57.58; H, 7.16; N, 5.50; S, 6.07. f . Methyl 6, 8-deoxy-6- (4-butyl-L-2-pyrrol id i necarboxami do) - 1 -thi o-D-erythro- -D-ga I acto-octopyranos i de hydroch I or i del A solution of 7.8 g. of methyl 6, 8-deoxy-6- ( 1 - ca rbo-benzoxy-4- butyl -L-2-pyrrol idi neca rboxami do) - 1 -thio-D-erythro- I g-D-gal acto-octopy ranos i de free base from Part in 200 ml . of methanol was shaken over 2 g. of 10% palladium on carbon under 40 lbs. of hydrogen pressure for 17 hours. The catalyst was removed by filtration 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. Dilution with 4 volumes of acetone precipitated methyl 6, 8-deoxy-6- (4-butyl-L-2-pyrrol idi neca rboxami do) - 1 - thi o-D-ery thro- g-D-gal acto-octopy ranosi de hydrochloride which was col lected by filtration and dried. The crystals, dried at 55° C. under vacuum, weighed 4.7 g. and melted at 188-194° C. The analytical sample obtained by recrystal 1 i zat i on from acetone melted at 197-199° C. and gave [ ] Q5-H50° (water, c = Ο.89).

Anal . Calcd. for C18H34N20eS. 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 8% of the antibacterial activity of 1 i ncomyci n by, _S. 1 utea assay.

By substituting the a-MTL by other alkyl or by cyclo-alkyl or aralkyl 6 -ami no-6, 8-d i deoxy- 1 - th? o-D-ery thro- -D-gal -acto-octanopy ranos i des wherein alkyl is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octa-decyl, nonadecyl, and eicosyl and the isomeric forms thereof; cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-methy 1 cycl openty 1 , 2, 3-d imethyl cyclobutyl, 2-methy 1 cycl obutyl , and 3-cycl openty 1 p ropyl ; and I 198 OA / methyl , the corresponding alkyl , cycloalkyl, and ara lkyl 6,8- d i deoxy-6- (l-carbobenzoxy-4-buty l-L-2-pyrrolidi necarboxami do) -1- thio-D-eryth ro-a-D-qa lacto-octopyranos i des and alkyl, cycloalkyl, and a ra 1 ky 1 6,8-d i deoxy-6- (4-buty 1 -L-2-py rro 1 i d i neca rboxami do) -1- th? o-D-eryth ro-a-D-qa lacto-octopyranos i des are obtained. For ex-',ι' ".. ample, by substituting the a- TL by ethyl, propyl, butyl, pentyl, and hexy 1 6-ami no-6,8-d? deoxy-1- th i o-D-e r yth ro-a-D-qa lac to- octo- pyranoside, there are obtai ned ethyl 6,8-d i deoxy-6- (1-ca rbobenz - bxy -4-buty l-L-2-pyrrolidi necarboxami do) -1- th ? o-D-e rythro- a-D-- , .' qa lacto-octopyranos ? de , propyl 6, 8-di deoxy-6- (l-carbobenzoxy-4- , '- buty 1 -L-2-py r ro 1 i d i necarboxami do) -1- th i o-D-eryth ro- x-D-qalacto-'■ ·' octopyranos i de , butyl 6,8-d i deoxy-6- (l-carbobenzoxy-4-buty 1 -L-2- . py r ro 1 i d i necarboxami do) -1-th i o-D-e rythro -a- D-qa lacto-octopy rano- side, penty 1 6,8-d i deoxy-6- (l-carbobenzoxy-4-buty 1 -L-2-pyrrol i - di necarboxami do) -1-th i o-D-eryth ro-a-D-qa lacto-octopyranos i de , hexy 1 6,8-d ί deoxy-6- (l-ca rbobenz ox y -4-buty 1 -L-2 -pyr rol i d ί neca r- boxami do) -1- th i o-D-ery thro-a-p-qa lacto-octopyranos i de, ethyl 6,8- , d i deoxy-6- (4-butyl-L-2-pyrrol idi necarboxami do) -1- thi o-D-e rythro- g-D-qa lacto-octopyranos i de, propyl 6, 8-di deoxy-6- (4-buty 1 -L-2-: · ; pyr ro 1 i di neca rboxami do) -1 -thi o-D-er th ro-a-D-qa lacto-octopyrano- ' s i de, buty 1 6,8-d i deoxy-6- (4-buty 1 -L-2 -pyr rol id i neca rboxami do) -1- . thi o-D-eryth ro-a-D-qa lacto-octopyranos i de, pentyl 6,8-d i deoxy-6- (4 -buty 1 -L-2-pyrro 1 i d i necarboxami do) -1-th i o-D-ery thro- - D-qa lacto-octopyranos i de, and hex 1 6,8-d i deoxy-6- (4-buty 1 -L-2- pyr ro 1 i - : d i neca rboxami do) -1* thi o-D-ery th ro-a-D-qa lacto-octopyranos ? de .

These alkyl, cycloalkyl, and aralkyl a-thiolinco- , saminides are obtained by hydrazinolysis of the corresponding S-alkyl, S-cycloalkyl , and S-aralkyl S-demethyllin-comycins which in turn are prepared by substituting the ethanethiol in preparation B-1 above by other alkyl mercaptans, for example, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pe tadecyl , hexadecyl, heptadecyl, octadecyl, non-adecyl, and eicosyl mercaptans and the isomeric forms thereof; by cycloadlkyl mercaptans, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-methylcyclopentyl, 2, 3-dimethyl-cyclobutyl, 2-methylcyclobutyl and 3-cyclopentylpropyl mercaptans; or by aralkyl mercaptans , for example, benzyl phenethyl, 3-phenylpropyl , and l-naphthylmethyl mercaptans* By substituting the ¾-butyl-l-carbobenz xy-L-prolineby other -alkyl-l-carbobenzoxy-L-prolines where the ¾-alkyl is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl and the isomeric forms thereof; by 4-cycloalkyl-l-carbobenzoxy-L- pro 1 i nes .where 4-cycloalkyl is cyclopropyl, cyclobutyl, cyclo-pentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-methy lcyclopent-y 1 j 2 ,3-d i methy 1 eye lobu ty 1 , 2-methy lcyc lobuty 1 , and 3_cyclopent-ylpropyl; and by 4-ara Iky 1-1-carbobenzoxy-L-prol i nes where 4-aralkyl is benzyl, phenethyl, J-pheny 1 p ropy 1 , and a-naphthyl-methyl, the cor esponding alkyl, cycloalkyl , and aralkyl 6,8-di -deoxy-6- (l-ca rbobenzoxy-4-a Iky 1 , 4-cycloalkyl, and 4-ara lkyl-L-2-pyrrol idineca rboxami do) -1 - th i o-D - e ry th ro-ct-D - qa lacto-octopy-ranosides, and the corresponding alkyl, cycloalkyl , and aralkyl-6- (4~a 1 ky 1 ■ 4-cycloalkyl, and 4-a ra 1 ky 1 -L -2-py r ro 1 i d i neca rboxa -mido)-l-thio-D-erythro-ct-D-ga lacto-octopyra nos i des are obtai ned. For example, by substituti ng the 4-buty 1 -1-carbobenzoxy-L-pro-1 i ne by 4-methyl-, 4-ethyl-, 4-propyl-, 4-pentyl-, and 4-hexyl-l-ca rbobenzoxy-L-pro 1 i ne, there are obtained methyl, ethyl , propyl, butyl, pentyl , and hexyl 6, 8-d i deoxy-6- (l-carbobenzoxy-4-methy 1 -L - 2-py rro 1 i d i neca rboxami do) -l-thio-D-erythro-a-p-qa lacto-octopy-ranosides; methyl , ethyl, propyl, butyl, pentyl, and hexyl 6,8-d i deoxy-6- (l-ca rbobenzoxy-4-ethy 1-L -2 -pyrrol idineca rboxami do) -1-th i o-D -e ryth ro-ct-D -qa lacto-octop ranos i des ; methyl, ethyl, propyl, butyl, pentyl, and hexyl 6,8-d i deoxy-6- (l-carbobenzoxy-4-propyl-L-2-pyrrol idi neca rboxami do) -1-th i o-D-erythro-ct-p-ga lacto-octopyra nos i des ; methyl , ethyl, propyl, butyl, pentyl, and hexyl 6,8-di deoxy-6- (l-carbobenzoxy-4-penty 1 - L-2-pyrrol id i" neca r-boxami do) -1- thi o-D-erythro-ct-D-qa lacto-octop ranos i des ; methy 1 , ethyl, propyl, butyl , pentyl, and hexyl 6,8-d i deoxy-6- (l -ca rbo-benz'oxy-4-hexy l-L-2-pyrrolidi neca rboxami do) -1-th io-D-ery thro-ct-D-ga lacto-octop ra nos i des ; methyl, ethyl, propyl, butyl, pentyl, and hexy 1 6,8-d i deoxy-6- (4 -me thy 1 -L-2-py r ro 1 i d i neca rboxami do ) -1-thio-D-erythro-ct-D-qa lacto-octopy ranos i des ; methyl, ethyl, propyl, butyl, pentyl, and hexyl 6, 8-d i deoxy-6- (4-ethy 1 -L- 2- py r ro 1 i d i neca rboxami do) -1- 1 h i o-D-ery th ro-a-D-qa lacto-octopyrano-sides; methyl, ethyl, propyl, butyl, pentyl, and hexyl 6,8-di-deoxy-6- ( -propyl-L-2~pyrrol idi neca rboxami do) -1-th i o-D-eryth ro-a-D-ga lacto-octop ranosi des ; methyl, ethyl, propyl, butyl, pentyl, and hexy 1 6,8-dideoxy-6-( -pentyl-L-2-pyrrol idi neca rboxami do) -1-th i o-D-ery thro- a-D-qa lacto-octopy ra nos i des ; and methyl, ethyl, propyl, butyl, pentyl, and hexyl 6,8-di deoxy-6- (4-hexy 1 -L-2-p r ro 1 i d i neca rboxami do) -1- th i o-D-eryth ro- t-p-qa lacto-octopyrano-s i des „ If desired, the 1-ca rbobenzoxy compounds prepared according I to Part f. can be halogenated by the procedure of Part A and the resulting 7"halo compound processed by the ensuing steps of the example to remove the 1-ca rbobenzoxy group and to substitute the proline nitrogen to give the final products of the example. 0¾έ Me thy 1 6,8 -deoxy-6- (l-methyl-4-butyl-L-2-pyrrolidineca rboxami do) -1- th i o-D-e ry th ro-a-D -qa lacto-octopyra nos i de A solution of 2.0 g. of methyl 6,8-deoxy-6- (4-buty 1 - L-2-py r ro 1 i d i neca rboxami do) -1-th i o-D-ery thro-ct-D-qa lacto-octo- , J pyranoside hydrochloride from Part and 2.0 ml. of 37$ formalin in I50 ml. of methanol was shaken over 500 mg. of 10$ palladium on carbon under 40 lbs. of hydrogen pressure for 3-5 hrs. Removal of the catalyst by fi ltration and the solvent by d i s ti 1 la t i on i n vacuo y i e lded pa rt i a 11 y crys ta 11 i ne met hy 1 6,8 deoxy-6- (l-methyl-4-butyl-L-2-pyrrolidineca rboxami do)-l-thio-D-e ry thro-g-D- qa lacto-octop ranosi de h drochloride which b 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 consisted chiefly of two materials, the cis and trans epimers of methyl 6, 8-deoxy-6- ( 1 -methyl -4-butyl -L-2-pyrrol ? di necarboxaml do) - 1 - th? o-D-erythro-a-D-gal acto-octo-pyranoside hydrochloride in a ratio of about 3 to 2.

K2 Gfc/. Separation of the cis and trans forms by chromatography The methyl 6, 8-deoxy-6- ( 1 -methyl -4-butyl -L-2-pyrrol i d i ne-carboxami do) - 1 - thi o-D-erythro-a-D-gal acto-octopyranos i de hydrochloride from Part f] were 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 silica gel and the solvent evaporated under vacuum leaving the antibiotic deposi.ted on the sil 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 by eluting with the same solvent and 20 ml. portions were collected. TLC of each fraction showed that fractions 31-38, 310 mg., were essential ly pure trans epimer and that fractions 49-74, 32 mg., were essentially pure cis epimer. Fractions 39-48 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 hydrochl ori de preci pi tated by addition of acetone. In this manner, there was obtained 50 mg. of methyl 6, 8-deoxy-6- (trans- 1 -methyl -4-butyl -L-2-pyrrol i di ne-carboxam i do) - 1 - thi o-D-erythro-a-D-gal acto-octopyranos i de hyd ro- o chloride, m.p. 135-137 , and about 150 mg. of methyl 6,8-deoxy-6-(cis-l-methyl-4-butyl-L-2-pyrrol idi necarboxami do- 1 - thi o-D- erythro-g-D- gal acto-octopy ranos i de hydrochloride, softening at 105° C. with further melting at 175-185° C The trans epimer recrysta 11 i zed from the same solvent melted at 139-141° C. and had the following analysis: Anal. Calcd. for CigH3aN20eS' HC1 C, 9.93; H, 8.16; N, 6.13; S, 7.02.

Found: (Corrected for 4.07% H20); C, 48.81; H, 8.54; N, 6.49; S, 6.67.

Similarly recrysta 11 i zat i on of the cis epimer gave a o o product softening at 108 C. and further at about 189 C. (sol-vated) with the- f ol 1 ow i ng analysis: Anal. Found (Corrected for 4.95% water); C, 50.27; H, 9.00; N, 6.05; S, 6.65.

The trans epimer was about 2.2 times as active as l inco-mycin by _S. 1 utea assay, about 2 times as active by the broth dilution assay, and 2.5 times as active in mice infected with . aureus .

The cis epimer was about 1/2 to 1/3 as active as the trans ep imer, . bei ng about equal to l incomycin.

LI Nl Methyl 6,8-deoxy-6- ( 1 - ethyl -4- butyl - L-2-pyrrol i di ne car boxam i do) - 1 - thi o-D-erythro-a-D-gal acto-octopy ranos i de hydro-chloride A mixture of 2.0 g. of methyl 6,8-deoxy-6- (4-butyl -L-2-pyrrol idi neca r boxam i do) - 1 -thio-D-erythro-g-D-galacto-octopyrano side hydrochloride from Part 1.5 ml. of acetal dehyde, 150 mg of 10% palladium on carbon in 150 ml. of methanol was shaken under 35 lbs. of hydrogen pressure for 5.5 hrs. The catalyst Was removed by filtration to give a residue consisting chiefly of the cis and trans epimers of methyl 6, 8-deoxy-6- ( 1 -ethy 1 -4-butyl -L-2-pyrrol i d i neca r boxam i do) - 1 -thi o-D-erythro-a-D-gal ac to- _ octopy ratios i de hydrochloride.

L2 tfgf. Separation of epimers As described in Part ^2, the mixture of epimers of Part (2 g. ) was chromatographed over 200 g. of si l ica gel using for el ution a solvent system of ethyl acetate, acetone, water (8:4: 1 ) . Fractions 53-42 by TLC were pure trans-epimer and were combined, fractions 49-64 were essential l y pure ci s-epimer and were al so combined. Fractions 43-48 were a mixture of the epimers which could be puri fied by rechromatography . Each epimer was di ssolved in a few drops of di l ute hydrochloric acid and the crystal l ine hydrochloric precipi tated on di lution with a large vol ume of ether.

The crude trans-epimer fraction of 415 mg. gave J40 mg. (15.4%) of crystal l i ne methyl 6,8-deoxy-6- (trans- 1 -ethyl -4-butyl -L-2-pyrrol i d i neca rboxam i do) - 1 - th i o-D-erythro-ct-D-gal acto- o octopyranoside hydrochloride, m.p. 144-151 C ecrysta 11 i za-tion from di l ute acetone ra i sed the m.p. to 148-151° C.

The ci s-epimer fraction of 645 mg. afforded 300 mg. (14.1%) of crystal 1 i ne methyl 6,8-deoxy-6- (ci s-1 -ethyl -4-butyl - L-2-pyrrol idi neca rboxam i do) - 1 - thi o-D-erythro-a-D-gal acto- octopy - o ranoside hydrochloride, m.p. 135-139 C. Recrystal 1 i zat i on o from di l ute acetone gave crystal s, m.p. 134^.138 C.

The trans epimer i somer showed about 1 -1.2 times the activi ty of l incomyci n by the S . 1 utea assay, 2-4 times the activi ty of 1 i ncomyci n agai nst Gram posi tive organi sms, and 8 times or more the activi ty of l i ncomyci n against Gram negative organi sms. I n mice against S.. aureus the trans-epimer was about 2 times as active as l incomycin. The ci s-epimer was about 1/2 as active as the trans epimer.

Separation of the cis- and trans- i somers is not a necessary step as the 7~chloro derivatives of the.mixed epimers are useful per se. It is desi rable,, however,, to keep the content of trans- isomer high as this is the most active form. By carrying out the process with this in mind mixed epimeri'c products containing a ratio of trans- and cis-epimers of 3:1 to 1:5 can readi ly be obtained. By substituting the formaldehyde and acetaldehyde of Parts i_f and ^ by other oxo compounds of the formula R4R5C0, for example, prop i ona 1 dehyde, acetone, buty ral dehyde, isobutyl methyl ketone, benza 1 dehyde, phenyl-aceta 1 dehyde, hydroc i rtnama 1 dehyde, acetophenone, prop i ophenone, buty ophenone, 3-methyl-4-phenyl- 2-butanone, 2-methyl-5-phenyl-j5- pentanone, 3-cyc lopentaneprop iona ldehyde, eye lohexaneaceta 1 -dehyde, eye 1 oheptaneca rboxa 1 dehyde, 2, 2- d imethy 1 eye 1 opropane-aceta 1 dehyde, 2, 2-d imethy lcyclopropyl methyl ketone, cyclopentyl methyl ketone, cyclobutyl methyl ketone, eye 1 obutanone, cyclo-hexanone, and 4-methyl cycl ohexanone, and using the appropriate al kyl, cycloalkyl, or aralkyl 6, 8- d i deoxy- 6- (4- a 1 ky 1 , 4-cyclo-al kyl-, or 4- a ra 1 ky 1 - L- 2- py r ro 1 i d i neca rboxam i do) - 1- th i o- D-erythro-g-D-qalacto-octopyranoside, there are obtained the corresponding al kyl, cycloalkyl, and aralkyl 6, 8- d i deoxy- 6- ( 1-R4R5CH-4- al ky 1 , 4- cycl oa 1 ky 1 , and 4-aral ky 1- L-2-pyrrol i d ine-ca rboxam i do) - 1- th i o- D-erythro- - D- qa 1 acto- octopy ranos i de wh i ch Rydon reagent on treatment with b+e«y — by the procedure of Part A gives the corresponding alkyl, cycloalkyl, and aralkyl 7~halo-6,7,8- trideoxy-6- ( 1- R4 R5CH-4- a 1 ky 1 - , 4- eye 1 oa 1 ky 1 - , and 4-a ra 1 ky 1 - L-2- pyr rol i d i neca rboxam i do)- 1- th i o- a- D- qa 1 acto- octo-pyranoside where R4R5CH- i s propyl, isopropyl, butyl, and 4-methyl-2-pentyl ; benzyl, phenethyl, 3- pheny 1 p ropy 1 , 1-phenyl-ethyl, 1- pheny 1 p ropy 1 , 1- pheny 1 buty 1 , 3_methy 1 -4- pheny 1 -2-butyl , methyl, 2- (2, 2-d i methyl cycl op ropy 1 )ethy 1 , 1 - (2, 2-d imethy 1 - cycl opropyl )ethyl , 1 -cycl opentyl ethyl , 1 -cycl obutyl ethyl , cyclobutyl, .cyclohexyl, and -methyl cycl ohexyl . By using formaldehyde and acetaldehyde or other alkanals, for example, p rop i ona 1 dehyde, butyral dehyde, val eral dehyde, or caproic aldehyde with an alkyl 6,8-d i deoxy-6- ( -al kyl -L-2-pyrrol idi ne carboxamido)- 1 - thi o-D-erythro-a-D-gal acto-octopyranosi de where alkyl and -.al kyl are methyl, ethyl, propyl, butyl, pentyl, or hexyl preferred starting compounds of the formula (D-erythro ) wherein X is hydroxy /end R, HRi, and R3 are alkyl of not more than six carbon atoms, advantageously of not more than twelve carbon atoms in the aggregate are obtained which on treatment with Rydon. reagent by the procedure of Part A give compounds according to Formula XXI V where X is halogen' and R, HRi, and R3 are alkyl as given above. The 6,8-dideoxy compounds of Formula XXIV as characterized above are active ant i bacter i a 1 s comparable with l incomycin. The 7_ a 1 o- 6, 7*8- 1 r i deoxy compounds of Formula XXIV as characterized above have the same spectrum of antibacterial activity but are substantial ly more active. Representative compounds of Formula XXIV wherein X is (L-threo) bromine /are given in the following table: TABLE I BROMINE (L-threo) COMPOUNDS OF FORMULA XX WHERE X T ' R HRi R3 4A methyl trans-ethyl methyl 4B methyl c i s-ethy 1 methyl 4C (7-bromo-7-deoxy- 1 i ncomyci n) methyl t rans-p ropy 1 methyl 4D (7-b romo-7-deoxy- al 1 ol i ncomyci n) . methyl ci s-p ropyl methyl 4E (7=bromo-7-deoxy- 1 i ncomyci n E ) methyl trans-propyl ethyl 4F (7-b romo-7-deoxy- '■ ~ al 1 ol i ncomyci n E ) methyl ci s-p ropyl ethyl 4G ethyl trans-propyl methyl 4H ethyl ci s-p ropyl methyl 41 methyl trans-butyl methyl 4J methyl cis- butyl methyl 4K methyl t rans-p ropyl ethyl 4L methyl c i s-p ropy 1 ethyl 4M ethyl trans-propyl ethyl 4N ethyl ci s-p ropyl ethyl 40 methyl trans-butyl ethyl 4P methyl ci s-butyl ethyl 4Q methyl trans-pentyl methyl 4R methyl c i s-penty 1 methyl 4S ethyl trans-butyl ethyl 4T ethyl ci s-butyl ethyl 4U methyl t rans-pentyl ethyl 4V methyl ci s-pentyl ethyl 4W ethyl t rans-pentyl methyl 4X ethyl ci s-pentyl methyl 4Y methyl trans-hexyl methyl 4Z methyl cis-hexyl methyl 4AA butyl trans-propyl methyl 4AB butyl ci s-p ropyl meth l 4AC ethyl trans-pentyl ethyl 4AD ethyl c i s-pentyl ethyl 4AE butyl trans-butyl ethyl 4AF butyl cis-butyl ethyl 4AG butyl t rans-pentyl methyl 4AH butyl ci s-pentyl methyl 4AI cycl ohexy 1 trans-propyl methyl 4AJ cycl ohexy 1 ci s-p ropyl methyl 4AK butyl trans-pentyl ethyl 4AL butyl ci s-pentyl ethyl 4AM pentyl t rans-pentyl ethyl 4AN penty 1 ci s-pentyl ethyl Intermediates for the preparation of the above compounds correspond to the above table where (1 ) X is hydroxy; (2) R3 is hydrogen; (3) X is hydroxy and R3 is hydrogen; (4) X i s hydroxy and R3 is carbobenzoxy ; (5) X is bromine and R3 is carbo- benzoxy; (6) X i.s hydroxy, R3 is ca rbobenzoxy, and HRi and H in the 4-position are replaced by the yl idene group, Ri; (7) X is bromine, R3 is carbobenzoxy, and HRi and H in the 4-position are replaced by the yl idene group, Ri; (8) X is hydroxy, R3 is hydrogen, and HRi and H in the 4-position are replaced by the yl idene group, Ri; (9) X is bromine, R3 hydrogen, and HRi and H in the 4-position are replaced by the yl idene group, Rx; (10) X is hydroxy, and HRi and H in the 4-position are replaced by the yl idene group, Ri; and (11) X is bromine and HRi and H in the 4-position are replaced by the yl idene group, Ri.

When the l i neomycin of Part A is replaced by alkyl, cycloalkyl, or aralkyl 6-am i no-6, 8-d i deoxy- 1 -thio-D-eryth'ro-g-D-gal acto-octopy ranos i de there are obtained compounds of the formula wherein R is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, penta-decyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl, or any of the i some ric forms thereof, cyclopropyl, cyclobutyl, cycl openty 1 , cyclohexyl, cycloheptyl, cyclooctyl., 2-methylcy-clopentyl, 2, 3-dimethyl cycl obutyl , 2-methyl cycl obutyl , and 3- and cycl opentyl propyl ;/benzyl , phenethyl, 3-phenyl propyl , and -naphthyl methyl which can be acylated by Part IE _ the procedures of €^«πτ ^«- to form the corresponding 7-halo-7-deoxyl i ncomyci n analogs, for example, the compounds of Form- ula XXIV as given and illustrated above, as well as the intermediates therefor.

SXAMPLE 2. 7-Chloro-7-deoxylincomycin D iethyl 7-chloro-6 , 7,8-trideoxy-6- ( trans-4-prop.yl-L-2-pyrrolidinecarboxamido)-l-thio-L-threo-a-D- galacto-octopyranoside .

- Chlorine was bubbled into a suspension of llg. of triphenyl- phosphine in 200 ml. of acetonitrile until an uptake of 2.84g. was noted. The reaction mixture was cooled to 26° and 1.72g. of -demethyllincomycin hydrochloride (lincomycin D ' hydrochloride) added. The solution was stirred at ambient temperature for 18 hrs. After addin 10ml. of methanol, the solvent was distilled in vacuo. The residue was stirred with 100 ml. , of ethyl acetater-ether (1:1). The mixture was filtered and the residue partitioned between 15 ml. of water. The ethyl acetate was washed once with 10 ml. water and the combined aqueous extracts back-washed with ethyl acetate. The aqueous solution afforded a residue of 3.05 g« on lyophiliza- tion. This solid was dissolved in methanol, lOg. of silica gel added and the solvent evaporated, to yield a free flowing column of 100 g. of silica g l and the column eluted with chloroform-methanol, 4:1. The fractions containing the product, as monitored by TLC were combined and evaporated to giye a crude. material weighing 1.27g. This solid was dissolved in acetone and acidified with dilute hydrochloric acid. 7-Chlor0-7-deoxy-N-demethyllincomycin hydrochloride, m.p. 212-216°, weighting 0.73g.» was deposited on cooling.

Recrystallization from acetone-water gave 550 mg. of hydrochloride, m.p. 217-221° dec.

A nal. Calcd. for C- „H¾_Cl N0cS'HCl: — — — J. ( yd d d C, 45.62; H, 7.2; N, 6.26.

Found: C, 45.89; H, 7.66; N, 6.55. 7-Chloro-7-deoxy-N-demethyllincomycin hydrochloride is about 8 times as active as lincomycin against Gram-positive bacteria and at least '8 t ries as active Gram-negative bacteria. It is about 2 times as active as 7-chloro-7-deoxylincomycin.

This is unexpected because -demethyllincomycin is less active than lincomycin.

By substituting bromine for chlorine, 7-bromo-7-deoxy-N-demethyllincomycin was obtained. In place of triphenylphosphine there can be substituted triphenylphosphite. Also, in that case, a methyl halide can be used in place of halogen.

The lincomycin D was prepared as described in U.S. Patent 3,329,568. • c u e Page V; +109° '·,;. and the fol lowing analysis: . ... '.·"'■ ■·■·-·■ ' '.•rCal cd; for CasHsaNaOeS: C, 57.07; -H, 7.27; N, 5.23· "■',·,' / ■ ·.' . Found: C, 56.84; H, 7, 8; N/ 5.37- · "·' . Par B. 7-Chl oro-7-deoxy-N-demethyl -N-ca rbobenzoxyl i ncomyc i n.' One gram of N-demethyl -N-ca rbobenzoxyl i ncomyci n was added to a SO : u ion of t i phenyl phosphi ne dichloride p repa red by add i ng ' 710 mg . o f e !-o;*'ine to 2.88 g. of tri phenyl phosphi ne in 25 ml . - of oceton i t r i 1 e A f er 2'"nrs. , 10 ml. of methanol ' was added and the so^l vent ^evapora ted ..

'The residue was chromatographed over' 100 g. of sil ica gel using chlor-■of o rm-methanol (4:l) for elution. A fraction of 717 nig. wa s ob ta i ned · wh i ch was a mixture of 7-chl oro-7-deoxy-N-demethy 1 -N-carbobenzoxy-.1 i .n comycin and t r i pheny 1 phosph i ne oxide. It was used/di ectly, in the.' nex t part.' ■:' · · ■ '.■ " ...' " '■ j ^ ■ Part C. . 7-Chl oro-7-deoxy-N-demethyl 1 i ncomyci n hyd roch.lpr i de.- ■ Crude 7-chl oro-7-deoxy-N-carbobenzoxy 1 i ncomyci n from above' (717 mg ) was dissolved in 25 ml. of methanol and 250 mg, of '104 pal ladium on charcoal added. The mixture was shaken under 40 lbs. of hydrogen pressure for'2 hrs. The catalyst was removed by f i 1 t ra i on and the sol ve n c d i s t i 11 ed _i_n vacuo. The. residue was purified by chromatography ove r s i 1 i ca' gel (100 g~! oT sil ica gel ; methanol -chl oroform l'-A for el ut i on), to gi ve 103 mg, of 7-chl oro-7-deoxy-N-demethy 11 i ncomyc i n hyd rochl o r i de , This product was dissolved in acetqne and acj d i f i ed wi th di lute hydrochloric acid. ' Crystall ine 7-chloro-7-deoxy-N-demethyl 1 i ncomyc i n hydrochloride; melting at 227-229° and weighing 95 mg., was obta i ned „. Standard curve, _S„ lutea assay, indicated antibacterial ac t i v i y 1,7 x 7-chl oro-7-deoxyi i neomycin.

Instead of separating the c i s and t rans isomers, the mixture can be . treated by the procedure of "Par ts B and ϋ to give methyl N-(l-car-bobenzoxy- - trans- and ci s -propyl -L-prol yl ) -7-ch loro-7-deoxy;-a- th ί ο· I I n cps am i n i de and methy _l ~- (4- 1 rans - and ci s- propyl - L-prol yl j) - 7- .·' ■ chj oro-7 -deoxy-a- thi ol i ncosami ni'de." The co respondi ng , S- anid -ana' and the co responding 7-bromo analogs can similarly be prepared Al ernatively,' me thy 1 7-hal o -7- deoxy-a- th i ol i ncosami ni de, or; a ana 1 og thereof, can be acylated wi h, a cis- and trans-l-carbobenzox;y- -a1 kyl , 4-cyc 1 oa 1 ky 1 , or 4-aral kyl -L-prol ine. ' The f ol 1 owi ng example' is i l lustrative. ■ ··'. "· .'■ . ■■ . ·.■' ■■'..;' '' ; '■>.. . ; , EXAMPLE 5 .; ■ ' ': !'■■■;■'' ·:". ; '' · > ' : ;': --ΐ;:-;-; ■ ;-. - " Pari A. Me thy 1. - (l-ca rbobenzoxy- - rans- and c i s -prop l - L-proly.l ) -7-chl oro-7-deoxy-l i ncosami ni de... , · ·'!"■'■■·". ■.. '■ ■·.,■. ;'■··: H2.N deoxy-6-( rans-4-propyl-L-2-pyrrolidinecarboxamido )-3Ahio-L-threo-g-D-galacto-octopyranoside ) and the cis isomer thereof.

The methyl, epi-a-thiolincosaminide is obtained by-the hydrazinolysis by the process of U.S.Patent 3,179,595 of epilincomycin which is prepared as follows: red i j LUG LliCTtt fen , EXAMPLE & 8 A „ 3, -0-1 sop ropy 1 i dene 1 i n corn c i n A solution of, 9.8 g. of li neomycin in 150 ml. of acetone i s added to a solution of 9.8 g. of p- to luenes u 1 fon i c acid mohohydrate in 100 ml. of acetone with good stirring and avoidance of exposure to moisture, The mixture is sti rred at ambient temperature for 1 hour, after which 100 ml. of anhydrous ether is added and sti rring is continued in an ice-bath for 0.5 hour. The mixture is fi ltered and the solid is dried i n vacuo at 50° C; yield 12-35 g. (85.5$) of 3>4-0-i sopropyl i denel i ncomyci n p-toluenesu lfonate . An additional 1..15 g- (7·4#) can be recovered from the mother liquors by adding 350 ml. of anhydrous ether to the mother liquor from the previous fi ltering operation and chi lling the solution for 1 hour. The 14.5 g. so obtained are suspended i n 200 ml. of ether and shaken vigorously with 125 ml. of.5$ potassium bicarbonate solution. The aqueous layer is back-extracted with two 100-m 1 , „ port i ons of ether. The ether extracts are washed with 50 ml. of saturated sodium chloride solution and then fi ltered through anhydrous sodium sulfate.

The ether is evaporated under vacuum, leaving 7-9 g.. (73.1$) of 3,4-0- i sopropy 1 i dene 1 ί ncomyc i n which is dissolved in 25 ml . of ether acetate and. concentrated to about 10 to 15 ml. The concentrate i s a 1 lowed to stand at room temperature for several hours and then refrigerated overnight. The crystals are fi ltered from the solution and washed sparingly with cold ethyl acetate; yield 4-55 g ·' (42.2$) of 3*4-0- i sopropy 1 idene 1 i ncomyci n having a melt- 25 i ng point of 126-128° C, and an optical rotation of [a]D 101-102° (c, 1, methylene chloride) .

B . 7_dehyd ro-3, 4-0- i sopropy 1 i dene 1 i ncomyci n To a solution of 6 g. (0.0135 moles) of i sopropy 1 i dene 1 i n-comycin in 75 ml* of pyridine was added 12 g. (excess) chromic oxide. The solution warms up about 20° C. After one hour the mixture was added to a solution containing 250 ml. each of ethyl ether and ethyl acetate. This was then fi ltered and evaporated to a syrup, 8.4 g. This syrup was distributed in a 500-transfer counte rcu r rent distribution using the system, water:ethyl acetate e thano 1 : c.yc 1 ohexa ne ( 1 : 1 : 1:1), 7"Dehyd ro-3* 4 -0- i sopropy 1 i dene -li neomycin was isolated as the peak fraction from tubes 330-380, K = 2 5.

Anal. Calcd. for C3iH3eN20eS: C, 56.72; H, 8.16; N, 6.30; S, 7.21.

Found: C, 56,37; H, 7.62; N, 6.5I; S, 6.84.

C. 3* -0- I sopropy 1 idene-epi lincomycin To 1.6 g. of Craig-pure 7~dehyd ro-3*4-0- i sopropy.l i dene 1 i n-comycin in 75 nil. of methanol was added 400 mg. of sodium boro-' on a rotary evaporator. The residue was added to 25 ml. of water and extracted three times with 25 ml. each of methylene chloride. The extract was back-washed wi th 15 ml . of water, then dried over magnesium chloride and evaporated to dryness. The, residue, 1.4 g., was distributed in a 500"transfer countercu rrent distribution using the solvent system, water:ethyl aceta te : e t hano 1 : eye lohexane (1:1:1:1), and a single peak which fit the theoretical was observed at K=1.05. The material in tubes 240 to 280 was isolated as a syrup * Anal . Calcd. for C2 i H38N20eS : 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 c romatography (TLC) showed that this material consisted of two substances. One was 3* -0- i sopropy 1 i dene 1 i neomycin; the other 3*4-0- i sopropy 1 i dene-epi 1 i ncomyci n, which moved s 1 i ght 1 s lower · ' .

D . Ep i 1 i ncomyci n The syrup from Part C was stored at room temperature 5 hrs. in .a solution containing 60 ml. of 0.25 ϋ hydrochloric acid and 40 ml, of ethanol. It was then kept at 0° C. for 4 days. Following neutralization with sodium bicarbonate, it was evaporated to 25 ml ., then extracted with chloroform. The extract was washed wi th a li ttle 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. lutea. The residue was chromatographed on a 14" x 3/4" Florisi l (a s ynthet i c s i 1 i ca te of the type described in U.S. Patent 2,393*625) column which was eluted gradientl'y with solvent which varied con t i nuous 1 y f rom 100$ Ske 11 ysolve B (technical hexane) to 100$ acetone, The total volume was 5000 ml . The two 198 OA Fraction I : Tubes 55-6 (.40 ml. cuts) Ep i 1 i ncomyc i n.

Assay 0 meg. /ml.

Anal . Cal cd. for Ci8rl34N20e S: C, 50.92; H, 8.55; N, 6.60; S, 7.56.

Found: C, 50.19; H, 7.91 ; N, 6.05; S, 6.42.

Fraction I I : Tubes 73-104. Lincomycin.

Assay 950 mcg./mg.

Example 9. 7_B romo-7-deoxyep i 1 i ncomyci n The procedure of Example 1 substituting the l incomycin by ep i 1 i ncomyci n yields 7- b romo -7-deoxyepi 1 i neomycin of the formul a as the free base and as the hydrochloride.

By substituting l incomycin in this example by l incomycin analogs of Formula I I wherein Z, R, Rx, Rs, R3 of the Ac group are as given and il lustrated abovej the corresponding 7- ha lo --7-deoxyep i 1 i ncomyci n analogs of the formula XXVI I I wherein Z, R, Ri, R∑ and R3 of the Ac :group are obtained. All the compounds that have been described above, therefore, have thei r counterpart i n . the oppos i te con iguration, that is con- As figuration derived from the 7_epi form, an inversion is effected by the substi tuti on of the 7-hydroxy by a Rydon reagent, then the epi -compounds which have the L-threo configuration, are u-aa -ev&ui both the D-erythro and L-threo forms are obtained depending on whether the normal 1 i ncomyci ns (D-erythro) or the epi - 1 i ncomyci ns (L-threo) are used.

Wh i le the processes of the invention have been described with reference to the preparation of specific compounds, i t is to be understood that the process is broadly appli cable to compounds of Formula I I. wherein Ac and R are radicals which are not reactive wi th Rydon reagent. Thus, in the broad process according to the invention, Ac can be hydrogen or any acyl that is non-reactive with Rydon reagent and R can be any a 1 ky 1 , i rrespective of the number of carbons, or any other radical, for example, aralkyl like benzyl, naphth lmethy 1 , and benzhydryl, or like hydrocarbon radicals that are non-reactive with Rydon reagent.

Claims (1)

1. WHAT IS CLAIMED ISi .1- A compound of the formula wherein R is alkyl of not more than 20 carbon atoms or cycloaltkyl of 3 to 8 carbon atoms and Ac is the acyl radical of a ¾-substituted-L-2-pyrrolidlnecarboxylic acid of the formula selected from the group consisting of wherein Rj. is selected from the grou consisting of alkylidene of not more than 20 carbon atoms, cycloalkylidene of from 3 to not more than 8 carbon atoms, and aralkylidene of not more than 12 carbon atoms; HR^ and HR^ are alkyl of not more than 20 carbon atoms, cycloalkyl of from 3 to not more than 8 carbon atoms, and aralkyl of not more than 12 carbon atoms.a d R^ is selected from the group consisting of hydrogen, and a protective group P. A. 25OIO/II removable by hydro genoly sis or alkyl ; and Halo designates chlorine or brominjfc ~2~ A compound of the formula wherein R, HR^ , are lower, alkyl , R^. is hydrogen or lower alkyl and Halo designates chlorine or bromine* P. Α. 25010/ϊΙ A compound according to claim 2 wherein R is methyl, HR is propyl, and ( 3 is methyl. •-4- ^A compound according to claim 2 wherein R Is methyl, HR is propyl, and is hydrogen, .5- A compound according to claim 2 wherein R Is methyl, HRi i s propyl , and R3 is ethyl . A compound according to claim 2 wherein R is ethyl, HRi is propyl, and 1 R^ is methyl. -7- A compound according to claim 2 wherein R is ethyl, HRX is propyl, and is hydrogen. -8- · A compound according to claim 2 wherein R is ethyl, H i s propyl, and