NO123608B - - Google Patents

Description

Process for the preparation of 7-chloro-7-deoxy-1-thio-α-D-galacto-octopyranose ides.

The present invention relates to the production of new 7-chloro-7-deoxy-lincomycin and 7-chloro-7-deoxyepilincomycin, and analogues and isomers thereof.

The new compounds produced by the present process have the following general formula: where R is alkyl with up to B carbon atoms, optionally alkyl-substituted cycloalkyl with 3 - B carbon atoms or aralkyl with up to 12 carbon atoms, preferably not more than 8 carbon atoms, and Y is the acyl radical of a 4 -substituted-L-2-pyrrolidinecarboxylic acid of the formula:

where R^ is the alkylidene with up to B carbon atoms, R^ and R_ are the alkylene with up to B carbon atoms, and R^ is hydrogen, methyl or ethyl.

Examples of optionally alkyl-substituted cycloalkyl are cyclopropylcyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-methylcyclopentyl, 2,3-dimethylcyclobuty1, 2-methyl-cyclobutyl and 3-cyclopentylpropyl.

Examples of aralkyl are benzyl, phenethyl, 3-phenylpropyl and 1-naphthylmethyl.

Examples of alkylidene, cycloalkylidene and aralkylidene (R ^ ) include methylene, ethylidene, propylidene, butylidene, pentylidene, hexylidene, heptylidene, octylidene, and the isomeric forms thereof, cyclopropylidene, cyclobutylidene, cyclopentylidene, cyclohexylidene, cycloheptylidene, cyclooctylidene, 2- cyclopropylethylidene, 3-cyclopentylpropylidene, benzylidene, 2-phenylethylidene, 3-phenylpropylidene, and 1-naphthylmethylene.

The new compounds produced according to the invention

of formula I as well as other related compounds, can be prepared by replacing with chlorine the 7-hydroxy group in a compound of the formula:

where R and Y are as indicated above. The replacement is effected by stirring 1 mol of the starting compound of formula II with at least 3 mol of thionyl chloride in the presence of a solvent at room temperature for a long time, whereby a compound of the formula is formed:

where R and Y are as indicated above, whereupon the reaction mixture is heated at a temperature between 50° and 100° C to replace the 7-hydroxy group with chlorine, and the compound formed is then subjected to basic hydrolysis at pH approx. 11 to remove the 3,4-0-cyclic sulphite group, and that a compound obtained with the group = R^ is optionally hydrogenated catalytically in a manner known per se, and/or the compound obtained is optionally transferred to an acid addition salt of i and familiar way.

When e.g. a compound of formula 11A (that is, a compound of formula II where Y is the rest of the acid according to formula A) is used as starting compound, a new compound of formula IA is obtained. When this compound or the starting compound (formula 11A) is hydrogenated with a catalyst capable of causing saturation of an olefinic double bond, the process yields a compound of formula IB as a mixture of the cis- and trans-epimers of the formulas:

which, if desired, can be separated by countercurrent partitioning or chromatography.

Preparation of the starting materials used in the present process is described in Belgian Patent No. 667,94B, Spanish Patent No. 316,092 and U.S. Pat. patent No. 3,179,565.

The starting compounds of formula II are prepared by

to acylate a compound of the formula:

where R is as indicated above, with a derivative of a 4-substituted-L-2-pyrrolidinecarboxylic acid of the formula A or B. This acylation and similar acylations discussed here can be carried out by methods previously known for the acylation of amino sugars. The starting acid of formula A can be prepared by reacting a 4-oxo compound of the formula: where Z is a protecting hydrocarbyloxycarbonyl group which can be removed by hydrogenolysis, trityl, i.e. triphenylmethyl, diphenyl-(p-methoxy-phenyl)-methyl, bis-(p-methoxy-phenyl)-phenyl-methyl, benzyl or p-nitrobenzyl, with a Wittig reagent, e.g. an alkylidentriphenylphosphorane (see, e.g., Wittig et al., Ber., 87, 1348 (1954); Trippett, Quarterly Reviews, XVII, No. 4, p. 406 (1963)). Examples of hydrocarbyloxycarbonyl groups (Z) are tertiary butoxycarbonyl, benzyloxycarbonyl groups of the formula: where W is hydrogen, nitro, methoxy, chlorine or bromine, e.g. carbobenzoxy, p-nitrocarbobenzoxy, p-bromo and p-chlorocarbobenzoxy and phenyloxycarbonyl groups of the formula:

where W is hydrogen, allyl or alkyl with up to 4 carbon atoms, such as phenyloxycarbonyl, p-tolyloxycarbonyl, p-ethylphenyloxycarbonyl1

and p-allylphenyloxycarbonyl and the like.

When carrying out this method, the 4-oxo-L-2-pyrrolidinecarboxylic acid (formula C) is added to a freshly prepared Wittig reagent. The Wittig reagent used here can generally be represented by the formula:

where R ^ is as indicated above. These Wittig reagents are prepared by reacting an alkyl, cycloalkyl or aralkyltriphenylphosphonium halide with a base such as sodium amide, sodium or potassium hydride, or sodium or potassium metallate of dimethylsulfoxide and the like. For example, the removal of hydrogen halide from alkyltriphenylphosphonium halide yields alkylidenetriphenylphosphorane. The preparation of phosphoranes is discussed in detail by Trippett, Quart. Fox. XVII, No. 4, p. 406 (1963). The reaction is generally carried out in an organic solvent, such as benzene, toluene, ether, dimethyl sulfoxide,

te tr ah yd rbf uranium: or li kn end at : temperatures between 1 O0'. D andS ■ •': ••'<•. the reflux temperature of the reaction mixture. Thus:: éxt:"' is-:1 held product-,éti; 4-alkylidene-?, -■ 4-cycloalkylidene- or 4-alkylidene a 1-protected-i-i-L-i-proline having the 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 obtained in a conventional manner, such as by recrystallization, chromatography 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 liberation of the amino acids from such compounds.By hydrogenating an acid of formula D in the presence of a catalyst, e.g., platinum, which is capable of effecting saturation of a double bond, but which is incapable of to cause hydrogenolysis, a compound of the formula is obtained:

Platinum deposited on a support, e.g. carbon or an anion exchange resin such as 'Towex-1', a cross-buried polystyrene-trimethylbenzylammonium resin in the hydroxy form', - is suitable. If desired, the starting compounds of formula V can be acylated with the acids of formula C, D or E under formation of compounds IIC, respectively HD and HE. The compound IIC can then be transferred to a compound HD by treatment with a wit'tx'g-reag e rfs rt»'g' 'for'bi-hdtiWtr'-'HU-"" 'kiarP is hydrogenated-! to- - the compound 11E' by the methods indicated above. The hydrogenation of both the acid and :>s-<:>'<:>'and the trans-epimers, which can be separated by counterstrSmsf ording or chromatography. The starting acids of formula B, where R^ is hydrogen, are obtained when an acid of formula D or E is subjected to hydrogenolysis over a palladium catalyst, e.g. palladium on carbon. Likewise, the compounds of formula IID and 11E are transferred to compounds of formula HB where R 1 is hydrogen, by the same method. The starting acids of the formula B where R^Br is hydrogen, as well as compounds of the formula HB where R^ is hydrogen, can be converted respectively to compounds of the formulas B and HB where R^ is HR^ by the methods indicated above. The starting acids of formula A are obtained by treating an acid of formula D with hydrogen bromide in acetic acid to remove the Z group and then replace the N-hydrogen with an HR^ group by the above-mentioned method. Compounds of the formula IID and HE are transferred to compounds of the formula HA and HB, respectively, by the same procedure.

Some of the starting compounds of formula II are obtained by biosynthesis. Lincomycin, methyl-6,B-dideoxy-6-(trans-1-methyl-4-propyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside, is obtained as a work-up product of a lincomycin-forming actinomycete according to U.S. Pat. patent 3,086,912. It has the following structural formula:

where R and R^ are methyl and -R^H is propyl. Lincomycin B, methyl-6,8-dideoxy-6-(trans-1-methyl-4-ethyl-L-2-pyrrolidine-carboxamido)-1-thio-D-erythro-α-D-galactb-octopyranoside (formula VI wherein R and R^

is methyl and -R^H is ethyl) is also a work-up product of the same microorganism when grown according to the method of the U.S. patent 3,086,912. Lincomycin C, (5-ethyl-S-demethy1-lincomycin ), ethyl-6,8-dideoxy-6-(trans-1-methyl-4-propyl-L-2-pyrrolidinecarbox-amido)-1-thio-D -erythro-α-D-galacto-octopyranoside (formula VI where R is ethyl, -R^H is propyl and R^ is methyl) is obtained when the method

in U.5. patent 3,086,912 is carried out in the presence of added ethionine. Preparation of lincomycin C is also described in U.S. Pat. patents 3,306,892, 3,316,243, 3,359,163 and 3,380,992. Lincomycin D, methyl-6,B-dideoxy-6-(trans-4-p^opyl-L-2-pyrrolidinecarboxamido)-1-thio-D-e^ythro-a-D-galacto-octopyranoside (formula VI where R is methyl, -R^H is propyl

and R^ is hydrogen) is obtained when the fermentation according to the U.S. patent 3,086,912 is performed in the presence of added α-MTL, methyl-6-amino-6,8-dideoxy-D-erythrp-1-thio-α-D-galacto-octopyranoside, a compound obtained by hydrazinolysis of lincomycin according to U.S. Pat. Patent No. 3,179,595, N-demethyl-lincomycin B, methyl-6,8-dideoxy-6-(trans-4-ethyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-oc-D- galacto-octopyranoside (formula VI where R is methyl, -RH is ethyl and R is hydrogen) is formed, also when α-MTL is added during the fermentation according to the U.S. patent 3,086,912. Likewise, lincomycin K is formed, ethyl-6,B-dideoxy-6-(trans-4-propyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside (formula VI where R is ethyl, -R^H

is propyl and R^ is hydrogen, when the fermentation according to the U.S. patent no. 3,086,912 is carried out in the presence of added α-ETL, ethyl-6-amino-6,8-dideoxy-1-thio-D-erythro-α-D-galacto-octopyranazide, a compound obtained by the hydrazinolysis of lincomycin C , (5-ethyl-S,N-didemethyl-lincomycin, B). < Ethyl-6,8-dideoxy-6-(trans-4-ethyl-L-2-pyrrolidine-carboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside (formula VI

where R is ethyl, -R^H is ethyl and R^ is hydrogen) is also obtained when a-ETL is added during the fermentation ifSlge U.S. patent No. 3,086,912.

The above-described N-desmethyl products obtained when α-MTL and α-ETL are added in the fermentation process according to U.S. Pat. patent 3,086,912 are examples of compound 11B where R 1 is hydrogen. In the process described above, replacement of N-hydrogen atoms gives compounds of formula 11B where R^ is HR^, e.g. methyl-6,8-dideoxy-6-(trans-1-ethyl-4-propyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside, ethyl-6,8-dideoxy- 6-(trans-1-methyl-4-ethyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside, ethyl-6,8-dideoxy-6-(trans-1-ethyl -4-ethyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside and methyl-6,B-dideoxy-6-(trans-1-ethyl-4-ethyl-L-2 -pyrrolidinecarbox-amido)-1-thio-D-erythro-α-D-galacto-octopyranoside.

Lincomycin D or any other of the starting compounds of formula II having the D-erythro configuration can be transferred to the L-threo configuration by oxidizing the 7-hydroxy group to a 7-oxo group and reducing this to a 7-hydroxy -group.

A suitable procedure for this purpose is illustrated in the following reaction sequence:

For example, lincomycin is transferred by treatment with acetone in the presence

of p-toluenesulfonic acid to 3,4-0-isopropylidenelincomycin which on oxidation with chromic acid gives 7-dehydro-3,4-0-isopropylidenelincomycin, methyl-6,B-dideoxy-3,4-0-isopropylidene-6-(trans -1-methyl-4-propyl-L-2-pyrrolidinecarboxamida)-1-thio-D-glycero-α-D-galacto-octanopyranos-7-uloside, which on treatment with sodium borohydride is converted to 7-epilincomycin, methyl-6,B -dideoxy-6-(trans-1-methyl-4-propyl-L-2-pyrrolidine-carboxamido)-1-thio-L-threo-α-D-galacto-octopyranoside.

Any of the starting compounds of formula II having

a D-erythro configuration can be transferred to the corresponding L-threo configuration by this method.

As the biosynthetically 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 or lower glycosides. It is also of course desirable to convert some of the compounds of formula II or V to higher or lower glycosides. This can be effectively carried out by reacting the compound to be converted with a mercaptan of the formula R^SH where R^ is an alkyl group of up to 8 carbon atoms, but is different from R, and. cyclize the formed dimercaptal. For example, compounds of formula II when reacted with a mercaptan of the formula RgSH give dithioacetals of the formula: which on treatment with acid or on heating, either in the presence of acid or not, are recycled to a compound of the formula:

The method can be applied directly to any of the starting products of formula II, ie IIA, IIB, IIC, IID and IIE. The products formed can be subjected to hydrazinolysis to form compounds of the formula:

which can be N-acylated as described above, with acids of formula A, B, C, D and E to give compounds of formula XII. The method can also be applied to the starting compounds of formula V. For example, α-MTL is converted by treatment with ethyl mercaptan followed by cyclization as described above, to α-ETL.

The mechanism by which thionyl chloride causes replacement of the 7-hydroxy group with chlorine is not fully understood, but is such that a change in configuration occurs. Thus, a 7-hydroxy compound of the D-erythro configuration gives a 7-chloro compound of the L-threo configuration. For example, certain 7-chloro-7-deoxylincomycin which is prepared from lincomycin having the D-erythro configuration has the L-threo configuration. However, it is provisionally established that the process goes through a number of intermediate products according to the following reaction sequence: where Y and R are as indicated above. The intermediate XVI Ia is the 3,4-cyclic sulphite of the starting material II and has the following formula:

where X is hydroxy. The intermediate XVIIb is the corresponding 3,4-cyclic sulfite of the 7-chloro compound (formula I) and has the formula

XVIIa where X is chlorine. The intermediate XVIII may be a bis-sulfite of the 3,4-cyclic sulfite of the 7-chloro compound having the formula:

If desired, each of the three intermediate products Vila, VHb and VIII

are isolated in the course of the reactions. However, all that is necessary to effect replacement of 7-hydroxy with chlorine is to mix the starting compound of formula II, preferably in the form of an acid addition salt, e.g. the hydrochloride, with thionyl chloride, preferably in the presence of an inert solvent, under gentle heating, preferably at reflux temperature, until the desired yield of the 7-hydroxy group with chlorine is effected. Preferably, the reaction is carried out in

an inert atmosphere, e.g. under nitrogen. Carbon tetrachloride can be used effectively as a solvent, but other inert solvents, such as chloroform, methylene chloride, ethylene chloride, ether,

benzene and the like can also be used. A satisfactory method is to stir the reaction mixture at room temperature for a considerable time, e.g. from approx. 1 to 1B hours, or as long as is necessary to obtain a reasonably clear solution, and then raise the temperature to between 50 and 100° C., e.g. to the reflux temperature (77° C for carbon tetrachloride). After the reaction is complete, usually after heating at reflux for approx. 1 to 5 hours, the reaction mixture is allowed to cool, preferably under nitrogen. Any material that is secreted during cooling is collected and tSrres. The solvent is removed by vacuum distillation at a temperature in the liquid preferably below approx. 35° C and the material which separates is collected and dried and treated with ethanol to convert any remaining sulphite intermediates to the desired product. The collected material can then be further purified by solvent extraction and/or recrystallization and can be recovered either as the free base or as an acid addition salt.

The ratio between the reagents can vary greatly. Stoichiometrically, however, at least three moles of thionyl chloride are required for each mole of starting material. A larger amount can be used, but usually it is not necessary or desirable to use more than approx. 10 times profit. Preferably, a surplus of approx. 2 to 3 times. The amount of solvent is not critical and can vary greatly as usual in the art. Usually, from approx. 15 to approx. 30 volumes of solvent for each part of solid starting compound should be sufficient. However, the ratio of solvent to thionyl chloride is important because of the solubility of the product in thionyl chloride. If the ratio of solvent to thionyl chloride (volume/volume) is high, the desired product precipitates upon cooling the reaction mixture and the preparation of the product is simplified. For example, with carbon tetrachloride, a mixture of the products will precipitate directly upon cooling the reaction mixture if the volume ratio of carbon tetrachloride to thionyl chloride is kept above approx. 10 to 1.

The compounds of formulas IA, IB, I1A, IIB and V exist either in the protonated or non-protonated form depending on the pH of the environment. When the protonated form is meant, the compound is referred to as an acid addition salt and when the non-protonated form is meant, it is referred to as the free base. The free bases can be converted to stable acid addition salts by neutralizing the free base with a suitable acid below approx. pH 7.0 and preferably to approx.

pH 2 to 6. Suitable acids for this purpose include hydrochloric acid, sulfuric acid, phosphoric acid, thiocyanic acid, fluorosilicic acid, hexafluoroarsenic acid, hexafluorophosphoric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid, fumaric acid, 4,4'-methylenebis-(3-hydroxy-2 -naphthoic acid, sulfuric acid, palmitic acid, mucic acid, camphoric acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, lauric acid, stearic acid, salicylic acid, 3-phenylsalicylic acid, 5-phenylsalicylic acid, 3-methylglutaric acid, ortho-sulfobenzoic acid, cyclopentanepropionic acid, 1,2-cyclohexanedicarboxylic acid, 4- cyclohexanecarboxylic acid, octadecenylsuccinic acid, octenylsuccinic acid, methanesulfonic acid, benzenesulfonic acid, heliantic acid, Reinecke's acid, dimethyldithiocarbamic acid, cyclohexylsulfamic acid, hexadecylsulfamic acid, octadecylsulfamic acid, sorbic acid, monochloroacetic acid, undecylenic acid, 4 '-hydroxyaZobenzene-4-sulfonic acid, octodecylsulfuric acid, pictic acid, benzoic acid, cinnamic acid and similar acids.

The acid addition salts can be used for the same purpose

as a free base or they can be used to purify this. For example, the free base can be transferred to a water-insoluble salt, such as picrate, which can be subjected to purification treatment, e.g. solvent extractions and washings, chromatography, fractional liquid-liquid extractions and Icrystallization, and then used to regenerate the free base form by treatment with alkali or to prepare another salt by. metathesis. Or the free base can be converted into a water-soluble salt, such as the hydrochloride or sulfate, and the aqueous solution of the salt can be extracted with various water-immiscible solvents for regeneration of the free base form by treatment of the thus extracted acid solution or transferred to another salt by metathesis. The free bases of formulas IA and IB can be used as buffers or antacids. The connections

of formula I reacts with isocyanates to form urethanes and can be used to modify urethane resins. The thiocyanic acid addition salt, when condensed with formaldehyde, forms resinous materials useful as pickling inhibitors according to U.S. Pat. patents 2,425,320 and 2,606,155. The free bases are also good carriers for toxic acids. For example, fluorosilicic acid addition salts are useful as mole impregnating agents according to U.S. Pat. patents 1,915,334 and 2,075,359, and hexafluoroarsenic acid and hexafluorophosphoric acid addition salts are useful as parasiticides.

Close analogues of 7-chloro-7-deoxy-lincomycin, that is

say where -R^H is cis- or trans-alkyl with up to 8 carbon atoms,

R^ is methyl or ethyl, R is alkyl with up to 8 carbon atoms, has antibacterial properties, and some are comparable to or superior to lincomycin and can be used for the same purposes as lincomycin. The corresponding compounds where R is hydrogen have similar antibacterial properties, and also have improved Gram-negative activity. Thus, 7-chloro-6,7,B-trideoxy-6-(trans-1-ethyl-4-butyl-L-2-pyrrolidinecarboxamido)-1-thio-L-threo-a-D-galacto-octopyranoside hydrochloride exhibits B times the activity of lincomycin against gram-positive bacteria and 16 - 64 times that of lincomycin against gram-negative bacteria. Other analogues and isomers have similar antibacterial properties, but to a lesser extent and can be used for the same purposes as lincomycin where larger amounts are not contraindicated.

For example, by the present method prepared: 7(S)-chloro-7-deoxylincomycin free Lase, a yellow amorphous solid, 7(S)-klOx'-7-cieoxylinr.amycin-hyJrokJ.crj d-solvate with 1 molecule of water .

Analysis - calculated for C.0H__CIN_Dr5«HC1*H_0: C 45.16;

1B 33 2 5 2

H 7.37; Cl 14.82; N 5.86; S 6, 10; h 2 O 3.77; Found: C 44.70;

H 7.65; Cl 14.27; U 5.78; 5 6.45?Ho0 3.85%.

Activity: Approx. 4-6 times lincomycin. Antibacterial spectrum:

As for lincomycin.

7(S)-chloro-7-d«oxyincomycin B hydrochloride.

Analysis - calculated for C ,^\\ yZl^^ Q^ S: C 45, 63;

H 7.21; M 6.26; S 7,18; Cl 15.05, Found: C 45.25; H 6.62; N 5.95;

S 7,10; Cl 16.45%. Corrected for 7.90 H20.

Methy-7-chloro-6,7,8-trideoxy-6-(l-methyl-trans-4--penyl-L-2-pyrrolidinecarboxamido)-1-thio-L-threo-a-E-galacto--oetc- pyranoside hydrochloride,

Analysis calculated for C^h^gC^N^O^S: C 47.33; H 7.98; N 5.52;5 6.70; Cl 14.82. Found: C 46.73; H 8.00; N 5.3-1; 5 6.26; Cl 13.91%. Corrected for 3.55% h^O.' Methyl-7-chloro-6,7,8-trideoxy-6-(trans-1-mcthyl-4-hexyJ.-L-2-pyrrolidinecarbox£3mido)-1-thio-L-threo-rt-D-gallateQ -octopyranoside hydrochloride,

Analysis - calculated for C2^H^gCl^N^OpS: C 47.33;

H 7.94; N 5.52; 5 6.32; Cl. 13.97. Found: L 46.73; H 8.00; N 5.34;

S 6.26; Cl 13.91% Corrected for 3.84% tt?0.

7 ( S )-chloro-7-deoxylincomycin C-liydrochloride,

Analysis - calculated for C 1 rjH^Cl^^S: C 4 7.99;

H 7.63; N 5.8?; 5 6.74; Cl 14.91. Found: C 43.95; H 7.81; N 5.41;

S 6.48; Cl 14.74%.

7(5)-Chloro-7-deoxy-N-dep,ethyl'incomycin-iiyl chloride,

melting point 217 - 221° C. split.

Analysis - calculated for C ,H_ . C1N„Cl5•HC1: C 45.62%;

J J 17 3 in 2 5

H 7.2%; N 6.26%; 5 7.175b. Found: C 45.89%; H 7.66%; N 6.55%;

S 7.17%.

Crystalline 7(S)-chloro-7-deoxy-i\]-dKnjsthyllinramyciri hydrochloride, melting point 227 - 229° ,C.

Methyl-7-chloro-6 , 7 , B-trideoy.y-6-(trans- and cis-4_

propyl-L-2-pyrrolidine-carboxamido)-1-thio-L-threo-α-D~cialacto-octopyranoside hydrochloride, melting point 226 - 234° C.

Analysis - calculated for C ,j ^H-^Cl-j^^- : C 45.63;

H 7.21; N 6.26; Found: C 45.85; H 7.51; N 5.86%. Corrected for 3.91% H20.

Methyl-7-chloro-6,7,B-trideoxy-6-(tran<3-4-bu+.y i-l.-2-pyrrolidinecarboxamido)-1-thio-L-threo-a-D-galacto-octopyranoside hydrochloride as a leathery yellow amorphous solid with mine L B times the activity of lincomycin.

Met hy 1-7-chloro r-6 , 7, B-trideoxy-6-(trans oy c.i s-4-pE-in tyi-L-2-pyrrolidinecarboxamido)-1-thio-L-thieo-a-D-galactc -oc topyranoside hydrochloride. Melting point 222 - 223° C. (splint.), jajn + 139°

(H 2 O).

Analysis - calculated for C9H<3>6C<12>f<g>2D5S : C ^f55'^»

H 7.63%; N 5.89%; S 6.74%. Found (corrected for 0.58% water):

C 47.85%; H 7.64%; N 6.01%; 5 6.B0%.

Methyl-7-chloro-6,7,8-trideoxy-6-(trans and cio--4-hexyl-L-2-pyrroliditecarboxamido)-1-th.i.o-L-threo-a-D-gnlncto-octo-pyranos 'iclihydrocl'6r'id'V-'; ' Melting point é;V9"2 r;22 V5 E'i'--!' !-1:"T;

l' : Analysis - calculated :- f or? Z^ qH^ qCI^ P^. ' > t'' AS, '07;s:a :'!

H 7.02; N 5.72; 5 6, 55 ; Cl 14.49. Canned: C 49, U7r H-7.64;; N 5;;3?t '

5 6,-51; Cl 14.41%. i •:} Methyl-7-chloro-6, 7, B-tridebxy-6- (trans and cis-4-bctyi-"-' L - 2 - p y r i:b 1 i d i n c ri r b 6 x a m i d i r) - 1 -1H i r. - L -1 h r e b - ei - D - g a i a ct o -oc tapyranofide hydrochloride, ' melting point 201 - 203° C. ' Methyi-7-klcr-6,7,B-trideoxy-6-(trans and cis-4-e t h yl- Il -!2 - p y r r o 1 idirc a'r'b'6 x åm i d d) -1 -thio-L-thréo-iz-D-galacto-octu-pyranoside hydrochloride, melting point 2' 4'Q - 242° "'C i"-1'" • ; Ariaiyse -'calculated for C ^ ^-H^gC^f^C1^ : C 44.33; H 6.9B; N 6.47; 5 7.4B. Found: C 44.21; H 7.25; N 6.00; S 7.26%. - thio-L-thfeb-a-D:-i-g alacto-octopyranoside-hydrbchloride-,: as a "leather-yellow amorphous solid with 8 times the activity of lincomycin' against gram-positive bacteria and 16 - 64 times the activity of lincomycin against gram- negative bacteria. Analysis - calculated for C2gh<l>2~Cl2N2rI^S: C 49.07; H 7.82; N 5.<f>7<;>2;''S 6.55';° Cl-i'4-r4i9V' Found: C 48.36; H 8(03; N 5.56';A S 6.43; Cl 15->Oi6%i.<;>,<;!V:>Corrected' fW b ,"02%^ 0V;'; '<; >" Methyl-7-chloro-6,7,B-trideoxy-6-(cis-1-ethyl-4-buty1-L-2-pyrolidinecarbbxarrrido-)-'1'iitTiio-'L-t1h'reo' -a'-D-galacto-octopyranoside-hydroYlbrid n m'e1d s aitrme'i an t'i'b'ak-ter'ieiller 'sp"ek*trum , "~

Analysis - Found: C 48.91; H 7.91; M 6.14; 5 5.97;

Cl 14.80%. Corrected, for 5.37% H^O.

7 ( S )-chloro-7-deoxy-f\i-ethyllincomycin-D-hydroxyl or id ,

Analysis - calculated for C.nH,DC1_N_0_5: C 47.99;

IN; Yes. <cb

H 7.63; N 5.89;' S 6.74; Cl 14.91. Found: C 48.20; H 7.81; N 6.07;

5 6.48; Cl 15.05%. Corrected for 4.57% h^O. Methyl-7-chloro-6,7,8-trideoxy~6-(trans-i-ethyl-4-hexyi-L-2-pyrrGlidincarboxamido)-1-thio-L-thr?.u-a-n-galacta-ccto-pyranoside -hydrochloride, Analysis - calculated for V.^^ H ^^ Cl^ i ^ U^ 5: C 5 1.05; H 8.18; N 5.41; 5 6.20; Cl Kt,70. Found: C 50.85; H 7.26; U 5.47; 5 6.79; Cl 13.43%. Corrected for 3.38% h^O.

Methyl-7-chloro-6,7,0-trideaxy-6-(i-isopropyl-4-penyl-L.-2-pyr rolidinc arbox amido ) - 1 -t hreo-L-1 hr eo-er -D - g alarta-oc tapy r an os id hydrochloride, melting point 77 - 00° C.

Methyl N-(L-prolyl)-7(S)-chloro-7-deoxylinco:;namine hydrochloride,

Analysis - calculated for C /j'^26^^2^2^5^: *~ ^

H 6.46; N 6.91; S 7.91; Cl 17.49. Found: C 41.74; IN! 7.01; ride 7.05;

S 8.04; Cl 17.36%.

7(R)-chloro-7-deoxylincomycin hydrochloride,

Analysis - calculated for C gH^^Cl^N^O.-S: C 46,C5;

H 7.43; N 6.07; 5 6.95; Cl 15.37. Found: C 46, 65 ; H 7.62; Nine 6.23;

5 6.80; Cl 15.35%. Corrected for 4.06% h^O.

The following examples illustrate the method and the products produced -by; this. Parts and percentages are given by weight and solvent ratios are given by volume where not otherwise. especially directed.

Example 1

7- chloro- 7- deoxvlincomycin fm ethyl- 7- chloro- 6. 7. B- trideoxy- 6-( trans - 1-methvl- 4- propyl- L- 2- pvrrolidinecarboxamido)- 1- thio- L- threo- α-D-qalacto-octopyranoside.

A. The free base

A suspension of 221.0 g (0.5 mol) of lincomycin hydrochloride in 5 L of carbon tetrachloride was stirred well at 25° C. under

nitrogen. 900 ml of thionyl chloride was added at once and stirring continued for 2 hours. During this time the solid dissolved and a clear solution was formed. The reaction mixture was heated under reflux for 2 hours, the heat source was then removed and nitrogen was bubbled into the amber solution until the temperature of the reaction mixture dropped to 25° C. Approx. 4 1 liquid was removed by vacuum distillation at a liquid temperature of below 35° C. The yellow solid that precipitated during this distillation was collected

and dried up. This solid was dissolved in approx. 300 ml of methanol, cooled to 25° C, made basic (pH 11) with dilute aqueous 2N sodium hydroxide solution, diluted to approx. 1200 ml with water and extracted well with ether. The ether extracts were combined, washed with a small amount of water, dried over anhydrous magnesium sulfate and filtered. Evaporation of an average portion of the combined ether extract gave 7-chloro-7-deoxylincomycin-free base as a yellow,

amorphous solid.

B. The hydrochloride

Addition of hydrogen chloride gas to the filtrate from Part A precipitated the precipitation of 7-chloro-7-deoxylincomycin hydrochloride which was separated and recrystallized from ethanol and ethyl acetate. A 32% yield of white, crystalline 7-chloro-7-deoxylincomycin hydrochloride containing approx. 1 molecular part of water was obtained. Analysis, calculated for C gH^C11^O^S•HC1*H^O: C 45.18; H 7.37; Cl 14.82; NINE 5.86; S 6.70; h^O 3.77. Found: C 44.70; H 7.65; Cl 14.27; N 5.78; S 6.45; H 2 O 3.85.

Activity: approx. 4 to 8 times lincomycin. Antibacterial spectrum: same as lincomycin....

Example 2

7- chlorine- 7- deoxylincomvcin- free base>

The procedure in Example 1, part A, was repeated except that methylene chloride was used instead of ether as extraction solvent and the combined extracts were filtered and evaporated to dryness. A 64% yield of 7-chloro-7-deoxylincomycin-free base was obtained as a yellow, amorphous solid. 15 g of this amorphous solid was adsorbed onto 30 g of silica gel in methylene chloride, dried and sieved onto a 75 mm diameter column of 1500 g silica gel. The column was then eluted with a mixture of methanol and chloroform in a ratio of 1:19, in 200 ml<1>s portions after a 2 1 course. Fractions 26, 27 and 28 were combined and evaporated to dryness to give 1.04 g of substantially pure 7-chloro-7-deoxylincomycin free base as an amorphous solid having an antibacterial spectrum of the same range and size as the 7-chloro-7-deoxylincomycin hydrochloride from Example 1.

Example 3

7- chloro- 7- deoxylincomycin C phethyl- 7- chloro- 6, 7. B- trideoxy- 6-( trans- 1-meth. vl- 4- propyl- L- 2- pyrrolidinecarboxamido)- 1- thio- L- threo- a- D-qalacto- octopyranoside^.

A. Chlorine substitute. John

A suspension of 1 g of lincomycin C hydrochloride in 25

ml of carbon tetrachloride and 4.5 ml of thionyl chloride were stirred under nitrogen for 2 hours at 25° C. A clear solution was obtained in the course of 15 minutes. The reaction mixture was then heated under reflux

for 2 hours and evaporated to dryness under vacuum to give a yellow solid which was dried under vacuum at 40° C. for 18 hours. The product was then dissolved in approx. 15 ml of hot ethanol, made basic with sodium hydroxide as in example 1, and diluted to 300 ml with water. The water solution was extracted 5 times with 100 ml portions of ether. The ether extracts were combined, dried over magnesium sulfate, filtered, saturated with hydrogen chloride gas and evaporated to a brown residue which, on recrystallization 2 times from a mixture of ethanol and ethyl acetate (dissolved in a minimal amount of ethanol and ethyl acetate added to bleach) gave 200 mg of 7- chloro-7-deoxylincomycin C hydrochloride as white crystals which had the same activity and spectrum as the 7-chloro-7-deoxylincomycin hydrochloride of Example 1.

Example 4

Methyl- 7- chloro- 6. 7. B- trideoxy- 6-( trans- 1- alkvl- 4- butyl- L- 2- pyrrolidine- carboxamido)- 1- thio- L- threo- a- D- qalacto- octopyranosides.

where R is methyl or ethyl; , 7y.-a r. xd \ >x. l > p.f. ::■ ni•.■■■ <

Part A ,. ^

One,- suspension j on ay 14 6c. mg methylr6:y8rdidee>xyT-6^;(trans-1-ethyl-4-butyl-L-2-pyr,rrolidinGarboxamido)^1-thio-D^érythro^a-D-galacto-octopyranoside free base in 3 ml of carbon tetrachloride and 0.7 ml of thionyl chloride was stirred at 25° C until a clear solution was obtained (about 15 minutes) and allowed to stand at 25° C for 2 hours. The reaction mixture was then heated under reflux for 2 hours and then evaporated to dryness under vacuum to a yellow solid which was worked up as described in Example 3. However, the product did not crystallize so the crystallizing solvents were evaporated, yielding 17 mg methyl-7-chloro-6,7,B-trideoxy-6-(trans-1-ethyl-4-butyl-L-2-pyrrolidinecarboxamido)-1-thio-L-threo-α-D-galacto-octopyranoside hydrochloride as a light brown, amorphous solid.

Instead of using the cis-epimer, methyl-7-chloro-6,7,B-trideoxy-6-(cis-1-ethyl-4-butyl-L-2-pyrrolidinecarbox-amido)-1-thio-L -threo-α-D-galacto-octopyranoside hydrochloride with the same antibacterial spectrum.

By using the 1-methyl analogues instead, methyl-7-chloro-6;,7,8-trideoxy-6-(cis- and -trans-1-methyl-4-butyl-L-2-pyrrolidinecarboxamido)-1 was obtained -thio-L-threo-α-D-galacto-octopyranoside hydrochlorides.

The cis- and tra.ns-epimers used as starting material

in the above example was produced as follows:

B. 4-butylidene-1-carbobenzoxy-L-proline and its cyclohexylamine salt. 19 g of sodium hydride as a 53% suspension in mineral oil was heated with 350 ml of dimethylsulfoxide at a temperature of 70-75° C until the reaction was complete (approx. 30 minutes).

After cooling to 32°C, 16.2 g of butyl-triphenylphosphonium bromide was added, and the resulting reaction mixture was stirred for 1 hour to ensure complete reaction. A solution of 26 g of 4-keto-1-carbobenzoxy-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 was added. This mixture was washed twice with 700 ml<1>s portions of ether and the ether was discarded after back-extraction 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 500 mL portions of ether. The combined ether extracts were washed successively with 250 ml water, three 250 ml portions of saturated aqueous sodium bisulfite and 250 ml water, and dried over anhydrous sodium sulfate. Evaporation of the solvent under vacuum gave 24 g of an oily residue which was 4-butylidene-1-carbobenzoxy-L-proline.

This residue was dissolved in 31 ml of acetonitrile and treated with 18 ml of dicyclohexylamine and cooled in a refrigerator. The crystals were collected, washed with acetonitrile and dried in vacuo,

whereby 21 g (46.8%) of crystalline dicyclohexylamine salt was obtained which melted at 136 - 140° C. After two recrystallizations from acetonitrile an analytical sample was obtained which melted at 142 - 144° C

and had a rotation of Md ~4° (c = °'99'CHC13>'

Analysis, calculated for C^^H^^<N>^<O>^:

C 71.86; H 9.15; N 5.78.

Found: C 71.69; H 9.30; N 5.74. 10 g of the dicyclohexylamine salt of 4-butylidene-1-carbobenzoxy-L-proline was shaken with ether and an excess of 5% aqueous potassium hydroxide until no solid substance remained. The layers were separated and each was backwashed. The aqueous, alkaline layer was combined with the backwash from the ether layer and acidified with 4N hydrochloric acid. The mixture was extracted repeatedly with ether and the ether extracts were combined, dried over sodium sulfate and evaporated in vacuo to give 6.3 g (93%) of 4-butylidene-1-carbobenzoxy-L-proline as an oil.

C. 4-Butyl-l-carbobenzoxy-L-proline.

The oil from part B was hydrogenated in 200 ml of methanol

□ v/er 2.1 g of 10% platinum on "Dowex-1" catalyst under 2.8 kg/cm<2>hydrogen pressure. The catalyst was removed by filtration and the filtrate was evaporated to give 6.3 g of 4-butyl-1-carbobenzoxy-L-proline as an oil. The product contained approx. 2 parts cis-4-butyl-1-carbobenzoxy-L-proline for every part trans-4-butyl-1-carbobenzoxy-L-proline.

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

By replacing the butyltriphenylphosphonium bromide in part

B with other substituted triphenylphosphonium bromides where the substituent is methyl, ethyl, propyl, pentyl, hexyl, heptyl, pctyl,

and the isomeric forms thereof, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-cyclopropylethy1 and 3-cyclopentylpropyl, benzyl, phenethyl, 3-f enylpropyl and 1 -n af thy Ime thy 1, the corresponding 4-alkylidene is obtained -p, 4-cycloalkylidene- and 4-aralkylidene-1-carbobenzoxy-L-prolines and the corresponding 4-alkyl-, 4-cycloalkyl- and 4-aralkyl-1-carbobenzoxy-L-prolines. When e.g. the butyltriphenylphosphonium bromide is substituted

with ethyl-, propyl-, isobutyl-, pentyl- and hexyl-triphenyl-1-phosphonium bromides, 4-ethylidene-1-carbobenzoxy-L-proline, 4-propylidene-1-carbobenzoxy-L-proline, 4-isobutylidene- 1-carboxybenzoxy-L-proline, . 4-pentylidene-1-carbobenzoxy-L-proline and 4-hexylidene-1-carbobenzoxy-L-proline and cis- and trans-4-ethyl-1-carbobenzoxy-L-proline, 4-propyl-1-carbobenzoxy-L -proline, 4-isobutyl-1-carbobenzoxy-L-proline, 4-pentyl-1-carbobenzoxy-L-proline and 4-hexyl-1-carbobenzoxy-L-proline. D. Methyl- 6- amino- 6. B- dideox. v- 1- thio- D- erythro- a- D- qalacto- octopyranoside ( a- MTL).

A solution of 40 g of lincomycin free base (U.S. patent 3,086,912) in 20 ml of hydrazine hydrate (98 - 100%) was refluxed 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 α-MTL free base after drying in vacuum at room temperature was 21 g (84%).

Recrystallization was carried out by dissolving α-MTL free base in hot dimethylformamide and adding an equal volume of ethylene glycol dimethyl ether.

Methyl-6-amino-6,8-dideoxy-1-thio-D-erythro-α-D-galacto-octopyranoside free base has a melting point of 225 - 228° C, an optical rotation of [gQ +276° (c = 0.768 , water) and a pHa' of 7.45.

Analysis, calculated for CgH^gNO^S:

C 42.7; H 7.56; N 5.53; S 12.66.

Found: C 42.6; H 7.49; N 5.75; 5 12.38.

By replacing lincomycin with other alkyl- or cycloalkyl- or aralkyl-6,B-dideoxy-6-(trans-1-methy1-4-propyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D- galacto-octopyrano sides where alkyl is ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and

the isomeric forms thereof, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-methyl-cyclopentyl, 2,3-dimethylcyclobuty1, 2-methyl-cyclobutyl and 3-cyclopentylpropyl and aralkyl is benzyl, phenethyl, 3-phenylpropyl and 1-n.aphthyl-methyl, the corresponding alkyl-, cycloalkyl- and aralkyl-6-amino-6,8-dideoxy-1-thio-D-erythro-a-D-g alacto-octopyranosides are obtained. For example by replacing the lincomycin with ethyl-, propyl-, butyl-, pentyl- and hexyl-6,8-dideoxy-6-(trans-1-methyl-4-propyl-L-2-pyrrolidinecarbox-amido)-1-thio -D-erythro-α-D-galacto-octopyranosides, ethyl-6-amino-6,8-dideoxy-1-thio-D-erythro-α-D-galacto-octopyranoside is obtained,

propyl-6-amino-6,8-dideoxy-1-thio-D-erythro-α-D-galacto-octopyranoside, butyl-6-amdnD-6,8-dideoxy-1-thio-D-erythro-α-D-galacto- octopyranoside, pentyl-6-amino-6,8-dideoxy-1-thio-D-erythro-α-D-galacto-octopyranoside and hexyl-6-amino-6,8-dideoxy-1-thio-D-erythro-α-D- galacto-octopyranoside.

E. Methvl- 6. 8- dideoxv- 6-( 1- carbobenzoxy- 4- butvl- L- 2- pvrrolidine-carboxamido)- 1- thio- D- erythro- a- D- qalacto- octopravnoside free base.

To a solution of 6.3 g of 4-butyl-1-carbobenzoxy-L-proline (the oil from part B) in 175 ml of distilled acetonitrile cooled to 0° C was added 3.46 ml of triethylamine followed by 3.34 ml of isobutyl- chloroformate. The mixture was stirred at 0°C (+ 3°) for 15 minutes. A solution of 6.2 g of oc-MTL free base from Part C in 85 mL of water was added and the reaction mixture was stirred at 0°C for 0.5 hours and at 25°C for 1 hour. The reaction product was then filtered off and dried, whereby 4.57 g (37.7%) of methyl-6,8-dideoxy-6-(1-carbobenzoxy-4-butyl-L-2-pyrrolidinecarboxamido)-1-thio- D-erythro-α-D-galacto-octopyranoside free base. The mother liquor was concentrated in vacuo and an additional 4.25 g (35.2%) of product was recovered. Recrystallization from acetonitrile gave crystals of methyl1-6,8-dideoxy-6-(1-carbobenzoxy-4-butyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside free base which melted at 194 - 196° C. Another recrystallization from acetonitrile gave an analytical sample with melting point .195.5 - 200° C, C«3D+ 111° (c = 0.98, MeOH).

Analysis, calculated for C^^H^QN^OgS:

C 57.75; H 7.46; N 5.13; S 5.93.

Found: C 57.58; H 7.16; N 5.50; 5 6.07.

F. Methyl- 6. 8- dideoxv- 6-( 4- butvl- L- 2- pyrrolidnecarboxamido)- 1- thio-D- erythro- a- D- qalacto- octopravnoside- hydrochloride.

'A solution of 7.B g methyl-6,8-dideoxy-6-(1-carbobenzoxy-4-butyl-L-2-pyrrolidiicarboxamido )-1 - thio-D-erythro-α-D-galacto-octopyranoside free base from part E in 200 ml of methanol was shaken over 2 g of 10% palladium on carbon under 2.8 kg/cm 2 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 yielded methyl-6,B-dideoxy-6-(4-butyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside hydrochloride which was collected 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 recrystallization from melted acetone

at 197 - 199° C and gave [V) 2 5 +150° (water, c 0.69).

Analysis, calculated for C gH^^N^O^S•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.

G1. Methyl- 6, B- dideoxy- 6-( 1- methyl- 4- butyl- L- 2- pyrrolidinecarboxamido)-1- thio- D- erythro-oc- D- qalacto- octopyranoside- hydrochloride.

A solution of 2.0 g of methyl-6,B-dideoxy-6-(4-butyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside hydrochloride from parts F and 2, 0 ml of 37% formalin in 150 ml methanol was shaken over 500 mg of 10% palladium on carbon under 2.8 kg/cm hydrogen pressure for 3.5 hours. Removal of the catalyst by filtration and the solvent by distillation in vacuo gave partially crystalline methyl-6,B-dideoxy-6-(1-methyl-4-butyl-L-2-pyrrolidinecarbox-amido)-1-thio-D-erythro -α-D-galacto-octopyranoside hydrochloride which by thin layer chromatography on silica gel using a mixture of ethyl acetate, acetone and water (8:4:1) for elution and potassium permanganate solution for detection consisted mainly of two materials, cis- and trans -the epimers of methyl-6,B-dideoxy-6-(1-methyl-4-butyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside hydrochloride in a ratio of approx. 3 to 2.

G2. Separation of the cis and trans forms by chromatography.

The methyl-6,B-dideoxy-6-(1-methyl-4-butyl-L-2-pyrrolidine-carboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside hydrochloride from part 51 was loaded into a mixture of methanol and methylene chloride (1:1) and 1.5 ml of triethylamine were added. To this solution was added 7 g of silica gel and the solvent was evaporated under vacuum leaving the antibiotic deposit on the silica gel which was sieved on top of a chromatographic column of 200 g of silica gel packed with a solvent mixture consisting of ethyl acetate, acetone and water in a ratio of 8:4:1. The column was developed by elution with the same solvent and 20 ml portions were collected. Thin layer chromatography of each fraction showed that fractions 31 - 38, 310 mg, were practically pure trans epimer, that fractions 49 - 74, 32 mg, were practically pure cis -epimer and that fractions 39 - 48 consisted of a mixture of the epimers . The latter could be separated further 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 way, 50 mg of methyl-6,B-dideoxy-6-(trans-1-methyl-4-butyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside hydrochloride were obtained with a melting point of 135 - 137° C, and approx. 150 mg methyl-6,B-dideoxy-6-(cis-1-methyl-4-butyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside hydrochloride, which softened at 105 ° C with further melting at 175 - 185 ° C

The trans-epimer recrystallized from the same solvent, melted at 139 - 141°C and had the following analysis:

Analysis, calculated for C^H^^<N>^<O>^<S*>HC1:

C 49.93; H 8.16; N 6.13; S 7.02.

Found: (corrected for 4.07% H20):

C 48.81; H 8.54; N 6.49; 5 6.67.

Corresponding recrystallization of the cis-epimer gave

a product which softened at 10B° C and further at approx. 189° C (with crystal water) with the following analysis:

Analysis, found: (corrected for 4.95% water):

C 50.27; H 9.00; N 6.05; 5 6.65.

The Tran.s epimer was approx. 2.2 times as active as lincomycin in S. lutea determination, approx. 2 times as active in the broth dilution assay and 2.5 times as active in mice infected with S. aureus.

The cis epimer was approx. 1/2 to 1/3 as active as the trans epimer, and was approximately equal to lincomycin.

H1. Methyl- 6. 8- dideoxy- 6-( 1- ethyl- 4- butyl- L- 2- pyrrolidinecarboxamido)-1- thio- D- erythro- a- D- qalacto- octopyranoside hydrochloride.

A mixture of 2.0 g of methyl 1-6,8-deoxy-6-(4-butyl-1_-2-pyrrolidinecarboxamido)-1-thio-D-erythro-oc-D-galcto-octopyranoside hydrochloride from Part F, 1.5 ml of acetaldehyde, 150 mg of 10% palladium on carbon in 150 ml of methanol were shaken under 2.4 kg/cm 2 hydrogen pressure for 5.5 hours. The catalyst was removed by filtration, yielding a residue consisting mainly of the cis- and trans-epimers of methyl1-6,B-dideoxy-6-(1-ethyl-4-butyl-L-2-pyrrolidine-carboxamido)-1 -thio-D-erythro-α-D-galacto-octopyranoside hydrochloride.

H2. Separation of the epimers.

As described in part G2, a mixture of the epimers from part H1 (2 g) was chromatographed over 200 g of silica gel, where a solvent system of ethyl acetate, acetone and water (8:4:1) was used for the elution. Fractions 33 - 42 by thin layer chromatography were pure trans-epimer and were combined, fractions 49 - 64 were practically pure cis-epimer and were also combined. Fractions 43 - 48 were a mixture of the epimers which could be purified by rechromatography. Each epimer was dissolved in a few drops of dilute hydrochloric acid and the crystalline hydrochloride precipitated by dilution with a larger volume of ether.

The crude trans-epimer fraction of 415 mg gave 340 mg

(15.4%) crystalline methyl-6,B-dideoxy-6-(trans-1-ethy1-4-butyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside hydrochloride with melting point 144 - 151° C. Recrystallization from dilute acetone raised the melting point to 148 - 151 C. The cis -epimer fraction of 645 mg gave 300 mg (14.1%) of crystalline methyl-6,8-dideoxy-6-( cis-1-ethyl-4-butyl-L-2-pyrrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside hydrochloride with m.p.

135 - 139 C. Recrystallization from dilute acetone gave crystals

with melting point 134 - 138° C.

Separation of the cis- and trans-isomers is not a necessary step as the 7-chloro derivatives of the mixed epimers are useful in and of themselves. However, it is desirable to keep the content of the trans isomer high, as this is the most active form. By carrying out the process with this in mind, mixed epimer products containing a ratio of trans and cis epimers of 3:1 to 1:5 can be readily obtained.

Example 5

Methvl- 7- chloro- 6. 7. 8- trideoxv- 6-( trans- 1- methvl- 4- propvl- L- 2- pyrro-lidincarboxamido)- 1- thio- D- ervthro- a- D- qalacto- octopyranoside (7-chloro-7-deoxy-epilincomycin).

A. 3. 4- 0- isopropylidene lincomycin.

A solution of 9.8 g of lincomycin in 150 ml of acetone

was added to a solution of 9.8 g of p-toluenesulfonic acid monohydrate in 100 ml of acetone with good stirring and avoiding exposure to moisture. The mixture was stirred at ambient temperature for 1 hour, after which 100 ml of anhydrous ether was added and stirring was continued in an ice bath for 0.5 hours. The mixture was filtered and the solid was dried in vacuo at 50°C, the yield was 13.35 g (85.5%) 3,4-O-isopropylidenelincomycin p-toluenesulfonate. A further 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 filtration operation and cooling the solution for 1 hour. They thus obtained 14.5 g

suspended in 250 ml ether and shaken vigorously with 125 ml 5% potassium bicarbonate solution. The aqueous layer is back-extracted with two 100 ml portions of ether. The ether extracts are washed with 50 ml of saturated sodium chloride solution and then filtered through anhydrous sodium sulphate. The ether is evaporated under vacuum, leaving 7.9 g (73.1%) of 3,4-0-isopropylidenelincomycin, which is dissolved in 25 ml of ether acetate and concentrated to approx. 10 - 15 ml. The concentrate is allowed to stand at room temperature for several hours and then placed in a refrigerator overnight. The crystals are filtered from the solution and washed sparingly with

cold ethyl acetate, whereby a yield of 4.55 g (42.2%) of 3,4-0-isopropylidenelincomycin with a melting point of 126 - 128° C is obtained

and an optical rotation of (V) 2 5 101 _ 102° (c = 1, methylene chloride).

D

B. 7- dehvdro- 3t4- 0- isopropvlidene lincomycin.

To a solution of 6 g (0.0135 mol) isopropylidene lincomycin in 75 ml pyridine was added 12 g (excess) chromium oxide. The solution heats up approx. 20° C. After t hour, the mixture was added to a solution containing 250 ml of ethyl ether and 250

ml of ethyl acetate.: This was then filtered and evaporated to a syrup weighing 8.4 g. This syrup was distributed in a 500-pass countercurrent distribution using the system water:ethyl acetate:ethanol:cyclohexane (1:1:1 :1). 7-dehydro-3,4-0-isopropylidene-lincomycin was isolated as the top fraction from rows 330 - 380, K = 2.45. Analysis, calculated for C^^H^<gN>^O^S: C 56.72; H 8, 16; N 6.30; 5 7.21.

Found: C 56.37; H 7.62; N 6.51; S 6.84.

C. 3, 4-0- isoprop. vliden- epilincomycin.

To 1.6 g of Craig-pure 7-dehydro-3,4-0-isopropylidenelincomycin in 75 ml of methanol was added 400 mg of sodium borohydride.

After 1.5 hours, this solution was evaporated to dryness on a rotary evaporator. The residue was added to 25 ml of water and extracted three times with 25 ml of methylene chloride each time. The extract was backwashed with 15 ml of water, then dried over magnesium chloride and evaporated to dryness. The residue, 1.4 g, was distributed in a 500-pass countercurrent distribution using the solvent system, water:ethyl acetate:ethanol:cyclohexane (1:1:1:1), and a single peak matching the theoretical was observed at K = 1.05. Materi-

alet in rows 240 - 280 was isolated as a syrup.

Analysis calculated for ^21^38^2^6^<:>

C 56.47; H. 8.58; N 6.27; 5 7.1B.

Found: C 56.24; H B, 54; N 6.13; 5 7.01.

Thin layer chromatography showed that this material consisted of two compounds. One was 3,4-0-isopropylidene-lincomycin,

the other 3,4-0-isopropylidene-epilincomycin, moved slightly longer.

D. Epilincomvicin.

The syrup from part C was stored at room temperature

for 5 hours in a solution containing 60 ml of 0.25N hydrochloric acid and 40

ml of ethanol. It was then kept at 0° C. for 4 days. After neutralization with sodium bicarbonate it was evaporated to 25 ml and then extracted with chloroform. The extract was washed with a little water and dried over magnesium sulfate and then evaporated to a residue. Thin-layer chromatography of the residue showed two compounds, both of which were active against S.lutea. The residue was chromatographed on a 355 x 18 mm "Florisil" (a synthetic silicate of the type described in U.S. Patent 2,393,625) column eluted gradiently with solvent varying continuously from 100% 115kellysolve B" (technical hexane) to 100 % acetone. The total volume was 5000 ml. The two connections were thus separated.

Fraction I: Ror 53 - 65 (40 ml's fractions)

Epilincomycin.

Content 450 mcg/ml.

Analysis, calculated for<C>^gH^^<N>^<0>^<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 II: RBr 73 - 104. Lincomycin.

a-"- Content 950 mcg/mg.

E. 7- epichlor- 7- deoxylincomycin• HC1

□ ,B5 g of 7-epilincomycin hydrochloride was suspended in 17 µl of carbon tetrachloride. 4.5 ml of thionyl chloride was added and the reaction mixture was stirred at 25° C. for 15 minutes. The solid dissolved to give a clear, colorless solution. The reaction was then heated under reflux for 2 hours, cooled to 25° C, evaporated under vacuum to a dry yellow residue. This crude product was dissolved in 10 ml ethanol, basified with 0.1N sodium hydroxide, diluted to 500 ml water and extracted four times with 50 mL portions of chloroform. The combined chloroform extracts were backwashed twice with 20 mL portions of saturated sodium chloride solution, filtered and evaporated under vacuum. The solid residue was suspended with 500 mL of ether, filtered, and hydrogen chloride gas rapidly into the filtrate. The solid precipitate was collected , dissolved in 3 ml of ethanol and reprecipitated by adding ether.

The solid material was collected and dried. One

30% yield (270 mg) 7-epichloro-7-deoxylincomycin which had approx.^^

to 3 times the activity of lincomycin, was obtained. Thin layer chromatography on silica gel (methanol:chloroform system, 1:6 volume ratio) showed a spot with an Rf of 0.44 compared with an Rf of

0.52 for 7-chloro-7-deoxylincomycin.

By replacing lincomycin in this example with lincomycin analogs of the formula II where Z, R, R^, R^, R_ i-Y group is as indicated and illustrated above, the corresponding 7-chloro-7-deoxyepilincomycin analogs of the formula are obtained :

where Z, R, R^fR2 and R^1 the Y group is as indicated and illustrated above. All the compounds described above therefore have their counterpart in the opposite configuration, that is, the configuration derived from the 7-epiform. As an inversion is effected by replacing the 7-hydroxy group with chlorine, the epi-\f or compounds having the L-threo configuration will be inverted to the D-erythro configuration. Thus, both the D-erythro and L-threo forms are obtained depending on whether either the normal lincomycins" (D-erythro) or the epi-lincomycins (L-threo) are used. :. -...... ■., , r, p.

Example 6.

7- chloro- 7- deoxvlincomvcin- hydrochloride.

10 g (0.0226 mol) of lincomycin hydrochloride, 200 ml of carbon tetrachloride and 10 ml of 50Cl, were stirred and heated under reflux for 4 hours. The reaction mixture was cooled to 25° C. and filtered. The yellow solid was dried under vacuum and then dissolved in approx. 10 ml of boiling ethanol. Ethyl acetate was added to blank and the solution was allowed to cool. The crystals of 7-chloro-7-deoxylincomycin hydrochloride thus formed were recovered in a yield of approx. 43%.

Claims (1)

Procedure for the preparation of 7-chloro-7-deoxy-1-thio-α-D-galacto-octopyranosides of the formula: where R is alkyl with up to 8 carbon atoms, optionally alkyl-substituted cycloalkyl with 3-8 carbon atoms or aralkyl with up to 12 carbon atoms, and Y is the acyl radical of a 4-substituted-L-2-pyrrolidinecarboxylic acid of the formula: where is the alkylidene with up to 8 carbon atoms, R.j and R_ are the alkylene with up to 8 carbon atoms, and R^ is hydrogen, methyl or ethyl, and acid addition salts thereof, characterized in that 1 mol of a compound of the formula: where R and Y are as stated above, is stirred with at least 3 mol of thionyl chloride in the presence of an inert solvent at room temperature for a longer time, whereby a compound of the formula is formed: where R and Y are as above, whereupon the reaction mixture OE OE is heated at a temperature between 50 C and 100 C to replace the 7-hydroxy group with chlorine, and the compound formed is then subjected to basic hydrolysis at pH approx. 11 to remove the 3,4-0-cyclic sulphite group, and that a compound obtained with the group = R^ is optionally hydrogenated catalytically in a manner known per se, and/or the compound obtained is optionally transferred to an acid addition salt of i and familiar way.