NO146472B - ANALOGY PROCEDURE FOR THE PREPARATION OF ERYTHROMYCIN DERIVATIVES - Google Patents

Description

This invention relates to the production of 4"-deoxy-4"-aminoerythromycin A derivatives.

Erythromycin is an antibiotic that is formed by

cultivation of a strain of Streptomyces erythreus in a suitable

medium as stated in US patent 2,653,899. erythromycin,

which is formed in two forms, A and B, is represented by the following structure:

The structure shows that the antibiotic comprises three main parts: a sugar fragment known as cladinose, another sugar part containing a basic amino substituent known as desosamine, and a 14-membered lactone ring designated as erythronolide A or B,

or as here, the macrolide ring. While the numbering system for the macrolide ring is unmarked numbers, desosamine uses marked numbers, and cladinose double-marked numbers.

Numerous derivatives of erythromycin have been prepared "in an attempt to modify its biological or pharmacodynamic properties.

US patent 3,417,077 describes the reaction product of erythromycin and ethylene carbonate as a highly active antibacterial agent. US Patent 3,884,903 states that 4"-deoxy-4"-erythromycin A and B derivatives are useful antibiotics.

Erythromycin, the 9-amino derivative of erythromycin A,

has been the subject of considerable research (British Patent 1,100,504, Tetrahedron Letters, 1645 (1967) and Croatica Chemica Acta, 39_, 273 (1967)) and some controversy exists as to its chemical structure (Tetrahedron Letters, 157 (1970 ) and British patent 1,341,022). Sulfonamide derivatives of erythromycylamine are described in US Patent 3,983,103 as useful antibacterial agents. Other derivatives are also described (Ryden, et al., J. Med. Chem., 16, 1059 (1973) and Massey, et al., J. Med. Chem., 17,

105 (1974)) as substances with in vitro and in vivo antibacterial action.

It has now been found that certain new 4"-deoxy-4"-amino-erythromycin A derivatives have excellent antibacterial properties. These compounds are represented by the formulas:

and pharmaceutically acceptable acid addition salts thereof, wherein is hydrogen or alkanoyl of 2 to 3 carbon atoms; R 2 is alkanoyl of 2 to 3 carbon atoms; R 1 is hydrogen; R. is hydrogen; or R2 and R^ together are together are A preferred group of compounds within this class of chemotherapeutic compounds are those of formula III, where R^ is hydrogen and R2 and R3 together are Another preferred group of compounds in this class of antibacterial compounds are those which has formula IV, where R,, R. and R. are each hydrogen, or where R, is hydrogen J- J <*>i and R^ and R^ together are ;The new compounds are prepared from intermediates with the formulas: and ; where R^, R2 and R., are as indicated above and Y' and Y" are each 0. These intermediates are prepared by reacting a compound with the respective formulas: and where Ac is alkanoyl with 2 to 3 carbon atoms with 1 mole each of dimethyl sulfoxide and trifluoroacetic anhydride in a reaction-inert solvent at about -30 to -65 C, followed by contacting the reaction mixture with at least 1 mole of triethylamine.;Removal of the alkanoyl moiety at the 2'-position in the intermediate ketones IV<1> (Y'=0) and III' (Y"=0) are made by a solvoly see reaction where the 2'-alkanoyl-4"-deoxy-4"-oxo-erythromycin A-related compound is stirred with an excess of methanol overnight at room temperature. Removal of the methanol and subsequent purification, when necessary, of the remaining product gives compounds of formula IV (Y'=0) and III' (Y"=0) where R is hydrogen. Several synthesis routes can be used in the preparation > of the compounds with formulas III and IV from the necessary ketones IV' (Y<1> = 0) and III' (Y" = 0). ;Preparation of the 4"-deoxy-4"-amino-erythromycin A compounds of formula III is carried out by condensation of the ketones II with the ammonium salt of a lower alkanoic acid and subsequent reduction of the imine formed in situ (Y"=NH). The designation " "lower alkanoic acid" denotes here an acid with 2 to 4 carbon atoms. In practice, a solution of the ketone II in a "lower alkanol, such as methanol or isopropanol, is treated with the ammonium salt of a lower alkanoic acid, such as acetic acid, and the cooled reaction mixture is treated with the reducing agent sodium cyanoborohydride. The reaction is allowed to proceed at room temperature for several hours before the reaction mixture is hydrolyzed and the product isolated. ;Although one mole of the ammonium alkanoate is required per mole of ketone, it is preferred that an excess as high as 10-fold be used to ensure complete and rapid formation of the imine. Such excess amounts seem to have little detrimental effect on the quality of the product. ;With respect to the amount of reducing agent used ;per m ol ketone, it is preferred that approx. 2 mol of sodium cyanoborohydride are used per moles of ketone. ;The reaction time will vary with the concentration, the reaction temperature and the inherent reactivity of the reagents. At room temperature, the preferred reaction temperature, the reaction is almost complete after 2-3 hours. When the lower alkanol solvent is methanol, as previously indicated, substantial solvolysis of an alkanoyl group in the 2' position takes place. In order to avoid the removal of such a group, it is preferred that isopropanol is used as a solvent in the reaction. The preferred ammonium alkanoate, as mentioned earlier, for this reaction is ammonium acetate. When isolating the desired 4"-deoxy-4"-amino-erythromycin A derivatives from any non-basic by-products or starting material, the basic nature of the final product is utilized. Thus, an aqueous solution of the product is extracted over a range of gradually increasing pH so that neutral or non-basic materials are extracted at lower pH values, and the product at a pH above 5. ;The extracting solvents, either ethyl acetate or diethyl ether, are washed back with saline solution and water, is dried over sodium sulfate, and the product is obtained by removing the solvent. Further purification, if necessary, can be carried out by column chromatography on silica gel according to known methods. As stated previously, solvolysis of the 2'-alkanoyl group from the appropriate 2'-alkanoyl-4"-deoxy-4"-amino-erythromycin A derivative can be accomplished by allowing a methanol solution of said compound to stand overnight at ambient temperature. ;In the reductive amination of the ketones of formula II where ;R and R^ together are ;and R^ is alkanoyl with 2 to 3 carbon atoms or hydrogen, it is seen that amines with both formulas III and IV are obtained. This is evident from the following scheme: The ainine products III and IV as stated above are suitably separated by selective crystallization from diethyl ether. Recrystallization of the mixture of III and IV as indicated above from acetone-water induces hemiketal formation in the amine of formula IV, resulting in the isolation of III as the sole product. The first direct synthesis route to the amine compounds of formula IV is the same route as discussed above and comprises condensation of the ketone IV<1> with an ammonium alkanoate followed by reduction of the in situ formed imine (Y'=NH) with sodium cyanoborohydric'.. Compounds of formula IV where R-^, R^ and R^ are as previously indicated, are also prepared by reduction of the above-mentioned imine using hydrogen and a suitable hydrogenation catalyst. Experimentally, the appropriate ketone (IV) is treated in a lower alkanol, such as methanol or isopropanol, with the ammonium salt of a lower alkanoic acid, such as acetic acid, and the hydrogenation catalyst, and the mixture is shaken in a hydrogen atmosphere until the reaction is nearly complete. ;Even if one mole of the ammonium alkanoate is required per moles of ketone, it is preferred that an excess, as much as 10-fold, is used to ensure complete and rapid formation of the imine. Such surplus quantities seem to have little detrimental effect on the quality of the product. The hydrogenation catalyst can be selected from a number of different agents. However, Raney nickel and 5-10% palladium-on-charcoal are preferred catalysts. These can be used in varying amounts depending on how quickly the turnover is to be completed. Amounts from 10-200% based on the weight of the IV can be suitably used. The pressure of hydrogen gas in the hydrogenation vessel also affects the reaction rate. It is preferred, in order to achieve a favorable reaction time, that an initial pressure of 3.5 kg/cm 2 is used. For practical reasons, it is also preferred that the reduction be carried out at ambient temperature. ;The reaction time is dependent on a number of factors, including temperature, pressure, concentration of the reaction components and the inherent reactivity of the reagents. Under the above reaction conditions, the conversion is complete within 12 to 24 hours. The product is isolated by filtering the used catalyst and removing the solvent in a vacuum. The remaining material is then treated with water, and the product is isolated from non-basic materials by extraction of the basic product from water at varying pH values as described above. When the lower alkanol solvent is methanol, as mentioned above, substantial solvolysis of any alkanoyl group in the 2' position occurs. In order to avoid the removal of such a group, it is preferred that isopropanol is used as solvent for the reaction. Another synthesis route to 4"-deoxy-4"-amino-erythromycin A antibacterial agents of formula IV comprises first converting the ketones of formula IV<1> (Y' =0) into an oxime or oxime derivative, i.e. Y' = N-OH and N-O-Cl CH-,J, followed by reduction of the oxime or its derivative. ;Reduction of the ketone derivatives (Y<1> = N-OH or N-O-Cit CH-,j) ;0 is carried out by catalytic hydrogenation in which a solution of the oxime or a derivative thereof in lower alkanol, such as isopropanol, and Raney nickel -catalyst is shaken in a hydrogen atmosphere at an initial pressure of o 70 kg/cm 2 at room temperature overnight. Filtration of the spent catalyst followed by removal of the solvent from the filtrate leads to the isolation of the desired 4"-deoxy-4"-amino antibacterial agent of formula IV. If methanol is used as solvent in this reduction, solvolysis of a 2'-alkanoyl group is likely. To avoid this side reaction, isopropanol is used. When utilizing the chemotherapeutic effect of the compounds of the formulas III and IV which form salts, it is of course preferred to use pharmaceutically acceptable salts. Although water insolubility, high toxicity or lack of crystalline nature may render certain salts unsuitable or less desirable for use as such for a given pharmaceutical purpose, the water insoluble or toxic salts may be converted into the corresponding pharmaceutically acceptable bases by cleavage of the salt as described above, or alternatively they can be converted to any desired pharmaceutically acceptable acid addition salt. Examples of acids having pharmaceutically acceptable anions are hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, sulfuric acid, sulfuric acid, acetic acid, lactic acid, citric acid, tartaric acid, succinic acid, maleic acid, gluconic acid and aspartic acid. As previously stated, the stereochemistry of the starting materials leading to the antibacterial agents is the same as in the natural material. The oxidation of the 4"-hydroxyl group to a ketone and the subsequent conversion of the ketone to the 4"-amines enables the stereochemistry of the 4"-substituent to change from what it was in the natural product. When the compounds IV (Y'=0 ) and III<*> (Y"=0) are converted to amines by one of the methods described above, it is thus possible that two epimeric amines are formed. Experimentally, it has been observed that both epimeric amines are present in the final product in varying proportions depending on the choice of synthesis method. If the isolated product consists predominantly of one of the epimers,

this epimer can be purified by repeated recrystallization from a suitable solvent to a constant melting point. The second epimer, the one present in smaller amounts in the initially isolated solid material, is the predominant product in the mother liquor. It can be extracted from this by methods known to those skilled in the art, e.g. by evaporation of the mother liquor and repeated recrystallization of the residue to a product with a constant melting point.

Although the mixture of epimers can be separated by methods known per se, for practical reasons it is appropriate to use the mixture as it is isolated from the reaction mixture. However, it is often appropriate to clean the mixture off

epimer by at least one recrystallization from a suitable solvent, by subjecting it to column or high pressure liquid chromatography or solvent partitioning or by trituration in a suitable solvent. This purification, which does not necessarily separate the epimers, leads to the removal of such foreign materials as starting materials and unwanted by-products.

The absolute stereochemical determination of the epimers has not been completed. However, both epimers of a given compound exhibit the same type of activity, e.g. as antibacterial agents.

The new 4"-deoxy-4"-amino-erythromycin A derivatives show in vitro activity against various Gram-positive microorganisms, e.g. Staphylococcus aureus and Streptococcus pyogenes, and against certain Gram-negative microorganisms such as those that have a spherical or ellipsoid shape (cocci). Their activity is easily demonstrated by in vitro tests against various microorganisms in a brain-heart infusion medium by the usual double serial dilution technique. Their in vitro activity makes them useful for local application in the form of ointments, creams, etc., for sterilization purposes, e.g. hospital equipment, and as industrial antimicrobial agents, e.g. for water treatment, slime control, paint and wood preservation.

For in vitro use, e.g. for local administration, will

it is often appropriate to mix the selected product with a pharmaceutically acceptable carrier such as a vegetable oil or mineral oil or an emollient cream. Likewise, the products can be dissolved or dispersed in liquid carriers or solvents such as water, alcohol, glycols or mixtures thereof or other pharmaceutically acceptable inert media, i.e. media that have no harmful effect on the active ingredient. For such purposes, it will usually be acceptable to use concentrations of the active ingredients of from approx. 0.01 to approx. 10% by weight, based on the total preparation.

Moreover, many of the new compounds and their acid addition salts are active against Gram-positive and certain Gram-negative microorganisms, e.g. Pasteurella multocida and Meisseria sicca,

in vivo by oral and/or parenteral administration to animals, including humans. Their in vivo activity is more limited with respect to organisms affected, and is determined by the usual method which involves infecting mice of approximately equal weight with the test organism and then treating them orally or subcutaneously with the test compound. In practice, the mice are given, e.g. 10, an intraperitoneal inoculation of suitable diluted cultures containing approximately 1 to 10 times the LD^00 (the lowest concentration of organisms necessary to produce 100% mortality). Control experiments are carried out at the same time with mice that receive inoculum in lower dilutions as a check for possible variations in the strength of the test organism. The test compound is administered 0.5 hour after inoculation, and repeated 4, 24 and 48 hours later. Surviving mice are kept for 4 days after the last treatment, and the number of survivors is recorded.

When used in vivo, these new compounds can be administered orally or parenterally, e.g. by subcutaneous or intramuscular injection, in a dose from approx. 1 to approx. 200 mg/kg body weight per day. The preferred dose range is from approx. 5 to approx. 100 mg/kg body weight per day, and a particularly preferred area is from approx. 5 to approx. 50 mg/kg body weight per day.

Carriers suitable for parenteral injection can

either be aqueous, such as water, isotonic saline, isotonic dextrose or Ringer's solution, or non-aqueous, such as fatty oils of vegetable origin (cottonseed, groundnut oil, corn, sesame), dimethyl sulfoxide or other non-aqueous carriers that will not affect the preparation's therapeutic effect and are non-toxic in the amount used (glycerol, propylene glycol, sorbitol). Preparations which are suitable for the preparation of solutions on the spot before administration can also be appropriately prepared.

Such preparations may contain liquid diluents, e.g. propylene glycol, diethyl carbonate, glycerol, sorbitol, etc.; buffering agents, hyaluronidase, local anesthetics and inorganic salts to provide desired pharmacological properties. These compounds can also be formulated with various pharmaceutically acceptable inert carriers including solid diluents, aqueous carriers and non-toxic organic solvents, for the preparation of capsules, tablets, pills, lozenges, dry mixes, suspensions, solutions, elixirs and parenteral solutions or suspensions. In general, the compounds are used in different dosage forms in concentrations varying from approx. 0.5 to approx. 90% by weight of the total preparation.

The following examples shall serve to further illustrate the invention.

Example 1

2'- acetyl- 4"- deoxy- 4"- amino- erythromycin A

A mixture of 14.0 g of 2'-acetyl-4"-deoxy-4"-oxo-erythromycin A-O-acetyl oxime and 60 g of isopropanol-washed Raney nickel in 400 ml of isopropanol is stirred in a hydrogen atmosphere at an initial pressure of 70 kg /cm 2 at room temperature. The catalyst is filtered off, and the filtrate is concentrated to a white foam. The residue is re-dissolved in 400 ml of isopropanol and mixed with 50 g of fresh isopropanol-washed Raney nickel. The hydrogenation is continued overnight at room temperature and an initial hydrogen pressure of 70 kg/cm<2>. The catalyst is filtered off, and the filtrate is concentrated in vacuo

to dryness to give 8.1 g of the desired product.

Example 2

4"- deoxy- 4"- amino- erythromycin A

A solution of 2.17 g of 2'-acetyl-4"-deoxy-4"-amino-erythromycin A in 50 ml of methanol is stirred at room temperature overnight. The solvent is removed under reduced pressure and the remaining foam is treated with a mixture of 50 ml of chloroform and 50 ml of water.

The pH in the aqueous layer is adjusted to 9.5, and the organic layer is separated. The chloroform layer is treated with fresh water, and the pH is adjusted to 4.0. The pH in the acidic, aqueous layer containing the product is gradually adjusted to 5, 6, 7, 8 and 9 by adding base, extraction being carried out at each pH with fresh chloroform. The extracts

at pH 6 and 7 contains the bulk of the product, and these are collected and treated with fresh water at pH 4. The aqueous layer is regulated again through pH 5, 6 and 7 and extracted at each pH with fresh chloroform. The chloroform extract at pH 6 is dried over sodium sulfate and concentrated to give 249 mg of the product as an epimeric mixture. NMR (6, CDCl 3 ): 3.30 (1H) s, 3.26 (2H) s, 2.30 (6H) s and 1.46 (3H) s.

In a similar way, 4"-deoxy-4"-amino-erythromycin A is prepared by methanol solvolysis of 2<1->propionyl-4"-deoxy-4"-amino-erythromycin A.

Example 3

4"- deoxy- 4"- amino- erythromycin A

To a stirred solution of 3.0 g of 4"-deoxy-4"-oxo-erythromycin A in 30 ml of methanol under a nitrogen atmosphere is added 3.16 g of dry ammonium acetate. After 5 minutes, 188 mg of sodium cyanoborohydride are washed into the reaction mixture with 5 ml of methanol, and the reaction mixture is stirred at room temperature overnight. The pale yellow solution is poured into 300 ml of water, and the pH is adjusted to 6.0. The aqueous solution is extracted at pH 6, 7, 7.5, 8, 9 and 10 using 125 ml of diethyl ether for each extraction. The extracts at pH 8, 9 and 10 are collected and washed with 125 ml of fresh water. The separated aqueous layer is extracted with ether (1 x 100 ml) at pH 7, ethyl acetate (1 x 100 ml) at pH 7, ether (1 x 100 ml)

at pH 7.5, ethyl acetate (1 x 100 ml) at pH 7.5 and ethyl acetate (1 x 100 ml) at pH 8, 9 and 10. The ethyl acetate extracts at pH 9

and 10 are collected, washed with a saturated saline solution and dried over sodium sulfate. Removal of the solvent in vacuo gives 30 mg of an epimeric mixture of the desired product as an ivory foam.

Example 4

4"- deoxy- 4"- amino- erythromycin A (single epimer)

A solution of 10.0 g of the epimeric mixture of 2'-acetyl-4"-deoxy-4"-amino-erythromycin A in 150 ml of methanol is stirred at room temperature under nitrogen for 72 hours. The solvent is removed in vacuo, and the residue is dissolved in a stirred mixture of 150 ml of water and 200 ml of chloroform. The aqueous layer is discarded, and 150 ml of fresh water is added. The pH in the aqueous layer is adjusted to 5, and the chloroform layer is separated. The pH in the aqueous phase is then adjusted to 5.5, 6, 7, 8 and 9, whereby extraction is carried out after each adjustment with 100 ml of fresh chloroform. The chloroform extracts from pH 6, 7 and 8 are combined, washed successively with water and a saturated saline solution and dried over sodium sulfate. Removal of the solvent under reduced pressure gives 2.9 g of an epimeric mixture of 4"-deoxy-4"-amino-erythromycin A. A 1.9 g sample of the mixture is triturated with diethyl ether which results in some of the undissolved foam crystallizes. The solid is filtered and dried to give 67 mg of a single epimer of 4"-deoxy-4"-amino-erythromycin A,

sm.p. 140-147°C.

Example 5

ll- acetyl- 4"- deoxy- 4"- amino- erythromycin A- 6, 9- hemiketal

305 mg of 85% sodium cyanoborohydride is added to a stirred solution of 4.4 g of 11-acetyl-4"-deoxy-4"-oxo-ervthromycin A-6,9-hemiketal and 4.38 g of ammonium acetate in 75 ml of methanol. After stirring at room temperature overnight, the reaction mixture is poured into 300 ml of water to which 250 ml of chloroform is then added. The pH of the aqueous layer is adjusted to 9.8, and the chloroform layer is separated. The aqueous layer is extracted again with chloroform, and the chloroform extracts are combined, dried over sodium sulfate, and concentrated to a white foam. The remaining foam is dissolved in a stirred mixture of 125 ml of water and

125 ml of fresh chloroform, and the pH is adjusted to 4.9. The chloroform is separated and discarded, and the aqueous layer is adjusted to pH 5, 6, 7

and 8, extracting with fresh chloroform after each adjustment. The extracts from the aqueous layer at pH 6 and 7 are collected, washed with a saturated salt solution and dried over sodium sulfate. Removal of the solvent gives 1.72 g of the desired product as a white foam. The product is dissolved in a minimal amount of diethyl ether and then treated with hexane until cloudy. The crystalline product formed is filtered and dried, 1.33 g, m.p. 204.5-206.5°C.

NMR (6, CDCl 3 ): 3.31 (2H) s, 3.28 (1H) s, 2.31 (6H) s, 2.11 (3H) s and 1.5 (3H) s.

Example b

4"- deoxy- 4"- amino- erythromycin A- 6, 9- hemiketal- 11, 12- carbonate ester

To 189 g of 4"-deoxy-4"-oxo-erythromycin A-6,9-hemiketal-11,12-carbonate ester in 1200 ml of methanol at room temperature, 19 3 g of ammonium acetate are added while stirring. After 5 minutes, the resulting solution is cooled to approx. -5°C and then treated with 13.4 g of 85% strength sodium cyanoborohydride in 200 ml of methanol over an addition period of 45 minutes. The cooling bath is removed, and the reaction mixture is stirred at room temperature overnight. The reaction mixture is reduced in volume to 800 ml in vacuo and added to a stirred mixture of 1800 ml of water and 900 ml of chloroform. The pH is adjusted from 6.2 to 4.3 with 6N hydrochloric acid, and the chloroform layer is separated. The chloroform is mixed with 1 liter of water, and the pH is adjusted to 9.5.

The organic phase is separated, dried over sodium sulfate and concentrated under reduced pressure to give 174 g of a white foam. The remaining material is dissolved in a mixture of 1 liter of water and 500 ml of ethyl acetate, and the pH is adjusted to 5.5. The ethyl acetate layer is separated, and the aqueous layer is adjusted to pH 5.7 and 9.5 successively, being extracted after each pH adjustment with 500 ml of fresh ethyl acetate. The ethyl acetate extract at pH 9.5 is dried over sodium sulfate and concentrated in vacuo to dryness, 130 g. 120 g of the remaining foam is dissolved in a mixture of 1 liter of water and 1 liter of methylene chloride. The pH of the aqueous layer is successively adjusted to 4.4,

4.9 and 9.4, extracting with 1 liter of fresh methylene chloride after each adjustment. The methylene chloride extract at pH 9.4 is dried over sodium sulfate and concentrated under reduced pressure to give 32 g of the product as a white foam. Crystallization from 250 ml of acetone-water (1:1, volume:volume) gives 28.5 g of the crystalline epimers.

NMR 100 Mz (6, CDCl 3 ): 5.20 (1H) m, 3.37 (1.5H) s, 3.34 (1.5H) s, 2.36 (6H) s, 1.66 (3H s and 1.41 (3H) s.

Example 1

Separation of the epimers of 4"-deoxy-4"-amino-erythromycin A-6,9-hemiketal-II,12-carbonate esters

On a high pressure liquid chromatography column (1.3 cm x 9 cm) packed with Gf 254 silica gel impregnated with formamide and eluted with chloroform, 200 mg of a mixture of the epimers is added.

A pressure of 16.8 kg/cm 2 at a rate of 4.76 ml per min., and

a fraction size of 10 ml is used. Fractions 14 to 21

and 24 to 36 are collected.

Fractions 14 to 21 are collected and concentrated to approx. 50 ml. Water (50 ml) is added and the pH is adjusted to 9.0. The chloroform layer is separated, dried over sodium sulfate and concentrated to give 106 mg of a white foam. Rubbing with diethyl ether causes the foam to crystallize. After stirring at room temperature for 1 hour, the crystalline product is filtered and dried, 31.7 mg,

sm.p. 194-196°C.

NMR 100 Mz (6, CDCl 3 ): 5.24 (1H) d, 5.00 (1H) t, 3.40 (3H) s,

2.40 (6H) s, 1.66 (3H) s and 1.40 (3H) S.

Fractions 24 to 36 are collected and worked up as described above to give 47.1 mg of product as a white foam, which is identical to the material from Example 12.

Example 8

To a suspension of 11.1 g of 2'-acetyl-4"-deoxy-4"-oxo-erythromycin A-6,9-hemiketal-11,12-carbonate ester in 300 ml of isopropanol at room temperature, while stirring, add 10.7 g ammonium acetate. After 5 minutes, 747 mg of sodium cyanoborohydride in 130 ml of isopropanol are added over a period of 30 minutes, and the resulting reaction mixture is stirred at room temperature overnight. The pale yellow solution is poured into 1100 ml of water to which 400 ml of diethyl ether is then added. The pH is adjusted to 4.5, and the ether layer is separated. The aqueous layer is basified to pH 9.5 and extracted (2 x 500 ml) with chloroform. The chloroform extracts are combined, dried over sodium sulfate and concentrated to give 7.5 g of a yellow foam. Recrystallization of the remaining material from diethyl ether gives 1.69 g which is retained with the mother liquor.

The mother liquor is treated with 75 ml of water, and the pH is adjusted to 5.0. The ether layer is replaced with 75 ml of fresh ether, and the pH is adjusted to 5.4. The ether layer is replaced with ethyl acetate, and the pH is raised to 10. The basic, aqueous layer is extracted with ethyl acetate (2 x 75 ml),

and the first ethyl acetate extract is dried over sodium sulfate and concentrated to dryness. The remaining foam (1.96 g) is added to a mixture of 75 ml of water and 50 ml of diethyl ether, and the pH is adjusted to 5.05. The ether is separated, and the aqueous layer is adjusted successively to pH 5.4, 6.0, 7.05 and 8.0, extraction being carried out after each pH adjustment with 50 ml of fresh diethyl ether. The pH is finally adjusted to 9.7, and the aqueous layer is extracted with 50 ml of ethyl acetate. The ether extract from pH 6.0 is mixed with 75 ml of water, and the pH is adjusted to 9.7. The ether layer is separated, dried and concentrated in vacuo to give 460 mg of a white foam.

NMR 100 Mz (6, CDCl 3 ): 5.20 (1H) h, 3.43 (2H) s, 3.40 (1H) s,

2.38 (6H) s, 2.16 (3H) s, 1.70 (3H) s and 1.54 (3H).

The NMR data show that the product is epimer of 2'-acetyl-4"-deoxy-4"-amino-erythromycin A-6,9-hemiketal-11,12-carbonate-

ester.

The above stated amount of 1.69 g of residual product is dissolved

in a mixture of 75 ml of water and 75 ml of diethyl ether, and the pH is adjusted to 4.7. The ether is separated, and the aqueous layer is further extracted with fresh ether (75 ml) at pH 5.05 and 5.4, and with ethyl acetate

(2 x 75 ml) at pH 9.7. The combined ethyl acetate extracts are dried

over sodium sulfate and concentrated under reduced pressure to give 1.26 g of white foam. Crystallization of this remaining material gives 411 mg of product, m.p. 193-196°C (decomposition). The mother liquor is concentrated to dryness, and the residue is dissolved in hot ethyl acetate. The solution is allowed to stand overnight at room temperature. The crystalline solids which are precipitated, filtered and dried, 182 mg,

sm.p. 198-202°C (dec) to give additional product.

NMR 100 Mz (6, CDCl 3 ): 5.10 (1H) h, 3.34 (2H) s, 3.30 (1H) s,

2.30 (6H) s, 2.08 (3H) s, 1.62 (3H) s and 1.48 (3H) s.

The NMR data show that the product is the epimers of 2'-acetyl-4"-deoxy-4"-amino-erythromycin A-11,12-carbonate esters.

Similarly, when example 8 is repeated, by

starting with 2<1->propionyl-4"-deoxy-4"-oxo-erythromycin A-6,9-hemiketal-11,12-carbonate ester, one obtains 2'-propionyl-4"-deoxy-4"-amino -erythromycin A-6,9-hemiketal-11,12-carbonate ester and 2<1->propiony1-4"-deoxy-4"-amino-erythromycin A-11,12-carbonate ester.

Example 9

A solution of 400 mg of 2'-acetyl-4"-deoxy-4"-amino-erythromycin A-6,9-hemiketal-11,12-carbonate ester in 20 ml of methanol is stirred overnight at room temperature. The reaction solution is poured into 100 ml of water, followed by the addition of 50 ml of ethyl acetate.

The pH is adjusted to 9.5, and the organic phase is separated. The extraction is repeated again with 50 ml of fresh ethyl acetate. The combined ethyl acetate extracts are dried over sodium sulfate and concentrated to give 392 mg of a white foam. Rubbing with diethyl ether and scraping with a glass rod causes crystallization. After standing at room temperature for 30 minutes, the crystalline solids are filtered and dried, 123 mg, and the mother liquor is retained. The product is identical by NMR to the material prepared in example li.

NMR 100 Mz (6, CDCl 3 ) : 3.26 (3H) s, 2.32 (6H) s, 1.61 (3H) s and

1.44 (3H) p.

The NMR data show that the crystalline product is a single epimer of 4"-deoxy-4"-amino-erythromycin A-11,12-carbonate ester.

The retained mother liquor is concentrated in vacuo to give

244 mg of a white foam.

The product is identical to the material from example 6.

The NMR data show that this product is a mixture of the epimers of 4"-deoxy-4"-amino-erythromycin A-6,9-hemiketal-11,12-carbonate ester and is identical to the product of Example 6.

Example, 1Q

By a method similar to that described in Example 9, methanolysis of 2'-propionyl-4"-deoxy-4"-amino-erythromycin A-6,9-hemiketal-11,12-carbonate ester, 4"-deoxy-4 "-amino-erythromycin A-11,12-carbonate ester and 4"-deoxy-4"-amino-erythromycin A-6,9-hemiketal-11,12-carbonate ester.

Example n

8 g of the epimeric mixture of 4"-deoxy-4"-amino-erythromycin A-11,12-carbonate ester from the non-crystalline product from Example 19 are dissolved in 50 ml of diethyl ether. The product is brought to crystallize by scraping with a glass rod. After stirring for 20 minutes, the crystalline product is filtered and dried, 1.91 g, m.p. 198.5-200°C.

NMR 100 Mz (6, CDCl 3 ): 3.26 (3H) s, 2.30 (6H) s, 1.61 (3H) s and 1.45 (3H) s.

The NMR data show that the crystalline product is a single epimer of 4"-deoxy-4"-amino-erythromycin A-11,12-carbonate ester and is identical to the ketone product from example g.

Example 12

1 g of the epimer from example n is dissolved in 20 ml of acetone

and heated at steam bath temperatures until the boiling point is reached.

Water (25 ml) is added and the resulting solution is stirred at room temperature. After stirring for 1 hour, the precipitate formed is filtered and dried to give 581 mg, m.p. 147-149°C.

NMR 100 Mz (6, CDCl 3 ): 5.12 (1H) d, 3.30 (3H) s, 2.30 (6H) s,

1.62 (3H) s and 1.36 (3H) s.

The NMR data show that the product is a single epimer of 4"-deoxy-4"-amino-erythromycin A-6,9-hemiketal-11,12-carbonate ester and is identical to the epimer in fractions 24-36 of Example 7,

Example 13

4"- deoxy- 4"- amino- erythromycin A

20 g of 4"-deoxy-4"-oxo-erythromycin A, 31.6 g of ammonium

acetate and 10 g of 10% palladium-on-charcoal in 200 ml of methanol are shaken at ambient temperature in a hydrogen atmosphere with an initial pressure of 3.5 kg/cm 2 overnight. The spent catalyst is filtered,

and the filtrate is concentrated to dryness in vacuo. The residue is distributed between water and chloroform at pH 5.5. The aqueous layer is separated,

The pH is adjusted to 9.6, and chloroform is added. The organic layer is separated, dried over sodium sulfate and concentrated to dryness under reduced pressure. The remaining white foam (19 g) is triturated with 150 ml of diethyl ether at room temperature for 30 minutes.

The resulting solids are filtered and dried to give 9.45 g

of a single epimer indistinguishable from that prepared in Example 4.

The diethyl ether filtrate is concentrated to dryness to give

6.89 g of product consisting of the second epimer plus some impurities.

Example 14

4"- deoxy- 4"- amino- erythromycin A

2 g 4"-deoxy-4"-oxo-erythromycin A, 3.1 g ammonium acetate

and 2.0 g of Raney nickel in 50 ml of methanol are shaken at room temperature in a hydrogen atmosphere at an initial pressure of 3.5 kg/cm<2>

overnight. An additional 3.16 g of ammonium acetate and 2.0 g of Raney nickel are added and the hydrogenation is continued for another 5 hours. The solids are filtered off, and the filtrate is concentrated to dryness in vacuo. The residue is stirred into a mixture of water and chloroform, and the pH is adjusted from 6.4 to 5.5. The aqueous phase is separated, the pH is adjusted to 9.6, and fresh chloroform is added. The chloroform extract is separated, dried over sodium sulfate and concentrated under reduced pressure to give 1.02 g of the product as a yellow foam. The dominant isomer has the opposite configuration at 4" compared to the compound according to example 4.

Claims (4)

1. Analogous method for the preparation of antibacterial 4"-amino-epimeric compounds of the formula: or the formula and pharmaceutically acceptable acid addition salts thereof, wherein R- is hydrogen or alkanoyl having from 2 to 3 carbon atoms; R 0 is alkanoyl having from 2 to 3 carbon atoms; R 1 is hydrogen; R 1 is hydrogen; or R2 and R^ together are and R 1 and R 4 together are characterized in that a compound with the formula: or the formula where R^, R? and R3 is as stated above, 0<3 Y' is NH, N-OH or N-OCCH3; and Y" is NH is reduced, and where (a) when Y" is NH and Y' is NH, N-OH or N-OCOCH3, the reduction is effected by catalytic hydrogenation, and (b) when Y' or Y" is NH, formed in situ from the ketones corresponding to the compounds of the formulas III' and IV' where respectively Y" and Y' are 0, by condensation of the ketone with an ammonium salt of an alkanoic acid, the reduction is carried out by a hydride reduction, and optionally, (c) when R , is hydrogen, it is converted to alkanoyl or when R-^ is alkanoyl, it is converted to hydrogen; (d) wherein a prepared compound of formula III wherein 1*2 and R., together are optionally converted to a compound of formula IV where R^ and R^ together are (e) the pharmaceutically acceptable acid addition salts are formed. 2. Process as stated in claim 1 for the production of the 4"-a- and -3-epimers of formula III where R, is hydrogen, and R2 and R^ together are , characterized by that a starting material of formula III' is used where R-^, R2 and R^ have the meanings given in the preamble, or R^ means alkanoyl which is converted to hydrogen. 3. Process as stated in claim 1 for the production of the 4"-a- and -p-epimers of formula IV where R^, R^ and R^ are each hydrogen, characterized in that starting materials of formula IV are used where R^ is hydrogen, or R 1 is alkanoyl which is converted to hydrogen. 4. Process as stated in claim 1 for the preparation of the 4"-a- and -(J-epimers of formula IV where R, is hydrogen, and R^ and R^ together are , characterized by conversion of a compound of formula III where R-^ is hydrogen or means alkanoyl which is converted to hydrogen, and R0 and R, together are