IE46661B1 - Erythromycin a derivatives - Google Patents

Abstract

New semi-synthetic derivatives of 4''-erythromycin A and their preparation are described. The new compounds of the invention correspond to the formulae I and II in which the symbols have the meaning given in Claim 1. The compounds of formulae I and II, in which X and Y are a pair H, NH2, possess antibacterial properties and can be used as medicinal products.

Description

This invention, relates to certain novel erythromycin antibacterial agents, to processes for their preparation, and to their use in curing certain bacterial infections.

In particular, the invention concerns certain 4-deoxy-45 amino-erythromycin A antibacterial agents.

Erythromycin is an antibiotic formed during the culturing of a strain of Streptomyces ervthreus in a suitable medium as taught in U.S. Patent No. 2,653,899. Erythromycin, which is produced in two forms, A and B, is represented by the following structure: Erythromycin R A -OH -H - 3 The structure reveals that the antibiotic is comprised of three main portions: a sugar fragment known as cladinose, a second sugar moiety containing a basic amino substituent known as desosamine and a fourteen membered lactone ring referred to as erythronolide A or B or, as herein described, the macrolide ring. While the numbering system of the macrolide ring is in unprimed numbers, that of the desosamine is in primed numbers and that of cladinose in double-primed numbers.

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

U.S. Patent No. 3,417,077 describes the reaction product of erythromycin and ethylene carbonate as a very active antibacterial agent. U.S. Patent No. 3,884,903 discloses 4-deoxy-4-oxo-erythromycin A and B derivatives as being useful as antibiotics.

Erythromycylamine, the 9-amino derivative of erythromycin A, has been the subject of considerable·’investigation (British Patent No. 1,100,504, Tetrahedron Letters, 1645 (1967) and Croatica Chemica Acta, 39, 273 (1967)) and some controversy as to its structural identity (Tetrahedron Letters, 157 (1970) and British Patent No. 1,341,022). Sulfonamide derivatives of erythromycylamine are reported in U.S. Patent No. 3,983,103 to be useful as antibacterial agents. Other derivatives are also reported (Ryden, et al, J. Med. Chem.. 16, 1059 (1973) and Massey, et al, J. Med. Chem., 17, 105 (1974)) to have in vitro and in vivo antibacterial activity.

It has now been discovered that certain novel 4deoxy-4~amino-erythromycin A derivatives are useful as antibacterial agents. Thus the invention provides compounds of the formulae: and the pharmaceutically acceptable acid addition salts thereof, wherein R and R are each hydrogen or alkanoyl X 4 of two or three carbon atoms; Rg is alkanoyl of two or 5 three carbon atoms; and R^ is hydrogen; or R2 and Rg taken together are ? or R, and R. taken °together -C- 3 4 are || -C46661 - 5 A preferred group of compounds within this class of chemotherapeutic agents are those of Formula III wherein Rg and Rg when taken together are II -c- .

A second preferred group of compounds in this class 5 of antibacterial agents are those of Formula IV wherein is hydrogen and also wherein Rg and when taken together are II -c- .

Useful intermediates leading to the antibacterial 10 agents of Formulae III and IV, and which are described and claimed in our copending divisional Patent Specification No. 46662 , are represented as follows: wherein R^ is hydrogen or alkanoyl of two or three carbon atoms; Rg is alkanoyl of two or three carbon atoms; Y is N-OH or 0 N-O-^CHg ; and Rg is hydrogen; or Rg and Rg when taken together are II -c- .

Preferred within this class of intermediates are those compounds of Formula I wherein R^ is hydrogen or acetyl.

A second group of preferred intermediates are those of Formula IX wherein R^ is hydrogen and also those· wherein R^ is acetyl.

Processes for preparing the intermediate compounds 15 of the formulae I and II are also described in the said divisional Patent Specification No. ‘JJ '-''jI. - 7 Throughout tho present invention, the stereochemical designation of the substituents on the sugars and macrolide ring, with the exception of epimerisation at the 4-position where noted, are those of the naturally occurring erythro5 mycin A.

In accordance with the processes employed for synthesizing the 4-deoxy-4-amino-erythromycin A antibacterial agents of the present invention, the following schemes, starting with a 2'-alkanoyl-erythromycin A, or a derivative thereof, are represented as follows: - 8 and KTiPFT ) II (R = Ac) These are described in detail in the said divisional Specification No. 46662.

The ketones of Formulae I (Y = 0) and II are useful intermediates leading to the 4-deoxy-4-amino-erythromycin A antibacterial agents of the present invention of formulae III and IV. Preferred as intermediates in this group 6 ϋ (51 are 2-0-acetyl-4-deoxy-4-oxo-erythromycin A 6,9-hemiketal 11,12-carbonate ester and 4-deoxy-4-oxo-erythromycin A 6,9-hemiketal 11,12-carbonate ester.

Several synthetic pathways can be employed in the preparation of the antibacterial agents of Formulae XII and IV from the requisite ketones I (Y = 0) and II.

Preparation of the 4-deoxy-4-amino-erythromycin A compounds of Formula III is carried out by the condensation of the ketones II with the ammonium salt of a lower alkanoic acid and the subsequent reduction of the in situ generated imine. The term lower alkanoic refers, in this instance, to an acid having two to four 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 treated with the reducing agent sodium cyanoborohydride. The reaction is allowed to proceed at room temperature for several hours before it is subsequently hydrolyzed and the product isolated.

Although one mole of the ammonium alkanoate is needed per mole of ketone, it is preferred that an excess, as great as ten-fold, be employed in order to ensure complete and rapid formation of the imine. Such excess amounts appear to have little deleterious effects on the quality of the product.

Regarding the amount of reducing agent to be employed per mole of ketone, it is preferred that about two moles of sodium cyanoborohydride per mole of ketone be used.

The reaction time will vary with concentration, reaction temperature and the inherent reactivity of the reagents. At room temperature, the preferred reaction temperature, the reaction is substantially complete after - 10 two to three hours.

When the lower alkanol solvent is methanol there is substantial solvolysis of any alkanoyl group at the 21 position. In order to avoid removal of such a moiety it is preferred that isopropanol be used as the reaction solvent.

The preferred ammonium alkanoate, as previously indicated, for this reaction is ammonium acetate.

In isolating the desired 4-deoxy-4-amino-erythro10 mycin A derivatives from any non-basic by-products or starting material, advantage is taken of the basic nature of the final product. Accordingly, an aqueous solution of the product is extracted over a range of gradually increasing pH such that neutral or non-basic materials are extracted at lower pH's and the product at a pH of greater than 5. The extracting solvents, either ethyl acetate or diethyl ether, are backwashed with brine and water, dried over sodium sulfate and the product obtained by removal of the solvent. Additional purification, if necessary, can be effected by column chromatography on silica gel according to known procedures.

Solvolysis of the 2'-alkanoyl group from the appropriate 2'-alkanoyl-4-deoxy-4-amino-erythromycin A derivative can be effected by allowing a methanol solution of said compound to stand overnight at ambient temperature.

During the reductive amination of ketones of Formula II wherein Rg and Rg when taken together are 9 and Rj is alkanoyl of two to three carbon atoms or hydrogen, it is noted that amines related to both Formulae III and IV are produced. This is represented by the following scheme: 4666 1 NH,OCOCH 4_3 NaCNBHg , 11 1 III (R2 + R3 = -c- ) The amine products III and IV as represented are conveniently separated by selective crystallization from diethyl ether. Recrystallization of the mixture of III and IV as represented 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 synthetic pathway to the amine compounds of Formula IV is the same route as discussed previously and comprises the condensation of the ketone I (Y = 0) with an ammonium alkanoate followed by reduction of the in situ generated imine, e.g. with sodium cyanoborohydride .

Compounds of Formula IV, wherein R^, R3 and R^ are as previously defined, are also prepared by the reduction of the aforementioned imine using hydrogen and an appropriate hydrogenation catalyst. Experimentally, the appropriate ketone (I) in a lower alkanol, such as methanol or isopropanol, is treated with the ammonium salt of a - 13 lower alkanoic acid, such as acetic acid, and the hydrogenation catalyst, and the mixture shaken in a hydrogen atmosphere until the reaction is essentially complete.

Although one mole of the ammonium alkanoate is needed per mole of ketone, it is preferred that an excess, as great as ten fold, be employed in order to ensure complete and rapid formation of the imine. Such excess amounts appear to have little deleterious effects on the quality of the product.

The hydrogenation catalyst can be selected from a wide range of agents; Raney nickel and 5-10 percent palladium-on-charcoal are, however, the preferred catalysts. These may be used in varying amounts depending on how fast the reaction is to be completed. Amounts from 10-200 percent of the weight of I can be employed effectively.

The pressure of the hydrogen gas in the hydrogenation vessel also influences the rate of reaction. It is preferred, for the convenience of reaction time, that an initial pressure of 50 p.s.i. be employed. It is also preferred, for convenience, that the reduction be carried out at ambient temperature.

Reaction time is dependent on a number of factors including temperature, pressure, concentration of the reactants and the inherent reactivity of the reagents.

Under the aforementioned preferred conditions the reaction is complete in 12 to 24 hours.

The product is isolated by filtration of the spent catalyst and removal of the solvent in vacuo. The residual material is subsequently treated with water and the product isolated from non-basic materials by extraction of the basic product from water at varying pH's previously described. - 14 As previously indicated, when the lower alkanol solvent is methanol there is substantial solvolysis of any alkanoyl group at the 2'-position. In order to avoid removal of such a moiety it is preferred that isopropanol be used as the reaction solvent.

The second synthetic route to the 4-deoxy-4-aminoerythromycin A antibacterial agents of Formula IV comprises initial conversion of the ketones of Formula I (Y = 0) to an oxime or oxime derivative, i.e., Y = N-OH and θ II N-O-CCHg , followed by reduction of the oxime or derivative thereof.

The oximes of the ketones I (Y = 0) are prepared by reacting said ketones with hydroxylamine hydrochloride and barium carbonate in methanol or isopropanol at room temperature. In practice, it is preferred that an excess of hydroxylamine be employed, and as much as a three-fold excess provides the desired intermediate in good yields. Employing ambient temperatures and an excess of the hydroxylamine allows for the preparation of the desired oxime derivative in a reaction period of one to three hour s. The barium carbonate is used in molar quantities twice that of the hydroxylamine hydrochloride employed.

The product is isolated by addition of the reaction mixture to water followed by basification to pH 9.5 and extraction with a water-immiscible solvent such as ethyl acetate.

Alternatively, the reaction mixture can be filtered and the filtrate concentrated in vacuo to dryness. The residue is subsequently partitioned between water at pH 9.0-9.5 and a water-immiscible solvent. 4666 1 - 15 Preparation of the O-acetyloxime compounds of Formula 0 I (Y = N-O-CCH^) is effected by acetylation of the corresponding oxime. Experimentally, one mole of the oxime is reacted with one mole of acetic anhydride in the presence of one mole of pyridine or triethylamine. The use of an excess of the anhydride and pyridine aid in the completion of the reaction and an excess of 30-40% is preferred.

The reaction is best conducted in an aprotic solvent such as benzene or ethyl acetate at room temperature overnight. On completion of the reaction, water is added, the pH adjusted to 9.0 and the product separated in the solvent layer .

The preferred oxime and oxime derivatives which are useful intermediates leading to the 4-deoxy-4-aminoerythromycin A antibacterial agents include 2'-0-acetyl4"-deoxy-4-oxo-erythromycin A oxime, 2-0-acetyl-4-deoxy4-oxo-erythromycin A O-acetyloxime, 4-deoxy-4-oxoerythromycin A oxime and 4 -deoxy-4''-oxo-erythromycin A O-acetyloxime.

Reduction of the ketone derivatives (Y = N-OH or N-O-CCH^) is carried out by catalytic hydrogenation wherein a solution of the oxime or derivative thereof in a lower alkanol, such as isopropanol, and a Raney nickel catalyst is shaken in a hydrogen atmosphere at an initial pressure of 1000 p.s.i. at room temperature overnight. Filtration of the spent catalyst followed by removal of the solvent from the filtrate provides for the isolation of the desired 4-deoxy-4-amino antibacterial agent related to Formula IV. If methanol is employed as the solvent in this reduction, solvolysis of a 2'-alkanoyl moiety is probable. In order - 16 to avoid this side-reaction, isopropanol is employed.

Preferred among these 4-deoxy-4-amino-erythromycin A antibacterial agents of Formulae III and IV are both epimers of 4-deoxy-4-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester and of 4-deoxy-4''-amino-erythromycin A, and of 4-deoxy-4-amino-erythromycin A 11,12-carbonate ester.

In the utilization of the chemotherapeutic activity of those compounds of Formulae III and IV of the present invention which form salts, it is preferred, of course, to use pharmaceutically acceptable salts. Although waterinsolubility, high toxicity, or lack of crystalline nature may make some particular salt species unsuitable or less desirable for use as such as a given pharmaceutical application, the water-insoluble or toxic salts can be converted to the corresponding pharmaceutically acceptable bases by decomposition of the salt as described above, or alternatively they can be converted to any desired pharmaceutically acceptable acid addition salt.

Examples of acids which provide pharmaceutically acceptable anions are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, or sulfurous, phosphoric, acetic, lactic, citric, tartaric, succinic, maleic, gluconic and aspartic acids.

As previously mentioned, the stereochemistry of the starting materials leading to the antibacterial agents of the present invention is that of the natural material.

The oxidation of the 4-hydroxyl group to a ketone and the subsequent conversion of said ketone to the 4-amines presents an opportunity for the stereochemistry of the 4-substituent to change from that of the natural product. Accordingly, when the compounds I (Y=0) and II are - 17 46661 converted to amines by one of the hereinbefore described procedures, it is possible that two epimeric amines are formed. Experimentally, it is observed that both epimeric amines are present in the final product in varying ratios depending on the choice of synthetic method. If the isolated product consists predominantly of one of the epimers, said epimer can be purified by repeated recrystallization from a suitable solvent to a constant melting point. The other epimer, the one present in smaller amounts in the originally isolated solid material, is the predominant product in the mother liquor. It can be recovered therefrom by methods known to those skilled in the art, as for example, the evaporation of the mother liquor and repeated recrystallization of the residue to a product of constant melting point.

Although said mixture of epimers can be separated by methods known to those skilled in the art, for practical reasons it is advantageous to use said mixture as it is isolated from the reaction. However, it is frequently advantageous to purify the mixture of epimers by at least one recrystallization from an appropriate solvent, subjecting it to column or high pressure liquid chromatography, solvent partitioning or trituration in an appropriate solvent. Said purification, while not necessarily separating the epimers, removes such extraneous materials as starting materials and undesirable by-products.

The absolute stereochemical assignment for the epimers has not been completed. Both epimers of a given compound, however, exhibit the same type of activity, e.g., as antibacterial agents.

The novel 4-deoxy-4-amino-erythromycin A derivatives described herein exhibit in vitro activity against - 18 a variety of Gram-positive microorganisms, .e.g... Staphylococcus aureus and Streptococcus pyogenes, and against certain Gram-negative microorganisms such as those of spherical or ellipsoidal shape (cocci). Their activity is readily demonstrated by in vitro tests against various microorganisms in a brain-heart infusion medium by the usual two-fold serial dilution technique. Their in vitro activity renders them useful for topical application in the form of ointments or creams; for sterilization, e_.g., of sick-room utensils; and as industrial antimicrobials, for example, in water treatment, slime control, paint and wood preservation.

For in vitro use, e.g., for topical application, it will often be convenient to compound the selected product with a pharmaceutically-acceptable carrier such as vegetable or mineral oil or an emollient cream. Similarly, the compounds may be dissolved or dispersed in liquid carriers or solvents, such as water, alcohol, glycols or mixtures thereof or other pharmaceutically-acceptable inert media; that is, media which have no harmful effect on the active ingredient. For such purposes, it will generally be acceptable to employ concentrations of active ingredients of from about 0.01 percent to about 10 percent by weight based on total composition.

Additionally, many compounds of this invention and their acid addition salts are active versus Gram-positive and certain Gram-negative microorganisms, e.g., Pasteurella multocida and Neisseria sicca, in vivo via the oral and/or parenteral routes of administration in animals, including man. Their in vivo activity is more limited as regards susceptible organisms and is determined by the usual procedure which comprises infecting mice of substantially - 19 uniform weight with the test organism and subsequently treating them orally or subcutaneously with the test compound. In practice, the mice, e.g., 10, are given an intraperitoneal inoculation of suitable diluted cultures containing approximately 1 to 10 times the Ι,ϋ^θθ (the lowest concentration of organisms required to produce 100% deaths) . Control tests are simultaneously run in which mice receive inoculum of lower dilutions as a check on possible variation in virulence of the test organism.

The test compound is administered 0.5 hour post-inoculation, and is repeated 4, 24 and 48 hours later. Surviving mice are held for four days after the last treatment and the number of survivors is noted.

When used in vivo, these novel compounds can be administered orally or parenterally, e.g., by subcutaneous or intramuscular injection, at a dosage of from about 1 mg.Ag· to about 200 mg ./kg- of body weight per day. The favored dosage range is from about 5 mg.Ag· to about 100 mg./kg. of body weight per day and the preferred range is from about 5 mg.Ag· to about 50 mg.Ag· of body weight per day.

Vehicles suitable for parenteral injection may be either aqueous such as water, isotonic saline, isotonic dextrose, Ringer's solution, or non-aqueous such as fatty oils of vegetable origin (cotton seed, peanut oil, corn, sesame), dimethylsulfoxide and other non-aqueous vehicles which will not interfere with therapeutic efficiency of the preparation and are non-toxic in the volume or proportion used (glycerol, propylene glycol, sorbitol) . Additionally, compositions suitable for extemporaneous preparation of solutions prior to administration may advantageously be made. Such compositions may include liquid diluents, for example, propylene glycol, diethyl carbonate, glycerol, sorbitol, etc.; buffering agents, hyaluronidase, local anaesthetics and inorganic salts to afford desirable pharmacological properties. These compounds may also be combined with various pharmaceutically-acceptable inert carriers including solid diluents, aqueous vehicles, non-toxic organic solvents in the form of capsules, tablets, lozenges, troches, dry mixes, suspensions, solutions, elixirs and parenteral solutions or suspensions. In general, the compounds are used in various dosage forms at concentration levels ranging from about 0.5 percent to about 90 percent by weight of the total composition.

Thus the invention also provides a pharmaceutical composition comprising a compound of the Formula (III) or (IV) as defined above or a pharmaceuticaliy-acceptable acid addition salt thereof together with a pharmaceutically acceptable diluent or carrier.

The following Examples are provided for the purpose of illustration.· The preparation bf relevant starting materials is described in Patent Specification No. . 46662.

Example 1 21-0-Acetyl-4-deoxy-4-amino-erythromvcin A A mixture of 14.0 g. of 2'-0-acetyl-4"-deoxy-4 oxo-erythromycin A O-acetyloxime and 60 g. of isopropanolwashed Raney nickel in 400 ml. of isopropanol is agitated in a hydrogen atmosphere at an initial pressure of 1000 p.s.i. overnight at room temperature. The catalyst is filtered off and the filtrate concentrated to a white - 21 foam. The residue is redissolved in 400 ml. of isopropanol and combined with 50 g. of fresh isopropanol-washed Raney nickel. The hydrogenation is continued overnight at room temperature and an initial hydrogen pressure of 1000 p.s.i.

The catalyst is filtered off and the filtrate concentrated in vacuo to dryness to give 8.1 g. of the desired product.

EXAMPLE 2 Starting with the appropriate 0-acetyloxime and employing the procedure of Example 1, the following 410 amino-erythromycin A compounds are prepared: H0 II CH CH,C46661 - 22 EXAMPLE 3 -Deoxy-411 -amino-erythromycin A A solution of 2.17 g. of 2'-0-acetyl-4-deoxy-4amino-erythromycin A in 50 ml. of methanol is stirred at room temperature overnight. The solvent is removed under reduced pressure and the residual foam treated with a mixture of 50 ml. of chloroform and 50, ml. of water.

The pH of the aqueous layer is adjusted to 9.5 and the organic layer separated. The chloroform layer is treated with fresh water and the pH adjusted to 4.0. The pH of the acid aqueous layer containing the product is gradually adjusted to 5, 6, 7, 8 and 9 by the addition of base, being extracted at each pH with fresh chloroform. The extracts at pH 6 and 7 contain the major portion of the product and these are combined and treated with fresh water at pH 4. The aqueous layer is again adjusted through pH 5, 6 and 7, being 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 (5, CDClg): 3.30 (IH)s, 3.26 (2H)s, 2.30 (6H)s and 1.46 (3H)s.

In a similar manner, 4-deoxy-4-amino-erythromycin A is prepared by the methanol solvolysis of 2'-0-propionyl4"-deoxy-4-amino-erythromycin A.

EXAMPLE 4 4-Deoxy-4-amino-erythromycin A To a stirring solution of 3.0 g. of 4-deoxy-4oxo-erythromycin A in 30 ml. of methanol under a nitrogen atmosphere is added 3.16 g. of dry ammonium acetate.

After 5 min. 188 mg. of sodium cyanoborohydride is washed - 23 into the reaction mixture with 5 ml. of methanol and the reaction mixture stirred at room temperature overnight.

The light yellow solution is poured into 300 ml. of water and the pH adjusted to 6.0. The aqueous layer 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 combined 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 (I 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 combined, washed with a saturated brine 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-colored foam.

EXAMPLE 5 4-Deoxv-4-amino-erythromycin A (single epimer) A solution of 10.0 g. of the epimeric mixture of 2'-0-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 stirring 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 of the aqueous layer is adjusted to 5 and the chloroform layer is separated. The pH of the aqueous phase is subsequently adjusted to 5.5, 6, 7, 8 and 9, being extracted after each adjustment with 100 ml. of fresh chloroform. The chloroform extracts from pH 6, 7 and 8 are combined, treated successively with water and a saturated brine solution and dried over sodium sulfate. - 24 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, causing some of the undissolved foam to crystallize. The solids are filtered off and dried to give 57 mg. of a single epimer of 4-deoxy-4-amino-erythromycin A, m.p. 140-147°C.

EXAMPLE 5 i .......... — i 11-O-Acetyl-4-deoxy-411 -amino-erythromycin A 6,9-hemiketal To a stirring solution of 4.4 g. of ll-0-acetyl-4·deoxy-4-oxo-erythromycin A 6,9-hemiketal and 4.38 g. of ammonium acetate in 75 ml. of methanol is added 305 mg. of 85% sodium cyanoborohydride. After stirring at room temperature overnight, the reaction mixture is poured into 300 ml. of water to which is then added 250 ml. of chloroform. The pH of the aqueous layer is adjusted to 9.8 and the chloroform layer separated. The aqueous layer is extracted with chloroform again, and the chloroform extracts are combined, dried over sodium sulfate and concentrated to a white foam. The residual foam is dissolved in a stirring mixture of 125 ml. of water and 125 ml. of fresh chloroform and the pH adjusted to 4.9.

The chloroform is separated and discarded, and the aqueous layer adjusted to pH 5, 6, 7 and 8, being extracted after each adjustment with fresh chloroform. The chloroform extracts following the adjustments at pH 6 and 7 are combined, washed with a saturated brine solution and dried over sodium sulfate. Removal of the solvent provides 1.72 g. of the desired product as a white foam.

The product is dissolved in a minimal amount of diethyl ether and is subsequently treated with hexane to turbidity. The crystalline product which forms is filtered and dried, 1.33 g., m.p. 204.5-206.5°C.

NMR (δ, CDCip: 3.31 (2H)s, 3.28 (lH)s, 2.31 (6H)s, 2.11 (3H)s and 1.5 (3H)s.

EXAMPLE 7 The procedure of Example 6 is repeated, starting with the appropriate 4-deoxy-4-oxo-erythromycin A and substituting isopropanol for methanol as the reaction solvent to give the following compounds: CH3C0 CH3C0 ch3c0 il ch3ch2c0 ί CH CH2CCH CH2C0 II CH CH C0 II ch3c46661 - 26 EXAMPLE 8 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-hemi5 ketal 11,12-carbonate ester in 1200 ml. of methanol at room | temperature is added with stirring 193 g. of ammonium S acetate. After 5 minutes the resulting solution is cooled to about -5°C. and is subsequently treated with 13.4 g. of 85% sodium cyanoborohydride in 200 ml. of methanol over a 45 minute addition period. The cooling bath is removed and the reaction mixture stirred at room temperature over' night.' The reaction mixture is reduced in volume to 800 ml. in vacuo and added to a stirring 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 separated. The chloroform is combined with 1 1 of water and the pH adjusted to 9.5. The organic phase is separated, dried over sodium sulfate and concentrated under reduced pressure to give 4.74 g. of a white foam. The residual material is dissolved in a mixture of 1 1. of water and 500 ml. of ethyl acetate and the pH adjusted to 5.5. The ethyl acetate layer is separated and the aqueous layer 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 residual foam is dissolved in a mixture of 1 1. of water and 1 1. of methylene chloride, the pH of the aqueous layer is adjusted to 4.4, 4.9 and 9.4 successively, being extracted after each adjustment with 1 1. of fresh methylene chloride. The methylene chloride extract at pH 9.4 is dried over sodium - 27 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, v:v) gives 28.5 g. of the crystalline epimers.

NMR 100 Mz (δ, CDC13): 5.20 (lH)m, 3.37 (1.5H)s, 3.34 (1.5H)s, 2.36 (6H)s, 1.66 (3Ii)s and 1.41 (3H)s.

EXAMPLE 9 Separation of the Epimers of 4-Deoxy-4-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester On to a high-pressure-liquid-chromatography column (1/2 x 9 cm.) packed with Gf 254 silica gel+ impregnated with formamide and eluted with chloroform is applied 200 mg. of an epimeric mixture of 4-deoxy-4-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester. A pressure of 240 p.s.i. is applied with a rate of 4.76 cc. per min. and a fraction size of 10 ml. is employed. Fractions 14 to 21 and 24 to 36 are collected.

Fractions 14 to 21 are combined and concentrated to about 50 ml. Water (50 ml.) is added and the pH adjusted to 9.0. The chloroform layer is separated, dried over sodium sulfate and concentrated to give 106 mg. of a white foam. Trituration with diethyl ether causes the foam to crystallize. After stirring at room temperature for one hour the crystalline product is filtered off and dried, 31.7 mg., m.p. 194-196°C.

NMR 100 Mz (δ, CDC13): 5.24 (lH)d, 5.00 (lH)t , 3.40 (3H)s, 2.40 (6H)s, 1.66 (3H)s and 1.40 (3H)s.

+Gf 254 silica gel is a silica gel which contains a zinc silicate phosphor indicator which absorbs 254 mu ultraviolet light. 6 6 61 Fractions 24 to 36 are combined and worked up as above to give 47.1 mg. of product as a white foam which is identical to the material from Example 14.

EXAMPLE 10 To a suspension of 11.1 g. of 2'-0-acetyl-4-deoxy4,'-oxo-erythromycin A 6,9-hemiketal 11,12-carbonate ester in 300 ml. of isopropanol at room temperature is added with stirring 10.7g. of ammonium acetate. After 5 min., 747 mg. of sodium cyanoborohydride in 130 ml. of iso10 propanol is 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 is then added 40ml. of diethyl ether. 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. Recrystall ization of the residual material from diethyl ether gives 1.69 g. which is retained along with the mother liquors.

The mother liquor is treated with 75 ml. of water, and the pH adjusted to 5.0. The ether layer is replaced with 75 ml. of fresh ether and the pH adjusted to 5.4.

The ether is replaced with ethyl acetate and the pH raised to 10. The basified aqueous layer is extracted (2 x 75 ml.) with ethyl acetate and the first ethyl acetate extract dried over sodium sulfate and concentrated to dryness. The residual foam (1.96 g.) is added to a mixture of 75 ml. of water and 50 ml. of diethyl ether and the pH adjusted to 5.05. The ether is separated and the aqueous layer adjusted successively to pH 5.4, 6.0, 7.05 and 8.0, being extracted after each pH adjustment - 29 with 50 ml. of fresh diethyl ether . The pH is finally adjusted to 9.7 and the aqueous layer extracted with 50 ml. of ethyl acetate. The ether extract carried out at pH 6.0 is combined with 75 ml. of water and the pH 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 (δ, CDC13): 5.20 (lH)t, 3.43 (2H)s, 3.40 (lH)s, 2.38 (6H)s, 2.16 (3H)s, 1.70 (3H)s and 1.54 (3H) .

The NMR data indicates the product to be the epimers of 21-0-acetyl-4-deoxy-411-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester.

The 1.69 g. indicated above is dissolved in a mixture of 75 ml. of water and 75 ml. of diethyl ether and the pH adjusted to 4.7. The ether is separated and the aqueous layer 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 a white foam. Crystallization of this residual material gives 411 mg. of product, m.p. 193-196°C. (dec.).

The mother liquor is concentrated to dryness, and the residue dissolved in hot ethyl acetate. The solution is allowed to stand overnight at room temperature. The crystalline solids which precipitate are filtered off and dried, 182 mg., m.p. 198-202°C. (dec.) to give additional product.

NMR 100 Mz (δ, CDC1 ): 5.10 (lH)t, 3.34 (2H)s, J 3.30 (lH)s, 2.30 (6H)s, 2.08 (3H)s, 1.62 (3H)s and 1.48 (3H)s.

The NMR data indicates the product to be the epimers of 21-0-acetyl-4-deoxy-4-amino-erythromycin A 11,12carbonate ester. 4666i - 30 In a similar manner, when Example 10 is repeated, starting with 21-0-propionyl-4-deoxy-4-oxo-erythromycin A 6,9-hemiketal 11,12-carbonate ester, there is obtained 2‘-0-propionyl-4-deoxy-4-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester and 21-0-propionyl-4-deoxy4-amino-erythromycin A 11,12-carbonate ester.

EXAMPLE 11 A solution of 400 mg. of 2'-0-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· 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. Trituration with diethyl ether and scratching with a glass rod affects crystallisation.

After standing at room temperature for 30 minutes, the crystalline solids are filtered off and dried, 123 mg., and the mother liquor is retained. The product is identical by NMR to material prepared in Example 13.

NMR 100 Mz (5, CDClg): 3.25 (3H)s, 2.32 (6H)s, 1.61 (3H)s and 1.44 (3H)s.

The NMR data indicates 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 with material from Example 8.

The NMR data indicates that this product is a mixture of the epimers of 4-deoxy-4-amino-erythromycin A 6,9hemiketal 11,12-carbonate ester, and identical with the product of Example 8.

EXAMPLE 12 In a manner similar to the procedure of Example 11, methanolysis of 2'-0-propionyl-4-deoxy-4-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester gives 4-deoxy-4-amino-erythromycin A 11,12-carbonate ester and 4-deoxy-4-amino-erythromycin A 6,9-hemiketal 11,12carbonate ester.

EXAMPLE 13 Eight grams of the epimeric mixture of 4-deoxy-4amino-erythromycin A 11,12-carbonate ester from the product of Example 8 is dissolved in 50 ml. of diethyl ether . The product is induced to crystallize by scratching with a glass rod. After 20 minutes' stirring, the crystalline product is filtered and dried, 1.91 g., m.p. 198.5-2OO°C.

NMR 100 Mz (5, CDC13): 3.26 (3H)s, 2.30 (6H)s, I. 61 (3H)s and 1.45 (3H)s.

The NMR data indicates that the crystalline product is a single epimer of 4-deoxy-4-amino-erythromycin A II, 12-carbonate ester and identical with the ketone product from Example 11.

EXAMPLE 14 One gram of the epimer of Example 13 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 stirred at room - 32 temperature. After one hour of stirring, the precipitate which forms is filtered off and dried to give 581 mg., m.p. 147-149°C.

NMR 100 Mz (S, CDClg): 5.12 (IH)d, 3.30 (3H)s, 2.30 (6H)s, 1.62 (3H)s and 1.36 (3H)s.

The NMR data indicates the product to be a single epimer of 4-deoxy-4-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester and identical to the epimer in fractions 24-36 of Example 9.

EXAMPLE 15 4-Deoxy-4-amino-ervthromycin A Twenty grams of 4-deoxy-4-oxo-erythromycin A, 31.6 g. of ammonium acetate and 10 g. of 10% palladium-oncharcoal in 200 ml. of methanol is shaken at ambient temperatures in a hydrogen atmosphere at an initial pressure of 50 p.s.i. overnight. The spent catalyst is filtered off and the filtrate concentrated to dryness in vacuo. The residue is partitioned between water-chloroform at a pH of 5.5. The aqueous layer is separated, the pH adjusted to 9.6 and chloroform added. The organic layer is separated, dried over sodium sulfate and concentrated under reduced pressure to dryness. The residual white foam (19 g.) is triturated with 150 ml. of diethyl ether at room temperature for 30 minutes. The resulting solids are filtered off and dried to give 9.45 g. of a single epimer indistinguishable from that in Example 5.

The diethyl ether filtrate is concentrated to dryness to give 6.89 g. of product consisting of the other epimer plus some impurities. - 33 46661 EXAMPLE 16 4-Deoxy-4-Amino-Erythromycin A Two grams of 4-deoxy-4-oxo-erythromycin A, 3.1 g. of 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 50 p.si. overnight.

An additional 3.16 g. of ammonium acetate and 2.0 g. of Raney nickel are added and the hydrogenation continued for an additional 5 hours. The solids are filtered and the filtrate concentrated to dryness in vacuo. The residue is added with stirring to a mixture of water-chloroform, and the pH adjusted from 6.4 to 5.5. The aqueous phase is separated, the pH adjusted to 9.6 and fresh chloroform 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 predominant isomer has the opposite configuration at 4 to the compound of Example 5.

EXAMPLE 17 4-Deoxy-4-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester L-aspartate To a solution of 1.0 g. of 4-deoxy-4-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester in 6 ml. of acetone warmed to 40°C. is added 20 ml. of water followed by 175 mg. of L-aspartic acid. The mixture is heated to reflux for 1,5 hours and is then filtered while hot.

The filtrate is concentrated by removal of the acetone and is subsequently freeze-dried to give 1.1 g. of the desired product as a white solid. - 34 EXAMPLE 18 4-Deoxy-4-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester dihydrochloride To 7.58 g. of 4-deoxy-4"-amino-erythromycin A 6,95 hemiketal 11,12-carbonate ester in 50 ml. of dry ethyl acetate is added 20 ml. of a IN ethyl acetate solution of hydrogen chloride, and the resulting solution concentrated to dryness under reduced pressure. The residual material is triturated with ether and filtered to give the desired salt.

By a similar procedure the other amine compounds of the present invention are converted to their di-acid addition salts.

EXAMPLE 19 4-Deoxy-4-amino-erythroniycin A 6,9-hemiketal 11,12-carbonate ester hydrochloride The procedure of Example 18 is repeated vzith the exception that 10 ml. of a IN ethyl acetate solution of hydrogen chloride is added. The solution is concen20 trated to dryness in vacuo and the residual monohydrochloride salt is triturated with ether and filtered.

By a similar procedure the other amine compounds of the present invention are converted to their mono-acid addition salts.

Claims (18)

1. CLAIMS;1. A 4-amino epimeric compound of the formula:- and the pharmaceutically acceptable acid addition salts 5 thereof, wherein and are each independently hydrogen or alkanoyl having two or three carbon atoms; R 2 is alkanoyl having two or three carbon atoms; and R^ is hydrogen; or R 2 and R 3 taken together are · or R 3 and R 4 taken together are ? - 36

2. A compound of the Formula (III) as claimed in Claim 1, wherein R 2 and R 3 taken together are II -C- .

3. The compound of the formula:-

4. The compound of the formula:- - 37 5. A compound of the formula (IV) as claimed in Claim 1, wherein R^ is hydrogen. 6. The compound of the formula :-

5. 7. The compound of the formula :-

6. 8. A compound of the formula IV as claimed in Claim 0 1, wherein R^ and R^ taken together are U 4 6 6 61 - 38

7. 9. The compound of the formula:-

8. 10. The compound of the formula:

9. 11. A process for preparing a compound of the formula (III) or (IV) as claimed in Claim 1, which comprises reacting a compound of the formula: respectively, wherein is hydrogen or alkanoyl of 2 or 3 carbon atoms, Rg is alkanoyl of 2 or 3 carbon atoms, Rg is hydrogen, and Y is 0, with an ammonium salt of an 5 alkanoic acid having from 2 to 4 carbon atoms, followed by reducing the thus-produced imine.

10. 12. A process for preparing a compound of the formula (XII) or formula (IV) as claimed in Claim 1, wherein R 2 and Rg, or Rg and R^, respectively, taken - 40 together represent il -c-, which comprises reacting a compound of the formula (II) as defined in claim 11, wherein R^ is hydrogen or alkanoyl 5 of 2 or 3 carbon atoms and R 2 and R 3 taken together represent -c-, with an ammonium salt of an alkanoic acid having 2 to 4 carbon atoms, followed by reducing the thus-produced imine to produce a mixture of the compound (III) and (IV), 10 followed by either separating these by selective crystallisation from diethyl ether, or, by recrystallising the mixture from acetone-water to recover the compound of the formula (III) as the sole product.

11. 13. A process for preparing a compound of the 15 formula (IV) as claimed in Claim 1, which comprises reducing a compound of the formula (I) as defined in Claim 11 but in which Y is -N-OH or -N-O-C-CH 3 .

12. 14. A process for preparing a compound of the 20 formula (III) or (IV) as claimed in Claim 1 in which R^ is hydrogen, which comprises treating the corresponding compound of the formula (III) or (IV) in which R^ is an alkanoyl group of 2 or 3 carbon atoms with methanol.

13. 15. A process as claimed in Claim 13 substantially 25 as hereinbefore described in either of Examples 1 and 2.

14. 16. A process as claimed in Claim 14 substantially as hereinbefore described in any one of Examples 3, 5, 11 and 12.

15. 17. A process as claimed in Claim 11 substantially 30 as hereinbefore described in any one of Examples 4, 6, 7, 15 and 16. - 41 46661

16. 18. A process as claimed in Claim 12 substantially as hereinbefore described in either of Examples 8 and 10.

17. 19. A compound of the formula (III) or (IV) as claimed in Claim 1 which has been prepared by a process 5 as claimed in any one of claims 11 to 18.

18. 20. A pharmaceutical composition comprising a compound of the formula (III) or (IV) as claimed in any one of Claims 1 to 10 and 19, or a pharmaceutically acceptable acid addition salt thereof, together with a 10 pharmaceutically acceptable diluent or carrier.