DK148036B - METHOD OF ANALOGUE FOR THE PREPARATION OF EPIMERE 4 '' - AMINO-ERYTHROMYCIN COMPOUNDS OR ACID ADDITION SALTS. - Google Patents

Description

148036

The present invention relates to an analogous process for the preparation of novel epimeric 4 "amino-erythromycin compounds of the formula: R1 1 <CH3'2. Ri fCH3> 2" vA \ Λ ° γ! ° Λ

R3O X or R30-L X

^ V χ0 ,,,, ί ° Ύ ° BX ^ NH2 l X ^ NH 2 'OCH ™ 7 t> CH, III υ ^ η3 IV 3 2 148036 or pharmaceutically acceptable acid addition salts thereof, each and each selected from the group consisting of hydrogen and alkanoyl having 2 3 2-3 C atoms, R is alkanoyl having 2-3 C atoms and R is hydrogen, 0 2 3 3 4 I * wherein R and R or R and R together may also be -C - or 2 3 a mixture of compounds of formulas III and IV wherein R and R are

ISLAND

3 4 H

R and R respectively are -C-.

Erythromycin is an antibiotic formed by growing a strain of Streptomyces erythreus in a suitable medium, as described in U.S. Patent No. 2,653,899. Erythromycin produced in two forms, A and B, is represented by the following structure: 8 · H? FH3 »2 - 10 6L 6JU 'H0 ,,," lil H0Si * "* 0 6” Π

I1 "W.

0 Z 'ΌΗ' 'also3

Erythromycin R

A -OH

B -H

The structure shows that the antibiotic is made up of three main parts, namely a sugar fragment known as cladinose, another sugar moiety containing a basic amino substituent known as desosamine and a 14-membered lactone ring referred to as erythronolide A or B or , as used herein, the macrolide ring. While the numbering system for the macrolide ring is in unmarked numbers, the numbering for the desos amine is in the marking and the numbering for cladinose is in double-labeled numbers.

Several derivatives of erythromycin have been prepared for the purpose of modifying its biological or pharmacodynamic properties.

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

Erythromycylamine, the 9-amino derivative of erythromycin Af has been the subject of considerable study [British Patent No. 1,100,504, Tetrahedron Letters, 1645 (1967) and Croatica Chemica Acta, 39, 273 (1967)] and to some controversy with for its structural identity [Tetrahedron Letters, 157 (1970) and British Patent Specification No. 1,341,022]. Sulfonamide derivatives of erythromycylamine have been reported to be useful as antibacterial agents in U.S. Patent No. 3,983,103. Other derivatives have also been reported to have antibacterial activity in vitro and in vivo [Ryden et al., J. Med.

Chem., 16, 1059 (1973) and Massey et al., J. Med. Chem., 17, 105 (1974)].

It has now been found that the novel epimeric 4'-amino erythromycin compounds of the above Formula III or IV or acid addition salts thereof are distinguished as antibacterial agents, e.g. possess a surprisingly better activity against Neisseria sicca than the known erythromycin compounds, as will be further demonstrated below.

A preferred group of compounds within this class 23 of antibacterial agents are those of Formula III wherein R and R are

II

together is -C-.

Another preferred group of compounds within said 4 class of antibacterial agents are those of Formula IV wherein R is 0

3 4 M

hydrogen, and also where R and R together are -C-.

The process according to the invention for the preparation of the present compounds is characterized in that a corresponding compound of the formula: R1 1 (CH3> 2 r1, M (CH3> 2 H ° 1 "γΝ 0 in i v ° v JX .... Αχ

aI X —.r 80 X

* ri | ζ * ^ fC

I Υ> νγ I γνγγ X ^ * ° X ^ r

Och3 6ch3 4 where R, R and R are as defined above, Y 'is NH, N-OH or N-OCOCHg, and Y' is NH, is reduced as (a) when Y 'is NH, N- OH or N-OCOCH 3, or Y "is NH, said reduction is carried out by catalytic reduction, or (b) when Y 'or Y" is NH, this fba is prepared in situ from the corresponding ketone by condensation of the ketone with the ammonium salt of an alkanoic acid, and the reduction is carried out by a hydride reduction or by catalytic hydrogenation and, if desired, (i) a won compound wherein R and / or R is hydrogen is alkanoylated to form a corresponding compound, wherein R 1 and / 4 or R are alkanoyl of 2 or 3 C atoms, or (ii) a won compound wherein R 1 is alkanoyl of 2-3 C atoms is converted to the corresponding compound wherein R 1 is hydrogen, and /or

(Iii) a won mixture of compounds of formulas III

2 3 3 4 and IV, with groups R and R, groups R and R respectively being 0

U

together are -C-, divided into the individual compounds by selective crystallization of diethyl ether, or recrystallized for recovery

ISLAND

2 3 W

of the compound alone, wherein R and R together are -C-, of acetone / water, and / or (iv) a won compound is transferred into an acid addition salt thereof.

The preparation of the ketone intermediate compounds useful for the present process (= 0 at the 4 "position) may be carried out by oxidation of the corresponding 4" OH compounds. This oxidation can be accomplished by reaction with trifluoroacetic anhydride and dimethyl sulfoxide followed by the addition of a tertiary amine such as triethylamine.

In practice, the trifluoroacetic anhydride and dimethyl sulfoxide are initially combined in a reaction-inert solvent at ca. -65 ° C. After 10-15 minutes, the above 4 "OH compounds are added at such a rate that the temperature is maintained at about -65 ° C and does not exceed -30 ° C. At temperatures above -30 ° C, the trifluoroacetic anhydride-dimethyl sulfoxide complex The reaction temperature is maintained between -30 and -65 ° C for about 15 minutes and then lowered to about -70 ° C. A tertiary amine is added at once and the reaction mixture is allowed to warm over a period of time. The reaction mixture is then treated with water and worked up.

With respect to the amounts of reactants, for each mole of 4 "-OH compound, one mole of each of trifluoroacetic anhydride and dimethyl sulfoxide is required. Tests have shown that it is advantageous to use an excess of 1-5 times the anhydride and dimethyl sulfoxide for The tertiary amine used must correspond to the molar amount of trifluoroacetic anhydride used.

The reaction-inert solvent used in this process must be one which significantly dissolves the reactants and does not to a greater extent react with any of the reactants or with the products formed. Since this oxidation process is carried out at -30 to -65 ° C, it is preferred that said solvent, in addition to having the above characteristics, have a freezing point below the reaction temperature. Such solvents or mixtures thereof meeting these criteria are toluene, methylene chloride, ethyl acetate, chloroform or tetrahydrofuran. Solvents which meet the above requirements but which have a freezing point above the reaction temperature can be used in smaller amounts in combination with one of the preferred solvents. The particularly preferred solvent for this process is methylene chloride.

The preferred compound prepared by this process is 2'-acetyl-4 "-deoxy-4" -oxo-erythromycin A-6,9-hemiketal-11,12-carbonate ester.

The reaction time is not critical and depends on the reaction temperature and the inherent reactivity of the starting reagents. At temperatures of approx. -30 to about -65 ° C, the reaction is completed in 15 to 30 minutes.

As to the order of addition of the reagents, it is preferred that the trifluoroacetic anhydride be combined with the dimethyl sulfoxide followed by the addition of the 4 "OH compound used. It is further proposed, as mentioned above, that the reaction temperature be kept below -30 ° C. in accordance with the teachings of Omura et al., J. Org. Chem., 41, 957 (1976).

Another oxidation method uses N-chlorosuccinimide and dimethyl sulfide as the oxidizing agent. In practice, these two reaes are first combined in a reaction-inert solvent at ca. 0 ° C. After 10-20 minutes, the temperature is lowered from 0 to -25 ° C and the 4 "OH compound is added, maintaining the above temperature. After 2-4 hours of reaction time, a tertiary amine such as triethylamine is added, the reaction mixture is hydrolyzed and worked up.

With respect to the amounts of reactants, for each mole of 4 "OH compound used, one mole of each of N-chlorosuccinimide and dimethyl sulfide is required. Tests have shown that it is advantageous to use an excess of 1-20 times the succinimide. and the sulfide reactants to accelerate the completion of the reaction The tertiary amine used must correspond to the molar amount of succinimide used.

The reaction-inert solvent used in this process must be such that it substantially dissolves the reactants and does not to any great extent react with neither the reactants nor the products produced. Since the reaction is carried out at from ca. 0 ° to approx. -25 ° C, it is preferred that the solvent, in addition to having the above characteristics, have a freezing point below the reaction temperature. Such solvents or mixtures thereof meeting these criteria are toluene, ethyl acetate, chloroform, methylene chloride or tetrahydrofuran. Solvents meeting the above requirements but having a freezing point above the reaction temperature may also be used in smaller amounts in combination with one or more of the preferred solvents.

The particularly preferred solvent for the process in question is toluene-benzene.

The preferred compound prepared by this process is 2'-acetyl-4 "-deoxy-4" -oxo-erythromycin A-6,9-hemiketal-11,12-carbonate ester.

The reaction time is not critical and depends on concentration, reaction temperature and the intrinsic reactivity of the reagents. By a reaction. The reaction time is from about 0 to -25 ° C.

2 to approx. 4 hours.

As to the order of addition, as mentioned above, it is preferred that the 4 "-OH compound be added to a premix of succinimide derivative and dimethyl sulfide.

Both of the above oxidation methods are considered unique due to the selectivity of the oxidation which takes place solely at the 4 "hydroxy substituent leaving other secondary alcohols in the molecule unaffected.

A ketone intermediate compound for the compounds of formula III, in which ketones R and R are each alkanoyl of 2-3 C atoms and R is hydrogen, can be obtained by treating a ketone intermediate compound for the compounds of formula IV, wherein R "*" is 2 alkanoyl having 2-3 C atoms, with an alkanoic anhydride (R0) and pyridine.

In practice, the latter ketone is contacted with an excess of the anhydride in pyridine as a solvent. It is preferred that as much as a four-fold excess of the anhydride be used in the reaction.

The reaction is conveniently carried out at room temperature. At these reaction temperatures, the reaction time is from ca. 12 to approx. 24 hours.

Removal of an alkanoyl group at the 2 'position of the ketone flour product compounds is accomplished by a solvolysis reaction consisting of stirring with an excess of methanol overnight at room temperature. Removal of the methanol and subsequent purification, if necessary, of the product obtained as the residue of evaporation provides the corresponding compound wherein R R is hydrogen.

Preferred ketone intermediates for the preparation of the compounds of formulas III and IV are 2, -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 routes can be used in the preparation of the compounds of formulas III and IV from the ketone intermediate compounds.

Preparation of the compounds of formula III is carried out by condensing the ketone compound with the ammonium salt of a lower alkanoic acid and subsequently reducing the in situ formed imine. The term "lower alkane" here refers to an acid having 2-4 C atoms.

In practice, a solution of the ketone 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 hydrolyzing and the product is isolated.

Although 1 mole of the ammonium alkanoate is required per moles of ketone, it is preferred to use an excess, as large as ten times, to ensure complete and rapid formation of the imine. Such surplus amounts appear to have little detrimental effect on the quality of the product.

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

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

When the lower alkanol solvent is methanol, as mentioned above, a significant solvolysis of any alkanoyl group occurs at the 2 'position. To avoid removal of such a group, it is preferred that isopropanol is used as the reaction solvent.

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

By isolating the desired compounds of formula III from the present non-basic by-products or starting material, the basic nature of the final product can be taken advantage of. An aqueous solution of the product is thus 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 higher than 5.

The extracting solvents, either ethyl acetate or diethyl ether, are washed back with brine and water, dried over sodium sulfate, and the product is recovered by removing the solvent. Further purification, if necessary, can be carried out by column chromatography on silica gel by methods known per se.

As mentioned above, solvolysis of the 2 * alkanoyl group of a compound of formula III can be carried out by leaving a methanol solution of said alkanoyl compound overnight at room temperature.

During the reductive amination of ketone intermediate compound · · 0 2 3 µl in series where R and R together are -C- and R is alkanoyl with 2-3 C atoms or hydrogen, it is observed that a mixture is formed 2 3 3

of amines of formulas III and IV, wherein R and R and R, respectively

0 4 li and R together are -C-. This can be represented by the following reaction scheme: 9 148036 R1 N (CH) / NH4OCOCH3 ° "\ 1 I NaCNBH.,> 0 '/ ^ N ai' ι \ λυυ 0> 0" ° x 'OCH3 (R2 + R3 =) 1 ^ (CH 3} 2

HO R1? (CH3> 2? R-0 * I

χγ "yK ογΝ row

° K> I l + ° <A I

* po h $ ^ Unt * f ° V

° / \ NH2 O / ¾1¾ III 7 OCH3 IV X ^ H3 (R2 + R3 = -å-) (R3 + R4 = - $ -)

The amine products III and IV shown are conveniently separated by selective crystallization of diethyl ether. Recrystallization of the mixture of III and IV of 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 for the amine compounds of formula IV is the same route as discussed above for the compounds of formula III and comprises condensation of the ketone intermediate compound for the compound of formula IV with an ammonium alkanoate followed by reduction of the in situ formed imine with sodium cyanoborohydride.

13 4

Compounds of formula IV wherein R, R and R are as defined above are also prepared by reducing the above imine using hydrogen and an appropriate hydrogenation catalyst. In practice, the ketone intermediate compound in question is lower alkanol, such as methanol or isopropanol, treated 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 substantially complete.

ίο 148036

Although 1 mole of the ammonium alkanoate is required per moles of ketone, it is preferred to use an excess, as large as ten times, to ensure complete and rapid formation of the imine. Such surplus quantities have been found to have little detrimental effect on the quality of the product.

The hydrogenation catalyst can be selected from a variety of agents. However, Raney nickel and 5-10 percent palladium-on-charcoal are the preferred catalysts. These can be used in varying amounts depending on how quickly the reaction is to be completed. Amounts of 10-200% by weight of the ketone can be used effectively.

The pressure of the gaseous hydrogen in the hydrogenation vessel also influences the reaction rate. For an appropriate reaction time, an initial pressure of 3.5 kg / cm is preferred. For simplicity, it is also preferred that the reduction be carried out at room temperature.

The reaction time depends on a number of factors including temperature, pressure, concentration of the reactants and the intrinsic reactivity of the reagents. Under the above preferred conditions, the reaction is completed in 12-24 hours.

The product is isolated by filtration of the spent catalyst and removal of the solvent in vacuo. The residual 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.

As discussed above, when the lower alkanol solvent is methanol, there is considerable solvolysis of any alkanoyl group at the 2 'position. In order to avoid removal of such a group, it is preferred that isopropanol is used as the reaction solvent.

The second synthetic route to the antibacterial 4 "amino-erythromycin compounds of formula IV initially involves conversion of the ketone intermediate compound to an oxime or an oxime derivative 0 <1 i.e. Y '= N-OH or NO-CCH followed by reduction of the oxime or derivative thereof.

Oxime past ndpl bpiup can be prepared by reacting said ketones with hydroxylamine hydrochloride and barium carbonate in methanol or isopropanol at room temperature. In practice, it is preferable to use an excess of hydroxylamine, and as much as an excess of 3 times the desired intermediate provides good yields. Use of room temperatures and an excess of the hydroxylamine allow preparation of the desired oxime derivative over a reaction time of 1 to 3 hours. The barium carbonate is used in molar amounts of twice the molar amount of the hydroxylamine hydrochloride used. The product is isolated by adding 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 to dryness in vacuo. The residue is then partitioned between water at pH 9.0-9.5 and a water-immiscible solvent. 0

Preparation of the O-acetyloxime compounds (Y YN-O-CCH ^) is carried out by acetylation of the corresponding oxime. According to experiments, 1 mole of the oxime is reacted with 1 mole of acetic anhydride in the presence of 1 mole of pyridine or triethylamine. The use of an excess of the anhydride or pyridine helps to complete the reaction and an excess of 30-40% is preferred. The reaction is best carried out in an aprotic solvent, such as benzene or ethyl acetate, at room temperature overnight. After the reaction is complete, water is added, the pH is adjusted to 9.0 and the product is separated into the solvent layer.

The preferred oxymers and oxime derivatives which are useful intermediates for the compounds of formula IV are 2'-acetyl-4 "-deoxy-4" -oxo-erythromycin A-oxime, 2β-acetyl-4 "-deoxy-4" - oxo-erythromycin AO-acetyloxime, 4 "-deoxy-4" -oxo-erythromycin A-oxime, and 4 "-deoxy-4" -oxo-erythromycin AO-acetyloxime.

Reduction of the oxime or oxime derivative is accomplished by catalytic hydrogenation, in which a solution of the oxime or derivative thereof in a lower alkanol such as isopropanol and a Raney-nickel catalyst are shaken in a hydrogen atmosphere at an initial pressure of 70.3 kg / ml. cm at room temperature overnight. Filtration of spent catalyst followed by removal of the solvent from the filtrate leads to isolation of the desired antibacterial 4 "amino erythromycin compound of formula IV. If methanol is used as a solvent in this reduction, solvolysis of a 2 'alkanoyl group is likely To avoid this side reaction, isopropanol is used.

Preferred among the present compounds of formulas 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

12 148036 and of 4 "-deoxy-4" -deoxy-4 "-amino-erythromycin A-11, 12-carbonate ester.

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

As mentioned above, the stereochemistry of the starting materials leading to the subject antibacterial compounds is the stereochemistry of the natural material. The oxidation of a 4 "hydroxy group to a ketone group and the subsequent conversion of the ketone compound to the 4" amino compound provide an opportunity for the stereochemistry of the 4 "substituent to be changed from the stereochemistry of the natural product. Consequently, when the ketone intermediate compounds are converted to amines by one of the methods described above, it is possible that two epimeric amines are formed and in experiments both amines are observed to be present in the final product in varying amounts depending on the choice of synthesis method. If the isolated product consists predominantly of one of the epimers, said epimer can be purified by repeated recrystallization of a suitable solvent to a constant melting point, the other epimer, that is, present in smaller amounts in the initially isolated solid material. is the predominant product in the mother liquor and can be extracted therefrom by methods known in the art, such as evaporation of the mother liquor and gent. again recrystallization of the evaporation residue into a product of constant melting point.

Although said mixture of epimers can be subdivided by methods known in the art per se, it is advantageous for practical reasons to use said mixture as isolated from the reaction. However, it is often advantageous to purify the mixture of epimers by subjecting the at least one recrystallization of a suitable solvent, subjecting the column or high pressure liquid chromatography, solvent partitioning or tearing to a suitable solvent. While said purification does not necessarily separate the epimers, it removes such foreign materials as starting materials and unwanted by-products.

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

They provided. compounds of formulas 13 and IV show in; we believe activity against various gram-positive microorganisms, e.g. Staphylococcus aureus and Streptococcus pyogenes, and against certain gram-negative microorganisms, such as those with spherical or ellipsoid form (cocci). Their activity can be readily detected by in vitro tests against various microorganisms in a brain-heart infusion medium by the usual dual-series dilution technique. Their in vitro activity makes them useful for local use, such as in the form of ointments or creams.

For in vitro use, e.g. for local use, it will often be convenient to combine the selected product with a pharmaceutically acceptable carrier such as vegetable oil or mineral oil or a softening cream. Similarly, the compounds 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 detrimental effect on the active ingredient. For such purposes, it will generally be acceptable to use active ingredient concentrations of from ca. 0.01% to approx. 10% based on the weight of the total composition.

Furthermore, many of the compounds of this invention and their acid addition salts are active against gram-positive and certain gram-negative microorganisms, e.g. Pasteurella multicida and Neisseria sicca, in vivo via oral and / or parenteral routes of administration to animals and humans. Their in vivo activity is more limited with respect to susceptible organisms and is determined by the usual method comprising infecting mice of substantially uniform weight with the test organism and subsequent treatment of the mice orally or subcutaneously with the test compound. .g. 10 mice, an intraperitoneal inoculation of appropriately diluted cultures containing approximately 1 to 10 times LD ^ 00 (the lowest concentration of organisms required to cause 100% death). Control trials are conducted simultaneously, at which mice receive inoculum of lower dilutions as a check for possible variation in virulence of the test organism. The test compound is administered 0.5 hours after inoculation and again 4, 24 and 48 hours later. Surviving mice are kept for four days after the last treatment and the number of survivors is noted.

When used in vivo, the subject compounds can be used orally or parenterally, e.g. by subcutaneous or intramuscular injection, at a dose of from ca. 1 mg / kg to approx. 200 mg / kg body weight per day. day. The most widely used dose range is from approx.

5 mg / kg to approx. 100 mg / kg body weight per day. day, and the preferred if-. advice is from approx. 5 mg / kg to approx. 50 mg / kg body weight per day. day.

Carriers suitable for parenteral injection may be either aqueous such as water, isotonic saline, isotonic dextrose or Ringer's solution, or non-aqueous such as vegetable oils (cotton seed, peanut oil, corn, sesame), dimethyl sulfoxide and other non-aqueous carriers which will not interfere with the therapeutic activity of the composition and which are non-toxic in the volume or amount ratio used (glycerol, propylene glycol, sorbitol). Furthermore, compositions suitable for preparing solutions immediately prior to use may be advantageously prepared. Such compositions may comprise liquid diluents, e.g. propylene glycol, diethyl carbonate, glycerol or sorbitol, buffering agents, hyaluronidase, local anesthetics and inorganic salts to provide desired pharmacological properties.

The present compounds may also be combined with various pharmaceutically acceptable inert carriers, including solid diluents, aqueous carriers and non-toxic organic solvents, in the form of capsules, tablets, cans, lozenges, 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 to approx. 90% by weight of the total composition.

In order to detect the particularly good antibacterial activity of the 4 "amino-erythromycin compounds of the invention, the minimum inhibitory concentration (MIC) in micrograms per ml is given below for a number of microorganisms for the compounds of formula III or IV, respectively. and a known closely related erythromycin A derivative.

148036 I. N (CHU) 2 = R1 I 3 2 H0y ^ i Λ κ2α> * γ ^ οΑ

r3 ° L X

* 'π Π "ο" γ ° γ' T- y.

Ill 'OCH3 MIC, mcg / ml .......

R = ^ NH ~ R = »1» NH „R = Λ / ν'ΝΗ0 R = ^ NH9 1 2 1 2 1 1 1 z

IC = H R = H R = CH3CO R = H

R + R3 = R2 + R3 = R2 + R3 = R2 = CH, CO

Microorganism _co_ -C0-. -c.0- ώ3 _ 3 _H - ii_ (1) Staph, aur. <0.10 <0.10 0.39 3.12 (2) "" <0.10 <0.10 0.39 12.5 (3) "" 200 200> 200> 200 (4) "" 200 200> 200> 200 (5) "" 0.10 <0.10 <0.1> 200 (6) "" 200 200> 200> 200 (7) "" 3.12 3.12 3.12 25 (8) Strp. fae. <0.10 <0.10 0.39 50 (9) Strp. pyog. <0.10 <0.10 $ 0.10 1.56 (10) "(11) Myco. Smeg. 50 50 100 0.39 (12) B. sub. $ 0.10 $ 0.10 <0.10 1, 56 (13) E.coll 3.12 1.56 3.12 100 (14) "" 3.12 1.56 3.12 200 (15) "" 3.12 3.12 6.25 200 (16) Ps. Air 50 50 200 200> 200 (17) Klebs. Pn. 12,5 6.25 12.5 200 (18) "6.25 6.25 50> 200 (19) Prot. Mira. 200 100 200 > 200 (20) Prot. Morning 50 50 200> 200 (21) Psalm Chol-6.25 6.25 6.25> 200 (22) Ps. Typhm 6.25 6.25 6.25 > 200 (23) "" 6.25 6.25 12.5 100 (24) Past, Mult. <0.10 <0.10 $ 0.1 25 (25) Serial Mar 25 25 50> 200 (26) ) Ent. Aero 6.25 6.25 50> 200 (27) Ent. Cloa. 25 25 25> 200 (28) Neiss. Sic. <0.10. $ 0.10 $ 0.1 0.20 16 148036. ϊ (ΟΗ3> 2 ° γλ. ° γ ^ 4 Æ 6.0 R ° '“' Γ Ηο'Τ κ3 °“ 4 \ IV 7 OCH3 MIC, mcg / ml R = «· * ΝΗ0 R = '*» »NH0 R = λλ-ΝΗ, R = mi NH,

1 Δ 1 Δ 1 I

R · 1 · = H R = H R = CH-.C0 R = H

3 3 3 4J 3 4 R-3 = H R3 = H R + R = R + R =

Microorganism r4 = H R4 = H -CO- -C0- (1) Staph, aur. 0.39 0.39 0.78 <0.10 (2) "" 0.78 0.78 0.78 <0.10 (3) ""> 200> 200> 200 200 (4) ""> 200 > 200> 200 200 (5) "" - - £ 0.39 0.10 (6) ""> 200> 200> 200> 200 (7) "" 3.12 6.25 25 6.25 (8) Strp. fae. 0.39 1.56 0.78 <0.10 (9) Strp. pyog. £ 0.10 £ 0.10 0.20 £ 0.10 (10) "" - - 200 (11) Myco. smeg. 100 200> 200 50 (12) B. sub. 1.56 0.39 0.78 <0.10 (13) E. coli 50 25 25 1.56 (14) "" 50 50 25 1.56 (15) "" 50 50 25 3.12 (16) ps. aerug. 200> 200> 200 100 (17) Klebs. pn. 200 100> 200 6.25 (18) ""> 200 200> 200 6.25 (19) Prot. mira. > 200> 200> 200 100 (20) Prot. bre. > 200> 200> 200 50 (21) Salmon. chol-su. 50 200 50 3.12 (22) Sal. typhm. 50 100 100 3.12 (23) "" 50 200 50 6.25 (24) Fits, multo. 1.56 1.56 0.39 £ 0.10 (25 Serr. Mar. 200> 200> 200 25 (26) Ent. Aero. 100> 200 100 6.25 (27) Ent. Cloa. 200> 200 100 12.5 (28) Neiss. 0.39 6.25 <0.10 <0.1 17 148036, n (ch3) 2 "" "J 1" "η- ^ ΝΛ- ^ R '° - r Ho'y .HO - ^ s.

II ^^^ OSO ^ CH.

o 7% 2 3 t OCH3

Comparison MIC, mcg / ml

Microorganism RJ. ^ R

(1) Staph, aur. 1.56 (2) "" 1.56 (3) ""> 50 (4) ""> 50 (5) "" 0.39 (6) ""> 50 (7) "" 50 (8) Strp 0.39 (9) Str. pyog. 0.20 (10) ""> 50 (11) Myco. smeg.> 50 (12) B. sub .20 (13) E. coli> 50 (14) ""> 50 (15) ""> 50 (16) Ps. Air.> 50 (17) Klebs. Pn.> 50 (18) ""> 50 (19) Prot. Mira.

(20) Prot. bre. > 50 (21) Salmon. chol-su.

(22) Sal. typhm. > 50 (23) "(24) Past, Mult. 12.5 (25) Serr. Mar.> 50 (26) Ent. Aero.> 50 (27) Ent. Cloa.> 50 (28) Neiss. Sic. 12.5

The process according to the invention is further described by the following examples.

18 148036

Example 1 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 was left stirring at room temperature overnight. The solvent was removed under reduced pressure and the foam recovered as evaporation residue was treated with a mixture of 50 ml of chloroform and 50 ml of water. The pH of the aqueous layer was adjusted to 9.5 and the organic layer was separated. The chloroform layer was treated with fresh water and the pH was adjusted to 4.0. The pH of the acidic aqueous layer containing the product was gradually adjusted to 5, 6, 7, 8 and 9 by the addition of base, extracting with fresh chloroform at each pH. The extracts at pH 6 and 7 contained the bulk of the product and these were combined and treated with fresh water at pH 4. The aqueous layer was again adjusted through pH 5, 6 and 7, with each chloroform extracted at each pH. The chloroform extract at pH 6 was dried over sodium sulfate and concentrated to give 249 mg of the product as an epimeric mixture.

NMR (δ, CDCl3): 3.30 (1H) s, 3.26 (2H) s, 2.30 (6H) s and 1.46 (3H) s.

Example 2 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 was added 3.16 g of dry ammonium acetate. After 5 minutes, 188 mg of sodium cyanoborohydride was washed into the reaction mixture with 5 ml of methanol and the reaction mixture was allowed to stir at room temperature overnight. The pale yellow solution was poured into 300 ml of water and the pH was adjusted to 6.0. The aqueous material was 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 were combined and washed with 125 ml of fresh water. The separated aqueous layer was extracted with ether (1X100 mL) at pH 7, ethyl acetate (1x100 mL) at pH 7, ether (1x100 mL) at pH 7.5, ethyl acetate (1x100 mL) at pH 7.5, and ethyl acetate (1x100 ml) at pH 8, 9 and 10. The ethyl acetate extracts at pH 9 and 10 were combined, washed with a saturated brine solution and dried over sodium sulfate. Removal of the solvent in vacuo gave 30 g of an epimeric mixture of the desired product as an ivory foam.

NMR (δ, CDCl3): 3.30 (3H) s, 2.27 (6H) s and 1.44 (3H) s.

Example 3 · 4 "-Deoxy-4" -amino-erythromycin A (single epimer).

A solution of 10.0 g of the epimer mixture of 2'-acetyl-4 "-deoxy-4" amino-erythromycin A in 150 ml of methanol was allowed to stir at room temperature under nitrogen for 72 hours. The solvent was removed in vacuo and the residue was dissolved in a stirred mixture of 150 ml of water and 200 ml of chloroform. The aqueous layer was discarded and 150 ml of fresh water was added. The pH of the aqueous layer was adjusted to 5 and the chloroform layer was separated. The pH of the aqueous phase was then adjusted to 5.5 - 6 - 7-8 and 9, after each setting extracted with 100 ml of fresh chloroform. The chloroform extracts from pH 6, 7 and 8 were combined, washed successively with water and a saturated brine solution, and dried over sodium sulfate. Removal of the solvent under reduced pressure gave 2.9 g of an epimeric mixture of 4 "-deoxy-4" amino-erythromycin A. A sample of 1.9 g of the mixture was grated with diethyl ether, which yielded some of it. unresolved foam to crystallize. The solids were filtered off and dried to give 67 mg of a single epimer of 4 "-deoxy-4" -amino-erythromycin A, m.p. 140-147 ° C.

Example 4 11-Acetyl-4 "-deoxy-4" -amino-erythromycin A-6,9-hemiketal.

To a stirred solution of 4.4 g of 11-acetyl-4 "-deoxy-4" -oxo-erythromycin A-6,9-hemiketal and 4.38 g of ammonium acetate in 75 ml of methanol was added 305 mg of 85% sodium cyanoborohydride. After stirring at room temperature overnight, the reaction mixture was poured into 300 ml of water, to which was then added 250 ml of chloroform. The pH of the aqueous layer was adjusted to 9.8 and the chloroform layer was separated. The aqueous layer was extracted with chloroform again and the chloroform extracts were combined, dried over sodium sulfate and concentrated to a white foam. The remaining foam was dissolved in a stirred mixture of 125 ml of water and 125 ml of fresh chloroform and the pH was adjusted to 4.9. The chloroform was separated and discarded, and the aqueous layer was adjusted to pH 5, 6, 7 and 8, with fresh chloroform extracted after each adjustment. The extracts from the aqueous material at pH 6 and 7 were combined, washed with saturated brine and dried over sodium sulfate.

20 148036

Removal of the solvent gave 1.72 g of the desired product as a white foam. The product was dissolved in a minimal amount of diethyl ether and then treated with hexane until cloudy. The crystalline product formed was filtered off and dried, 1.33 g, m.p. 204.5 to 206.5 ° C.

NMR (δ, CDCl3): 3.31 (2H) s, 3.28 (1H) s, 2.31 (6H) s, 2.11 (3H) s and 1.5 (3H) s.

Example 5 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 were added 193 g of ammonium acetate at room temperature with stirring. After 5 minutes, the resulting solution was cooled to ca. -5 ° C and then treated with 13.4 g of 85% sodium cyanoborohydride in 200 ml of methanol over a 45 minute addition period. The cooling bath was removed and the reaction mixture was allowed to stir at room temperature overnight. The reaction mixture was 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 was adjusted from 6.2 to 4.3 with 6N hydrochloric acid and the chloroform layer was separated. The chloroform was combined with 1 liter of water and the pH was adjusted to 9.5. The organic phase was separated, dried over sodium sulfate and concentrated under reduced pressure to give 174 g of a white foam. This residual material was dissolved in a mixture of 1 liter of water and 500 ml of ethyl acetate and the pH was adjusted to 5.5. The ethyl acetate layer was separated and the aqueous layer was successively adjusted to pH 5.7 and 9.5, extracting with 500 ml of fresh ethyl acetate after each pH adjustment. The ethyl acetate extracts at pH 9.5 were dried over sodium sulfate and concentrated in vacuo to dryness to give 130 g. Of the foam obtained as evaporation residue, 120 g was dissolved in a mixture of 1 liter of water and 1 liter of methylene chloride. The pH of the aqueous layer was 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 was dried over sodium sulfate and concentrated under reduced pressure to give 32 g of the product as a white foam. Crystallization of 250 ml of acetone-water (1: 1, v / v) gave 28.5 g of the crystalline epimers.

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

148036 21

Example 6

Separation of the epimers of 4 "-deoxy-4" -amino-erythromycin A-6,9-hemiketal-11,12-carbonate ester.

On a high pressure liquid chromatography column (1/2 "x 9 cm) packed with Gf 254 silica gel impregnated with formamide and eluted with chloro-2 form was applied 200 mg. A pressure of 16.87 kg / cm was applied at a rapid rate. at a rate of 4.76 cm per minute and a fraction size of 10 ml was used. Fractions 14 to 21 and 24 to 36 were collected.

Fractions 14 to 21 were pooled and concentrated to ca.

50 ml. Water (50 ml) was added and the pH was adjusted to 9.0. The chloroform layer was separated, dried over sodium sulfate and concentrated to give 106 mg of a white foam. Diethyl ether tearing caused the foam to crystallize. After stirring at room temperature for 1 hour, the crystalline product was filtered off and dried. 31.7 mg, m.p. 194-196 ° C.

NMR 100 Mz (δ, 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 were pooled and worked up as above, yielding 47.1 mg of product as a white foam identical to the material of Example 10.

Example 7

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

The mother liquor was treated with 75 ml of water and the pH was adjusted to 5.0. The ether layer was replaced with 75 ml of fresh ether and the pH was adjusted to 5.4. The ether was replaced with ethyl acetate and the pH was raised to 10. The basic aqueous layer was extracted with ethyl acetate (2 x 75 mL) and the first ethyl acetate extract was dried over sodium sulfate and concentrated to dryness. The foam obtained as evaporation residue (1.96 g) was added to a mixture of 75 ml of water and 50 ml of diethyl ether and the pH was adjusted to 5.05. The ether was separated and the aqueous layer was successively adjusted to pH 5.4 - 6.0 - 7.05 and 8.0, extracting with 50 ml of fresh diethyl ether after each pH adjustment. The pH was finally adjusted to 9.7 and the aqueous layer was extracted with 50 ml of ethyl acetate. The ether extract from pH 6.0 was combined with 75 ml of water and the pH was adjusted to 9.7. The ether layer was separated, dried and concentrated in vacuo to give 460 mg of a white foam.

NMR 100 Mz (δ, CDCl 3): 5.20 (1H) t, 3.43 (2H) s, 3.40 (1H) s, 2.38 (6H) s, 2.16 (3H) s, 1 , 70 (3H) s and 1.54 (3H).

These NMR data show that the product was the epimers of 2'-acetyl-4 "-deoxy-4" amino-erythromycin A-6,9-hemiketal-11,12-carbonates.

The above 1.69 g was dissolved in a mixture of 75 ml of water and 75 ml of diethyl ether and the pH was adjusted to 4.7. The ether was separated and the aqueous layer was 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 were dried over sodium sulfate and concentrated under reduced pressure to give 1.26 g of a white foam. Crystallization of this residue gave 411 mg of product, m.p. 193-196 ° C (dec.). The mother liquor was concentrated to dryness and the residue was dissolved in hot ethyl acetate. The solution was left to stand overnight at room temperature. The crystalline solids which precipitated were filtered off and dried to give 182 mg of additional product, m.p. 198-202 ° C (dec.).

NMR 100 Mz (δ, CDCl 3): 5.10 (1H) t, 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.

These NMR data indicated that the product was the epimers of 2'-acetyl-4 "-deoxy-4" amino-erythromycin A-11,12-carbonate ester.

23 148036

Example 8

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 was left stirring overnight at room temperature. The reaction solution was poured into 100 ml of water followed by the addition of 50 ml of ethyl acetate, the pH was adjusted to 9.5 and the organic phase was separated, and the extraction was repeated with 50 ml of fresh ethyl acetate. and concentrated to give 392 mg of a white foam. Tearing with diethyl ether and scraping with a glass rod produced crystallisation. according to NMR identical to material prepared in Example 9, NMR 100 Mz (δ, CDCl 3): 3.26 (3H) s, 2.32 (6H) s, 1.61 (3H) s and 1.44 (3H) s .

These NMR data showed that the crystalline product was a single epimer of 4 "-deoxy-4" -amino-erythromycin A-11,12-carbonate ester.

The preserved mother liquor was concentrated in vacuo to give 244 mg of a white foam.

NMR data showed that this product was a mixture of the epimers of 4 "-deoxy-4" amino-erythromycin A-6,9-hemiketal-11, 12-carbonate ester and identical to the product of Example 5 .

Example 9

From the epimeric mixture of 4 "-deoxy-4" -amino-erythromycin A-11,12 carbonate ester from the non-crystalline product of Example 5, 8 g was dissolved in 50 ml of diethyl ether. The product was crystallized by scraping with a glass rod. After 20 minutes of stirring, the crystalline product was filtered off and dried. 1.91 g, m.p. 198.5 to 200 ° C.

24 NMR 100 Μζ (δ, CDCl3): 3.26 (3H) s, 2.30 (6H) s, 1.61 (3H) s and 1.45 (3H) s.

These NMR data showed that the crystalline product was a single epimer of 4 "deoxy-4" amino erythromycin A-11, 12 carbonates and identical to the ketone product of Example 8.

Example 10

Of the epimer of Example 9, 1 g was dissolved in 20 ml of acetone and heated at steam bath temperatures until the boiling point was reached. Water (25 ml) was added and the resulting solution was allowed to stir at room temperature. After stirring for 1 hour, the precipitate formed was filtered off and dried to give 581 mg, m.p. 147-149 ° C.

NMR 100 Μζ (δ, CDCl3): 5.12 (1H) d, 3.30 (3H) s, 2.30 (6H) s, 1.62 (3H) s and 1.36 (3H) s.

These NMR data showed that the product was 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 6 .

Example 11 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 were shaken at room temperature in a hydrogen atmosphere at an initial pressure of 2 3.5 kg / cm overnight. The spent catalyst was filtered off and the filtrate was concentrated to dryness in vacuo. The evaporation residue was partitioned between water chloroform at a pH of 5.5.

The aqueous layer was separated, the pH was adjusted to 9.6, and chloroform was added. The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure to dryness. The white foam (19 g) obtained as evaporation residue was grated with 150 µl of diethyl ether at room temperature for 30 minutes. The resulting solids were filtered off and dried, yielding 9.45 g of a single epimer indistinguishable from that obtained in Example 3.

Claims (3)

An analogous process for preparing epimeric 4 "amino-erythromycin compounds of the formula: 1 N (CH3) 2 ^ CH3} 2, ΐχύ .y \ 3. I or r3o-1 Jl, ι »Ί p«, n * »'*] rV _ C) 1 Γ ° ν V ° iv V" 8 nh2 o \ Χυνη2' S) CH- / T> CH3 III 3 IV or pharmaceutically acceptable acid addition salts thereof, wherein and 4 R are each selected from the group consisting of hydrogen and alkanoyl having 2 to 2-3 C atoms, R being alkanoyl having 2-3 C atoms, and R being hydrogen, 0 2 3 3 4 M wherein R and R or R and R taken together may also be -C-, or 2 3 a mixture of compounds of formulas III and IV wherein R and R, R 3 and R together are -C- , characterized in that a corresponding compound of the formula:! N (CH3) 2 1 j> <Cl3 3> 2 H ° 1 VS 0 I vy ΕΧγ '„/ S» ···· 0 TT π now R3 ° JI or HoJ In VVr XXrY> Y ° / f '' DCH3 0cH3