CS241099B2 - Process for the preparation of 4 "-epi-9-deoxo-9a-methyl-9a-aza-9a-bomoerythromycin A - Google Patents

Abstract

A method for preparing 4“-epi-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A or its pharmaceutically acceptable salts, characterized in that 4“-deoxy-4“-oxo-9- -deoxo-9a-methyl-9a-aza-9a-homoerythromycin A is hydrogenated over a noble metal catalyst or Raney nickel in an inert solvent at a temperature of 20 to 100 °C. The compound of the invention and its pharmaceutically acceptable salts can be used as an antibacterial agent.

Description

The present invention relates to the antibacterial 4'-epi-19-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A, its pharmaceutically acceptable salts and intermediates useful in the preparation from erythromycin A.

Erythromycin A is a well-known macrolide antibiotic of formula I that has found significant clinical use.

The therapeutically useful compound is the 4'-epimer of the previously reported erythromycin A of formula II (R = methyl and III (R = hydrogen))

Some of the novel intermediates are also 4''-epimers of previously known compounds. Thus, 4"-epi-9-deoxo-9a-aza-9a-homoerythromycin A is the 4"-epimer of the above compound of formula III, and 4"-epierythromycin A oxime is the 4"-epimer of the erythromycin A oxime described by Djokic et al. in U.S. Patent No. 3,478,014.

The subject of the present invention is a process for the preparation of 4“-epi-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A of formula IV or its pharmaceutically acceptable salts, which is characterized in that 4“-deoxy-4“-oxo-9-deoxo-9a-methyl-9a-aza-homoerythromycin A is hydrogenated over a noble metal catalyst or Raney nickel in an inert solvent at a temperature of 20 to 100 ^C, C.

which are the subject of Belgian patent No. 892,357.

In the Belgian patent, the compound of formula II is called the N-methyl derivative of "11-aza-10-deoxo-10-dihydroethythromycin A", a name previously used by Kobrehel et al., U.S. Patent No. 4,328,334, for the precursor of this compound of formula III.

Since the erythromycin A derivative has a ring extension (homo, nitrogen is replaced by carbon (azay), we prefer to name it 9-deoxo-9a-aza-9a-homoerythromycin A. This compound can also be named as a 10-aza-14-hexadecanolide derivative.

(IV) R = methyl, Z = Z ¹ = hydrogen atom, (V) R = hydrogen atom, Z and Z ¹ together form an oxygen atom, (Ví) R = Z = Zi = hydrogen atom.

The therapeutically active compound IV of the invention exhibits a relatively broad spectrum of antibacterial activity, which includes strains susceptible to erythromycin A and, in addition, fully encompasses the major respiratory pathogen Hemophilus influenzae. It exhibits high oral absorption and an excellent in vivo half-life, which makes compound IV particularly valuable for the treatment of bacterial infections in mammals.

The intermediates in the synthesis of 4“-epi-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (IV) are:

a) A compound selected from the group consisting of 4'-epi-9a-aza-9a-homoerythromycin A and its 9-deoxo-derivative of formulae V and VI above.

bj 4“-Epierythromycin A-oxime.

cj A compound selected from the group consisting of 9a-benzyloxycarbonyl-9-deo«o-4“-deoxy-4“-oxo-9a-aza-9a-homoerythromycin A of formula VII; 9-deoxo-4“-deoxy-4“-oxO'-9a-methyl-9a-aza-9a-homoerythromycin A of formula (Vila) and their corresponding 2‘-O-alkamoyl derivatives with up to 3 carbon atoms of formulas VIII and Vlila. Acetyl is preferably used as the 2‘-O-alkamoyl derivative with 2 to 3 carbon atoms.

(VII) Ri = benzyloxycarbonyl, R ² = H (VIII) Ri = benzyloxycarbonyl,

R ² = (C ₂ —C 3 ) alkanoyl (VIIIa) R 1 = methyl, R ² = H (VIIIa) R 1 = methyl, R ² = alkanoyl with 2 to carbon atoms.

d) A compound selected from the group consisting of 2'-O-acetyl- and 2'-O-propionyl-9-deoxo-9a-benzoyldoxycarbonyl-9a-aza-9a-homoerythromycin A of formula IX. The 2'O-acetyl derivative of formula IX is particularly valuable.

(IX) Ri — benzyloxycarbonyl,

R ² = alkanoyl with 2 to 3 carbon atoms.

e) A compound from the group consisting of 4“-epi-9-deoxo-9a-hydroxy-9a-aza-9a-homoerythromycin A 3′-N-oxide and 4“-epi~9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A 3′-N-oxide, of formulas (Xj and (XI).

(X) R ³ = hydroxyl (XI) R ³ = methyl.

The antibacterial compound of the present invention, 4'-epi-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (IV), is readily prepared by a number of routes from erythromycin A. These routes, which variously lead through novel and known compounds as intermediates, include the following transformations:

(A) epimerization at C-4" (BJ ring expansion with introduction of a nitrogen atom at position 9a (C) removal of the 9-oxo group (D) methylation of 9a-N together with optional or necessary introduction or removal of protecting groups.

The following transformations performed in one or more sequences are preferred: (A)(B)(C)(D), (B)(A)(C)(D) or (B)(C)(D)(A).

The various intermediates and final products are isolated by standard methods (e.g. extraction, precipitation, evaporation, chromatography, crystallization).

(A)(B)(C)(D)

The operational sequence (A)(B)(C)(D) involves first converting erythromycin A (I) to 4-epierythromycin A by the method of Sciavolino et al. (supra). 4-Epi-erythromycin A is then converted in almost quantitative yield to 4'-epi-erythromycin A oxime by reaction with hydroxylamine or, preferably, a hydroxylammonium salt such as the hydrochloride.

Under the presently found preferred conditions, at least one molar equivalent, usually an excess, for example 10 to 30 equivalents of hydroxylamine, is used in an excess of a weakly basic amine (preferably pyridine) as solvent at a temperature of from 0 to 50 ^° C, preferably at room temperature.

The resulting 4"-epi-erythromycin oxime is rearranged to the 4"-epi-9a-aza-9a-homoderivative (V)' by a Beckman rearrangement. Preferred conditions employ an excess (e.g. 3 to 4 molar equivalents) of an organic sulfonyl chloride, preferably methanesulfonyl chloride, which is reacted with the oxime (in the form of the free base or in the form of an acid salt) in a mixture of a lower ketone (e.g. methyl ethyl ketone, acetone) and water containing a large excess of sodium bicarbonate at a temperature of from 0 to 50°C, preferably from 0 to 30°C.

The amidic carbonyl at the C-9 position of the compound of formula (V) is then preferably reduced to the corresponding dihydro derivative, for example 4'-epi-9-deoxo-9a-aza-9a-homoerythromycin A (VI) with sodium borohydride (preferably using an excess to force the reaction to complete in a suitable time, but using at least two equivalents).

The reduction is carried out in a suitable protic solvent such as a lower alkanol (preferably methanol) at a temperature of 0 to 50°C (preferably at or below 38°C). Excess NaBH4 is carefully decomposed by adding dilute acid to the reaction mixture.

Final methylation to form the compound

IV is carried out by reductive methylation using formaldehyde in the presence of a reducing agent such as hydrogen and a noble metal catalyst, sodium cyanoborohydride or, preferably, formic acid.

The reaction is preferably carried out with at least one equivalent of formaldehyde and one equivalent of formic acid in an inert solvent at 20 to 100°C.

The preferred solvent is chloroform.

In this solvent, the reactants are preferably mixed at room temperature and then heated to reflux to complete the reaction.

Alternatively, methylation of the compound of formula (VJ) to (IV) is carried out by oxidative protection of the dimethylamino group to the corresponding N-oxide (with simultaneous formation of the 9a-N-hydroxy derivative), methylation with methyl iodide with simultaneous (at least partial) 9a-N-deoxygenation and reduction of the resulting 9a-methyl-3'-N-oxide.

The oxidation of the compound of formula VI is readily carried out by reaction with hydrogen peroxide, generally in excess of the minimum required two molar equivalents in a reaction-inert organic solvent at a temperature of 10 to 50°C, preferably at room temperature.

In this way, 9α-hydroxy-3′-N-oxide (X) is formed. This compound is methylated and deoxygenated to the compound of formula (XI) with methyl iodide, preferably in a reaction-inert organic solvent, for example methylene chloride, at a temperature of 0 to 50 °C (preferably at room temperature), most preferably in the presence of a base insoluble in the solvent which neutralizes the resulting acid (for example, H2O if methyl iodide is used as the methylating agent).

With methylene chloride as solvent, potassium carbonate is the preferred base. Thus, the excess base and the resulting potassium iodide are completely removed by simple filtration before isolation of 9α-methyl-3'-N-oxide (XI). Finally, removal of the 3'-N-oxide group is easily accomplished by hydrogenation over a noble metal catalyst or Raney nickel.

In this hydrogenation, the temperature and pressure are not critical, for example the temperature may range from 0 to 100°C and the pressure from less than 0.1 MPa to 10 MPa or more.

Most preferably, room temperature and a slight overpressure of, for example, 0.2 to 0.8 MPa are used. Suitable noble metal catalysts are palladium, rhodium and platinum supported or unsupported on supports commonly known in the art of catalytic hydrogenation. Preferred catalysts are palladium on carbon and Raney nickel. (B)(A)(C)(D)

The reaction sequence (B)(A)(C)(D) involves first converting erythromycin A (I) to 9-deoxo-9a-aza-9a-homoerythromycin (III) via erythromycin A oxime and 9a-aza-9a-homoerythromycin, following the method of Kobrehel et al. (supra).

In this context, the novel process described above for 4'-epi-erythromycin A oxime is advantageously used for the preparation of the intermediate erythromycin A oxime.

The 2'-hydroxy group of compound (III) is first protected in the form of its acetate or propionate. The acylation is selectively carried out by reacting compound III with a limited excess of acetic or propionic anhydride in an inert solvent (e.g. methylene chloride) at a temperature of 0 to 30°C (preferably at room temperature).

A limited excess of anhydride is used to compensate for reagent consumed in side reactions, for example, undesired acylation of other groups, especially the 9α-nitrogen.

The resulting 2'-alkanoyl derivative with 2 to 3 carbon atoms is then protected at the 9a-nitrogen with a benzyloxycarbonyl group. This compound IX is formed by reacting the above 2'-ester with carbobenzoxychloride in a reaction-inert solvent in the presence of a base. Schotten-Baumann conditions are particularly advantageous, for example the reaction of the 2'-ester with the acid chloride is carried out under suitable alkaline conditions, for example in aqueous tetrahydrofuran, while the pH is maintained at

7.5 to 8.5 with dilute sodium hydroxide during the addition of the acid chloride and during the course of the reaction.

The temperature is not critical, but generally a range of 0 to 50°C is used, preferably at room temperature.

The C-4'-hydroxyl in compound IX is then oxidized to C-4'-oxo compound VIII by the action of a mixture of oxalyl chloride and dimethyl sulfoxide at low temperature (-40 to -80 °C) in an organic solvent inert to the reaction (for example, methylene chloride), after which the reaction mixture is treated in the cold with an excess of a tertiary amine (for example, triethylamine).

The alkanoate protecting group is then removed by solvolysis, preferably by contacting it with excess methanol at a temperature of 0 to 100°C, to give compound (VII).

Hydrogenation over Raney nickel using the conditions described above converts compound VII to 4'-epi-9-deoxo-9a-aza-9a-homoerythromycin A (VI). This compound is in turn converted to the 9a-N-methyl derivative (IV) by one of the alternative methods described above.

(B)(C)(D)(A)

This reaction sequence involves first converting erythromycin A to the above compound of formula II by the procedure of the above-cited patent (co-pending application) using the methods described in detail in the "Preparations" section below. The C-4" epimerization is then carried out by the procedures and methods described above.

The 2'-hydroxy group is protected by acylation, the 4''-hydroxy group is oxidized to the 4''-oxo group, preferably by replacing trifluoroacetic anhydride with oxalyl chloride.

The protecting acyl group is removed and the 4"-oxo group is catalytically hydrogenated to the desired 4"-epimeric hydroxy group. In this case, the preferred catalyst is Raney nickel.

Since compound IV of the present invention contains two basic nitrogen atoms, pharmaceutically acceptable mono- and di-acid addition salts of the free base IV are formed depending on whether one equivalent of acid or at least two equivalents of acid are used.

Salts are generally formed by combining reactants in a solvent inert to the reaction and, if the salt does not precipitate directly, it is isolated by concentration and/or addition of a non-solvent.

Suitable, pharmaceutically acceptable addition salts are compounds with HCl, HBr, HNO3, H2SO4, HO2CCH2CH2COZH, cis- and trans-HO21CCHCHCH2H, CH3SO3H and p-CH3C&H6SOaH, although the salts are not limited to these acids.

The antibacterial activity of the compounds of formula IV is demonstrated by measuring the minimum inhibitory concentration (MIC) in µg/ml against various microorganisms in brain-heart infusion medium. Generally, two-fold dilutions of the test compound are used, with the original concentration of the test substance ranging from 50 to 200 µg/ml.

The susceptibility (MIC) of a test organism is the lowest concentration of a compound capable of producing complete growth inhibition observed with the naked eye.

A comparison of the activity of 4“-epi-9-deoxo-9a-methyl-9a-aza-9a ^- homoerythromycin A IV with the control erythromycin A is shown in Table I.

Table I

In vitro activity of compound (IV)

Repeated MIC values day 1 day 2

A B A B

Staph. aur. 005 0.015 0.20 0.05 0.39 052 0.10 0.20 0.10 0.39 400 3.12 3.12 6.25 12.5 Staph. epi. 111 0.05 0.10 0.05 0.20 Shard, faec. 008 0.78 1.36 0.78 0.78 Shred. pyog. 203 0.025 0.0.25 0.025 0.025 Shard, pneumo. 012 0.025 0.025 0.025 0.025 E. coli 125 (a) 6.25 (a) 6.25 129 (a) 1.56 (a) 6.25 266 (a) 3.12 (a) 6.25 470 3.12 0.78 3.12 0.78 Gossip. Mr. 009 (a) 12.5 (a) 12.5 031 (a) 12.5 (a) 12.5 Oxygen glue 024 (a) 12.5 (a) 12.5 Past. mult. 001 1.56 0.10 1.56 0.10 Serr. mar. 017 (a) 50.00 (a) 50.00 Not at all. 000 1.56 0.20 3.12 0.39 Ent. aerog. 040 (a) 12.5 (a) 12.5 Ent. cloac. 009 (a) 25.00 (a) 25.00

Lil

2.4Ί 0 9 9

AND

Prov. stru. 013 - (a) H. influ. 012 /.· ‘ 3.12 036 ‘ 6.25 038 6.25 .042 1.53 051 3.12 073 3.12 078 1.56 081 3.12

Repeated MIC values day 1 day 2;

B • i 'V? A - - B 50.00 “ ’ ’ '('ay / .'.50,00 0.39 1.56 . 0.39 0.39 3.12 0.39 0.39 3.12 0.78 0.39 1.56 0.33 0.39 3.12 0.78 0.39 3.12 0.78 0.39 1.56 0.39 0.39 3.12 0.78

(a) greater than 50 A erythromycin A control

B compound (IV)

Compound IV was additionally tested in vivo by the well-known mouse protection assay or by the microbiological assay to determine serum levels in various mammals (e.g., mice, rats, dogs). Using rats as the test species, Compound IV is very well absorbed upon oral dosing, yielding high and long-lasting serum levels.

For the treatment of systemic infections in mammals, including humans, caused by susceptible microorganisms, Compound IV is administered in an amount of 2.5 to 100 mg/kg per day, preferably 5 to 50 mg/kg/day, in divided doses or preferably in a single dose per day. Variations in dosage will depend on the individual being treated and the susceptibility of the microorganism. These compounds are administered orally or parenterally, preferably orally.

The susceptibility of a microorganism isolated in a hospital is routinely tested in clinical laboratories by well-known plate methods: Compound IV is generally the compound of choice, as evidenced by the relatively large zone of inhibition against the bacteria causing the infections being treated.

The preparation of optimal dosage forms is carried out by methods commonly known in the art of pharmacy. For oral administration, the compounds are formulated either alone or in combination with pharmaceutical carriers such as inert solid diluents, aqueous solutions or non-toxic organic solvents in dosage forms such as gelatin capsules, tablets, powders, cachets, etc. Such carriers include water, ethanol, benzyl alcohol, glycerin, propylene glycol, vegetable oils, lactose, starch, talc, gelatins, gums and other well-known carriers.

Parenteral dosage forms require, for the above systemic use, to be dissolved or suspended in a pharmaceutically acceptable carrier such as water, saline, sesame oil, and the like.

Agents that improve the suspendability and dispersion qualities of the parenteral form may also be added.

For the typical treatment of infections in animals, including humans, caused by susceptible microorganisms, compound IV is formulated by methods well known in the pharmaceutical art into lotions, ointments, creams, gels, etc. in concentrations ranging from 5 to 200 mg/cm ³ of dosage form, preferably ranging from 10 to 100 mg/cm ³ . The dosage form is applied to the site of infection generally at least once daily.

The present invention is further illustrated in the following examples. It is understood, however, that the invention is not limited to the particular details of these examples.

Unless otherwise stated, all operations are performed at room temperature, all solvents are evaporated under vacuum from the bath to 40°C or less, all temperatures are in degrees Celsius, all thin layer chromatography is performed on commercial silica gel plates (using the eluent indicated in parentheses), all solvent ratios are by volume.

Example 1

4"-epi-erythromycin A oxime [Oxime 4 ^: '-epimer (I)]

4"-Epi-erythromycin A (50 g, 0.0040 mol) was dissolved in 265 ml of pyridine. Hydroxylamine hydrochloride (112.2 g, 1.615 mol) was added and the suspension was stirred for 16 hours. The reaction mixture was evaporated to a thick slurry, diluted with 300 ml of isopropanol, mixed well, filtered and washed with 3 X 100 ml of isopropanol.

The filtrate and washings were combined, evaporated to a water-soluble foam, triturated with ether to give the crude title compound as the hydrochloride (100 g). This was purified by partitioning between CH2Cl2 and aqueous sodium bicarbonate adjusted to pH 9.5 with dilute sodium hydroxide.

The aqueous phase was separated, washed with ethyl acetate and then ether. All organic phases were combined, dried over sodium sulfate and evaporated to give the title compound as a white foam (59.5 g), thin layer chromatography Rf 0.5 (60:10:1 CH2Cl2:CH3OH:conc. NH4OH);

*Hnmr (CDGb) <5:

2.31 (6H, s, (CH3)2N— j,

3.32 {3Ή, s, cladinose CHsO—).

Example 2

4"-epi-9a-aza-9a-homoerythromycin A (V)

The product prepared in the previous example (59.2 g, 0.0787 mol) was dissolved in 400 ml of acetone. A suspension of NaHCO3 (60 g) in 225 ml of water was added. Methanesulfonyl chloride (36.3 g, 24.5 mol) in 50 ml of acetone was then added portionwise over 10 minutes, the temperature being maintained below 30 °C by means of a cooling bath.

The mixture is stirred for 4.5 hours, the acetone is evaporated, dichloromethane (400 ml) is added to the residue and the pH is adjusted to 5.6 with 6N hydrochloric acid. The aqueous phase is separated, washed with two more portions of dichloromethane and adjusted to 9.5 by adding 6N NaOH. The alkaline solution is extracted 2X with fresh dichloromethane, 1X ethyl acetate and 1X ether.

The alkaline organic extracts were combined, dried over sodium sulfate and evaporated to give the title compound as a foam, yield 41 g, thin layer chromatography, Rf 0.4 (60:10:1, CH2 Cl2 : CH3 OH : conc. NH4 OH);

U-inmr (CDCl3) δ:

2.27 (6H, s, (CPU)zN—),

3.29 (3Ή, s, cladinose CHsO);

¹³ Cnmr (CDCl3, (CH3Si internal standard) ppm:

177.24 (lactone C=O),

163.53 (amide C=O),

102.29 and 96.24 (C-3, C-5),

40.22 (CH3)21N-).

Example 3

2'-O-acetyl-9-,deo'Xo-aza-9a-homoerythromycin A [2'-O-acetate (III)]

9-Deoxo-9a-aza-9a-homoerythromycin A (10 g, 0.0136 mol; (III), U.S. Patent No. 4,323,334) was dissolved in 150 mL of dichloromethane. Acetic anhydride (1.39 g, 1.28 mL, 0.0136 mol) was added and the mixture was stirred for 3 hours. Acetylation was monitored by thin layer chromatography and 0.25 mL of acetic anhydride and then 0.5 mL of acetic anhydride were added to complete the reaction, and the reaction mixture was further stirred for 1.5 hours and then 1 hour.

The reaction mixture is diluted with water and the pH is adjusted to 11 with dilute sodium hydroxide solution. The organic phase is evaporated to obtain

11.5 g foam. The foamy product (10 g) was chromatographed on 300 g silica gel using a 9:1 mixture of CH2Cl2:CH3OH as eluent for monitoring. ¹

The less polar impurities (3.6 g) eluted first and then the purified title product, isolated as a foam of 2 g, was thin layer chromatographed, Rf 0.2 (90 : 10 : 1 CH2Cl2 : CH3OH : conc. NH4OH); -d-inmr (CDCl3) δ:

2.02 (3H, s, C-2')

O

II

--O-C-CH3),

2.28 (-6H, s, (CH3)2N—),

3.35 (3H, s, cladinose CH 2 O—).

Using the same method, replacing propionic anhydride with acetic anhydride, the corresponding 2'-O-propionyl derivative is prepared.

Example 4

2'-O-,acetyl-9-deoxo-9a-benzyl-xycarbonyl-9a-aza-9a-homoerythromyclin A [(IX), R ² -= acetyl.l]

The product prepared in the previous example (1.7 g, 0.00219 mol) was dissolved in 70 ml of a 5:2 mixture of tetrahydrofuran and water. The pH was adjusted to 8 with dilute sodium hydroxide. Carbobenzoxy chloride (0.51 g, 0.427 ml, 0.003 mol) was added and the mixture was stirred for 2 hours while adding more dilute sodium hydroxide, maintaining the pH at 8.

Since the reaction was incomplete according to thin layer chromatography, additional carbobenzoxychloride (0.3 ml) was added and the reaction was continued for another 3 hours, maintaining the pH at 8. The reaction mixture was rinsed with water and ethyl acetate, the pH was adjusted to 9.6 and the aqueous phase was washed with dichloromethane.

The organic phase is combined, dried over sodium sulfate and evaporated. 2.4 g of a foam is obtained, which is chromatographed on 85 g of silica gel eluting with 170:10:1 CH3Cl2:CH3OH:conc. NH4OH. The pure fractions are combined, evaporated to a foam, shaken into dichloromethane and concentrated until the product begins to crystallize. 1.2 g is obtained, m.p. 122 degrees C. thin layer chromatography Rf = 6.4 (90:10:1 CH3Cl2:CH3OH:conc. NH4OH);

Uínmr (CDCi.j í:

2.00 (3H. s, C-2'

O

II —O-C-CHs),

2.27 (6H, s, (CH3)2N—),

3.35 (3H, s, clavicle, nose GHsO—);

¹³ Cnm>r (CDCb, (CH3Si internal standard) ppm:

176.31 lactone C=O,

169.36 (C-2' ester C = Oj,

157.10 (carbamate C=O),

137.0, 127.55 and 127.92 (aromatic ring), 40.6 ((CH3)aN_).

In the same manner, the 2'-O-propionyl derivative from the previous example is converted into the corresponding 2'-O-propionyl-9a-benzyloxycarbonyl derivative.

Example 5

2'-O-acetyl-9a-benzyloxycarbonyl-9-deoxo-4"-deo>xy-4"-oixo-9a-aza-9a-homoerythromycin A [(Vini), R ² = acetyl]

Oxalyl chloride (4.37 g, 3.0 ml, 0.0344 mol) was dissolved in 25 ml of dichloromethane and cooled to 60 °C. Dimethyl sulfoxide (6.70 g, 6.0.9 ml, 0.0836 mol) in 9 ml of dichloromethane was added. The reaction mixture was maintained at -60 °C for 10 minutes and at this temperature the product from the previous example (5.2 g, 0.005172 mol) in 16 ml of dichloromethane was added.

After a further 215 minutes at -60°C, triethylamine (17.3 g, 23.9 ml, 0.172 mol) was added and the mixture was warmed to room temperature, diluted with 50 ml of water and excess sodium bicarbonate. The organic phase was separated, dried over sodium sulfate and evaporated to give the title product as a sticky foam.

Yield 6.8 g, thin layer chromatography Rf 0.6 (90:10:1 CH2Cl2:CH3OH:conc. NH4OH);

1H nmr (CDCl3) <5:

2.05 (3H, s, C-2*)

O

II —O—C—CH3),

2.25 (6H, s, (CH 3 ) N—),

3.32 (3H, s, cladinose CH3O— j,

7.37 (5H, s, aromatic protons);

MS: major peaks at m/e 536 and 518 [N-benzyloxycarbonyl aglycone ion (minus both sugars cleavage at C-1", C-5)], 200 (basic peak, fragment derived from desosamine), 125 (fragment derived from neutral sugar). This intermediate is preferably used immediately in the next step.

In the same manner, the corresponding 2'-O-proponyl-4'-O'Xo derivative is prepared from the 2'-O-propionyl compound of the previous example.

Example 6

9a-benzyloxycarbonyl-9-deoxo-4"-deoxy-4"-oxo-9a-aza-9a-homoerythromycin A (VII) •The product prepared according to the previous example 1.0 g is stirred for 65 hours in 25 ml of methanol and then evaporated to a foam. The foam is dissolved in dichloromethane, washed with saturated sodium bicarbonate solution and evaporated again to a second foam. The second foam is chromatographed on 20 g of silica gel using a 13:1 mixture of CH2Cl2:OH3OH as eluent.

The fractions containing pure product were combined and evaporated to give the title compound as a foam.

Yield 336 mg, thin layer chromatography, Rf 0.4 (90:10:1 CH2Cl2:CH3OH:conc. NH1OH;

¹³ Cnmr (CDCb, (CH3Cl2 internal standard) ppm: ₍

210.87 (C-4", C = O),

176.03 (lactone C=O),

157.41 (carbamate C=O),

138.31, 128.2 and 128.0 (aromatic ring),

1104.15 and 96.83 (C-3, C-5).

Alternatively, the compound prepared in the previous example (6 g) was stirred for 116 hours and refluxed for 4 ^hours and evaporated to give the title product as a sticky foam (6.2 g), which thin layer chromatography (Rf and eluents as above) showed to be of sufficient purity for direct use in the next step.

In the same manner, the same product is prepared by solvolysis of the 2'-O-propionyl ester from the previous example.

Example 7

4"-epi-9-deoxo-9a-aza-9a-homoerythromycin A (Vij

Method A

The product prepared in Example 2 (40 g) was dissolved in 600 ml of methanol. NaBH4 (45 g) was added over 45 minutes, maintaining the temperature below 38°C. The reaction mixture was stirred for 64 hours and evaporated to give a thick slurry containing excess borohydride and boronic ester complex with the product.

The product thus obtained is partitioned between 500 ml of dichloromethane and water and the following sequence is repeated three times, the pH is adjusted to a constant value of 2.5 with dilute hydrochloric acid, the mixture is stirred intensively for 25 minutes and the aqueous phase is separated, combined with 500 ml of fresh dichloromethane, the pH is adjusted to 9.5 with dilute NaOH and the dichloromethane phase is separated.

The dichloromethane phase at pH 9.5 is mixed with 500 ml of fresh water and the sequence is repeated. On the third repetition, the dichloromethane phase at pH 9.5 is dried over sodium sulfate and evaporated in vacuo. The crude title product is obtained as a foam (34 g), which is recrystallized from 150 ml of hot isopropyl ether, cooled, and diluted with 300 ml of pentane. The purified title product is obtained, 25.8 g as white crystals.

Thin layer chromatography Rf = 0.5 (9:1, chloroform: diethylamine), Rf = 0.1 (90:10:1 CH2Cl2: CH3OH: conc. NH3OH), mp 170-180 °C;

Hinrin (CDCl) <5:

2.26 (SH, s, (CH3)2—N—),

3.29 (31-Ϊ, s, cladinose CHsO—);

¹³ Cnmr (CDCl, (CH3Cl2Si internal standard) ppm:

179.44 (lactone C=O),

103.57 and 96.70 (C-3, C-5),

41.50 ( _CH3 )2—N--).

Method B

The unchromatographed title product of the previous example (6.2 g) was dissolved in 200 ml of ethanol and hydrogenated over 12.5 g of Raney nickel for 18 h at 0.34 MPa. The reaction mixture was filtered, 20 g of fresh Raney nickel was added and hydrogenation was continued for another 4 h. Filtration was repeated and fresh catalyst was added and hydrogenation was continued for another 16 h.

Filtration and evaporation of the filtrate gave the crude title product as a white foam. The crude product was partitioned between dichloromethane and saturated sodium bicarbonate and the organic phase was separated, dried over sodium sulfate and evaporated. The title product was obtained as a second white foam (3.6 g) which was crystallized as described above. The purified title product was obtained, 955 mg, with physical properties identical to that of the product prepared according to Method A.

Example 8

4 ⁱ '-e.pi-9-deo.xo-9a-hydroxy-9a-aza-9a-homoerythromycin A 3'-N-oxide (X)

While stirring under nitrogen, the product from the previous example (3.0 g) was dissolved in 15 ml of a 1:1 mixture of tetrahydrofuran and methanol. 30% hydrogen peroxide (5 ml) was added. After 30 minutes, another 30% hydrogen peroxide (2.5 ml) was added. After another 30 minutes, the reaction mixture was carefully poured into a 1:1 mixture of CH2Cl2 and water containing Na2SO4 (exothermic reaction).

The pH of the reaction mixture is 9. The aqueous phase is washed with fresh dichloromethane and then with ethyl acetate. The organic phases are combined, dried over sodium sulfate and evaporated to give the title product.

Yield 2.7 g. Thin layer chromatography Rf 0.15 (60:10:1 CH2Cl2:CH3OH:conc. NH4OH);

H nmr (CDCl) δ:

3.21 (6H, s, (CH3J2N—, O),

3.33 (3H, s, cladinose CH',0—);

MS: major peaks at m/e 576 (ion from desosamine fragmentation at C-5). 418 (N-hydroxyaglycone ion-minus both sugars). Both peaks are diagnostic for the —N—OH portion of the aglycone molecule.

Example 9

4"-epi-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A 3 ^! -N-oxide (XI)

The product from the previous example (2.6 g, 0.0034 mol) was dissolved in 100 ml of dichloromethane. K2CO3 (37.5 g, 0.271 mol) was added with vigorous stirring followed by methyl iodide (19.3 g, 8.5 ml, 0.136 mol) and the reaction mixture was stirred for 20 hours. After filtration and evaporation in vacuo, the product was obtained as a foam.

Yield 2.9 g, thin layer chromatography Rf 0.3 (60:10:1 CH2Cl2:CH3OH:conc. NH4OH), Rf 0.15 (90:10:1 CH3Cl2:CH3OH:conc. NH3OH).

The title product prepared in this manner (2.8 g) was further purified by chromatography on 85 g of silica gel using a mixture of 90:10:1 CH2Cl2:CH3OH:conc. NH4OH as eluent. Minor more polar impurities were thus removed. 0.87 g of product was isolated;

Hínmr (CDCh) 5:

2.32 (3Ή, s, aglycone

CHs—N—),

3.20 (6H, sj (CH2)2N- -> O),

3.37 (3H, s, cladinose CH3O—),

Ex.ad 10 '-epi-9-deoxo-9a-methyl-9a-,aza-9a-homo' erythromycin A (IV)

Method A

The product prepared according to Example 7 (0.706 g, 0.96 mmol] was dissolved in 20 ml of chloroform. Formaldehyde (37%, 0.078 ml) was added followed by formic acid (0.03 ml) and the reaction mixture was stirred for 4 hours and then heated to reflux for 7 hours.

The reaction mixture was cooled, 30 ml of water was added and the pH was adjusted to 9 with 6 N NaOH. The organic phase was separated, dried over sodium sulfate and evaporated in vacuo to give the title product as a white foam.

Yield 0.7 g of product, which after crystallization from a hot mixture of ethanol and water gives 302 mg of product, m.p. 153 “C and recrystallization from a hot mixture of ethanol and water gives 246 mg of product, m.p. 155 °C. Thin layer chromatography Rf = 0.55 (60 : 10 : 1 CH2Cl2: CHaOH : conc. NH4OH), Rf 0.6 (9:1 CHCl3: diethylamine);

⁴ H nmr (CDCl 3 ) <S:

2.29 (9H, broad s, aglycone N—CHs and desosamine (CH3)aN—),

3.31 (3H, s, cladinose CH3O—);

¹³ Cnmr (CDCl3, CDCl3 internal standard) ppm:

178.89 (lactone C=O),

102.63 and 95.15 (C-3, C-5),

40.38 [(CH3)21N-];

MS: main peaks at m/e 590 (M-methyl aglycone-desosamine ion by cleavage of cladinose at C-1"), 416 [N-methyl aglycone ion (minus both sugars by cleavage at C-1", C-5)], 158 (main peak, fragment derived from desosamine).

Method B

The unchromatographed product of the title of the previous example (0.242 g) and 10% palladium on carbon (0.4 g) were mixed in 15 ml of 9151% ethanol and the mixture was hydrogenated for 1 hour at 0.34 MPa.

The catalyst was filtered off and the filtrate was evaporated to give the title compound as a white foam, 160 mg, which, after crystallization from ether/pentene, 124 mg, and recrystallization from ethanol/water, 95 mg, gave a product with physical properties identical to that prepared by Method A.

Method C

The chromatographically purified title product of the previous example (319 mg) and Raney nickel (1.5 g, 50% wet) were mixed in 20 ml of ethanol and hydrogenated at 0.34 MPa for 1.5 hours.

The catalyst is filtered off and the mother liquors are evaporated to dryness. 205 mg of the title product is obtained, which is identical in its physical properties to the product prepared by method A.

Example 11

2'-O-acetyl-9-deoxo-9α-methyl-9α-aza-9α-homoerythromyein A

The product from Preparation 5 (2.5 g, 3.34 mmol) was stirred with acetic anhydride (0.339 mL, 3.50 mmol) in 30 mL of dichloromethane for 4 h. The reaction mixture was evaporated in vacuo and the residue was dissolved in 50 mL of ethyl acetate, mixed with 50 mL of water and adjusted to pH 9.5 with 1 N NaOH.

The aqueous phase was separated and washed with 20 ml of fresh ethyl acetate. The organic phases were combined, dried over sodium sulfate, evaporated and dissolved in 30 ml of chloroform and evaporated again to give the title product as a dry solid.

Yield 2.82 g, ⁴ Hnmr (CDCl3 includes δ

3.31 (C 4 "-OCH 3 ), 2.28 (N—CH 3 ), 2.25 [N— — (CH 3 ) 2 ], and 2.0 (2'—OCOCH 3 ).

Example 12

2'-0-acetyl-4"-deoxy-4"-oxo-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (Vlila)

The title product of the previous example (2.5 g, 3.2 mmol) and dimethyl sulfoxide (0.38 mL, 5.23 mmol) were dissolved in 90 mL of dichloromethane and cooled to -70°C. While maintaining the temperature below -50 ^° C, trifluoroacetic anhydride (0.72 mL, 4.95 mmol) was added via syringe and the mixture was stirred at -60°C for 50 minutes.

Triethylamine (1.54 mL, 11 mmol) was added via syringe and the temperature was maintained below -50° C. during the addition. The mixture was then warmed to 0° C., diluted with water, and the pH was adjusted to 9.5 by addition of dilute sodium hydroxide.

The organic phase was separated, dried over sodium sulfate to give the title product as a foam (2.5 g). The foam was chromatographed on silica gel with a 10:1 mixture of chloroform and methanol and three fractions were collected by thin layer chromatography.

The purest product from fraction 1, 1.7 g, was dissolved in chloroform, diluted with water, adjusted to pH 4 with dilute hydrochloric acid, and the aqueous phase was separated, diluted with fresh chloroform, adjusted to pH 8 with dilute sodium hydroxide, and the organic phase was separated.

The last aqueous phase is extracted with three portions of fresh chloroform. The last four phases are combined, washed again with water, dried over sodium sulfate and evaporated in vacuo to give the title product.

241093

Yield 0.98 g, thin layer chromatography Rf = 0.7 (5:1: 0.1 CHCl 3 : Cl 2 OH : NH 4 OH);

⁴ HNMR (CDCl3) S (ppm):

2.05 (s, 3H, COCHS),

2.26 [s, 61H, N(CH3)2],

2.33 (d, 3iH, NCH3) and

3.33 (d, 3H, OCH 3 ).

Example 13

4"-deoxy-4"-oxO'-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (Vila)

The title compound of the previous example (0.93 g) was dissolved in methanol. After 20 minutes, the mixture was evaporated to give the title product.

Yield 0.74 g, MS 746.4, 588.4, 573.4, 413.3, 108.1, 125.1;

¹ H NMR (CDCl 3 ) <5 (ppm):

5.5 (t, 1H, Cl-H),

4.6 (q, 1H, C5'-H),

3.35 (s, 3H, OCH3),

2.38 (s, 3Ή, NCH3),

2.30 (s, 6H, N( _CH3 )2).

Example 1 4

4"-epi-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A (IV)

The title product of the previous example (0.25 g) and 250 mg of Raney nickel were combined in 20 ml of ethanol and hydrogenated at 3.4 MPa for 4 hours.

The catalyst was filtered off and evaporated in vacuo to give an oil which crystallized on standing. The title product was isolated by trituration in isopropyl ether and filtration. Yield 0.13 g of product with the same properties as the product of Example 10.

P r e p a r a t i o n 1

4“-epi-erythromycin A 'Suspense 100 g Raney ¹ nickel in 1 liter absolute ethanol containing 100 g 4“-deoxy-4'-oxoerythromycin A (U.S. Patent No. 4,510,220) is shaken under hydrogen overnight at room temperature and 3.4 MPa pressure. The spent catalyst is filtered off through diatomaceous earth and the filtrate is concentrated in vacuo to 300 ml. Water (700 ml) is added and the concentrated filtrate and the resulting milky solution are heated on a steam bath. A small amount of ethanol is added to prevent the formation of a gummy product during precipitation from the hot solution.

After stirring for two hours at room temperature, the product was filtered off and dried. 57.6 g of product was obtained and the filtrate was concentrated in vacuo until cloudy. The mixture was then stirred for one hour, then filtered off and dried. Yield 21.4 g.

The crystalline fractions obtained are combined and have a melting point of 141-144° C. Their ⁴ HNMR spectrum (CDCl3) shows absorption at 3.3 (3H, s), 2.3 (6H, s) and 1.4 (3H, s) ppm.

Preparation 2

Erythromycin A oxime hydrochloride

Erythromycin A (506 g, 0.681 mol) was dissolved in pyridine (2.787 kg, 2.850 L, 35.29 mol) under nitrogen. Hydroxylamine hydrochloride (1.183 kg, 17.02 mol) was added and the mixture was stirred for 22 hours before being evaporated to a slurry consistency which was filtered, rinsing with isopropanol.

The combined filtrates were re-evaporated to a thick waxy mass which crystallized by trituration with 2 l of water. Product 615 g (slightly moist, used in the next step without drying). Thin layer chromatography Rf 0.45 (60:10:1, CH2Cl2:CH3OH:conc. NH4OH).

By the same procedure, 5 g of erythromycin A was converted to the dry title product. Yield 4.5 g, at least 95% pure by ¹³ CNMR. Recrystallization of 1 g from 10 ml of methanol and 30 ml of isopropyl ether gave 725 mg, mp 187 °C (dec.), [literature, mp 188-191 °C, Massey et al., Tetrahedron Letters, pp. 157-160, 1970);

!3CNMR(DMSO-d6, (CH3Si internal standard) ppm:

174.35 (lactone C = O, ,168.78 (C = N—),

101.0, and 95.46 (C-3, C-5).

Preparation 3

9a-aza-9a-homoerythromycin A

Following the procedure of Example 2, the slightly moist title product of the previous Preparation 2 (615 g, calculated to 506 g, 0.613 mol dry matter) was converted to the crystalline title product with the evolution of gas observed upon addition of sodium bicarbonate. Yield 416 g;

¹³ CNMR (CDCb, CDCb internal standard) ppm:

177.54 (lactone C=O),

163.76 (amidic C=O),

102.28 and 94.20 (C-3, C-5),

40.13 [(GH3)2N-].

Preparation 4

9-deoxo-9a-aza-9a-hO'moerythromycin A

By reduction of NaBPU according to the procedure of Kobrehe241099 la et al. above, the title product of the previous preparation is converted to the title compound.

Preparation 5

9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A

Claims (1)

Subject Process for the preparation of 4'-epi-9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A or a pharmaceutically acceptable salt thereof, characterized in that it is hydrogenated with 4'-deoxy-4'-oxo-9-deoxo-9a by methyl-9α-aza-9α-homoerythromyl Following the procedure of Example 10 above, the product prepared in the preceding preparation (21.1 g, 0.0287 mol) was converted to the title compound, which was first isolated as a white foam and crystallized from of a mixture of hot ethanol and water gave 18.0 g of product, mp 130 ° C. The invention is based on a noble metal or Raney nickel catalyst in an inert solvent at a temperature of 20 to 100 ° C, and the compound obtained is optionally converted to a pharmaceutically acceptable salt thereof. Severography, n. P., Plant 7, Most Price 2,40 Kčs