DK152924B - 20-Iminotylosine derivatives or pharmaceutically acceptable acid addition salts thereof and pharmaceutical compositions comprising the compounds - Google Patents

Description

DK 152924B

in

The present invention relates to novel 20-imino-tylosin derivatives of the general formula I as defined in claim 1 or pharmaceutically acceptable acid addition salts thereof and to pharmaceutical compositions containing the compounds.

Demycinosyl and (mycinosyloxy) -tylsine and their acyl derivatives are known. For example, published UK patent application 2077730 discloses 23-de (mycinosyloxy) tylosin and its esters, and published UK patent application 10 2077731 discloses 23-demycinosyl tylosin and its esters.

Furthermore, it is known, cf. the descriptions of Danish patent applications Nos. 2555/81, Nos. 2556/81 and Nos.

5536/82 and the description of EP Application No. 70170, compounds similar to those of formula I, but wherein the 20 group is a group -CHO or -Cl 2 OH.

The novel 20-imino-tylosin derivatives of the present invention have effective and broad-spectrum antibacterial activity, especially against Gram-positive strains and are better absorbed than the compounds known in the art to date.

Compared to the aforementioned known compounds, wherein the 20 group is -CHO or -CH2OH, the compounds of this invention are distinguished by providing higher blood levels, as demonstrated below.

The compounds of the present invention have the general formula I:% aV>.

30 23 SE r T3'2 w L5? O 9¾

16 5'6 1 ,, ^ vs2 ^ - ^ rC £ L

«17 I \> ^ 5 ^ A ^ OR2 35 CEj ^ \ *“ 3 2

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where: is hydrogen or (C 2 -C 5) alkanoyl, R 2 is (C 2

R 5 is hydrogen or (C 2 -C 6) alkanoyl, or 5 R 2 and R 2 together form a carbonyl group linking the 3 "and 4" hydroxy groups, R 1 is hydrogen or (C 2 -C 4) alkanoyl,

R 9 is hydroxymethyl or acetoxymethyl and R 1 is

ISLAND

10 / --- \ = N-N SO o N- / 2 or are pharmaceutically acceptable acid addition salts thereof.

Although no stereochemical configuration is specified for the above structure, it should be understood that the stereochemical configuration is identical to the tylosin configuration.

The present compounds are capable of forming non-toxic, pharmaceutically acceptable acid addition salts with inorganic acids as a result of the dimethylamino group at the 3 'position. By "non-toxic, pharmaceutically acceptable acid addition salts" is meant those salts which do not exhibit toxic manifestations at normal therapeutic doses. Examples of such salts are those formed with such acids as salt, sulfur, phosphorus, lemon, vinegar, propionic, wine, maleic, benzoic, cyclopropyl, carboxyl, adamantylcarboxyl, , laurylsulfonic, glycoheptonic, stearic or lactobionic acid.

Acid addition salts can be prepared by the methods commonly used in the art, such as by adding a stoichiometric amount of acid to a solution of the antibiotic in a non-reactive organic solvent and isolating the salt by methods known in the art, such as

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3 precipitates the salt with a solvent in which the salt is not very soluble, e.g. diethylet-here. A non-reactive organic solvent is one which does not react with the antibacterial substance, acid or salt.

Compounds of formula I wherein R 9 is CK 2 OH are referred to as derivatives of 23-demycinosyl-tylosin or 23-O-demycinosyl-tylosin or DMT.

The present compounds are also useful in the form of their esters. Among the esters, they are preferred, wherein R 1, R 3 and R 4 are hydrogen and R 2 is acetyl, propionyl, n-butyryl or isovaleryl.) Where acetyl, R 3 and R 4 are hydrogen and R 2 is acetyl, propionyl. , n-butyryl or isovaleryl, 15.) wherein R 1 and R 3 are hydrogen and R 2 and R 4 are independently acetyl, propionyl, n-butyryl or isovaleryl.) wherein R 1 and R 4 are hydrogen and R 2 and R 3 are independently acetyl, propionyl, n-butyryl or isovaleryl;) wherein R 1 is hydrogen and R 2, R 3 and R 4 are independently acetyl, propionyl, n-butyryl or isovaleryl, or R 2 and R 3 together are a bridging carbonyl. - group.

25

Examples of preferred compounds of the present invention are the following (the numbers in brackets refer to the Table below for the examples containing physical data): 23-demycinosyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) -30 imino] -4 "- O-isovaleryl-tylosin (1), 4 "-O-acetyl-23-demycinosyl-20-deoxo-20 - [(4,4-dioxothio-morpholinyl) imino] -tylosin (6), 2 ', 4" -di-O-acetyl-23-demycinosyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin. (3), 4

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2 '-O-acetyl-3 ", 4" -O-carbonyl-23-demycinosyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] -ylosin (5) or 3 ", 4H-O- carbonyl-23-demycinosyl-20-deoxo-20- [(4,4-dioxo-thiomorpholinyl) imino] -tylosin (2), or pharmaceutically acceptable acid addition salts thereof.

The present compounds elicit antibacterial response in mammals with a bacterial infection. In order to induce an antibacterial effect, the subject compounds may be used orally, topically, intramuscularly or intravenously. Use can be made by any of the conventional methods, i. when used see tablets, capsules, suspensions, solutions, creams, ointments or injections. Each of the dosage forms can be built up using non-toxic, pharmaceutically acceptable excipients commonly known in the art. The compounds of this invention are preferably used in an amount of from ca. 5 mg to approx. 500 mg, preferably 5-50 mg per kg per day in a single dose or divided doses.

The compounds of the invention are antibacterial agents which exhibit a broad spectrum of activity against gram-positive strains and with distinct activity against many strains of Staphylococcus, Streptococcus, Bacillus and Sarcina.

The antibacterial activity of the subject compounds is determined by testing the compounds against various pathogens using standard anti-biotic dilution samples in Mueller-Hinton agar, the activity being expressed as the minimum inhibitory concentration (MIC, mcg / ml , 24 hours). The geometric mean MIC values for many of the compounds in question range from 0.125 to 2.0.

The serum levels of the subject compounds can be determined by applying the compounds either intravenously, subcutaneously or orally to the test animals. Generally 5

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serum levels are expressed as areas under the curve within a specified time period. The test compounds are generally used intravenously at doses of 12.5 mg / kg or subcutaneously in doses of either 100 mg / kg or 400 mg / kg.

The acute intravenous toxicities for the compounds of the invention are determined in mice and expressed as the dose of ID 50 which results in the death of 50% of the animals.

Below are a number of test results.

Compound A: 23-Demycinosyl-20-deoxy-20 [(4,4-dioxothio-morpholinyl) imino] tylosin lactobionate.

Compound B: 23-Demycinosyl'-20-deoxy-20 [(4,4-dioxothio-morpholinyl) imino] -4 "-O-isovaleryl-tylosin.

15 MIC Values

Organism A. B.. .

Bacillus subtilis ATCC 6633 0.25 0.125

Staphylococcus aureus Ziegler 0.5 0.125

Staphylococcus aureus 209P 0.5 0.125

Streptococcus faecalis 373 0.5 0.125

Streptococcus agalactiae Crisp 0.5 0.25

Streotococcus faecalis Z 2.0 0.5

Streptococcus Lancefield 0.5 0.0625 Group C Thacker

Streptococcus Lancefield 1.0 0.25

Group D 72052310

Streptococcus Lancefield 0.5 0.125

Group G Hamer ^ Streptococcus pneumoniae McCollo 0.125 0.0313

Streptococcus pneumoniae Fox 0.125 0.0625

Streptococcus pyogenes Harper 0.5 0.125

Streptococcus viridans 1578 0.125 0.125

Escherichia coli ATCC 10536 ^ 64> 64 ^ Klebsiella pneumoniae. Adler. 17> 6.4> 64 ....

LD ^ q (mg / kg) of Compound A: 375.

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Serum levels of the subject compounds were determined on male mice (Carworth CF-1) weighing 18-20 g each. Groups of six mice received intravenous aqueous solutions of the drugs as single bolus doses of 5,100 mg / kg. After 1, 3, 5, 10.20, 30, 45, 60, 90, 120 and 180 minutes, the mice were killed by cutting the jugu larva and the blood was allowed to flow into the tube (8 mm in diameter), that had been standing in an ice-water bath. At each time point, blood from the group of mice was collected. Samples 10 were allowed to solidify at room temperature for 15 minutes. After solidification, the samples were centrifuged for 10 minutes. Serum was removed and introduced into another test tube in an ice-water bath until antibiotic levels were determined by a cylinder beaker method using either Bacil-15 lus subtilis ATCC 6633 or Micrococcus luteus ATCC 9431 as test organisms. Stock solutions of each antibiotic were prepared daily in the appropriate vehicle and diluted in homologous serum to produce a standard curve. Serum samples, if necessary diluted in the appropriate serum, were compared with a standard curve of known strength by standard test methods. All tests were performed at least twice.

AUC values (areas under the curve) were based on serum level versus time data according to the trapezoidal rule.

As a comparison compound, the known compound was used 3-O-acetyl-O- (4-O-isovaleryl-αL-mycarosyl) - (1-4) -O- (8-D-mycaminosyl) - (1-5) -tylonolid.

The test compounds, with reference to Formula I, were the following (the numbering being similar to that used in the table after the examples): 35 7

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No. »I r2 r3%

Known CH,

together- I J

• lignincs-coc ^ COCR-j-CH Η H CHjOH O

CH, 5 ___: _____ CH, I 3 3 COCH3 COCH2CH h H CH2OH = N-i (^ 02 CH3 10 ch3

I J

7 coch3 coch2ch h COCH3 ch2oh, jj-iQo2 CH3 T2 15 9 -C (0) CH3 C (0) CH2CH h H ch2ococh3 = n-nQso2 ch3 2 H H CHjOH = N-Cs02 o 20 ______ · ·.

Serum levels and AUC (0-3h)

Compound AUC

No. 25 known comparative equations 28.3.

• 8 40.1 -7 80.5 9 61.8 30 2 45.0 a) Administered intravenously to vehicle mouse 0 at a concentration of 10 mg / ml.

b) The vehicle was a mixture of 115 ml of polyethylene (PEG) 40 castor oil and 1 g of lactic acid USP in 1 liter of distilled water.

8

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The present compounds can be prepared by various known multitrine syntheses from tylosin or any of the acylated tylosin derivatives or the demycinosyl tylosins known from the above-mentioned British patent applications. The methods described below can be used to prepare the compounds of the invention. Possible sequences for these processes in the preparation of the desired products are discussed in Schemes A to H below. It is furthermore apparent from these Schemes A to H that the various hydroxy groups or alkanoyloxy groups contained in the compounds of formula I have different reactivity. Depending on the desired end products, it is necessary to protect / deprotect certain groups during the multi-step synthesis and / or to carry out the necessary steps of the synthesis in a certain order.

Compounds of Formula If wherein R 9 is hydroxymethyl (the other substituents being as defined for Formula I) may be prepared by hydrolytic removal of 4 '' - oxo-mycinosyl sugar from a compound of the general formula: O '21 22 120 CB,, J | I 58-¾ 25% 7 Vi 2 '7¾ y wr JU “s 4 · 7 *« = 316 5 · ^

30 17 CF

Where R 1, R 2 / R 3, R 2 and R 2 are as defined for formula I. The removal of the sugar groups at the 23-position can be carried out, for example, with methanol, optionally in the presence of NaOH. Depending on the reaction conditions, any acyl groups at positions 3, 2 ', 3 "and 4" are also removed in this reaction.

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9

The starting compound for this process can be prepared by subjecting a corresponding tylosin derivative containing the groups R 2 / R 2, R 2 and R 5 to a Pfitzner-Moffatt oxidation.

For the preparation of 23-demycinosyl-tylosin (a suitable intermediate for several of the processes disclosed herein), tylosin or an ester thereof can be subjected to Pfitzner-Moffatt oxidation and then a hydrolytic removal of 4 '' - oxo-mycinosyl sugar as discussed above. .

The preparation of the compounds of the general formula 1 can be illustrated by the following reaction schemes in which acyl means (C 2 -C 4 -) alkanoyl and imino means the group / - \, = N-N SO 2): \ _ /

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10

Scheme A Tylosin

(1) protect 4 ,,, - OH

5 ψ 41 1 '- (OH-protected) -tylosin (2 *) /

I (2) I Acyl acyl 2'- ^ 2'-OH

and 4 "-OH 21-acyl-41 '1- (OH protected) tylosin

^ (3) ^ acyls 4 * '- OH

4 "-acyl-2'-acyl-4''1- (OH-protected) -tylosin

15 I

(4) In remote 4 '' '- protecting group 4 "-acyl-2'-acyl-tylosin (5) J, derivatize 20-aldehyde group 20-imino-20-deoxo-4" -acyl-2'-acyl -tylosin 20 (6) j, Pfitzner-Moffatt oxidation 4 '' - oxo-20-imino-20-deoxo-4 "-acyl-2'-acyl-tylosin (7) cleave 23 sugar and remove 25v 2'-acyl group 23-demycinosyl-20-imino-20-deoxo-4 '-acyl-tylosin

The preparation begins by reacting tylosin with a suitable reagent to effect protection of the 4 '' 1 -30 hydroxy group (Step A1). Many different conventional hydroxy protecting groups can be used for this purpose (see, for example, U.S. Patent No. 4,205,163), a highly preferred 4111 hydroxy protecting group 35

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has been found to be the tert-butyldimethylsilyl group. As with other such protecting groups, it is most convenient to add it to the 4 '' hydroxy group by reacting the tylosin with tert-butyldimethylsilyl chloride in the presence of an acid acceptor such as imidazole, 4-dimethylaminopyridine, triethylamine or pyridine. As the reaction medium, an anhydrous solvent such as dimethylformamide, dichloromethane or tetrahydrofuran is preferably used. The reaction proceeds at temperatures in the region of approx.

10 to 50 ° C, with room temperature being satisfactory in most cases. Typical reaction times range from approx.

12 to approx. 48 hours.

Step A2 involves introducing an acyl group at the Ί-hydroxy position. It is necessary to block this hydroxy group prior to introducing any acyl group at the 3 "or 4" position. Of course, when the acyl group to be introduced at the 4 "position is identical to the 2'-acyl group, both can be introduced simultaneously, essentially by combining Step A2 and Step A3 (as shown in Scheme A as Step A2 ") by simply increasing the molar amounts of the acylating agent and adding a base such as pyridine as a catalyst. Selective acylation of the 2'-hydroxy group may be by the conventional methods known for such acylation of common macrolide antibiotics." The acylating agent is suitable for the process of carboxylic acids, acid halides and acid anhydrides corresponding to the acyl groups. Acetic anhydride is preferably used as a result of the obtained yield and the reaction specificity. The range of about 10-50 ° C, with room temperature being preferred Typical reaction times vary within the range of about 10-48 hours, depending on the nature of the application. ended • ft reactants.

35

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12

Step A3 relates to the introduction of the 4 "acyl group. The 4" hydroxy group of the tylosin derivative which is acylated and protected at the 2 'and 4 "positions is generally acylated more easily than the 3-hydroxy group. When the 4" hydroxy group is acylated. , a small amount of 3,4 "diacyl derivative is sometimes produced as a by-product according to the acylating agent used.

The corresponding acid halides, acid anhydrides or mixed anhydrides with pivalic acid are suitable for use as the reactive derivatives of carboxylic acid compound in the process of this step. When an acid halide of a carboxylic acid or a mixed acid anhydride is used as the acylating agent, the reaction of Step A3 is carried out in the presence of a basic reagent. Preferred basic reagents are pyridine, 4-dimethylaminopyridine, picoline, piperidine and triethylamine or mixtures thereof. A mixture of triethylamine and 4-dimethylaminopyridine is particularly preferred. The reaction is generally carried out in an inert organic solvent such as benzene, toluene, chloroform, dichloromethane, tetrahydrofuran or a mixture thereof. The basic reagent itself can be used as the solvent for the reaction. The temperature range is typically between -20 ° C and 50 ° C, while a higher reaction temperature promotes by-product formation. The preferred reaction temperature is generally between -10 ° C and room temperature. Optionally, the 3 "hydroxy group can also be acylated at this step. This 3" hydroxy group is a tertiary alcohol which reacts only under certain conditions. The 3-hydroxy group should be blocked, preferably using the trimethylsilyl ether derivative, prior to the addition of the 3 "acyl group. Generally, the conditions should be more stringent, i.e. at higher temperatures, namely 60-10 Typically, as an acylating agent, an acyl chloride and as the basic agent are tribenzylamine, any non-polar, organic 13

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nical solvent is suitable for carrying out the reaction. When the 3-position is blocked during a synthesis sequence, it must be unblocked at a later stage after the addition of the 3 "acyl group. This typically occurs after completion of Step 5 of Step A3 or at any other convenient step during the synthesis.

In Step A4, the 4 "hydroxy protecting group is removed.

Of course, the exact conditions for removal depend on the nature of the protecting group introduced in Step A1. Such methods are well known in the art. When the 4 "hydroxy protecting group is used as the highly preferred tert-butyldimethylsilyl group, the removal of this group is conveniently carried out using tetra-n-butylammonium fluoride or a similar fluoride ion source.

Typically, as an reaction medium, an anhydrous solvent such as tetrahydrofuran or diethyl ether is used. A non-reactive atmospheric atmosphere, such as argon, prevents by-products. Typical reaction temperatures are in the range of 0-50 ° C, while typical reaction times are in the range of 1-24 hours.

In Step A5, the 20-aldehyde group of the compound is derivatized into the desired 20-imino-20-deoxo ~ 4 "acyl-2'-acyl-tylosin derivative. This is done by reacting the product of Step A4 with a" 1-amino reactant " The reaction is carried out x generally in a nonpolar, anhydrous organic solvent such as benzene, toluene, chloroform, dichloromethane, tetrahydrofuran or a mixture thereof. within the range of about 0-50 ° C, with room temperature being preferred Reaction times vary from 12 hours to 10 days depending on the reactants used.

35

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14

Step A6 involves conversion of the 4, M hydroxy group to a 4 "oxo group. This conversion is via a Pfitzner-Moffatt oxidation which uses a combination of diethylcarbodiimide or dicyclohexylcarbodiimide with an organic base such as pyridine, Diethylcarbodiimide is preferred because of the water solubility of its by-products. Typically, 3 equivalents of diethylcarbodiimide, 1 equivalent of organic base and 0.5 equivalent of trifluoroacetic acid are used. Typical solvents, for the range of 10-50 ° C, and typical times range from 2 to 12 hours.

In Step 7, both the mycinosyl sugar and the 2'-acyl group are removed to give the desired 23-demycinosyl-20-imino-20-deoxo-4 ”-acyl-tylosin. This is typically done by dissolving the compound in a mixture of methanol and silica gel and stirring at a temperature of 0-50 ° C (preferably room temperature) for a period of 1-5 days.

20 25 30 35

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15

Scheme B Tylosin 5 (1) ^ derivatizes 20-aldehyde group 20-imino-20-deoxytylosin

(2) In protecting 4-OH

4 * »1_ (OH protected) -20-imino-20-deoxytylosin 10

/ (3) J, acyl 2 * -OH

(3 ·) 2'-acyl-4 '' (OH-protected) -20-imino-20-decacyls 2'-oxo-tylosin

/ And 4 "-OH

(4) acyls 4 "-OH

* (and optionally 3 "-OH) 4" -acyl-21-acyl-4 "'- (OH protected) -20-imino-20-deoxo-1-tylosin remove 4' '' protecting group 20 4" - acyl-2'-acyl-20-imino-20-deoxo-tylosin (6) J'Pfitzner-Moffatt oxidation 4 '' - oxo-2'-acyl-4 "-acyl-20-imino-20-deoxo -ylosin 25 (7) remove mycinose sugar and 2'-acyl-group 2 3 -demycinosyl-20-imino-20-deoxo-14 "-acyl-tylosin

Scheme B uses the same basic reaction step as Ske-50 ma A, but the order has changed.

Step B1 of the process begins by reacting tylosin with a "1-amino reactant" of the formula: h2 - r5 where is as defined above, to derivatize the 20-aldehyde group to a 20-imino-20-deoxo group and then is produced to produce a 20-imino-20-deoxytylosin. The reaction conditions used in this Step B1 are essentially 16

DK 152924 B

the same as those used in Step A5 described above.

The 20-1mino-20-deoxo-tylosin is then reacted in Step B2 with a suitable reagent to protect the 4 * * -hydroxy group, thereby producing a 4'11-hydroxy-protected-tetra-20-imino-20-deoxy tylosin. Step B2 typically uses such reaction conditions as those described for Step A1 of Scheme A. The 41'1 hydroxy protecting group may be selected from the many known in the macrolide range, but as noted above is tert-butyldimethylsilyl. group strongly preferred. . .

Protection of the 20-aldehyde as the 20-imino-20-deoxo derivative also has the advantage of eliminating aldehyde-derived by-products formed under the protection of the 41 '' -hydroxy-15, thereby obtaining significantly higher yields of the desired 4 * 1 *. -hydroxy protected derivatives.

Step B3 of Scheme B acylates the 21-hydroxy group of the compound formed in the previous Step B2 to provide a 2, -acyl-411-hydroxy-protected-20-imino-20-deoxytylosin. The sectional conditions of this acylation step are substantially similar to those described above for Step A2 of Scheme A. As noted above, when the 2'and 4 "acyl groups are identical, the acylations of Steps B3 and B4 can be combined in a single Step B3 'by simply increasing the molar amounts of the acylating agent and adding a base such as pyridine as a catalyst.

The 2'-Acyl-4 '' - hydroxy protected-20-imino-20-deoxytylosin is then acylated at the 4 "hydroxy position (and optionally the 3" position) in Step B4 to form a 4 "-30 acyl-2 '-acyl-4' 'hydroxy protected-20-imino-20-deoxytylosin. The reaction conditions for the 4 "and 3" acylations are essentially the same as those described above for Step A3 of Scheme. A. As indicated for Scheme A, the 3-hydroxy group is preferably blocked intermediately before the addition of the 3 "acyl group.

Step B5 of Scheme B causes removal of the 4 "hydroxy protecting group to form the desired 17

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4 "-acyl-2, -acyl-20-imino-20-deoxo-tylosin. This reaction is carried out under the same conditions as those used for Step A4 of Scheme A.

Step B6 of Scheme B which converts the 4n-acyl-2'-acyl-20-imino-5-20-deoxo-tylosin to a 4 ", oxo-2, -acyl-4" -acyl-20-imino-20 -deoxo-tylosin, is carried out by a Pfitzner-Moffatt oxidation under substantially the same conditions as described above for Step A6 of Scheme A.

The final step of Scheme B, namely Step B7, causes removal of the 2'-acyl group and mycinose sugar in substantially the same manner as described for Step A7.

Schedule C

Tylosin 15 (1) In derivatives, the 20-aldehyde group 20-imino-20-deoxo-tylosin (2) In acyls 3-, 2'-, 4 "- and 4" - OH-20 in the groups 3,2 ' , 4 ", 4''1-tetraacyl-20-imino-20-deoxytylosin (3) I add new 4" acyl group and trans.

In acyls formerly 4 "acyl to 3" OH

4 "-acyl-3,2 ', 3", 4 "- tetraacyl-20-imino-20-deoxytylosin

25 I

(4) remove the 4 "1-, 3- and optionally the 2'-acyl groups 4" -acyl-3 "-acyl- (optionally 2'-acyl) -20-imino-20-deoxytylosin Ψ · ( 5) in Pfitzner-Moffatt oxidation 411 in oxo (optionally 2'-acyl) -4 "-acyl-3" -acyl-20-imino-20-deoxo-tylosin (6) In cleaved mycosine sugar and ammonia using 2'-acyl group 2 3-demycinosyl-4 "-acyl-3" -acyl-20-imino-20-deoxytylosin 18

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Scheme C begins, as Scheme B, to convert tylosin into a 20-imino-20-deoxo-tylosin by reaction with a "1-imino reactant" of the formula: H2 - R5 where is as defined above. Reaction conditions for this Step Cl are essentially the same as described for Step B1 of Scheme B.

Step 2 of Scheme C involves acylating the 3-, 2'-, 4 "and 4 '* 1 hydroxy groups simultaneously. This is done using 10 reaction times and temperatures similar to those described for Steps A2 and A3 in Scheme A and Stages B3 and B4 of Scheme B, but the molar amounts of acylating agent and basic agent are greatly increased, usually to about 5-20 equivalents. This step thus produces 3.2 ', 4 ", 4'" - tetraacyl-20-imino-20-deoxo-tylosin.

Step 3 of Scheme C interchanges the 4 "acyl group with a new 4" acyl group and transacylates the old 4 "acyl group to the 3" hydroxyl. This transacylation is made possible by the reactivity differences between the secondary 4 "hydroxy group and the tertiary 3" cis hydroxy group. [See, for example, Jaret et al., J. Chem.Soc., (C), 1374 (1973)]. A large molar excess (typically 5-10 equivalents) of the new acylating agent is used, as well as temperatures ranging from 80 ° C to the reflux temperature of the solvent. As the solvent, pyridine, which also acts as a basic agent, is typically used, so that the reaction is carried out at ca. 110 ° C (reflux of pyridine). Typical reaction times vary within the range of approx. 12-24 hours. Thus, this Step C3 provides a 4 "acyl-3,2 ', 3", 4 "' tetraacyl-20-imine-20-deoxytylosin wherein the 4" acyl group differs from 3.2 " The 3 ", 4" 'acyl groups.

In Step C4, removal of the 4 "-, 3- and optionally 2'-acyl groups takes place by the addition of an organic base, typically triethylamine. Typical solvents are such as methanol and typical temperatures are in the range of 25 ° C. The reaction is followed to determine the completion of the removal of the 4 "', 3' and optionally 2'-acyl groups and formation thereof.

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19 desired 4 "-acyl-3 ', - acyl- (optionally 2'-acyl) -20-amino-20-deoxytylosin.

Step C5, the Pfitzner-Moffatt oxidation, is carried out in substantially the same manner as described above for Step A6 of Scheme A.

In Step C6, the mycinosis sugar and, if necessary, the 2'-acyl groups are removed to form the desired 23-demycinosyl-4 "-acyl-3" -acyl-20-imino-20-deoxo-tylosin. This step is carried out by the method described above for Step A7 of Scheme A.

10 Schedule D

2 '-acyl-4' '- (OH-protected-20-imino-20-deoxytylosin (prepared as in Scheme B, Steps 1 to 3) (1) Prepare 3 ", 4" carbonate

V

2 '-acyl-41' 1- (OH-protected) -3 ", 4" -carbonyl-2Q-imino-20-deoxo-tylosin (2) remove 4'1 'protecting group 4/2' - acyl-3 ", 4" carbonyl-20-imino-20-deoxytylosin (3). Pfitzner-Moffatt Oxidation 25 ^4 '' -oxo-21-acyl-3 ", 4" -carbonyl-20-imino-2Q-deoxytylosin (4) remove mycinose sugar and 2'-30V acyl group 23-demycinosyl -3 ", 4" -carbonyl-20-imino-2Q-deoxytylosin 35 Scheme D begins as Scheme B with converting tylosin to a 2'-acyl-4 "-hydroxy protected-20-imino-20 deoxo-tylosin using the methods described above for Step B1 to B3. 2'-Acyl-4-hydroxy protected

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The -20-imino-20-deoxo-tylosin is then converted to a 2'-acyl-4, -, hydroxy protected-3 ", 4" -carbonyl-20-imino-20-de-oxo-tylosin in Step D1. This conversion is typically accomplished using Ν, Ν'-carbonyldiimidazole in an inert solvent, such as anhydrous dichloromethane. Typical times range from 12-30 hours and typical temperatures range from approx. 0-30 ° C.

Step D2 of Scheme D causes removal of the 4 "hydroxy group to form the desired 2'-acyl-3", 10 4 "carbonyl-20-imino-20-deoxytylosin. This reaction is carried out under the same conditions as used in Step A4 of Schedule A.

Step D3 of Scheme D, the Pfitzner-Moffatt oxidation, is carried out in substantially the same manner as described for Step A6 of Scheme A.

In Step D4, the mycinose sugar and 2'-acyl groups are removed as described for Step A7 of Scheme A.

20 21

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Schedule E 5

tylosin

(1) [protect 4 '' '- OH

4111 - (OH-protected)-tylosin

10 I

(2) then 3,20-hemiacetal and V acyls 2 (W2'- and 4 "-OH

ISLAND

IIS-CH3 15 Protect- CH3 IN (CH2) 9 -CN ^ v ^ -Q TacOwA / 32 ™ · 20 20 3 (formula shown for clarity) (representative acyl groups) 25 20.2 ', 4 " -triacyl-4 '' - (OH protected) - tylosin-3,20-hemiacetal (3) distant 4 '' '- OH-protecting group 20,2', 4 "-triacyl-tylosin-3, 20-hemiacetal (41 µ Pfitzner-Moffatt oxidation 4 '' - oxo-20,2 ', 4 "triacyl-tylosin-3,20-hemiacetal (5) remove mycinose sugar and 20-, 2 35 ψ and 4" -acyl groups 23-O-demycinosyl-tylosin (23-DMT) (61µ derivatives 20-aldehyde group 20-imino-20-deoxo-23-demycinosyl-tylosin 22

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The first step (Step E1) of Scheme E, like Scheme A, begins by converting tylosin into a 4 "-OH protected tylosin. The reagents and reaction conditions are identical to those described for Step A1 of Scheme A.

Step E2 of Scheme E converts the 4, M -OH-protected tylosin to a 2,2 ', 4 "-triacyl-4'" - (OH-protected) -tylo-sin-3,20-hemiacetal under simultaneous formation of the 3,20-hemi-acetal and acylation of the 20-, 2'- and 4 "-hydroxy groups.

This is done by using the acyl anhydride in molar excess in the presence of a base. Preferred bases are those inorganic bases, such as anhydrous potassium carbonate, which are also used in molar excess amounts. Reaction temperatures are in the range of ca. 50-100 ° C and typical reaction times are in the range 5-12 15 hours.

In Step E3 of Scheme E, the 4 "hydroxy protecting group is removed to form a 20,21,4" triacyl-tylosin-3,20-hemiacetal. This is done in essentially the same manner as described for Step A4 of Scheme A.

Step E4 of Scheme E is a Pfitzner-Moffatt oxidation which produces a 4, M-oxo-20,2 ', 4 "triacyl-tylosin-3,20-hemiacetal. This step is carried out in substantially the same manner. as described for Step A6 of Schedule A.

In Step E5, the mycinose sugar and the 20-, 2'- and 25 "-acyl groups are all simultaneously removed from the 4", - oxo-20,2,4,4 "-tri-acyl-tylosin-3,20-hemiacetal, thereby forming 23 -dymycinosyl-tylosin of the formula: 30 CH

cb3 and XJ

Y J S (CH3) 2 CE => CK3 23

DK 152924B

and hereinafter sometimes abbreviated to 23-DMT. The removal is typically accomplished by treating 4 '"-oxo-20, 2' / 4" -triacyl-tylosin-3/20-hemiacetal with 1-5% methanol sodium hydroxide for 15-60 minutes at room temperature, followed by of solution in an alcoholic solvent containing a small amount of an organic base (typically methanol containing triethylamine) and heating at reflux temperature for 12-24 hours.

In Step E6 of Scheme E, the 20-aldehyde group of the 23-10 demycinosyl-tolosine is derivatized by reaction with a "1-amino reactant" by the procedure described for Step B1 of Scheme B to provide a 20-imino-20-dehydrogenase. oxo-23-demycinosyl-tylosin.

15 24 '

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Schedule F

23-demycinosyl-tylosin (23-DMT) 5 1 (1) ψ protected 23-OH group

23- (OH-protected) -DMT

(2) acyls

10 v 2'-OH

2'-acyl-23- (protected-OH) -23-DMT

(3) acyls V 4 "-OH

4 "-acyl-2'-acyl-23- (OH-protected) -23-DMT

((4) \ acyl 3-OH

3-Acyl-4 "-acyl-2β-acyl'-23- (OH-protected) -er-DMT

(5) remove 23-OH-protecting group (5) remove 23-OH-protecting group protecting group 2'-acyl-4 "-acyl-23-DMT 3-acyl-4" -acyl-21

// acyl-23-DMT

2 ^ (6 *) / optionally remove / (6) optionally remove ^ 21-acyl> γ 2'-acyl

4 "-acyl-23-DMT 3-acyl-4" -acyl-23-DMT

(7 ') Derivatives 20- (7) Derivatives 20-3Q', Aldehyde Group ^ Aldehyde Group 20-Imino-20-deoxo-4'-acyl-20'-imino-20-deoxo-3- (optionally (2-acyl) -23-DMT acyl-4 "-acyl - (optionally 2'-acyl) -23-DMT

DK 152924B

Scheme F begins with protecting the 23-hydroxy group of 23-demycinosyl-tylosin. This hydroxy protection can take place in a variety of ways well known in the art, but typically occurs using the preferred protecting group, the tert-butyldimethylsilyl group. As with other such protecting groups, it is most conveniently added to the 23-hydroxy group by reacting the 23-de-mycinosyl tylosin with tert-butyldimethylsilyl chloride in the presence of an acid acceptor such as imidazole, 4-dimethyte aminopyridine, triethylamine or pyridine. As the reaction medium, an anhydrous solvent such as dimethylformamide, dichloromethane or tetrahydrofuran is preferably used. The reaction proceeds at temperatures within the range of approx. 10-50 ° C, with room temperature being satisfactory in most cases. Typical reaction times range from approx. 12 to approx. 48 hours.

Step F2 of Scheme F involves acylation of the 2-hydroxy group. This is done in essentially the same manner as described for Step A2 of Scheme A.

In Step F3 of Scheme F, the 21-acyl-23- (QH-protected) -23-demycinosyl tylosin is acylated at the 4 "hydroxy position to give the 4" -acyl-2-acyl-23- (OH-) protected) -23-demycinosyl-tylosin. This acylation proceeds in the same manner as described for Step A3 of Scheme A.

Likewise, the acylations of Steps F2 and F3 can be combined in a single step if the 2 'and 4 "acyl groups are identical. This is done by simply increasing the molar amounts of the acylating agent and adding a base catalyst such as pyridine.

The acylation steps F2, F3 and F4 can be combined in a single step if the 3-, 2'- and 4 "acyl groups are identical. This is done by increasing the molar amounts of the acylating agent and adding a base such as pyridine, triethylamine, 4 -dimethylaminopyridine or mixtures thereof.

Step F4 of Scheme F acylates the 3-hydroxy group to form a 3-acyl-4 "-acyl-2'-acyl-23-hydroxy-protected -23-demycinosyl-tylosin. Typically, 3-7 equivalents of an acylating agent, f. eg acetic anhydride, together

DK 152924 B

26 with excess equivalents of an organic base such as pyridine or 4-dimethylaminopyridine. A mixture of solvents such as triethylamine and methylene chloride is often used as the reaction medium. Temperatures vary in the range of 10-50 ° C, and reaction times in the range of ca. 18-24 hours.

In Step 5 of Scheme F, the 23-hydroxy protecting group is removed to form a 3-acyl-4 "-acyl-2'-acyl-23-de-mycinosyl tylosin. The conditions for removal vary according to the nature of the protecting groups used. When the tert-butyldimethylsilyl group is used, removal can conveniently be effected by treating with 80% acetic acid in water Typical temperatures are in the range of 10-50 ° C with room temperature being the preferred reaction times within the range of about 1- 5 hours, with about 2 hours generally being sufficient for the completion of the reaction.

Step F6 of Scheme F is an optional removal of the 2'-acyl group. Depending on the nature of the acyl group, removal is typically done by dissolving the component in methanol and stirring at a temperature of 0-50 ° C, preferably room temperature, for 1-5 days to form a 3-acyl-4 "acyl acid. 23-demycinosyl-tylosin.

In Step F7 of Scheme F, the 20-aldehyde group of the 3-25 acyl-4 "-acyl-23-demycinosyl tylosin is derivatized by reaction with a" 1-amino reactant "by the methods described for Step B1 of Scheme B - the desired 20-imino-20-deoxo-3-acyl-4 "-acyl-23-O-demycinosyl-tylosin, which may optionally contain the 2'-acyl group.

The implementation of Steps F5 ', F6' and F7 'in Scheme F

using 4 '-acyl-2 * -acyl-23-hydroxy-protected-23-demycinosyl-tylosin in substantially the same manner as described for Steps F5, F6 and F7 yields a 21-acyl-4 "-acyl-23, respectively. -demycinosyl-tylosin, a 4 "-acyl-23-demycinosyl-tylosin, and the 20-imino-20-deoxo-4" -acyl-23-demycinosyl-tylosin, which may optionally contain the 2'-acyl group.

Alternatively, steps F1 to F6 may be carried out using 20-imino-20-deoxo-23-demycinosyl-tylosin 27

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as starting material, thereby obtaining the same desired products.

Scheme G 5 23-demycinosyl-tylosin (23-DMT)

(1) v derivatize 20-aldehyde group 10-imino-20-deoxo-23-DMT

(2) N, acyls 3-, 23-, 2'- and 4 "-OH groups 3,23,2 ', 4" -tetraacyl-20-imino-20-deoxo-23-DMT

15 (3) adds new 4 "acyl and transacyls v

formerly 4 "acyl to 3" acyl 4 "acyl-3,23,2 ', 3" tetraacyl-20-imino-20-deoxo-23-DMT

(4 ') remove 2'-, 3- and (4) remove 2'- and 23-

V

20v 23-acyl acyl groups 4 "-acyl-3" -acyl-20-4 "-acyl-3,3" -diacyl-20-imino-20-deoxo-2,3-DMT imino-20-deoxo-2 2 3-DMT 1

In Step G of Scheme G, the 20-aldehyde group of 23-25 demycinosyl-tylosin is derivatized by reaction with an "1-amino reactant" by the methods described in detail for Step B1 of Scheme B, whereby a 20-imino-20 23-demycinosyl-tylosin.

Step G2 of Scheme G simultaneously involves acylation of the 3-, 23-, 2'- and 4 "-hydroxy groups to form a 3,23,2 ', 4" -tetraacyl-20-imino-20-deoxo-23- demycinasyl-tylosin. This may conveniently be done by adding a large molar excess (typically 5-15 equivalents) of the acylating agent and a large molar excess (again 5-15 equivalents) of a base. Suitable bases are the organic bases such as pyridine, triethylamine and 4β-dimethylaminopyridine. Solvents include such as methylene chloride, chloroform and mixtures thereof. Typical reaction times

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which is in the range of 12-36 hours, while typical temperatures are in the range of approx. 10-50 ° C, with room temperature being the most preferred.

In Step G3 of Scheme G, a new 4 "acyl group is added and 5 the previous 4" acyl group is transacylated to the 3 "acyl group. This transacylation takes place in substantially the same manner as described for Step C3 of Scheme C and leads to a 4 "acyl-3 / 23,2,3,3-tetraacyl-20-imino-20-deoxo-23-demycinosyl-tylosin.

In Step G4 of Scheme G, the 2'- and 23-acyl groups are removed to form a 4n-acyl-3,3-, diacyl-20-imino-20-de-oxo-23-demycinosyl-tylosin. This removal is done by dissolving the compound in methanol and triethylamine and gradually heating from room temperature to ca. 60-70 ° C. The completion of the removal is ascertained by thin layer chromatography, at which time the reaction mixture is worked up and the desired product is isolated.

As an alternative to Step G4, the reaction may be allowed to proceed to effect removal of the 3-acyl group 2Q as well as of the 2'- and 23-acyl groups. This is shown in a Step G4 'of Scheme G. The conditions are identical to G4 except for the reaction time, and a 4 "acyl-3" acyl-20- ± mino-20-deoxo-23-0- demycinosyfc-tylosin.

Alternatively, steps G2, G3, G4 and G4 'of Scheme G may be used

is carried out using 23-demycinosyl-tylosin as starting material, thereby forming either a 4 "-acyl-3" -acyl-23-demycinosyl-tylosin or a 4 "-acyl-3,3" -diacyl-23-demycinosyl tylosin which can then be derivatized at the 20-aldehyde position as described in Step B1 of Scheme B. This pathway results in the same compounds prepared in Scheme G.

35 29

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Schedule 'H

23-demycinosyl-tylosin (23-DMT).

5 (1) derivatizes 20-aldehyde group

20 O-imino-20-deoxo-2 3 -DMT

(2). protect 23-OH

20-imino-20-deoxo-23- (OH-protected) -23-DMT

10

(3). J, acyl 21-OH

2'-acyl-20-imino-20-deoxo-23- (protected-OH) -23-DMT

(4) acyl 4 "-OH

21-acyl-4 "-acyl-20-imino-2Q-deoxo-23- (OH protected) -

23-DMT

(5 ') v protect 3-OH (5) v acyls 3-OH

2'-acyl-4 "-acyl-3- (OH-protected-3-acyl-21-acyl-4" -acyl) -20-imino-20-deoxo-23-20-imino-20 -deoxo-23

20 (OH protected) .- 23-DMT (OH protected) -23-DMT

(6 ') j, acyls 3 "-OH (6) acyls 3" -OH

2-acyl-3 "-acyl-4" -acyl-3- 3-acyl-2β-acyl-3 "-acyl- (OH-protected) -20-imino-20-4" -acyl-20-imino -20-de-deoxo-23- (OH-protected) -23-oxo-23- (OH-protected) -

DMT 23-DMT

(7'1j, remove 23-OH- and 3- (7) <] / remove 23-OH-be-OH-protecting groups protecting group 2 '-acyl-3 "-acyl-4" -acyl-20-3 -acyl-2 '-acyl-3 "-a (1-4" -acyl)

imino-20-deoxo-2 3-DMT 20-imino-20-deoxo-23-DMT

(8 ') optionally remove (8) ^ optionally remove 2'-acyl 2'-acyl 3 "-acyl-4" -acyl-20-imino-3-acyl-3 "- acyl-4" - iacyl-

20-deoxo-23-DMT 20-imino-20-deoxo-23-DMT

DK 152924 B

In Step H1 of Scheme H, the 20-aldehyde group of 23-demycinosyl-tylosin is derivatized by reaction with a "1-amino reactant" by the methods disclosed for Step B1 of Scheme B to form a 20-imino-20 23-demyci-5-nosyl-tylosin.

Step H2 of Scheme H involves the protection of the 23-hydroxy group. Any conventional hydroxy protecting group may be used for this purpose, but as discussed above in Step A1 of Scheme A, a very preferred group is the tert-butyldimethylsilyl group. Reaction conditions for this protection are essentially as described for Step A1 of Scheme A.

Step H3 of Scheme H involves acylating the 2'-hydroxy group of the 20-imino-20-deoxo-23-hydroxy protected-23-15 demycinosyl tylosin to form a 2'-acyl-20-imino-20-deoxo -2 3-hydroxy protected-2 3-demycinosyl-tylozine. This is carried out in substantially the same manner as described for Step A2 of Scheme A.

In Step H4 of Scheme H, the 2, -acyl-20-imino-20-20 deoxo-23-hydroxy protected-23-demycinosyl tylosin is acylated at the 4 "hydroxy position to form a 4" acyl-2'-acyl 20-Imino-20-deoxo-2,3-hydroxy-protected-23-demycinosyl-tylosin. This acylation proceeds in the same manner as described for Step A3 of Scheme A.

When the 2'and 4 "acyl groups are identical, the acylations in Steps H3 and H4 can be combined. This is done in substantially the same manner as described for Scheme F-

Step H5 of Skama H acylates the 3-hydroxy group to form a 3-acyl-2, -acyl-4,12-acyl-2Q-imino-20-deoxo-23-hydroxy-protected-23-demycinosyl-tylosin. Reaction conditions for this step are essentially the same as described for Step F4 of Scheme F.

When the desired 2 ', 4' and 3-acyl groups in the final product are identical, steps H3, H4 and H5 can be combined to form the desired product in a single step.

This is done as described above for process F.

. DK 152924B

31

As an alternative, the 3-hydroxy group is protected rather than acylated (Step H5 '). This is preferably done in the same manner as described for Step A3 of Scheme A, to form a 3-hydroxy-protected-2'-acyl-4 "-acyl-20-imino-20-deoxo-23-hydroxy-protected ~ 23-demycinosyl tylosin.

Step H6 acylates the 3 "hydroxy group. An acyl chloride is typically used as an acyl chloride and tribenzylamine as the basic agent. Any non-polar organic solvent is suitable for carrying out the reaction.

In Steps H7 and H7 'of Process H, the 23-hydroxy protecting group is removed. Due to the presence of the 3-acyl group in Step H7, a milder deprotection medium such as 80% acetic acid in water must be used. When the removal of the 3-hydroxy protecting group is desired, as in Step H71, the deprotection is carried out using fluoride ion as described for Step A4 of Scheme A.

Steps H8 and IB 'involve the optional removal of the 2'-ac # L group. When such removal is desired, it is carried out in substantially the same manner as described for Step 7 of Scheme B to form either the desired 3-acyl-3 "-acyl-4" -acyl-20-imino-20-deoxo -23-demycinosyl-tylosin or a 3 "-acyl-4,12-acyl-20-imino-20'-deoxo-23-demycino-silyl-tylosin.

Alternatively, steps H2 to H8 can be carried out using 23-demycinosyl tylosin as starting material to form compounds which can then be derivatized to the 20-imino 20-deoxo compounds.

30 32

DK 152924B

In all of the above schemes, the 3 "acyl compounds can be prepared by modifying the process to include the transacylation method described for Step C3 of Scheme C and Step G3 of Scheme G.

The following examples illustrate the present invention. In these examples, "Rotation" denotes optical rotation, "UV" denotes ultraviolet spectra, "IR" denotes infrared spectra, and "NMR" denotes nuclear magnetic resonance spectra.

Example 1 A. 4 "- O- (tert-Butyldimethylsilyl) tylosin.

Tylosin (25 g) and imidazole (18.6 g) were dissolved in dry dimethylformamide (250 ml) and tert-butyldimethylsilyl chloride (19.7 g) was added. The solution was left at 25 ° C for 19 hours. It was then evaporated to dryness and the residue was taken up in chloroform, washed with water, dried (MgSO4) and filtered. The filtrate was evaporated to dryness and the residue was triturated with hot hexane (3x1 liter). The insoluble residue was then chromatographed on a silica gel column (160 x 5 cm) using 1.5% methanol in chloroform as eluent to give 4 "- O- (tert-butyldimethylsilyl) tylosin as an eluent. colorless amorphous solid having the following characteristics: 25 30 35 33

DK 152924B

Rotation: [<x] R6 -41.8 ° (CHCl3), UV: Xmax (CF3CH2OH) 284 na (ε 22.616), IR: (CHCl3) 3500, 2980, 2950, 2910, 1722, 1682, 1600, 1320 , 1262, 1220, 1050 cm -1, NMR: 6R (CDCl3) 0.14 (3H, s, 4 '' - SiCH3), 0.17 (3H, s, 4 '' - SiCH3), 97 (9H, s, 4 »1 * -SiC (CH3) 3), 1.80 (3H, d, J13 22 1.5Hz, 22-CH3), 2.50 (6H, s, 3 '-N ( CH3) 2 (3.51 (3H, s, 2 "-C) CH3), 3.62 (3H, s, 3'1'-OCH3), 4.23 (1H, 2, 7, 5Hz, Ηχ ·), 4.62 (1H, d, JlfI 2iII 7.5Hz, δ,,), 5.95 (1H, dq, J13'22 1'5 Ez 'J13 ^ lOHz, Hi3) / 6 , 25 (1H, d, 10 Ί) _ of 11 15 H- ^ q) 1 ^ (1H, d, J ^ Q 15Hz, H. ^) ΟξΓ 9.77 (1H, s, H 2 O).

B. 2-O-00171-411'-O- (tert-butyldimethylsilyl) -tylosin.

4'1'-O- (tert-Butyldimethylsilyl) tylosin (15 g) was dissolved in dry acetone (500 ml) and acetic anhydride (7.4 g) was added. The mixture was left at 25 ° C for 17 hours. The solution was then evaporated to dryness and the residue was azeotroped with toluene to give 21-O-acetyl-4 "-O- (tert-butyldimethylsilyl) tylosin as a colorless amorphous solid.

An analytical sample was purified by chromatography on a silica gel column (70 x 2.5 cm) using 20¾ acetone in hexane as the eluent. The product had the following characteristics:

Rotation: [<x] R6 -45.4 ° (CH3OH), UV: Xmax (CF3CH2OH) 285nm (ε 22.784), IR: (CDCl3) 3530, 2980, 2960, 2920 ,, 1743, 1720, 1680, 1590, 1230, 1160, 1045 cm -1, NMR: (CDCl3), 0.10 (3H, s, 4 '' - SiCH3), 0.13 (3H, s, 4 '' -SiCH-j), , 94 (9H, s, 4'1'-SiC (CH3) 3), 1.78 (3H, d, J13 22 1.5H; 22-CH3), 2.06 (3H, s, 2'- OCOCH3), 2.38 (6H, s, 3'-NiCH4), 3.48 (3H, s, 2 '' - OCH3), 3.59 (3H, s, 3 '' - 0CH3), 4 , 27 (M, d,

Jl »2 '^ 1i®i») / 4.60 (1H, d, 1.12 «11 8Hz, H ^, ()' 5.92 (1H, dq, J13 14 10.5Hz, J13 22 1.5Hz, H13), 6.25 UH, d, 35 ^ 10 11 16Hz, H ^ q), 7.31 (1H, d, J ^ q 15Hz, H ^^) and 9.63 (1H, s , H2 O) · 34

DK 152924B

C. 2'-O-Acetyl-41 '' - O- (tert-butyldimethylsilyl) -4 "-O-isovaleryl-tylosin.

2'-O-Acetyl-41'1-O- (tert-butyldimethylsilyl) tylozine (prepared as in Part B of this example) (15.6 g), 4-dimethylaminopyridine (1.85 g) and triethylamine (30 ml) was dissolved in dry dichloromethane (1 liter). Iso-Valeri anhydride (2.82 g) in dry dichloromethane (200 ml) was added dropwise with stirring at 25 ° C over 1 hour. The solution was then stirred for an additional 16 hours at 25 ° C. The solution was washed with water, dried (MgSO 4), filtered and evaporated to dryness. The evaporation residue was then chromatographed on a silica gel column (160 x 5 cm) using 30% ethyl acetate in dichloromethane as the eluent, whereupon, during the elution, 2 * -O 2 acetyl-4 - (tert-butyldimethylsilyl) -4-, - 0-isovaleryl-tylosin as a colorless amorphous solid having the following characteristics:

Rotation: [α] 6 -51.7 ° (CHCl1), UV: (CF, CH ~QH) 285 nm

D J IftcLX <3 Z

(ε 23.323), IR: δ (CDCl3) 3520, 2980, 2950, 2900, 1740, 1720, 1675, 1590, 1235, 1160, 1050 cm, NMR: (CDCl3) 0.10 (3H, s, 4 '' -SiCH 3), 0.13 (3H, s, 4 '' -SiCH 3, 0.94 (9H, s, 4 "-SiC (CH 3) 3), 0.98 (6H, d, J6Hz, 4 "-OCOCH2CH (CH3) 2), 1.78 (3H, d, J13 22.1, 5Hz, 22-CH3), 2.06 (3H, s, 2'-OCOCH-j), 2 , 40 (6H, s, 3'-N (CH 3) 2), 3.48 (3H, s, 2 "- OCH-j), 3.58 (3H, s, 25 3 '" - OCH 3), 4.25 (1H, d, J · 2, 7.5 Hz, δ), 4.59 (1H, d,

Jl111 2 '* * t *) / 5.91 (1H, dq, 22 ^ rr ^ 13 14 10.5Hz, H13), 6.24 (1H, d, J10 n 15.5Hz, H10), 7.31 (1H, 'd, J10 11 15'5Hz' Hn) and 9.65 (1H, s, H2q), and unreacted 2'-O-acetyl-41 '' - O- (tert-butyldimethylsilyl) tylosin.

D. 2'-O-Acetyl-4 "-O-isovaleryl-tylosin.

2'-O-Acetyl-4''1-O- (tert-butyldimethylsilyl) -4 "-O-isovaleryl-tylosin (prepared as in Part C of this Example) (8.25 g) and anhydrous tetra-n -butylammonium fluoride (obtained by azeotroping the trihydrate (2.2 g) with

DK 152924B

(Toluene) was dissolved in dry tetrahydrofuran (400 ml) and the solution was left at 25 ° C for 16 hours under dry argon gas. The solution was then evaporated to dryness and the residue was taken up in dichloromethane, washed with water, dried (MgSO4), filtered and evaporated to dryness. The evaporation residue was chromatographed on a silica gel column (160 x 2.5 cm) using 40% acetone in hexane as eluent to give the product 2, -O-acetyl-4 "-0-isovaleryl-tylosin 10 as a colorless amorphous solid with the following characteristics: or

Rotation: [α]--66.6 ° (CHCl1), UV: (CH-, ΟΗ) 282 nm O IudA o (ε 22.641), IR: δ (CDCl,) 3550, 2980, 2950, 2900, ula X O 1740, 1735, 1730, 1680, 1600, 1248, 1175, 1065 cm, NMR: δΗ (CDCl3), 0.98 (6H, d, J 6Hz, 4 "-OCOCH2CH (CH3) 2_), l , 78 (3H, d, J13 22.5HHz, 22-CH3), 2.07 (3H, s, 2'-OCOCH3), 2.41 (6H, s, 3'-N (CH3) 2), 3.50 (3H, s, 2 '' - OCH 3), 3.63 (3H, s, 3 '' - OCH 3), 5.95 (1H, dq, J13 22 l, 5Hz, J13 14 10.5Hz , H13), 6.31 (1H, d, J ^ q 15.5Hz, H ^ q), 7.36 (1H, d, J ^ q 15.5Hz, 20 Η1χ) and 9.70 (1H, s , H2q).

E. 21-O-Acetyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] -4 "-O-isovaleryl-tylosin.

2, -O-Acetyl-4 "-O-isovaleryl-tylosin (prepared as in Part D of this example) (1.8 g) and 1N-amino-4,4-dioxo-thio-morpholine (458 mg) was dissolved in dry dichloromethane (50 ml) and the mixture was stirred at 25 ° C for 212 hours.

The solution was evaporated to dryness and the residue was chromatographed on a silica gel column r (110 x 30 2.5 cm) at a maximum flow rate using 30% acetone in hexane as the eluent to give 2'-O-acetyl acid. 20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] -4 "-O-isovaleryl-tylosin as a colorless amorphous solid having the following characteristics:

DK 152924B

Rotation: [α] 6 -71.3 ° (CHCl 3), UV: λ (CH 3 OH) 240 nm (ε 8.033), 283nm (ε 23.102), IR: (CDCl CD) 3510, 2980, max j 2950, 2900 , 1740, 1720, 1680, 1600, 1223, 1192, 1172, 1130, 1060 cm -1, NMR: (CDCl3) 0.98 (6H, d, J 6Hz), 4 "- 50COCH? CH (CH?) ?), 1.78 (3H, d, J13 22 1.5Hz, 22-CH3), 2.06 (3H, s, 2'-OCOCH3), 2.40 (6H, s, 3'-N (CH3) ) 2), 3.48 (3H, s, 2 'OCH 3), 3.62 (3H, s, 3' '- OCH 3), 5.90 (1H, dq, J13 22 1.5Hz, J13.14 6.25 (1H, d, Jio, ii 15Hz, H10), 7.97 (1H, m, H2O), and 7.34 (1H, d, J10 u 15Hz, Hn).

F. 20-Deoxo-20-E (4,4-dioxothiomorpholinyl) imino] -4'-oxo-4 "-O-isovaleryl-tylosin.

21-O-Acetyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] -4 "-O-isovaleryl-tylosin (1.4 g) and dicyclohexylcarbodiimide (737 mg) were dissolved in a mixture of dry dimethyl sulfoxide (3 ml) and dry benzene (20 ml), pyridine (93 mg) and trifluoroacetic acid (65 mg) were added and the mixture was stirred at 25 ° C for 5 hours. in vacuo, and the residue was taken up in dichloromethane and washed with water. The dichloromethane layer was dried (MgSO4), filtered and evaporated to dryness to give the crude title product.

G. 23-O-Demycinosyl-20-deoxo - [(4,4-dioxothiomorpholinyl) imino] -4 "-O-isovaleryl-tylosin.

The 4 "ketone (prepared in section F) was dissolved in methanol (250 ml) and silica gel (14 g) was added. The mixture was stirred at 25 ° C for 168 hours. The silica gel was filtered off and washed with methanol. Total methanol filtrates were evaporated to dryness and the residue was chromatographed on a silica gel column (160 x 2.5 cm) using 35% acetone in hexane as the eluent. The appropriate fractions were pooled and chromatographed on silica gel plates (20 x 20 cm). (250) 35 using 50% acetone in hexane as the eluent, the most polar band yielding 23-O-demycinosyl-20-dehydrogenase.

DK 152924 B

37 oxo-20 - [(4,4-dioxothiomorpholinyl) imino] -4 "-O-isovaleryl · tylosin as a colorless amorphous solid having the following characteristics:

Rotation: [α] 6 -61.8 ° (CHCl ,,), UV: (CH, CH ,OH) 236 D * 5 ΙΠ 3.X j ώ 5 nm (ε 7.145), 284nm (ε 21.172), IR: v (CDCl 3) 3610, 2980, 2950, 2900, 1725, 1683, 1598, 1315, 1192, 1172, 1130, 1060, 1030 cm -1, NMR: <H (CDCl3) 1.00 (6H, d, J 6Hz, 4 "-OCOCH2CH (CH3) 2), 1.84 (3H, d, J13 22 1.5Hz, 22-CH3), 2.54 (6H, s, 3'-N (CH3) 2) ), 4.31 (1H, d, 2, 7.5Hz, 1H), 5.95 (1H, dq, J13 ^ 22 1.5Hz, J13 / 14 10Hz 'H13), 6.33 ( 1H, d, 0 "i0 15.5Hz, H-q) r 7.06 (1H, t, 2q 5Hz, H2q) and 7.40 (1H: a'Jio, u 15'5H2 'Hll> ·

Example 2 A. 23-0- (tert-Butyldimethylsilyl) -23-0-demycinosyl tylosin.

23-O-Demycinosyl-tylosin (20 g) and imidazole (3.7 g) were dissolved in dry dimethylformamide (200 ml). A solution of tert-butyldimethylsilyl chloride (4.1 g) in dry dimethylformamide (100 ml) was added dropwise over 2 hours and the mixture was stirred at 25 ° C for an additional 16 hours. The reaction mixture was worked up and the product purified as in Example 1, Section A, thereby obtaining 23-0- (tert-rbutyldimethylsilyl) -23-0-demycinosyl tylosin.

B. 2'-O-Acetyl-23-O- (tert-butyldimethylsilyl) -23-O-demycinosyl-tylosin.

23-O- (tert-Butyldimethylsilyl) -23-O-demycinosyl-tylosin (prepared as in Section A of this Example) 30 (10 g) was treated with acetic anhydride (4.8 g) in dry acetone (100 ml) substantially the same as described in Section B of Example 1, thereby obtaining 2'-O-acetyl-23-O- (tert-butyldimethylsilyl) -23-O-demycinosyl tylosin.

35 38

DK 152924B

C. 2 ', 4 "-Di-O-acetyl-23-O- (tert-butyldimethylsilyl) -23-O-demycinosyl-tylosin.

2'-O-Acetyl-23-O- (tert-butyldimethylsilyl) -23-O-demycinosyl-tylosin (prepared as in section B of this example) (8g), 4-dimethylaminopyridine (1/1g) and Triethylamine (10 ml) was dissolved in dry dichloromethane (100 ml). Acetic anhydride (0.9 g) in dry dichloromethane (50 ml) was added as described in section C of Example 1 and the reaction mixture worked up and the product purified as described in Example 1 to give 2 ', 4 "-di -O-acetyl-23-O- (tert-butyldimethylsilyl) -23-O-demycinosyl-tylosin.

D. 2 ', 4H-Di-O-acetyl-23-O-demycinosyl-tylosin.

2 ', 4 "-Di-O-acetyl-23-O- (tert-butyldimethylsilyl) - 23-O-demycinosyl-tylosin (prepared as in section C of this example) (5g) was dissolved in 80% acetic acid water (50 ml) and the solution was allowed to stand at 25 ° C for 2 hours, the pH was adjusted to 9.5 with dilute 20 aqueous sodium carbonate and the aqueous layer was extracted with dichloromethane. The latter was washed with water, dried (MgSO4), filtered and evaporated to dryness. The product was chromatographed on a preparative HPLC apparatus using a silica gel cartridge and 20% acetone in hexane as eluent to give 2 ', 4 "-di-0-acetyl -23-0-demycinosyl-tylosin.

E. 4 "-O-Acetyl-23-O-demycinosyl-tylosin.

2 ', 4 "-Di-O-acetyl-23-O-demycinosyl-tylosin (prepared as in Section D of this example) (3g) was dissolved in methanol (100ml) and the solution was stirred at room temperature. The solution was evaporated to dryness and the product was chromatographed on a preparative HPLC apparatus using a silica gel cartridge and 20% acetone in hexane as eluent to give 4 "-O-acetyl-23 -O-demycinosyl-tylosin.

DK 152924 B

39 F. 4 "-O-Acetyl-23-O-demycinosyl-20-deoxo-2 (> - [(4,4-dioxo-thiomorpholinyl) imino] tylosin.

4 "-O-Acetyl-23-O-demycinosyl-tylosin (prepared as in section E of this example) (2 g) and 1N-amino-4,4-5 dioxothiomorpholine (383 mg) was dissolved in dry dichloromethane (50 ml) and the mixture was stirred at 25 ° C for 72 h. The solution was evaporated to dryness and the residue was chromatographed on a preparative HPLC apparatus using a silica gel cartridge and 30% acetone in hexane as eluent to give 4 "- O-acetyl-23-O-demycinosyl-20-'deoxo-20 - '((4,4-dioxothio-morpholinyl) imino] tylosin, [a] + (CHCl4): -70.1 (6.25 mg / 2ml).

Example 3 A. 3,2 ', 4 "-Tri-O-acetyl-23-O- (tert-butyldimethylsilyl) -2,3-O-demycino syl-tylosin.

21,4 "-Di-O-acetyl-23-O- (tert-butyldimethylsilyl) -23-O-demycinosyl-tylosin (prepared as in Section C of Example 2) (5 g), 4-dimethylaminopyridine (3, 35 g) and 20 triethylamine (5 ml) were dissolved in dry dichloromethane (100 ml).

Acetic anhydride (2.7 g) was added and the mixture was left at 25 ° C for 16 hours. The reaction mixture was worked up and purified as described in section C of Example 1 to give 3.2 ', 4 "-O-acetyl-23-O- (tert-butyldimethylsilyl) -23-O-denycinosyl-tylosin.

B. 3,2 ', 4 "-Tri-O-acetyl-23-O-demycinosyl-tylosin.

3,2 ', 4 "-Tri-O-acetyl-23-O- (tert-butyl dimethylsilyL) -23-O-demycinosyl-tylosin (prepared as in section A of this example) (3g) was dissolved in 80 % acetic acid-water 30 (50 ml) and the reaction was carried out and the product purified as described in Section D of Example 2 to give 3,2 ', 4 "tri-O-acetyl-23-O-demycinosyl tylosin.

C. 3,4 "-Di-O-acetyl-23-O-demycinosyl-tylosin.

3,2 ', 4 "-Tri-O-acetyl-23-O-demycinosyl-tylosin 40

DK 152924B

(prepared as in section B of this example) (2 g) was dissolved in methanol (50 ml) and the reaction was carried out and the product purified as in section E of Example 2 to give 3,4 "di-O-acetyl -23-0-demycinosyl-tylosin.

D. 3,4 "-Di-O-acetyl-23-O-demycinosyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin.

3,4 "-Di-O-acetyl-2,3-O-demycinosyl-tylosin (prepared as in section C of this example) (1g) and 1-N-10 amino-4,4-dioxothiomorpholine (160 mg) was dissolved in dry dichloromethane (50 ml) and the reaction was carried out and the product purified as described in section F of Example 2 to give 3,4 "di-O-acetyl-23-O-demycinosyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin [alpha] 6 (CHCl 3): - 97.9 (9.05 mg / 2ml).

Example 4 A. 20-Deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin. Tylosin (30 g) and 1-N-amino-4,4-dioxothiomorpholine 20 (4.92 g) were dissolved in absolute ethanol (310 ml) and the mixture was stirred at 25 ° C for 42 hours. The solution was evaporated to dryness. The residue was then chromatographed on a silica gel column (120 x 5 cm) using 2% methanol in chloroform as the eluent followed by gene chromatography of the overlapping fractions on a silica gel column (120 x 5 cm) using 1.5% methanol in chloroform as eluent to give 20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin as a colorless amorphous solid having the following characteristics:

Rotation: [α] 6 -56.8 ° (CHOU), UV: (CFoCHoOH) 235

J (JIUciX JL · nm (ε 6.626), 286nm (ε 21.765), IR: (CDCl1) 3580, 2980, 2950, 2900, 1710, 1675, 1585, 1305, 1160, 1120, 1040 cm "1, NMR: H (CDCl3) 0.94 (3H, t, Jlg 1? 7Hz, 17-CH3), 35.02 (3H, d, J4 χ8 6Hz, 18-CH3), 1.80 (3H, d, J13) 22, 1.5Hz, 22-CH 3), 2.50 (6H, s, 3'N (CH 3) 2), 3.50 (3H, s, 2 '' - 0CH 3), 3.63

DK 152924 B

41 (3H, s, 31 '1-OCH 3), 4.29 (111, (1, 2, 7Hz, HI)), 4.58 (1H, d, 1 H, 21, 1 71 71 111) ) / 5.94 (1H, dq, J- ^ 3 ^ 22 * ^ Ez> * ^ 13 14 10Hz, H13), 6.30 (1H, d, J1Q 15ι 15.5Hz, H1Q), 7.01 ( 1H, t, J19,20 5HZ / H 2 O 2 g 7/37 (1H / d, J10, 11 15'5Hz / Hn) · 5 B. 4'1'-O- (tert-Butyldimethylsilyl) -20-deoxo -20 - [(4,4-dioxothiomorpholinyl) imino] -tylosin.

20-Deoxo-20 - [(4,4-dioxothiomorpholinyl) iminoj-tylosin (prepared as in Part A of this example) (3 g) and 10 imidazole (975 mg) were dissolved in dry dimethylformamide (45 ml), and tert-butyldimethylsilyl chloride (2.16 g) was added. The mixture was stirred at 25 ° C for 18 hours under dry argon gas. The solution was evaporated to dryness and the residue was taken up in dichloromethane, washed with water, dried (MgSO4), filtered and evaporated. The residue was then chromatographed on a silica gel column (30 x 5 cm) using 30% acetone in hexane as the eluent to give 4 ": -O- (tert-butyldimethylsilyl) -20-deoxo-20 - [(4.4 (dioxothio-morpholinyl) imino] tylosin as a colorless amorphous solid with the following characteristics:

Rotation: [<x] 36 -47.1 ° (CHCl1), UV: λ (CF, CH₂OH) 235 V λ ITla.X j Δ, nm (ε 6.710), 286 nm (ε 23.082), IR: ( CDCl1 3500, 2970, 2940, 2900, 1740, 1680, 1595, 1315, 1260, 1130, 1052 cm -1, NMR: δ (CDCl 3) 0.09 (3H, s, 4 '"- SiCH 3) , 0.12 (3H, s, 4'-SiCl3), 0.94 (9H, s, 4'1'-SiC (CH3) 3), 1.79 (3H, d, J13 22 λ'5Εζ ' 22-CH 3), 2.50 (6H, s, 3, -N (CH 3) 2), 3.50 (3H, s, 2, - OCH 3), 3.61 (3H, s, 3 '* -OCH3), 4.28 (1Hrd., Jx, 8Hz, Ηχι), 4.62 (1H, d, Jlt,, f2,,, 8Hz, δ,,), 5.95 (1H, dq, J13 22 30 1.5Hz, J13 14 10Hz, H 1), 6.28 (1H, d, J 10 u 15.5 Hz, H 2), 7.00 (1H, 1'19 19 5Hz, H 2 O) and 7 , 36 (lHfd, J10 n 15.5Ήζ, Η ^.

r. 2'-O-Acetyl-4-O- (tert-butyldimethylsilyl) -20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin.

4 "-O- (tert-Butyldimethylsilyl) -20-deoxo-20 -: [(4,4-42)

DK 152924B

dioxothiomorpholinyl) imino] tylosin (prepared as in section B of this example) (1.41 g) was dissolved in dry acetone (30 ml) and acetic anhydride (0.5 ml) was added. The mixture was left at 25 ° C for 40 hours.

The solution was evaporated to dryness and the residue was azeotroped with toluene. The residue was taken up in a mixture of dichloromethane-water and the pH was adjusted to 9.5 with dilute aqueous sodium hydroxide. The dichloromethane layer was washed with water, dried (MgSO 4), filtered and evaporated to dryness to give 2'-O-acetyl-4 '"-O- (tert-butyldimethylsilyl) -20-deoxo-20 - [(4, An analytical sample (200 mg) prepared by chromatography on a silica gel column (60 x 2 cm) using 30% acetone in hexane as the eluent had the following characteristics:

Rotation: [α] 6 -52.2 ° (CHCl,), UV: (CF-, CHOH) 234

U J IuaX O Z

nm (ε 6.369), 286 nm (ε 22.441), IR: v (CDCl3) 3500, max o 2975, 2950, 2900, 1750, 1715, 1680, 1598, 1318, 1240, 20 1130, 1055 cm -1, NMR: δΗ (CDCl3) 0.09 (3H, s, 4 '- SiC (CH3) 3), 0.12 (3H, s, 4' '-SiCH3), 0.92 (9H, s, 4 -SiC (CH3) 3), 1.77 (3H, d, J13 22 1.5Hz, 22-CH3), 2.07 (3H, s, 2'-OCOCH3), 2.40 (6H, s , 3'-N (CH 3) 2, 3.49 (3H, S, 2 + - OCH 3), 3.61 (3H, s, 3 '- OCH 3), 4.34 (1H, d, J ·, ^ 2, βΗζ, Η ^), 4.62 (1H, d, ti · 2> ·· 3Hz, H ^ ((·), 5.96 (1H, dg, J ^ 3 22 1.5Hz) , J13.14 10'5KZ 'h13 ^ 6'31 (1H'd'J10, ll 15Hz / h10), 6.99 (1H, t, J19 ^ 20 5Hz, H2O) and 7.38 (1H, d, J10 n 15Ηζ, Ηχι).

D. 2'-O-Acetyl-4 '' - O- (tert-butyldimethylsilyl) -3 ", 4" -0-30 carbonyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imine] -tylosin.

2'-O-Acetyl-4 '' - O- (tert-butyldimethylsilyl) -20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin (prepared as in section C of this example) (1.2 g) became 43

DK 152924B

dissolved in dry dichloromethane (50 ml) and NjN'-carbonyldiimidazole (178 mg) was added. The mixture was stirred at 25 ° C for 20 hours. The solution was evaporated to dryness and the residue was chromatographed on a silica gel column (200 g) using 15% acetone in dichloromethane as eluent to give 2'-O-acetyl-4 "1-O- (tert-butyldimethylsilyl) - 3 ", 4" -O-carbonyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin as a colorless amorphous solid having the following characteristics:

Rotation: [α] 26 -46.3 ° (CHCl 3), UV: λ max (CH 3 OH) 283 nm (ε 20,900), NMR: δΗ (CDCl 3) 0.10 (3H, s, 4 '"- SiCH 3), , 14 (3H, s, 4 ·· ′ -SiCH 3), 0.93 (9H, s, 4 ′ -SiC (CH 3) 3), 1.52 (3H, s, 3 ”-CH 3), 1, 77 (3H, d, J13 22 1.5Hz, 22-CH3), 2.00 (3H, s, 21-OCOCH3), 2.40 (6H, s, 3'-N (CH3) 2) , 48 (3H, s, 2 '"- OCH 3), 3.61 (3H, s, 31" -OCH 3), 4.60 (1H, d, Jin 2 "8Hz, Hin), 5.91 (1H , dq, J13 22 1.5Hz, J13 χ4 10Hz, H13), 6.24 (1H, d, J10 11 15Hz 'E10) r 6.94 (1H' '' J19.20 5Hz 'H20} ° g 7, 32 (1H ^ d, J1Q n 15Hz, H1; l).

E. 2 * -O-Acetyl-3 ", 4" -O-carbonyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin.

21-O-Acetyl-411'-O- (tert-butyldimethylsilyl) -3 ", 4" -O-carbonyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] -25 tylosin (prepared as in section D of this example) (1.05 g) was dissolved in anhydrous tetrahydrofuran (25 ml) and tetra-n-butylammonium fluoride trihydrate (0.32 g) was added. The mixture was stirred at 25 ° C for 5 hours. The solution was evaporated to dryness and evaporation residue was taken up in dichloromethane water. The dichloromethane layer was washed with water, dried (MgSO4), filtered and evaporated to dryness. Chromatography of the residue on a silica gel column (100 g) with 75% acetone in dichloromethane as eluent gave 2 1 -O-acetyl-3 ", 4" -O- 35 carbonyl-20-deoxo-20 - [(4.4 -dioxothiomorpholinyl) imino] -ylosin as a colorless amorphous solid with the following characteristics: »

DK 152924B

44

Rotation: [et]; 6 -54.7 ° C (CHCl3), UV: λ (CH, OH) 280 nm (ε 11.100), NMR: (CD3COCD3) f 1.56 (3H, s, 3 " -CH3), 1.86 (3H, d, J13 22 1.5Hz, 22-CH3), 2.01 (3H, s, 2'-OCOCH 3), 2.44 (6H, s, 3'-N ( CH3) 2) / 3.47 (3H, s, 2 '' - OCH 3), 3.53 (3H, s, 5 3 '' - OCH 3), 5.90 (1H, dq, J13 22 l / 5Hz , J13 14 10Hz, H1), 6.54 (1H, d, J10 / 15Hz, H1Q), 7.04 (1H, t, Jlg ^ 20 5Hz, H2 ()) and 7.25 (1H, d, J10 to 15Hz, χιχ).

F. 2'-Acetyl-4 '' '- oxo-3 ", 4" -carbonyl-20-deoxo-20- [(4,4-dioxothiomorpholinyl) imino] tylosin.

A solution of 2'-acetyl-3 ", 4" -O-carbonyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin (558 mg) in 2 ml of dry DMSO (dimethylsulfoxide) and 4 Dry benzene (ml) was treated at room temperature with anhydrous pyridine (0.04 ml), freshly distilled trifluoroacetic acid (0.02 ml) and dicyclohexylcarbodiimide (310 mg). The mixture was stirred under N 2 for 4 hours and the solids were filtered off and washed with more benzene. The organic filtrate was washed three times with distilled water 20 (3 x 5 ml) and once with saturated brine and dried (MgSO4).

Removal of the solvent gave the title compound which was used for the next step without further purification.

G. 23-Demycinosyl-3 ", 4" -O-carbonyl-20-deoxo-20- [(4,4-dioxothiomorpholinyl) imino] tylosin.

The oxidation product obtained in section F was dissolved in 10 ml of methanol and the solution was treated with silica gel (5 g). The mixture was stirred at room temperature for 4 days. Silica gel was filtered off and washed well with 30% methanol in CHCl 3 · The combined filtrates were evaporated to dryness and the residue was purified on 100 g of silica gel column. Elution with 5% methanol in CHCl3 gave 23-demycinosyl-3 ", 4" -O-carbonyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin.

35

DK 152924B

45 Η. 2 '-Acetyl-3 ", 4" -O-carbonyl-23-demycinosyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin.

A solution of 3 ", 4" -O-carbonyl-23-demycinosyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] tylosin (100 mg) and acetic anhydride in dry methylene chloride (5 ml) stirred at room temperature overnight. Azeotropic distillation of the reaction mixture with benzene gave 2'-acetyl-3 ", 4" -O-carbonyl-23-demycinosyl-20-deoxo-20- 26 [(4,4-dioxothiomorpholinyl) imino] tylosin. [α] β (CHCl3): 70 -71.8 (6.80 mg / 2ml.).

In the table below, physical data for the compounds of general formula I are prepared by the preparation methods described in the above examples.

15 20 25 30 35 46

DK 152924B

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DK 152924 B

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DK 152924B

The following composition examples illustrate pharmaceutical compositions containing the subject compounds. In these compositions, 23-demycinosyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] -4 "-O-isovaleryl-tylosin can be replaced by an equivalent amount of any other of the present invention compounds.

Composition 1

Capsule 10 23-demycinosyl-20-deoxo-20- 250.00 mg [(4,4-dioxothiomorpholinyl) imino I -4 "-O-isovaleryl-tylosin

Lactose 248.75 mg

Magnesium stearate 1.25 mg 500.00 mg

The active ingredient and lactose were mixed. Magnesium stearate was added and mixed. Filling in capsule.

20

Composition 2

Oral Suspension (to give a dose of 125 mg / 5 ml) 23-demycinosyl-20-deoxo-20- 25.00 g - [(4,4-dioxothiomorpholinyl) imino] -

Magnesium-aluminum silicate 9.50 g

Sodium carboxymethyl cellulose U.S.P. 2.50 g

Flavor sufficient _Λ Color "30

Methylparaben, U.S.P. 0.90 g

Propylparaben, U.S.P., 0.20 g

Polysorbate 80, U.S.P. 1.00 g

Sorbitol Solution, U.S.P. 500.00 g

Water to 1000.00 ml '45

DK 152924B

1. 200 ml of water was heated to boiling and dissolved half of the parabens. It was cooled to ca.

70 ° C, after which Polysorbate 80 was mixed. The silicate was stirred and stirred until there was a uniform smooth suspension.

2. An additional 200 ml of water was heated to boiling and dissolved in the rest of the parabens. CMC was distributed therein until a smooth gel was obtained. The sorbitol solution was mixed. Then the sodium citrate was dissolved.

3. The product of Step 2 was slowly added with constant stirring to the product of Step 1. The mixture was cooled to 25 ° C. The active ingredient, flavor and colorant were added and mixed thoroughly. Sufficient water was added to obtain a total volume of 1000 ml.

Composition 3

Topical Salve 23-demycinosyl-20-deoxo-20-10 g [(4,4-dioxothiomorpholinyl) imino] -4 "-O-isovaleryl tylosin Petrolatum 990 g 1000 g

Petrolatum was melted. The antibiotic was stirred with ca. 10% petrolatum and passed through a 30 colloid mill. The ground porridge was mixed with the rest of the molten petrolatum. Recommendation for cooling.

35

DK 152924B

50

Composition 4

Topical Creme 23-demycinosyl-20-deoxo-20-10 g [(4,4-dioxothiomorpholinyl) imino] -54-O-isovaleryl-tylosin

Stearic acid 200 g

Sorbitan Monostearate 104 g

Sorbitan Monoleate 20 g

Polyoxyethylene Sorbitan Monolaurate 56 g 10 Water to 100 ml 1. Stearic acid, sorbitan monostearate, sorbitan monoleate and polyoxyethylene sorbitan monolaurate were heated to 65 ° C.

2. Approx. 90% of the water was heated to 70 ° C.

3. The water from Step 2 was added to the mixture from Step 1, 20 and mixed to form a cream base.

4. The active ingredient was stirred for approx. 10% of the water and passed through a colloid mill.

25 5. The ground porridge was added to the molten base and mixed. Recommendation for cooling.

30 35

Claims (4)

23. Ra ° -T \ -v Wherein I is hydrogen or (C 2 -C 5) alkanoyl R 2 is (C 2 -C 5) alkanoyl, R 8 is hydrogen or (C 2 -C 4) alkanoyl, or R 2 and R 9 are together a carbonyl group connecting the 3 "-20 and 4" hydroxy groups, R 2 is hydrogen or (C 2 -C 4) alkanoyl, R 9 is hydroxymethyl or acetoxymethyl, and R 5 is / - \ -NN -SO 25 - / or are pharmaceutically acceptable acid addition salts thereof. Tylosin derivative according to claim 1, characterized in that R 1 'R 3 R 4 is hydrogen and R 2 is acetyl, propionyl, n-butytyl or isovaleryl, or R 1 is acetyl, R 1 and R 2 are hydrogen and R 2 is acetyl. , propionyl, n-butyryl or isovaleryl, or R 2 and R 9 are hydrogen and R 9 and R 2 are independently acetyl, propionyl, n-butyryl or isovaleryl, or R 2 and R 2 are hydrogen and R 9 and R 9 are independently acetyl, propionyl, n-butyryl or isovaleryl, or DK 152924Β is hydrogen, and R The tylosin derivative of claim 1, characterized in that it is 23-demycinosyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] -4 "-O-isovaleryl-tylosin, 4 "-O-acetyl-23-demycinosyl-20-deoxo-20 - [(4,4-dioxothiomorpholinyl) imino] -tylosin, 2 ', 4" -di-O-acetyl-23-demycinosyl-20-deoxo 20 - [(4,4-dioxo thiomorpholinyl) imino] tylosin, 21-O-acetyl-3 ", 4" -O-carbonyl-23-demycinosyl-20-deoxo-20- [(4,4-dioxothiomorpholinyl) imino] tylosin or 3 ", 4" -O-carbonyl-23 -demycinosyl-20-deoxo-20-15 t (4,4-dioxothiomorpholinyl) imino] tylosin, or a pharmaceutically acceptable acid addition salt of any of these compounds. Pharmaceutical composition, characterized in that it contains a compound of the general formula 2g or a pharmaceutically acceptable acid addition salt thereof as defined in any one of claims 1-3, if desired in the form of a shaped dosage unit. 25 30 35