JPS6155518B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to new derivatives of the antibiotic tylosin. More specifically, the present invention provides the formula (In the formula, R ₁ is a hydrogen atom or a lower alkanoyl group, one of A ₁ and A ₂ is R ₂ group, the other is R ₃ group, R ₂
and R ₃ represents an alkanoyl group having 2 to 6 carbon atoms, except when R ₁ represents a hydrogen atom, A ₁ represents an acetyl group, and A ₂ represents an isovaleryl group. ) or a salt thereof. The above salts are pharmaceutically acceptable salts.
Such suitable salts include salts with inorganic acids such as hydrochloric, sulfuric, and phosphoric acids, and salts with organic acids such as acetic, propionic, tartaric, citric, succinic, malic, aspartic, and glutamic acids. is included. Other non-toxic salts are also included. The above-mentioned novel compound [1] not only has antibacterial activity equivalent to that of the known antibiotic Tylosin, but also possesses all macrolide-resistant bacteria, such as macrolide-resistant group A bacteria (erythromycin, oleandomycin). , 16-membered ring macrolide antibiotic-resistant patient isolates) It has strong antibacterial activity against group B bacteria and group C bacteria, and is reported to be effective against macrolide antibiotic-resistant bacteria.
4″-O-acyltylosin, 3-O-acetyl or -propionyl-4″-O-acyltylosin (JP-A-52-139088, JP-A-53-137982) is more effective against these resistant bacteria. It has antibacterial properties. Moreover, it lacks the bitter taste that is typical of tylosin, making it useful as a syrup for children who cannot take tablets or capsules, and is an antibacterial agent that is expected to have an extremely excellent clinical effect in treating infections.
It is also useful as a therapeutic agent for animals and a feed additive. Tylosin has five hydroxyl groups at the 3-position, 2'-position, 3''-position, 4''-position and 4-position. 3 of these
It is said that the hydroxyl groups at the 2', 4', and 4' positions are easily acylated, and the 3' hydroxyl group is inactive. Even if the hydroxyl group at the 3″ position could be acylated, it would be practically impossible using conventional acylation methods because highly reactive hydroxyl groups exist at positions other than the 3″ position. When acylating the hydroxyl group at the 3″ position, the present inventor has previously developed a method for acylating the hydroxyl group at the 3″ position, especially the 3″ position, the 2′ position, and the 4″ position.
Focusing on protecting the hydroxyl group at the 3'-position with a protecting group that can be selectively removed after acylation of the hydroxyl group at the 3'-position, we continued various studies and found that a lower alkanoyl group was used as a protecting group for the 2'-position hydroxyl group. We learned that it is preferable to use a lower alkanoyl group, a halogenated acetyl group, or a trimethylsilyl group as a protecting group for the hydroxyl group at the 4-position.Also, the hydroxyl group at the 3-position can be protected in the presence of an aliphatic carboxylic acid anhydride and an inorganic base. Let it react,

It was discovered that protection can be achieved by forming a ring as shown in the following formula, and based on these various findings, the present invention was completed. The object compound [1] of the present invention is produced by the following method. [A] Compound [1] in which R ₁ is a hydrogen atom, A ₁ is an R ₂ group, and A ₂ is an R ₃ group, that is, the formula (wherein R ₂ and R ₃ mean the same groups as above). The above target compound [1a] can be obtained by reacting tylosin or tylosin with the 4-position hydroxyl group protected with an aliphatic carboxylic acid anhydride in the presence of an inorganic base. (In the formula, R ₄ is lower alkanoyl or halo-lower alkanoyl, R ₂ means the same group as above) is obtained, and the compound [2]
was reacted with an aliphatic carboxylic acid anhydride under heating in the presence of a tertiary organic amine in an inert organic solvent to obtain the formula (wherein R ₂ , R ₃ and R ₄ mean the same groups as above) is obtained, the compound [3] is treated with ammonia in methanol or ethanol, and then heated in methanol. It can be obtained by The introduction of protecting groups into the 3-, 2'-, and 4-position hydroxyl groups of tylosin is carried out by reacting an aliphatic carboxylic acid anhydride in the presence of an inorganic base. The above aliphatic carboxylic acid anhydride [(R ₂ ) ₂ O]
Examples include anhydrides of lower fatty acids such as acetic anhydride, propionic anhydride, butyric anhydride, and isovaleric anhydride. Inorganic bases include alkali hydroxides such as potassium hydroxide, sodium hydroxide, alkali carbonates such as potassium carbonate, sodium carbonate,
Examples include alkali hydrogen carbonate, such as sodium bicarbonate, and alkali carbonate is particularly preferred. The above protecting group introduction reaction usually takes 30 to 100 minutes.
It is carried out under heating at a temperature of preferably 40 to 60°C. Since the progress of the reaction can be tracked by thin layer chromatography using silica gel or the like, the reaction can be appropriately terminated after waiting for the disappearance of tylosin. Through the above reaction, an acyl group is introduced into the aldehyde group at position 20, and the ring is closed via the carbon atom at position 20 and the oxygen atom at position 3, which not only protects the hydroxyl group at position 3 but also protects the hydroxyl group at position 2'. , 4″ position and 4 are also acylated at the same time. This protection of 3 and 20 positions is also excellent in selective reactions as a protecting group.
Furthermore, since it has excellent stability, it is an extremely excellent protecting group for the 3-position hydroxyl group. In the introduction of the above-mentioned protecting group, only the hydroxyl group at the 4-position is protected with a halo-lower alkanoyl group, and then the remaining hydroxyl groups at the 3-, 2'-, and 4''-positions are acylated by the above-mentioned method of introducing a protecting group. Preferred examples of halo-lower alkanoyl groups include chloroacetyl, dichloroacetyl, and trichloroacetyl groups.Introduction of this protecting group is carried out in an inert organic solvent such as dichloromethane with a tertiary organic amine such as pyridine, etc. The reaction is carried out by reacting 1.2 to 1.5 times the mole of chlorinated aliphatic carboxylic acid halide in the presence of 4-O-halo lower alkanoyl tylosine obtained in this way. The hydroxyl group at the 2′-position is protected, and the hydroxyl group at the 4″-position is acylated. As a result of the introduction of the protecting group as described above, the hydroxyl groups at the 3-, 2'-, and 4-positions were protected, and the hydroxyl group at the 4''-position was acylated. Collect the product [2] from the reaction solution. can be collected by pouring the reaction solution into water, adjusting the pH of the aqueous layer to 8 to 10, and extracting with a suitable non-hydrophilic organic solvent.If further purification is required, silica gel, activated It can be separated and purified by chromatography using an adsorbent such as alumina or adsorption resin and eluting with a suitable solvent, such as a benzene-acetone solvent.Next, compound [2] is 3″-acylated. , by reacting an aliphatic carboxylic acid anhydride under heating in the presence of a tertiary organic amine. The above aliphatic carboxylic acid anhydride [(R ₃ ) ₂ O]
Examples include anhydrides of aliphatic carboxylic acids having 2 to 6 carbon atoms, such as acetic anhydride, propionic anhydride, butyric anhydride, isobutyric anhydride, valeric anhydride, isovaleric anhydride, and hexanoic anhydride. Examples of tertiary organic amines include pyridine, picoline,
Pyridine compounds such as collidine are preferred, but the present invention is not limited thereto, and other known tertiary organic amines can also be selected as appropriate. The heating temperature is usually 50 to 120°C, preferably 80 to 100°C. The reaction time varies mainly depending on the heating temperature, but since the reaction can be monitored by thin layer chromatography using silica gel or the like, the end point of the reaction can be appropriately determined based on the disappearance of compound [2]. It is usually carried out for a period of 1 to 100 hours. As a result of the above reaction, the previously existing acyl group (R ₂ ) at the 4″ position is transferred to the 3″ position, and the acyl group (R ₃ ) resulting from the above acylation reaction is introduced into the 4″ position. Reaction Compound [3] can be collected and purified from the solution in the same manner as the above-mentioned process for obtaining compound [2].Next, the protecting group of compound [3] is removed. By treating compound [3] with an ammonia-containing methanol or ethanol solution, the protecting groups at positions 3 and 20 and the protecting group at position 4 are eliminated. This elimination reaction proceeds satisfactorily at room temperature. The reaction can be tracked by thin layer chromatography such as chromatography, so the reaction can be appropriately terminated after the disappearance of compound [3].The product obtained by distilling off ammonia and alcohol from the reaction solution thus obtained teeth,
The 2'-position acyl group is eliminated by heat treatment in methanol which may contain water. Heating is carried out under reflux of methanol. Since the reaction can be tracked by thin layer chromatography using silica gel or the like, the reaction may be terminated as appropriate. The product obtained by distilling methanol off from the reaction solution is separated and purified as described below to obtain the desired compound [1a]
can be obtained. For example, the desired target compound [1a] is separated by means such as concentration, extraction, washing, dissolution, recrystallization, and chromatography using an adsorbent such as silica gel, activated alumina, and adsorption resin. Can be purified. [B] Compound [1] in which R ₁ is a hydrogen atom, A ₁ is an R ₃ group, and A ₂ is an R ₂ group, that is, the formula (wherein R ₂ and R ₃ mean the same groups as above). The above target compound [1b] is the compound [2]
was reacted with an aliphatic carboxylic acid halide under heating in the presence of a tertiary organic amine in an inert organic solvent to obtain the formula (wherein R ₂ , R ₃ and R ₄ mean the same groups as above) is obtained, the compound [4] is treated with ammonia in methanol or ethanol, and then heated in methanol. It can be obtained by The above compound [2] is 3″-acylated with an aliphatic carboxylic acid halide. This reaction is carried out by reacting the corresponding aliphatic carboxylic acid halide under heating in the presence of a tertiary organic amine in an inert organic solvent. The inert organic solvent is usually acetone, methyl ethyl ketone, ethyl acetate, dimethoxyethane, tetrahydrofuran, dioxane, benzene, toluene, dichloroethane, etc. The tertiary organic amine is usually pyridine, Pyridine compounds such as picoline and collidine are used, but other known tertiary organic amines such as triethylamine, dimethylaniline, diethylaniline, N-methylpiperidine, N-methylmorpholine, quinoline, isoquinoline, tribenzylamine are used. Corresponding aliphatic carboxylic acid halides include aliphatic carboxylic acid halides having 2 to 6 carbon atoms,
Examples include acetyl chloride, propionyl chloride, butyryl chloride, isobutyryl chloride, valeryl chloride, isovaleryl chloride, hexanoyl chloride, and the like. The heating temperature is usually in the range of 50 to 120°C. The reaction time varies mainly depending on the reaction temperature, but since the progress of the reaction can be monitored by thin layer chromatography such as silica gel, the end point of the reaction may be appropriately determined within the range of 1 to 150 hours. When the reaction solvent is a hydrophilic organic solvent, the compound [4] thus obtained can be precipitated by adjusting the pH of the reaction solution in water to 8 to 10 with an alkali, and then taken as is, or
When the reaction solvent is a non-hydrophilic organic solvent,
It can be collected by pouring the reaction solution into water, adjusting the pH of the aqueous system to 8-10, and extracting with a suitable non-hydrophilic organic solvent. If further purification is required, it can be separated and purified by chromatography using an adsorbent such as silica gel, activated alumina, or adsorption resin and eluting with a suitable solvent, for example a benzene-acetone solvent. The protecting groups at the 3-, 2'- and 4-positions of the reaction product [4] can be removed to obtain the target product [1b] in the same manner as in step A above. The desired target product [1b] can be obtained by separating and purifying the product from which methanol has been distilled off as described below. [C] R ₁ is lower alkanoyl group, A ₁ is R ₂ group, A ₂
Compound [1] in which is R ₃ group, that is, the formula (In the formula, R′ ₁ is a lower alkanoyl group, R ₂ and
R ₃ means the same group as above). The above target compound [1c] has the formula (In the formula, R ₅ is an alkanoyl group having 2 to 6 carbon atoms, and R ₆ is a hydrogen atom, an alkanoyl group having 2 to 6 carbon atoms, or a halo-lower alkanoyl group). was acylated with an aliphatic carboxylic acid halide in the presence of a tertiary organic amine in an inert organic solvent to give the formula (In the formula, R ₇ is an alkanoyl group having 2 to 6 carbon atoms or a halo-lower alkanoyl group, R' ₁ , R ₂ and
R ₅ represents the same group as above), and the compound [6] was acylated with an aliphatic carboxylic acid anhydride under heating in the presence of a base to obtain a compound represented by the formula (wherein R′ ₁ , R ₂ , R ₃ , R ₅ and R ₇ represent the same groups as above), the compound [7] was treated with ammonia in methanol or ethanol, and then Obtained by heat treatment in methanol. As the above starting material [5], commonly known
2'-O-acyltyrosine is used. This acyl group is a group that is eliminated later, but it is an alkanoyl group having 2 to 6 carbon atoms, and is acetyl,
Propionyl, butyryl groups, etc. are preferred. The hydroxyl group at position 4 of the above 2'-O-acyltylosine is protected with an alkanoyl group having 2 to 6 carbon atoms, a halo-lower alkanoyl group, especially a halo-lower alkanoyl group, such as chloroacetyl, dichloroacetyl, trichloroacetyl group, etc. However, it does not necessarily protect you. Using the corresponding aliphatic carboxylic acid halide,
4″-acylation, and this reaction is carried out by reacting the corresponding aliphatic carboxylic acid halide in the presence of a tertiary organic amine in an inert organic solvent. Acetone, methyl ethyl ketone, ethyl acetate, tetrahydrofuran, dioxane, benzene, toluene, dichloroethane, etc. are used.As the tertiary organic amine, pyridine compounds such as pyridine, picoline, and collidine are usually used, but other known Tertiary organic amines such as triethylamine, dimethylaniline, diethylaniline, N-methylpiperidine, N-methylmorpholine, quinoline, isoquinoline, etc. may also be used.As aliphatic carboxylic acid halides, those having 2 to 6 carbon atoms may also be used. It is an aliphatic carboxylic acid halide, such as acetyl chloride, propionyl chloride, butyryl chloride, isobutyryl chloride, valeryl chloride, isovaleryl chloride, hexanoyl chloride, etc., but the 3-position acyl group is more Since lower-grade compounds are preferred, acetyl chloride, propionyl chloride, etc. are usually particularly preferred.Since the reaction temperature usually proceeds at room temperature, there is no need to heat unless the reaction rate is particularly slow. ~50℃.The reaction time can be tracked using thin layer chromatography such as silica gel, so the reaction time is usually 1.
The end point of the reaction may be appropriately determined within the range of ~10 hours. In the above acylation reaction, not only the 3- and 4-position hydroxyl groups are acylated, but also the 4-position hydroxyl group is acylated. Therefore, depending on the number of hydroxyl groups to be acylated, aliphatic carbon The amount of acid halide used may be changed as appropriate. Also, if you want to introduce different acyl groups into the 3- and 4-position hydroxyl groups, first reduce the amount of aliphatic carboxylic acid halide used and carry out the reaction in the same way.
After obtaining the 4″-acyl compound, it can be obtained by reacting the desired different aliphatic carboxylic acid again and performing the same treatment. Compound [6] obtained in this way is obtained when the reaction solvent is hydrophilic In the case of a neutral organic solvent, the reaction solution can be precipitated in water by adjusting the pH to 8 to 10 with an alkali, and then taken as is, or
When the reaction solvent is a non-hydrophilic organic solvent,
It can be collected by pouring the reaction solution into water, adjusting the pH of the aqueous system to 8-10, and extracting with a suitable non-hydrophilic organic solvent. If further purification is required, it can be separated and purified by chromatography using an adsorbent such as silica gel, activated alumina, or adsorption resin and eluting with a suitable solvent, for example a benzene-acetone solvent. Next, the product [6] is 3''-acylated, and this reaction is carried out by reacting an aliphatic carboxylic acid anhydride under heating in the presence of a base.The base may be an alkali carbonate, e.g. Suitable examples include potassium carbonate, sodium carbonate, tertiary organic amines, such as pyridine compounds such as pyridine, picoline, and collidine, but are not limited to these, and other known alkali hydroxides, alkali hydrogen carbonates, Tertiary organic amines may also be used. Examples of aliphatic carboxylic acid anhydrides include the compounds listed in Step A above.
Heating temperature is usually 50-120℃, preferably 80-100℃
It is carried out in the range of °C. The reaction time varies mainly depending on the heating temperature, but since the progress of the reaction can be monitored by thin layer chromatography using silica gel or the like, the end point of the reaction can be appropriately determined by waiting for the disappearance of compound [6]. Usually 1~
It will be conducted over a period of 100 hours. As a result of the above reaction, the previously existing acyl group (R ₂ ) at the 4″ position is transferred to the 3″ position, and the acyl group (R ₃ ) resulting from the above acylation reaction is introduced into the 4″ position. Reaction Compound [7] can be collected and purified from the solution in the same manner as described in the step of obtaining compound [4].Next, the protecting group of compound [7] is removed. However, by first treating compound [7] with ammonia-containing methanol or ethanol, the protecting group at position 4 is eliminated. This elimination reaction proceeds satisfactorily at room temperature. The reaction is performed using a thin layer chromatograph such as silica gel. Since it can be tracked by E, the reaction can be terminated appropriately after waiting for the disappearance of compound [7].The product obtained by distilling off ammonia and alcohol from the reaction solution obtained in this way is
The 2'-position acyl group is eliminated by heat treatment in methanol which may contain water. Heating is usually carried out under refluxing methanol. Since the reaction can be tracked by thin layer chromatography using silica gel or the like, the reaction may be terminated as appropriate. The product obtained by distilling methanol off from the reaction solution is separated and purified as described below to obtain the desired compound [1c]
can be obtained. [D] R ₁ is lower alkanoyl group, A ₁ is R ₃ group, A ₂
Compound [1] in which is R ₂ group, that is, the formula (In the formula, R′ ₁ , R ₂ and R ₃ represent the same groups as above.) The above target compound [1d] is compound [6]
is reacted with an aliphatic carboxylic acid halide under heating in the presence of a tertiary organic amine in an inert organic solvent to obtain the formula (wherein R′ ₁ , R ₂ , R ₃ , R ₅ and R ₇ are the same groups as above) was obtained, the compound [8] was treated with ammonia in methanol or ethanol, and then Obtained by heat treatment in methanol. Compound [8] is obtained by 3''-acylation with an aliphatic carboxylic acid halide, which can be carried out in the same manner as the 3''-acylation of compound [2] in step A above. Next, the protecting groups at the 2′ and 4″ positions of compound [8] are removed to obtain the desired target compound [1d] in the same manner as in the method for removing the protecting groups of compound [7] above. The desired target compound [1d] can be obtained by separating and purifying the product from which methanol has been distilled off as described below.The target compound [1] thus obtained is added to the reaction solution. In order to collect macrolide antibiotics, known methods for separating and purifying macrolide antibiotics, such as concentration, extraction, washing, dissolution transfer, and recrystallization, and chromatography using adsorbents such as silica gel, activated alumina, and adsorption resins are required. This can be carried out by using graphical means, etc. Next, the results of measuring the minimum inhibitory concentration (MIC) of the target compound [1] of the present invention for microbial growth are measured in the first
Enter as shown in the table. This result shows that the compound [1] of the present invention is effective against macrolide-resistant group A bacteria.

【table】

[Table] Next, production examples of the target compound [1] of the present invention will be specifically explained with reference to Examples. Unless otherwise specified, Rf values in the examples were measured by thin layer chromatography (TLC) using the following carrier and developing solvent. Support: Silica gel 60Art5721 manufactured by Merck & Co., Ltd.Developing solvent A: n-hexane-acetone-benzene-ethyl acetate-methanol (30:10:25:20:10) B: benzene-acetone (3:1) C: benzene-acetone ( 4:1) Example 1 3″-O-acetyl-4″-O-butyryltylosin 10g of tylosin is dissolved in 20ml of acetic anhydride, 7g of potassium carbonate is added, and the mixture is heated and stirred at 60°C for 24 hours. Pour the reaction solution into 200ml of water and add aqueous ammonia.
After adjusting the pH to 9.5, extract twice with 100 ml of chloroform. After drying the extract over anhydrous magnesium sulfate, it was dried under reduced pressure to give 20・2′・4″・4-tetra-O
10.2 g of crude product of -acetyl-3.20-O-cyclo-tylosin is obtained. TLC; Rf _A = 0.75, Rf _B = 0.49, Rf _C = 0.34 (TLC of tylosin; Rf _A = 0.20, Rf _B = 0.01, Rf _C =
0.01) Mass; 1083 (M ⁺ ), 1024 (M ⁺ -59) Dissolve 10 g of the above product in 50 ml of dry pyridine,
Add 4 ml of butyric anhydride to this, and heat and stir at 100°C for 4 days. Pour the reaction solution into 400 ml of water and extract twice with 200 ml of chloroform. After drying the extract over anhydrous magnesium sulfate, it was dried under reduced pressure to give a size of 20, 2′, 3″, and 4
-Tetra-O-acetyl-3.20-O-cyclo-4''-O-butyryltylosin crude product 9.8g is obtained. TLC; Rf _B = 0.77, Rf _C = 0.61 This is mixed with benzene-acetone (15: Perform silica gel column chromatography eluting with step 1), and dry the elution fraction with the above Rf value under reduced pressure to obtain 3.5 g of purified product. Dissolve this in 30 ml of methanol, and add 20 ml of ammonia-saturated methanol to this solution.
ml and stirred at room temperature for 8 hours. water in reaction solution
Add 300 ml and extract twice with 150 ml of chloroform. The extract is dried over anhydrous magnesium sulfate and then dried under reduced pressure. Dissolve the residue in 150ml of methanol,
After heating under reflux for 12 hours, the mixture was dried under reduced pressure. The residue was subjected to silica gel column chromatography eluting with benzene-acetone (9:1) and benzene-acetone (7:1), and the former eluted fraction was dried under reduced pressure to give 3″·4-di-O-acetyl. −4″−O
-Butyryltylosin (TLC; Rf _A = 0.71) 400
mg, the latter elution fraction was dried under reduced pressure to obtain the desired 3″-O
-Acetyl-4″-O-butyryltylosin 2.5g
get. TLC; Rf _A = 0.57 Mass; 922 (M ⁺ −87−18) NMR (100MHz in CDCl ₃ ); 1.39 (3″CH ₃ ),
1.76 ( _12CH3 ), 1.96 (3″OAc), 2.51 (3′N
( _CH3 ) ₂ , 3.44( _2OCH3 ), 3.56(3
OCH ₃ ), 9.57 (20CHO) ppm Example 2 3″-O-acetyl-4″-O-propionyltylosin In Example 1, using propionic anhydride instead of butyric anhydride, 20·2′·3″・4-tetra-
O-acetyl-3.20-O-cyclo-4″-O-propionyltylosin (TLC; Rf _B =0.75, Rf _C
=0.59) to the desired 3″-O-acetyl-
4″-O-propionyltylosin is obtained. TLC; Rf _A = 0.55 Mass; 1013 (M ⁺ ) NMR (100MHz in CDCl ₃ ); 1.39 (3″ CH ₃ ),
1.76 ( _12CH3 ), 1.96 (3″OAc), 2.51 (3′N
(CH ₃ ) ₂ ), 3.44 (2 OCH ₃ ), 3.56 (3
OCH ₃ ), 9.56 (20CHO) ppm Example 3 3″・4″-di-O-acetyltylosin In Example 1, using acetic anhydride instead of butyric anhydride, 20・2′・3″・4″・4-Penta-O-
The desired 3″·4″-di-O-acetyltylosin is obtained via acetyl-3·20-O-cyclo-tylosin (TLC; Rf _B =0.71, Rf _C =0.55). TLC; Rf _A = 0.53 Mass; 981 (M ⁺ −18) NMR (100MHz in CDCl ₃ ); 1.39 (3″CH ₃ ),
1.75 ( _12CH3 ), 1.97 (3″OAc), 2.11
(4″OAc), 2.51 (3′N( _CH3 ) ₂ ), 3.44
(2″OCH ₃ ), 3.56 (3COH ₃ ), 9.56
(20CHO) ppm Example 4 3″-O-acetyl-4″-O-hexanoyltylosine In Example 1, using hexanoic anhydride instead of butyric anhydride, 20・2′・3″・4-titra -O
-acetyl-3.20-O-cyclo-4″-O-hexanoyltylosin (TLC; Rf _B = 0.80, Rf _C =
0.65) via the desired 3″-O-acetyl-4″-
O-hexanoyltylosine is obtained. TLC; Rf _A = 0.61 Mass; 9.22 (M ⁺ −115−18), 390 NMR (100MHz in CDCl ₃ ); 1.39 (3″CH ₃ ),
1.76 ( _12CH3 ), 1.96 (3″OAc), 2.52 (3′N
( _CH3 ) ₂ ), 3.43( _2OCH3 ), 3.55(3
OCH ₃ ), 9.55 (20CHO) ppm Example 5 4″-O-isovaleryl-3-O-propionyl tylosin 50 g of tylosin was dissolved in 139.8 ml of propionic anhydride, and 37.7 g of anhydrous potassium carbonate was added thereto.
Stir at 60°C for 24 hours. Pour the reaction solution into 500 ml of water, adjust the pH to 9.5 with aqueous ammonia, and extract twice with 300 ml of chloroform. Wash the extract with water,
After drying over anhydrous magnesium sulfate, it is dried under reduced pressure to obtain 48.5 g of crude product of 3,20-O-cyclo-20,2',4'',4-tetra-O-propionyltylosin. TLC; Rf _A = 0.84 , Rf _B =0.74, Rf _C =0.57 Dissolve 10 g of the above product in 50 ml of dry pyridine,
Add 4.5 ml of isovaleric anhydride to this and heat to 100°C.
Heat and stir for 5 days. Pyridine was distilled off from the reaction solution under reduced pressure, and the remaining liquid was poured into 200 ml of water, and chloroform was added.
Extract twice with 200ml. The extract was washed with water and aqueous ammonia (PH9.0) in that order, dried over anhydrous magnesium sulfate, and dried under reduced pressure to give 3.20-O-cyclo-
10.2 g of crude product of 4″-O-isovaleryl-20・2′・3″・4-tetra-O-propionyltylosin
get. TLC; Rf _B =0.87, Rf _C =0.78 Dissolve this in 50 ml of methanol, add 50 ml of ammonia-saturated methanol, and stir at room temperature for 15 hours. Pour the reaction solution into 500ml of water and add chloroform.
Extract twice with 300ml. The extract was dried under reduced pressure, and the residue was dissolved in 100 ml of methanol and heated under reflux for 17 hours. The reaction solution was dried under reduced pressure to obtain 9.5 g of product. This was mixed with benzene-acetone (15:1-7:
Perform silica gel column chromatography using eluting step 1) to obtain 4″-O-isovaleryl-3″・4
-di-O-propionyltylosin (TLC; Rf _A
=0.79) 100mg and the desired 4″-O-isovaleryl-
Obtain 2.7 g of 3″-O-propionyltyrosine. TLC; Rf _A = 0.60 Mass; 981 (M ⁺ −74), 954 (M ⁺ −101) NMR (100 MHz in CDCl ₃ ); 1.39 (3″ CH ₃ ) ,
1.75 (12CH ₃ ), 2.51 (3′N(CH ₃ ) ₂ ), 3.43 (2
OCH ₃ ), 3.55 (3 OCH ₃ ), 9.56
(20CHO) ppm Example 6 4″-O-butyryl-3″-O-propionyltylosin In Example 5, butyric anhydride was used instead of isovaleric anhydride, and 4″-O-butyryl-3.
20-O-cyclo-20・2′・3″・4-tetra-O
−Propionyltylosin [TLC; Rf _B =0.87,
Rf _C = 0.78, NMR (100MHz in CDCl ₃ ); 1.39
(3″CH ₃ ), 1.92 (12CH ₃ ), 2.36 (3′N(CH ₃ ) ₂ ),
3.39( _2OCH3 ), 3.46( _3OCH3 ) ppm] to obtain the desired 4''-O-butyryl-3''-O-propionyltylosin. TLC; Rf _A = 0.58 Mass; 954 (M ⁺ −87) NMR (100MHz in CDCl ₃ ); 1.39 (3″CH ₃ ),
1.76 (12CH ₃ ), 2.51 (3′N(CH ₃ ) ₂ ), 3.44 (2
OCH ₃ ), 3.56 (3 OCH ₃ ), 9.56
(20CHO) ppm Example 7 3″·4″-di-O-propionyltylosin In Example 5, propionic anhydride was used instead of isovaleric anhydride, and 3·20-O-cyclo-
20・2′・3″・4″・4-penta-O-propionyltylosin [TLC; Rf _B =0.86, Rf _C =0.76,
NMR (100MHz in CDCl ₃ ); 1.39 (3″CH ₃ ),
1.92 (12CH ₃ ), 2.36 (3′N(CH ₃ ) ₂ ), 3.39 (2
OCH ₃ ), 3.46 (3 OCH ₃ ) ppm] to obtain the desired 3″·4″-di-O-propionyltylosin. TLC; Rf _A = 0.58 Mass; 954 (M ⁺ −73) NMR (100MHz in CDCl ₃ ); 1.39 (3″CH ₃ ),
1.75 (12CH ₃ ), 2.51 (3′N(CH ₃ ) ₂ ), 3.44 (2
OCH ₃ ), 3.56 (3 OCH ₃ ), 9.55
(20CHO) ppm Example 8 3″·4″-di-O-butyryltylosin 10g of tylosin is dissolved in 28ml of butyric anhydride, 7.5g of anhydrous potassium carbonate is added thereto, and the mixture is stirred at 60°C for 24 hours. Pour the reaction solution into 100 ml of water and extract twice with 100 ml of chloroform. The extract was washed with water, dried over anhydrous magnesium sulfate, and dried under reduced pressure to give 20.
2′・4″・4-tetra-O-butyryl-3・20-
9.8 g of crude powder of O-cyclo-tylosin is obtained. TLC; Rf _B =0.86, Rf _C =0.74 This was dissolved in 50 ml of dry pyridine, 4 ml of butyric anhydride was added thereto, and the mixture was heated and stirred at 100°C for 102 hours.
Pour the reaction solution into 400ml of water, and add 200ml of chloroform.
Extract times. The extract was dried over anhydrous sodium sulfate and then dried under reduced pressure to obtain 8.9 g of a crude product of 20, 2', 3'', 4'', 4-penta-O-butyryl-3, 20-O-cyclo-tylosin. . TLC; Rf _B = 0.89, Rf _C = 0.83 This was subjected to silica gel column chromatography eluting with benzene-acetone (19:1), and the eluted fraction with the above Rf value was dried under reduced pressure to yield 2.9 g of purified product. get. Dissolve this in 25 ml of methanol, add 25 ml of ammonia-saturated methanol, and stir at room temperature for 10 hours. Add 250ml of water to the reaction solution and add 150ml of chloroform.
Extract twice. The extract is dried over anhydrous sodium sulfate and then dried under reduced pressure. Pour the residue into 150ml of methanol
After heating under reflux for 17 hours, it was dried under reduced pressure. The residue was dissolved in benzene-acetone (15:1-7:
Perform silica gel column chromatography eluting with step 1) to obtain 2.3 g of the desired 3''/4''-di-O-butyryltyrosine. TLC; Rf _A = 0.60 Mass; 1055 (M ⁺ ) NMR (100MHz in CDCl ₃ ); 1.39 (3″CH ₃ ),
1.76 (12CH ₃ ), 2.51 (3′N(CH ₃ ) ₂ ), 3.44 (2
OCH ₃ ), 3.56 (3 OCH ₃ ), 9.56
(20CHO) ppm Example 9 4″-O-acetyl-3″-O-isovaleryltylosin 20·2′·4″·4-tetra-O described in Example 1
-acetyl-3・20-O-cyclo-tylosin 10
Dissolve g in 70 ml of dry dioxane and add γ-collidine.
Add 13.42 ml and 11.29 ml of isovaleryl chloride, and heat and stir at 90°C for 45 hours. Pour the reaction mixture into ice water.
Pour into 300ml and extract twice with 200ml of chloroform. The extract was washed with 0.1N hydrochloric acid, diluted aqueous ammonia, and water in that order, dried over anhydrous magnesium sulfate, and dried under reduced pressure to give 20.2'.4".4-tetra-O-acetyl-3.20-O. 10.2 g of crude powder of -cyclo-3''-O-isovaleryltylosin is obtained. TLC; Rf _B = 0.76, Rf _C = 0.61 This was subjected to silica gel column chromatography eluting with benzene-acetone (15:1), and the fraction having the above Rf value was dried under reduced pressure to obtain 4.1 g of purified product. . Dissolve this in 100 ml of methanol, add 100 ml of ammonia-saturated methanol, and stir at room temperature for 12 hours. Pour the reaction solution into 500 ml of ice water and extract twice with 300 ml of chloroform. The extract is dried over anhydrous magnesium sulfate and then dried under reduced pressure. The residue was dissolved in 100 ml of methanol, heated under reflux for 18 hours, and then dried under reduced pressure. The residue was dissolved in benzene-acetone (7:
Perform silica gel column chromatography eluting with step 1), collect the corresponding active fractions, dry them under reduced pressure, and sequentially obtain 4″·4-di-O-acetyl-3″-
O-isovaleryltylosin (TLC; Rf _A =
0.73) 0.2g and the desired 4″-O-acetyl-
Obtain 3.2 g of 3″-O-isovaleryltyrosine. TLC; Rf _A = 0.57 Mass; 1041 (M ⁺ ) NMR (100MHz in CDCl ₃ ); 1.39 (3″ CH ₃ ),
1.77 ( _12CH3 ), 2.11 (4″OAc), 2.51 (3′N
(CH ₃ ) ₂ ), 3.44 (2 OCH ₃ ), 3.56 (3
OCH ₃ ), 9.56 (20CHO) ppm Example 10 3.3″-di-O-acetyl-4″-O-butyryltylosin Dissolve 10g of tylosin in 50ml of dry acetone,
Add 8.5 ml of acetic anhydride to this and stir at room temperature for 4 hours. Pour the reaction solution into 200ml of water and add ammonia water PH.
After adjusting to 9.5, extract twice with 200 ml of chloroform. After drying the extract with anhydrous magnesium sulfate,
Dry under reduced pressure to obtain 10.2 g of 2'-O-acetyltylosin.
get. TLC; Rf _B = 0.12, Rf _C = 0.06 (of tylosin
TLC; Rf _A = 0.20, Rf _B = 0.01, Rf _C = 0.01) Dissolve 10 g of the above product in 50 ml of dry acetone,
8.05ml dry pyridine and acetyl chloride
Add 6.4 ml and stir at 45°C for 150 minutes. Pour the reaction solution into 200ml of water, adjust the pH to 9.5 with aqueous ammonia, remove the precipitate, and add 3・2′・4″・4−
8.44 g of tetra-O-acetyltyrosine are obtained. TLC; Rf _B =0.40, Rf _C =0.22 2.0 g of the above product is dissolved in 10 ml of dry pyridine, 1 ml of butyric anhydride is added thereto, and the mixture is heated and stirred at 100° C. for 4 days. Pour the reaction solution into 50 ml of water, adjust the pH to 9.5 with aqueous ammonia, and then extract with 50 ml of chloroform. The extract was washed twice with 50 ml of 0.1N hydrochloric acid and once with dilute aqueous ammonia, dried over anhydrous magnesium sulfate, and dried under reduced pressure to give 3・2′・3″-4-tetra-O-acetyl-4″. A crude product of -O-butyryltylosin is obtained. TLC; Rf _B =0.81, Rf _C =0.66 Dissolve this in 10 ml of methanol, add 10 ml of ammonia-saturated methanol, and stir for 3 hours under ice cooling. Pour the reaction solution into 100ml of water and add 100ml of chloroform.
Extract with The extract is dried over anhydrous magnesium sulfate and then dried under reduced pressure. The residue was dissolved in 50 ml of methanol, heated under reflux for 17 hours, and then dried under reduced pressure to give 3.
A crude product of 3''-di-O-acetyl-4''-O-butyryltylosin is obtained. This was purified by silica gel column chromatography eluting with benzene-acetone (10:1) to obtain 1.2 g of purified product. TLC; Rf _A = 0.73 Example 11 3·3″-di-O-acetyl-4″-O-isovaleryltylosin In Example 10, using isovaleric anhydride instead of butyric anhydride, 3·3″-di-O-acetyl-4″-O-isovaleryltylosin ″-di-O-acetyl-
4″-O-isovaleryltylosin is obtained. TLC; Rf _A =0.76 Example 12 3·3″·4-tri-O-acetyltylosin 3·2′·4″·4- obtained in Example 10 Dissolve 1 g of tetra-O-acetyltylosin in 5 ml of dry pyridine and add 0.5 ml of acetic anhydride.
Heat and stir at 100℃ for 2 days. Pour the reaction solution into 30 ml of water, adjust the pH to 9.5 with aqueous ammonia, and extract with 30 ml of chloroform. Add the extract to 0.1N hydrochloric acid
Wash twice with 30 ml and once with dilute ammonia water, dry over anhydrous magnesium sulfate, and dry under reduced pressure. Dissolve the residue in 10 ml of methanol, add 5 ml of ammonia-saturated methanol, and stir for 3 hours under ice cooling. Pour the reaction solution into 100 ml of water and extract with 100 ml of chloroform. The extract is dried over anhydrous magnesium sulfate and then dried under reduced pressure. Dissolve the residue in 30 ml of methanol,
After heating under reflux for an hour, drying under reduced pressure
A crude product of tri-O-acetyltylosin is obtained. This was purified by silica gel column chromatography eluting with benzene-acetone (7:1) to obtain 700 mg of purified product. TLC; Rf _A = 0.54 Mass; 982 (M ⁺ −59), 922, 753, 563, 391,
372, 174, 169

Claims (1)

[Claims] 1 formula (In the formula, R ₁ is a hydrogen atom or a lower alkanoyl group, one of A ₁ and A ₂ is R ₂ group, the other is R ₃ group, R ₂
and R ₃ represents an alkanoyl group having 2 to 6 carbon atoms, except when R ₁ represents a hydrogen atom, A ₁ represents an acetyl group, and A ₂ represents an isovaleryl group. ) or its salt. 2. The compound or salt thereof according to claim 1, wherein R ₁ is a hydrogen atom. 3. The compound or a salt thereof according to claim 2, wherein A ₁ is an acetyl, propionyl, butyryl or isovaleryl group, and A ₂ is an acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl or hexanoyl group. 4 3″・4″-di-O-acetyltylosin, 3″-
O-acetyl-4″-O-propionyltylosin, 3″-O-acetyl-4″-O-butyryltylosin, 3″-O-acetyl-4″-O-hexanoyltylosin, 3″・4″- Di-O-propionyltylosin, 4″-O-butyryl-3″-O-propionyltylosin, 4″-O-isovaleryl-3″-O-
Propionyltylosin, 3″/4″-di-O-butyryltylosin or 4″-O-acetyl-3″-O
- The compound according to claim 3, which is isovaleryltylosin, or a salt thereof. 5. The compound according to claim 1, wherein R ₁ is a lower alkanoyl group. 6. The compound or salt thereof according to claim 5, wherein the lower alkanoyl group is an acetyl or propionyl group. 7. The compound or a salt thereof according to claim 6, wherein A ₁ is an acetyl, propionyl, butyryl, or isovaleryl group, and A ₂ is an acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, or hexanoyl group. 8 Claim 7 which is 3.3″-di-O-acetyl-4″-O-butyryltylosin or 3.3″-di-O-acetyl-4″-O-isovaleryltylosin Compounds or salts thereof.