JPH0510355B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a new derivative of the macrolide antibiotic tylosin, and particularly to a new tylosin derivative produced by a chemical method. [Prior Art] Tylosin belongs to the oldest class of macrolide antibiotics, and is widely used as a treatment for animal infections and as a feed additive. In recent years, various derivatives have been proposed using chemical or biological conversion methods in order to enhance their antibacterial activity and, at the same time, their ability to be absorbed and excreted in vivo. Those belonging to the former include, in particular, various acyl derivatives of the hydroxyl group at the 4'' position of tylosin, such as those disclosed in JP-A-53-137982, JP-A-54-66692, JP-A-54-70291, etc. For example, Japanese Patent Publication No. 55-23272 can be mentioned. [Problems to be Solved by the Invention] All of the above-mentioned known tylosin derivatives are more susceptible to various pathogenic bacteria in in vitro tests than tylosin. However, drugs with high antibacterial activity have poor in vivo stability (for example, they are easily absorbed by mammalian liver homogenates). On the other hand, those with improved in-vivo stability have low antibacterial activity against certain clinically isolated drug-resistant bacteria.
It has the disadvantage that its use as a therapeutic agent for infection is limited. Therefore, the present invention provides a new tylosin derivative that exhibits strong antibacterial activity against the drug-resistant bacteria and is stable against degradation in mouse liver homogenate. [Means for Solving the Problems] As a result of intensive research into the production of various derivatives of tylosin and the correlation between their antibacterial activity against drug-resistant bacterial strains, the present inventors found that tylosin and its third derivative - Among the 4″0-acyl derivatives, 4″-side chain compounds having an acyl group, a nuclear-substituted pheniaacetyl group substituted by a fuso atom, and a benzylsulfonyl group are effective against susceptible bacteria as well as The present invention was completed based on the discovery that it exhibits strong antibacterial activity even against drug-resistant bacterial strains and is stable against degradation by mouse liver homogenate. Therefore, the present invention, the following formula In the formula, R represents a hydrogen atom, an acetyl group or a propiniol group, Y represents a group -CO- or _-SO2- , and Z represents a fuso atom or an acetyl group bonded to the 2 or 4 position of the benzyl group. The present invention provides a tylosin derivative represented by: Specific examples of such derivatives include those in which the hydrogen atom of the trihydroxyl group in the aglycone moiety of tylosin is replaced with an acetyl group or a propiniol group.
Acetylphenylacetyl group, 2-acetylphenylacetyl group, 4-fluorophenylacetyl group, 2-fluorophenylacetyl group, 4-acetylbenzylsulfonyl group, 2-acetylbenzylsulfonyl group, 4-fluorobenzylsulfonyl group and tylosin derivatives substituted with a 2-fluorobenzylsulfonyl group. These derivatives exhibit strong antibacterial activity against various pathogenic microorganisms such as Gram-positive bacteria, Gram-negative bacteria, and mycoplasma, and are especially effective against drug-resistant strains of Staphylococcus aureus. It not only has similar activity against susceptible bacteria, but also shows stability against decomposition by mouse liver homogenate, making it useful as a medicine, veterinary drug, feed additive, etc. Its activity is demonstrated by the following test. Antibacterial activity Table 1 below shows the results measured by the tube dilution method using Brain Heart Infusion Broth (PH7.5) as a medium.

【table】

[Table] Stability test on mouse liver homogenate The liver of an ICR mouse was homogenized with a 5-fold volume of 0.1M phosphate buffer (PH7.2) using a Potter homogenizer (3000 rpm, 10 min). After adding 1 ml of analyte 500 μg/ml (10% methanol water) to the supernatant (1 ml) and reacting at 37°C for 1 hour,
After heating at 100℃ for 3 minutes, add 0.1M phosphate buffer (PH9.0)
Add 1 ml and extract with 1 ml of ethyl acetate. Silica gel thin layer chromatography of this organic layer (chloroform/methanol/ammonia = 15/1.2/
0.1), the production ratio of the unchanged substance and the hydrolyzed product was determined using a chromatoscanner (283 nm), and the production of the hydrolyzate was expressed as a percentage. The result is the second
Shown in the table.

[Table] From the above results, the compound of the present invention has high antibacterial activity against macrolide-susceptible strains and its resistant bacteria, and has high stability in a hydrolysis test using mammalian liver homogenate. , can be an excellent infectious disease treatment. Note that previous proposals by the present inventors (for example,
53-137982), as the most effective tylosin derivative against macrolide-resistant bacteria,
4″-O-phenylthioacetyltylosin is mentioned.However, although this derivative has high antibacterial activity in vitro, 4″-O-
In infection treatment experiments in mice, the phenylthioacetyl group at the − position was 100% hydrolyzed.
Satisfactory results have not been obtained. On the other hand, it can be seen that the phenylacetyl derivatives at the 4''-O-position, which show good results in the stability test using liver homogenate, generally have low antibacterial activity against resistant bacteria.The compound represented by the above formula () is , tylosin or 3-O-acyl tylosin (Special Publication No. 1983-
13711, etc.) as a starting material, the hydroxyl groups at the 3-position, 2'-position and/or 4-position are protected by a method known per se, for example, as described in the above-mentioned JP-A-53-137982, and then 4 It can be produced by acylating the ``-O--position with a desired acylating agent, and then removing the hydroxyl-protecting groups at the 3-, 2'-, and/or 4-positions by partial hydrolysis. , More advantageously, the following formula is obtained by selectively acetylating the hydroxyl group at the 2'-position of tylosin (2-position of mycaminose) using acetyl chloride or acetic anhydride, etc. In the formula, Y represents a group -CO- or _-SO2- ,
Z represents a fuso atom or acetyl group bonded to the 2- or 4-position of the benzyl group.
4″-O- position and 4- of O-acetyltyrosine
Based on O-position

[Formula] is induced and further by partial hydrolysis, an acetyl group at the 2'-O-position and a group at the 4-O-position.

To make [formula] go away It can be manufactured more easily. This base

The reaction for introducing the formula can be carried out according to a known acylation method. For example, the reaction may be carried out in the absence of a solvent or using a suitable inert solvent such as methylene chloride, chloroform, ethyl acetate, acetone, benzene, toluene, tetrahydrofuran, acetonitrile, etc., generally from about -30°C to refluxing the reaction mixture. temperature,
Preferably, it can be carried out at a temperature of -20 to 60°C. At this time, when tylosin is used as a starting material, it is desirable to carry out the reaction at a temperature below room temperature, particularly in order to avoid the by-product of O-acylated product at the 3-position of tylosin. In the above reaction, reactive derivatives of the acid of formula () used as acylating agents include halides (especially chlorides), acid anhydrides or mixed acid anhydrides (for example, combinations of acids of formula () and pivalic acid). anhydrous). The amount of the reactive derivative of such acid used is not strictly limited, but is generally from 1 to 1 mole of 2'-O-acetyltyrosine.
It can be used in an amount of 50 mol, preferably 2 to 20 mol. Moreover, the above acylation reaction can be carried out in the presence of an acid binder, if necessary. Acid binders that can be used include, for example, pyridine, collidine, N
- Organic bases such as methylpiperidine, triethylamine, dimethylaniline and the like may be mentioned.
The amount of the base to be used is generally 2 to 50 equivalents, preferably 2 to 30 equivalents, per mole of 2'-O-acetyltyrosine, but in the case of a liquid base such as pyridine, it may be used in large excess. By using it, it can be used as a substitute for a solvent. Thus, the 4″- of 2′-O-acetyltyrosine
groups at O-position and 4-O-position

A compound into which the formula is introduced can be produced. The compound can be separated from the reaction mixture by methods known per se and, after or without separation, further subjected to the following partial hydrolysis. That is, the 2'-O position of the compound
Selective removal of the acyl group at the - and 4-O- positions is carried out by using an organic solvent that is miscible with water and dissolves the compound, and if necessary, dissolving or suspending the compound in water. After that, 2′-O was added in advance under reflux.
After the -position acetyl group is eliminated, the reaction solution is allowed to cool and is further treated by adding a base to eliminate the 4-O-position acyl group. Note that the organic solvents used in this reaction include lower alkanols such as methanol and ethanol;
Preferred examples include ethers such as tetrahydrofuran and dioxane. In addition, as a base to be added after cooling the reaction solution,
Ammonia, methylamine, dimethylamine, etc. are used. The amount of these bases added varies depending on the type of base used and is not critical, but it is advantageous from the viewpoint of selectivity of the elimination reaction and reaction operation to set the base concentration in the range of 1 to 10 weight percent. It is. This elimination reaction of the 4-O-position acyl group is -
At a temperature of 10-40°C, preferably 0-5°C, about 1-40°C
It can be carried out under stirring for 48 hours. The thus produced derivative of the present invention represented by the formula () can be isolated and purified from the reaction solution by various chromatography treatments known per se. Hereinafter, the present invention will be explained in more detail with reference to Examples. In addition, in the examples, the formula The macrocyclic lactone part of

It is abbreviated as [formula]. Example 1 2′-O-acetyl-4″-4-O-di(4-fluorophenylacetyl)tylosin 4-fluorophenyl acetic acid 5.0g (32mmol),
4.5 ml (32 mmol) of triethylamine was dissolved in 50 ml of methylene chloride. After cooling this to -15°C, 4.0 ml (32 mmol) of pivalic acid chloride was added dropwise over 5 minutes, and the mixture was further stirred for 15 minutes. 9 ml of pyridine
(110 mmol) and 2'-O-acetyltylosin
5.0g (5.2mmol) was added and stirred at 5°C for 30 hours. Add sodium hydrogen carbonate aqueous solution to the reaction solution,
The organic layer was washed with an aqueous sodium chloride solution and then dried over anhydrous sodium sulfate. After concentration under reduced pressure, toluene was added again and concentrated under reduced pressure to remove pyridine. The residue was subjected to column chromatography on 150 g of silica gel (benzene/acetone (7/1)), and then chromatographed on silica gel TLC developed with benzene/acetone (3/1).
Concentrate the elution section showing sulfuric acid coloring at an Rf value of 0.47 under reduced pressure.
The residue was washed with hexane to obtain 3.6 g of the title compound as a white powder. (56%)

[Table] Example 2 4″-O-(4-fluorophenylacetyl)tylosin 3.6 g of 2'-O-acetyl-4''-4-O-di(4-fluorophenylacetyl)tylosin was dissolved in 100 ml of methanol and refluxed for 15 hours. The reaction solution was concentrated to 40 ml and diluted to 17% under ice cooling. 60 ml of ammonia/methanol and 8 ml of water were added and stirred at 10°C for 7 hours. After adding 25 ml of benzene, it was concentrated under reduced pressure. Ethyl acetate was added to the residue, water was separated, and then dried over anhydrous sodium sulfate. Concentrated under reduced pressure. The resulting residue was subjected to column chromatography (benzene/acetone (3/1)) using 130 g of silica gel, and the elution section showing sulfuric acid coloring at an Rf value of 0.27 was determined by silica gel TLC developed with benzene/acetone (3/2). The resulting white powder was dissolved in 20 ml of benzene, insoluble materials were filtered off, and the mixture was poured into 150 ml of hexane for precipitation to obtain 1.36 g of the title compound as a white powder.
(44%)

【table】

[Table] Example 3 2'-O-acetyl-4''-O-(4-fluorobenzylsulfonyl)-4-O-chloroacetyltylosin 1.0 g (0.97 mmol) of 2'-O-acetyl-4-O-chloroacetyl tylosin was dissolved in 5 methylene chloride.
ml and 2 ml of pyridine, 350 mg (1.6 mmol) of 4-fluorobenzylsulfonyl chloride was added thereto while cooling to -20°C, and the mixture was stirred for 1 hour.
The reaction solution was transferred to an aqueous sodium hydrogen carbonate solution,
The organic layer was washed with an aqueous sodium chloride solution and then dried over anhydrous sodium sulfate. After concentration under reduced pressure, toluene was added again and concentration under reduced pressure was performed to distill off pyridine.
The residue was subjected to column chromatography on 30 g of silica gel (benzene/acetone (6/1)), and then chromatographed on silica gel TLC developed with benzene/acetone (3/1).
The eluted fraction showing sulfuric acid coloring at an Rf value of 0.48 was concentrated under reduced pressure to obtain 1.0 g of the title compound as a white powder.
(83%) Example 4 4″-O-(4-fluorobenzylsulfonyl)
Tylosin Dissolve 1.0 g of 2'-O-acetyl-4''-O-(4-fluorobenzylsulfonyl)-4-O-chloroacetyltylosin in 20 ml of methanol,
Refluxed for an hour. After concentrating the reaction solution under reduced pressure, silica gel 30
Column chromatography (benzene/acetone (3/1)) with benzene/acetone (2/1)
The eluted fraction showing sulfuric acid coloring at an Rf value of 0.29 was collected by developing silica gel TLC and reprecipitated from benzene/hexane to obtain 492 mg of the title compound as a white powder. (55%)

[Table] 3.50 3H s 2〓OCH ₃

[Table] Example 5 2'-O-acetyl-4''-O-(4-acetylphenylacetyl)-4-O-chloroacetyltylosin 4-acetylphenyl acetic acid 485 mg (2.7 mmol)
7 ml of methylene chloride, 0.38 ml of triethylamine
(2.7 mmol), and 0.33 ml (2.7 mmol) of pivalic acid chloride was added dropwise while cooling to -15°C. After stirring for 15 minutes, 0.8 ml (10 mmol) of pyridine, 2'-O-
Acetyl-4-O-chloroacetyltylosin
900 mg (0.87 mmol) was added and stirred at 7°C for 3 hours. An aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After concentration under reduced pressure, toluene was added again and concentrated under reduced pressure to remove pyridine.The residue was subjected to column chromatography on 30 g of silica gel (benzene/acetone (6/1)) and silica gel developed with benzene/acetone (3/1). The elution fractions showing sulfuric acid coloration with an Rf value of 0.30 were combined by TLC and concentrated under reduced pressure.
700 mg of the title compound was obtained as a white powder. (67
%)

【table】

[Table] Example 6 4″-O-(4-acetylphenylacetyl)tylosin 700 mg of 2'-O-acetyl-4''-O-(4-acetylphenylacetyl)-4-O-chloroacetyltylosin was dissolved in 15 ml of methanol and refluxed for 10 hours. After concentrating the reaction solution, column chromatography on 25 g of silica gel was performed. The eluate fraction showing sulfuric acid coloring at an Rf value of 0.46 was subjected to graphie (chloroform/methanol (40/1)) and silica gel TLC developed with chloroform/methanol (10/1) and concentrated under reduced pressure to obtain a white powder as a residue. was washed with isopropyl ether to obtain 380 mg of the title compound (60%).

【table】

[Table] Example 7 2'-O-acetyl-4''-O-(2-fluorophenylacetyl)-4-O-chloroacetyltylosin 2-fluorophenyl acetic acid 1.0g (6.5mmol)
Dissolved in 15ml of methylene chloride, triethylamine
After adding 0.9ml (6.5mmol), cool to -15℃.
0.8 ml (6.5 mmol) of pivalic acid chloride was added dropwise. After stirring for 20 minutes, 1.8 ml of pyridine, 2'-O
-acetyl-4

Claims (1)

[Claims] 1 formula In the formula, R represents a hydrogen atom, an acetyl group or a propionyl group, Y represents a group -CO- or _-SO2- , and Z represents a fuso atom or an acetyl group bonded to the 2 or 4 position of the benzyl group. A tylosin derivative represented by 2. The tylosin derivative according to claim 1, wherein R in formula () is a hydrogen atom.