JPH0510356B2 - - 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 drug for treating 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.
The drawback was that its use as an infection treatment drug was 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, 23-demycin, Among the 4″-O-acyl derivatives of siloxytylosin and their 3-O-acyl derivatives, nuclear-substituted phenylacetyl groups substituted with an acetyl group, a methoxy group, a methylthio group, a methanesulfonyl group, or a benzoyl group are used. have
The present invention was completed by discovering that 4″-side chain compounds exhibit strong antibacterial activity against not only susceptible bacteria but also drug-resistant bacterial strains, and are stable against decomposition by mouse liver homogenate. Therefore, the present invention provides the following formula In the formula, R represents a hydrogen atom, an acetyl group, or a propionyl group; or a D-mycinose residue (formula), provided that when X represents an acetyl group, Y represents a hydrogen atom. Specific examples of the derivatives include 4″-O-(4-methanesulfonylphenylacetyl)tylosin, 3-O-acetyl-4″-O-(4-methanesulfonylphenylacetyl)tylosin, 3 -O-propionyl-4″-O-(4-methanesulfonylphenylacetyl)tylosin, 4″-O-(2-methanesulfonylphenylacetyl)tylosin, 3-O-acetyl-4″-O-(4 -methoxyphenylacetyl)tylosin, 3-O-propionyl-4''-O-(4-methoxyphenylacetyl)tylosin, 4''-O-(2-methoxyphenylacetyl)tylosin, 3-O-acetyl- 4″-O-(4-methylthiophenyl acetyl)tylosin, 3-O-propionyl-4″-O-(4-methylthiophenyl acetyl)tylosin, 3-O-acetyl-4″-O-(2- methylthiophenyl acetyl) tylosin, 3-O-acetyl-4″-O-(4-benzoylphenylacetyl) tylosin, 4″-O-(2-benzoylphenylacetyl)
Tylosin, 4″-O-(4-benzoylphenylacetyl)-
23-Demaicinosiloxytylosin, 4″-O-(4-acetylphenylacetyl)-23
- Demysinosiloxytylosin, Among the above derivatives, especially tylosin 4″-
Those in which the core substituent of the O-phenylacetyl group is bonded to the 4-position of the phenyl group are preferred from the viewpoint of antibacterial activity against drug-resistant bacterial strains and stability against degradation in mammalian liver homogenates. All of these derivatives exhibit strong antibacterial activity against various pathogenic microorganisms such as Gram-positive bacteria, Gram-negative bacteria, and mycoplasma .
Not only does it have the same activity against drug-resistant strains of P. aureus as it does against susceptible strains, but it also shows stability against degradation by mouse liver homogenate, making it useful as a pharmaceutical, veterinary drug, feed additive, etc. Useful. 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] [Table] [Table] Stability test on mouse liver homogenate ICR mouse liver was homogenized with 5 times the volume of 0.1M phosphate buffer (PH7.2) using a Potter homogenizer (3000 rpm, 10 min). And so. Add 1 ml of analyte 500 μg/ml (10% methanol water) to the supernatant (1 ml), react at 37°C for 1 hour, heat at 100°C for 3 minutes, and then 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 second result is
Shown in the table. [Table] From the above results, the compound of the present invention has high antibacterial activity against macrolide-susceptible bacteria and macrolide-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 (control compound 2). However, the derivative
Despite its high antibacterial activity in vitro, the phenylthioacetyl group at the 4″-O-position is removed by esterases in vivo (especially in the liver).
As 100% hydrolyzed, experimental infection treatment in mice has not yielded satisfactory results.
On the other hand, the -O-phenylacetyl derivative at the 4'' position (control compound 1), which showed good results in the stability experiment using liver homogenate, is generally seen to have low antibacterial activity against resistant bacteria.The above formula () The compound of the present invention represented by is tylosin or 3-O-acyltylosin (see Japanese Patent Publication No. 53-13711, etc.), or demysinosiloxytylosin (hereinafter referred to as YT3927: see Japanese Patent Application Laid-Open No. 57-85399, etc.). ) as the starting material,
For example, after protecting the hydroxyl group at the 3-position and/or 2'-position and, if necessary, the 4-position, by a method known per se as described in JP-A-53-137982,
Produced by acylating the 4″-O-position with a desired acylating agent, and then removing the hydroxyl protecting group at the 3-position and/or 2′-position and, if necessary, the 4-position by partial hydrolysis. Also, more advantageously, tylosin or YT3927
After selectively acetylating the hydroxyl group at the 2'-position (2-position of mycaminose) using acetyl chloride or acetic anhydride, the following formula In the formula, X represents the same meaning as above, and by reacting with a reactive derivative of the acid represented by
4″-O-position and 4 of O-acetyltylosin
in O-position or 4″- of 2′-O-acetyl YT3927
It can be produced by introducing a group [formula] at the O-position and then selectively eliminating the substituents at the 2'-O-position and the 4-O-position. This reaction for introducing the group [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 It can be carried out at reflux temperature, preferably 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 reactive derivatives of such acids used is not strictly limited, but is generally 1 to 50 moles, preferably 2 moles per mole of 2'-O-acetyl tyrosine or 2'-O-acetyl YT3927. It can be used in the range of ~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 used is 2'-O-acetyl tyrosine or 2'-O-acetyl YT3927,
Generally 2 to 50 equivalents per mole, preferably 2 to 30
However, in the case of liquid bases such as pyridine, they can be used in place of solvents by using them in large excess. Thus, the 4″- of 2′-O-acetyltyrosine
A derivative in which a nuclear-substituted phenylacetyl group is introduced at the O-position and the 4-O-position or at the 4″-O-position of 2′-O-acetyl YT3972 can be produced. It can be separated by methods known per se and, after or without separation, is further subjected to the following partial hydrolysis: the 2'-O- position and/or the 4-O position of the compound. Selective removal of the acyl group at the -position uses an organic solvent that is miscible with water and dissolves the compound, and if necessary, after dissolving or suspending the compound in the solvent with water, under reflux. After the acetyl group at the 2'-O-position has been eliminated in advance, the acyl group at the 4-O-position can be eliminated by allowing the reaction solution to cool if necessary and further treating the reaction solution by adding a base. The organic solvent used in this reaction is
Suitable examples include lower alkanols such as methanol and ethanol, and 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 from the viewpoint of selectivity of the elimination reaction and reaction operation, it is best to set the base concentration in the range of 1 to 10% by weight. It's advantageous. This elimination reaction of the 4-O-position acyl group is carried out at a temperature of -10 to 40°C, preferably 0 to 5°C, for about 1
Can be carried out under stirring for ~48 hours. The derivative of the present invention represented by the formula () produced in this way is
It 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 moiety of tylosin is shown as
It is abbreviated as "[expression]". Example 1 Production of 2′-O-acetyl-4-O-chloroacetyl-4″-O-(4-methylsulfonylphenylacetyl)tylosin Paramethylsulfonylphenyl acetic acid 500mg
(2.3 mmol) was dissolved in 5 ml of ethyl acetate and 0.33 ml (2.3 mmol) of triethylamine, and cooled to -15°C.
Add 0.29 ml (2.3 mmol) of pivalic acid chloride to 20
Stir for a minute. Raise the temperature to 0℃ and add pyridine 0.8
ml (10 mmol) and 1.2 g (1.16 mmol) of 2'-O-acetyl-4-O-chloroacetyltylosin were added and stirred for 2 hours. An aqueous sodium bicarbonate solution and 1 ml of methanol were added to the reaction mixture, and after stirring for 20 minutes, the organic layer was separated, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Toluene was added to the residue, and the mixture was again concentrated under reduced pressure to remove pyridine. The residue was subjected to column chromatography using 40 g of silica gel (developing solvent: benzene/acetone (5/1)).
Silica gel developed with benzene/acetone (2/1)
The elution fractions showing coloration with sulfuric acid at an Rf value of 0.50 by TLC were combined and concentrated under reduced pressure to obtain 1.1 g of the title compound.
Yield 80%. NMR (CDCl ₃ ) main peaks are shown below. [Table] Example 2 Production of 4″-O-(4-methylsulfonylphenylacetyl)tylosin 1.1 g of 2'-O-acetyl-4-O-chloroacetyl-4''-O-(4-methylsulfonylphenylacetyl)tylosin was dissolved in 30 ml of methanol and stirred at 60°C for 40 hours. The methanol was distilled off under reduced pressure. After evaporation, the residue was subjected to silica gel 25g column chromatography (developing solvent: benzene/acetone (3/1)), and sulfuric acid coloration was shown at an Rf value of 0.23 on silica gel TLC developed with benzene/acetone (3/2). The fractions were combined and concentrated under reduced pressure. The white powder residue was washed with 2 ml of isopropyl ether to obtain 730 mg of the title compound. Yield 71%. mp: 121-126°C [α] _D : -40.7° (c 1.0 , CH ₃ OH) UV: λ ^CH3OH _nax 283.5nm (ε 20000) 223.5nm (ε 14000) IR: ν ^KBr _nax cm ^-1
1720 (ester, aldehyde) 1675 (conjugated ketone) 1590 (double bond) 1305, 1145 (sulfone) The main NMR (CDCl ₃ ) peaks are shown below. [Table] Example 3 4″-O-(4-methylthiophenyl acetyl)
Manufacture of tylosin Paramethylthiophenyl acetic acid 420mg (2.3mmol)
5 ml of ethyl acetate, 0.33 ml of triethylamine
(2.3 mmol), 0.29 ml (2.3 mmol) of pivalic acid chloride was added to the mixture under cooling at -15°C, and the mixture was stirred for 15 minutes. 0.8 ml of pyridine and 0.8 g (0.84 mmol) of 2'-O-acetyltyrosine were added, and the mixture was stirred at room temperature for 20 hours. The reaction solution was washed with an aqueous sodium hydrogen carbonate solution and an aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. Toluene was added to the residue and concentrated under reduced pressure again to remove pyridine, and 2'-O-
A crude powder of acetyl-4'',4-di-O-(4-methylthiophenylacetyl)tylosin was obtained.
Add this to 40 ml of 9%-ammonia methanol and 5 ml of water.
The mixture was dissolved in water and allowed to stand at room temperature for 6 hours. After adding benzene, methanol was distilled off under reduced pressure.
Benzene was added to the residue, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was precipitated from benzene-hexane to give 2′-O-acetyl-4″-O-(4-methylthiophenyl acetyl).
A coarse powder of tylosin was obtained. Add this to methanol 40
ml and stirred and refluxed for 20 hours. After concentrating the reaction solution under reduced pressure, the residue was subjected to column chromatography on 25 g of silica gel (developing solvent benzene/acetone (5/1
~3/1), and the elution fractions showing sulfuric acid coloring at an Rf value of 0.50 were combined by silica gel TLC developed with benzene/acetone (3/2) and concentrated under reduced pressure. The residue was washed with isopropyl ether to obtain 405 mg of the title compound. Yield 45%. mp: 105~108℃ [α] _D : −35.1° (c 1.0, CH ₃ OH) UV: λ ^CH3OH _nax 281nm (ε 19000) 262.5nm (ε 19400) IR:
ν _nax cm ^-1 1720 (ester, aldehyde) 1675 (conjugated ketone) 1590 (double bond) NMR (CDCl ₃ ) main peaks are shown below. [Table] Example 4 Production of 2'-O-acetyl YT3927 YT3927 300mg (0.41mmol) and acetone 3ml,
0.1 ml of acetic anhydride was added, and the mixture was stirred at room temperature overnight.
The reaction solution was adjusted to pH 10 by adding water and aqueous ammonia, extracted with ethyl acetate twice, washed with water, dried over sodium sulfate, and concentrated under reduced pressure. The title compound was obtained quantitatively. Example 5 Production of 2′-O-acetyl-4″-O-(4-acetylphenylacetyl) YT3927 Paraacetylphenyl acetic acid 242 mg (1.36 mmol),
Ethyl acetate 2.5ml, triethylamine 0.19ml
(1.36 mmol) was cooled to −15° C., 0.17 ml (1.36 mmol) of pivalic acid chloride was added thereto, and the mixture was stirred for 30 minutes. Add 0.4 ml of pyridine and 310 mg (0.41 mmol) of 2'-O-acetyl YT3927 and cool on ice.
Stir for hours. 1 ml of methanol and aqueous sodium hydrogen carbonate solution were added and stirred for 30 minutes. The organic layer was separated, washed with an aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Toluene was added again and the mixture was concentrated under reduced pressure to remove pyridine. The residue was subjected to column chromatography using 9 g of silica gel (developing solvent: benzene/acetone (8/1)).
Silica gel developed with benzene/acetone (3/1)
The elution fraction showing sulfuric acid coloring at an Rf value of 0.54 was collected by TLC to obtain 240 mg of the title compound. Yield 63%. Example 6 4″-O-(4-O-acetylphenylacetyl)
Manufacture of YT3927 2'-O-acetyl-4''-O-(4-acetylphenyl acetyl) YT3927 240mg methanol 10
ml and refluxed for 20 hours. After distilling off methanol under reduced pressure, the residue was subjected to column chromatography on 5 g of silica gel (developing solvent: benzene/acetone (4/1)).
The elution fractions showing sulfuric acid coloration at an Rf value of 0.49 were combined and concentrated under reduced pressure using silica gel TLC developed with benzene/acetone (2/1), and the residual powder was washed with isopropyl ether to give 170 mg of the title. The compound was obtained. Yield 74%. mp: 102~104.5℃ [α] _D : −51.6° (c 1.0, CH ₃ OH) UV: λ ^CH3OH _nax 283.5nm (ε 2000) 253nm (ε 19000) IR: ν ^KBr _nax cm ^-1
1720 (ester, aldehyde) 1680 (conjugated ketone) 1595 (double bond) The main NMR (CDCl ₃ ) peaks are shown below. [Table] [Table] Example 7 Production of 2'-O-acetyl-4''-O-(4-benzoylphenylacetyl)-4-O-chloroacetyltylosin Parabenzoyl phenyl acetic acid 2.1g (8.7mmol)
Dissolve in 20 ml of ethyl acetate and add triethylamine.
1.21 ml (8.7 mmol) was added. After cooling to −15℃,
1.07 ml (8.7 mmol) of pivalic acid chloride was added dropwise and stirred for 20 minutes. 3 ml (38 mmol) of pyridine,
3.0 g (2.9 mmol) of 2'-O-acetyl-4-O-chloroacetyltylosin was added, and the mixture was stirred for 2 hours under ice cooling. After the reaction, 1 ml of methanol and aqueous sodium hydrogen carbonate solution were added and stirred for 1 hour. The organic layer was separated, washed with water, and dried over anhydrous sodium sulfate. After concentration under reduced pressure, toluene was added again and concentrated under reduced pressure to remove pyridine. Silica gel 200 to remove residue
column chromatography (benzene/acetone (6/1)) and benzene/acetone (2/1).
The fractions showing coloration with sulfuric acid at an Rf value of 0.56 on developed silica gel TLC were collected and concentrated under reduced pressure to obtain 3.0 g of the title compound. Yield 82%. NMR (CDCl ₃ ) main peaks are shown below. [Table] [Table] Example 8 4″-O-(4-benzoylphenylacetyl)
Manufacture of tylosin Dissolve 3.0 g of 2'-O-acetyl-4''-O-(4-benzoylphenylacetyl)-4-O-chloroacetyltylosin in 60 ml of methanol,
Refluxed for an hour. After methanol was distilled off under reduced pressure, the residue was subjected to column chromatography (benzene/acetone (3/1)) using 70 g of silica gel.
Rf value by silica gel TLC developed with acetone (3/2)
The fraction showing sulfuric acid coloration at 0.26 was concentrated under reduced pressure, and the residual solid was washed with isopropyl ether to obtain 1.8 g of the title compound. Yield 66%. mp107.5−109.5℃ [α] _D : −36.2° (c 1.0, CH ₃ OH) UV: λ ^CH3OH _nax 272nm (ε 22000) IR: ν ^KBr _nax cm ^-1
1720 (ester, aldehyde) 1670 (conjugated ketone) 1650 (benzoyl) 1590 (double bond) NMR (CDCl ₃ ) main peaks are shown below. [Table] [Table] Example 9 2'-O-acetyl-4-chloroacetyl-
Production of 4″-O-(4-methoxyphenylacetyl)tylosin NMR (CDCl ₃ ) main peaks are shown below. [Table] [Table] Example 10 Production of 4″-O-(4-methoxyphenylacetyl)tylosin mp: 110~111℃ [α] ²⁴ _D : −43.6° (c 1.0, CH ₃ OH) UV: λ ^CH3OH _nax 284nm (ε 19000) 227nm (ε 8700) IR: ν ^KBr _nax
1725cm ^-1 (ester, aldehyde) 1675cm ^-1 (conjugated ketone) 1590cm ^-1 (double bond) The main NMR (CDCl ₃ ) peaks are shown below. [Table] [Table]

Claims (1)

[Claims] 1 formula In the formula, R represents a hydrogen atom, an acetyl group, or a propionyl group; or a D-mycinose residue (formula), provided that when X represents an acetyl group, Y represents a hydrogen atom. 2. The tylosin derivative according to claim 1, wherein R in formula () is a hydrogen atom. 3. The tylosin derivative according to claim 1 or 2, wherein Y in formula () is a D-mycinose residue. 4 Claims 1 and 2 in which X in formula () is bonded to the 4-position of the phenylacetyl group
The tylosin derivative according to item 1 or 3.