JPS631956B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides novel aminoglycoside derivatives, more specifically, the formula (In the formula, R ₁ and R ₂ are the same or different and represent a hydrogen atom or a hydroxyl group, and n represents an integer of 1 to 3. The same applies hereinafter.) The present invention relates to an aminoglycoside derivative or an acid addition salt thereof. The compound () of the present invention has excellent antibacterial activity and is a useful compound as an antibacterial agent. Research to improve antibiotics involves, for example, strengthening their antibacterial activity, expanding their antibacterial spectrum, and sustaining their antibacterial activity in vivo through partial changes in their chemical structures. The present inventors previously obtained derivatives exhibiting excellent antibacterial activity through biological conversion of the partial structure of the antibiotic kanamycin. The compound of the present invention () could be obtained. The compound of the present invention () can be produced by the reaction steps shown in the diagram below. (In the above formula, R ₃ is a hydrogen atom, R ₄ and
R ₅ is a monovalent amino group protecting group, or R ₃ and R ₄
Alternatively, R ₃ and R ₅ together represent a divalent amino protecting group. (The same applies hereinafter) In the above, the protecting group for a monovalent amino group means a protecting group introduced by a reaction between a compound () and a protecting group introducing reagent commonly used in the fields of amino acid and peptide chemistry. group, methoxycarbonyl group, ethoxycarbonyl group, phenacylcarbonyl group, tert-butoxycarbonyl group, benzyloxycarbonyl group, p-
nitrobenzyloxycarbonyl group, etc., and 2
The protective group for the valent amino group is, for example, a phthaloyl group. In addition, as reactive derivatives at the carboxyl group of compound (), p-nitrophenol, N-
Activated esters such as esters with hydroxysuccinimide etc.; Mixed acid anhydrides such as carbonic acid monoesters and lower alkanoic acids; Acid halides such as acid chlorides and acid bromides; Acid anhydrides: obtained by reaction with carbonyldiimidazole. Examples include active amides. Next, each step will be explained in detail. First step: The introduction of a protecting group into the amino group at the 6' position of the compound () is preferably carried out in a solvent such as water, methanol, ethanol, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, dimethylformamide, or a mixed solvent thereof. This can be carried out by a conventional method at around room temperature. Second step: The reaction between compound () and compound () or its reactive derivative in the carboxyl group is preferably carried out in a solvent such as water, methanol, ethanol, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, dimethylformamide, or a mixed solvent thereof. can be carried out at around room temperature. When compound () is used as a free acid, a condensing agent such as N.N'-dicyclohexylcarbodiimide may be used. Third step: Various methods can be used to remove the protecting group of the amino group of the compound () depending on the nature of the protecting group.
Conventional methods used in the fields of amino acid and peptide chemistry can be applied as well as the introduction of protecting groups. For example, when the protecting group is a benzyloxycarbonyl group, a catalyst such as platinum, palladium on carbon, or Raney nickel is used in a solvent such as water, methanol, ethanol, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, or dimethylformamide, or a mixed solvent thereof. , hydrochloric acid, acetic acid, etc., by a conventional catalytic reduction operation. Isolation and purification of the compound () thus produced can be carried out by conventional operations such as column or thin layer chromatography, vacuum concentration, and the like. Acid addition salts of compound () include hydrochloric acid, sulfuric acid,
Examples include salts with acids such as hydrobromic acid, tartaric acid, citric acid, succinic acid, maleic acid, and fumaric acid. The thus obtained compound () of the present invention exhibits superior antibacterial activity compared to the starting material, and is useful in the treatment of various bacterial infections.

【table】

[Table] Compound () used as a raw material in the present invention can be produced by contacting kanamycin A or kanamycin B with a gentamicin-producing strain belonging to the genus Micromonospora or its mutant strain (Japanese Patent Application Laid-Open No. 53−3188). Next, the present invention will be explained in more detail with reference to Examples. Example 1 (a) 3′・4′-dideoxy-4″-C-methyl-3″-
Cupric acetate hydrate [Cu
Add 221 mg of (OAc) 2.5H ₂ O, stir at room temperature for 30 minutes, and then add benzyl p-nitrophenyl _.
336 mg of carbonate was added and stirred overnight at the same temperature. To this reaction solution were added 0.5 ml of concentrated ammonia water and 20 ml of water, and after stirring for 30 minutes, hydrogen sulfide was blown into the solution, and perlite was added and filtered.
After adjusting the pH of the solution to 5 with 1N hydrochloric acid, the tetrahydrofuran was distilled off under reduced pressure, and the remaining aqueous solution was chromatographed on a column containing 70 ml of Amberlite CG-50 (NH ₄ ⁺ type), and the fraction containing the product was collected. and concentrated under reduced pressure to give 6'-N-benzyloxycarbonyl-3',4'-dideoxy-
332 mg of 4″-C-methyl-3″-N-methylkanamycin A or its 4″ epimer was obtained. NMR (D ₂ O) spectrum δ: 1.29 (4″-C- CH ₃ , s, 3H) 2.57 (3″-N- CH ₃ , s, 3H) 7.43 (

[Formula] s, 5H) FD-MS 615 (M・H) ⁺ (b) Dissolve 285 mg of the product in (a) in a mixture of 1.2 ml of water and 3.6 ml of ethylene glycol dimethyl ether, then L-(-)- Add 196 mg of γ-benzyloxycarbonylamino-α-hydroxybutyric acid N-hydroxysuccinimide ester,
Stir overnight at room temperature. After concentrating the reaction solution under reduced pressure, the residue was dissolved in 5.0 ml of 50% dioxane, 0.1 ml of acetic acid was added, and using 10% palladium on carbon,
The protective group was removed by catalytic reduction. After the reaction was completed, the catalyst was removed, concentrated, dissolved in 5 ml of water, and chromatographed on a column containing 40 ml of Amberlite IRC-50 (NH ₄ ⁺ type), eluted with 0.8N ammonia water, and the fraction containing the product was collected. The mixture was collected and concentrated under reduced pressure to obtain 264.4 mg of crude product. 198 mg of the crude product obtained in this way was added to 1 ml of Amberlite CG-50 (NH ₄ ⁺ form).
It was adsorbed onto a cm x 50 cm column. After thoroughly washing the resin with water, it was further washed with 500 ml of 0.05N ammonia water, and concentration gradient fractionation was performed using 1 part of 0.05N ammonia water and 1 part of 0.8N ammonia water, and each fraction (5.5 ml each) was subjected to silica gel thin layer chromatography. Graphie [Silica gel plate: Kieselgel 60F ₂₅₄ , developing solvent: chloroform: methanol: 28% ammonia water (20:
15:8)], and fractions containing the target compound were collected, concentrated under reduced pressure, and then dried under vacuum to obtain 19 mg of a crude product. Add this to silica gel (Wakogel C-200)
Charge a 6 mm x 300 mm column and develop with a solvent of the same composition as used for the above thin layer chromatography, detect each fraction (0.5 ml each) using the above silica gel thin layer chromatography, and concentrate the fraction containing the target compound. The mixture was dried to obtain 16 mg of crude powder. This coarse powder was mixed with Dowex 1×2 (OH-
Charge it to a 6 mm x 200 mm column (type), elute with water, collect the fractions containing the target compound, and freeze-dry to obtain 11 mg of pure 1-N-
[L-(-)-γ-amino-α-hydroxybutyryl]-3′・4′-dideoxy-4″-C-methyl-
A powder of 3''-N-methylkanamycin A or its 4'' epimer was obtained. IR spectrum ν ^KBr _nax cm ^-1 : 820, 1025 1045, 1145 1320, 1375 1475, 1555 1640, 2925 3350, NMR (D ₂ O) spectrum δ: 1.30 (4″-C- CH ₃ , s, 3H) 2.57 (3″-N- CH ₃ , s, 3H) 5.14 (1″ anomeric proton, d, 1H) 5.34 (1′ anomeric proton, d, 1H) FD-MS 582 (M・H) ⁺ Implementation Example 2 (a) Water containing 1.5 g of 4″-C-methyl-3″-N-methylkanamycin A or its 4″ epimer 30
ml and 60 ml of tetrahydrofuran, 600 mg of cupric acetate hydrate was added thereto, and the mixture was stirred at room temperature for 30 minutes, then 1.0 g of benzyl p-nitrophenyl carbonate was added, and the mixture was stirred overnight. Add 60 ml of water and 1 ml of concentrated ammonia water to the reaction solution, stir for 30 minutes, then blow in hydrogen sulfide.
I added perlite. The solution was adjusted to pH5 with 1N hydrochloric acid, concentrated under reduced pressure, and the residue was dissolved in water.
Dissolve Amberlite CG-50 (NH ₄ ⁺
Column chromatography is carried out using a type (type), and fractions containing the product are collected and concentrated under reduced pressure.
6′-N-benzyloxycarbonyl-4″-C-
1.185 g of methyl-3''-N-methylkanamycin A or its 4'' epimer was obtained. NMR (D ₂ O) spectrum δ: 1.28 (4″-C- CH ₃ , s, 3H) 2.52 (3″-N- CH ₃ , s, 3H) 7.41 (

[Formula] s, 5H) FD-MS 647 (M・H) ⁺ (b) Dissolve 1.376 g of the product in (a) in a mixture of 20 ml of water and 20 ml of ethylene glycol dimethyl ether, L-(-)-γ- A solution of 20 ml of ethylene glycol dimethyl ether containing 0.9 g of benzyloxycarbonylamino-α-hydroxybutyric acid N-hydroxysuccinimide ester was added dropwise over 30 minutes and stirred overnight at room temperature. After concentrating the reaction solution under reduced pressure, the residue was dissolved in 50% dioxane 30
ml, 0.5 ml of acetic acid was added, and the protective group was removed by catalytic reduction using 10% palladium on carbon.
After the reaction was completed, the catalyst was removed, the dioxane in the solution was distilled off, and the pH was adjusted to 7, followed by Amberlite.
It was adsorbed onto a 20 mm x 460 mm column of CG-50 (NH ₄ ⁺ type). After thoroughly washing the resin with water and further washing with 500 ml of 0.05N ammonia water, concentration gradient fractionation was performed using 1 part of 0.05N ammonia water and 1 part of 0.8N ammonia water, and the fraction containing the target compound was subjected to silica gel thin layer chromatography. [Silica gel plate; Kieselgel 60F ₂₅₄ ,
Developing solvent; n-butanol: ethanol: chloroform: 28% aqueous ammonia (4:5:2:
8)], and fractions containing the target compound were collected and concentrated to dryness to obtain 271 mg of crude powder. Spread this coarse powder onto 10mm of silica gel (Wakogel C-200).
It was charged to a x1000 mm column, developed and fractionated with a solvent having the same composition as used in the thin layer chromatography described above. The eluate was detected by the silica gel thin layer chromatography described above, and fractions containing the target compound were collected and concentrated to dryness to obtain 251 mg of crude powder. This coarse powder contains a small amount of by-products, so this is used as Amberlite CG-
50 (NH ₄ ⁺ type) on a 10 mm x 500 mm column, perform concentration gradient fractionation using 1 part of water and 1 part of 0.8N ammonia water, and eluate (10 ml of each fraction) was subjected to the silica gel thin layer chromatography described above. The fractions containing the target compound were collected and concentrated to dryness to obtain 242 mg of a single target compound as a white powder on thin layer chromatography. This is 6mm x 170mm of Dowex 1 x 2 (OH ^- type).
After charging the column and eluting with water, it was detected using the silica gel thin layer chromatography described above, and the fractions containing the target compound were collected, concentrated under reduced pressure, and lyophilized to obtain pure 1-N-[L-(-)-γ- 209 mg of white powder of amino-α-hydroxybutyryl]-4″-C-methyl-3″-N-methylkanamycin A or its 4″ epimer was obtained. IR spectrum ν ^KBr _nax cm ⁻¹ : 820, 1025 1050, 1145 1330, 1375 1475, 1545 1565, 1645 2925, 3360 NMR (D ₂ O) spectrum δ: 1.28 (4″-C- CH ₃ , s, 3H) 2.51 (3″-N- CH ₃ , s, 3H) 5.14 (1″ anomeric proton, d, 1H) 5.38 (1′ anomeric proton, d, 1H) FD-MS 614 (M・H) ⁺ Elemental analysis value (C ₂₄ H ₄₇ N ₅ O ₁₃・2H ₂ O) C(%) H(%) N(%) Theoretical value 44.37 7.91 10.78 Experimental value 44.08 7.90 10.48

【table】

[Table] Reference Example 4 Production of epimer of ``-C-methyl-3''-N-methylkanamycin A or its 4'' 5% dextrin, 3.5% defatted soybean flour Essanmeat special grade (trade name), and 0.7% calcium carbonate. 100 ml of the liquid medium (PH7.5) was dispensed into 50 ml flasks and sterilized. Micromonospora sp. A loopful of the -41 strain was inoculated and shaken at 29°C for 48 to 72 hours to obtain a seed culture medium. Separately, 100 ml of main culture medium was prepared in a 500 ml flask, and 1 ml of the above seed mother culture was inoculated into it. The composition of this main culture medium was 5% dextrin, 3.5% defatted soybean flour Ssanmeat special grade, and calcium carbonate.
0.7%, cobalt chloride 0.000025% (PH7.5),
It was used after being sterilized in an autoclave at 120°C for 20 minutes. 24 hours after inoculation, Kanamycin A, which had been sterilized separately, was added at 1000 mcg (titer) per ml of the medium. The culture solution for 600 flasks obtained by shaking culture at 29°C for 120 hours after addition was PHed with 4N hydrochloric acid.
After adjusting to 2.0, the bacterial cells were separated. The pH of the solution was readjusted to 7.0 with 4N sodium hydroxide, and the solution was passed through a column filled with Amberlite IRC-50 (NH ₄ ⁺ type) 4 to adsorb the target compound. The resin was thoroughly washed with water and eluted with 12 parts of 1N aqueous ammonia, and the eluate was concentrated under reduced pressure and dried to obtain a crude eluate. This crude eluate was added to Amberlite CG-50.
(NH ₄ ⁺ form) 1.6 is adsorbed on a column packed with
After washing the resin with water, a concentration gradient elution operation was performed using water 7 and 0.7N ammonia water 7, and each fraction (15 ml each) was subjected to paper disc method and silica gel thin layer chromatography using Escherichia coli K-12 as the test bacteria. E [manufactured by Merck & Co., Ltd., Kieselgel]
60F ₂₅₄ (Product name) Thickness 0.25mm, Developing solvent: Chloroform: Methanol: 28% ammonia water (20:
After developing for 2 hours at 15:8), ninhydrin color development Rf value
0.11]. The target compound fraction is eluted together with the added kanamycin A. This eluate was concentrated to dryness and the target compound obtained was roughly separated using a silica gel column [Wakogel C-200, (trade name)]
Attached to 20mm x 1200mm, developing solvent: chloroform:
Elution fractionation was performed with methanol:28% ammonia water (20:15:8), and detection was performed using silica gel thin layer chromatography in the same manner as above.The fractions containing the target compound were collected, and concentrated to dryness to obtain the crude fraction. 2g was obtained. This fraction still contains Kanamycin A, so in order to remove it, it was again applied to a silica gel column (Wako Gel C-200) 20 mm x 1000 mm, and eluted and fractionated using the same developing solvent as in the above silica gel column chromatography. It was detected using the silica gel thin layer chromatography described above. The eluted target compound was collected and concentrated under reduced pressure, then dried to yield 462 mg of white powder.
I got it. This coarse powder is used as Amberlite CG-50.
(NH ₄ ⁺ type) adsorbed on a 20 mm x 500 mm column and water 1
A concentration gradient elution operation was performed using 1 part of 0.7N ammonia water, and each fraction (15 ml each) was detected by the thin layer chromatography described above. This target compound elution fraction was concentrated to dryness to obtain 388 mg of crude powder. For further purification, 300 mg of this crude powder was charged to a Dowex 1 x 2 (OH ^- type) 8 mm x 300 mm column and eluted with water. The eluted target compound fraction was detected and collected using the above-mentioned silica gel thin layer chromatography, and this was lyophilized to yield 249 mg.
A white powder of pure target compound was obtained. The free base (lyophilized product) of this product showed the following physical and chemical properties. Basic white powder (vacuum dried at 80℃ for 18 hours) Solubility: Extremely soluble in water. It is poorly soluble in methanol and ethanol, and insoluble in organic solvents such as acetone, chloroform, benzene, ethyl acetate, butyl acetate, ether, and n-hexane. Elemental analysis value (as C ₂₀ H ₄₀ N ₄ O ₁₁・H ₂ O) C H N Theoretical value (%) 45.28 7.98 10.56 Experimental value (%) 45.33 7.93 10.39 Melting point: 168-170℃ Optical rotation: [α] ²⁵ _D +163.6゜ (C=0.5, H ₂ O) Ultraviolet absorption spectrum: terminal absorption Infrared absorption spectrum (KBr) absorption maximum (cm ^-1 ) 1045, 1145, 1360, 1450, 1595, 1635, 2910,
3375 NMR spectrum (in heavy water): Characteristic peak 1.38ppm...Tertiary 4''-C-methyl, 2.72ppm...
…3″-N-methyl, 5.13ppm……1″-anomeric proton, 5.28ppm……1′-anomeric proton Mass spectrum: Main ion peaks (m/
e) 110, 125, 126, 128, 130, 145, 146, 162,
163, 174, 189, 190, 191, 192, 205, 233,
245, 246, 264, 275, 306, 334, 352, 362,
388, 446, 513 (M+1) Rf value by thin layer chromatography:

[Table] The above physical and chemical properties, especially typical fragment ion peaks and
Based on the NMR results and the fact that it is derived from kanamycin A, it is recognized to be 4''-C-methyl-3''-N-methylkanamycin A or its 4'' epimer.

Claims (1)

[Claims] 1 formula (In the formula, R ₁ and R ₂ are the same or different and represent a hydrogen atom or a hydroxyl group, and n represents an integer of 1 to 3.) An aminoglycoside derivative or an acid addition salt thereof. 2 1-N-[L-(-)-γ-amino-α-hydroxybutyryl]-3′・4′-dideoxy-4″-C-
The aminoglycoside derivative according to claim 1, which is an epimer of methyl-3''-N-methylkanamycin A or its 4''. 3 1-N-[L-(-)-γ-amino-α-hydroxybutyryl]-4″-C-methyl-3″-N-methylkanamycin A or its 4″ epimer Aminoglycoside derivative according to item 1.