JPH0367076B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to 6'-N-formimidoyl amikacin and 6'-N-acetimidoyl amikacin, which are novel compounds widely effective against resistant bacteria as semisynthetic aminoglycoside antibiotics, and their acid addition salts. It is. The present inventors have already synthesized various kanamycin derivatives including 6'-N-methylamikacin as a compound that inhibits resistant bacteria having 6'-acetyltransferred oxygen (Journal of Antibiotics, Vol. 28, p. 486). , 1975). Furthermore, the present inventors have introduced a formimidoyl group and an acetimidoyl group into the 6'-N-amino group of amikacin, which has been widely used against various resistant bacteria including those with 6'-acetyl transfer oxygen. The present invention was completed by synthesizing an effective new compound. General formula () in the present invention: The physicochemical and biological properties of the new compound represented by the formula [wherein R represents a hydrogen atom or a methyl group] are as follows. (a) Disulfate of 6′-N-formimidoyl amikacin [when R is H in general formula ()]
Hemihydrate is a white powder with a decomposition point of 209-213℃,
[α] ²³ _D +46° (c1.0, water) was shown. The elemental analysis values showed C32.91%, _H5.87 %, _N10.31 %, _S8.01 %, and the theoretical value of _C23H44N6O13・_2H2SO4・₃ / _2H2O (C33 .05%, H6.15%, N10.05%, S7.67
%). m/mass spectrometry (SIMS)
z613 (MH ⁺ ) was shown. Cellulose thin layer chromatography allows propanol, pyridine,
Using a mixture of acetic acid and water (15:10:3:12 volume) as the developing solvent, a single spot (ninhydrin coloring) was observed at Rf0.12. (b) The disulfate dihydrate of 6'-N-acetimidoyl amikacin [when R is CH ₃ in the general formula ()] is a white powder with a decomposition point of 204 to 207.
℃, [α] ²¹ _D +47° (c1.0, water). Elemental analysis values are C33.89%, H6.47%, N10.01%, S7.25
The theoretical values _{of C24H46N6O13 ・ 2H2SO4}・_2H2O (C33.56% _, H6.34%, _N9.79 %, S7.47
%). m/mass spectrometry (SIMS)
z627 (MH ⁺ ) was shown. The cellulose thin layer chromatography described above showed an Rf of 0.12. 6′-N-formimidoyl amikacin disulfate hemihydrate (FI-AK) obtained in the present invention
and 6'-N-acetimidoyl amikacin.
The antibacterial spectrum of disulfate dihydrate (AI-AK) is shown in Table 1 in comparison with that of amikacin sulfate (AK). Minimum inhibitory concentrations (MICs) are expressed as free base equivalents and were measured on Mueller-Hinton agar plates.

【table】

【table】

[Table] Both 6'-N-formimidoyl amikacin and 6'-N-acetimidoyl amikacin in the present invention were tested in acute toxicity tests (14-day observation) by intravenous administration to mice in the form of sulfate. As a result, all mice survived at a dose of 200 mg/Kg (free base equivalent), indicating extremely low toxicity. 6'-N-formimidoyl amikacin and 6'-N-acetimidoyl amikacin in the present invention can be obtained as a free base, a hydrate, or a carbonate, and may be obtained as a pharmaceutically acceptable acid salt by a conventional method. It is more preferred with regard to their stability to form any non-toxic acid addition salts thereof. As the acid to be added, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid, and organic acids such as acetic acid, malic acid, citric acid, ascorbic acid, and methanesulfonic acid are used. General formula () according to the invention: The production of 6'-N-formimidoyl amikacin and 6'-N-acetimidoyl amikacin represented by [wherein R represents a hydrogen atom or a methyl group] starts from kanamycin, and the 6'-position Only the amino group of is protected with a known amino protecting group, and then the amino groups at the 3- and 3″-positions are protected with a known amino protecting group different from the amino protecting group used for the 6′-position amino group, The remaining amino group at position 1 is acylated with 4-amino-2-hydroxybutyric acid with or without protection of the amino group or a reaction derivative thereof, and only the amino protecting group at position 6' is selectively removed. The following general formula (): [In the formula, A is a hydrogen atom and B is a monovalent amino-protecting group, or A and B jointly represent a divalent amino-protecting group] 3,3'' of amikacin ,4-tri-N-protected form, and its
This can be carried out by introducing a formimidoyl group or an acetimidoyl group into the amino group at the 6' position, and then removing the amino protecting groups at the 3, 3'' and 4 positions. A preferred method for producing the compound will be described. As the amino protecting group that protects the amino group of kanamycin, which is the raw material, a normal amino protecting group is used. That is, tert-butoxycarbonyl is used as the monovalent amino protecting group. groups, alkoxycarbonyl groups such as tertiary amyloxycarbonyl groups, cycloalkyloxycarbonyl groups such as cyclohexyloxycarbonyl groups, aralkyloxycarbonyl groups such as benzyloxycarbonyl groups and paramethoxybenzyloxycarbonyl groups, trifluoroacetyl groups, ortho Examples include acyl groups such as nitrophenoxyacetyl groups, which can also be protected in the form of Schiff bases with phthaloyl groups as divalent amino protecting groups, and aldehydes such as salicylaldehyde. A known amino protecting group can be introduced by a method known in peptide synthesis, for example, using an acid halide, an acid azide, an active ester, an acid anhydride, etc. For the protection of the amino group of kanamycin in the present invention. In this case, a formimidoyl group or acetimidoyl group must be introduced at the 6' position, and a 4-amino-2hydroxybutyryl group must be introduced at the amino group at the 1st position, so each amino group is different. It is preferable that the amino protecting group can be removed separately. Therefore, for example, the known substance 6'-N-benzyloxycarbonyl kanamycin (Hiroshi Kawaguchi et al.: Journal of Antibiotics, 25)
Vol., p. 695, 1972), a tert-butoxycarbonyl group was introduced into the amino group at the 3-position, a trifluoroacetyl group was subsequently introduced into the amino group at the 3''-position, and then a trifluoroacetyl group was introduced into the amino group at the 1-position. The amino group at the 4-position was acylated with 4-amino-2-hydroxybutyric acid, and only the amino-protecting group at the 6'-position was selectively removed to obtain the 3,
Obtaining the 3'',4-tri-N-protected form is the most preferred example for producing the compounds of the present invention.
In order to selectively protect the amino group at the 3-position of 6'-N-protected kanamycin, the inventors'
The method of forming a zinc complex described in Japanese Patent Publication No. 55-64598 is effectively used, and the method of selectively protecting the amino group at the 3″ position is also described in
The method of introducing a dihalogenated or trihalogenated alkanoyl group such as a trifluoroacetyl group described in Japanese Patent No. 164696 is preferred. The reaction is to acylate the amino group at position 1 of kanamycin, in which the amino groups at positions 3, 6', and 3'' are protected, with 4-amino-2-hydroxybutyric acid, which has a protected or unprotected amino group. , dicyclohexylcarbodiimide method, mixed acid anhydride method, azide method, active ester method, and any other known peptide synthesis method.
When introducing a formimidoyl group or an acetimidoyl group to the amino group at the 6' position of the 3″,4-tri-N-protected product, the following general formula (): R′O C R ² = Ethylformimidate hydrochloride ^, benzylform Imidate hydrochloride or methylacetimidate hydrochloride is preferably used, and the reaction can be carried out by a known method of reacting in an organic solvent such as dioxane or methanol, or in an aqueous solution.For the introduction of formimidoyl group, Therefore, a reaction temperature of 30°C or lower is preferable, and a high temperature of around 60°C is preferable for the introduction of an acetimidoyl group.Next, the method for producing the compound of the present invention will be explained with reference to Examples.Example 1 3, Synthesis of 3″,4-tri-N-tert-butoxycarbonylamikacin: (a) 2.0 g (3.14 mmol) of 6′-N-benzyloxycarbonyl kanamycin (monohydrate) at 90%
% dimethyl sulfoxide, 3.4 g (15.5 mmol) of zinc acetate [Zn(OCOCH ₃ ) _{2.2H 2} O] was added, and the mixture was stirred at room temperature for 5 hours. To this, tert-butyl S-4,6-dimethylpyrimide-2
A solution of 1.16 g (4.85 mmol) of -ylthiocarbonate dissolved in 2 ml of dimethyl sulfoxide was added, and the mixture was stirred at 50°C for 6 hours. After washing this reaction solution with 50 ml of n-hexane, 300 ml of saturated aqueous sodium chloride solution was added, the pH was adjusted to 11 with 17% aqueous ammonia, and the mixture was extracted twice with 100 ml of butanol. The butanol layer was dehydrated with anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and then purified by column chromatography on silica gel (Wako Gel C-200, 200 g) (developed with chloroform-methanol-concentrated aqueous ammonia, 6:6:1). and 6′−
N-benzyloxycarbonyl-3-N-tert-butoxycarbonyl kanamycin white powder
1.57g was obtained. Yield 70%. (b) Dissolve 750 ml (1.04 mmol) of 6'-N-benzyloxycarbonyl-3-N-tert-butoxycarbonyl kanamycin obtained in the previous section (a) in 10 ml of dimethyl sulfoxide, and add 0.19 ml (1.56 mmol) of ethyl trifluoroacetate. ) and
Stirred at room temperature for 1 hour. After the reaction, 0.22 ml (1.56 mmol) of triethylamine and (S)-4-
A solution of 491 mg (1.56 mmol) of N-hydroxysuccinimide ester of tert-butoxycarbonylamino-2-hydroxybutyric acid dissolved in 1 ml of tetrahydrofuran was added and stirred at room temperature for 4 hours to perform 1-N-acylation. Ta. After the reaction, 100 ml of water was added and the resulting precipitate was collected. The precipitate was dissolved in 10 ml of 90% trifluoroacetic acid and stirred at room temperature for 1 hour to remove the tert-butoxycarbonyl group. The reaction solution was concentrated under reduced pressure, dissolved in 10 ml of water, adjusted to pH 11 with 17% aqueous ammonia, and stirred at room temperature for 15 hours to remove trifluoroacetyl groups.
After adding 50 ml of water to the reaction solution and adjusting the pH to 6.5 with 6N hydrochloric acid, it was passed through a column (inner diameter 12 mm) filled with 20 ml of Amberlite CG-50 (NH ₄ ⁺ ), and 90 ml of water,
Washing with 90 ml of 0.1M ammonia followed by elution with 0.3M ammonia (3 ml fractions). Min.13−25
The mixture was combined and concentrated to dryness to obtain 457 ml of white powder of 6'-N-benzyloxycarbonylamikacin.
Yield 61%. (c) 400 mg (0.556 mmol) of 6'-N-benzyloxycarbonylamikacin obtained in the previous section (b) was dissolved in a mixture of 5 ml of water and 5 ml of methanol, and 0.23 ml (1.67 mmol) of triethylamine and tert-butyl S- A solution of 600 mg (2.50 mmol) of 4,6-dimethylpyrimid-2-ylthiocarbonate dissolved in 5 ml of methanol was added and heated at 60°C for 16 hours.
Stir for hours. After concentrating the reaction solution to dryness and washing with 30 ml of water and n-hexane, silica gel (Wako Gel C-200, 50 g) was added.
column chromatography (chloroform-
6′-N
-benzyloxycarbonyl-3,3″,4-
408 mg of white powder of tri-N-tert-butoxycarbonylamikacin was obtained. Yield 72%. (d) 350 mg (0.343 mmol) of 6′-N-benzyloxycarbonyl-3,3″,4-tri-N-tert-butoxycarbonylamikacin obtained in the previous section (c) was mixed with 30 ml of 90% methanol water and 0.1 acetic acid. ml of the mixture, added 30 mg of 5% palladium-carbon, and stirred at room temperature in a hydrogen stream for 4 hours to perform hydrogenolysis.The reaction solution was concentrated to dryness and subjected to column chromatography on silica gel (Wako Gel C-200, 30 g). Purified with graphie (developed with chloroform-methanol-concentrated ammonia water, 30:10:1),
268 mg of white powder of 3,3″,4-tri-N-tert-butoxycarbonylamikacin was obtained. Yield
88%. Example 2 Synthesis of 6′-N-formimidoyl amikacin: (a) 3,3″,4-tri-tri-amikacin obtained in Example 1(d)
N-tert-butoxycarbonylamikacin 150mg
(0.169 mmol) was dissolved in 20 ml of anhydrous methanol, and 56 mg of ethylformimidate hydrochloride was dissolved in 20 ml of anhydrous methanol.
A solution of (0.507 mmol) dissolved in 1 ml of anhydrous methanol was added dropwise under ice cooling, and the mixture was stirred at room temperature for 16 hours. After concentrating the reaction solution to dryness, extracting with 20 ml of ethyl acetate and concentrating to dryness again.
4-tri-N-tert-butoxycarbonyl-
130 mg of white powder of 6'-N-formimidoyl amikacin hydrochloride was obtained. Yield 81%. (b) 3,3″,4-tri-N- obtained in the previous section (a)
120 mg (0.126 mmol) of tert-butoxycarbonyl-6'-N-formimidoyl amikacin hydrochloride was dissolved in 5 ml of 90% trifluoroacetic acid and stirred for 2 hours under ice cooling. The reaction solution was concentrated to dryness, and water 5
Amberlite IRA-400 in ml
It was passed through a column packed with 10 ml of (SO ₄ ^2- ) (1 ml fraction) and converted to sulfate. Fraction 4-11 was synthesized and lyophilized to obtain 78 mg of white powder of 6'-N-formimidoyl amikacin sulfate. Yield 74
%. Example 3 Synthesis of 6′-N-acetimidoyl amikacin: (a) 3,3″,4-tri-tri-amikacin obtained in Example 1(d)
N-tert-butoxycarbonylamikacin 200mg
(0.226 mmol) was dissolved in 20 ml of anhydrous methanol, and 74 mg of methyl acetimidate hydrochloride was added to this.
A solution of (0.678 mmol) dissolved in 1 ml of anhydrous methanol was added, and the mixture was stirred at 60°C for 3 hours. The reaction solution was concentrated to dryness, and silica gel (Wakogel C-
6'-N-acetimidoyl-3,3'',4 was purified by column chromatography (developed with chloroform-methanol, 2:1) of
112 mg of white powder of -tri-N-tert-butoxycarbonylamikacin hydrochloride was obtained. Yield 51%. (b) Add 100 mg (0.104 mmol) of 6′-N-acetimidoyl-3,3″,4-tri-N-tert-butoxycarbonylamikacin hydrochloride obtained in the previous section (a) to 5 ml of 90% trifluoroacetic acid. The mixture was stirred for 2 hours under ice-cooling.The reaction solution was concentrated to dryness and diluted with water 5.
Amberlite IRA-400 in ml
It was passed through a column packed with 10 ml of (SO ₄ ^2- ) (1 ml fraction) and converted to sulfate. Fractions 3-9 were combined and lyophilized to obtain 69 mg of a white powder of 6'-N-acetimidoyl amikacin sulfate. Yield 77%.

Claims (1)

[Claims] 1 General formula (): 6'-N-formimidoyl and 6'-N-acetimidoyl derivatives of amikacin represented by the formula [wherein R represents a hydrogen atom or a methyl group] and acid addition salts thereof. 2. The compound according to claim 1, which is 6'-N-formimidoyl amikacin [when R is a hydrogen atom in the general formula ()]. 3. The compound according to claim 1, which is 6'-N-acetimidoyl amikacin [when R in the general formula () is a methyl group].