JPS6043356B2 - Kanamycin C derivatives effective against resistant bacteria and their production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is novel and widely effective against bacteria resistant to aminoglycoside antibiotics, and is a namycin C derivative having the following general formula (1) [
1-N-(L-4-amino-2-hydroxybutyryl)-kanamycin C (in the formula, R1 and R2 each represent a hydroxyl group) , 1-N-(L-amino-2-hydroxybutyryl)-3″-deoxykanamycin C (where R1 is a hydrogen atom and R2 is a hydroxyl group) and 1-N-(L-4-amino-2- Hydroxybutyryl)-3″●4″-dideoxykanamycin C
(When R1 and R2 both represent hydrogen atoms in the formula)
The present invention also relates to a method for producing 1-N-(L-4-amino-2-hydroxybutyryl)kanamycin C or a deoxy derivative thereof from kanamycin C or a deoxy derivative thereof.

The present inventors have proceeded with their research based on the knowledge obtained from the study of the resistance mechanism of amino acid glycoside antibiotics by various inactivating enzymes, which have been clarified by the present inventors.
Namycin B is effective against resistant bacteria that produce monophosphotransferase.
various deokine derivatives (e.g. Japanese Patent Publication No. 50-7595
No., Patent No. 7946P) was synthesized [Hamao Umezawa: Advances in Carbohydrate Chemistry●
and Biochemistry, Atsushi Volume, 183 pages (197
Sho) and drag●action●and drag・
Resistance●in●Bacteria, Volume 2, Page 211 (1
97 Kui)].

In particular, 3'',4''-dideoxykanamycin B has already been widely used to treat infections caused by various resistant bacteria including Pseudomonas aeruginosa. However, the growth of resistant bacteria that produce these deoxy derivatives of kanamycin B, 6''-acetyl transfer derivatives, and 7-nucleotidyl transfer enzymes cannot be prevented. Therefore, the present inventors have conducted research to develop 6''-acetyl transfer derivatives of kanamycin B or its deoxy derivatives. By substituting the amino group at the `` position with a hydroxyl group, we succeeded in synthesizing kanamycin C and its deoxy derivatives which are essentially not inactivated by 6''-acetyl transferase (Japanese Patent Application No. 51-6975 Wani). Subsequently, we conducted research on the synthesis of derivatives that are effective against bacteria resistant to 2''-nucleotidyl transferase, for which kanamycin C and its deoxy derivatives are ineffective, and broadly inhibit bacteria resistant to aminoglycoside antibiotics. 1 described in the present invention
The present invention was completed by accomplishing the synthesis of -N-(L-4-amino-2-hydroxybutyryl)kanamycin C and its deoxy derivative. In other words, the object of the present invention is to eliminate the enzymes that are essentially inactive not only by 3'' monophosphotransferase, which is widely distributed in bacteria resistant to aminoglycoside antibiotics, but also by 6''-acetyltransferase and 2''-nucleotidyltransferase. Our objective is to provide a novel namycin C derivative with extremely low toxicity that is effective against resistant bacteria without activation.1-N-(L-4-amino-2-hydroxybutyryl) of kanamycin A or kanamycin B. Derivatives have already been completed by Hiroshi Kawaguchi et al.
However, these derivatives are essentially different from the kanamycin C derivatives according to the present invention in that they cannot inhibit 6''-acetyltransferase-resistant bacteria.

Furthermore, in the production method described in the above-mentioned Japanese Patent Application Laid-Open No. 2003-100003, the starting material, kanamycin A or kanamycin B, has its 6"-amino group protected in advance with a known amino protecting group, and then the L-4-amino-2 - In the production of the present invention,
The amino group of kanamycin C or its deoxy derivative as a starting material is significantly different in that it is reacted directly with the reactive derivative of L-4-amino-2-hydroxybutyric acid without protection. The first gist of the present invention is to provide kanamycin C or its deoxy derivative, that is, kanamycin C (in the formula When R1 and R2 both represent a hydroxyl group), 3''-deoxykanamycin C (where R1 is a hydrogen atom and R2 represents a hydroxyl group) or 3''-4-dideoxykanamycin C (when R1 and R2 both represent a hydroxyl group) ) are used as starting materials, their 1-position amino group is acylated with an amino-protected form of L-4-amino-2-hydroxybutyric acid, and the acyl group of the acylated product is further amino-protected. By removing the group, the above general formula (
A compound of the general formula (1) (wherein R1 and R2 are as defined above) is produced, and if desired, is reacted with a conventional acid to form an acid addition salt. (having the same meaning) is a method for producing kanamycin C derivatives or acid addition salts thereof.

Next, the method of the present invention will be described in detail.

Kanamycin C shown by the general formula (■) used as a starting material (where R1 and R2 both represent a hydroxyl group), 3''-deoxykanamycin C (where R1 represents a hydrogen atom and R2 represents a hydroxyl group) ) or 3'',4''-dideoxykanamycin C (in the formula, R1 and R2 both represent hydrogen atoms), not only the free base but also an appropriate acid addition salt can be used. After adjusting the pH to 6 to 8, preferably 6.5 to 7.0 using a common acid or alkali such as hydrochloric acid, sulfuric acid, sodium hydroxide, or potassium hydroxide, L-4 with protected amino groups is prepared. The amino group at position 1 of the starting material was acylated by adding a solution of -amino-2-hydroxybutyric acid or a reactive derivative acting equivalently thereto. As the amino protecting group, a known amino protecting group commonly used in conventional peptide synthesis is used, but a reaction is performed to remove the amino protecting group from the acyl group of the acylated product obtained in this acylation reaction. It must be possible to easily eliminate it under reaction conditions or operations that do not substantially break the amide bond between L-4-amino-2-hydroxybutyric acid and the amino group at position 1 of kanamycin C or its deoxy derivative. .

That is, monovalent amino protecting groups that can be used for this purpose include alkyloxycarbonyl groups such as tert-butyloxycarbonyl group and tert-amyloxycarbonyl group, cycloalkyloxycarbonyl groups such as cyclohexyloxycarbonyl group, and benzyloxycarbonyl group. and aralkylcarbonyl groups such as para-methoxybenzyloxycarbonyl groups, and as divalent amino protecting groups, Schiff bases such as salicylidene groups are preferably used.
The reaction of acylating the amino group at the 1-position of kanamycin C of the general formula (■) or its deoxy derivative with L-4-amino-2-hydroxybutyric acid with the amino group protected can be performed using the dicyclohexylcarbodiimide method or the mixed acid anhydride method. Any known amide synthesis method such as , azide method or active ester method can be carried out using L-4-amino-2-hydroxybutyric acid in the form of its reactive derivative. However, since kanamycin C or its derivative, which is difficult to dissolve in organic solvents other than water, is used as a starting material without a protective group, it is preferable to use an active ester method in a water-containing solvent as the acylation method. For example, as an active ester obtained by a conventional method, L-4-tert-butyloxycarbonylamino-2-
Using N-hydroxysuccinimide of hydroxybutyric acid, 1 to 2 moles of it are dissolved in a water-miscible solvent, preferably dimethoxyethane, dimethylformamide, etc., and the pH is adjusted to 6 to 8. It was added to an aqueous solution under stirring at room temperature and allowed to react.
The reaction time is several hours, preferably 5 to 6 hours. The acylation reaction product thus produced was treated without purification in an aqueous solution of trifluoroacetic acid, acetic acid, or a dilute solution of hydrochloric acid to remove the tert-butyloxycarbonyl group, which is an amino protecting group.

The method for removing the amino protecting group from the acylation reaction product is carried out by a conventional method. That is, all of the above amino protecting groups are removed by weak acid hydrolysis,
Further, when the protecting group is an aralkyloxycarbonyl group, it can be easily removed by ordinary catalytic reduction. In addition to the 1-N-(L-4-amino-2-hydroxybutyryl derivative of kanamycin C or its deoxy derivative), the product obtained by removing the amino protecting group from the acylation reaction product as described above contains In addition, a positional isomer in which the amino group at the 7 or 3 position is acylated and a terminally reacted kanamycin C or its deoxy derivative coexist.

The target 1-N-acyl derivative is separated from this mixture using a cation exchanger having a carboxyl group as an active group, such as Amperlite CG-50 (manufactured by Rohm & Haas, USA), CM-Sephadex. C-25(
(manufactured by Pharmacia, Sweden), ion exchange chromatography using CM-cellulose, ion exclusion chromatography using strong anion exchange resins such as Dowex 1-X2 (manufactured by Dow Chemical, USA), columns using silica gel, etc. It is efficiently carried out by chromatography, etc. In particular, chromatography using a weak cation exchange resin Amperlite CG-50 (NH4 type), which has a carboxyl group as an active group, and elution with dilute ammonia water is recommended. recovered in pure form. The second gist of the present invention is a novel compound, a kanamycin C derivative represented by the general formula (1) (in the formula, R1 and R2 each represent a hydroxyl group or a hydrogen atom) or an acid addition salt thereof. .

Novel compound 1-N-(L-4-amino-2-hydroxybutyryl)kanamycin Cll-N- obtained by the present invention
(L-4-amino-2-hydroxybutyryl)-3″-
The physicochemical and biological properties of deoxykanamycin C and 1-N-(L-4-amino-2-hydroxybutyryl)-3'',4''-dideoxykanamycin C are as follows.

1-N-(L-4-amino-2-hydroxybutyryl)
Kanamycin C is a white powder with no clear melting point16
Decomposes at 7-180℃, [α]? +91 (C1, water) is shown.

The elemental analysis value is the theoretical value of C22Hl3N5Ol3・11.0 (C43.77%, H7.5l%, Nll.6O%)
matches. Silica gel (Art.
Butanol●
Ethanol ● Chloroform ● 17% ammonia water (4:
5:2:8 volume ratio) as a developing solvent and RfO. l & chloroform ● methanol, 28% ammonia water/water (1:4
:2:1 volume ratio) and RfO. A single spot (ninhydrin coloring) is shown at 19. 1-N-(L-4-amino-2-
hydroxybutyryl)-3″-deoxykanamycin C
is a white powder that does not show a clear melting point and decomposes at 151-160°C and shows [α]r+83 point (C1, water).

The elemental analysis value is C22H43N5Ol. - It corresponds to the theoretical value of H2O (C44.96%, H7.72%, Nll.92%). In silica gel thin layer chromatography, RfO. 22, the latter with RfO. 24
shows a single spot. 1-N-(L-4-amino-2
-Hydroxybutyryl)-3''/4''-dideoxykanamycin C is a white powder with no clear melting point and 142~
It decomposes at 158°C and shows [α] 8+76 (C1, water).

The elemental analysis values match the theoretical values of C22H48N5Oll.H2O (C46.22%, 7.94%, Nl 2.25%). In silica gel thin layer chromatography, RfO. 3l, the latter with RfO. A single spot is shown at 3O. The structures of these kanamycin C derivatives were confirmed by proton and carbon nuclear magnetic resonance and acid hydrolysis, respectively. 1-N-(L-4-amino-2-hydroxybutyryl)kanamycin C (AlIB
-KC) 1-N-(L-4-amino-2-hydroxybutyryl)-3″-deoxykanamycin C (AH
The antibacterial spectra of 1-N-(L-4-amino-2-hydroxybutyryl)-3″●4″-dideoxykanamycin C (abbreviated as AHB-DDKC) are shown in Table 1. It is widely effective against bacteria resistant to aminoglycoside antibiotics.

In addition, the acute toxicity of intravenous injection in mice is extremely low, and none of them die from administration of 400m91k9.
It is used to treat infections caused by various Gram-positive bacteria. According to the present invention, the target kanamycin C derivative of general formula (1) is usually obtained as a free base, a hydrate or a carbonate, but any non-toxic acid addition salt can be obtained by reacting with an acid in a conventional manner. It can be done.

As the acid to be added, inorganic acids such as hydrochloric 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. Next, the method of the present invention will be explained by giving examples.

Example 11 Synthesis of N-(L-4-amino-2-hydroxybutyryl)kanamycin C: Kanamycin C free base (-112H20) 500 mg (1.02 mmol)
was dissolved in 10 ml of water, and 3.2 ml of 1N hydrochloric acid was added to adjust the pH to 6.35.

A solution of 500 mg (1.58 mmol) of N-hydroxysuccinimide of L-4-tert-butyloxycarbonylamino-2-hydroxybutyric acid dissolved in 5 ml of dimethylformamide was added to this solution for 5 minutes under stirring at room temperature. The mixture was added dropwise, and the mixture was further stirred for 6 hours. The generated 1-N-(
After concentrating the reaction solution containing L-4-tert-butyloxycarbonylamino-2-hydroxybutyryl)-kanamycin C to dryness under reduced pressure, 16 mL of 90% trifluoroacetic acid was added and reacted at room temperature for 1 hour to remove the amino protecting group. (tert-butyloxycarbonyl group) was eliminated. The reaction solution was concentrated to dryness under reduced pressure and washed twice with 20 ml of ether to give 1-N-
1.55y of a crude white powder of (L-4-amino-2-hydroxybutyryl)kanamycin C was obtained. This crude product was dissolved in 15 mt of water, and purified with 1N aqueous ammonia.
The target substance was adsorbed by passing through a column (inner diameter 20Tfr!!i) filled with 190ml of Amperlite CG-50 (Nlll type), washed with water (660mt), and then 0.1N ammonia water (1940ml). and 0.4
It was eluted with N ammonia water (1540 ml) and fractionated into 20 ml portions. Fraction 75 of the elution part of 0.1N ammonia water
~106 was collected and concentrated to dryness under reduced pressure, and 199 mg of unreacted kanamycin C was recovered (recovery rate 40%).
.

Fractions 152 to 175 of the eluted portion of 0.4N ammonia water were collected and concentrated to dryness under reduced pressure to obtain white powder 2 containing the target product.
Gained 14m9.

This was suspended in 2 ml of a mixture of chloroform-methanol-17% ammonia water (1:4:2 volume), and silica gel (
A tower filled with 50y (CC-17) manufactured by Mallinckrodt Co., Ltd. (USA)
(inner diameter 14 wm), expanded with the above solution mixture,
Fractionation was carried out by ml. Fractions 86 to 114 were collected and concentrated to dryness under reduced pressure, dissolved in water and adsorbed in a 5 mL column of Amperlite CG-50 (NH4 type) in the same manner as above.
Elution with 0.5N aqueous ammonia gave 49ml of 1-N-(L-4-amino-2-hydroxybutyryl)kanamycin C as a white powder. Yield 8.0%. Example 21-N-
(L-4-amino-2-hydroxybutyryl)-3″-
Synthesis of Deoxykanamycin C: 3″-Deoxykanamycin C Free Base (-112H20) 440i(i).
Dissolve 92 mmol) in 8.8 mL of water, add 1N hydrochloric acid 2
．． 757 nL was added to adjust the pH to 6.6O.

To this solution, 1 powder of a solution of 449 mg (1.42 mmol) of N-hydroxysuccinimide of L-4-tert-butyloxycarbonylamino-2-hydroxybutyric acid dissolved in 4.7 ml of dimethylformamide was added under stirring at room temperature. The mixture was added dropwise over time, and the mixture was further stirred for 6 hours. Generated 1-N
-(L-4-tert-butyloxycarbonylamino-2-
hydroxybutyryl)-3″-deoxykanamycin C
After the reaction solution containing the above was concentrated to dryness under reduced pressure, 11 ml of 90% trifluoroacetic acid was added, and the mixture was allowed to react at room temperature for 1 hour to remove the amino protecting group (tert-butyloxycarbonyl group). The reaction solution was concentrated to dryness under reduced pressure and washed twice with 20 mt of ether to obtain 2.12y of a crude white powder. Dissolve this crude product in 15 ml of water, add 1 part of ammonia water to P.
H7.8, Amperlite CG-50 (NHl type)
The target substance was adsorbed by passing through a column packed with 220 mt (inner diameter 20w0n), and after washing with water (1040 mt), 0.
It was eluted with 2N ammonia water (1340ml) and 0.5N ammonia water (1320ml) and fractionated into 20mt portions. Fractions 71 to 80 of the elution part of 0.2N ammonia water
was collected and concentrated to dryness under reduced pressure to obtain 149 m9 of unreacted 3″
-Deoxykanamycin C was recovered (recovery rate 34%)
).

Fractions 134 to 145 of the eluted portion of 0.5N ammonia water were collected and concentrated to dryness under reduced pressure to obtain white powder 1 containing the target product.
Gained 55m9.

This was suspended in 2 ml of a mixture of chloroform-ethanol-17% aqueous ammonia (1:4:2 volume), and silica gel (
The mixture was poured into a column (inner diameter 1 h) containing 25 g of CC-7), expanded with the above solvent mixture, and fractionated into 3.2 ml portions. Fraction 9
9-140 was collected and concentrated to dryness under reduced pressure, dissolved in water, and adsorbed on a 5 nt column of Amperlite CG-50 (NH4 type) in the same manner as above, and eluted with 0.5N aqueous ammonia to obtain 1-1- N-(L-4-amino-2-hydroxybutyryl)-3″-deoxykanamycin C white powder 46
I got mg. Yield 8.5%. Example 3 1-N-(L-4-amino-2-hydroxybutyryl)
-3″・4″-Synthesis of dideoxykanamycin C: 3″
・4″-dideoxykanamycin C free base (-112
1 (20) 340 mg (4). 74 mmol) to 7.5
Dissolve in ml of water and add 2.45ml of 1N hydrochloric acid to adjust the pH.
The temperature was adjusted to 6.7O.

To this solution, a solution of 373 mg (1.18 mmol) of N-hydroxysuccinimide of L-4-tert-butyloxycarbonylamino-2-hydroxybutyric acid dissolved in 3.75 ml of dimethylformamide was added for 5 minutes under stirring at room temperature. The mixture was added dropwise over a period of time, and the mixture was further stirred for 6 hours. After concentrating this reaction solution to dryness under reduced pressure, 8.5% of 90% trifluoroacetic acid was added.
m1 was added and reacted at room temperature for 1 hour to remove the amino protecting group. The reaction solution was concentrated to dryness under reduced pressure and washed twice with 20 mL of ether to obtain 1.46y of a white powder as a crude product. This crude product was dissolved in 14 mL of water, the pH was adjusted to 7.8 with 1N ammonia water, and the pH was adjusted to 7.8 with 1N ammonia water.
Pass 220 ml of (Hi type) through a packed column (inner diameter 20w!n) to adsorb the target substance, wash with water (1040 ml), then add 0.2N ammonia water (1560 ml) and 0.5
Elute with N ammonia water (1460ml), 20TrL
It was fractionated one by one. Fractions 74 to 93 of the 0.2N ammonia water eluate were collected and concentrated to dryness under reduced pressure to recover 153 mg of unreacted 3''/4''-dideoxykanamycin C (recovery rate 45%).

Fractions 152 to 168 of the eluted portion of 0.5N ammonia water were collected and concentrated to dryness under reduced pressure to obtain white powder 1 containing the target product.
Gained 16m9.

This was suspended in 2 ml of a mixture of chloroform-ethanol-17% ammonia water (1:4:2 volume), and silica gel (
The mixture was placed in a column (inner diameter 1 h) packed with CC-7) 25f, expanded with the above solvent mixture, and fractionated into 3.2 ml portions. Fraction 7
1 to 96 were collected and concentrated to dryness under reduced pressure, and this was dissolved in water to obtain Amperlite CG-50 (NH4 type) in the same manner as above.
The mixture was adsorbed in a 5 ml column and eluted with 0.5N aqueous ammonia to obtain 35 mg of white powder of 1-N-(L-4-amino-2-hydroxybutyryl)-3''●4''-dideoxykanamycin C. Yield 8.3%. Example 4 The following experiment was conducted to examine the relationship between the pH of the acylation reaction and the yield of 1-N-(L-4-amino-2-hydroxybutyryl)kanamycin C.

Kanamycin C free base (-112H20) 50m9 each
(0.10 mmol) was dissolved in 0.5 to 1.0 mL of water, and 1N hydrochloric acid was added appropriately to prepare aqueous solutions of various pH values. A solution of 49 mg (0.15 mmol) of N-hydroxysuccinimide of 2-hydroxybutyric acid dissolved in 0.5 ml of dimethylformamide was added dropwise with stirring at room temperature, and the mixture was stirred for 6 hours. After concentrating the reaction solution to dryness under reduced pressure,
1.2 to 1.4 ml of 90% trifluoroacetic acid was added, reacted at room temperature for 1 hour, concentrated to dryness under reduced pressure, and washed with ether to obtain a crude product. This crude product was chromatographed in the same manner as in Example 1 using a 20 mt column (inner diameter 127 r$L) of Amperlite CG-50 (N1 (type 4)) to remove unreacted pure Namycin C. Collect 1
A crude powder containing -N-(L-4-amino-2-hydroxybutyryl)kanamycin C was obtained. 1- in this coarse powder
N-(L-4-amino-2-hydroxybutyryl)kanamycin C was quantified by measuring the antibacterial activity by a conventional cylinder plate method using Bacillus subtilis PCI2m strain as the test bacterium. The reaction conditions and the yield of kanamycin C in the final reaction are shown below.
Since ".4"-dideoxykanamycin C is a new compound, an example of its synthesis is shown below as a reference example (see Japanese Patent Application No. 51-69759 (Japanese Unexamined Patent Publication No. 52-153942)). In Reference Example 1, BOC represents a tert-butyloxycarbonyl group. Reference Example 1 Synthesis of 3″-deoxykanamycin C: (a) 6″-N-■°C-3″-deoxykanamycin B
Preparation of 3″-deoxykanamycin B2.Oy (4.3
A solution of t-butyl 1S-4,6-dimethylpyrimidol-2-ylthiocarbonate 1.03y (4.7 mmol) dissolved in 40 ml of dioxane was added to a solution of t-butyl 1S-4,6-dimethylpyrimidol-2-ylthiocarbonate (4.7 mmol) dissolved in 40 ml of water, and the mixture was heated at room temperature. After stirring for 2 hours, the reaction solution was concentrated to dryness under reduced pressure.

Dissolve this in 32ml of water and use Amperlite CG-50.
1 (NHi type) was adsorbed in a column filled with 160 mL,
After washing with 800ml of water, 0.1N ammonia water (80ml)
0 ml) and fractionated into 15 ml. Fraction 26-4
2 was collected and concentrated to dryness under reduced pressure to obtain 1.06y of 6-N-BOC-3''-deoxykanamycin B as a white powder.
Yield 44%. Further elute with 0.5N ammonia water and
52 mg of 3''-deoxykanamycin B was recovered. Recovery rate 23%. (b) Preparation of penta-N monoprotector 6''-N-BOC-3''-deoxykanamycin, B2
llm9 (0.37 mmol) was dissolved in 5 ml of methanol, 2.5 ml of acetic anhydride was added, and the mixture was reacted at room temperature for 5 hours. Water was added to the reaction solution and the mixture was concentrated to dryness under reduced pressure.
mg of 6″-N-BOC-tetra N-acetyl 3″
-Deoxykanamycin B powder! Obtained.

(c) Powder 23 of penta-N monoprotected form obtained in the pre-preparation step (part) of 6″-amino form
Dissolve 5 mg in 2 ml of 90% trifluoroacetic acid aqueous solution,
Leave the solution at room temperature to remove the BOC group. The residue was concentrated to dryness under reduced pressure and washed with about 2 ml of ether to obtain 227 Tng of Tetra N-acetyl compound, ie, a white powder of 1,3,2'',3''-Tetra N-acetyl-3''-deoxykanamycin B.

(d) Preparation of 6″-alcohol form and removal of protecting group 193 m9 of powder of 6″-amino form obtained in step (c) was dissolved in 3.27711 ml of a 33% aqueous acetic acid solution, and nitrite was added under stirring under ice cooling. Aqueous soda solution (265 mg/3.2 ml) and 1.6 mt of acetic acid were added, and the mixture was further stirred for 1 hour under ice cooling and at room temperature for 16 hours. During this reaction, the amino group at the 6″ position was converted to a hydroxyl group.The reaction solution was once concentrated to dryness under reduced pressure to form a solid (240
mg) was obtained. This solid containing 1,3,7,3''-tetra-N-acetyl-3''-deoxykanamycin C was dissolved in 4 mL of caustic soda and heated under reflux for 7 hours to remove the acetyl group.

Add 200ml of water to the reaction solution, and add Amperlite CG.
-50 (70% NH4 type) was adsorbed in a column (inner diameter 1.6 cm) packed with 50 ml, and after washing with water (250 ml), 0
．． It was eluted with 5N ammonia water and fractionated into 10ml volumes.

Fractions 5 and 59 were combined and concentrated to dryness under reduced pressure.
- A crude powder of deoxykanamycin C was obtained. Dissolve this coarse powder in 2 mL of water and use Amperlite CG-50 again.
(NH4 type) 10mt column (inner diameter 0.75C77) was adsorbed, washed with water (30m1), 0.1N (45m1)
, 0.2N (457rL1) of ammonia water and fractionated into Lml. Fractions 78 to 91 were combined and concentrated to dryness under reduced pressure, and purified powder 5 of 3″-deoxykanamycin C was obtained.
47 rLg (0.11 mmol) was obtained. Yield 45%.
Example 2 Synthesis of 3″-4″-dideoxykanamycin C: a) 6
Preparation of ``-N-benzyloxycarbonyl-3'', 4''-dideoxykanamycin B 3.53y (30 mmol) of 3'', 4''-dideoxykanamycin B was added to 135 m
The mixture was dissolved in ice, and 5.61 da (33 mmol) of benzyloxycarbonyl chloride was added dropwise to the mixture under ice cooling over 1 hour while stirring.

After the addition, the mixture was further stirred at room temperature for 1 hour, the resulting precipitate was removed, and the resulting liquid was washed with 135 ml of ether. The aqueous layer was neutralized with aqueous ammonia and concentrated under reduced pressure, and the concentrated solution was passed through and adsorbed in a 480 ml column of Amperlite CG-50 (NHl type), washed with water (1920 ml), and eluted with 0.1N aqueous ammonia. Throw away the first 960m1, and then move on to the next 78m1.
0mt was collected and concentrated to dryness under reduced pressure to obtain 5.43y of white powder of 6''-N-benzyloxycarbonyl-3''●4''-dideoxykanamycin B. Yield 31%.
．． After elution with 5N ammonia water, 2.7 g of 3''/4''-dideoxykanamycin B was recovered. Recovery rate 20%
. b) Preparation of penta-N monoprotector 6″-N-benzyloxycarbonyl-3″●4″-dideoxykanamycin Bl.59 (2.72 mmol)
160 ml of acetic anhydride and 16 y of sodium acetate were added to the solution, and after heating and refluxing (110°C) for 2 hours, it was concentrated to dryness under reduced pressure to give a solution of approx.
Extracted with ml of acetone, and the extract was concentrated to dryness under reduced pressure (
2.5y).

This was dissolved in 10 ml of chloroform, and silica gel (
A column (inner diameter 2.6C77) filled with 150f of Wako Pure Chemical Wako Gel C-200 was poured into a column (inner diameter 2.6C77),
1) followed by chloroform-methanol (30
: 1 volume) 900m11 Chloroform-methanol (15
: 1 volume) 900 m11 Chloroform-methanol (1 volume)
:1 volume) and fractionated into approximately 14 ml. Fraction 9
1 to 149 were combined and concentrated to dryness under reduced pressure to give 1.80y of 6″-
N-benzyloxycarbonyltetra N-acetyl-
A white powder of Tetra 0-acetyl-3''/4''-dideoxykanamycin B was obtained. (c) Preparation of 6″-amino compound 1.18y of the white powder obtained in the previous step (portion) was dissolved in a mixture of 20ml of methanol and 5ml of water, 1.61y of 5% palladium-barium carbonate was added, hydrogen gas, Flowing 4
Intermediate catalytic reduction (elimination of benzyloxycarbonyl group)
After removing the catalyst, it was concentrated to dryness under reduced pressure to obtain Tetra N-
Acetyl Tetra 0-acetyl form, i.e. 1,3,
2″・3″-Tetra N-acetyl 5・2″″・4″
942 m9 of white powder of ".6""-tetra,0-acetyl-3".4""-dideoxykanamycin B was obtained.

(d) The white powder (942 m9) obtained in step (c) before the removal of 6″-alcohol product and protection was dissolved in 16 ml of a 33% acetic acid aqueous solution, and the mixture was stirred under ice cooling with a sodium nitrite aqueous solution (1. 24y/16ml) and 8ml of acetic acid,
The mixture was further stirred for 1 hour under ice cooling and for 3 hours at room temperature.

Claims (1)

[Claims] 1. A kanamycin C derivative or an acid addition salt thereof represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (in the formula, R_1 and R_2 each represent a hydroxyl group or a hydrogen atom). 2 1-N-(L-4-amino-2-hydroxybutyryl) kanamycin C (when both R_1 and R_2 are hydroxyl groups), the kanamycin C derivative according to claim 1. 3. The kanamycin according to claim 1, which is 1-N-(L-4-amino-2-hydroxybutyryl)-3'-deoxykanamycin C (when R_1 is a hydrogen atom and R_2 is a hydroxyl group) C derivative. 4 1-N-(L-4-amino-2-hydroxy▲Mathematical formulas, chemical formulas, tables, etc.▼(I) Butyryl)-3',4'-dideoxykanamycin C (
The kanamycin C derivative according to claim 1, wherein R_1 and R_2 are both hydrogen atoms. 5. The kanamycin C derivative according to claim 2, which is an acid addition salt of 1-N-(L-4-amino-2-hydroxybutyryl)kanamycin C. 6. The kanamycin C derivative according to claim 3, which is an acid addition salt of 1-N-(L-4-amino-2-hydroxybutyryl)-3'-deoxykanamycin C. 7. The kanamycin C derivative according to claim 4, which is an acid addition salt of 1-N-(L-4-amino-2-hydroxybutyryl)-3',4'-dideoxykanamycin C. 8 General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R_1 and R_2 each represent a hydroxyl group or a hydrogen atom). -2-Hydroxybutyric acid is acylated by reacting with an amino-protected form or a reaction derivative thereof, and then the amino-protecting group of the acyl group of the acylated product is removed to form the following general formula (I). The following formula ▲ includes mathematical formulas, chemical formulas, tables, etc. ▼ (in which R_1 and R_2 has the same meaning as above)) or an acid addition salt thereof. 9 Kanamycin C or Kanamycin C derivative and L-
9. The production method according to claim 8, wherein the reaction derivative of 4-amino-2-hydroxybutyric acid is reacted in an aqueous solution at a pH in the range of 6 to 8.