JPS631319B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing an aminoglycoside antibiotic protected derivative in which an amino group or an alkylamino group at a specific position of an aminoglycoside antibiotic is selectively protected with an acyl group. The present invention also relates to a method for selectively protecting amino groups or alkylamino groups at specific positions of aminoglycoside antibiotics. The present invention mainly includes:
It is applied to the production of amino-protected derivatives of aminoglycoside antibiotics consisting of deoxystreptamine having a 3-aminoglycosyl group at the 6-position. Aminoglycoside antibiotics are substances containing a plurality of relatively highly reactive amino groups and hydroxyl groups. A number of semi-synthetic aminoglycoside antibiotics have been produced that are derived from aminoglycoside antibiotics. In this semi-synthetic production, it is often necessary or preferable to selectively protect the amino group or hydroxyl group, or both, at a specific position of the starting aminoglycoside antibiotic in advance with an appropriate protecting group. be done. Conventionally, various methods of protecting hydroxyl groups have been successful in selectively protecting amino groups and/or hydroxyl groups in aminoglycoside antibiotics. It is difficult or requires complicated operations to selectively protect only the amino group or alkylamino group at a specific position because there is not much difference in the reactivity of each amino group or alkylamino group. As seen in the 6'-position amino group of kanamycin A, the amino group or methylamino group bonded to a certain carbon atom is bonded to only one carbon atom in the molecule. Since they are relatively more reactive than amino groups or methylamino groups bonded to a certain carbon atom that are bonded to higher carbon atoms, the former amino groups or alkylamino groups are somewhat preferentially used over the latter. Reacts with a protecting group-introducing agent, and thus a protected derivative in which the amino group or methylamino group of the former is selectively protected (especially in the case of protection with an acyl group) may be obtained in higher yield than other protected derivatives. . In addition, some of the present inventors have recently discovered that when a pair of amino groups and hydroxyl groups in an aminoglycoside antibiotic molecule are adjacent in the three-dimensional structure of the molecule, only they can be combined into a cyclic carbamate. They discovered that the above pair of amino groups and hydroxyl groups could be protected simultaneously without blocking other amino groups (U.S. Patent No.
3925354 and 3965089). Recently, Nagabutsuyan et al.
No. 153944 or US Patent Application SN.697297) discloses 4-0-(aminoglycosyl)-6-0- represented by kanamycin, gentamicin, sisomicin, etc.
For aminoglycoside antibiotics belonging to the (aminoglycosyl)-2-deoxystreptamines, copper (), nickel (), cobalt (),
When a salt of a divalent transition metal (M ⁺⁺ ) selected from the group consisting of cadmium (M++) is allowed to act in an appropriate organic solvent, a specific position in a vicinal relationship in the antibiotic molecule is activated. It has been found that the divalent metal cation is complexly bonded between a pair of amino groups and hydroxyl groups to form a metal salt complex. In this way the amino group is occluded by a divalent metal cation. Therefore, when the complex is subsequently treated with an acylating agent, only the amino groups that are not blocked by the complex bond of the divalent metal cation are acylated, so that selective protection by the acyl group can be obtained. This is kanamycin A.
This will be explained using an example. That is, divalent metal cations (M ⁺⁺ ) of copper (), nickel (), cobalt () or cadmium () to kanamycin A
When acting, As shown in the above formula (), the divalent metal cation (M ⁺⁺ ) interacts with the 1-position amino group of the kanamycin A molecule.
A complex bond forms between the 2″ hydroxyl group and between the 3″ amino group and the 4″ hydroxyl group (therefore, kanamycin A1
per mole metal salt requires a minimum of 2 moles). As a result, the amino groups at the 1st and 3″ positions are blocked simultaneously,
When the complex of formula () is then treated with an acylating agent, only the amino group at the 3-position and the amino group at the 6'-position that are not blocked by the above-mentioned metal cation are acylated. It was confirmed that an -N-acylated product can be obtained. (See Journal of the American Chemical Society, Vol. 100, pp. 5253-5254, 1978). While recognizing the above facts, the present inventors further studied in detail the interactions between various metal cations and aminoglycoside antibiotics such as kanamycin A and kanamycin B, and their derivatives. As a result, the divalent zinc cation behaves differently than the divalent nickel, divalent cobalt, divalent copper, and divalent cadmium cations described above, but as a result, the 3-aminoglycosyl group ( or 3-alkylaminoglycosyl group) to 6
For aminoglycoside compounds having deoxystreptamine as a partial structure (for example, kanamycin A, B, C), the 1-position amino group or alkylamino group and the 3-aminoglycosyl group (or 3-alkylaminoglycosyl It has been discovered that zinc cations strongly block both the 3-amino group (or alkylamino group) of For example, when kanamycin B is reacted with cations such as nickel, cobalt, copper, and cadmium, a metal salt complex as shown in formula () is expected to be formed, according to Nagabutsuyan et al. This is the above-mentioned "Journal of American,"
This is clear from the facts described in "Chemical Society." This is because Kanamycin B has three pairs of amino groups and hydroxyl groups in a vicinal relationship between the 1 and 2'' positions, between the 2' and 3' positions, and between the 3'' and 4'' positions. However, in reality, when kanamycin B interacts with zinc cations, the complex formed is
The space between the 2'-amino group and the 3'-hydroxyl group is not blocked by the zinc cation. Or even if it is blocked, the force is extremely small. Therefore, if the complex of kanamycin B and zinc cation is subsequently acylated with benzyloxycarbonyl group, 3.
A tri-3.2'.6'-N-acylated derivative in which three amino groups at the 2' and 6' positions are acylated is obtained in a relatively high yield. In that case, 3・6′-G-N-
Virtually no acylated derivatives are obtained (Example 19
reference). This fact suggests that zinc cations behave differently from the four metal cations mentioned above. Furthermore, to give an example, kanamycin A
When acylated with a benzyloxycarbonyl group after acting with a zinc cation (see the above formula ()), if the zinc cation is present in a ratio of slightly more than 1 mole to 1 mole of kanamycin A, The fact is that 3,6'-di-N-benzyloxycarbonylkanamycin A is formed as the main acylation product. Moreover, what should be noted at this time is that due to this acylation reaction, the 1,3,6',3''-tetra-N-benzyloxycarbonyl derivative of Kanamycin A and Kanamycin A, which is an unreacted raw material, are simultaneously produced to a certain extent. However, only a very small amount of the tri-N-benzyloxycarbonyl derivative, which was expected to be formed from the reaction mechanism, was formed (see Example 7).
The above-mentioned Nagabushiyan et al. (Japanese Patent Application Laid-Open No. 153944/1986) specifies that the salts of metals such as copper, nickel, and cobalt must be used in an amount of at least 2 molar ratios, as clearly deduced from the formula (). As mentioned in the book's explanation, the zinc cation is different from this and exhibits its blocking effect when the molar ratio is more than 1 molar.In fact, nickel salt acts on kanamycin A in an amount of slightly more than 1 molar. When the complex formed is subsequently acylated with a benzyloxycarbonyl group, 3.
6'-di-N-benzyloxycarbonylkanamycin A is obtained only in a very low yield. (See Example 7). From the above facts, it is recognized that the zinc () cation has a different complex formation mechanism or complex stability strength than the nickel (), cobalt (), copper () and cadmium () cations. Furthermore, zinc cations are supplied in the form of zinc salts, which have the advantage of being inexpensive and unlikely to become a source of pollution. Finally, according to the present inventors, the 3-aminoglycosyl group or the 3-alkylaminoglycosyl group was
When a zinc cation is applied to an aminoglycoside antibiotic (which can also have an aminoglycosyl group at the 4-position) in a suitable solvent, the amino acid at a specific position determined depending on the type of aminoglycoside antibiotic is It was found that the group and the hydroxyl group were simultaneously complexed with the zinc cation and thereby occluded, and the complex thus formed between the aminoglycoside antibiotic and the zinc cation was used in polypeptide synthesis. In order to introduce an acyl group for protecting amino groups, when an acylating agent is reacted, at least one amino group of the aminoglycoside antibiotic that is not blocked by a complex bond with a zinc cation is acylated and protected. Furthermore, when the zinc cation is subsequently removed from the acylated product (complex with zinc cation) by an appropriate means, the amino group that initially does not form a complex with the zinc cation is selectively protected by the acyl group. It has been found that protected derivatives of aminoglycoside antibiotics can be obtained. Therefore, the gist of the present invention is to provide a zinc complex produced by the action of a zinc cation on an aminoglycoside antibiotic consisting of deoxystreptamine with a 3-aminoglycosyl group or a 3-alkylaminoglycosyl group at the 6-position. , reacted with an acylating agent to introduce an acyl protecting group for the amino group,
This method produces a zinc complex of the N-acylated derivative of the aminoglycoside antibiotic complexed with a zinc cation, and then removes the zinc cation from the zinc complex of the N-acylated derivative. The present invention provides a method for producing a protected derivative of the aminoglycoside antibiotic in which a group is selectively protected with an acyl group. Now, the method of the present invention uses aminocasin, whose efficacy as a pharmaceutical has been rapidly proven in recent years (Journal of Antibiotics, Vol. 25, 695-708).
Page, 1972), one of the kanamycins.
In the synthesis of -N-aminoacylated derivatives, it is useful for preparing a protected form in which amino groups other than the amino group at the 1-position of a raw material aminoglycoside antibiotic are protected with an acyl group. These 1-N-aminoacylated derivatives range widely, including kanamycin A, kanamycin B, kanamycin C, gentamicins, sisomicin, and their various deoxy derivatives or N-alkyl derivatives, but the common thing is that the amino acid at the 1-position The group is acylated. (U.S. Patent No. 3781268, No. 3939143, No.
3940382, No. 4001208, etc.). This 1-N-
Acylation imparts antibacterial activity against resistant bacteria that the parent substance lacks, and in many cases, the antibacterial activity against various bacteria is greater than that of the parent substance. Next, implementation of the method of the present invention will be described in detail. In the present invention, aminoglycoside antibiotics that are to be reacted with zinc cations to produce zinc complexes (also referred to as zinc complex salts) have a 3-aminoglycosyl group or a 3-alkylaminoglycosyl group as a substituent on the 6-position OH group. It consists of deoxystreptamine, which has OH at the 4-position
The group can also have an aminoglycosyl group as a substituent. Examples of such aminoglycoside antibiotics for use in the present invention include kanamycin A, 6'-N-alkyl kanamycin A, especially 6'-
N-Methylkanamycin A, 3'-deoxykanamycin A, 6'-N-methyl-3'-deoxykanamycin A, 4'-deoxykanamycin A, 6'-N
-Kanamycin A group of methyl-4'-deoxykanamycin A; kanamycin B, 3'-deoxykanamycin B (tobramycin), 4'-deoxykanamycin B, 3'/4'-dioxykanamycin B
(dibekacin), 3', 4'-dideoxy-3'-enokanamycin B, kanamycin B group of 6'-N-methyl-3', 4'-dideoxykanamycin B; kanamycin C, 3'-deoxykanamycin C, 3′・
Kanamycin C of 4'-dideoxykanamycin C
Group: Gentamicin A, B, C: Known compounds such as berdamycin and sisomicin. Regarding currently unknown aminoglycoside antibiotics that will be discovered in the future, as long as they belong to aminoglycoside antibiotics whose basic skeleton is deoxystreptamine having a 3-aminoglycosyl substituent or 3-alkylaminoglycosyl group on the 6-position OH group. too,
The present invention is also applicable to new semi-synthetic aminoglycoside antibiotics that may be synthesized in the future by chemical conversion of known aminoglycoside antibiotics. A particularly suitable example of an aminoglycoside antibiotic to which the present invention is applied is the following general formula () [In the formula, R ¹ is a hydroxyl group or an amino group, R ² and R ³ are each hydrogen or a hydroxyl group, and R ⁴ is a hydroxyl group or an amino group is an alkylamino group having 1 to 4 carbon atoms, especially a methylamino group. kanamycin A, kanamycin B, kanamycin C, deoxy derivatives of these kanamycins, or 6'-N-alkyl derivatives thereof. In the present invention, in order to form a complex by acting a zinc cation on the above aminoglycoside antibiotic, the free base or acid addition salt of the above aminoglycoside antibiotic is dissolved or suspended in an appropriate organic solvent or a water-containing organic solvent. , wherein a suitable zinc salt is added to the aminoglycoside antibiotic for at least 1 hour.
Add in molar amounts. As organic solvents used, all customary organic solvents can be used, provided that the zinc complex formed after addition of the zinc salt is at least partially dissolved. However, it is not desirable to use large amounts of polar organic solvents and especially water. This is because it reduces the stability of the zinc complex and makes the subsequent acylation or amino protection reaction incomplete. Therefore, it is preferable to use a solvent such as dimethyl sulfoxide, which has a large dissolving power, for forming the complex, but water-containing dimethyl sulfoxide,
Dimethylformamide, aqueous dimethylformamide, a mixture of dimethyl sulfoxide and tetrahydrofuran, tetrahydrofuran, aqueous tetrahydrofuran, methanol, ethanol, and lower alkanols such as aqueous methanol can also be used. Zinc cations are supplied to the complex formation site in the form of zinc salts. As the zinc salt, those produced by the reaction of ordinary inorganic acids or organic acids with zinc cations can be used. However, as complexes containing amino groups, complex salts of free amino groups and metals are generally more stable than complex salts of ammonium-type amines and metals, so weak acid salts such as zinc acetate are preferable. If a strong acid salt such as zinc chloride is used, a complex will be formed as it is, but it is preferable to add a weakly alkaline sodium acetate or the like at the same time. Also, for the same reason, acid addition salts of aminoglycoside antibiotics,
For example, when hydrochloride is used, it is desirable to add a base such as sodium acetate or sodium hydroxide as a neutralizing agent. At this time, care must be taken since zinc hydroxide will precipitate if an unnecessarily large amount of the above neutralizing agent is used. For example, when using the tetrahydrochloride salt of the aminoglycoside antibiotic mentioned above, 4 moles of sodium hydroxide should be added. In addition, the molar ratio of zinc salt and aminoglycoside antibiotic is as follows:
Although the complex formation reaction proceeds, since this complex formation is an equilibrium reaction, it is desirable to use a larger number of moles. The number of moles that actually gives a good yield is 2.3
-6 moles, but it is practically preferable to use 4 to 5 moles. The time required for complex formation after addition of the zinc salt depends on the solvent used, but ranges from instantaneous (in the case of a water-containing solvent) to 20 hours, and the reaction is usually carried out at room temperature. At this time, it may be cooled or heated. An acylating agent is added to the solution or suspension containing the aminoglycoside antibiotic zinc complex thus obtained. The acylating agent used in the present invention is an alkanoyl group, an aroyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, It is a common protecting reagent used to attach acyl groups that are aryloxycarbonyl, alkylsulfonyl, aralkylsulfonyl or arylsulfonyl groups. This acylating agent has the following general formula _a R ⁵ COOH (a) [wherein R ⁵ is hydrogen, an alkyl group, especially a carbon number 1]
-6 alkyl or aryl group, especially phenyl group, or aralkyl group, which may further have a substituent], or its acid halide, acid anhydride, or active ester or the chloroformate of the formula R ⁵ O−CO−Cl _b or the p-nitrophenyl carbonate of the formula R ⁵ O−CO−O−C ₆ H ₅ −p−NO _{2 c} or the formula activated esters with N-hydroxysuccinimide such as or azidoformates of the formula R ⁵ O-CO-N _{3 e} , where R ⁵ has the meaning given above.
or the following general formula _f R ⁶ SO ₃ H _f [wherein R ⁶ is an alkyl group, especially an alkyl group having 1 to 6 carbon atoms, an aryl group, especially a phenyl group or an aralkyl group, especially a benzyl group] phenylalkyl group such as a phenylalkyl group] or an acid halide, acid anhydride or active ester thereof. Therefore, the acylation reaction for protecting an amino group is acylation in a broad sense, such as formylation, acetylation, propionylation, trifluoroacetylation, benzyloxycarbonylation, p-methoxybenzyloxycarbonylation. , t-butoxycarbonylation, phenoxycarbonylation, tosylation, and the like. Specific acylating agents include acetoxyformyl, p-nitrophenyl formate, acetic anhydride, acetyl chloride, propionic anhydride, p-nitrophenol ester of trifluoroacetic acid, ethyl trifluoroacetate, and N-benzyloxycarbonyl. Oxysuccinimide (typical active ester), N-benzyloxycarbonyloxyphthalimide, benzyloxycarbonyl chloride, p-
Methoxybenzyloxycarbonyloxy p-nitrophenyl, t-butoxycarbonyl azide,
Phenoxycarbonyl chloride, tosyl chloride, etc. are used. These acylating reagents may be added as such or after being dissolved in a solvent system such as tetrahydrofuran, dimethyl sulfoxide, or a mixture of both. The amount added is usually the same number of moles as the amino group to be reacted or slightly in excess, but up to three times the number of moles of the amino group may be used depending on the case. The time required for addition may range from adding at once to adding over 2 to 3 hours, but is usually in the range of 30 minutes to 1 hour. The reaction temperature is between -20℃ and 100℃, but usually 0℃.
- Perform at room temperature. Sometimes, the temperature during addition of the acylating reagent is low and the reaction temperature is gradually increased. Usually, this acylation reaction can be carried out in the solvent used when forming the above zinc complex. In the method of the present invention, after the above-mentioned acylation reaction, the produced aminoglycoside antibiotic N-
The zinc cation is removed from the complex of the acylated derivative and the zinc cation (decomposition of the complex), thereby separating and forming the N-acylated or N-protected derivative of the aminoglycoside antibiotic. As a method for removing zinc cations from the above complex, zinc cations are insoluble in the acylation reaction solution containing the above complex or in an organic solvent solution obtained by dissolving the above complex in another organic solvent. There is a first method in which a zinc precipitant having the effect of converting zinc compounds into zinc sulfide, zinc hydroxide or zinc carbonate is added and allowed to act. Alternatively, the acylation reaction solution or the organic solvent solution obtained by dissolution may be concentrated or dried by evaporation of the solvent, or a diluent liquid may be added to produce an oily to solid precipitate or residue. A second method for recovering the desired N-acylated product from the compound can also be used. The diluent liquid herein is an organic liquid or water that is incapable of dissolving the above-mentioned zinc complex or the aminoglycoside antibiotic N-acylated derivative contained therein at all or to a small extent. Zinc precipitants used in this first method include hydrogen sulfide, alkali metal sulfides such as sodium sulfide, ammonium sulfide, alkaline earth metal sulfides such as calcium sulfide, and alkali metal carbonates such as sodium carbonate. In some cases, the zinc cations can be removed from the complex by simply adding water. According to this first method, when a zinc precipitant is added to the solution of the complex, the zinc cations precipitate out as an insoluble zinc compound relatively quickly, and this precipitate is removed. The N-acylated derivative of the aminoglycoside antibiotic remaining in the solution is recovered in a conventional manner, for example by concentration of the solution or by extraction. And refine if necessary. For its purification, for example, silica gel column chromatography can be used. According to the second method, an oily to solid precipitate or residue is first obtained by concentrating or drying the acylation reaction solution (or other organic solvent solution). When a poorly volatile solvent such as dimethyl sulfoxide is used as a solvent for the acylation reaction, a diluent solvent such as ethyl ether is added and the hardly volatile solvent is dissolved (or diluted) in ethyl ether to achieve the desired N-acylation. A solid containing the product zinc complex can be precipitated. These oils and solids are generally caused by complexes between N-acylated derivatives of aminoglycoside antibiotics and zinc cations, and by the rupture of some of the complex bonds upon removal of the solvent. A mixture of the free N-acylated derivative, the accompanying zinc salt, the zinc salt added as an excess and present from the beginning, and some residual amount (or sometimes none) of the organic solvent used. Consists of. (b) A polar organic liquid that has the effect of breaking the complex bond of zinc cations in the zinc complex contained in this oily to solid mixture, and that can dissolve zinc salts, is used as an aminoglycoside antibiotic. When an organic liquid, a mixture of such organic liquids or a water-containing organic liquid, or water, which does not dissolve the substance N-acylated derivative, is added or mixed,
Since the zinc complex is decomposed and the released zinc cation is dissolved and extracted in the form of zinc salt, the N-acylated derivative remains as an insoluble residue and can be collected. This can be further purified if desired by redissolving in an organic solvent. Polar organic liquids that can be used for the above purpose include, for example, methanol, ethanol, liquid ammonia, ethylamine, triethylamine. (b) A polar organic solvent that has the effect of breaking the complex bonds of zinc cations in the zinc complex contained in the oily or solid mixture, but cannot dissolve the zinc salt, but can be used for the desired purpose. When a polar organic solvent (which may contain water) that can dissolve the aminoglycoside antibiotic N-acylated derivative is added and mixed, the zinc complex decomposes and the liberated N-
The acylated derivative can be dissolved and extracted in the organic solvent and thus separated from the zinc salt which remains insoluble. The N-acylated derivative solution thus obtained is purified and concentrated if desired.
The desired N-acylated derivative can be obtained. (c) The above-mentioned oil or solid obtained by concentrating or evaporating to dryness the above-mentioned acylation reaction solution or an extract obtained by extracting the acylation product therefrom with an organic solvent, or by adding a diluent liquid. If the entire mixture is water-soluble or partially water-soluble, the entire mixture is redissolved in a suitable water-containing organic solvent, and from this solution the zinc salt and the desired nitrogen are extracted by chromatography. -acylated derivatives can be collected separately.
For this purpose, the inventors have found that it is possible to utilize cation or anion exchange resins, chelate exchange resins, or water-insoluble polymeric substances such as chitin and chitosan having groups capable of bonding with metals. Types of cation exchange resins that can be used include:
There are those having a carboxyl group (-CO ₂ H) and those having a sulfonic acid group (-SO ₃ H) as an exchange group. First, when using a cation exchange resin having a carboxyl group, the above-mentioned solid mixture is mixed with a suitable water-containing organic solvent such as water-methanol (the water content varies from 10 to 90% depending on the case) or water-dioxane ( Water content is the same as above) and charged to a column containing the above exchange resin, first washed well with the above water-containing organic solvent, and then developed using the above water-containing organic solvent containing an acid or base as a developing solvent. do. As the acid, a weak organic acid such as acetic acid or an inorganic acid such as dilute hydrochloric acid is mainly used, and as the base, ammonium hydroxide is mainly used. The amount of acid or base contained in the developing solvent is 0.01 to 5% (weight), respectively.
is appropriate. Zinc cation and target N-
Since the acylated derivative has a different binding force to the resin, the two are separated during the development operation due to the difference in adsorption force. The fraction containing the N-acylated derivative thus separated and eluted from the zinc salt is collected and concentrated to obtain the desired N-acylated derivative. Even when using an ion exchange resin having a sulfonic acid group as an exchange group, the mechanism is exactly the same as above, and therefore, the same treatment can be performed. When using weakly basic or strongly basic anion exchange resins, N-acylated derivatives have amino groups that cannot be acylated, so they are ionically repelled and generally adsorbed by the above weakly or weakly basic anion exchange resins. Instead, the N-acylated derivative is eluted by developing with a suitable aqueous solution. At that time, zinc cations often remain in the resin. Furthermore, since chelate exchange resins can bind zinc cations due to their metal chelating ability, a solution of the oily or solid mixture in a water-containing organic solvent is first charged to a column of chelate exchange resin, and then this column is charged with a suitable solvent system. By developing with , only the N-acylated product can be preferentially eluted. Furthermore, water-insoluble polymeric substances such as chitin or chitosan having the property of binding to metals can also be used in the same manner as the above-mentioned chelate exchange resin. (d) The acylation reaction solution subjected to the above acylation protection reaction is directly applied to a column of the above-mentioned cation or anion exchange resin, chelate exchange resin, or a water-insoluble polymer substance having a group capable of binding to a metal. Adsorption is carried out by flowing the column, and then the column thus charged is treated with aqueous organic solvent, if necessary.
The third step is to wash, then develop with the water-containing organic solvent containing or not containing an acid or base as described in the previous section (c), and then operate in the same manner as in the previous section (c).
The method can also be employed for desorption of zinc cations from zinc complexes and recovery of the desired aminoglycoside antibiotic N-acylated derivatives. (e) When the desired aminoglycoside antibiotic N-acylated derivative itself is substantially insoluble in water, the N-acylated derivative produced by the acylation reaction
- A fourth method can be performed in which the acylation reaction solution containing the zinc complex of the acylated derivative and the zinc cation is directly treated with water (addition of water, mixing). Examples of aminoglycoside antibiotic N-acylated derivatives that are substantially water-insoluble include 3,2',6'-tri-N-benzyloxycarbonyldibekacin (Example 23).
In this case, if water was directly added and mixed with the acylation reaction solution, the complex bonds of the zinc complex would be broken and the N-acylated derivative would precipitate as a solid, and the zinc salt generated from the eliminated zinc cation would dissolve. What remains is that the almost pure N-acylated derivative can be separated from the zinc salt. In the method of the present invention, as described above, the zinc complex of an aminoglycoside antibiotic is subjected to acylation or amino group protection reaction, and the mono-, di- or tri-N-acyl formed thereby. Since the zinc complex of the acylated derivative has a complex bond with the zinc cation used, if the desired N-acylated derivative is insoluble or slightly soluble in water (Example 23), the zinc complex after the acylation reaction is By treating the acylation reaction solution with water, zinc cations are desorbed, dissolved in water, and removed, and a water-insoluble N-acylated derivative is precipitated as a solid. Therefore, this water-insoluble material can be used as a starting material. The following desired reaction can be carried out using the compound as a compound (in the case of the previous item (e)). However, in general,
Since the aminoglycoside antibiotic N-acylated derivative itself has water solubility or partial water solubility,
If the acylation reaction solution is simply mixed with water and treated with water, the yield of the desired N-acylated substance will be significantly reduced. Therefore, the zinc complex of the aminoglycoside antibiotic N-acylated derivative produced in the acylation reaction is first separated from the acylation reaction solution.
Next, this is dissolved in water or a water-containing organic solvent,
It is better to remove only the zinc cations from the solution (in the case of the previous sections (b) and (c)). One of the generally accepted and simple methods for eliminating zinc cations is a method in which hydrogen sulfide or sulfide is used as a precipitant to precipitate zinc cations as zinc sulfide (an example of the case (a) above). However, zinc sulfide often turns into a colloid, making it difficult to remove, and hydrogen sulfide or sulfides used as precipitants emit a bad odor, so they are not suitable for industrial use. Therefore, the present inventors have conducted various studies on methods for desorbing zinc cations using other methods that do not involve sulfides.
(d)) was found to be effective. These methods (c) and (d) are easy to operate, have good separation efficiency, and have high yields, so they are extremely advantageous in terms of industrialization. In the method of the present invention, as described above,
Aminoglycoside antibiotic N-acylated derivatives are obtained in which amino groups or alkylamino groups other than the amino groups or alkylamino groups at the 1- and 3''-positions are protected. This compound is then converted into 1-acylated derivatives by conventional methods. N-acylation followed by removal of the above amino protecting group by conventional methods yields semi-synthetic 1-N-acylated aminoglycoside antibiotics useful as antibacterial agents. 1-N-acylated aminoglycoside antibiotics. The synthesis of kanamycin A will be explained using kanamycin A as an example.Since the amino groups at the 1- and 3''-positions of kanamycin A are first blocked by forming a complex with a zinc cation, a suitable zinc complex is added to the zinc complex according to the present invention. By acting with an acylating reagent or other amino group-protecting reagent, the amino groups at the 3- and 6'-positions are protected with an acyl group or other protecting group. After that, after removing the zinc cation that has taken part in the complex formation, when an acylating reagent having an acyl group to be introduced into the 1-position amino group is reacted, this acyl group is transferred to the free 1- and 3''-position amino groups. However, since the 1-position amino group is generally much more reactive than the 3″-position amino group, the desired 1-N
- Acylkanamycin A is obtained in good yield. In this way, the desired 1-N-acyl kanamycin A can be obtained in a higher yield than when the acylating reagent is directly applied to kanamycin A or kanamycin A whose 6'-amino group has been protected in advance. It is clear that When the method of the present invention is applied to kanamycins of the above general formula (), the following formula () in which some or all of the amino groups other than the amino groups at the 1st and 3'' positions are protected: [In the formula, R ¹ a represents a -OH group, -NH ₂ group, -NHCOR ⁵ group, -NHCO・OR ⁵ group, or -NHSO ₂ R ⁶ group, and R ⁴ a represents -OH group, -NHCOR ⁵ group,

[Formula] -NHCO・OR ⁵ groups,

[Formula] -NHSO ₂ R ⁶ groups or

[Formula] represents a group, R ² and R ³ have the same meaning as in formula (), R ⁷ represents a -COR ⁵ group, a -CO・OR ⁵ group, or a -SO ₂ R ⁶ group, and R ⁵ and R ⁶ has the same meaning as in formula (a)-( _f ), R ⁸ is an alkyl group]
Acylated derivatives are obtained. That is, when this method is applied to kanamycins, a substance () in which all of the amino groups or alkylamino groups other than the 1st and 3'' positions are protected by the above-mentioned groups is usually obtained. When the protected acyl group to be introduced is sterically large (for example, t-butoxycarbonyl group), or even if the protected acyl group to be introduced is of a normal size, the number of moles used is reduced or the reaction time is When shortened, a protected substance with fewer introduced acyl groups can be obtained.
Since the amino group at the 6'-position or the methylamino group at the 6'-position is more reactive than other amino groups, a substance in which only the amino group at the 6'-position or the methylamino group at the 6'-position is protected can be obtained. Compound () is an important intermediate in the synthesis of various derivatives. A semi-synthetic 1-N-acylated aminoglycoside antibiotic that is effective against resistant bacteria by acylating the amino group at the 1-position of the compound () and then removing the protecting group of the amino group or methylamino group other than the acyl group at the 1-position. When combined with a reaction leading to a substance, the value of the compound () as a synthetic intermediate increases in particular. For example, when bonding an acyl group, such as (S)-4-benzyloxycarbonylamino-2-hydroxybutyryl group, to compound (), various active esters of substituted butyric acid, such as N-hydroxy succinimide,
An ether with N-hydroxyphthalimide or p-nitrophenol may be prepared and reacted with the compound (2) in a suitable solvent such as aqueous tetrahydrofuran. Thereafter, the benzyloxycarbonyl group and the protecting group R ⁷ are removed by conventional methods such as hydrolysis with acids, bases, catalytic reduction with metals, or radical reduction with sodium in liquid ammonia, resulting in (S)- The general formula () is an aminoglycoxide antibiotic effective against resistant bacteria that has a 4-amino-2-hydroxybutyryl group attached to it. A compound of [wherein R ₁ , R ₂ , R ₃ and R ₄ represent the same meanings as shown in formula () and n=1 to 3] can be obtained. Next, the present invention will be explained with reference to examples. Example 1 Preparation of 3.6'-di-N-benzyloxycarbonylkanamycin A (i) 2.0 g (4.13 mmol) of kanamycin A (free base) was suspended in a mixture of 50 ml of dimethyl sulfoxide and 20 ml of tetrahydrofuran, and zinc acetate was added. Add 4 g (18.1 mmol) of ( ) dihydrate,
The reaction mixture was stirred at room temperature until it became a homogeneous solution. It took about 4 to 5 hours for the suspended kanamycin A to form a zinc complex and dissolve. Next, this solution was cooled to 0°C, and N-benzyloxycarbonyloxysuccinimide was added to it.

[Formula] 2.37 g (9.5 mmol) was added to 40 ml of a mixture of tetrahydrofuran and dimethyl sulfoxide (1:1).
A cold solution (0° C.) dissolved in 100% was gradually added over about 1 hour, and then the reaction solution was left at room temperature for 4 hours. During this time, the zinc complex of kanamycin A underwent benzyloxycarbonylation (acylation according to the invention). This reaction solution was analyzed in a thin layer chromatogram using the lower layer of chloroform methanol-28% aqueous ammonia (1:1:1) as the developing solvent.
A main spot was observed at Rf0.23, but only two to three spots due to other by-products were faintly observed above it. (ii) This reaction solution was poured into 500 ml of ethyl ether, and the precipitated oily substance was washed several times with ether to obtain 8.8 g of a hard syrupy substance. (iii) Next, the following methods (A) to (J) were carried out to remove zinc salts from this syrupy substance. (A) A method using a weakly acidic cation exchange resin (Amberlite CG50 resin (H ⁺ type), manufactured by Rohm and Haas) that has a carboxy group (-CO ₂ H) as a functional group. 60 ml of Amberlite CG50 (H ⁺ type) resin was thoroughly treated in advance with water-dioxane (2:1), and this was packed into a column, where 1 g of the syrupy substance mentioned above was mixed with water-dioxane (1:1). The dissolved solution was charged and developed with water-dioxane (2:1) containing 1% acetic acid. It is the target substance. 3,6'-di-N-benzyloxycarbonylkanamycin A, which is active in ninhydrin, was eluted first, followed by zinc acetate, which is active in coloring with diphenylcarbazide.
The former fraction was collected, concentrated, and the concentrate was washed with ether to give 340 mg (81%) of a white solid.
I got it. [α] ²⁵ _D +76° (c1, hydrous dimethylformamide = 1:2) Elemental analysis C ₃₄ H ₄₈ N ₄ O ₁₅・2CH ₃ CO ₂ H−
As H ₂ O Theoretical value: C, 51.23; H, 6.56; N, 6.29% Analytical value: C, 51.02; H, 6.71; N, 6.22% (B) Weakly acidic cation exchange resin with carboxylate group as a functional group (amber light
Method using CG50 resin (NH ₄ ⁺ type)). Amberlite CG50 ( _NH4 ⁺ type) resin 60ml
A solution prepared by dissolving 1 g of the syrupy substance obtained in Example 1(ii) in water-dioxane (1:1) was charged, and the mixture was subjected to gradient development with water-dioxane (1:1) containing 0 to 0.1N ammonia. . Zinc ions are not eluted, and the target substance 3.
6'-di-N-benzyloxycarbonylkanamycin A is eluted. Concentrate the eluate and
328 mg (89%) of a white solid was obtained. [α] ²⁵ _D +86゜ (c1, hydrous dimethylformamide = 1:2) Elemental analysis C ₃₄ H ₄₈ N ₄ O ₁₅・1/2H ₂ CO ₃ Theoretical value: C, 52.87; H, 6.30; N, 7.15 % Analysis value: C, 52.50; H, 6.59; N, 7.00% (C) Use a cation exchange resin with a strong acidic group (-SO ₃ H) as a functional group, Dowex 50W x 2 resin, manufactured by Dow Chemical Company) Method. The syrup-like material obtained in Example 1(ii) 1 was placed in a column containing 30 ml of Dowex 50W x 2 (H ⁺ form) resin pretreated with water-dioxane (2:1).
Dissolve g in water-dioxane (2:1), charge the solution, and dissolve in water-dioxane (2:1).
The eluate was washed with a solution until the eluate became neutral. Then water containing 0-1 normal ammonia.
When gradient development is performed with a dioxane (2:1) solution, the target substance is obtained. A portion containing 3,6'-di-N-benzyloxycarbonylkanamycin A was eluted. The eluate was concentrated to dryness under reduced pressure to obtain the same white solid 311 as in Example 1(iii)(B).
mg (84%). (D) Another method using Dowex 50W x 2 resins. The syrupy substance obtained in Example 1(ii) 1 was placed in a column containing 30 ml of Dowex 50W x 2 (H ⁺ form) resin pretreated with water-methanol (3:1).
Dissolve g in water-methanol (3:1), charge the solution, and dissolve in water-methanol (3:1).
After thorough washing with water, gradient development was carried out with water-methanol (3:1) containing 0-6N hydrochloric acid.
Collect the fraction containing the desired 3,6'-di-N-penzyloxycarbonylkanamycin A, add a strongly basic anion exchange resin, such as Dowex 1×2 resin (OH type), until the mixture becomes slightly acidic. After filtration, the solution was concentrated to dryness to obtain 285 mg of the target substance as dihydrochloride.
(72%). [α] ²⁵ _D +79° (c1, hydrous dimethylformamide = 1:2) (E) A method using an anion exchange resin (Dowex 1×2 resin, manufactured by Dow Chemical Company) having a strong basic group as a functional group. The syrupy material 1 obtained in Example 1(ii) was placed in a column containing 30 ml of Dowex 1x2 resin (OH type) pretreated with water-dioxane (1:1).
g was charged and developed relatively quickly with water-dioxane (1:1). The portion containing the target substance was collected and concentrated to dryness to obtain 305 mg (83%) of a white solid similar to Example 1(iii)(B). (F) A method using an anion exchange resin (Dowex WGR resin, manufactured by Dow Chemical Company) that has a weakly basic group as a functional group. The syrupy material 1 obtained in Example 1(ii) was placed in a column containing 50 ml of Dowex WGR resin (base form) pretreated with water-dioxane (2:1).
A solution obtained by dissolving g in water-dioxane (2:1) was charged and developed with water-dioxane (2:1). It is the target substance. 3・6′-G-N
-Benzyloxycarbonylkanamycin A
Since the zinc ions are eluted with a small amount of zinc ions, this part is collected and concentrated to dryness to form a white solid with 450%
I got mg. This substance was prepared as is, and 1-N-((S)-4-amino-2
-Hydroxybutyryl) Kanamycin A can be used as a raw material in the production of Kanamycin A, and at this time, the inclusion of a small amount of zinc ions in the substance does not cause any problems. (G) A method using a chelate resin (Dowex A1 resin, manufactured by Dow Chemical Company) that has a weakly acidic group as a functional group. Dowex A1 resin pretreated with water-dioxane (1:1) containing 1% ammonia
Dissolve 1 g of the syrupy substance obtained in Example 1 (ii) in a 50 ml column in water-dioxane (1:1), charge it, and decant with a water-dioxane (1:1) mixture containing 0-1N ammonia. Expanded. Only the portion containing 3,6'-di-N-benzyloxycarbonylkanamycin A, which is the target substance, is gradually eluted. The following treatment was carried out in the same manner as in method (B) to obtain 272 mg (74%) of a white solid. (H) Method using chitosane. A column was filled with 100 ml of chitosan that had been sufficiently pretreated with water-methanol (3:1), and the syrupy substance 1 obtained in Example 1(ii) above was added to the column.
g was dissolved in water-methanol (3:1), charged, and developed with water-methanol (3:1). The target substance, 3,6'-di-N-benzyloxycarbonylkanamycin A, was eluted first, and zinc acetate was eluted very late. The former was collected and concentrated, and the residue was charged to a column containing Amberlite CG50 resin (NH ₄ ⁺ type) that had been previously treated with water-dioxane (1:1), and the column was thoroughly washed with water-dioxane (1:1). After that, gradient development was carried out with water-dioxane (1:1) containing 0-0.1N ammonia, and the ninhydrin-active fraction was collected and concentrated to give the same white solid as Example 1(iii)(B).
Obtained 301 mg (82%). (I) A method using a polymer substance having a carboxyl group as a functional group (CM-Sephadex C-25, manufactured by Pharmacia Fine Chemicals). CM-Sephadex C-25 in advance
(NH ⁺ ₄ type) resin 40ml water-dioxane (1:
1), packed into a column, and added 1 g of the syrupy substance obtained in Example 1(ii) with water.
The solution dissolved in dioxane (1:1) was charged, washed with 200 ml of water-dioxane (1:1), and then decanted with water-dioxane (1:1) containing 0-0.1N ammonia. Zinc ions are not eluted and target 3,6'-di-N
-Benzyloxycarbonylkanamycin A
is eluted. The eluate was concentrated and used in Example 1(iii).
303 mg (82%) of a white solid identical to (B) was obtained. (J) Method using hydrogen sulfide as a zinc precipitant Dissolve 1 g of the syrupy substance obtained in Example 1 (ii) in 20 ml of water-methanol (1:1), add aqueous ammonia, and then fully introduce hydrogen sulfide. did. The mixture containing the zinc sulfide precipitate was filtered through a glass filter filled with Celite as a supernatant, and the liquid was concentrated under reduced pressure, and the resulting syrupy substance was thoroughly washed with ether. The obtained solid residue was dissolved in water-dioxane (1:1),
Column 30 of Amberlite 1RA900 (OH type) (strong basic resin; manufactured by Rohm and Haas)
ml, developed with water-dioxane (1:1), collected the part containing 3,6'-di-N-benzyloxycarbonylkanamycin A, and concentrated to dryness. Same as Example 1(iii)(B). white solid 235
mg (64%). Example 2 Production of 3,6'-di-N-benzyloxycarbonylkanamycin A 500 mg (equivalent to 1 mol) of kanamycin A (free base) was suspended in 15 ml of dimethyl sulfoxide, and 420 mg (equivalent to 3 mol) of zinc chloride was suspended in 15 ml of dimethyl sulfoxide. ) and 840 mg (equivalent to 6 moles) of sodium acetate trihydrate,
After stirring at room temperature for 10 hours, add N-benzyloxycarbonyloxyphthalimide.

[Formula] A solution of 675 mg (equivalent to 2.2 moles) dissolved in 10 ml of dimethyl sulfoxide was gradually added over about 1 hour, and then 4
It was left at room temperature for an hour. Thereafter, it was treated in exactly the same manner as in Example 1 (ii) and (iii) (I),
598 mg (74%) of 3,6'-di-N-benzyloxycarbonylkanamycin A as a white solid was obtained. Example 3 Production of 3,6'-di-N-benzyloxycarbonyl kanamycin A 600 mg (0.95 mmol) of kanamycin A tetrahydrochloride and 150 mg (3.8 mmol) of sodium hydroxide were stirred and reacted in 15 ml of dimethyl sulfoxide for 1 hour. Then, 1 g (4.55 mmol) of zinc acetate dihydrate was added thereto, and stirring was continued for an additional 5 hours. N
-Benzyloxycarbonyloxysuccinimide 545 mg (2.2 mmol) dimethyl sulfoxide-
A solution in 5 ml of tetrahydrofuran (1:1) was added over 30 minutes and the mixture was stirred at room temperature overnight. Ethyl ether was added to the reaction mixture, the precipitate was collected, and the precipitate was treated in the same manner as in Example 1(iii)(H) to obtain 581 mg (78%) of a white solid. Example 4 Preparation of 3,6'-di-N-benzyloxycarbonylkanamycin A (i) 500 mg (1.03 mmol) of kanamycin A (free base) was dissolved in water-dimethyl sulfoxide (1:9).
1 g of zinc acetate dihydrate dissolved in 20 ml of a mixture of
(4.55 mmol) and then N-benzyloxycarbonyloxysuccinimide 590 mg (2.4
mmol) and left overnight at room temperature. A large amount of ethyl ether was added and the precipitated water-rich layer was separated and washed several times with ether.
A syrupy layer was obtained. (ii) This syrupy substance was dissolved in water-methanol (3:1), and the solution was charged to a 200 ml chitosan column and developed with water-methanol (3:1). After collecting the active part of ninhydrin and concentrating, the concentrated liquid was converted into Amberlite CG50 resin (NH ₄ ⁺ type)
of water-dioxane (1:
After thoroughly washing the column with the mixture of 1), water containing 0 to 0.1N ammonia-dioxane (1:1)
The part containing the target substance was collected and concentrated to obtain 494 mg of the same white solid as Example 1(iii)(B).
(61%). Example 5 Production of 3,6'-di-N-benzyloxycarbonylkanamycin A A mixture of 500 mg (1.03 mmol) of kanamycin A (free base) and water-tetrahydrofuran (1:3)
Add 20 ml of zinc acetate dihydrate and 1 g of zinc acetate dihydrate (4.55
590 mg (2.4 mmol) of N-benzyloxycarbonyloxysuccinimide
was added and left overnight at room temperature. After concentrating the reaction solution under reduced pressure, the residue was charged to a 200 ml chitosan column.
Thereafter, the treatment was carried out in exactly the same manner as in Example 4, and white solid 414
mg (51%). Example 6 Preparation of 3,6'-di-N-benzyloxycarbonyl kanamycin A 500 mg (1.03 mmol) of kanamycin A (free base) was dissolved in 15 ml of water-methanol (1:7), and 1.5 ml of zinc acetate dihydrate was added. After obtaining g, N-benzyloxycarbonyloxysuccinimide 590mg
7 ml of a tetrahydrofuran solution containing (2.4 mmol) was added and left overnight at room temperature. After concentrating the reaction solution, the residue was charged to a 200 ml chitosan column and treated in the same manner as in Example 4 to obtain 442 mg of white solid.
(55%). Example 7 Production of 3,6'-di-N-benzyloxycarbonyl kanamycin A 500 mg (equivalent to 1 mole) of kanamycin A (free base) was suspended in 20 ml of dimethyl sulfoxide,
Add to this 272 mg of zinc acetate dihydrate (equivalent to 1.2 moles)
was added, and stirring was continued for 10 hours at room temperature. To the resulting almost transparent solution, 540 mg (equivalent to 2.1 moles) of N-benzyloxycarbonyloxysuccinimide was gradually added in small portions over about 2 hours. Thereafter, it was further left at room temperature overnight. A large amount of ether was added to this solution, and a precipitate was collected, which was further washed several times with ether. The resulting hard syrupy substance is chloroform-
In a thin layer chromatogram developed with methanol-28% ammonia (1:1:1, lower layer), 1,3,6',3''-tetra-N-benzyloxycarbonylkanamycin A was detected at Rf0.4. Weak spot (sulfuric acid was sprayed onto a thin laminate plate and then ignited to develop color), weak spot at Rf0.28, and 3-6'-di-N-benzyloxycarbonylkanamycin A targeted at Rf0.23. The strongest spot, Rf0.12
A weak spot for 6'-N-benzyloxycarbonyl kanamycin A was added, as well as a very weak spot for unreacted kanamycin A at Rf0. but
Almost no spots corresponding to tri-N-benzyloxycarbonylkanamycin A appearing between Rf0.28 and 0.4 were observed. This hard syrupy substance was dissolved in water-dioxane (1:1) and then dissolved in water-dioxane (1:1).
CM-Sephadex C-25 (NH ₄ ⁺
Type) resin was charged to 100 ml and treated in the same manner as in Example 1(iii)(I). This treatment removes zinc ions and at the same time removes the target substance 3,6'-di-N-
Benzyloxycarbonylkanamycin A was separated from other products as a white solid, 412 mg (51%).
Obtained. For comparison, if the above reaction is carried out in exactly the same manner as above using 500 mg (equivalent to 1 mol) of kanamycin A (free base) and 308 mg (equivalent to 1.2 mol) of nickel acetate tetrahydrate as starting materials, the desired reaction will be achieved. 59mg of the substance 3,6'-N-benzyloxycarbonylkanamycin A as a white solid similar to the above.
(7.3%) was obtained. Example 8 Preparation of 3,6'-di-N-(p-methoxybenzyloxycarbonyl) kanamycin A 500 mg (1.03 mmol) of kanamycin A (free base) was suspended in 12 ml of dimethyl sulfoxide and 1 g of zinc acetate dihydrate was added. (4.55 _mmol ) was added to a homogeneous solution prepared in exactly the _same manner as in Example 1 _, and p _- methoxybenzyloxycarbonyloxy- _p -nitrophenyl ( _{pCH3OC6H4CH2OCOOC6H4pNO2 )}
789 mg (2.6 mmol) dimethyl sulfoxide 10
ml solution was added for about 30 minutes and then left overnight. Thereafter, it was treated in the same manner as in Example 1 (ii) and (iii) (B) to obtain 722 mg (83%) of a white solid. [α] ²⁵ _D +87゜(c1,
Hydrous dimethylformamide = 1:2) Elemental analysis C ₃₆ H ₅₂ N ₄ O ₁₇・1/2H ₂ CO ₃ Theoretical value: C, 51.95; H, 6.33; N, 6.64% Analytical value: C, 51.56; H, 6.41; N, 6.53% Example 9 Preparation of 6'-N-(t-butoxycarbonyl) kanamycin A 500 mg (1.03 mmol) of kanamycin A (free base) was suspended in 12 ml of dimethyl sulfoxide, and zinc acetate dihydrate was added to the suspension. 220 mg (1.54 mmol) of t-butoxycarbonyl azide was added to a homogeneous solution prepared in the same manner as in Example 1(i) with the addition of 1 g of t-butoxycarbonyl azide, and the mixture was stirred overnight at room temperature. The mixture was treated in the same manner as in Example 1 (ii) and (iii) (B) to obtain 627 mg of a white solid. [α] ²⁵ _D +96゜(c1,
water-containing dimethylformamide = 1:2). Example 10 Preparation of 3,6'-di-N-trifluoroacetyl kanamycin A 500 mg (1.03 mmol) of kanamycin A (free base) was suspended in 12 ml of dimethyl sulfoxide, and 1 g (4.55 mmol) of zinc acetate dihydrate was suspended in 12 ml of dimethyl sulfoxide. ) was added to a homogeneous solution prepared in the same manner as in Example 1(i), and paranitrophenol ester of trifluoroacetic acid 1.2
g (5.1 mmol) in 10 ml of dimethyl sulfoxide
A solution dissolved in was added, and the mixture was stirred at room temperature overnight. Thereafter, the mixture was treated with ethyl ether in the same manner as in Example 1(ii), and the ether-insoluble material was treated in the same manner as in Example 1(iii)(A) to obtain 590 mg (70%) of a white solid. [α] ²⁵ _D +81゜ (c1, hydrous dimethylformamide = 1:2) Elemental analysis C ₂₂ H ₃₄ N ₄ O ₁₃ F ₆・2CH ₃ CO ₂ H・H ₂ O
Theoretical value C, 38.33; H, 5.44; N, 6.88% F, 13.99% Analytical value C, 38.03; H, 5.48; N, 6.54% Example 11 3.6'-di-N-phenoxycarbonylkanamycin Preparation of A 500 mg (1.03 mmol) of Kanamycin A (free base) was suspended in a mixture of 15 ml of dimethyl sulfoxide and 5 ml of tetrahydrofuran, and 1 g (4.55 mmol) of zinc acetate dihydrate was added thereto, as in Example 1(i). A homogeneous solution prepared in the same manner was cooled to 0°C, and phenoxycarbonyl chloride (C ₆ H ₅ OCOCl) was added to it.
A solution of 400 mg (2.55 mmol) in tetrahydrofuran (3 ml) cooled to 0°C was gradually added at the same temperature, and the temperature of the reaction solution was brought to room temperature for about 1 hour. Example 1 after being left at room temperature for another 3 hours
Treated with ethyl ether in the same manner as in (ii), and treated the ether-insoluble matter in the same manner as in Example 1 (ii) (A) to produce a white solid.
Obtained 625 mg (70%). [α] ²⁵ _D +73° (c1, hydrous dimethylformamide = 1:2) Elemental analysis C ₃₂ H ₄₄ N ₄ O ₁₅・2CH ₃ CO ₂ H・H ₂ O Theoretical value: C, 50.11; H, 6.31; N, 6.49% Analytical value: C, 49.77; H, 6.60; N, 6.11% Example 12 Production of 3,6'-di-N-acetyl kanamycin A 500 mg (1.03 mmol) of kanamycin A (free base) was dissolved in dimethyl sulfoxide. 1 g (4.55 mmol) of zinc acetate dihydrate was added to this, and 260 mg (2.6 mmol) of acetic anhydride was dissolved in 10 ml of dimethyl sulfoxide to a homogeneous solution prepared in the same manner as in Example 1(i). The solution was added and stirred at room temperature overnight. Thereafter, it was treated in exactly the same manner as in Example 1 (ii) and (iii) (A) to obtain 525 mg (72%) of a white solid. [α] ²⁵ _D +93゜(c1,
Hydrous dimethylformamide = 1:2) Elemental analysis C ₂₂ H ₄₀ N ₄ O ₁₃・2CH ₃ CO ₂ H・H ₂ O Theoretical value: C, 44.19; H, 7.13; N, 7.93% Analysis value: C, 44.20 ; H, 7.07; N, 7.85% Example 13 Production of 3,6'-di-N-formyl kanamycin A 500 mg (equivalent to 1 mol) of kanamycin A (free base) was suspended in 12 ml of dimethyl sulfoxide, and Paranitrophenyl formate (OHCO-
C ₆ H ₄ pNO ₂ ) 690 mg (equivalent to 4 moles) was added and stirred overnight at room temperature. Thereafter, the treatment was carried out in the same manner as in Example 1 (ii) and (iii) (H) to collect the ninhydrin active fraction, and after introducing carbon dioxide gas, it was concentrated to give 430 mg (67 mg) of a white solid.
%) was obtained. [α] ²⁵ _D +101゜ (c1, water) Elemental analysis C ₂₀ H ₃₆ N ₄ O ₁₃・H ₂ CO ₃・H ₂ O Theoretical value: C, 40.64; H, 6.50; N, 9.03% Analysis value: C, 40.43; H, 6.47; N, 8.83% Example 14 Preparation of 3,6'-di-N-tosylkanamycin A 500 mg (1.03 mmol) of kanamycin A (free base) was suspended in 15 ml of dimethyl sulfoxide. 1 g (4.55 mmol) of zinc acetate dihydrate was added and stirred until a homogeneous solution was obtained. Tosyl chloride
A solution of 400 mg (2.1 mmol) dissolved in 7 ml of tetrahydrofuran was gradually added and left at room temperature for 1 hour. Furthermore, a solution of 200 mg of tosyl chloride dissolved in 3.5 ml of tenalahydrofuran was added and left to stand for 2 hours. Thereafter, it was treated in exactly the same manner as in Example 1 (ii) and (iii) (A) to obtain 270 mg (28%) of a white solid. [α] ²⁵ _D +
68゜(c1, hydrous dimethylformamide = 1:2) Elemental analysis C ₃₂ H ₄₈ N ₄ O ₁₅ S ₂・2CH ₃ CO ₂ H・H ₂ O
Theoretical value: C, 46.44; H, 6.28; N, 6.02; S, 6.89% Analytical value: C, 46.31; H, 5.98; N, 6.31; S, 6.55% When this reaction is performed without zinc acetate, the above solid are rarely collected. Example 15 3,6'-di-N-benzyloxycarbonyl-
Production of 6'-N-methylkanamycin A 6-N-methylkanamycin A (free base) 500
mg (1.0 mmol) dimethyl sulfoxide 12 ml
To this was added 1 g (4.55 mmol) of zinc acetate dihydrate, and the mixture was stirred until a homogeneous solution was obtained. Add 550 mg (2.2 mmol) of N-benzyloxycarbonyloxysuccinimide to this solution in 5 ml of dimethyl sulfoxide-tetrahydrofuran (1:1).
The solution was added for 30 minutes and then left at room temperature overnight. Thereafter, it was treated in exactly the same manner as in Example 1 (ii) and (iii) (A) to obtain 720 mg (79%) of a white solid.
[α] ²⁵ _D +74゜(c1, hydrated dimethylformamide =
1:2) This substance was then treated in the same manner as in Reference Example 1 to obtain 1-N-((S)-4-amino-2-hydroxybutyryl)-6'-N-methylkanamycin A. Example 16 3,6'-di-N-benzyloxycarbonyl-
Production of 3'-deoxykanamycin A 3'-deoxykanamycin A (free base) 500mg
(1.07 mmol) was suspended in 12 ml of dimethyl sulfoxide, and 1 g (4.55 mmol) of zinc acetate dihydrate was added thereto and stirred until a homogeneous solution was obtained. Add 610 mg (2.45 mmol) of N-benzyloxycarbonyloxysuccinimide to this solution in 5 ml of dimethyl sulfoxide-tetrahydrofuran (1:1).
A solution dissolved in was added and left overnight at room temperature. Thereafter, it was treated in exactly the same manner as in Example 1 (ii) and (iii) (A) to obtain 765 mg (82%) of a white solid. [α] ²⁵ _D +76° (c1, hydrous dimethylformamide = 1:2) Elemental analysis C ₃₄ H ₄₈ N ₄ O ₁₄・2CH ₃ CO ₂ H・H ₂ O Theoretical value: C, 52.16; H, 6.68; N, 6.40% Analysis value: C, 51.99; H, 6.75; N, 6.20% This substance was treated in the same manner as in Reference Example 1 to produce 1-N-((S)-4-amino-2-hydroxybutylene). Riru)-3'-deoxykanamycin A was obtained. Example 17 3,6'-di-N-benzyloxycarbonyl-
3'-deoxy-6'-N-methylkanamycin A
Production of 3'-deoxy-6'-N-methylkanamycin A
500 mg (1.04 mmol) of the free base was suspended in 12 ml of dimer sulfoxide, and 1 g (4.55 mmol) of zinc acetate dihydrate was added to the resulting homogeneous solution, which contained 595 mg (2.4 mmol) of N-benzyloxycarbonyloxysuccinimide. A solution of 5 ml of dimethyl sulfoxide-tetrahydrofuran (1:1) was added thereto, and the mixture was left at room temperature overnight. Thereafter, it was treated in exactly the same manner as in Example 1 (ii) and (iii) (A) to obtain 737 mg (80%) of a white solid. [α] ²⁵ _D +73° (c1, hydrous dimethylformamide = 1:2) This substance was treated in the same manner as in Reference Example 1 to convert it into 1-N-((S)-4-amino-2-hydroxybutyryl. )-3'-deoxy-6'-N-methylkanamycin A was obtained. Example 18 3,6'-di-N-benzyloxycarbonyl-
Production of 4'-deoxykanamycin A 4'-deoxykanamycin A (free base) (Naito et al., Journal of Antibiotics, Vol. 27)
838-849, 1974); Miyake, Tsuchiya, Umezawa, Umezawa,
500 mg (1.07
mmol) was suspended in 12 ml of dimethyl sulfoxide, 1 g (4.55 mmol) of zinc acetate dihydrate was added thereto, and the mixture was stirred until a homogeneous solution was obtained. A solution of 580 mg (2.3 mmol) of N-benzyloxycarbonyloxysuccinimide dissolved in 4 ml of dimethyl sulfoxide was gradually added to this solution over an hour or so, and then allowed to stand overnight at room temperature. Hereinafter, Example 1
(ii) and (iii) Treated in exactly the same manner as (A), 666 mg of white solid
(71%). [α] ²⁵ _D +77゜ (c1, hydrous dimethylformamide = 1:2) Elemental analysis C ₃₄ H ₄₈ N ₄ O ₁₈・2CH ₃ CO ₂ H・H ₂ O Theoretical value: C, 52.16; H, 6.68; N, 6.40% Analytical value: C, 51.77; H, 6.79; N, 6.31% Example 19 Production of 3, 2', 6'-tri-N-benzyloxycarbonyl kanamycin B Kanamycin B (free base) 500 mg (1.03 mmol) was suspended in a mixture of 12 ml of dimethyl sulfoxide and 4 ml of tetrahydrofuran, 1 g (4.55 mmol) of zinc acetate dihydrate was added thereto, and the mixture was stirred until a homogeneous solution was obtained. This solution was cooled to 0°C, and 825 mg (3.3 mmol) of N-benzyloxycarbonyloxysuccinimide was added to a mixture of tetrahydrofuran and dimethyl sulfoxide (1:1) for 10 minutes.
ml of the cold solution was gradually added over a period of about 1 hour, and then allowed to stand at 0° C. for 2 hours and at room temperature overnight.
Thereafter, it was treated in exactly the same manner as in Example 1 (ii) and (iii) (A) to obtain 740 mg (70%) of a white solid. [α] ²⁵ _D +63゜ (c1, hydrous dimethylformamide = 1:2) Elemental analysis C ₄₂ H ₅₅ N ₅ O ₁₆・2CH ₃ CO ₂ H・H ₂ O Theoretical value: C, 53.95; H, 6.40; N, 6.84% Analysis value: C, 53.66; H, 6.67; N, 6.63% This substance was treated in the same manner as in Reference Example 1 to produce 1-N-((S)-4-amino-2-hydroxybutylene). kanamycin B. Example 20 Preparation of 3.2'.6'-tri-N-benzyloxycarbonyl topramycin 480 mg (1.03 mmol) of topramycin (free base) was suspended in 12 ml of dimethyl sulfoxide.
To this was added 1 g (4.55 mmol) of zinc acetate dihydrate, and the mixture was stirred for 1 hour. Add 850mg of N-benzyloxycarbonyloxysuccinimide to this solution.
A solution of (3.4 mmol) dissolved in 10 ml of a mixture of tetrahydrofuran and dimethyl sulfoxide (1:1) was gradually added over about 1 hour, and then left overnight at room temperature. The reaction solution was treated with a large amount of ethyl ether in the same manner as in Example 1(ii) to obtain a hard syrup-like substance. Thereafter, the same treatment as in Example 1(iii)(A) was carried out (however, water-dioxane=2:1 was changed to 1:1) to obtain 810 mg (78%) of a white solid. [α] ²⁵ _D +65° (c1, hydrous dimethylformamide = 1:2) Elemental analysis C ₄₂ H ₅₅ N ₅ O ₁₅・2CH ₃ CO ₂ H・H ₂ O Theoretical value: C, 54.81; H, 6.50; N, 6.95% Analysis value: C, 54.77; H, 6.71; N, 6.88% This substance was treated in the same manner as in Reference Example 1 to produce 1-N-((S)-4-amino-2-hydroxybutylene). Lil) got the Tobra machine. Example 21 Preparation of 3,2',6'-tri-N-benzyloxycarbonyl-6'-N-methyltobramycin 500 mg of 6'-N-methyltobramycin free base
(1.04 mmol) was treated in exactly the same manner as in Example 20 to obtain 890 mg (84%) of a white solid. [α] ²⁵ _D +63゜(c1,
Hydrous dimethylformamide = 1:2) Example 22 Production of 3,2',6'-tri-N-benzyloxycarbonyl-4'-deoxykanamycin B 4'-deoxykanamycin B (Miyake, Tsuchiya, Umezawa, Umezawa, Bulletin of Chemical Society of Japan, vol. 50, pp. 2362-2368, 1977
Example: 480 mg (1.03 mmol) (free base)
It was treated in exactly the same manner as in Example 20 to obtain 815 mg (79%) of a white solid. [α] ²⁵ _D +63° (c1, hydrous dimethylformamide = 1:2) Example 23 Production of 3,2',6'-tri-N-benzyloxycarbonyl dibekacin Add dimethyl to 600 mg (1.33 mmol) of dibekacin free base 15 ml of sulfoxide was added and stirred. 1.4 g (6.4 mmol) of zinc acetate dihydrate was added to the resulting solution and stirred. 1.1 g of N-benzyloxycarbonyloxysuccinimide was added to the resulting solution.
A solution of (4.4 mmol) dissolved in 12 ml of dimethyl sulfoxide was gradually added over about 1 hour, and then left overnight at room temperature. A large amount of ethyl ether was added to the reaction solution to form an oily precipitate (mainly containing the desired product and dimethyl sulfoxide), which was further washed with ethyl ether to obtain a hard syrup-like substance. This was repeated and washed with water. Excess zinc acetate was removed by water treatment and the complex was also decomposed and removed with water. 1.1 g of water-insoluble solid was obtained. This solid is chloroform-methanol-18% ammonia (1:
A single point is given to Rf0.3 in the thin-layer chromatogram using a 1:1 lower layer) as the developing system, indicating that almost pure 3,2',6'-tri-N contains a small amount of zinc. -benzyloxycarbonyldibekacin. This substance was then treated in the same manner as in Reference Example 1 to obtain 1
-N-((S)-4-amino-2-hydroxybutyryl)-dibekacin. The same treatment as in Example 20 was carried out to purify the previously obtained crude 3,2',6'-tri-N-benzyloxycarbonyldibekacin. [α] ²⁵ _D +68° (c1, hydrous dimethylformamide = 1:2). Example 24 Preparation of 3,2',6'-tri-N-benzyloxycarbonyl-6'-N-methyldibekacin 500 mg of 6'-N-methyldibekacin free base
(1.07 mmol) and zinc acetate dihydrate 1.2 g (5.45
to which 910 mg (3.6 mmol) of N-benzyloxycarbonyloxysuccinimide was gradually added over about 30 minutes, and the solution was left overnight at room temperature. Thereafter, treatment was carried out in exactly the same manner as in Example 23 to obtain 910 mg of almost pure title substance. This substance was then treated in the same manner as in Reference Example 1 to obtain 1-N-
((S)-4-amino-2-hydroxybutyryl)-
This could lead to 6'-N-methyldibekacin. Example 25 Preparation of 3.2'-di-N.benzyloxycarbonylkanamycin C. Kanamycin C (free base) 500 mg (1.03 mmol) was treated in the same manner as in Example 1(i), (ii) and (iii)(A) to obtain 730 mg (79%) of a white solid. [α] ²⁵ _D +75゜(c1,
Water-containing dimethylformamide = 1:2) This substance could be converted into 1-N-((S)-4-amino-2-hydroxybutyryl)kanamycin C by subsequently treating in the same manner as in Reference Example 1. Example 26 6'-N-benzyloxycarbonyl kanamycin A 500 mg (1.03 mmol) of kanamycin A (free base) was suspended in 20 ml of dimethyl sulfoxide, and 0.5 g (2.3 mmol) of zinc acetate dihydrate was added thereto to create a homogeneous solution. Stir until . 283 mg (1.13 mmol) of N-benzyloxycarbonyloxysuccinimide was added to this solution and left overnight at room temperature. Hereinafter, Example 1(ii) and (iii)(I)
The mixture was treated in the same manner as above to obtain 556 mg of a white solid. [α] ²⁵ _D
+92° (c1, water) Example 27 6'-N-benzyloxycarbonyl dibekacin Dibekacin (free base) 500 mg (1 mole equivalent)
was dissolved in 12 ml of dimethyl sulfoxide, 0.7 g of zinc acetate dihydrate and 305 mg (equivalent to 1.1 mm) of N-benzyloxycarbonyloxysuccinimide were added and treated in the same manner as in Example 26 to obtain 382 mg of a white solid. [α] ²⁵ _D +105° (c0.5, water) Example 28 Production of 3,2',6'-tri-N-benzyloxycarbonyl-3',4'-dideoxy-3'-enokanamycin B. 3′・4′-dideoxy-3′-enokanamycin B
(Nishimura, Tsuchiya, Umezawa, Umezawa, Bulletin of Chemical Society of Japan Volume 50, 1580-
1583, 1977) (free base) was dissolved in 12 ml of dimethyl sulfoxide, 1 g (4.55 mmol) of zinc acetate dihydrate was added thereto, and the mixture was stirred for 1 hour. Add 870 mg (3.49 mg) of N-benzyloxycarbonyloxysuccinimide to this solution.
After 30 minutes of gradual addition of 1 mmol), the reaction solution was left overnight and treated with a large amount of ethyl ether in the same manner as in Example 1(ii) to obtain a hard syrup-like substance. Thereafter, the treatment was carried out in the same manner as in Example 1 (iii) (B) (however, water-dioxane = 2:1 was changed to 1:2) to obtain a white solid 784
I got mg. [α] ²⁵ _D +30° (c1, hydrous dimethylformamide = 1:2) Example 29 Production of 3,2',6'-tri-N-benzyloxycarbonylsisomicin Sisomicin (free base) 500 mg (1.12 mmol)
was treated in exactly the same manner as in Example 28 with 1 g (4.55 mmol) of zinc acetate dihydrate and 870 mg (3.49 mmol) of N-benzyloxycarbonyloxysuccinimide in dimethyl sulfoxide (12 ml) to obtain 780 mg of a white solid. [α] ²⁵ _D +110° (c1, hydrous dimethylformamide = 1:2) Example 30 Production of 3,2',6'-tri-N-benzyloxycarbonylgentamicin 500 mg of gentamicin mixture (free base) was dissolved in dimethyl sulfoxide. (12ml) medium zinc acetate dihydrate 1
g and 870 mg of N-benzyloxycarbonyloxysuccinimide in exactly the same manner as in Example 32 to obtain 787 mg of a white solid. Example 31 3,6'-bis(N-t-butoxycarbonyl)
Production of Kanamycin A Kanamycin A (10.0g) and zinc acetate dihydrate (27g) were dissolved in dimethyl sulfoxide (DMF).
(180 ml) at room temperature for 2 hours, and then added t-
Butyl S-(4,6-dimethylpyrimidine-2-
Add thiol carbonate (24g) and 50℃
The mixture was stirred for 40 hours. The solvent was distilled off under reduced pressure, and the residue was dissolved in a mixture of methanol (300 ml) and water (300 ml).
(H ⁺ form) (650 ml) was passed through the column. After washing the column with a mixture of water and methanol (1:1), it was eluted with 0.8N aqueous ammonia, and the crude target product 13.5
I got g. This was subjected to silica gel chromatography [Wakogel C-200, 300 g, developing solvent chloroform-methanol-concentrated aqueous ammonia (9:9:
1.5)], 11.0g of the title target compound
I got it. mp237-246℃ (decomposition) [α] ²³ _D +89.9゜ (c1.0, DMSO) SIMS: 685 (M+1) Example 32 3・2′・6′-Tris(N-t-butoxycarbonyl)kanamycin Production of B Kanamycin B (1.06 g) and zinc acetate dihydrate (2.4 g) were stirred in a mixture of water (2 ml) and DMF (20 ml) at room temperature for 2 hours, and then di-t-butyl-
Dicarbonate (1.65 g) was added and stirred at 30°C for 20 hours. Concentrate the reaction solution to 1/4 volume, and add n-
Butanol (40 ml), concentrated aqueous ammonia (20 ml), and saturated brine (10 ml) were added, and the mixture was stirred at room temperature for 30 minutes. Furthermore, the aqueous layer was extracted with 15 ml of n-butanol. Combine the butanol extracts and apply silica gel chromatography [Wakogel C-200, 30g,
Purification using a developing solvent of chloroform-methanol-concentrated aqueous ammonia (9:6:0.3) yields the title substance.
0.71g was obtained. mp178～188℃ [α] ²³ _D +78.9° (c1.0, DMSO) SIMS: 784 (M+1) Example 33 3・2′・6′-Tris(Nt-butoxycarbonyl)-3′・Production of 4'-dideoxykanamycin B 3',4'-dideoxykanamycin B (1.0g) and zinc acetate dihydrate (2.4g) were dissolved in water (2ml).
After stirring at room temperature for 2 hours in a mixture of 20 ml of DMF,
Di-t-butyl dicarbonate (1.65 g) was added and stirred at 30°C for 20 hours. The reaction solution was concentrated to 1/4 volume, and n-butanol (40 ml), concentrated aqueous ammonia (20 ml), and saturated brine (10 ml) were added thereto, and the mixture was stirred at room temperature for 30 minutes. The aqueous layer was extracted again with n-butanol (15ml). The butanol extracts were combined and purified by silica gel chromatography [Wako Gel C200, 30 g, developing solvent chloroform-methanol-concentrated aqueous ammonia (9:6:0.1)] to obtain 1.26 g of the title substance. mp163-170℃ [α] ²³ _D +78゜ (c1.0, DMSO) SIMS: 752 (M+1) Reference example 1 1-N-((S)-4-amino-2-hydroxybutyryl (kanamycin A (amikacin) ) Production of 3,6'-di-N-benzyloxycarbonylkanamycin A acetate 55 mg (0.062
mmol) in water-tetrahydrofuran (2:5)
Dissolved in 1.5 ml of anhydrous sodium carbonate 13 mg (0.12
After adding (S)-4-benzyloxycarbonylamino-2-hydroxybutyric acid (N-
Hydroxysuccinimide ester 23mg
(0.066 mmol) was added and left at room temperature for 10 hours.
After concentrating the reaction solution, add 4 ml of hydrous dioxane (1:1).
After adding a little acetic acid to make the liquid slightly acidic, palladium black was added and the mixture was reduced with hydrogen at normal pressure for 1 hour. After filtering and concentrating the reaction solution, the concentrate was charged to a column of CM Sephadex C-25 (NH ¹⁺ ₄ type) (manufactured by Pharmacia Fine Chemical Co., Ltd.).
Slope development was carried out with 0.5 normal ammonia water. The eluate containing the target substance was collected and concentrated to dryness to obtain 24 mg (60%) of the title substance as a monocarbonate. This substance matched the standard in terms of physical properties and antibacterial activity. Reference Example 2 Production of 1-N-(DL-isoseryl)dibekacin 58 mg (0.06
mmol) in water-tetrahydrofuran (2:5)
Dissolved in 1.5 ml of anhydrous sodium carbonate 13 mg (0.12
After adding 21 mg (0.063 mmol) of N-hydroxysuccinimide ester of N-benzyloxycarbonyl-DL-isoserine, the mixture was left at room temperature for 10 hours. Thereafter, the same treatment as in Reference Example 1 was carried out to obtain 21 mg of the title substance as a monocarbonate.
(59%) obtained. This substance matched the standard in terms of physical properties and antibacterial activity.

Claims (1)

[Claims] 1 A zinc complex produced by the action of a zinc cation on an aminoglycoside antibiotic consisting of deoxystreptamine having a 3-aminoglycosyl group or a 3-alkylaminoglycosyl group at the 6-position,
reaction with an acylating agent that introduces an acyl protecting group for the amino group, thereby forming a zinc complex of the N-acylated derivative of the aminoglycoside antibiotic complexed with the zinc cation;
A method for producing a protected derivative of the aminoglycoside antibiotic in which an amino group is selectively protected with an acyl group, the method comprising removing a zinc cation from a zinc complex of the acylated derivative.