JPS632268B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to formimidoylistamycin A, a novel compound useful as a semisynthetic aminoglycoside antibiotic, and to a method for producing the compound. The present inventors conducted research focusing on the effectiveness of formimidoylhoteimycin A (SF-2052 substance, Journal of Antibiotics, Vol. 32, pp. 1354-1356, 1979), and the present inventors Istamycin B (patent application No. 54-52517) discovered by
Formimidoylistamycin B was synthesized by formimidoylating the amino group of the glycine moiety of hortimycin A (Japanese Patent Application No. 1984-41184). It was confirmed that it has antibacterial properties and low toxicity. Therefore, the present inventors further developed formimidoyl istamycin A by forming the amino group of the glycine moiety of istamycin A.
were synthesized, and the present invention was completed. The gist of the first invention is that a new compound is prepared by the following formula () Formimidoylistamycin A represented by
and its acid addition salts. The physicochemical and biological properties of this new compound are as follows. Formimidoylistamycin A disulfate trihydrate is a white powder with a decomposition point of 202-208°C.
[α] ²⁷ _D +82° (c1, water) is shown. The elemental analysis value is
Showing C32.34%, H6.52%, N11.82%, S9.69%,
Theoretical value of C ₁₈ H ₃₆ N ₆ O ₅・2H ₂ SO ₄・3H ₂ O (C32.42
%, H6.95%, N12.60%, S9.62%).
Thin layer chromatography of cellulose shows a single spot (ninhydrin coloring) at Rf0.39 using propanol/pyridine/acetic acid/water (15:10:3:12 volume ratio) as the developing solvent. The antibacterial spectrum of formimidoylistamycin A disulfate trihydrate obtained in the present invention is as shown in Table 1, and is similar to that of istamycin A.

【table】

【table】

[Table] In an acute toxicity test of intravenous administration of formimidoylistamycin A (disulfate trihydrate) to mice, the mice survived at a dose of 300 mg/Kg. The formimidoylistamycin A according to the first invention can be obtained as a free base or as a hydrate or carbonate, but with the addition of a pharmaceutically acceptable acid by conventional methods to any other non-toxic acid. Addition salts are more preferred in terms of stability of the substance. As the acid to be added, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid, and organic acids such as acetic acid, malic acid, citric acid, ascorbic acid, and methanesulfonic acid are used. The gist of the second invention is the following formula () Istamycin A ₀ expressed as
117912) as a raw material to produce formimidoylistamycin A. That is, in the method of the present invention, istamycin A ₀
The amino group or methylamino group at the 1-position, 2'-position and 6'-position of is simultaneously protected with a known amino protecting group to form the following formula () A compound represented by [in the formula R represents a monovalent amino protecting group] is produced, and this compound is reacted with glycine or a reactive derivative thereof whose amino group has been previously protected with an amino protecting group different from the above. The methylamino group at the 4-position of the compound () is acylated to form the following formula (). [In the formula, R is the same as above, A is a hydrogen atom,
B is a monovalent amino protecting group, or A, B
together represent one divalent amino-protecting group], and then selectively remove only the amino-protecting group of the glycine moiety [in formula (), R is Same as above,
A compound in which the amino group of glycine is converted to an amidine group by reacting with an imino ether such as an alkyl or aralkyl formimidate [formula () corresponding to the case where A and B are both hydrogen atoms]
, R is the same as above, A is a hydrogen atom, and B is a formimidoyl group (-CH=NH)], and then the 1-, 2'-, and
By removing the amino protecting group at the 6′ position, the following formula () can be obtained. Formimidoylistamycin A represented by
or its acid addition salt can be produced. Next, embodiments of the method of the present invention will be described. Although the formula and the conformation of the cyclitol moiety in the formula described to explain the method of the present invention are not yet clear, the formula is described according to the conformation of formimidoylistamycin A (formula) for convenience. This method, in short, istamycin A ₀ (formula)
Formimidoylistamycin A according to the present invention is synthesized by starting from , and acylating its 4-position methylamino group with formimidoylglycine. Istamycin A ₀
When acylating the methylamino group at the 4-position, it is preferable to protect the 1- and 2'-position amino groups and the 6'-position methylamino group with an amino protecting group in advance. Typical monovalent amino protecting groups that protect these amino groups and methylamino groups include alkoxycarbonyl groups such as tert-butoxycarbonyl group and tert-amyloxycarbonyl group, and cycloalkyloxycarbonyl groups such as cyclohexyloxycarbonyl group. group, an aralkyloxycarbonyl group such as a benzyloxycarbonyl group, an acyl group such as a trifluoroacetyl group and an orthonitrophenoxyacetyl group, and the like. These amino-protecting groups can be introduced by known methods used in peptide synthesis, etc., using known amino-protecting group-introducing agents in the form of acid halides, acid azides, active esters, acid anhydrides, and the like. By using these amino protecting group introducing agents in the range of 2.5 to 3.5 molar equivalents to istamycin A ₀ , istamycin
Due to the difference in reactivity of each amino group and methylamino group of A ₀ , the 1,2',6'-tri-N-protected product can be preferentially obtained. Next, the methylamino group at the 4-position of this 1,2',6'-tri-N-protected product is glycylated, and this glycylation reaction can be carried out using the dicyclohexylcarbodiimide method, mixed acid anhydride method, azide method, Any known peptide synthesis method, such as the active ester method, can be carried out in the presence of glycine or a reactive derivative thereof. The amino groups of the glycine used are preferably protected with an amino protecting group in advance for this acylation reaction, and the amino groups at the 1- and 2'-positions and the methylamino groups at the 6'-position of istamycin A ₀ It must be an amino-protecting group that can be easily removed in distinction from the amino-protecting group that protected the group. Therefore, it is selected from among the above-mentioned amino protecting groups and divalent amino protecting groups such as Schiff bases. For example, the methylamino group at the 4-position of 1,2',6'-tri-N-benzyloxycarbonyl istamycin A ₀ is acylated with N-hydroxysuccinimide ester of N-tert-butoxycarbonylglycine. Compounds with protected amino groups and methylamino groups (in formula (),
When R is a benzyloxycarbonyl group, A is a hydrogen atom, and B is a tert-butoxycarbonyl group)
is one of the preferred intermediates in the present invention. The acylation reaction with glycine is preferably carried out in an organic solvent such as dioxane using the active ester method at a temperature of 40-60°C. This produces the compound of formula () above. Next, by selectively deprotecting only the amino group of the glycine moiety of the above-mentioned compound () in which the amino group and methylamino group are protected, the 1,2',6'-tri-N- A protected body (corresponding to the case where R represents an amino protecting group and A and B are both hydrogen atoms in the formula ()) is obtained. At this time, in the case of an aralkyloxycarbonyl group, the reaction of removing the protecting group from the amino group of the glycine moiety of the compound of formula () is carried out by hydrogenolysis using palladium, platinum oxide, etc. as a catalyst; In the case of amino protecting groups, this is generally carried out by hydrolysis by treatment in an aqueous solution such as trifluoroacetic acid, acetic acid, or a dilute solution such as hydrochloric acid. Furthermore, the amino group of the glycine moiety of the 1,2',6'-tri-N-protected form of istamycin A thus obtained is converted to an amidine group. Hydrochlorides of iminoethers of the formula R′OCH=NH ₂ Cl, where R′ is a lower alkyl group or an aralkyl group such as benzyl group; iminoethers such as ethylformimidate hydrochloride and benzylformimidate hydrochloride; The reaction can be carried out by a known method using a hydrochloride of , in an organic solvent such as dioxane or methanol, or in an aqueous solution at a temperature of 30° C. or lower. Obtained formimidoylistamycin A
The hydrochloride of the 1,2',6'-tri-N-protected product is purified by column chromatography using silica gel or the like. Next, the amino protecting group is removed by a known deprotection method as described above, whereby the desired formimidoylistamycin A can be obtained in the form of any acid salt. Next, the present invention will be explained with reference to Examples. Example 1 (a) Synthesis of 1,2',6'-tri-N-benzyloxycarbonyl Istamycin A ₀ (when R is a benzyloxycarbonyl group in the formula): Istamycin A ₀ 1.0 g (3.0 mmol) Dissolve in 40ml of methanol and add 0.72ml of triethylamine.
ml, then add 2.1 g of N-benzyloxycarbonyloxysuccinimide while stirring.
A solution of (8.1 mmol) dissolved in 12 ml of methanol was added, and the mixture was stirred at room temperature for 2 hours. After the reaction solution was concentrated to dryness, it was dissolved in 60 ml of chloroform, washed with 60 ml of water, and the chloroform layer was concentrated to dryness to obtain 2.17 g of crude powder. This was dissolved in 3 ml of dichloromethane, poured into a column filled with 55 g of silica gel (CC-7 manufactured by Mallinckrodt), and dichloromethane/ethanol (150:1) was added.
The mixture was washed with 560 ml of a mixture of (20:1) and then developed and eluted with a mixture of (20:1) to obtain 931 mg of the title compound as a powder. Yield 42%. (b) 1,2',6'-tri-N-benzyloxycarbonyl-2''-N-tert-butoxycarbonyl istamycin A (in the formula, R is a benzyloxycarbonyl group, A is a hydrogen atom, and B is a Synthesis of 3-butoxycarbonyl group): 1,2',6'-tri-N-benzyloxycarbonyl istamycin A obtained in (a) ₀ 931 mg
(1.27 mmol) in 30 ml of dioxane,
A solution of 0.42 ml of triethylamine and 830 mg (3.0 mmol) of N-hydroxysuccinimide ester of N-tert-butoxycarbonylglycine dissolved in 5 ml of dioxane was added, and the mixture was stirred at 60°C for 4.5 hours. The reaction solution was concentrated to dryness to obtain a crude powder. Dissolve this in 5 ml of dichloromethane and add 150 g of silica gel (CC-7 manufactured by Mallinckrodt).
The crude powder obtained by elution and elution with a mixture of dichloromethane and ethanol (200:1) was further dissolved in 5 ml of methanol and added to Cephadex LH20 (manufactured by Pharmacia).
The mixture was purified by applying it to a 100 ml column and developing with methanol to obtain 438 mg of the title compound as a powder. Yield 39%. (c) Formimidoylistamycin A (formula)
Synthesis: Add 388 mg (0.44 mmol) of 1,2',6'-tri-N-benzyloxycarbonyl-2''-N-tert-butoxycarbonyl Istamycin A obtained in (b) to 5 ml of 90% trifluoroacetic acid aqueous solution. After stirring, leave at room temperature for 45 minutes, concentrate to dryness, and remove ether.
After washing with 20 ml, 375 mg of trifluoroacetate of 1,2',6'-tri-N-benzyloxycarbonylistamycin A was obtained. Add this to methanol
A solution of 426 mg (2.5 mmol) of benzylformimidate hydrochloride dissolved in 10 ml of methanol was dissolved in a mixture of 60 ml of water and 8 ml of water, and the pH was adjusted to 8.5 with 0.5 N potassium hydroxide under ice-cooling.
It was added dropwise over a period of minutes. The reaction solution was further stirred for 1 hour under ice cooling (formimidoyl group -CH=NH
(introduction reaction), the pH was adjusted to 3.7 with 1N hydrochloric acid, and the mixture was concentrated to dryness. Dissolve the residue in 100ml of chloroform,
After washing twice with 30 ml of water, the chloroform layer was concentrated to dryness to obtain 411 mg of crude powder. This was dissolved in 3 ml of a mixture of chloroform and methanol (20:1), and silica gel (CC-
7) Pour into a column packed with 20 g and purify by chromatography developed with the same solvent to obtain 1, 2',
6'-tri-N-benzyloxycarbonyl-
193 mg of powder of 2''-N-formimidoylistamycin A hydrochloride was obtained. Yield: 54%. 193 mg (0.24 mmol) of this powder was dissolved in 12 ml of a mixture of methanol, acetic acid, and water (2:1:1). , 100 mg of 5% palladium on carbon was added, and deprotection was carried out by hydrogenolysis at room temperature in a hydrogen stream for 4 hours. The catalyst was removed and concentrated to dryness. The residue was dissolved in 1 ml of water, and Amberlite IRA-400 (SO ₄
The mixture was poured into a 12 ml column (type (manufactured by Rohm & Haas)), developed with water for salt exchange, and concentrated to dryness to obtain 134 mg of a powder of the disulfate trihydrate of the title compound. Yield 82%.

Claims (1)

[Claims] Linear formula () Formimidoylistamycin A represented by
and its acid addition salts. Quadratic formula () The amino group or methylamino group at the 1-position, 2'-position and 6'-position of istamycin A ₀ represented by is simultaneously protected with a known amino protecting group, and the following formula () is obtained. A compound represented by [in the formula R represents a monovalent amino protecting group] is produced, and this compound is reacted with glycine or a reactive derivative thereof whose amino group is protected with an amino protecting group different from the above. By acylating the 4-position methylamino group of compound (),
The following formula () [In the formula, R is the same as above, A is a hydrogen atom, and B
is a monovalent amino protecting group, or A and B together represent one divalent amino protecting group]
A compound represented by is produced, and then only the amino protecting group of the glycine moiety is selectively removed [In formula (), R is the same as above, A, B
When both become hydrogen atoms], this compound is then reacted with iminoether to convert the amino group of the glycine moiety into an amidine group [In formula (), R is the same as above. In this case, A is a hydrogen atom and B is a formimidoyl group (-CH=NH).
The following formula () is characterized in that the amino protecting groups at the 1-, 2'- and 6'-positions are subsequently removed. Formimidoylistamycin A represented by
manufacturing method.