DE2741431C3 - IN- (L-4-amino-2-hydroxybutyryl) -3'-deoxykanamycin-C, IN- (L-4-amino-2-hydroxybutyryl) -3 ', 4'-dideoxykanamycin-C and their acid addition salts, processes for their preparation and antibacterial compositions containing these compounds - Google Patents

Description

A) the 1-amino group of 3'-deoxykanamine-C or 3 ', 4'-Dideoxykanamycin-C by reaction with a protected on the amino group

th derivative of L-4-amino-2-hydroxy-butyric acid or a functional equivalent acylated therefrom,

B) the amino protecting group from the one obtained

1-N-acylation product is split off and

C) the compounds obtained are optionally converted into their acid addition salts

3. Antibacterial compositions, characterized in that they contain at least one compound according to claim 1 together with customary carriers and diluents.

The invention relates to

l-N- (L-4-amino-2-hydroxybutyryl) -3'-deoxykanamycin-C,

l-N- (L-4-amino-2-hydroxybutyryl) -3 ', 4'-dideoxykanamycin-C

and their acid addition salts, a process for producing these compounds and these compounds containing antibacterial compositions.

It is already known that butirosin-B, ie 1 -N- (L-4-amino-2-hydroxybutyryl) -ribostamycin can be prepared by a fei mentation method, see Tetrahedron Letters, 28 (1971), pp. 2617-2620 and that IN- (L-4-amino-2-hydroxybutyryl) derivatives of Kanamycin-A and Kanamycin-B have been synthesized, see US Pat. No. 3,781,268. IN- (L-4-Amino-2-hydroxybutyryl) - derivatives of some other aminoglycoside antibiotics have also been synthesized, see British Patent 14 26 908. Furthermore, deoxy derivatives of kanamycins were also synthesized on the basis of the earlier discoveries made by H. U me ζ awa et. al. regarding the mechanism of resistance of bacteria to aminoglycoside antibiotics as a result of the various inactivating enzymes produced by the resistant bacteria. For example, 3 ', 4'-dideoxykanamycin-B and 3'-deoxykanamycin-B were synthesized, which are active against the resistant bacteria which produce aminoglycoside 3'-phosphotransferase, see US Pat. No. 3,753,973 and 3,929,762 and H. Umezawa "Advances in Carbohydrate Chemistry and Biochemistry", 30 (1974), p. 183 and "Drug Action and Drug Resistance in Bacertia", 2 (1975), p. 211.3 ', 4'-Dideoxykanamycin-B has been used in therapeutic treatment widely used by infections caused by a variety of resistant bacteria including Pseudomonas aeruginosa. However, it has been found that these deoxy derivatives of K.anamycin-B do not inhibit the growth of such resistant bacteria which are capable of producing aminoglycodide-e'-acetyltransferase and 2 "-nucleotidyltransferase. As a result of further investigations, the applicant succeeded to synthetically convert the 6'-amino group of kanamycin-B or its deoxy derivatives into the hydroxyl group and thereby produce kanamycin-C and its deoxy derivatives, which of course cannot be inactivated by the 6'-acetyltransferase, see DE-OS 27 24 597.
However, kanamycin-C, 3'-deoxykanamycin-C and 3 ', 4'-dideoxykanamycin-C obtained in this way are unable to inhibit the growth of those resistant bacteria which produce 2 "-nucleotidyltransferase.

The object of the invention was therefore to provide such new kanamycin derivatives which are by nature not due to the strong presence of bacteria in a large number of aminoglycoside-resistant bacteria 3'-Phosphotransferase can be inactivated, and also not by the 6'-Acetyltransferase and the 2 "-Nucleotidyltransferase can be inactivated, so that the new compounds not only are active against the aminoglycoside-resistant, 3'-phosphotransferase-producing bacteria, but also against the 6'-acetyltransferase producing, aminoglycoside-resistant bacteria and the 2 "-nucleotidyltransferase producing, aminoglycoside-resistant bacteria are active and yet have a very low level of activity Have toxicity, as well as a process for the preparation of these Kanamycin-C derivatives, which in simple Can be done wisely and with reasonable effectiveness.

Based on investigations by the applicant it has now been found that l-N- (L-4-amino-2-hydroxybutyryl) derivatives of 3'-deoxykanamycin-C and 3 ', 4'-dideoxykanamycin-C and their acid addition salts to a wide variety of aminoglycoside resistant bacteria including the 2 "nucleotidyl transferase producing bacteria are active.

3 4

The compounds according to the invention have the following general formula:

CH ₂ OH
HO

CO

(L) CHOH CH ₂ CH ₂ NH ₂

in which, in the case of the 3'-deoxykanamycin C derivative, R _{1 is} a hydrogen atom and R _{2 is} a hydroxyl group, and in the case of the 3 ', 4'-dideoxykanamycin C derivative, R 1 and R 2 are each hydrogen atoms.

The chemical, physical and biological properties of the compounds according to the invention are as follows:

IN- (L-4-amino-2-hydroxybutyryl) -3'-deoxykanamycin-C is a substance in the form of a colorless powder with no defined melting point, but which decomposes at 151-160 ° C. It shows a specific optical rotation [a] | ⁷ + 83 ° (c = 1, water). Its elemental analysis agrees with the theoretical values for

C ₂₂ H ₄₃ N ₅ O ₁₂ • H ₂ O

matched (C = 44.96%; H = 7.72%; N = 11.92%). This substance gives a single, compared to ninhydrin positive spot at Rf = 0.22 in the aforementioned thin layer chromatography on silica gel and the development with butanol-ethanol-chloroform ■ 17% aqueous ammonia (4: 5: 2: 8 by volume) as developing solvent and at Rf = 0.24 with the same thin layer chromatography and development with chloroform-methanol-28% aqueous ammonia (1: 4: 2 by volume).

IN- (L-4-amino-2-hydroxybutyryl) -3 ', 4'-dideoxykanamycin-C is also a substance in the form of a colorless powder with no defined melting point, but which decomposes at 142-158 ° C. It shows a specific, optical rotation [α]! "+ 76 ° (c = 1, water). Its elementary analysis agrees with the theoretical values for C22H43N5O11 · H2O (C = 46.22%; H = 7.94%; N = 12.25%). It gives a single, ninhydrin-positive spot at Rf = 0.31 in the aforementioned thin-layer chromatography on silica gel and development with the first-mentioned developer solvent and at Rf = 0.30 in the same thin-layer chromatography and development The molecular structure of these new kanamycin C derivatives was identified by acid hydrolysis and by the absorption spectra of the nuclear magnetic resonance of ¹ H and ¹³ C.
The minimum inhibitory concentrations ^ g / ml) of 1 -N- (L-4-amino-2-hydroxybutyryl) -3'-deoxykanamycin-C (hereinafter abbreviated as AHB-DKC) and of IN- (L-4-amino -2-hydroxybutyryl) -3 ', 4'-dideoxykanamycin-C (hereinafter abbreviated as AHB-DDKC) against various microorganisms were determined by the serial dilution method on nutrient agar medium at 37 ° C, the assessment being carried out after an incubation of 18 hours . For comparison, the minimum inhibitory concentrations of IN- (L-4-amino-2-hydroxybutyryl) -kanamycin-A (amikacin) were also determined in the same manner described above

The antibacterial spectra of these substances are compiled in Table I below.

Table I.

Minimum inhibitory concentration in [ig / m \ AHB-DKC AHB-DDKC Amikacin 6.25 12.5 (Comparison) Test organisms 12.5 6.25 Staphylococcus aureus FDA 209P 6.25 12.5 0.39 Mycobacterium smegmatis ATCC 607 6.25 12.5 <0.20 Escherichia coli NIHJ 6.25 12.5 0.78 Escherichia coli K-12 6.25 6.25 0.78 Escherichia coli K-12 R5 0.39 Escherichia coli K-12 ML 1629 0.78

continuation

Minimum Heram concentrations in [/ g / ml AHB-DKC AHB-DDKC Amikacin 12.5 12.5 (Comparison) Test organisms 6.25 25th Escherichia coli K-12 ML 1630 12.5 25th 0.78 Escherichia coli K-12 ML 1410 12.5 12.5 0.78 Escherichia coli K-12 ML 1410 R 81 6.25 6.25 1.56 Escherichia coii LA 290 R55 3.13 12.5 0.78 Escherichia coli LA 290 R56 6.25 12.5 0.39 Escherichia coli LA 290 R64 12.5 25th 0.39 Escherichia coli W 677 3.13 6.25 <0.20 Escherichia coli Jr66 / W677 6.25 25th 1.56 Klebsieila pneumoniae PCI 602 6.25 12.5 0.39 Klebsiella pneumoniae 22 No. 3038 50 100 1.56 Pseudomonas aeruginosa A3 50 100 3.13 Pseudomonas aeruginosa No. 12th 50 100 3.13 Pseudomonas aeruginosa TI-13 100 100 3.13 Pseudomonas aeruginosa GN 315 50 100 100 Pseudomonas aeruginosa 99 6.25 Pseudomonas aeruginosa H 11 12.5

It can be seen from Table I that the novel compounds of the invention inhibit the growth of numerous types of bacterial strains. The new compounds according to the invention also have an extremely low acute toxicity in animals and in humans. It has been estimated that AHB-DKC has an LD ₅₀ of greater than 400 mg / kg when injected intravenously into mice. The new compounds according to the invention can therefore be used as chemotherapeutic agents for the therapeutic treatment of infections which are caused by gram-negative and gram-positive bacteria.

IN- (L-4-amino-2-hydroxybutyryl) -kanamycin-A
(Amikacin) and IN- (L-4-amino-2-hydroxybutyryl) -kanamycin-B were described by Kawaguchi et. al., US Pat. No. 3,781,268, and these known compounds are acetylated by a 6'-acetyltransferase, w-.ilche from aminoglycoside-resistant strains such as Pseudomonas aeruginosa GN 315, see M. Yagisawa et. al., J. Antibiot, 28 (1975), p. 486, and in this respect they differ from the novel compounds according to the invention, which are not inactivated by 6'-acetyltransferase.

The novel compounds of the invention can easily be converted into a pharmaceutically acceptable form Acid addition salt are converted, e.g. B. in the hydrochloride, sulfate, phosphate, nitrate, acetate, maleate, Fumarate, succinate, tartrate, oxalate, citrate, ascorbate, methanesulfonate, ethanesulfonate and the like, and by reacting the free base in the form of the 1 -N- (L-4-amino-2-hydroxybutyryl) derivative of

3'-Deoxykanamycin-C or 3 ', 4'-Dideoxykanamycin-C with the appropriate acid in an aqueous medium. The new kanamycin C compounds according to the invention and their pharmaceutically acceptable acid addition salts can be administered orally, intraperitoneally, intravenously, subcutaneously, or intramuscularly using a any pharmaceutical form known per se in the art for such application and can be applied in a manner similar to the known Kanamycins. For example, the new, Compounds of the invention orally using any pharmaceutical per se known form for oral administration. Examples of pharmaceutical forms for the Oral application are powders, capsules, tablets, syrups and the like. A suitable dosage of the new, Compounds according to the invention for the effective treatment of bacterial infections lie in one Range from 0.5 to 4 g per person per day when given orally. It is preferred that this dosage is administered orally in three or four portions per day. The new compounds according to the invention can also be administered by intramuscular injection at a dose of 200 to 2000 mg per person applied twice to four times a day. In addition, the new compounds according to the invention to an ointment for external application, which contains the active compound at a concentration of 0.5 contains up to 5% by weight in connection with a known ointment base such as polyethylene glycol will. In addition, the new compounds according to the invention for the sterilization of surgical Instruments usable.

The invention further relates to antibacterial compositions which

l-N- (L-4-amino-2-hydroxybutyryl) -

3'-deoxykanamycin-C or

l-N- (L-4-amino-2-hydroxybutyryl) -3 ', 4'-dideoxykanamycin-C

or an acid addition salt thereof in an antibacterially effective amount to inhibit the growth of bacteria in combination with a carrier or diluent for the active ingredient.
The invention also relates to a method for

Preparation of the compounds according to the invention, the " is characterized in that it comprises the following stages:

Acylation of the 1-amino group of 3'-deoxykanamycin-C or 3 ', 4'-Dideoxykanamycin-C by reaction with one protected on the amino group Derivative of L-4-amino-2-hydroxybutyric acid or a functional equivalent thereof per se known manner with formation of the 1-N-acylation product the starting compound and removal of the amino protective group from the 1-N-acylation product obtained in a manner known per se.

The process according to the invention can, as a further stage, convert the compound thus obtained into Form of the base with a pharmaceutically acceptable acid to form the corresponding, pharmaceutically acceptable acid addition salt.

Protecting group for the L-4-amino-2-hydroxybutyric acid can be a monovalent amino protecting group like an alkyloxycarbonyl, cycloalkyloxycarbonyl or aralkyloxycarbonyl group or a divalent group Amino protective group such as a phthaloyl group or a Schiff base group of the formula N = CHR, wherein R represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, or an aryl group such as Is phenyl group.

Such amino protective groups are known amino protective groups as they are usually can be used in the conventional synthesis of peptides. However, it must be used Amino protecting group must be such that it can be readily released in such a manner and under such reaction conditions can be removed which between the L-4-amino-2-hydroxybutyryl substituent and the Amino bond present in the 1-amino group of the aminoglycosidic unit of the acylation product obtained Do not interrupt the removal of the amino protective group from the 1-N-aminoalkanoyl substituent of the acylation product is carried out.

The introduction of the amino protective group in the L-4-amino-2-hydroxybutyric acid can be carried out by reaction the acid with a suitable reagent to introduce the amino protecting group, which is in the form an acid halide. an acid azide, an active ester or an acid anhydride is achieved are, as described for example in US-PS 39 29 762 and 39 39 143 and in GB-PS 14 26 908 is.

The starting compounds 3'-deoxykanamycin-C used in the process according to the invention and 3'.4'-Dideoxykanamycin-C and a process for their preparation are in DE-OS 27 24 597 described.

The acylation stage of the process according to the invention can generally be carried out as this is described in GB-PS 14 26 908 or US-PS 40 01 208

The acylation product obtained in this way is in the form of the mixed acylation products, which the desired l-N- (L-4-protected amino-2-hydroxybutyryl) derivative as well as the others which are not desired mono-N-acylated, di-N-acylated and poly-N-acylated Products include one or more of the 1-amino, 3-amino, 2'-amino and possibly 3 "amino groups of the kanamycin C moiety are acylated. The acylation product, i.e. the mixture of Acylation products can be used as such directly in the second stage of the process according to the invention Removal of the amino protective group are treated, this being known in itself for peptide synthesis Manner is carried out. All amino protective groups of the aforementioned kind can easily be replaced by weak ones Acid hydrolysis using an aqueous solution of trifluoroacetic acid or acetic acid or dilute hydrochloric acid. When the amino protecting group is an aralkyloxycarbonyl group, this can also be removed by ordinary hydrogenolysis.

That obtained in the second stage of the process according to the invention, freed from the protective groups The acylation product is also in the form of the mixed products, this being the desired one 1-N-Acylierup.gsprodukt as well as other acylated derivatives of the starting kanamycin-C compound together with unreacted starting kanamycin C-compound. To isolate the desired 1-N-acylation product from the mixed Chromatographic separation can be carried out on products, e.g. B. an ion exchange chromatography using a cation exchange resin containing carboxyl functions and carboxymethyl cellulose or by ion exchange chromatography using a strong Anion exchange resin or by column chromatography using silica gel. on in this way, the isolation and recovery of the desired product can be achieved with high efficiency will. In chromatography and the use of a weak cation exchange resin containing carboxyl functions dilute aqueous ammonia is used as the developer solvent. Here it is possible to use both the desired end products and unreacted starting kanamycin C compound in an effective manner and in a pure state. Depending on the chromatographic used Method is the desired Kanamycin-C derivative in the form of its free base, the hydrate or the Carbonates won.

The invention is explained in more detail with reference to the following examples.

example 1

Synthesis of l-N- (L-4-amino-2-hydroxybutyryl) -3'-deoxykanamycin-C

A solution of 440 mg (0.92 mmol) of the free base 3'-deoxykanamycin-C (hemihydrate). in 8.8 ml of water were adjusted to pH = 6.60 by adding 2.75 ml of 1N hydrochloric acid a solution of 449 mg (1.42 mmol) of the N-hydroxysuccinimide ester dropwise over 10 minutes of L ^ -tert-butoxycarbonylamino ^ -hydroxybutyric acid in 4.7 ml of dimethylformamide added at ambient temperature with stirring The mixture was stirred for an additional 6 hours at ambient temperature to induce acylation Reaction solution which contains the l-N- (L-4-tert-butoxycarbonylamino-2-hydroxybutyryl) -3'-deoxykanamycin-C was concentrated to dryness under reduced pressure. The solid residue was with 11 ml of an aqueous solution of 90% trifluoroacetic acid mixed and the mixture brought to ambient temperature for removal to occur the tert-butoxycarbonyl-amino protecting group stirred. The reaction solution was under reduced Pressure was reduced to dryness and the residue was removed

twice with 20 m! Washed ethyl ether, with 2.12 g of a crude product of l-N- (L-4-amino-2-hydroxybutyryl) -3'-deoxykanamycin-C as a colorless powder were obtained.

This crude product was taken up in 15 ml of water, and the resulting aqueous solution was adjusted to pH 7.8 by adding 1N aqueous ammonia, and then through a column with an inner diameter of 20 mm with 220 ml of carboxyl group-containing cation exchange resin in the NH -i-form sent to carry out the adsorption of the desired product. After the column was washed with 1040 ml of water, it was washed with 1340 ml of 0.2 N aqueous ammonia and then with 32Om! 0.5 N aqueous ammonia eluted. The eluate was collected in 20 ml fractions. Fractions 71-80 of the eluate obtained using 0.2N aqueous ammonia were combined and concentrated to dryness under reduced pressure to recover 149 mg of unreacted 3'-desgxykanarnycin-C (yield 34%). Fractions 134-145 of the eluate obtained using 0.5N aqueous ammonia were also combined and concentrated to dryness under reduced pressure to give 155 mg of a colorless powder containing the desired product. This powder was suspended in 2 ml of a mixed solvent of chloroform-ethanol-17% aqueous ammonia (1: 4: 2 by volume), and the suspension thus obtained was passed through a column having an inner diameter of 10 mm with 25 g of silica! Hindu sent, this was then developed with the aforementioned mixed solvents. The dispensed liquid was collected in 3.2 ml fractions. The combined fractions 99-140 were concentrated to dryness under reduced pressure and the solid residue was taken up in water and _{chromatographed on a column of 5 ml of a carboxyl group-containing cation exchange resin in the NH 4} form using 0.5N aqueous ammonia as the eluent . The eluate was concentrated to dryness under reduced pressure. IN- (L-4-amino-2-hydroxybutyryl) -3'-deoxykanamycin-C was obtained as a colorless powder in a yield of 46 mg = 8.5%. This powder had no definite melting point but decomposed at 151-160 ⁰ C. [ «] '"' + 83 ° (c = 1, water).

Example 2

Synthesis of i-N- (L-4-Aiiiifiü-2-iiyuruxybuiyry!) - 3 ', 4'-dideoxykanamycin-C

A solution of 340 mg (0.74 mmol) of the free base 3 \ 4'-Dideoxykanamycin-C (hemihydrate) in 7.5 ml of water was adjusted to pH 6.70 by adding 2.45 ml of 1N Hydrochloric acid adjusted To this solution was added dropwise a solution of 373 mg (1.18 mMo!) of the N-hydroxysuccinimide ester of L-4-tert-butoxycarbonylamino-2-hydroxybutyric acid in 3.75 ml of dimethylformamide at ambient temperature with stirring. The mixture thus obtained became further stirred for 6 hours to bring about the acylation. The l-N- (L-4-tert.-

Butoxycarbonylamino-2-hydroxybutyryl) -3 ', 4'-dideoxykanamycin-C contained reaction solution concentrated to dryness under reduced pressure, and the solid residue was washed with 8.5 ml of an aqueous Mixed solution of 90% trifluoroacetic acid. The mixture was left for 1 hour at ambient temperature stirred to bring about the removal of the tert-butoxycarbonyl-amino protective group. The reaction mixture was then concentrated to dryness under reduced pressure and the residue became washed twice with 20 ml of ethyl ether, whereby 1.46 g of a crude product of l-N- (L-4-amino-2-hydroxybutyryl) -3 ', 4'-dideoxykanamycin-C were obtained as a colorless powder.

This crude product was taken up in 14 ml of water and the aqueous solution obtained was adjusted to pH 7.8 by adding 1N aqueous ammonia. The solution was then through a column with an inner diameter of 20 mm with 220 ml of carboxyl groups containing calcium ion exchange resin sent through in the NH-i form for adsorption of the active compounds. after the The column was washed with 1040 ml of water, it was washed with 1560 ml of 0.2 N aqueous ammonia and then eluted with 1460 ml of 0.5N aqueous ammonia, the eluate being collected in 20 ml fractions became. Fractions 74-93 of the eluate obtained with 0.2N aqueous ammonia were mixed together combined and concentrated to dryness under reduced pressure, with 143 mg of unreacted 3 ', 4'-Dideoxykanamycin-C (Yield of recovery 45%) were recovered. Fractions 152-168 of the obtained with 0.5N aqueous ammonia Eluates were also combined with one another and concentrated to dryness under reduced pressure, whereby 116 mg of a colorless powder containing the desired product were obtained.

This powder was suspended in 2 ml of a mixed solvent of chloroform-ethanol-17% aqueous ammonia (1: 4: 2 by volume), and the resulting suspension was applied to a column having an inner diameter of 10 mm of 25 g of silica gel, this was then eluted with the aforementioned mixed solvent. The discharged liquid was collected in 3.2 ml fractions, and fractions 71-96 were combined and concentrated to dryness under reduced pressure. The residue was dissolved in water and the solution was chromatographed on a column of 5 ml of carboxyl group-containing cation exchange resin in the NH4 form in the same manner as before using 0.5N aqueous ammonia as the eluent. The eluate was concentrated to dryness under reduced pressure, whereby 35 mg of IN- (L-4-amino-2-hydroxybutyryl) -3 ', 4'-dideoxykanamycin-C were obtained as a colorless powder in a yield of 8.3%. This product did not show a definite melting point but decomposed at 142-158 ° C.
[a] g> + 76 ° (c =!, water).

Claims (2)

Patent claims: 1.1 -N- (L-4-amino-2-hydroxybutyryl) -3'-deoxykanamycin-C,
IN- (L-4-amino-2-hydroxybutyryl) -3 ', 4'-dideoxykanamycin-C
and their acid addition salts. 2. Process for the preparation of the compounds according to claim 1, characterized in that one each in a manner known per se