NO139562B - ANALOGICAL PROCEDURE FOR PREPARING ANTIBIOTIC DERIVATIVES XK-62-2 - Google Patents

Description

The present invention relates to the production of terra-

therapeutically active l-N-[L-(-)-α-hydroxy-Y-aminobutyryl]-XK-62-2 with the formula:

or pharmaceutically acceptable acid addition salts thereof.

Antibiotic XK-62-2 and its production by

fermentation is described in British Patent No. 1,399•578. As stated in this patent, antibiotic XK-62-2 is readily produced by cultivation of actinomycetes such as Micromonospora sagamiensis, Micromono-

spora echinospora and Micromonospora purpurea, by methods usually used in the cultivation of actinomycetes. More specifically, strains of the above-mentioned microorganisms are inoculated into a liquid medium containing a carbon source that the microorganism can utilize,

such as sugars, hydrocarbons, alcohols, organic acids, etc.; inorganic or organic nitrogen sources and inorganic salts and growth inhibiting factors and grown at 25-^0°C for 2-12

days. The isolation and purification of XK-62-2 is carried out by a suitable combination of adsorption and desorption from ion out-

exchange resins and activated carbon and column chromatography using cellulose, "Sephadex"® and silica gel. In this way, XK-62-2 can be obtained in the form of the sulfate or in the free form.

XK-62-2 is a basic substance and is obtained as a white powder. XK-62-2 has the molecular formula<n><c>2<oH>4l<N>5°7 and molecular weight ^63. The substance is easily soluble in water and methanol, poorly soluble in ethanol and acetone and insoluble in chloroform, benzene, ethyl acetate and n-hexane.

The derivatives of XK-62-2 produced according to the invention have

a strong antibacterial activity against a number of gram-positive and gram-negative bacteria and in particular has a significant antibacterial activity against those bacteria which are resistant to known aminoglycoside antibiotics. As a result of this, the manufactured antibiotics are useful for cleaning and sterilizing laboratory glassware and surgical instruments and can also be used in combination with various soaps for hygiene purposes and for cleaning and sterilizing hospital rooms and areas used for manufacturing amber foods. Furthermore, the derivatives are expected to be effective in the treatment of various infections, such as urinary tract infections eTP: pg respiratory tract infections, caused

- of "different phlogogenic bacteria.

The drawing shows

fig. 1 the infrared absorption spectrum of 1-N-[L-(-)-α-hydroxy-Y-aminobutyryl]-XK-62-2 (compound I);

fig. 2 The NMR spectrum of 1-N-fL-(-)-α-hydroxy-γ-amino-butyryl]-XK-62-2.

According to the present invention, the compound of formula I is prepared by a compound of the formula:

is acylated with an acylating agent of the formula: 1 2 Y means hydrogen, and Y means a protecting group selected from 12 .. where R and R can be the same or different and means H, OH, N0~, Cl, Br, I, alkyl of 1-5 carbon atoms or alkoxy of 3 12 1-5 carbon atoms, and R means H, Cl, Br or I , or Y and Y together form a phthaloyl group, to form a compound of the formula:

1 2

where Y and Y have the above meaning, after which the protecting groups Y 1 and Y 2 are removed in a manner known per se, and, if desired, the resulting compound is transferred to a pharmaceutically acceptable acid addition salt.

The acylating agent is derived from α-hydroxy-γ-aminobutyric acid.

Acylation methods are described in M. Bodansky et. al: Synthesis, page 435 (1972) and in M. Bodansky et al: Peptide Synthesis, page 75-135 (1966) (John Wiley & Sons., U.S.A.).

The acylation reaction in the present method is preferably carried out using an acylating agent with the formula: 12.

where Y and Y have the above meaning.

In this case, a compound with the formula:

where Y 3 and Y 2 have the meaning stated above, is previously reacted with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide to produce a compound with the above formula, which can be isolated and reacted with XK-62-2.

Alternatively, a compound is reacted with the formula:

where Y 1 and Y 2 have the above meaning, N-hydroxysuccinimide and dicyclohexylcarbodiimide, and the resulting reaction mixture is reacted with XK-62-2.

In another embodiment, the acylation reaction can be carried out by adding dicyclohexylcarbodiimide to a mixture of XK-62-2 and a compound with the formula:

It should be understood that the above-mentioned reactions can be used where the acylation reaction of XK-62-2 is carried out by any other method, and a suitable method can be chosen depending on the particular acylating agent used.

Usually 0.4-2.5 mol is used, preferably 0.7-1.5

mol, of the acylating agent per mole XK-62-2. The reaction is carried out at a temperature between -50 and 50°C, preferably between -20 and 20°C, for from 15 minutes to 24 hours, preferably 5-15 hours.

Suitable solvents for the reaction can be selected from tetrahydrofuran, dimethylacetamide, dimethylformamide, lower alcohols, dioxane, ethylene glycol dimethyl ether, pyridine, water and mixtures thereof; A mixture of ethyl alcohol and water is preferably used in a volume ratio of 2:1.

The intermediate IA for the desired product prepared in the manner indicated above can be isolated and purified for use in the second reaction. However, it is preferred to use the reaction mixture as it is, without isolation and purification of the acylated compounds. In the subsequent reaction, the compounds obtained from the intermediate are isolated and purified. The latter method is advantageous in that it simplifies the procedure and increases the yield.

If necessary, the intermediate IA, i.e. containing a protecting group, can be easily isolated and purified by any of the known methods, e.g. column chromatography using an adsorbent such as ion exchange resins, silica gel, alumina, cellulose, "Sephadex"®, etc., and thin layer chromatography using silica gel, alumina, cellulose, etc.

The intermediate IA, either isolated or in mixture, re-

1 2 is then set to remove the protective groups Y and Y in a known manner and form the desired compound of formula I as stated above.

Removal of the protecting group is carried out in a known manner. E.g. where the protecting group is a phthaloyl group, the removal is carried out with hydrazine; where the protecting group is a methoxycarbonyl group or ethoxycarbonyl group, the removal is carried out with barium hydroxide; where the protecting group is a tertiary butoxycarbonyl group, the removal is carried out with formic acid or trifluoroacetic acid; where the protecting group is an o-nitrophenylsulfenyl group, the removal is carried out with acetic acid or hydrochloric acid; and where the protecting group is a chloroacetyl group, the removal is carried out with 3-nitropyridine-2-thione (reported by K. Undheim et al: Journal of the Chemical Society, Parkin Transactions, Part I, page 829 (1973)).

It is preferred that the protecting group is

12 in

where R and R have the above meaning, and the removal is carried out by hydrogenolysis in the presence of a metal catalyst selected from palladium, platinum, rhodium and. Raney nickel, preferably palladium on a support of activated carbon, in at least one solvent selected from tetrahydrofuran, dimethylacetamide, dimethylformamide, lower alcohols, dioxane, ethylene glycol dimethyl ether, pyridine and water, preferably a mixture of water and methanol in a 1:1 volume ratio ; in the presence of a small amount of hydrochloric acid, hydrobromic acid, hydroiodic acid or acetic acid, preferably acetic acid, and at room temperature and atmospheric pressure.

It should be understood that any suitable compound can be used in the above-mentioned method to protect the amino groups in α-hydroxy-γ-aminobutyric acid, which is the precursor to a compound of the formula:

where Y , Y and Z have the above meaning. Such a technique is well known. Introduction of an easily removable protecting group is a technique frequently used in peptide synthesis. The protection of amino groups with such protecting groups and their removal is described in M. Bodansky et al: Peptide Synthesis, pages 21-41 (1966) (John Wiley & Sons, Inc., U.S.A.);

and A. Kapoor: Journal of Pharmaceutical Sciences, Vol. 59,

pages 1-27 (1970).

Compound I, either prepared from an isolated and purified intermediate or an impure medium, is isolated and purified from the reaction mixture in a known manner. E.g. the compounds are isolated and purified by column chromatography using an adsorbent such as ion exchange resins, silica gel, alumina, cellulose, etc., and thin layer chromatography using silica gel, alumina, cellulose, etc.

If desired, compound I can be converted into pharmaceutically acceptable, non-toxic acid addition salts (mono-, di-, tri-, tetra- or penta-salts) according to conventional methods.

According to the invention, non-toxic acids include inorganic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, carbonic acid, etc., and organic acids such as acetic acid, fumaric acid, malic acid, citric acid, mandelic acid, tartaric acid, ascorbic acid, etc. The methods for the preparation of the acid addition salts are well known in the art. The semi-synthetic derivative 1 has an excellent antibacterial activity. It is particularly noteworthy that it has a strong antibacterial activity against strains of Escherichia coli with an R factor showing resistance to known aminoglycoside antibiotics.

Table I shows the antibacterial spectrum of kanamycin A, "Gentamicin C-, L cl", XK-62-2 and 1-N-(L(-)-ct-hydroxy-y-amino-butyryl) XK-62-2 (compound I) against various gram-positive and gram-negative bacteria, measured by the agar dilution method at pH 8.0.

By comparing the minimal inhibitory concentration listed in Table I, it is clear that compound I has a strong antibacterial activity. Characteristically, the compound shows particularly strong antibacterial activity against Escherichia coli KY 8327 and 8348.

In the above table, Escherichia coli produces

KY 8327 and KY 8348 gentamicin adenyltransferase and gentamicin acetyltransferase type I intracellularly, respectively. The former bacterium inactivates kanamycin and gentamicins by adenylation, and the latter inactivates gentamicins by acetylation.

The antibacterial spectrum of 1-N-[L-(-)-α-hydroxy~Y-aminobutyryl]-XK-62-2 in comparison with kanamycin A, gentamicin complex (C,, C, and C„) and XK -62-2, measured by the agar dilution method at pH 7.2, is shown in Table II below.

1: produces gentamicin adenyltransferase

2: produces gentamicin-adenyltransferase and neomycin-kanamycin-phosph otr ansferase type- II

3: produces gentamicin acetyltransferase type I

4: produces neomycin-kanamycin phosphotransferase type I

5: produces kanamycin acetyltransferase

6: produces gentamicin acetyltransferase type I and neomycin-kanamycin phosphotransferase type I'

7: produces neomycin-kanamycin-phosphotransferase type I and

type II and streptomycin phosphotransferase

8: probably produces kanamycin acetyltransferase 9: produces gentamicin acetyltransferase type II

The above-mentioned enzymes are produced intracellularly and with the enzyme the bacteria inactivate the antibiotics.

From the above table I, it appears that 1-N-(L(-)-α-hydroxy-Y-aminobutyryl)-XK-62-2 has a very strong antibacterial activity against various bacteria with resistance to at least one of gentamicin- antibiotics and XK-62-2, aom produces gentamicin adenyltransferase and/or gentamicin acetyltransferase type I and type II intracellularly and thereby inactivates gentamicin antibiotics and XK-62-2.

Furthermore, in various tests with regard to the protection against infections caused by the strains of the above-mentioned resistant bacteria, the prepared compound has been shown to preserve the α-hydroxy-γ-aminobutyryl group in vivo and to exhibit much higher antibacterial activity than gentamicin antibiotics and XK-62-2.

From DE-OS 2,234,315, a method is known for introducing the L-(-)-α-hydroxy-Y-aminobutyryl group (hereinafter referred to as "HABA") onto the amino group in the 1-position in kanamycin A or kanamycin B ( in the following denoted respectively "KMA" and KMB") for the formation of 1-N-HABA-KMA and 1-N-HABA-KMB, respectively. In the following table III, data for comparison of the antibacterial spectrum against 63 microorganisms st for the compound I prepared according to the invention (1-N-HABA-XK-62-2) and the known compound 1-N-HABA-KMA. The minimal inhibitory concentration (hereinafter referred to as "MIC") is measured by the same method as explained for Table II above.

From Table III it appears that 1-N-HABA-XK-62-2 is significantly better than 1-N-HABA-KMA because it is more effective against 30 strains and equivalent to 1-N-HABA-KMA against the other strains . When assessing antibacterial activity, a difference of one order of magnitude (2- or 1/2-fold) in MIC is considered to be insignificant and a difference of more than two orders of magnitude (4- or 1M-fold) in MIC is considered to be significant.

Furthermore, l-N-HABA-XK-62-2 has the following special features.

It is known that an aminoglycoside antibiotic urn so in has a. primary ammonium group in the 6' position, is inactivated by a microorganism containing acetyltransferase. 1-N-HABA-KMA and 1-N-HABA-KMB have a primary amino group in the 6' position. It is therefore assumed that these antibiotics are inactivated by a microorganism containing acetyltransferase and that they are ineffective against such a microorganism. On the other hand, since amino groups in the 6' position of 1-N-HABA-XK-62-2 are protected by a methyl group, this antibiotic is not inactivated by a microorganism containing acetyltransferase and is therefore effective against such a microorganism. This is proven by the fact that 1-N-HABA-XK-62-2 has a higher antibacterial activity than 1-N-HABA-KMA against a microorganism containing kanamycin acetyltransferase.

In Table III, Escherichia coli KY Z-3^3, Pseudomonas aeruginosa KY 8510, Pseudomonas aeruginosa KY 8516 and Serratia marcescens POE 1065 are microorganisms that contain kanamycin acetyltransferase. From the MIC data given in Table III, it appears that 1-N-HABA-XK-62-2 is more effective than 1-N-HABA-KMA against these four microorganisms.

Although there are no experimental data in the case of 1-N-HABA-KMB, it is naturally to be expected that 1-N-HABA-KMB with a primary amino group in the 6' position is less effective than 1-N-HABA-XK-62 -2 against the four above-mentioned microorganisms.

It is also known that an aminoglycoside antibiotic urn having a hydroxy group in the 3' position is inactivated by a microorganism containing phosphotransferase, KMA, KMB, 1-N-HABA-KMA and 1-N-HABA-KMB have a hydroxy group in the 3' position. It is therefore assumed that these antibiotics are phosphorylated by a microorganism containing phosphotransferase, and that they are ineffective against such a microorganism. Indeed, it is stated in Table IV 1 of US Patent No. 3,781,268 that the MICs of KMA and BBK-26 (1-N-HABA-KMB) against Pseudomonas aeruginosa D113, which contain phosphorylase, are >50 and 50, respectively. The high MIC values show that KMA and 1-N-HABA-KMB are ineffective against Ps. aeruginosa D113.

On the other hand, it is believed that an aminoglycoside antibiotic urn without a hydroxy group in the 3' position, such as XK-62-2 and l-N-HABA-XK-62-2, is not phosphorylated by a microorganism containing phosphorylase, and that the antibiotic is effective against such a microorganism. E.g. are Escherichia coli KY 8349 and Pseudomonas aeruginosa KY 8512 in the preceding Table II microorganisms containing neomycin-kanamycin phosphotransferase type I. It is clear that XK-62-2 and l-N-HABA-XK-62-2 are effective against these microorganisms as The MIC of XK-62-2 against these microorganisms is 0.4 and 0.78, respectively, and the MIC of l-N-HABA-XK-62-2 against it is 0.2 and 0.78, respectively. On

on the other hand, it is clear that KMA opposite it is respectively

>100 and 12.5.

From the foregoing tables it is clear that compound I exhibits remarkably strong antibacterial activity against a variety of gram-positive bacteria and gram-negative bacteria, including those resistant to aminoglycoside antibiotics. It is therefore expected to be effective in the treatment of various infections in humans and animals, produced by such phlogogenic bacteria. E.g. compound I is expected to be effective in the treatment of urinary tract infections and respiratory tract infections caused by Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and strains of the genus Proteus.

When administering the compound or its acid addition salts, parenteral administration is preferred with an effective dose of 1.6-6 mg/kg per day.

The acute toxicity (LD^) of l-N-[L-(-)-α-hydroxy-Y-aminobutyryl]-XK-62-2 in mice is 250 mg/kg by intravenous administration, while the toxicity of XK-62-2 and the gentamicin complex (a mixture of C^ C and C^) are 93 mg/kg and 72 mg/kg, respectively.

The method according to the invention is illustrated in more detail by the following representative examples.

Example 1

Preparation_of_l-N;[L-(-);a-h^

A sample of XK-62-2 (2.778 g, 6.0 mmol) is dissolved in

30 ml anhydrous 50? tetrahydrofuran. To the solution is added a solution of the N-hydroxysuccinimide ester of L-(-)-α-hydroxy-γ-carbobenzoxyaminobutyric acid (the preparation of this compound from L-(-)-α-hydroxy-γ-aminobutyric acid is described in the Journal of Antibiotics, Vol. XXV, pages 695-708 (1972).

The preparation of L-(-)-α-hydroxy-Y-aminobutyric acid is described

in Tetrahedron Letters, pages 2625-2628 (197U). (2.94 g, 84 mmol) in 20 ml of tetrahydrofuran with stirring, while maintaining the temperature between -5 and 0°C. The addition is completed in one hour and the mixture is allowed to react overnight.

By silica gel thin-layer chromatography (developing agent: isopropanol/concentrated ammonia water/chloroform in the volume ratio 2:1:2, coloring agent: ninhydride) the presence of the intermediate product, i.e. l-N-[L-(-)-a-hydroxy-y -carbobenzoxyaminobutyryl]-XK-62-2, (Rf 0.81), together with i.a. a small amount of unreacted XK-62-2. The reaction mixture is concentrated under reduced pressure whereby a faint yellow residue is obtained. This mixture containing the intermediate is dissolved in 40 ml of aqueous. 50% methanol. 0.3 ml of acetic acid is added to the solution and the mixture is subjected to hydrogenolysis in the presence of 250 mg of 5$ palladium-on-activated carbon at room temperature and atmospheric pressure for 6 hours. The presence of compound I (Rf 0.40) is confirmed by silicon dioxide gel thin-layer chromatography (under the same conditions as in example 1). The catalyst is removed by filtration and the filtrate is concentrated under reduced pressure. To the resulting residue is added 15 ml of water to dissolve

of the residue and the solution is passed through a column (diameter 2.5 cm) of "Alberlite^CG - 50" (ammonium form, 150 ml). The column is washed with 200 ml of water. Elution is then carried out with 0.2 N aqueous ammonia and the eluate is taken up fractionally in 10 ml portions.

To obtain the desired compound, fractions No. 225-250 are collected. These fractions are combined and concentrated to dryness under reduced pressure to give 0.69 g of compound I as a colorless product.

Analysis of compound I shows the following:

Melting point: 120-124°C

Specific rotation: (a)^9 =.+99.0° (C = 0.10, water)

Infrared absorption spectrum (KBr) (cm<-1>): 3700-3100, 2940, 1640, 1565, 1480, 1385, 1340, 1282, lill, 1054, 1022, 973, 8l6, 700-600 (Fig. 3).

NMR spectrum (in deuterium oxide) 6 (in ppm from DSS):

1»20 (3H, singlet), 2.36 (3H, singlet), 2.52 (3-H, singlet),

5.14 (1H, doublet, J=4.0 Hz), 5.22 (1H, doublet, J=4.Q Hz) (fig.4).

Elemental analysis:

Calculated for C^H^gNgC^ • 1/2H2CC>3: C 49.39% - H 8.29% - N 14.11% Found: C 48.96% - H 8.37% - N 13.95 %

From the above it is confirmed that compound I has

the previously stated structural formula.

Example 2

Preparation of the sulfate of compound I where 1 mole of compounds

0.1 mol of 1-N-[L-(-)-α-hydroxy-Y-aminobutyryl]-XK-62-2 (compound I) is dissolved in 200 ml of water. A solution of 1 mol of sulfuric acid in 50 ml is added to the solution while cooling. After 30 minutes, ethanol is added to the solution to form a precipitate, until precipitation is complete. The precipitate is separated by filtration and dried to obtain a white powder.

Analysis of the powder shows the following:

Melting point: 242.8°C.

computers; n

Specific rotation: (a)n^ = +95.-9 (C = 1.0, water)

Infrared absorption spectrum (KBr) (cm _ i): 3400, 1625, 1122. NMR spectrum (in deuterium-icksyd) 6 (in ppm from DSS):

1.33 (3H, singlet), 2.77 (3H, singlet), 2.9.3 (3H, singlet),

5.20 (1H, doublet, J = 3.9 Hz), 5.93 (1H, doublet, J=3.9 Hz). Elemental analysis:

Calculated for C24<H>48<N>6°9'1 H2SCVH20: C 3l+>95%, H 7.14%,

N 10.04%, S 9.40%;

Found: C 34.82%, H 6.70%, N 10.15%, S 9.68%.

From the above analysis, the powder is identified as the sulfate of compound I with the molecular formula

<C>24<H>48<N>6°9 2 <H>2<S0>4 <H>2<0>'

Claims (1)

Analogous process for the preparation of therapeutically active l-N-[L-(-)-ct-hydroxy-Y-aminobutyryl]-XK-62-2 with the formula: the formula: or pharmaceutically acceptable acid addition salts thereof, characterized in that a compound with the formula: acylated with an acylating agent with the formula: 1 2 Y means hydrogen, and Y means a protecting group selected from where. Æ'..and R can be the same or different and mean H, OH, ;.;Cli Br, I, alkyl with 1-5 carbon atoms or alkoxy with ^—■•' 3 12 1-5 carbon atoms, and R means H, Cl, Br or I, or Y and Y together form a phthaloyl group, to form a compound of the formula: 1 2 where Y and Y have the above meaning, after which the protecting groups Y 1 and Y 2 are removed in a manner known per se, and, if desired, the resulting compound is transferred to a pharmaceutically acceptable acid addition salt.