CS195712B2 - Method of producing novel aminoglycosides of 2-deoxystreptsmine - Google Patents

Abstract

1464401 Silylated aminoglycoside PFIZER Ltd 24 Oct 1975 [26 Oct 1974] 46412/74 Heading C3S [Also in Division C2] The penta - N - acetyl - octa - O - trimethylsilyl derivative of 1 - N - [(S) - 4 - amino - 2- hydroxybutyl] - kanamycin A is prepared by treating 1 - N - [(S) - 4 - amino - 2 - hydroxybutyl]-kanamycin A with acetic anhydride in methanol at room temperature for 24 hours, followed by a 2 : 1 mixture of hexamethyldisilazane and trimethylchlorosilane at room temperature for 24 hours.

Description

BACKGROUND OF THE INVENTION The present invention relates to antibacterial agents, and more particularly to a class of novel 2-deoxystreptamine amnoglycosides which are antibacterially active, as well as to a process for the preparation thereof.

Numerous naturally occurring 2-deoxystreptamine aminoglycosides have a generally tricyclic structure, which can be described by the following general formula:

wherein ring A is a hexopyranose skeleton containing an amino group at the 2 'and / or 6' position, ring V is a 2-deoxystreptamine group and ring C represents a glycosyl group linked by a glycosidic bond to either the 5 or 6 position of the streptamine ring hydroxyl group.

The novel antibacterial agents of the invention represent the group of aminoglycosides 2195712

-deoxystreptamine bearing a (3-hydroxy-ω-aminoalkyl substituent on the amino group at the 1-position and a glycosyl group attached at the 5 or 6 position of the streptamine ring B. These compounds are effective in the treatment of a number of infections caused by Gram positive and Gram-negative bacteria. animals and humans, and have some advantages over using 2-deoxystreptamine aminoglycosides with an unsubstituted amino group at position 1 of the 2-deoxystreptamine ring B, such as natural kanamycin A and B, neomycin and ribostamycin.

Accordingly, the present invention provides novel compounds of Formula I

cunhr ¹ nh _h > —O · i

HO- \> О ^И horVo ^ oi ^ (i) in which

R ¹ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms,

R ² is amino or hydroxyl, one of R ³ and R ⁴ is hydrogen and the other of the symbols represents a glycosyl group wherein in the case when R ³ glycosyl group, this group corresponds to the formula

OH

OH and when R ^{4 represents a} glycosyl group, this group corresponds to the formula

and n is 1, 2 or 3, and pharmaceutically acceptable acid addition salts thereof.

A particular subgroup of the compounds of the invention are those compounds of formula (I) wherein R ¹ is hydrogen and n is 1 or 2.

A preferred group of compounds of the invention are those compounds of formula I wherein R ³ is hydrogen and R ⁴ is a 3-amino-3-deoxy-α-D-glucopyranosyl group, i.e. kanamycin derivatives A and B. wherein the N-hydroxy-1-aminoalkyl group on N ¹ has the (S) n configuration and is 2 or 3. R ¹ is preferably a hydrogen atom or a methyl group.

Particularly preferred individual compounds of the invention are 1-N- [(S) -4-amino-2-hydroxybutyl] kanamycin A and 1-N - [(S) -5-amino-2-hydroxypentyl] kanamycin A.

Pharmaceutically acceptable acid addition salts of the compounds of the invention are those formed with acids leading to non-toxic acid addition salts containing pharmaceutically acceptable anions such as hydrochlorides, hydrobromides, hydroiodides, sulfates or bisulfates, phosphates or acid phosphates, acetates, maleates, fumarates, oxalates, lactates, tartrates, citrates, gluconates, saccharates, p-toluenesulfonates and salts with carbonic acid.

The novel compounds of the formula I are prepared according to the invention by the preparation of compounds of the formula vzorce

CHNHRNlk

O OH ~ с ~ сн (сн ^ - ^ хнн ^ (и) in which

R ¹ , R ² , R ³ , R ⁴ and n are as defined above, and

X is CH? or CO, in a suitable solvent, are reacted with a reducing agent to reduce the amide bond on the N ¹ nitrogen and any carborinyl groups present as X.

Optionally, this process may be supplemented by optional preformation of a suitable acid addition salt to achieve solubility of the compounds of Formula II in organic solvents. This reaction can be carried out, for example, by dissolving the compound of formula II at a temperature generally between room temperature and 0 ° C in excess of anhydrous trifluoroacetic acid. Excess acid is removed by evaporation of the reaction mixture under vacuum to dryness, the salt is dissolved in an anhydrous organic solvent inert under the reaction conditions, for example tetrahydrofuran or dimethoxyethane, and a reducing agent, for example diborane, is conveniently added as a solution in tetrahydrofuran. . The reducing agent is generally used in excess and is usually operated at a temperature ranging between room temperature and the boiling point of the reaction mixture, depending on the nature of the individual reactants and the solvent used. When X is CO, naturally enough reducing agent is used to ensure the reduction of the two amide carbonyl groups. When the reaction is carried out in tetrahydrofuran at 50 ° C using diborane, it is substantially complete in 24 hours. The product is then conveniently recovered by adding water to the reaction mixture to quench unreacted diborane and evaporating the organic solvent in vacuo. The pH of the remaining aqueous solution is adjusted to 5 and the crude product can then be separated from the unreacted starting material and by-products by conventional chromatographic techniques.

Many of the compounds of formula (II) wherein X is CH2 are known antibiotics as previously described. For example, N1- (4-amino-2-hydroxybutyryl) kanamycin A, also abbreviated BB-K8, is described in U.S. Patent 3,781,268. Other examples of compounds are described in U.S. Patent 3,781,268. 268, 3,541,078 and 3,860,574, and in published West German application Nos. 2,350,203 and 2,322,576. N- (5-amino-2-hydroxyvaleryl) - and 1 N- (3-amino-2-hydroxypropionyl) derivatives kanamycin A and V are disclosed in West German Patent Application No. 2 408 666 and in J. Antibiotics 1974, 27, 851. 6'-N-alkyl derivatives are described in West German Patent Application No. 2,350,169 and in J. Antlbiotics 1975, 28, 483.

Compounds of formula II wherein X is CO may be prepared by acylating the 1-amino group of 2-deoxystreptamine aminoglycosides with methods analogous to those used to prepare compounds of formula II wherein X is CH 2 with the proviso that the acylating agent uses a reactive acid derivative of the formula

OH

HOOC-CH (CH 2) _n -CONH 2

The novel compounds of formula (I) according to the invention may exist in various conformational forms and the invention is not limited to any of these forms. Generally, both rings А and В are in "chair" form and all residues R ² , OR ³ , OR ⁴ , amino and hydroxyl groups have an equatorial position relative to rings A and B. Furthermore, the glycosidic bond between the hexopyranosyl ring A and the 2-deoxystreptamine В ring is most commonly QF-linkage due к first ^from said rings, especially in cases where the compounds of formula II are derived from naturally occurring 2-deoxystreptamine aminoglycosides. The β-hydroxy-ω-aminoalkyl group on the N ¹ nitrogen may further exist in the S or R configuration or as a mixture of both optical isomers.

The in vitro evaluation of the antibacterial activity of the compounds of the invention is performed by determining the minimum inhibitory concentration of the test compound in a suitable environment that does not give rise to growth of the test microorganism. In practice, agar plates supplemented with test compound at the appropriate concentration are inoculated with a standard number of cells of the test microorganism and each plate is then incubated at 37 ° C for 24 hours. It is then determined whether or not bacteria have been grown on the test plates and the respective values of minimum inhibitory concentrations are determined. Microorganisms used in these assays include strains of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus faecalis.

More potent compounds are also evaluated in in vivo assays in which test compounds are administered subcutaneously to mice exposed to Escherichia coli infection. Each of the test compounds is administered in a series of different doses to groups of mice and its efficacy is expressed as providing 50% protection against the lethal effect of Escherichia coli within 72 hours.

For use in human medicine, the antibacterially active compounds of the invention may be administered alone, but are generally administered in admixture with a pharmaceutically acceptable carrier selected with respect to the intended route of administration and standard pharmaceutical practice. For example, the disclosed compounds may be administered orally in the form of tablets containing, for example, starch or lactose as the carrier, or in the form of capsules containing either the active ingredients alone or mixtures thereof with carriers and excipients, or elixirs or suspensions containing flavorings. additives or dyes. The active compounds may also be administered by parenteral, e.g., in, travenous, intramuscular or subcutaneous injection. For parenteral administration, the active compounds according to the invention are most preferably used in the form of sterile aqueous solutions which may contain other soluble substances, for example, an appropriate amount of salts or glucose, for isotonization of the solution.

When administered in human medicine, the antibacterially active compounds of the invention are expected to be administered: at daily dosages comparable to aminoglycoside antibacterial agents used in the art, for example at doses of from 0.1 to 50 mg / kg (in divided doses) when administered parenteral or in doses of 10 to 100 mg / kg (in divided doses) when administered orally. Thus, it is expected that tablets or capsules for oral administration up to four times a day will contain from 0.1 to 1 g of active ingredient, while unit doses for parenteral administration will contain from 10 to 500 mg of active ingredient. It will be for the physician to determine the appropriate dose that will be most appropriate for the patient and which will vary depending on the patient's age, weight and response. The above dosages are exemplary dosages for the average patient. Of course, there may be individual instances where higher or lower dosages will be required, and these are also within the scope of the invention. The invention is illustrated by the following non-limiting examples. Temperatures in these examples are given in degrees Celsius.

Example 1

150 mg of 1-N- [(S) -4-amino-2-hydroxybutyryl] -canamycin A, prepared as described in U.S. Pat. No. 3,781,268, are dissolved in 10 ml of anhydrous trifluoroacetic acid at 0 ° C. The solution was evaporated in vacuo to dryness and the residue was dried under high vacuum at 20 ° C for 15 minutes. The glassy residue is taken up in 5 ml of tetrahydrofuran and 20 ml of a 1M solution of diborane in tetrahydrofuran are added in portions under nitrogen. The resulting clear solution was heated to 50 ° C for 3 hours, then allowed to stand at room temperature for 16 hours, then heated to 50 ° C for a further 3 hours. Excess diborane was decomposed by careful addition of a few drops of water and the organic solvent was evaporated under reduced pressure. The residue is taken up in 10 ml of water and basified with N / 10 aqueous sodium hydroxide. The pH of the aqueous solution was adjusted to 5 by the addition of 2N hydrochloric acid, and the resulting solution was chromatographed on a 50 ml Amberlite CG 50 ion exchange column. The column is washed with distilled water to remove inorganic solids and then eluted with an aqueous solution of ammonium hydroxide at increasing concentrations ranging from 0.1 to 1.0 N. Fractions containing the desired product according to thin-layer chromatography are combined and evaporated in vacuo. vacuum. 75 mg (yield 50%) of 1-N - [(S) -4-amino-2-hydroxybutyl] -canamycin A are obtained.

Thin-layer electrophoresis

The product has an _{Rf of} 0.6. A mixture of equal parts of acetic acid and formic acid of pH 2 is used as the electrolyte and a voltage difference of 900 V is applied to the opposite ends of the silica gel coated plates (length 20 cm) for 45 minutes. Detection is accomplished by drying the plate, spraying with a cyclohexane solution of tert-butyl hypochlorite, then drying and developing upon cooling with a solution of starch and potassium iodide. Under these conditions, the reference standard sample has 1-N- [(S) -4-amino-2-hydroxybutyryl] cyanamycin AR _f 1.0 and kanamycin AR _f 0.9.

Infrared spectrum

It shows the disappearance of the amide carbonyl band found at 1635 cm ^-1 in the spectrum of 1 N - [(S) -4-amino-2-hydroxybutyryl] kanamycin A.

Optical rotation [ _.alpha. ] _{D @} ²⁵ = + 73 DEG (c = 1.0 in water).

Mass spectrometry (field ionization)

The spectrum contains a strong M + 1 signal at m / e 572.

A sample of the product was converted to a volatile penta-N-acetyl-octa-O-trimethylsilyl derivative by reaction with acetic anhydride in methanol at room temperature for 24 hours, followed by reaction with a mixture of hexamethyldisilazane and trimethylchlorosilane at room temperature for 24 hours.

For C56H119N5O17Si8 calculated M ⁺ 1357, found M ⁺ 1357.

For C22H45N5O12.2 1/2 H2CO3 calculated:

40.5 ° C, 6.9% H, 9.6% N, found:

40.1% C, 6.7% H, 9.6% N.

Example 2

100 mg of butirosine (1 N - [(S) -4-amino-2-hydroxybutyryl] ribostamycin) as the free base was dissolved in 5 ml of anhydrous trifluoroacetic acid at room temperature and evaporated to dryness to leave the trifluoroacetic acid salt in vacuo to dryness. The residue is taken up in 10 ml of dry dimethyl ether of diethylene glycol and 10 ml of a 1M solution of diborane in tetrahydrofuran is added to give a clear solution which is left to stand at room temperature for 18 hours, followed by an additional 5 ml of solution. The excess diborane was quenched by carefully adding a few drops of water and the organic solvents were evaporated in vacuo at 50 ° C. The residue was basified with a few drops of 2 N sodium hydroxide solution and then added The pH is adjusted to 5 with 2 N hydrochloric acid. The product is isolated by column chromatography A mberlite CG 50 as described in the previous example, evaporation of the fractions containing the product in pure form in vacuo gave 1-N- [(S) -4-amino-2-hydroxybutyl] -ribostamycin.

Thin-layer electrophoresis

The product has an _{Rf of} 0.5. The conditions are as described in the previous example. Butirosine having an R _{f of} 1.0 is used as reference standard.

Example 3

Following the procedure described in Example 1, 0.35 g of 1-N-J (S) -5-amino-2-hydroxylvaleryl] kanamycin A was converted to the trifluoroacetic acid salt, reduced and chromatographed. 0.12 g (35%) of 1-N - [(S) -5-amino-2-hydroxypentyl] -canamycin A is obtained.

Thin-layer electrophoresis

The product has an R ₍ 0.7). It is operated under the conditions described in Example 1. The starting material used as a reference standard has an R _{f of} 1.0.

He attaches

Following the procedure described in Example 1, reduction of 0.15 g of N- (3-amino-2-hydroxypropionyl) kanamycin A yields 0.04 g (27%) of 1-N- (3-amino-2 ^: hydroxypropyl) kanamycin A.

Thin-layer electrophoresis

The product has an _{Rf of} 0.6. The starting material used as the reference standard has an R _{f of} 1.0.

Example 5

The procedure described in Example 1 similarly reduced 1-N - [(S) -4-amino-2-hydroxybutyryl] -canamycin B to give 1-N - [(S-4-amino-2-hydroxybutyl) kanamycin B.

Example 6

6‘-N-methyl-1-N - [(S) -4-amino-2-hydroxybutyryl] -canamycin A (prepared according to the procedure described by H. Umezawa et al. In J. Antlbiotics, 1975, 28, 483). gives 6‘-N-methyl-1-N- [(S) -4-amino-2-hydroxybutyl] kanamycin A by the reduction described in Example 1

Thin-layer electrophoresis

The product has an Rf of 0.7. The starting material used as the reference standard has an R _{f of} 1.0 and the kanamycin A has an Rf of 1.03.

The results of the in vitro antibacterial activity assays of the compounds of the previous examples, using the methods described above, are shown in the following table:

TABLE

In vitro potency example of minimum inhibitory concentration in µg / ml.

No. Escherichia Klebsiella Proteus Pseudomonas Staphylococcus coli pneumoniae mirabilis aeruginosa aureus

1 6.2 3.1 3.1 1.6 1.6 2 6.2 6.2 25 12.5 12.5 3 6.2 3.1 12.5 3.1 1.6 4 12.5 6.2 12.5 3.1 3.1

In addition, the activity of Example 1 was tested in vivo using the procedure described above. This compound shows a PD 50 against Escherichia coli of 3.8 mg / kg in mice.

Claims (1)

OBJECT OF THE INVENTION A process for the preparation of the novel 2-deoxystreptamine aminoglycosides of the formula I CH.NHRNH HIM OH NHCHHMCH2H (I) ve. which. R 1 represents a hydrogen atom or a C 1 -C 3 alkyl group, R2 represents a hydroxyl group or amino this, one of R ³ and R 4 is hydrogen. and the other of these symbols represents a glycosyl group, wherein when R ^{3 is a} glycosyl group, the group corresponds to the formula OH OH and when R4 represents a glycosyl group, this group corresponds to the formula and n has a value of 1, 2 or 3, and their pharmaceutically acceptable acid addition salts, characterized in that the compounds of formula II CHNHR ^ NK'O OH HO O \ flHrt-CHÍCHjn.XCH no R ^ o O? ^(II) wherein R 1, R 2, R 3, r 4 and n are as defined above and X is CH 2 or CO, reduced by reaction with diborane in an anhydrous organic solvent, then water is added to the reaction mixture.