IE42452B1 - Aminoglycosides - 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

PATENT APPLICATION BY (71) PFIZER LIMITED, A BRITISH COMPANY OF RAMSGATE ROAD, SANDWICH, KENT, ENGLAND.

Pnce This invention relates to antibacterial agents and is particularly concerned with a class of novel antibacterial 2-deoxystreptamine aminoglycosides and with methods for the preparation of such compounds.

Many naturally-occurring 2-deoxystreptamine aminoglycosides have in common a three ring structure which may be represented by the general formulas where the ring A is the skeleton of a hexopyranose group 10 having an amino group in the 2' and/or 6'- positions, the ring B is the 2-deoxystreptamine group and the ring C represents a glycosyl group attached by a glycosidic linkage to either the 5- or the 6- position of the streptamine ring, the other position being occupied by a hydroxyl group. i - 3 The novel antibacterial agents of the invention are a series of 2-deoxystreptamine aminoglycosides having a B-hydroxy-ω-aminoalkyl substituent on the 1amino group and having a glycosyl group attached to the 5 or 6- position of the streptamine ring B. Such compounds are effective in treating a variety of gram-positive or gram-negative bacterial infections, including urinary tract infections, in animals, including humans, and possess advantages in use over 2-deoxystreptamine aminoglycosides having an unsubstituted amino group in the 1- position of the 2-deoxystreptamine ring B, such as the naturally-occurring kanamycin A and B, neomycins and ribostamycin.

According to the invention, therefore, there are provided novel compounds having the general formula: where R3 represents a hydrogen atom or a lower alkyl group; π R represents an amino or hydroxyl group; one of R3 and R^ represents a hydrogen atom, while the other represents a glycosyl group as hereinafter defined; and n is 1, 2 or 3; and their pharmaceutically-acceptable acid addition salts. 34 5.3 - 4 4 When R represents a glycosyl group such group is defined as a single hexopyranosyl group containing two or more hydroxy groups and preferably containing an amino group, for example R^ may be a 3-amino-35 deoxy-a-D-glucopyranosyl group as found in kanamycin 3 A and B. When R represents a glycosyl group such group is a pentofuranosyl group, optionally linked to a further hexopyranosyl group by a further glycosidic linkage. For example R may he a β-D-ribofuranosyl 10 group as found in ribostamycin. rt »* The expression lower alkyl group means a group containing from 1 to 4 carbon atoms which may be straight or branched-chain.

One particular group of compounds according to 15 the invention comprises the group in which R^ is a hydrogen atom and n is 1 or 2.

A preferred class of compounds according to the invention comprises compounds in which R is a hydrogen 4 atom and R is a 3-amino-3~deoxy-a-D-glucopyranosyl 20 group, i.e. derivatives of kanamycin A and B. Also preferred are compounds in which the g-hydroxy-ωaminoalkyl group at the 1-N position has the (S) configuration and n is 2 or 3. R^ is preferably a hydrogen atom or a methyl group.

Particularly preferred individual compounds accord ing to the invention include 1-N-/7(S)-4-amino-2hydroxy-buty127-kanamycin A and 1-N-£(S)-5-amino-2hydroxy-pentyl]7-kanamycin A.

Pharmaceutically-acceptable acid addition salts of the compounds of the invention are those formed from acids which form non-toxic acid addition salts containing pharmaceutically-acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, sulphate, or bisulphate, phosphate or acid phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate 2 4 5 2 - 5 saccharate, β-toluene-sulphonate and carbonate salts.

The novel compounds of formula (I) may be prepared according to the invention from compounds of the formula: 4 where R to R and n are as previously defined and X is CH2 or CO, by reacting with a reducing agent(preferably in a suitable solvent) in order to effect reduction of the amide link at N-l, and X in the case where X is a carbonyl group, and isolating the compound of formula (I).

This process entails, as an optional initial step, formation of a suitable acid addition salt in order to render the compounds of formula (II) soluble in organic solvents. Such a reaction may be performed, for example, by dissolving the compound of formula (II) in anhydrous trifluoroacetic acid, the latter being used in excess at a temperature generally between room temperature and 0°C. Excess acid is removed by evapo20 rating to dryness under vacuum. The salt is then dissolved in an anhydrous, reaction-inert, organic solvent, for example, tetrahydrofuran or 1,2-dimethoxyethane and treated with the reducing agent, for example, diborane, conveniently added as a solution of diborane in tetrahydrofuran, generally In excess at a temperature generally between room temperature and reflux temperature, depending on the nature of the particular reactants and solvent employed. In the case - 6 where X is CO, sufficient reducing agent is naturally used to ensure reduction of both amide carbonyl groups.

The reaction is substantially complete within 24 hours when it is performed in tetrahydrofuran at 50°C with an excess of diborane; the product is then conveniently isolated by the addition of water to destroy unreacted diborane and removal of the organic solvent by evaporation under vacuum. The pH of the remaining aqueous solution is adjusted to 5 and the crude product may then be purified from unreaeted starting material and by-products by a conventional chromatographic technique.

Many of the compounds of formula (II) where X is CH, are known antibiotics previously disclosed; for example N-l-(4-amino-2-hydroxy-butyryl) kanamycin A, which is also referred to as BB-K8, is disclosed in U.S. Patent Specification No. 3781268. Other examples are described in U.S. Patent Specifications Nos. 3781268 3541078 and 3860574 and in published West German Patent Applications Nos. 2350203 and 2322576. The l*-N-(5-amino2-hydroxy-valeryl) and 1-N-(3-amino-2-hydroxy-propionyl) derivatives of kanamycin A and B are described in published West German Patent Application No. 2408666 and in J. Antibiotics 1974, 27, 851. 6'-N-Alkyl deri25 vatives are described in published West German Patent Application No. 2350169 and in J. Antibiotics, 1975, 28, 483, Compounds of formula (II) where X is CO may be derived by acylation of the l-amino group of 2-deoxy30 streptamine aminoglycosides by methods analogous to those used in the preparation of compounds of formula (II) where X is CH, but using as acylating agent a reactive derivative of an acid of the formula: OH I HOOC CHCCH^^CONH, 3 4 5 2 - 7 The novel compounds of formula (I) according to the invention may exist in various conformational forms, and the invention is not limited to any one such form thereof.

Generally the rings A and B are each in the chair form, and each of the moieties R?, OR^ and OR4 and the amino and hydroxyl groups is disposed equatorially with respect to the rings A and B. Furthermore, the glycosidic linkage between the hexopyranosyl ring A and the 2deoxystreptamine ring B is more usually an «-linkage with respect to the former, particularly when the compounds of formula (II) are derived from naturallyoccurring 2-deoxystreptamine aminoglycosides. Additionally the B-hydroxy-oi-aminoalkyl group at N-l may exist in the S or R configuration or may be present as a mixture of both optical isomers.

The in vitro evaluation of the compounds of the invention as antibacterial agents has been performed by determining the minimum inhibitory concentration (M.I.C.) of the test compound in a suitable medium at which growth of the particular micro-organism fails to occur. In practice, agar plates, each having incorporated therein the test compound at a particular concentration, are inoculated with a standard number of cells of the test micro-organism and each plate is then incubated for 24 hours at 37°C. The plates are then observed for the presence or absence of the growth of bacteria and the appropriate M.I.C. value noted. Microorganisms used in such tests have included strains of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis. Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus faecalis.

In vivo evaluation of the compounds has also been carried out for the more active compounds, by administering the compounds subscutaneously to mice which are - 8 exposed to a strain of Escherichia coli. Each compound is administered at a series of dosage levels to groups of mice and its activity is determined as the level at which it gives 50% protection, against the lethal effect of the Escherichia coli organism over a period of 72 hours.

By virtue of their antibacterial activity the compounds of the invention are useful for treating grampositive and gram-negative bacterial infections. For human use, the antibacterial compounds of the invention can be administered alone, but will generally be administered in admixture with a pharmaceutically acceptable carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, they may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsules either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavouring or colouring agents. They may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, they are best used in the form of a sterile aqueous solution which may contain other solutes, for example, enough salts or glucose to make the solut25 ion isotonic.

For administration to human patients, it is expected that the daily dosage level of the antibacterial compounds of the invention will be comparable with that of aminoglycoside antibacterial agents currently in use, e.g. from 0.1 to 50 mg/kg (in divided doses) when administered by the parenteral routes, or from 10 to 100 mg/kg (in divided doses) when administered by the oral route. Thus tablets or capsules of the compounds can be expected to contain from 0.1 to 1 g of active compound for administration orally up to 4 times a day, while dosage units for parenteral administration will contain 2 4 5 2 - 9 from 10 to 500 mg. of active compound. The physician in any event will determine the actual dosage which will be most suitable for an individual patient and it will vary with age, the weight and response of the particular patient. The above dosages are exemplary of the average host. There can, of course, be individual cases where higher or lower dosage ranges are merited, and such are within the scope of this invention.

The following are Examples of the preparation of novel compounds according to the invention. Temperatures are given in °C; Amberlite is a registered trade mark.

EXAMPLE 1 1-N-Z7 (S)-4-Amino~2-hydroxybutyrylJ7''kanamycin A (BB-K8, prepared as described in U.S. Patent Specification No. 378126&X15O mg) was dissolved in anhydrous trifluoroacetic acid (10 ml) at 0°C. The solution was evaporated to dryness in vacuo and dried under high vacuum at 20°C for 15 minutes to yield a glassy solid. This was taken up in dry tetrahydrofuran (5 ml) and a 1M solution of diborane in tetrahydrofuran (20 ml) was added in portions, under an atmosphere of nitrogen. The resulting clear solution was heated at 50° for 3 hours, allowed to stand at room temperature for 16 hours and heated for a further three hours at 50°. The excess diborane was destroyed by the cautious addition of a few drops of water and the organic solvent was then removed by evaporation under reduced pressure. The residue was taken up in water (10 ml) and basified with N/10 aqueous sodium hydroxide. The pH of the resulting solution was adjusted to 5 by the addition of 2N hydrochloric acid. The solution was then chromatographed on a column containing Amberlite CG 50 ion-exchange resin (50 ml), in the ammonium-ion form, eluting in turn with distilled water to remove inorganic solids, and then with a gradient of aqueous ammonium hydroxide of - 10 increasing concentration from 0.1 to 1.0N. Fractions containing the product (as monitored by thin layer chromatography) were combined and evaporated in vacuo to give l-N-/~(S)-4—amino-2-hydroxybutyl~7~kanamycin A (75 mg, 50% yield).

Thin layer electrophoresis. Rf = 0.6 (The electrolyte was an eguipart mixture of acetic and formic acids, giving a pH value of 2, and a potential difference of 900 volts was applied across the ends of the 20 cm silica-coated plate for 45 minutes. Detection was performed by drying the plate, spraying with a cyclohexane solution of tertiary-butyl hypochlorite and then drying, cooling and developing the plate with starch-potassium iodide solution. Under these conditions the reference standard BB-K8 gave an Rf value of 1.0 and kanamycin A an Rf value of 0.9).

Infra-red spectrum confirmed the loss of the amide carbonyl absorption band observed in BB-K8 at 1635 cm Optical Rotation /~a + 73° (c 1.0, H20).

Mass spectrometry (field desorption) showed a strong P+1 peak at m/e 572.

A sample was converted to the volatile penta-Nacetyl-octa-O-trimethylsilyl derivative by treatment with acetic anhydride in methanol at room temperature for 24 hours, followed by reaction with a 2:1 mixture of hexamethyldisilazane and trimethylchlorosilane at room temperature for 24 hours. M+ found 1357. CggHj^gN^O^ySig requires M+ 1357.

Analysis:- Found: C, 40.1; H, 6.7; N, 9.6% C22H45N5°12‘2^H2CO3 retIuires c/ 40.5; H, 6.9; N, 9.6% EXAMPLE 2 Butirosin (1-N-/7 (Ξ)-4-amino-2-hydroxybutyrylZ7 ribostamycin), as free base, (100 mg) was dissolved in 3 4 5 2 - 11 anhydrous trifluoroacetic acid (5 ml) at room temperature. Excess acid was removed by evaporation to dryness under vacuum to yield trifluoroacetate salt as a glass. This was taken up in dry diethylene glycol dimethyl ether (diglyme) (10 ml) and a IM solution of diborane in tetrahydrofuran (10 ml) was added to give a clear solution, which was allowed to stand for 18 hours at room temperature. A further 5 ml diborane solution was added and the solution kept at room temperature for a further 24 hours. Excess diborane was destroyed by the cautious addition of a few drops of water and the organic solvents were removed under vacuum at 50°. The residue was basified with a few drops of 2N sodium hydroxide solution and the pH adjusted to 5 by the addition of 2N hydrochloric acid. The product was isolated by ion-exchange chromatography on Amberlite CG 50 resin, as described in the foregoing Example, fractions containing the product in pure form were combined and evaporated under vacuum Lo give l-N-/~(E)4-amino-2-hydroxy-butyl T-ribostamycin.

Thin layer electrophoresis. Rf = 0.5 (The conditions were as previously described, but irosin was used as the reference standard with an Rf value of 1.0).

EXAMPLE 3 l-N-/“(S)-5-Amino-2-hydroxyl-valerylJ7“kanamycin A (0.35 g) was converted to the trifluoroaoetate salt, reduced and chromatographed as described for Example 1 to give l-N-/~(S)-5-amino-2-hydroxy-pentyl~7~kanamycin A. (0.12 g, 35%).

Thin layer electrophoresis. Rf = 0.7 (Conditions as described for Example 1, the starting material was used as the reference standard with an Rf of 1.0). - 12 EXAMPLE 4 1-N-(3-Amino-2-hydroxy-propionyl)-kanamycin A (0.15 g) was similarly reduced by the method of Example 1 to give 1-N-(3-amino-2-hydroxy-propyl)-kanamycin A (0.04 g, 27%).

Thin layer electrophoresis. Rf = 0.6 (Conditions as described for Example 1, the starting material was used as the reference standard with an Rf of 1.0).

EXAMPLE 5 1-N-/7(S)-4-Amino-2-hydroxy-butyryl27-kanamycin B was similarly reduced by the method of Example 1 to yield l-N-/~(S)-4-amino-2-hydroxy-butyl 7-kanamycin B. Thin layer electrophoresis. Rf = 0.6 (Conditions as described for Example 1, the starting material was used as the reference standard with an Rf of 1.0'. Kanamycin B gave an Rf of 0.45).

EXAMPLE 6 61-N-Methyl-l-N-/7(S)-4-amino-2-hydroxy-butyryl2720 kanamycin A (prepared as described by H. Umezawa et. al. in J. Antibiotics, 1975, 28, 483) was reduced as described for Example 1 to yield 6 *-N-methyl-l-N/~(S) 4-amino-2-hydroxy-butyl 7-kanamycin A.

Thin layer electrophoresis. Rf = 0.7 (Conditions as described for Example 1, the starting material was used as the reference standard with an Rf of 1.0 and kanamycin A gave an Rf value of 1.03).

Results of the testing of the compounds of the Examples for anti-bacterial activity in vitro by the methods previously described are given in the following Table:4 2 4 5 2 - 13 TABLE: tn vi Lro ad i yj ty Example No. M.I.C.'s pg/ml E. Coli Klebsiella Proteus mirabi- lis Pseudomonas aeruginosa Staphyl- ococcus aureus pneumoniae 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. J 5 3.1 1.6 6.2 0.8 l.G 6 6.2 3.1 6.2 3.1 3.1 Additionally the compounds of Example 1 has been tested for in vivo activity by the methods previously described. The Ρϋ,-θ against E. Coli in mice was 3.8 mg/kg.

Claims (18)

1. CLAIM S:Compounds of the general formula: where R^ represents a hydrogen atom or a lower alkyl 5 group; R represents an amino or hydroxyl group; 3 4 one of R and R represents a hydrogen atom, while the other represents a glycosyl group as herein defined; 10 and n is 1, 2 or 3; and their pharmaceutically-acceptable acid addition salts.

2. Compounds according to claim 1 in which R^ is a hydrogen atom and n is 1 or 2. 15 3. Compounds according to claim 1 or 2 in which

3. 4 R is a hydrogen atom and R is a 3-amino~3-deoxy-a-Dglucopyranosyl group.

4. Compounds according to claim 1 or 2 in which R^ is a hydrogen atom and R^ is a β-D-ribofuranosyl 20 group.

5. Compounds according to any of claims 1, 3 and 4 in which R^ is a hydrogen atom.

6. Compounds according to any of claims 1, 3 and 4 2 4 5 2 - 15 10 4 in which R 3 is a methyl group.

7. l-N-ZZ(S)-4-amino-2-hydroxy-butylJ7“kanamycin A.

8. 1-N-(3-amino-2-hydroxy-propyl)-kanamycin A.

9. 1-N-/7 (S)—5—am.ino-2—hydroxy-pentyl J7~ kanamycin A.

10. l-N-/]~ (S)-4-amino-2-hydroxy-butylJ7~ kanamycin B.

11. 6'-N-Methyl-l-N-/~(S)-4-amino-2-hydroxybutyl7~kanamycin A.

12. 1-N-Z. (S) -4-Amino-2-hydroxy-butyl_’7 - ribostamycin.

13. A process for preparing compounds of formula (I) as defined in claim 1 which comprises reducing a compound of the formula: where R 3 to and n are as defined in claim 1 and X is CH 2 or CO, and isolating the compound of formula (I).

14. A process as claimed in claim 13 in which 20 the compound of formula (II) is reduced with diborane to give the compound of formula (I).

15. A process as claimed in claim 13 substantially as described in any one of the Examples. -

16. 16. A compound of formula (I) as defined in claim 1 which has been prepared by a process as claimed in any one of claims 13 to 15.

17. Pharmaceutical compositions comprising a 5 compound as claimed in any of claims 1 to 12 together with a pharmaceutically acceptable carrier.

18. A method of treating gram-positive and gramnegative antibacterial infections in non-human animals which comprises administering to the animal an 10 effective amount of a compound as claimed in any of claims 1 to 12 either alone or in the form of a pharmaceutical composition as claimed in claim 17.