CA1039275A - 2-hydroxy-w-aminoalkyl-derivatives of aminoglycoside antibiotics - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Compounds of the general Formula:
where R1 represents a hydrogen atom or a lower alkyl groups R2 represents an amino or hydroxyl groups one of R3 and R4 represents a hydrogen atom, while the other represents a glycosyl group as herein define and n is 1, 2 or 3;
and their pharmaceutically acceptable acid addition salts.

Description

~3~3Z7~
~ , This invention rela-tes 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 amino-glycosides have in common a three ring structure which may be represented by the general formula:

4, ~ _ O ~ 2 ~ H
where the ring A is the skeleton of a hexopyranose group hav-ing 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 posi-tion being occupied by a hydroxyl group.
The novel antibacterial agents of the invention are a series of 2-deoxystreptamine aminoglycosides having a ~-hydroxy-~-aminoalkyl-substituent on the l-amino group and having a glycosyl group attached to the 5- or 6-posi.tion o~
the streptamine ring B. Such compounds are effec~ive in ~ 3~7~
trea~ing a variety o~ gram-pos~tlve or gram-negative bacterial infectlons, including urinary tract in~ect~ons, ln anlmal~, including humans and possass advantages :Ln uae over 2-deoxy-~treptamine amlnoglyco~ides havlng an un~ub~tituted amino group in the l-positlon of the 2-deoxy~tr~pt~m~ne r~ng ~, ~uch a~ the naturally-occurrlng kanamycin A and ~, neomycln~
and ribostamycln.
According to the invention, therefore, there i~
provided a proces.~ for preparing novel 2--deoxy~treptamine-10 aminoglycosides of the general formul~: :
~H2NHRl H

HO ~ O ~ N~CH2CH(CH2)nNH2 HO~R2 R30~'oR
...(I)and their pharmaceutically-acceptable ~alts;
wherein Rl represents a hydrogen atom or an alkyl group having from 1 to 4 carbon atoms;
R2 represents an amino or a hydroxyl group;
one o~ R3 an~ R4 represents a hydrogen atom, while the other represents a glycosyl group; and n is 1, 2 or 3;
wherein R3, when it is a glycosyl group, is o~ the 20 formula: ~O-CH2 ~

HO-CH ~ or ~ ~ ~ OX

HO OH ~
H~ N%2 and R , when it is a glycosyl group, is of the ~orm~la:

~ -3- :

, ,~

~3~2t7~
~o HO-~ < ~ _ CH2 H2~ OH
whl~h ~omprla~s reducing a compou~d of the ~ormulao C~2NHRl NH~

HO ~ O ~ NN~-CN~cN2)n 1XN~2 ... ~II) :
where Rl to R4 and n are a~ previou~ly defined and X 19 CH2 or CO, and isolating the compound of Formula (I).
The term lower alkyl group means a group contain-ing from 1 to 4 carbon atoms whlch may be ~traight or branch--3a-~ t~
ed chain.
One particular group of compounds according to the invention comprises the group in which Rl is a hydrogen atom and n is 1 or 2.
A preferred class of compounds according to the in-vention comprises compounds in which R3 is a hydro~en atom and R4 is a 3~amino-3-deoxy-a-D-glucopyranosyl group, i.e., derivatives of kanamycin A and B. Also preferred are com-pounds in which the ~-hydroxy-~-aminoalkyl group at the l-N
lG position has the (S) configuration and n is 2 or 3. Rl is preferably a hydrogen atom or a methyl group.
Particularly preferred individual compounds accord-ing to the invention include l-N-[(S)-4-amino-2-hydroxy-butyl~-kanamycin A and l-N-~(S)-5~amino-2-hydroxy-pentyl]-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 pharma-ceutically-acceptable anions such as the hydrochloride, hydro-bromide, hydroiodide, sulphate or bisulphate, phosphate or acid phosphate, acetate, maleate, fumarate, oxalate, lactate, tartrate, citrate, gluconate, saccharate, p-toluene sulphon-ate and carbonate salts.
The novel compounds of Formula (I) may be prepared according to the invention from compounds of the formula:

(~H2NHRl H2 ~ ~' H ~ O ~ NH-~-CH(CH~) YNH

H R R O OR

.

:~3~
where Rl to ~ and n are as previously defined and X is CH2 or C0, by reacting with a reducing agent 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.
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 gen-erally between room temperature and 0C. Excess acid is re-moved by evaporating to dryness under vacuum. The salt is then dissolved in an anhydrous, reaction-inert, organic solvent, for example, tetrahydrofuran or dimethoxyethane and treated with the reducing agent, for example, diborane, con-veniently 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 where X is C0, sufficient reducing agent is naturally used to ensure reduckion of both amide carbonyl groups.
The reaction i9 substantially complete within 24 hours when it is performed in tetrahydrofuran at 50 with an excess o~ diborane; the product is then conveniently isolated by the addition of water to destroy unreacted diborane and re-- moval of the organic solvent by evaporation under vacuum.
The pH of the remaining a~ueous solution is adjusted to pH 5 and the crude product may then be purified from unreacted starting material and bi-products by a conventional chromat~graphic technique.

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Many of the compounds of Formula (II) where X is CH2 are known antibiotics previously disclosed, for example N-1-(4-amino-2-hydroxy-butyryl) kanamycin A, which is also referred to as BB-K8, is disclosed in United States Patent

3,781,268. Other examples are described in United States Patents Nos. 3,781,268, 3,541,078 and 3,860,574 and in published West German Patent Application Nos. 2,350,203 and 2,322,576. The l-N-(5-amino-2-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. 2,408,666 and in J. Antibiotics 1974, 27, 851. 6'-N-Alkyl derivatives are described in published West German Patent Application No. 2,350r169 and in J. Antibiotics, 1975, 28, 483.
Compound~ of Formula (II) where X is CO may be derived by acylation of the l-amino group of 2-deoxy-streptamine aminoglycosides by methods analogous to those used in the preparation of compounds of Formula (II) where X is CH2 but using as acylating agent a reactive derivative of an acid of the formula:
OH
HOOC CH(CH2)n_lcONH2 ~he novel compounds of Formula (I) according to ~;
the invention may exist in various conformational forms and the invbntion 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 R2, oR3 and oR4 and the amino and hydroxyl groups is disposed equatorially with respect to the rings A and B. Furthermore, the glycosidic linkage between khe hexopyranosyl ring A and the 2-deoxystreptamine ring B is ., ~ . . .
. . . .
,: . . . .
~, :

~ 3~
more usually an ~-linkaye with respect to the former, par-ticularly when the compounds o~ Yormula (II~ are derived from naturally-occurring 2-deoxystreptamine aminoglycosides.
Additionally the ~-hydroxy-~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 ln 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 o~ 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 37C. The plates are then observed for the presence or absence of the growth of bacteria and the appropriate M.I.C. value noted. Micro-organisms used in such tests have included strains of Escherichla coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Stre~ococcus faecalis.
In vlvo evaluation of the compounds has also been carried out for the more active compounds, by administering the compounds subcutaneously to mice which are 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 o~ 72 hours.
For human use, the antibacterial compounds of the 0 invention can be administered alone, but will generally be 7~
administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. ~or 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 flavoring or coloring agents. They may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administra- `
tion, they are best use~ in the form of a sterile aqueous solution which may contain other solutes, for example, enough `
salts or glucose to make the solution isotonic.
Fo~ administration to human patients, it is expect-ed that the daily dosage level of the antibacterial compounds o~ the invention will be comparable with that of aminoglyco-side 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 admin-istration will contain 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. Temperature~ are given in C; I'Amberlite" is a registered trade mark.

l-N-[(S) 4-Amino-2-hydroxybutyryl]-kanamycin A
~BB-K8, prepared as described in United States Patent 3,781,268) (150 mg.) was dissolved in anhydrous trifluoro-acetic acid (10 ml.) at 0C. The solution was evaporated to dryness in vacuo and dried under high uacuum at 20C., for 15 minutes to yield a glassy solid. This was taken UE~ in dry tetrahydrofuran (5 ml.) and a lM 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 tempera- :
ture for 16 hours and heated for a further three hours at 50. The excess diborane was destroyed by the cautioùs addi- ~
tion 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 solu-tion was adjusted to 5 by the addition of 2N hydrochloric acid. The solution was then chromatographed on a column con-taining 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 increasing concentration from 0.1 to l.ON. Fractions containing the product (as monitored by thin layer chromatography) were combined and evaporated ln vacuo to give l-N-l(S)-4-amino-2-hydroxybutyl~ kanamycin A
(75 mg., 50% yield).
_g_ ~, .

~3~

Thin layer elec-trophoresis. Rf = 0.6 (The electrolyte was an equipart mix~ure 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 S 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, cool-ing and developing the plate with starch~potassium iodide solution~ Under these conditions the r~ference standard BB-K8 gave a~ 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-X8 at 1635 cm~l.
Optical Rotation [~]D25 ~ 73~ (c 1.0, H2O).
Mass spectrometry (field desorption) showed a strong P~l peak at m/e 572.
A sample was converted to the volatile penta-N-acetyl-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 hexamethyl-disilazane and trimethylchlorosilane at room temperature for 24 hours. M~ found 1357.C56HllgN5O17Si8 reqUire Analysis:- Found: C, 40.1; M, 6.7; M, 9.6%
22 45N5O12.2 1/2H2CO3 requires: C, 40.5; H, 6 9; N 9 6%

Butirosin (l-N~(S)-4-amino-2-hydroxybutyryl]ribo- ;
stamycin), as free base, (100 mg.) was dissolved in anhydrous trifluoroacetic acid (5 ml.) at room temperature. Excess acid was removed by evaporation to dryness under vacuum to yield the trifluoroacetate salt as a glass. This was taken -~

: ~ . .

~39~

up in dry diethylene glycol dimethyl ether (diglyme) (10 ml.) and a lM solution of diborane in tetrahydrofuran (lO 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 adjus~ed to 5 by the addition of 2N hydrochlor:ic 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 to give l~N-[(S)-4-amino-2-hydroxy butyl]ribostamycin.
Thin layer electrophoresis. Rf = 0.5 (The conditions were as previously described, butirosin was used as the reference standard with an Rf value of l.O).

l-N-[(S)-5-Amino-2 hydroxyl-valery]-kanamycin A
(0.35 g.) was converted to the trifluoroacetate salt, re-duced and chromatographed as described for Example l to give l-N-[(S)-5-amino-2-hydroxy-pentyl]-kanamycin A. (0.12 g. 35~).
Th n layer electrophoresis. Rf = 0.7 (Conditions as described for Example l, the start-ing material was used as the reference standard with an Rf of l.O).

l-N-(3-Amino-2-hydroxy-propionyl)-kanamycin A

: .

(0.15 g.) was similarly reduced by the method of Example 1 to give l-N-t3-amino-2-hydroxy-pro~x~kanamycln A. (0.04 g, 27%).
Thin layer electrophoresis. Rf = 0.6 (Conditions as described for E,xample 1, the start-ing material was used as the reference standard with an Rf of 1.0).

l-N-[(S)-4-Amino-2-hydroxy-butyryl]-kanamycin B was similarly reduced by the method of Example 1 to yield l-N-[(S)-4-amino-2-hydroxy-butyl]-kanamycin B.

6'-N-Methyl-l-N-[(S)-4-amino-2-hydroxy-butyryl]-kanamycin A ~prepared as described by H. Umezawa et. al~, in ~. Antibiotics, 1975, 28, 483) was reduced as described ?
for Example 1 to yield ~'-N-methyl-l-N-[tS)-4-amino-2-hydroxy-butyl]-kanamycin A.
Thin layer electrophoresis. Rf = 0.7 tConditions as described for Example 1, the start-ing 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 ln vitro by the methods previously described are given in the following Table:-, . . .
. .

7~
TABLE: In vitro activity Ex- M.I.C.~s ~g./ml.
ample . .
No.
E. KlebsieLla Proteus Pseudomonas ~ lo-Coli pneumonlae mirabl- eru~inos3 coccus lis aureus 1 6.2 3.1 3.11.6 1.6 2 6.2 6.2 2512.5 12.5 3 6.2 3.1 12.53.1 1.6 412.5 6.2 12.53.1 3.1 Additionally the compound of Example 1 has been tested for ln vivo activity by the methods previously de-scribed. The PD50 against E. Coli in mice was 3.8 mg~/kg.

Claims (6)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing novel 2-deoxystreptamina-aminoglycosides of the general Formula:
.. (I) and their pharmaceutically-acceptable salts;
wherein R1 represents a hydrogen atom or an alkyl group having from 1 to 4 carbon atoms;
R2 represents an amino or a hydroxyl group;
one of R3 and R4 represents a hydrogen atom, while the other represents a glycosyl group; and n is 1, 2 or 3;
wherein R3, when it is a glycosyl group, is of the formula or and R4, when it is a glycosyl group, is of the formula:

which comprises reducing a compound of the Formula:

.. (II) whare R1 to R4 and n are as previously defined and X is CH2 or CO, and isolating the compound of Formula (I).

2. A process as claimed in claim 1, in which the compound of Formula (II) is reduced with diborane to give the compound of Formula (I).

3. A process as claimed in claim 1, in which R3 is a hydrogen atom and R4 is a 3-amino-3-dioxy-.alpha.-D-glucopyranosyl group.

4. A process as claimed in claim 1 or 2, in which R4 is a hydrogen atom and R3 is a .beta.-D-ribofuranosyl group.

5. A process according to claim 1, in which R1 is a hydrogen atom or a methyl group.

6. A process according to claim 1, in which the compound of Formula (I) is:
1-N-[(S)-4-amino-2-hydroxy-butyl]-kanamycin A, 1-N-(3-amino-2-hydroxy-propyl)-kanamycin A, 1-N-[(S)-5-amino-2-hydroxy-pentyl]-kanamycin A, 1-N-[(S)-4-amino-2-hydroxy-butyl]-kanamycin B, 6'-N-Methyl-1-N-[(S)-4-amino-2-hydroxy-butyl]-kanamycin A or 1 N-[(S)-4-amino-2-hydroxy-butyl]-ribostamycin and is prepared by reduction of 1-N-[(S)-4-amino-2-hydroxy butyryl]-kanamycin A, 1-N-(3-amino-2-hydroxy-propionyl)-kanamycin A, 1-N-[(S)-5-amino-2-hydroxy-valeryl]-kanamycin A, 1-N-[(S)-4-amino-2-hydroxy butyryl]-kanamycin B
6'-N-methyl-1-N-[(S)-4-amino-2-hydroxy-butyryl]-kanamycin A or 1-N-[(S)-4-amino-2-hydroxy butyryl]-ribostamycin respectively,