NO140426B - ANALOGICAL PROCEDURE FOR THE PREPARATION OF ANTIBIOTIC ACTIVE DERIVATIVES OF AMINOCYCLITOLS. - Google Patents

Description

The present invention relates to an analogous process for the production of antibacterially active 1-N-substituted derivatives of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols where the 1-N-substituent is an aminohydroxyacyl group, and non-toxic acid addition salts of such connections.

US patent 3 780 018 describes a method whereby 1-(L-(-)-γ-amino-α-hydroxybutyryl)-gentamicin C. and 2'-(L-(-)-Y-amino-α- hydroxybutyry])-gentamicin is produced by reacting gentamicin with a blocked active ester of L-(-)-y ~ amino-α-hydroxybutyric acid followed by deblocking via known methods and separation of the reaction mixture by chromatography. US patent document 3 796 698 describes a process for the production of T-(L-(-)-Y -amino-α-hydroxybutyryl)-gentamicin C2 and US patent document 3 796 699 describes a process for the production of 1 -(L-(-)-γ-amino-α-hydroxybutyryl)-gentamicin C .

The new antibacterial agents produced according to the present invention have surprisingly unexpected properties. In general, these new compounds are active against bacteria and/or protozoan strains. Furthermore, they are active against many bacteria that have become essentially insensitive to the non-derivative antibiotics.

The invention thus relates to an analogue process for the preparation of antibiotically active 1-N-substituted derivatives of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitolene gentamicin B, gentamicin B^.# gentamicin , gentamicin C^a, sisomicin, verdamicin , Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B and Antibiotic G-52, in which the 1-N substituent is denoted by X and consists of

S -3-amino-2-hydroxypropionyl or S -4-amino-2-hydroxy-butyryl, with the proviso that in the case of gentamicin C1

and gentamicin C. In 3., X is S -3-amino-2-hydroxypropionyl,

and pharmaceutically acceptable acid addition salts thereof.

A preferred group of compounds consists of 1-N-substituted derivatives of the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols gentamicin B, gentamicin B^, sisomicin and verdamicin, where the 1-N substituent is part of the group consisting of S-3-amino-2-hydroxypropionyl and S-4-amino-2-hydroxybutyryl, and pharmaceutically acceptable acid addition salts thereof.

Within this group of compounds, the following specifically indicated compounds are 1-N-(S-4-amino-2-hydroxybutyryl)-gentamicin B, 1-N-(S-3-amino-2-hydroxypropionyl)-gentamicin B, 1 -N-(S-4-amino-2-hydroxybutyryl)-gentamicin B^, 1-N-(S-3-amino-2-hydroxypropionyl)-sisomicin, 1-N-(S-3-amino-2- hydroxypropionyl)-verdamicin and the pharmaceutically acceptable acid addition salts thereof preferred.

The compounds 1-N-(S-4-amino-2-hydroxybutyryl)-gentamicin

B, 1-N-(S-3-amino-2-hydroxypropionyl)-gentamicin B, 1-N-(S-3-amino-2-hydroxypropionyl)-sisomicin, 1-N-(S-3-amino- 2-hydroxypropionyl)-verdamicin and the pharmaceutically acceptable acid addition salts thereof represent a particularly preferred group of compounds and the compounds 1-N-(S-4-amino-2-hydroxybutyryl)-gentamicin B, 1-N-(S-3-amino -2-hydroxypropionyl)-gentamicin B and the pharmaceutically acceptable acid addition salts thereof are most preferred.

The compounds produced according to the invention are defined by the following structural formula I, II and III, where X is the 1-N-substituted as previously indicated.

where Y is an aminoglycosyl function selected from the group consisting of: 1-N-X-Antibiotic 66-40D of formula II and 1-N-X-gentamicin A and 1-N-X-Antibiotic 66-40B of formula III where Y' is

The non-derivative (parent) antibiotics (defined by the structural formulas I, II and III given above where X is hydrogen) are all known in the art.

Antibiotics 66-40B and 66-40D are produced together with sisomicin, as the main product of the fermentation of Micromonospora inyoens-sis (described in British Patent 1 274 518). Antibiotic 66-40B and 66-40D can be separated from the fermentation medium using special chromatographic separation techniques as described in Belgian Patent Document 811 370.

The analog method according to the invention is characterized by using one of the following method alternatives A) or B): A) one of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diamino-cyclitols which can have amino-protecting groups in any position other than the 1-position, is treated with an acid of the formula in which X' is a group as defined for X, in which the amino group and/or the hydroxy group may be protected, in the presence of a carbodiimide, or with a reactive derivative of the above-mentioned acid, or B) that one of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diamino-cyclitols which is partially neutralized by the formation of an acid addition salt, is treated with an acid of the formula

in which X<1> is a group as defined for X, in which the amino group and/or the hydroxy group may be protected,

in the presence of a carbodiimide or with a reactive deriv

the above-mentioned acid,

which method (A) or (B) is followed by the removal of all protective groups present in the molecule and the isolation of the derivative as such, or as a pharmaceutically acceptable acid addition salt.

In process alternative A, it is generally preferred to use the starting compounds which have a 6'-CH2-NH2 group in the form of their 6'-N-protected derivatives. Examples of preferred protected groups are trifluoroacetyl and t-butoxycarbonyl. Gentamicin C. can advantageously be used as the 2',3-di-N-protected derivative. Gentamicin B1, verdamicin, Antibiotic G-418, Antibiotic JI-20B and Antibiotic G-52 can be used in protected form but can also advantageously be used in free form.

In method alternative B), specific acylation is achieved at the amino group in the 1-position rather than arbitrary acylation by using 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol which is partially neutralized by forming an acid addition salt.

Protonation of an amino group provides the same effect as the presence of a "normal chemical" blocking group. It has surprisingly been found that the amino group in the 1-position of a 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol is the last to be protonated by the addition of acid, or if a peracid addition salt is treated with a base, the first which is deprotonated. Thus, "blocking groups" (in the form of protons) can easily be introduced into the molecule by adding a desired amount of acid to the aminoglycoside or by adding a desired amount of base to a peracid addition salt thereof. This method alternative draws advantagefay. these discoveries thus provide a convenient method for the preparation of 1-N-acyl derivatives of the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols using partially neutralized and therefore partially blocked starting materials.

As used herein, the term "partially neutralized

in forming an acid addition salt" that each mole of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol has bound to it less than the stoichiometric number of moles of acid necessary to form the peracid addition salt. Furthermore, this expression indicates that each mole of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol has at least one mole of acid associated with it.

For example, one equivalent of gentamicin Cj has five amino groups that require five equivalents of acid to form the peracid addition salt. The new method is performed on an acid addition salt of gentamicin having less than five equivalents and at least one equivalent of the acid (eg, 4.5. 4.0, 3.5, 3.0, 2.5, 2.0, 1.5 or 1.0 equivalents of acid).

In the preferred feature of this method, the starting compound is neutralized with (n-1) equivalents of acid, n being the number of amino groups in the molecule. Thus (n-1) amino groups neutralize by forming an acid addition salt. However, it will be understood that the method can also advantageously be carried out on partially neutralized starting compounds in which more or less than (n-1) equivalents of acid give rise to the acid addition salt within the above stated limits. Expressed in pH ranges, the method is carried out in a range from 5.0 to 9.0, preferably from 5.0 to 8.0. The most preferred range for the reaction medium is a pH from 6.5 to 7.5, especially 6.8 to 7.2.

The term "acid addition salt" includes such salts which can be formed between the basic 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol and an acid regardless of whether the acid can be designated as inorganic or organic. Examples of acids covered by the term are sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, acetic acid, propionic acid, succinic acid, oxalic acid, cyclopropylcarboxylic acid, trimethylacetic acid, maleic acid, benzoic acid, phenylacetic acid, trifluoroacetic acid or the like. If it is desired to use scmet starting material an acid addition salt where (n-1) amino groups are protonated, this compound is preferably formed in situ by reacting a per-acid addition salt with an equivalent of a strong base, e.g. triethylamine.

It is also possible to use partially neutralized starting materials which have chemically blocking groups. However, this is not necessary.

The hydroxyaminoacylating agents used in the methods according to the invention are preferably used in the form of reactive derivatives, e.g. in the form of active esters, anhydrides, azides or imidazole derivatives. Furthermore, it is preferred that the amino group in the hydroxyaminoacylating agent is blocked. Thus, when the desired hydroxyaminoacyl product is the iso-serinyl derivative, the preferred acylating agent will be taken from

where the carboxyl function is preferably activated in the form of an active ester (leaving group Lg) and the amino group is preferably blocked (blocking group Bg) with a benzyloxycarbonyl group which is easily removed by hydrogenolysis or a t-butoxycarbonyl group or a trifluoroacetyl group, the former being suitably removed by acid and the latter with base.

If pharmaceutically acceptable acid addition salts of the compounds according to the present invention are desired, these can be prepared by adjusting an aqueous solution of the agent to pH 4.0 followed by lyophilization. The salts thus formed are the functional equivalents of the free nitrogen base. Examples of acids that can be used for salt formation are sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, acetic acid, propionic acid, succinic acid, oxalic acid or maleic acid.

As used herein, the terms "blocking group" or "protecting group" denote groups which render the blocked or protected amino groups inert to subsequent desired chemical treatment, but which can be easily removed after completion of the synthesis sequence without cleavage of the desired N-aminohydroxyacyl group.

Amino protecting groups are generally known in the art. For the present method, however, trifluoroacetyl, 2,2,2-trichloroethoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl and 4-methoxy-benzyloxycarbonyl groups are preferred groups. Particularly preferred among the foregoing are trifluoroacetyl, t-butoxycarbonyl and benzyloxycarbonyl groups.

In the blocking process, the blocking group is usually used in the form of an acid imidazole derivative, an acid azide or as active esters such as ethylthioltrifluoroacetate, t-butoxycarbonylazide, N-benzyloxycarbonyloxysuccinimide or p-nitrophenyltrichloroethylcarbonate.

The following examples illustrate the invention.

Example 1

1- N-( S- 4- amino- hydroxypropionyl)- sisomycin

A. S-4-trifluoroacetamido-2-hydroxypropionic acid

Dissolve 5 g of S-4-benzyloxycarbonylamino-2-hydroxy-propionic acid in a mixture of 80 ml of dioxane and 20 ml of water. Add 200 mg of 30% palladium-on-carbon and hydrogenate at ambient temperature and 3.4 atmospheres for 3 hours. Remove the catalyst by filtration and concentrate the filtrate to a residue in vacuo. Dry the residue in high vacuum for 72 hours. Dissolve the dried residue in 30 ml of cold trifluoroacetic anhydride while stirring. Stir the mixture for 3 hours at ambient temperature and then concentrate in vacuo to a residue. Triturate the residue with benzene to give a gray solid which is isolated by filtration, washed with benzene and dried to give the product of this step.

B.

Dissolve 20 millimoles of the product according to step A in 50 ml of ethyl acetate, add 2.31 g of N-t-hydroxysuccinimide with stirring and cool the resulting solution in an ice bath. Add 4.3 g of dicyclohexylcarbodiimide to the solution and stir the reaction mixture for 3 hours at room temperature. Filter to remove the precipitate and concentrate the filtrate to dryness to give the title compound which is dried under high vacuum and used in step D.

C. 6'- N- trifTuoracéty1s in somiein

Dissolve 20 g of sisomicin in 1.2 liters of anhydrous methanol and add dropwise a solution of 6 ml of ethylthioltrifluoroacetate in 60 ml of methanol during 3 hours with stirring. Allow the reaction to proceed for 18 hours at room temperature and remove the solvent in vacuo to leave a residue of 23.8 g of the product with approx. 95% purity and with the following physiochemical properties: -Mass spectral data: m/e 543 M+, other definitive peaks at m/e 413, 395, 385, 362, 223 and 126.

NMR (60 MHZ, D 2 O) δ 5.37 doublet, J=2Hz, H-1<1>; 5^12 doublet, J=4Hz, H-l"; 4.96 broad singlet, H-4'; 2.57, singlet, N-GH3;

1.26 singlet, C-CH3.

D. 1-N-(S-4-amino-2-hydroxybutyryl)-sisomycin

Dissolve 814 mg (1.5 mmol) of 6'-N-trifluoroacetylsisomicin in 12 ml of 50% aqueous methanol and add dropwise while stirring a

solution of 1.5 mmol of the product according to step B dissolved in 3 ml of dimethylformamide. Stir it. resulting mixture at room temperature for 18 hours and concentrate in vacuo to a residue. Dissolve the residue in 10 ml of concentrated ammonium hydroxide and allow to stand for 2 hours (to remove the trifluoroacetyl groups). Evaporate the solvent to give the title product as a residue. Chromatograph the residue on a silica gel column using the lower phase of a chloroform methanol:ammonium hydroxide (2:1:1) solvent system as eluent. Fractions containing the title product are collected and concentrated to a residue which is dissolved in water and lyophilized to form an amorphous white solid.

1-N-(S-3-amino-2-hydroxypropionyl)-sisomicin, (c)^6 + 139°

(C, 0.3 in water) Anal. Calculated for C22<H>42°9<N>6<*> ^ <H>2C03'

C=47.74; h = 7.66; N = 14.85; found C = 47.39; H = 7.14; N = 15.16 Pmr. 100 MHz D20 6 1.22 (S), C-Me, 6 2.60 (S) N-Me 4.22 (Q),

J=4Hz, 7.5Hz; 65.09 (d) , H^,, ^« 2». 58 4'° Hz i 6 5'34 <d> ' Hi" <J>l',2'<=2Hz>

Prepare in a similar way:

1-N-(S-3-amino-2-hydroxypropionyl)-gentamicin C, , Pmr 100 MHz,

D20: 6 1.22 C-Me; 2.59 N-Me; (a) £° + 115.4° (C, 0.3 in water);

5.09 H-l", Jin 2„=4Hz, 5.17 H-l', J^, 2.= 3.5 Hz Anal.

Calculated <fir C22H44<0>9<N>6•<H>20-C02; C = 45.01; H = 7.39; N = 13.70; found C = 44.90; H = 7.81; N = 13.68%

1-N-(S-3-amino-2-hydroxypropionyl)-gentamicin B, Pmr 100 MHz,

D20 + DC1; 6 1.18 C-Me (S); 2.77 N-Me (S); 6 4.35 (Q), J=4.5, 8.0; H-1', 6 5.01 (d), Jlf 2'=4'° Hz; H-1"' 5'43 <d>' Jr<f2" =3.5 Hz

Example 2

1- N-( S- 4- amino- 2- hydroxybutyryl)- gentamicin B

A. 6'-N-t-butoxycarbonylgentamicin B

Dissolve 1 g of gentamicin B in 30 ml of 50% aqueous methanol

and cool to 5° C. Add 0.297 g of t-butoxycarbonylazide dropwise with stirring followed by 0.186 ml of triethylamine and stir the resulting mixture for 18 hours. Evaporate the reaction mixture in vacuo to a residue and chromatograph the residue on 100 g silica gel using a lower phase of a 2:1:1 chloroform:methanol:concentrated ammonium hydroxide solvent system as eluent. Collect 2 ml fractions and monitor the column effluent by TLC. Combine fractions containing similar material (fractions 180 -

230) and evaporate to give 0.830 g of 6<1->N-t-butoxycarbonylgentamicin B with the following physical constants: Pmr (60 MHz, D2O) 6 1.21 (3H, s, C-CH3), 1.42 (9H, s, C(CH3)3), 2.53 (3H, s, N-CH3),

5.2 (1H, d, J=4.5 Hz, H-l"), 5.23 (1H (1H, d, J=3.0 Hz, H-l') PPM, Calculated for C24<H>46N4612 "C02-H20; C = 46.57; H = 7.51; N, 8.6% Found C = 46.80; H = 7.82; N = 8.54% fa)<26> + 124° (C, 1 in methanol).

B. 1-N-(S-4-amino-2-hydroxybutyryl)-6'-N-t-butoxycarbonyl-gentamicin B

Dissolve 0.582 g of 6<1->N-t-butoxycarbonyl-gentamicin Bi in 25 ml of methanol:water (1:3). Add a solution of 0.334 g of N-(S-4-carbo-benzyloxyamino-2-hydroxybutyryloxy)-succinimide in 5 ml of dimethylformamide dropwise with stirring. Stir the reaction mixture at 5° C. for 5 hours and then evaporate to dryness in vacuo. Chromatograph the residue on 60 g of silica gel using the lower phase of a

2:1:1 chloroform:methanol:ammonium hydroxide solvent system as eluent. Collect 3 ml fractions and monitor contents by TLC. Combine fractions 160 - 235 and evaporate to a residue of 0.280 g which migrates as a single spot on TLC. Dissolve the residue in 8 ml of methanol and 10 ml of water and hydrogenate at 3.7 atmospheres over 60 mg of 5% palladium-on-charcoal catalyst. Remove the catalytic converter

by filtration, evaporate the filtrate to a residue and chromatograph on 20 g of silica gel using the lower phase of a 1:1:1 chloroform:methanol:ammonium hydroxide solvent system as eluent. Combine fractions 79 - 127 (2 ml each) consisting of 1-N-(S-4-amino-2-hydroxybutyryl)-6'-N-t-butoxycarbonyl-gentamicin B. Yield: 86 mg.

Pmr <S 1.21 (3H, s, C-CH3) , 1.43 (9H, s, C-(CH3)3), 2.51 (3H, s, N-CH3), 5.08 (1H , d, J = 4Hz, H-l"), 5.25 (1H, d, J= 3.5 Hz, H-l')

PPM.

C. 1- N-( S- 4- amino- 2- hydroxybutyryl)- gentamicin B

Dissolve the product from step B in 0.3 ml of trifluoroacetic acid and after 5 minutes add 30 ml of ethyl ether. A precipitate of the compound of this example as its trifluoroacetate salt forms and is recovered by filtration. Dissolve the precipitate in water and pass the solution through a basic ion exchange resin column in hydroxydione form to convert the acid addition salt to the free base. Freeze

the column effluent while forming the compound of this example

as' a white, amorphous solid. Yield: 72 mg.

Pmr D20 100 MHz: 6 1.14 c-methyl (S), 2.45 (S) N-methyl, 5.04 (d),

H-l", Jin „»4.0 Hz; 5.30 (d) , H-l', ^,^,=3.5 Hz.

Example 3 1-N-(S-3-amino-2-hydroxypropionyl)-gentamicin C^

A. 1-N-(S-3-carbobenzyloxyamino-2-hydroxypropionyl)-2'-3-di-N-trifluoroacetylgentamicin Cj^

Dissolve 0.84 g of 2',3-di-N-trifluoroacetyl-gentamicin Cl in 40

ml of tetrahydrofuran and add a solution of 1.19 g of N-(S-3-carbo-benzyloxyamino-2-hydroxypropionyloxy)-succinimide in 24 ml of tetrahydro-

furan dropwise and with stirring. Stir the reaction mixture for 24 h

hours and then evaporate to a residue. Chromatograph the residue on silica gel using the lower phase of a chloroform:methanol:concentrated ammonium hydroxide:water (2:1:0.2:0.8) v/v solvent system as eluent. Monitor the chromatography by TLC and combine fractions containing the same product to form a major product of the reaction with the following constants: mp. 125 - 131° C, (a)p<6>° = +93° (C<H>3OH) Anal: Calculated (for dihydrate) C = 47.47; H = 6.20; N = 9.23%. Found: C =

47.85; H = 6.67; N = 9.08%.

B. 1-N-(S-3-carbobenzyloxyamino-2-hydroxypropionyl)-

gentamicin C-^

Dissolve 0.55 g of the product according to step A in 55 ml

methanol and add 25 ml of concentrated ammonium hydroxide while stirring. Stir for 3 days at which time TLC on silica gel-

plates using the lower phase of a 1:1:1 mixture of chloroform:methanol:concentrated ammonium hydroxide solvent system which elicits essentially complete removal of the trifluoroacetyl blocking groups. Evaporate the solution to dryness in vacuo. Yield 0.2 g. Sm.p. 109 - 112°C; (a)<26>° = +73° (H 2 O).

C. 1-N-(S-3-amino-2-hydroxypropionyl)-gentamicin C-^

Dissolve 0.153 g of the redisolate from step B in 8 ml of acetic acid

and add 0.05 g of 10% palladium-on-carbon catalyst. Hydro-

ner solution at 4.0 atmospheres and at 25°C until TLC (using the solvent system used in step B) indicates complete conversion to the title product (ie, 16 hours to 3 days). Remove the catalyst by filtration and evaporate the

ning to a residue in vacuum. Chromatograph the residue over 7 g of silica gel using the lower phase of a 2:1:1 mixture of chloroform:methanol:ammonium hydroxide as eluent. Monitor the column with TLC, combine similar materials and thereby obtain the product according to the present example. Yield 112 mg. Sm.p.

109 - 119° C, (ot)2<6>° = +98° (H 2 O) . Analysis calculated (monohydrate)

C = 49.47; H = 8.65; N = 14.42% Found: C = 49.24; H = 8.53;

N = 14.10%.

Example 4

1- N-( S- 4- amino- 2- hydroxybutyryl)- verdamicin

A. 1-N-(S-4-phthalimido-2-hydroxybutyryl)-verdamicin

Dissolve 4.61 g of verdamicin in 50 ml of a methanol:water (1:3) solution and cool to 0 - 5° C. Add a solution of 3.81 g of N-(S-4-phthalimido-2-hydroxybutyryloxy) -succinimide in 20 ml of dimethylformamide drop by drop while stirring to the antibiotic solution. Stir for a further 16 hours and then concentrate to a residue in vacuo. Chromatograph the residue on a 250 g silica gel column using the lower phase of a 2:1:1 mixture of chloroform:methanol:concentrated ammonium hydroxide as eluent. OMonitor the eluate by TLC and combine fractions containing equal material. Evaporate the combined fractions containing the major product to give 1-N-(S-4-phthalimido-2-hydroxybutyryl)-verdamicin and an isomeric clay.

B. 1-N-(S-4-amino-2-hydroxybutyryl)-verdamicin

Dissolve 4.0 g of the product from the previous step in 35 ml of ethanol and add 1.0 g of hydrazine hydrate. Boil the solution under reflux for 3 hours and then evaporate to dryness in vacuo. Chromatograph the residue over 160 g of silica gel, eluting with the lower phase of a 1:1:1 (v/v) mixture of chloroform:methanol:concentrated ammonium hydroxide. Combine and evaporate the fractions containing the major component of the reaction as shown by TLC to give the compound of this example as a white amorphous solid.

CMR (25.2 MH, D20) 147.2, 101.5, 100.5, 99.0, 86.2, 83.9,

75.2, 70.9, 68.9, 68.3, 64.6, 51.2, 49.6, 46.4, 43.0, 35.6,32.8,

23.4, 21.7, 16.8.

Prepare in a similar way:

(s-4-amino-2-hydroxybutyryl)-gentamicin B1

PMR (60 MH , D 2 O) 6 1.28, p, C-Me; 1.26, d, CH-CH3; 1.7-2.3.m, 2xH-3'" and H-2"eq; 2.74, p, N-Me; 5.18, d, J=4Hz, H-1", 5.53,

d, J=3H , H-1' ppm. 1-N-(S-4-amino-2-hydroxybutyryl)-antibiotic JI-20B m.p. 128-138°C; IVf£ + 126.4 (C , 0.28 in water). PMR (100 MH , D 2 O) 6 1.12, p, C-Me; 1.21, d, CH-Me; 2.48, p, N-Me; 5.08, d,

J=3H , H-1"; 5.25, d, J=4Hz, H-1' ppm and 1-N-(S-3-amino-2-hydroxypropionyl)-gentamicin B1 PMR (D20): 6 1.08 (3H , d, J= 7.0 Hz» CHCH3),

1.20 (3H, s, C-CH3), 1.38 (1H, q,J= 13.0 Hz, H-2ax,),1.93 (1H,

m, H2e ), 2.48 (1H, d, J=10.5 Hz, H-3"), 2.5 (3H, s, N-CH^), 3.93-

4.28 LI, 2H, H-5" and H-2"'), 5.05 (1H, d, J= 4.0 Hz, H-1"), 5.25

e.g

(1H, d, J= 3.0 Hz, H-1').

CMR (D20),6 (rel TMS, PPM): 179.1, 102.4, 99.1, 89.6, 80.3, 76.1,

75.1, 74.7, 73.5, 73.1, 72.3, 69.3, 68.6, 64.1, 50.2, 49.4, 46.7, 46.1, 38.0, 22.7, 16.9.

Example 5

1. N-( S- 3- t- butoxycarbonylamino- 2- hydroxypropionyloxy)- succinimide A. S- 3- t- butoxycarbonylamino- 2- hydroxypropionic acid

Dissolve 8.0 g of S-3-carbobenzyloxyamino-2-hydroxypropion-

acid in -10 ml dioxane-water (4:1). Add 200 mg palladium-on-carbon catalyst and hydrogenate at 3.4 atmospheres for 3 hours.

Filter from the catalyst and evaporate the filtrate to a residue comprising S-3-amino-2-hydroxypropionic acid. Dissolve the acid in 40 ml of methanol and 8.5 ml of triethylamino and cool the solution in an ice bath and add 4.76 g of t-butoxycarbonyl azide. Allow the mixture to stand overnight with gradual warming to room temperature. Evaporate most of the methanol, dilute with water and acidify with dilute hydrochloric acid to pH 3. Extract the aqueous mixture several times with ethyl acetate, combine and dry the ethyl acetate extracts and

steam to a residue in vacuo.' Recrystallize the residue from ethyl acetate-hexane to give the title compound of this example. Yield 3.5 g. Sm.p. 92 - 94° C, (a)^ 6°, + 14.9°

(c = 0.68, <H>20), Calculated for CgH15<N0>5 required: C = 46.82;

H = 7.37; N = 6.83%. Found: C = 46.81; H = 7.61; N = 6.63%.

B. N-(S-3-t-butoxycarbonylamino-2-hydroxypropionyloxy)-succinimide

Dissolve 2.3 g of S-3-t-butoxycarbonylamino-2-hydroxypropionic acid in a mixture of 25 ml of tetrahydrofuran and 60 ml of ethyl acetate. Add with stirring 1.42 g of N-hydroxysuccinimide followed by

a solution of 2.51 g of dicyclohexyl carbodiimide in 10 ml of ethyl acetate. Allow the mixture to stir for 16 hours and then filter off the precipitated solid. Evaporate the filtrate to form the product of this example. Yield 3.56 g.

2. 2', 3-di-N-trifluoroacetyl-Gentamicin

A. 2'-N-trifluoroacetyl-Gentamicin

Dissolve 1.7 g of gentamicin C-^ in 20 ml of methanol, cool the mixture to 4°C and add 0.46 ml (0.563 g) of ethyl thiol trifluoroacetate with stirring. Allow the reaction to proceed for 2 hours and then concentrate the solution to a residue in vacuo. Chromatograph the product on 80 g of silica gel G using the lower phase of a mixture of chloroform:methanol:water:ammonium hydroxide in the volume ratio 10:5:4:1 as eluent. Combine the fractions containing the major component and concentrate to give 1.4 g of the title compound, m.p. 108 - 111° C, (a)^° = +128° (c = 0.3%, H 2 O). Analysis for C23<H>42<N>5<0>8<F>3<«H>20 (required

C = 46.69%, H = 7.50%, N = 11.84%, F = 9.63%. Found: C = 46.66%, H = 7.65%, N = 11.60%, F = 9.24%.

B. 2', 3-di-N-trifluoroacetyl-gentamicin C-^

Dissolve 0.66 g of the product according to step A in 10 ml of methanol, cool the mixture to 4° C and add 0.148 ml (0.182 g) of ethyl thiol trifluoroacetate dissolved in 3 ml of methanol. Stir the reaction mixture for approx. 16 hours and concentrate to a residue in vacuo. Chromatograph the product on 30 g of silica gel as described in step A. Monitor the column by thin layer chromatography, combine the appropriate fractions and concentrate to give 0.32 g of the title compound, m.p. 121 - 129° C, (ct}<26>° = 121<0> (c = 0.3%, H20) . Analysis for C25<H>41<N>5</>9<F>6 requires C =44.84%, H=6.1%, N= 10.46% Found: C = 44.94%, H = 6.35%, N = 10.17%.

Example 6

1- N-( S- 4- amino- 2- hydroxybutyryl)- gentamicin C,

X Say

A. 1-N-(S-4-benzyloxycarbonylamino-2-hydroxybutyryl)-gentamicin C,

Dissolve 2.8 g (4 mmol) gentamicin C, -sulphate in 30 ml

x 3.

water and add 15 ml of methanol. Add 0.56 mL (4 mmol) of triethylamine and stir for 10 minutes. Add a solution containing 4 mmol of N-(S-4-benzyloxycarbonylamino-2-hydroxybutyryloxy)-succi-

nimid in 20 ml of dry dimethylformamide dropwise with stirring to the antibiotic solution.

Stir the mixture overnight (16 hours) at ambient temperature. Thin-layer chromatography of the reaction mixture via TLC

on silica gel using the lower phase of a solvent subsystem consisting of chloroform:methanol:ammonium hydroxide (1:1:1), shows the presence of a plurality of minor components and one major component. Concentrate the reaction mixture to a residue in vacuo and triturate the residue with methanol to form 3.2 g of white solid material containing all the previously observed components by thin-layer chromatography in .

Chromatograph 150 ml of the product on 50 g of silica gel

using the lower phase of a solvent system consisting of chloroform:methanol:ammonium hydroxide (2:1:1). Collect fractions containing the main component and lyophilize under the

nelsis of 1-N-(S-4-benzyloxycarbonylamino-2-hydroxybutyryl)-gentamicin Cla. Yield 70 mg. NMR (D 2 O) <5 1.15 (3H, s, C-CH 3 ),

2.49 (3H, s, NCH3), 4.10 (1H, dd, J = 8.0, 4.0 Hz, side chain H-2), 7.36 (5H, m, phenyl).

B. 1-N-(S-4-amino-2-hydroxybutyryl)-gentamicin C^

Dissolve the product according to step A in a mixture consisting of

of 12 mL of methanol and 3 mL of water, add 20 mg of 10% palladium-on-carbon and hydrogenate at 4.0 atm at room temperature. After 3 hours, the reaction is essentially complete. Remove the catalytic converter

by filtration and lyophilized the filtrate to form 46 mg of 1-N-(S-4-amino-2-hydroxybutyryl)-gentamicin. NMR (D2O)

<S 1.17 (3H, s, C-CH3), 2.48 (3H, s, NCH3), 4.22 (1H, dd, J =

9.5 Hz, 4.0 Hz, side chain CHOH), 5.04 (2H, m, H-l' and H-l"). Mass spectral data: (M-H 2 O) m/e 532.

Example 7

1- N-( S- 4- amino- 2- hydroxybutyryl)- gentamicin B

A. 1-N-(S-4-benzyloxycarbonylamino-2-hydroxybutyryl)-gentamicin B

Dissolve 3.39 g of gentamicin B-sulphate in 48.4 ml of water and dilute with 23.7 ml of methanol. Add 0.7 ml of triethylamine dropwise while stirring. Dissolve 1.67 g of N-(S-4-benzyloxycarbonylamino-2-hydroxybutyryloxy)-succinimide in dimethylformamide and add the solution dropwise while stirring to the antibiotic solution. Stir the resulting solution at room temperature for 18 hours and then concentrate it to a residue in vacuo. Dissolve the residue in water and treat with dilute barium hydroxide solution while stirring until the pH reaches approx. 8.0. Remove the precipitated barium sulfate by filtration using a filter aid. Wash the precipitate with water, combine the filtrate and washings and concentrate to dryness in vacuo. Chromatograph the residue on a column containing 600 g of silica gel using the lower phase of a solvent system consisting of chloroform:methanol:ammonium hydroxide (1:1:1) as eluent. Collect the material eluting immediately before gentamicin B and concentrate the combined fractions to dryness to give 1-N-(S-4-benzyloxycarbonylamino-2-hydroxybutyryl)-gentamicin B as an amorphous solid. Yield 0.2 g.

NMR (D 2 O), δ 1.27 (3H, s, C-CH 3 ), 2.51 (3H, s, NCH 3 ), 5.08

(1H, d, J = 4 Hz), 5.25 (1H, d, J = 3.5 Hz).

B. 1- N-( S- 4- amino- 2- hydroxybutyryl)- gentamicin B

Dissolve the product according to step A in a mixture consisting of 20 ml of water and 8 ml of methanol. Hydrogenate the product in the presence of 60 mg of 5% palladium-on-carbon at 3.4 atmospheres and room temperature for 3 hours. Remove the catalyst by filtration using a filter aid f. Wash the filter cake with water and combine the filtrate and the wash water. Concentrate the combined filtrate and washings to dryness in vacuo. Chromatograph the residue on a silica gel column containing 10 g of silica gel using a solution consisting of chloroform:methanol:ammonium hydroxide (1:2:1) as eluent. Fractions containing the most polar components are collected, concentrated and lyophilized to form 1-N-(S-4-amino-2-hydroxybutyryl)-gentamicin B. Yield 12 mg.

Pmr D20 100 MHz: 6 1.14 c-methyl (S), 2.45 (S), N-methyl, 5.04 (d), H-1", J 2„=4.0 Hz; 5, 30 (d), H-1', Jq•, 2'=3.5 Hz.

The compounds produced according to the present invention are broad-spectrum antibacterial agents that show activity against organisms that are resistant to their 1-N-unsubstituted precursors. Thus, the compounds according to the present invention can be used alone or in combination with other antibiotic agents to prevent the growth or reduce the number of bacteria in different environments. The compounds according to the present invention are more active against many organisms that inactivate antibiotics by acetylation of the 3-amino group and/or by adenylation of the 2"-hydroxyl group. Thus, the compounds according to the invention have the ability to become antibacterial agents and can be used for the same purposes as their non-derivative (parent) antibiotics, for example they can be used as a bacteriostatic rinse agent for glassware in hospitals, surgical instruments, bathtubs or to clean areas where laboratory animals are housed or the like.

Minimum inhibitory concentration (MIC) in vitro.

Names of compounds in Table I:

a) known compounds

Compound 1: 1-N-(S-4-amino-2-hydroxybutyryl) gentamicin

C. (US Patent 3,796,699)

Compound 2: 1-N-(S-4-amino-2-hydroxybutyryl) gentamicin

C1 (US Patent 3,780,018)

b) New connections

Compound 3: 1-N-(S-3-amino-2-hydroxypropionyl) gentamicin C1

Compound 4: 1-N-(S-3-amino-2-hydroxypropionyl) gentamicin B Compound 5: 1-N-(S-4-amino-2-hydroxybutyryl) gentamicin B1 Compound 6: 1-N-(S- 4-amino-2-hydroxybutyryl) gentamicin B Compound 7: 1-N-(S-4-amino-2-hydroxybutyryl) antibiotic

JI-20B

Compound 8: 1-N-(S-3-amino-2-hydroxypropionyl) sisomicin Compound 9: 1-N-(S-3-amino-2-hydroxypropionyl) verdamicin

Test method used to obtain the results indicated in Table I.

Sterilize 200 ml Mueller-Hinton cast adjusted to pH 7.2. Transfer 5 ml of the sterile medium to each of 120 sterile, 16 x 150 mm test tubes with cotton plugs. Arrange the tubes in 10 groups of 12 tubes each. Add to each tube 10 4 cells of one of the bacterial strains to be tested. Add to each group an aqueous solution of the antibiotic to be tested, forming the following final concentration per tube: 50 mcg/ml, 25 mcg/ml, 10 mcg/ml, 5 mcg/ml, 2 mcg/ml, 1 mcg/ml, 0.5 mcg/ml, 0.2 mcg/ml, 0.1 mcg /ml, 0.05 mcg/ml, 0.01 mcg/ml, 0.005 mcg/ml and 0.0 mcg/ml. (control). Incubate the tubes for 24 hours at 37°C. Visually determine for each group of tubes the lowest concentration of antibiotic that inhibits bacterial growth and the highest concentration of antibiotic that allows bacterial growth.

Determine the minimum inhibitory concentration for testing the antibiotics against each of the tested bacteria by calculating the midpoint between the two values found.

In general, the administered dose of the compounds according to the invention will depend on the age and weight of the animal to be treated, the method of administration and the type and degree of bacterial infection to be prevented or reduced. In general, the dose used of the derivatives of 4,6-di-(aminoglycosyl)-1,3-diamino-cyclitols to combat a given bacterial infection will be equal to the dose of the 1-N-unsubstituted-4,6-di-( aminoglycosyl)-1,3-diaminocyclitols.

The compounds prepared according to the present invention can be administered orally. It can also be applied topically in the form of ointments, both hydrophilic and hydrophobic, in the form of lotions which may be aqueous, non-aqueous or of the emulsion type or in the form of creams. Pharmaceutical carriers which can be used in the preparation of such formulations include, for example, such substances as water, oils, fats, polyesters, polyols and the like.

For oral administration, the compounds according to the invention can be composed in the form of tablets, capsules, elixirs or the like or can also be mixed with animal feed. It is in these dosage forms that the antibacterial agents are most effective for the treatment of bacterial infections in the gastrointestinal system which infections cause diarrhoea.

In general, the topical preparations will contain from 0.1

to 3.0 g of the compounds according to the invention per 100 g ointment, cream or lotion. The topical preparations are usually gently applied to wounds from two to five times a day. day.

The antibacterial agents produced according to the present invention can be used in liquid form such as solutions, suspensions and the like for use in the ear and eye and can also be administered parenterally via intramuscular injection. The injectable solution or suspension will usually be administered with from 1 mg to 15 mg of antibacterial agent per body weight per

day divided into 2 to 4 doses. The exact dose depends on the degree and extent of the injection, the susceptibility of the infecting organism to the antibacterial agent and the individual characteristics of the animal being treated.

Claims (2)

1. Analogous process for the preparation of antibiotically active 1-N-substituted derivatives of 4,6-di-(aminoglycosyl)-1,3-diaminocyclitolene gentamicin B, gentamicin B^, gentamicin , gentamicin C1 I a, sisomicin, verdamicin, Antibiotic G -418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B and Antibiotic G-52, in which the 1-N-substituent is denoted by X and consists of S-3-amino-2-hydroxypropionyl or S-4-amino-2-hydroxybutyryl, with the proviso that in the case of gentamicin and gentami cin C. la, X is S-3-amino-2-hydroxypropionyl, and pharmaceutically acceptable acid addition salts thereof, characterized in that one of the following process alternatives A) or B) is used: A) one of the above-mentioned 4,6-di-(aminoglycosyl)-1,3-diamino-cyclitols which may have amino -protecting groups in any position other than the 1-position are treated with an acid of the formula HO - X' in which X' is a group as defined for X, in which the amino group and/or the hydroxy group may be protected, in the presence of a carbodiimide, or with a reactive derivative of the above-mentioned acid or, B) that one of the above-mentioned 4,6 -di-(aminoglycosyl)-1,3-diaminocyclitols which have been partially neutralized by formation of an acid addition salt are treated with an acid of the formula HO - X' wherein X' is a group as defined for X, wherein the amino group and/or the hydroxy group may be protected, in the presence of a carbodiimide or with a reactive derivative of the above specified acid, which method (A) or (B) is followed by the removal of all protecting groups present in the molecule and the isolation of the derivative as such, or as a pharmaceutically acceptable acid addition salt 2. Method according to claim 1, characterized in that 1-N-(S-3-amino-2-hydroxypropionyl) gentamicin B is prepared and isolated as such, or as a pharmaceutically acceptable acid addition salt.