FI60714C - FOERFARANDE FOER FRAMSTAELLNING AV SAOSOM LAEKEMEDEL ANVAENDBARA 1-N-SUBSTITUERADE DERIVAT AV 4,6-DI- (AMINOGLYCOSYL) -1,3-DIAMINOCYCLITOLER - Google Patents

Description

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Patent and Registry Board 'An * desert * n published and issued report -3 (32) (33) (31) Pjrydattjr etuoikeus— Begird priority 19-03-7¾ 19-03-7¾ USA (US) U52586, U52571 31.07.71 * Switzerland Switzerland (CH) 10601 / 7¾

Toteennäytetty-Styrkt (71) Oy Kolster Ab, Lönnrotsgatan 19 B, 00120 Helsinki 12, Suomi-Finland (FI) (72) John Jessen Wright, Orange, New Jersey, Peter John Lovell Daniels,

Cedar Grove, New Jersey, USA (US) (5¾) Process for the preparation of 1-N-substituted derivatives of U, 6-di- (aminoglycosyl) -1,3-diaminocyclitols as a drug - Menetelmä lääkeaineina käytettävien U, 6-di- (aminoglycosyl) -1,3-diaminocyclitoline 1-N-substituent

The invention relates to a process for the preparation of medicaments useful as 1-N-substituted derivatives of the 4,6-di (aminoglycosyl) -1,3-diaminocyclitols gentamicin B, gentamicin, gentamicin C1 gentamicin Cla, sisomicin, verdamicin, antibiotic cum G-418, antibiotic JI-20A, antibiotic JI-20B, and antibiotic G-52, wherein the 1-N substituent is designated X and is S-3-amino-2-hydroxypropionyl or S-4-amino-2 -hydroxybutylryl, with the proviso that for a derivative of gentamicin and gentamicin C-α, X has the meaning of S-3-amino-2-hydroxypropionyl, and of pharmaceutically acceptable acid addition salts of these derivatives.

U.S. Patent No. 3,780,018 discloses a process wherein 1- [1- (-) - ") f-amino - ^ - hydroxybutyrryl-gentamicin and 2 '- [1- (-) -T ^ -amino- ^ -hydroxybutyrryl] Gentamicin is prepared by reacting gentamicin with a blocked, active ester of 6071 4 L ~ (-) - amino - hydroxybutyric acid and then deblocking by generally known methods and separating the reaction mixture by chromatography. U.S. Patent 3,796,698 discloses a process for the preparation of 1- / L- (-) - γ'-amino- <X-hydroxy-butyryl / -gentamicin C2 and US Patent 3,796,699 disclose a process for the preparation of 1- / L- (-) - amino - <A-hydroxybutryl7-gentamicin Cla.

A particularly suitable group of compounds consists of 1-N-substituted derivatives of the 4,6-di (aminoglycosyl) -1,3-diaminocyclitolines gentamicin B, gentamicin B 2, sisomicin and verdamicin, the 1-N substituent being S -3-amino-2-hydroxypropionyl or S-4-amino-2-hydroxybutylryl, and the pharmaceutically acceptable acid addition salts thereof.

Within this group of compounds, 1-N- (S-4-amino-2-hydroxy-butyryl) -gentamicin B, 1-N- (S-3-amino-2-hydroxypropionyl) -gentamicin B, 1-N- (S-4-Amino-2-hydroxybutyrryl) -gentamicin β, 1-N- (S-3-amino-2-hydroxypropionyl) -isomicin, 1-N- (S-3-amino-2-hydroxypropionyl) - verdamicin and the pharmaceutically acceptable acid addition salts thereof are particularly suitable.

The compounds 1-N- (S-4-amino-2-hydroxybutyrryl) -gentamicin B, 1- N- (S-3-amino-2-hydroxypropionyl) -gentamicin B, 1-N- (S-3-amino) 2-hydroxypropionyl) -isomicin, 1-N- (S-3-amino-2-hydroxypropionyl) -verdamicin and the pharmaceutically acceptable acid addition salts represent a particularly suitable 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 particularly advantageous.

The novel 1-N-substituted 4,6-di (aminoglycosyl) -1,3-diamino-cyclitol derivatives have the general formula b

a-c-c W

C--

NOx

/ \ 0H 7

ψ- 0.- I

f 0 H0Ar \ NHCH31 "V- \ p" 2 "| /.,

H jCOH

X 60 has the meaning given above and substituents a-f and the dashed line in the 4'-5 'position has the meaning given in the following table.

(6 ') (6 ") (6 *) (4') (3 ') (2') 41-51

Antibiotic a b c d e f binding

Sisomicin H H NH2 H H NH2 double

Verdamicin H CH3 NH2 H H NH2 double

Antibiotic G-52 H H NHCH3 H H NH2 double

Gentamicin B H H NH2 OH OH OH single

Gentamicin Cla H H NH2 H H NH2 single

Antibiotic JI-20A H H NH2 OH OH NH2 single

Antibiotic JI-20B H CH3 NH2 OH OH NH2 single

Antibiotic G-418 OH CH3 H OH OH NH2 single

Gentamicin NHCH3 CH3 H H H NH2 single

Gentamicin Βχ NH2 CH3 H OH OH OH single

The univatized (maternal) antibiotics are all known.

The process according to the invention is characterized in that one (A) is treated with one of the said 4,6-di (aminoglycosyl) -1,3-diaminocyclitols, which can have amino protecting groups at any position other than the 1 position, with an acid of the formula HO-X 'where X' has the meaning given to X, wherein the amino group and / or hydroxy group may be protected, in the presence of a carbodium moiety or with a reaction-prone derivative of said acid, or (B) treating one of said 4 The 6-di (aminoglycosyl) - 1,3-diaminocyclitols, which are partially neutralized by forming an acid addition salt, with an acid of the formula HO-X 'wherein X * is as defined for X, wherein the amino group and / or the hydroxy group can be be protected, in the presence of a carbodiimide such as dicyclohexylcarbodiimide, or with a reaction-prone derivative of said acid, and after step (A) or (B) remove all present protecting groups and isolate the derivative as usual or in the form of a pharmaceutical good. edible acid addition salt.

For process A, it is generally particularly suitable to use the starting compounds having a 6'-C 1 -N 2 group in the form of their 6'-N protected derivatives. Examples of particularly suitable protecting groups are trifluoroacetyl and t-butoxycarbonyl. Gentamicin can advantageously be used as the 2 ', 3-di-N protected derivative. Gentamicin B 2, verdamicin, antibiotic G-418, antibiotic JI-20B and antibiotic G-52 can be used in their protected form, but they can also be used in their free form.

In Method B of the invention, specific acylation is performed at the amino group at the 1-position, and not random acylation, using 4,6-di (aminoglycosyl) -1,3-diaminocyclytol, which is partially neutralized by formation of an acid. addition salt.

Protonation of an amino group gives the same effect as the presence of a "conventional chemical" blocking group. Unexpectedly, it has been found that the amino group at the 1-position of a 4,6-di (aminoglycosyl) -1,3-diaminocyclitol is the last protonated upon the addition of acid or, if treated with a base addition salt with a base, the first which is deprotonated. "Blocking groups" (in the form of protons) can thus be readily introduced into the molecule by simply adding a desired amount of base to a peracid addition salt thereof. The process of the invention takes advantage of these findings and thus provides a convenient method for preparing the 1-N-acyl derivatives of the 4,6-di (aminoglycosyl) -1,3-diaminocyclitols using partially neutralized starting compounds.

The term "partially neutralized by formation of an acid addition salt" means that each mole of 4,6-di (aminoglycosyl) -1,3-diaminocyclitol has been associated with less than the stoichiometric number of moles of acid required to form the acid addition salt. This term further implies that each mole of 4,6-di (aminoglycosyl) - 1,3-diaminocyclitol has at least one mole of associated acid.

Thus, for example, one equivalent of gentamicin C, which has five amino groups, would require five equivalents of acid to form the acid addition salt. The process of the invention is carried out on an acid addition salt of gentamicin C C having less than five equivalents of acid and at least one equivalent (4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5 or 1.0 equivalents of acid).

In the most preferred embodiment of the process, the starting compound is neutralized with (n-1) equivalents of acid, where n is the 60714 number of amino groups in the molecule. Thus, (n-1) amino groups are neutralized by formation of an acid addition salt. However, it should be noted that the process can advantageously also be carried out on partially neutralized starting compounds, where more or less than (n-1) equivalents of acid give rise to the formation of the acid addition salt within the above limits. Expressed in pH range, the process is performed in a range of 5.0-9.0, preferably 5.0-8.0.

The most suitable range in the reaction medium is pH 6.5-7.5, especially 6.8-7.2.

The term "acid addition salt" encompasses 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 considered inorganic or organic. Examples of acids of this type include sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, acetic acid, propionic acid, succinic acid, oxalic acid, cyclopropylcarbonic acid, trimethylacetic acid, maleic acid, benzoic acid, phenylacetic acid, trifluoroacetic acid and the like. If such starting material is desired to use an acid addition salt, where (n-1) amino groups are protected, this compound is advantageously prepared in situ, reacting a peracid addition salt with an equivalent of a strong base, such as triethylamine.

It is also possible to use partially neutralized exit compounds which exhibit chemical blocking groups. However, this is generally not necessary,

The hydroxy aminoacylating agents used in the process of the invention are preferably used in the form of reaction-prone derivatives, for example in the form of active esters, anhydrides, azides or imidazole derivatives. Furthermore, it is particularly convenient that the amino group of the hydroxy aminoacylating agent is blocked.

When the desired hydroxy aminoacyl product is the isoserinyl derivative, the most appropriate acylating agent should be of the formula

O

Wherein the carboxy 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 readily removed by hydrogenolysis, or a t-butoxycarbonyl group or a trifluoroacetyl group, the former being preferably removed with acid and the latter with base.

6 60714

Pharmaceutically acceptable acid addition salts of the novel compounds can be prepared by installing an aqueous solution of said agent of pH 4.0, and then lyophilizing it. The salts thus obtained are functional equivalents to 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.

The terms "blocking group" or "protecting group" refer to the group which makes the blocked or protected amino groups inert against subsequent desired chemical treatment, but which can be readily removed at the end of the synthesis without cleaving the desired N-aminohydroxyacyl group.

Amino protecting groups are generally known. However, in connection with the invention, trifluoroacetyl, 2,2,2-trichloroethoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl and 4-methoxybenzyloxycarbonyl groups are the most suitable. Particularly suitable thereof are trifluoroacetyl, t-butoxycarbonyl and benzyloxycarbonyl.

In the blocking process, the protecting group is usually used in the form of an acid imidazole derivative, an acid azide or in the form of active esters such as ethyl thiol trifluoroacetate, t-butoxycarbonyl azide, N-benzyl oxycarbonyloxysuccinimide or p-nitrophenyltrichloroethyl chloride. Blocking groups can thus be described as derived from a compound BgLg, where Bg is the blocking group, as the acid moiety of an active ester, and Lg is a leaving group, such as imidazole.

The compounds of the present invention are broad-spectrum antibacterial agents which advantageously exhibit activity against many organisms, especially gram-negative organisms, which are resistant to their 1-N unsubstituted precursors. The compounds obtained according to the invention can thus be used separately or in combination with other antibiotic agents to prevent the growth or reduction of the number of bacteria in different environments.

Results of Pharmacological Tests Table I shows the minimum inhibitory concentration (MIC) for some compounds obtained according to the invention. The tests were performed in Mueller-Hinton culture medium (pH 7.2) using standard methods. The following compounds were tested: V 60714 (a) Known Compounds Compound 1: 1-N- (S-4-amino-2-hydroxybutyrryl) -gentamicin C Ca (U.S. Patent 3,796,699) Compound 2: 1-N- ( S-4-Amino-2-hydroxybutryryl) -gentamicin (U.S. Patent 3,780,018) (b) New Compounds Compound 3: 1-N- (S-3-amino-2-hydroxypropionyl) -gentamicin

Compound 4: 1-N- (S-3-amino-2-hydroxypropionyl) -gentamicin B

Compound 5: 1-N- (S-4-amino-2-hydroxybutyrryl) -gentamicin

Compound 6: 1-N- (S-4-amino-2-hydroxybutyrryl) -gentamicin B

Compound 7: 1-N- (S-4-amino-2-hydroxybutyrryl) antibiotic JI-20B

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

Test Method

Prepare and sterilize 2000 ml Mueller-Hinton nutrient liquid with pH = 7.2. Transfer 5 ml of the sterile medium to 120 sterile test tubes (16 x 150 mm) with cotton stoppers. Arrange test tubes> 4 in 10 groups of 12 test tubes each. Add to each test tube 10 cells of the bacterial strain to be used. Add to each batch of test tube an aqueous solution of the antibiotic to be tested; use the following concentrations: 50, 25, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.01, 0.005 and 0.000 (control) µg / ml. Incubate the test tubes for 24 hours at 37 ° C. Visually determine for each group of test tubes the lowest concentration of antibiotic that inhibits bacterial growth and the highest concentration that still allows growth.

Determine the minimum inhibitory concentration (MIC) by calculating the mean of these two values.

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9 60714

The invention is further illustrated by the following examples, where the temperatures indicated are degrees Celsius.

Example 1 1-N- (S-3-amino-2-hydroxypropionyl) -isomicin A. S-3-trifluoroacetamido-2-hydroxypropionic acid Dissolve 5 g of S-3-benzyloxycarbonylamino-2-hydroxybutyric acid in a mixture of 80 ml of dioxane and 20 ml of water. Add 200 mg of 30% palladium / carbon and hydrate for 3 hours at room temperature under a pressure of 3.4 at. Remove the catalyst by filtration and concentrate the filtrate to a residue in vacuo. Dry the ci ters toad in high vacuum for 72 hours. Dissolve the dried ether in 30 ml of potassium trifluoroacetic anhydride with stirring. Stir the solution for three hours at room temperature and concentrate in vacuo to a residue. Triturate the ether with benzene to form a grass solid, which is isolated by filtration, washed with benzene and dried to form the intended product.

B. N- (S-3-Trifluoroacetamido-2-hydroxypropionyloxy) -succinimide Dissolve 20 mmol of the product from step A in 50 ml of ethyl acetate, add 2.31 g of N-hydroxysuccinimide with stirring and cool the resulting solution in an ice bath. Add 4.3 g of dicyclohexylcarbodiimide to the solution and stir the mixture for three hours at room temperature. Filter to eliminate the precipitate and concentrate the filtrate to dryness to give the title compound, which is dried in vacuo and used in step D.

C. 6'-N-Trifluoroacetylsisomicin Dissolve 20 g of sisomicin in 1.2 liters of anhydrous methanol and drop dropwise a solution of 6 ml of ethyl thiol trifluoroacetate in 60 ml of methanol over a period of three hours with stirring. Allow the reaction to proceed for 18 hours at room temperature and remove the solvent in vacuo to give 23.8 g of product with a degree of purity of about 95% and the following physicochemical properties: Mass spectrum data: m / e 543 M +, other determined peaks at m / e 413 , 395, 385, 362, 223 and 126. NMR (60 MHz) δ 5.37 doublet, J = 2Hz, H-1 '; 5.12 doublet, J = 4Hz, H-1 "; 4.96 broad singlet, H-4 '; 2.57, singlet, N-CH 2; 1.26 singlet? C-CH 2.

D. 1-N- (S-3-amino-2-hydroxypropionyl) -isomicin Dissolve 814 mg (1.5 mmol) of 61-N-trifluoroacetylisomicin in 12 ml of 50% methanol in water and add dropwise with stirring of 1.5 mmol of product from step B, dissolved in 3 ml of dimethylformamide. Stir the formed mixture for 18 hours at room temperature and concentrate in vacuo to an ether. Dissolve the ether residue in 10 ml of concentrated sodium hydroxide and store the mixture for two hours (to eliminate the trifluoroacetyl groups). Evaporate the solvent to form the title product in the form of an ether residue. Chromatograph the ether on a silica gel column using the lower phase of the solvent system chloroform / methanol / ammonium hydroxide (2: 1: 1) as eluent. Fractions containing the title product are pooled and concentrated to an ether, which is dissolved in water and lyophilized to an amorphous white solid. 1- N- (S-3-amino-2-hydroxypropionyl) -isomicin, +139 (C = 0.3 in water) C22H42 ° 9N6-1 / 2 H2CO3:

Calculated: C 47.74 H 7.66 N 14.85%

Found: C 47.39 H 7.14 N 15.16% PMR 100 MHz D20 in ~ 1.22 (S), C-Me, 2.60 (S) N-Me 4.22 (Q), J = 4Hz, 7.5Hz; £ 5.09 (d), Hin 2 "= 4.0 Hz; £ 5.34 (d), Ηχ, 2 '= 2 Hz' In the same way, the following compounds can be prepared: 1N- (S-3-amino-2-hydroxypropionyl) -gentamicin C, PMR 100 MHz, Do0: . 2 c = 1.22 C-Me; 2.59 N-Me; + 115.4 (C, 0.3 in water); 5.09 H-1 ", J1 (, 21, = 4Hz, 5.17 H-1", 2, = 3.5 Hz C22H44 ° 9N6, H20, C ° 2

Calculated: C 45.01 H 7.39 N 13.70%

Found: C 44.98 H 7.81 N 13.68%, and 1- N- (S-3-amino-2-hydroxypropionyl) -gentamicin B, PMR 100 MHz, D20 + DC1; (f 1.18 C-Me (S); 2.77 N-Me (S); 4.35 (Q), J = 4.5, 8.0; H-1 ', <f 5.01 (d), J1 (2, = 4.0 Hz; H1 ", 5.43 (d), JlM 2" = 3.5 Hz. Example 2

1- N- (S-4-amino-2-hydroxybutyl) -gentamicin B A. 6'-N-t-butoxycarbonylgentamicin B

Dissolve 1 g of gentamicin B in 30 ml of 50% ethanol and cool to 5 °. Add dropwise 0.297 g of t-butoxycarbonyl azide with stirring and then 0.186 ml of triethylamine, stirring the resulting mixture for 18 hours. Evaporate the reaction mixture in vacuo to an ether and chromatograph the ether on 100 g of silica gel using the lower phase of the solvent system chloroform / methanol / concentrated ammonium hydroxide as eluent. Collect 2 ml fractions and monitor the flow from the column by TLC. Combine fractions containing equal materials (fractions n 60714

(180--230) and evaporated to give 0.830 g of 6'-Nt-butoxy-carbonylgentamicin B with the following physical constants: PMR (60MHz, D20) 4 * 1.21 (3H, s, C-CH3), 1, 42 (9H, s, C (CH3) 3), 2.53 (3H, s, N-CH3), 5.2 (1H, d, J = 4.5Hz, H1 "), 5.23 (1H, d, J = 3.0 Hz, H-1 ') PPM

C24H46N4 ° 12'C02 * H2 °!

Calculated: C 46.57 H 7.51 N 8.69%

Found: C 46.80 H 7.82 N 8.54% + 124 ° (C, 1 in methanol).

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

Dissolve 0.582 g of 61-N-t-butoxycarbonylgentamicin B in 25 ml of methanol / water (1: 3). Add, while stirring, a solution of 0.334 g of N- (S-4-carbobenzyloxyamino-2-hydroxybutryloxy) -succinimide in 5 ml of dimethylformamide. Stir the reaction mixture for 5 hours at 5 ° and then evaporate to dryness in vacuo. Chromatograph the ether on 60 silica gel using the lower phase of the solvent system chloroform / methanol / ammonium hydroxide (2: 1: 1) as eluent. Collect 3 ml fractions and monitor the contents with TLC. Combine fractions 160-235 and evaporate to a residue of 0.280 g, which migrates in the form of a single spot by TLC. Dissolve the ether residue in 8 ml of methanol and 10 ml of water and hydrate under a pressure of 3.7 at over 60 mg of 5% palladium / carbon catalyst. Remove the catalyst by filtration, evaporate the filtrate to an ether and chromatograph on 20 g of silica gel using the lower phase of the solvent system chloroform / methanol / ammonium hydroxide (1: 1: 1) as eluent. Combine fractions 79-127 (each 2 ml), consisting of 1-N- (S-4-amino-2-hydroxybutyrryl) -61-N-t-butoxycarbonyl-gentamicin B.

Yield 86 mg. PMR £ 1.21 (3H, s, C-CH 3), 1.43 (9H, s, C- (CH 3) 3), 2.51 (3H, s, N-CH 3), 5.08 (1H, d, J = 4Hz, H1 "), 5.25 (1H, d, J = 3.5Hz, H-1 ') PPM.

C. 1-N- (S-4-amino-3-hydroxybutyrryl) -gentamicin B

Dissolve the product from step B in 0.3 ml of trifluoroacetic acid and add 30 ml of ethyl ether after five minutes. A precipitate of the compound from this example is formed in the form of the trifluoroacetate salt and is obtained by filtration. Dissolve the precipitate in water and pass the solution through a column of a basic ion exchange resin in the hydroxide form to be transferred to the free base. Lyophilize the column effluent 12 to form the compound of this example in the form of a white amorphous substance. Yield 72 mg. PMR D20 100 MHz: ζ 1.14 C-methyl (S), 2.45 (S) N-methyl, 5.04 (d), H (D) H-1 ', Jx, 2, = 3.5 Hz.

Example 3 1-N- (S-3-amino-2-hydroxypropionyl) -gentamicin A. 1-N- (S-3-carbobenzyloxyamino-2-hydroxypropionyl) -21-3-di-N-trifluoroacetylgentamicin Dissolve 0.84 g of 2 ', 3-di-N-trifluoroacetyl-gentamicin in 40 ml of tetrahydrofuran and dropwise adding a solution of 1.19 g of N- (S-3-carbobenzyloxyamino-2-hydroxypropionyloxy) succinimide in 24 ml of tetrahydrofuran. Stir the mixture for 24 hours and evaporate to a residue. Chromatograph the ether on silica gel using the lower phase of the solvent system chloroform / methanol / concentrated ammonium hydroxide / water (volume 2: 1: 0.2: 0.8) as eluent. monitor the chromatogram by TLC and combine equal fractions to form the main product with the following constants:

Melting point 125-131 °, + 93 ° (CH 3 OH)

Calculated (for dihydrate): C 47.47 H 6.30 N 9.23%

Found: C 47.85 H 6.67 N 9.08% B. 1-N- (S-3-carbobenzyloxyamino-2-hydroxypropionyl) -gentamicin Dissolve 0.55 g of the product from step A in 55 ml of methanol and add 25 ml of concentrated ammonium hydroxide. Stir for three days, using TLC on silica gel plates using the lower phase of the solvent system chloroform / methanol / concentrated ammonium hydroxide (1: 1: 1), which induces essentially complete elimination of the trifluoroacetyl blocking groups. Evaporate the solution to dryness in vacuo. Yield 0.2 g, mp 109-112 °; Zc * / p6 = + 73 ° (H2 O).

C. 1-N- (S-3-amino-2-hydroxypropionyl) -gentamicin Dissolve 0.153 g of the ether from step B in 8 ml of acetic acid and add 0.05 g of 10% palladium / carbon catalyst. Hydrate the solution under a pressure of 4.0 at 25 ° until TLC (using the solvent system of step B) indicates the complete transfer to the title compound (i.e., 16 hours to 3 days), remove the catalyst by filtration and evaporate the solution to a remained in vacuo. Chromatograph the ether over 13 g of silica gel using the lower phase of the chloroform / methanol / ammonium hydroxide (2: 1: 1) mixture as eluent. Monitor the pillar using TLC and combine equal materials to form the title compound. Yield 112 mg. Mp 109-119 °, / c </ 26 = + 98 ° (H + Q)

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-hydroxybutyrryl) -verdamicin A. 1-N- (S-4-phthalimido-2-hydroxybutyrryl) -verdamicin Dissolve 4.61 g of verdamicin in 50 ml of a methanol aqueous solution (1: 3) and cool to 0-5 °. Drop by dropwise a solution of 3.81 g of N- (S-4-phthalimido-2-hydroxybutryloxy) succinimide in 20 ml of dimethylformamide with stirring to the antibiotic solution. Stir for a further 16 hours and concentrate to a residue in vacuo. Chromatograph the ether on a column of 250 g of silica gel using the lower phase of a (2: 1: 1) mixture of chloroform, methanol and concentrated ammonium hydroxide as eluent. monitor the eluate by TLC and combine fractions containing equal materials. Evaporate the combined fractions containing the main product to form 1-N- (S-4-phthalimido-2-hydroxybutyrryl) -verdamicin and an isomer thereof, B. 1-N- (S-4-amino-2-hydroxybutyrryl) ) -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, reflux the solution for three hours and then evaporate to dryness in vacuo. Chromatograph the ether over 160 g of silica gel and elute with the lower phase of a (1: 1: 1) mixture (volume) of chloroform, methanol and concentrated ammonium hydroxide. Combine and evaporate the fractions containing the major component from the reaction, as determined by TLC, to give the title compound as a white amorphous substance. 1-N- (S-4-amino-2-hydroxybutyl) -verdamicin has the following data: CMR (25.2 MHz, D 2 O) 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.

In this way, 1-N- (S-4-amino-2-hydroxybutyryl) -gentamicin B ^ is obtained by this method, which has the following data: PMR (60 MHz, D20): / l, 28, s, C-Me; 1.26, d, CH-CH 3; 1.7-2.3, m, 2xH-3 "and H-s" eq; 2.74, s, N-Me; 5.18, d, J = 4Hz, H1 ", 5.53, d, 14 6071 4 J = 3Hz, H-1J ppm, and 1-N- (S-4-amino-2-hydroxybutyrryl) antibiotic JI -20B, exhibiting the following data: mp 128-138 °; C = 7p +126.4 (C, 0.28 in water) PMR (100 MHz, D2O): 1.12, s, C Me; 1.21, d, CH-Me; 2.48, s, N- Me; 5.08, d, J = 3Hz, H1 "; 5.25, d, J = 4Hz, H-1 * ppm.

By analogy, N- (S-3-phthalimido-2-hydroxypropionyloxy) succinimide can be prepared.

1-N- (S-3-amino-2-hydroxypropionyl) -gentamicin, which has the following data: PMR (D20): δ 1.08 (3H, d, J = 7.0 Hz, CHCH 3), 1.20 (3H, s, C-CH 3), 1.38 (1H, g, J = 13.0 Hz, H-2ax); 1.93 (1H, m, H2eg), 2.48 (1H, d, J = 10.5 Hz, H-3 "), 2.5 (3H, s, N-CH-.), 3.93 -4.28 (m, 2H, H-5 "and H-2"), 5.05 (1H, d, J = 4.0 Hz, H1 "), 5.25 (1H, d, J = 3 , 0 Hz, H-1 '). CMR (D20) (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-hydroxypropionyloxyl) succinimide A. S-3-t-butoxycarbonylamino-2-hydroxypropionic acid Dissolve 8.0 g of S-3-carbobenzyloxyamino-2-hydroxypropionic acid in 110 ml of dioxane / water (4: 1). Add 200 mg of palladium / carbon catalyst and hydrate for 3 hours at 3.4 at. Filtrate 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 triethylamine, cool the solution in an ice bath and add 4.76 g of t-butoxy-carbonylazide. Store the mixture overnight under gradual heating to room temperature, evaporate the bulk of the methanol, dilute with water and acidify with dilute hydrochloric acid to pH 3. Extract the water mixture several ginger with ethyl acetate, combine and dry the ethyl acetate extracts and evaporate to an ether solution in vacuo. Recrystallize the residue from ethyl acetate / hexane to give the title compound. Yield 3.5 g. Melting point 92-94 °, + 14.9 ° (c = 0.68, H 2 O).

C8H15N ° 5

Calculated: 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 6071 4 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.

With stirring, add 1.42 g of N-hydroxysuccinimide and then a solution of 2.51 g of dicyclohexylcarbodiimide in 10 ml of ethyl acetate.

Stir the mixture for 16 hours and filter out the precipitated solids. Evaporate the filtrate to give the title compound. Yield 3.56 g.

2. 21,3-di-N-trifluoroacetyl-gentamicin A. 2'-N-trifluoroacetyl-gentamicin Dissolve 1.7 g of gentamicin in 20 ml of methanol, cool the mixture to 4 ° and add 0.46 ml (0.563 g) of ethyl thiol trifluoroacetate with stirring. Allow the reaction to continue for two hours and 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 and ammonium hydroxide in the volume ratio of 10: 5: 4: 1 as eluent. Combine the fractions containing the main component and concentrate to give 1.4 g of the title compound, mp 108-111 °, / e * + 128 ° (c = 0.3%, 2 O).

C23H42N5 ° 8F3, H2 °

Calculated: 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 Dissolve 0.66 g of the product from step A in 10 ml of methanol, cool the mixture to 4 ° and add 0.182 g of ethyl thiol trifluoroacetate, dissolved in 3 ml of methanol. Stir the mixture for 16 hours and concentrate to a residue in vacuo. Chromatograph the product on 30 g of silica gel according to step A. Monitor the column by thin layer chromatography, combine the corresponding fractions and concentrate to give 0.32 g of the title compound, m.p. ).

C H N O F ^ 25 41 5 9 6

Calculated: C 44.84 H 6.17 N 10.46%

Found: C 44.94 H 6.35 N 10.17%

Example 6 1-N- (S-4-amino-2-hydroxybutyrryl) -gentamicin A. 1-N- (S-4-benzyloxycarbonylamino-2-hydroxybutyrryl) -gentamicin C gentamicin C 2 -sulfate in 30 ml of water and add 15 ml of methanol. Add 0.56 ml (4 mmol) of triethylamine and stir for 10 minutes. Add dropwise a solution containing 4 mmol of N- (S-4-benzyloxycarbonylamino-2-hydroxybutyrloxy) succinimide in 20 ml of dry dimethylformamide to the antibiotic solution with stirring. Stir the mixture overnight (16 hours) at room temperature. Thin layer chromatography of the reaction mixture via TLC on silica gel using the lower phase of a solvent system comprising chloroform, methanol and ammonium hydroxide (1: 1: 1) shows the presence of a number of smaller components and a larger component. Concentrate the reaction mixture to an ether in vacuo and triturate it with methanol to give 3.2 g of white solids containing all of the components observed in chromatography.

Chromatograph 150 mg of the product on 50 g of silica gel using the lower phase of a solvent system, consisting of chloroform, methanol and ammonium hydroxide (2: 1: 1). Combine the fractions containing the major component and lyophilize to form 1-N- (S-4-benzyloxycarbonylamino-2-hydroxybutyrryl) -gentamicin Cla. Yield 70 mg. NMR (D 2 O) δ 1.15 (3H, s, C-CH 2); 2.49 (3H, s, NCH 3); 4.10 (1H, dd, J = 8.0, 4.0Hz, side chain H-2); 7.36 (5H, m, phenyl).

B. 1-N- (S-4-Amino-2-hydroxybutyrryl) -gentamicin C 2a Dissolve the product from step A in a mixture consisting of 12 ml of methanol and 3 ml of water, add 20 mg of 10% palladium / boil and hydrate at 4.0 at and room temperature. After three hours, the reaction is essentially complete. Remove the catalyst by filtration and lyophilize the filtrate to give 46 mg.

1-N- (S-4-amino-2-hydroxybutyrryl) -gentamicin C ^ aH NMR (DjO) 1, 1.17 (3H, s, C-CH 2.48 (3H, s, NCH. 4.22 (1H, dd, J = 9.5Hz, 4.0Hz, side chain CHOH); 5.04 (2H, m, H-1 'and H-1 "). Mass spectrum data: (M-H 2 O) m / e 532.

Example 7

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

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

Dissolve 3.39 g of gentamicin B sulfate in 48.4 ml of water and dilute with 23.7 ml of methanol. Add, while stirring, 0.7 ml of triethylamine. Dissolve 1.67 g of N- (S-4-benzyloxycarbonylamino-2-hydroxy-butyryloxy) -succinimide in dimethylformamide and, with stirring, drop the solution dropwise into the antibiotic solution. Stir the solution for 18 hours at room temperature and concentrate to a residue in vacuo. Dissolve the ether in water and treat with barium hydroxide solution with stirring until the pH reaches about 8.0. Remove the barium sulphate formed by filtration using a filter aid. Wash the precipitate with water, combine the filtrate and washing liquids and concentrate to dryness in vacuo. Chromatograph the ether on a column containing 600 g of silica gel using the lower phase of a solvent system consisting of chloroform, methanol and ammonium hydroxide (1: 1: 1) as eluents. Combine the material eluted immediately before gentamicin B and concentrate the pooled fractions to form 1-N- (5-4-benzyloxycarbonylamino-2-hydroxybutyrryl) -gentamicin B in the form of an amorphous substance. 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 = 4Hz); 5.25 (1H, d, J = 3.5 Hz)

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

Dissolve the product from step A in a mixture of 20 ml of water and 8 ml of methanol. Hydrate the product in the presence of 60 mg of 5% palladium / carbon at 3.4 at and room temperature for three hours.

Remove the catalyst by filtration through a filter aid. Wash the filter cake and combine the filtrate and washing liquids. 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 and ammonium hydroxide (1: 2: 1) as eluents. The fractions containing the most polar component are pooled, concentrated and lyophilized to form 1-N- (S-4-amino-2-hydroxybutyrryl) -gentamicin B. Yield 12 mg.

Data: see Example 2.

Claims (1)

ice 6071 4 Analogy procedure for preparation of as-drug 1-N-substituted derivatives of 4,6-di (aminoglycosyl) -1,3-diamino-cyclitols gentamicin B, gentamicin B], gentamicin C C, gentamicin C ^ a, sisomicin, verdamicin, antibiotic G-418, antibiotic JI-20A, antibiotic JI-20B, and antibiotic G-52, wherein the 1-N substituent Sr is X and is S-3-amino-2-hydroxypropionyl or S -4-amino-2-hydroxybutryryl, with the proviso that for a derivative of gentamicin and gentamicin C Ca, X has the meaning of S-3-amino-2-hydroxypropionyl, and of pharmaceutically acceptable acid addition salts of these derivatives, treating (A) one of said 4,6-di (aminoglycosyl) -1,3-diaminocyclitols, which may have amino protecting groups at any position other than the 1 position, with an acid of the formula HO-X * wherein X 'has the meaning given for X, wherein the amino group and / or hydroxy group may be protected, in the presence of a carbodiimide and or (B) treating one of said 4,6- (di (aminoglycosyl) - 1,3-diaminocyclitols, which is partially neutralized by forming an acid addition salt, with an acid having the formula HO-X 'where X' is as defined for X, wherein the amino group and / or hydroxy group may be protected, in the presence of a carbodiimide such as dicyclohexylcarbodiimide, or with a reaction-prone derivative of said acid, and after step (A) or ( B) removes all the protecting groups present and isolates the derivative as usual or in the form of a pharmaceutically acceptable acid addition salt.