DK156070B - METHOD OF ANALOGY FOR THE PREPARATION OF ANTIBIOTIC ACTIVE DERIVATIVES OF AMINOCYCLITOLS - Google Patents

Description

1 DK 156070 B

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

U.S. Patent No. 3,780,018 discloses a process in which 1- [L - (-) - γ-amino-α-hydroxybutyryl-3gentamicin and 2 [L - (-) - γ-amino-α-rhydroxybutyryl] gentamicin are prepared by reaction of gentamicin with a blocked active ester of L - (-) - γ-amino-α-hydroxybutyric acid followed by removal of the blocked group by methods known in the art and separation of the reaction mixture by chromatographic route. U.S. Pat.

2 DK 156070B

No. 3,796,698 discloses a process for the preparation of 1 - [L - (-) - γ-amino-α-hydroxybutyryl] gentamicin C2, and U.S. Pat.

3,796,699 discloses a process for the preparation of 1- [L - (-) - γ-amino-α-hydroxybutyryl] -gentamic in.

The novel antibacterial agents prepared according to the invention have surprisingly unexpected properties. Generally, these compounds are active against bacterial strains and / or protozoan strains. Furthermore, they are active against many bacteria which have become essentially insensitive to. the non-derived antibiotics.

The invention thus relates to an analogous process for the preparation of antibiotically active 1-N-substituted derivatives of the 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols, gentamicin B, gentamicin, gentamicin, gentamicin C, sisomicin, verdamicin, antibiotic G-418, antibiotic 66-40B, antibiotic 66-40D, antibiotic JI-20A, antibiotic JI-20B, and antibiotic G-52, wherein the 1-N substituent is X and is either 1-3-amino-2-hydroxypropionyl or S-4-amino-2-hydroxybutyryl, with the proviso that in the case of gentamicin, gentamicin C. and gentamicin CO, X is S-3-amino-2-hydroxypropionyl, as well as the pharmaceutically acceptable acid addition salts of these compounds. .

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

Particularly preferred from this group of compounds are the following specially named compounds: 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 B1, 1- N- (S-3-amino-2-hydroxypropionyl) sisomicin, 1-N- (S-3-amino-2-hydroxypropionyl) verdamicin and the pharmaceutically acceptable acid addition salts thereof.

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-

3 DK 156070B

2-hydroxypropionyl) gentamicin B and the pharmaceutically acceptable acid addition salts thereof are the most preferred.

The compounds of the invention have the following structural formulas I, II and III, wherein X represents the 1-N substituent as set forth above.

NH2

. jA \ NHX

y-0 — K ^ QH y i

XX I

wherein Y is an aminoglycosyl group selected from the following: CH3 ch2nh2 nh2-I- / 'V (in 1N-X-K OH A_ (in 1-N "X" nn \ l / L gentamicin B) hINL-J / "gentamicin B ')

OH OH

ch3 ch3nh-- ch2nh2 (i 1-N-X-IX) (- - (i 1-N-X-I-f gentamicin C-)] [gentamicin C,) nh2 nh2 ch3 --NH2

Ni i / 1- ϋ i-η - * -,] f verdamicin) NH2 4

DK 156070 B

CH2NH2 CH2NH2 XI, / L (i 1-NX- ho \? H (± 1 in sisomicin) | antibiotic JI-20A) nh2 nh2 CH3 - nh2 ch2nhch3 wn \? H y / - (i 1-NX-H0 ] -f antibiotic N- <- NH2 JI-20B)> JjH antibiotic ^ 2 G-52) and ch3 HO--

I ^ NI

(i-N-X antibiotic G-418) nh2 1-N-X antibiotic 66-40D of the following formula II CH0NH0 NH2 NH2 h ° kS> lo

OH

and i-N-X-gentamicin A and 1-N-X-antibiotic 66-40B of the following formula III

5

DK 156070 B

NH2

λ-k HHX

* .- o — Κ ^ ΐ_ρ i / ^ Vj wherein Y 'represents

A

- in 1-N-X antibiotic 66--40B.

NH2

The non-derived (parent) antibiotics (defined by the preceding structural formulas I, II and III, wherein X represents hydrogen), are all known in the art.

Antibiotic 66-40B and antibiotic 66-40D are produced together with sisomicin, as the main product of the fermentation of Micromonospora inyoensis (described in British Pat.

1,274,518). Antibiotic 66-40B and antibiotic 66-40D can be separated from the fermentation medium using special chromatographic separation techniques as described in Belgian Patent Specification No. 811,370.

The analogous process of the invention is characterized by using one of the following process alternatives A) or B): A) one of the 4,6-di- (aminoglycosyl) -1,3-diamino-cyclitols mentioned above, which can be having amino protecting groups at any position other than position 1, treated with an acid of the formula HO-X 'wherein X' is a group as defined for X wherein the amino group and / or hydroxyl group may be protected, in the vicinity of Λ 1 Π Λ «· Ρ / —V 4- •« »ΚλαΙ ί · Ι» W 1 Λ 1 1 Λν Λ ·! · 1 Ttt ^ / - »V * 4 ΤΤΑ 4- 6

DK 156070 B

of the above acid, or B) one of the above 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols, which is partially neutralized by formation of an acid addition salt, is treated with an acid of the formula HO-X 'wherein X 'is a group as defined for X wherein the amino group and / or hydroxy group may be protected in the presence of a carbodiimide, or with a reactive derivative of the above acid, which method A) or B) is followed by removal of all protecting groups present. in the molecule, and 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 having a 6'-C1 -NI2 group in the form of their 6'-N protected derivatives. Examples of preferred protecting groups are trifluoroacetyl and t-butoxycarbonyl. Gentamicin can advantageously be used as the 2 ', 3-di-N protected derivative. Gentamicin B 1, verdamicin, antibiotic G-418, antibiotic JI-20B and antibiotic G-52 can be used in their protected form, but can also be used in free form.

In Method Alternative B), specific acylation is achieved at the amino group at position 1 rather than random acylation using 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol, which is partially neutralized by the formation of an acid addition salt.

Protonation of an amino group gives the same effect * as the presence of a "usual chemical" blocking group. Surprisingly, it has been found that the amino group at position 1 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 base, the first , which is deprotonized. Thus, "blocking groups" (in the form of protons) can be readily introduced into the molecule by adding a desired amount of acid to the aminoglycoside or by adding a desired base amount to a peracid addition salt thereof. This process alternative is advantageous because of these discoveries, and thus provides a convenient way to prepare 1-N-acyl derivatives of the 4,6-di- (aminoglycosyl) -1,3-diaminocyclic tolols using partially neutralized and therefore partially blocked output connections.

7 DK 156070B

As used herein, the term "partially neutralized by the formation of an acid addition salt" means that each mole of 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol is thereby associated with less than the stoichiometric number of moles of acid required. to form the peracid addition salt. Furthermore, this term means that each mole of 4,6-di- (α-minoglycosyl) -1,3-diaminocyclitol has at least one mole of acid attached to it.

Eg. would 1 equivalent gentamicin with 5 amino groups would require 5 equivalents of acid to form the peracid addition salt. The process of the invention is carried out on an acid addition salt of gentamicin having less than 5 equivalents and at least 1 equivalent acid (e.g., 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1, 5 or 1.0 equivalents of acid).

According to a preferred embodiment of the analogous process of the invention, the starting material is neutralized with (n-1) equivalents of acid, where n is the 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 understood that the process can also be advantageously carried out on partially neutralized starting materials in which more or less than (n-1) equivalents of acid cause formation of the acid addition salt within the foregoing limits. Expressed in pH ranges, the process can be carried out in a range of 5.0-9.0, preferably 5.0-8.0. The most preferred range for the reaction medium is pH 6.5-7.5, especially 6.8-7.2.

The term "acid addition salt" includes such salts which may be formed between the basic 4,6-di- (aminoglycosyl) -1,3-diaminocyclitol and an acid regardless of whether the acid may be referred to as inorganic or organic. Examples of acids encompassed 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 as an starting material an acid addition salt in which (n-1) amino groups are protonated, this compound is prepared in situ by reacting a peracid addition salt with 1 equivalent strong base, e.g. triethylamine.

It is also possible to use partially neutralized starting compounds having chemical blocking groups. However, this is not necessary.

The hydroxyamino acylating agents used in the methods of the invention are preferably used in the form of reactive derivatives,

8 DK 156070 B

eg. in the form of active esters, anhydrides, azides or imidazole derivatives. It is further preferred that the amino group of the hydroxyamino acylating agent is blocked. When the desired hydroxyaminoacyl product is the iso-serinyl derivative, the preferred acylating agent will take the following form

Lg-C-CH-CH-I in 2 OH NH-Bg wherein the carboxyl group is preferably activated in the form of an active ester (leaving group Lg) and the amino group is preferably blocked (blocking group Bg) by a benzyloxycarbonyl group which is readily removed by hydrogenolysis , or a t-butoxycarbonyl group or a trifluoroacetyl group, the former being conveniently removed with acid and the latter with base.

If pharmaceutically acceptable acid addition salts of the compounds of the invention are desired, they may be prepared by adjusting an aqueous solution of the pH 4.0 agent followed by lyophilization.

The salts thus formed are functional equivalents to the free nitrogen base. Examples of acids which can be used for salt formation are sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, acetic acid, propionic acid, succinic acid, oxalic acid and maleic acid.

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

Amino protecting groups are well known in the art. However, for the present method trifluoroacetyl, 2,2,2-trichloroethoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl and 4-methoxybenzyloxycarbonyl groups are preferred groups. Particularly preferred of the above 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 1 esters such as ethyl thiol trifluoroacetate, t-butoxycarbonylazide, N-benzyloxycarbonyloxysuccinimide or p-nitrophenyltrichloroethyl carbon. ;

V

9 DK 156070 B

The following examples further explain the invention.

Example 1 1-N- (S-3-Amino-2-hydroxy-propionyl) sisomicin.

A. S-3-Trifluoroacetamido-2-hydroxy, propionic acid.

Dissolve 5 g of S-3-benzyloxycarbonylamino-2-hydroxypropyric acid in a mixture of 80 ml of dioxane and 20 ml of water. 200 ml of 30% palladium on charcoal is added and hydrogenated for 3 hours at ambient temperature and 3.4 atmospheres. The catalyst is removed by filtration and the filtrate is concentrated to a residue in vacuo. The residue is dried in high vacuum for 72 hours. The dried residue is dissolved in 30 ml of cold trifluoroacetic anhydride with stirring. The solution is stirred for 3 hours at ambient temperature and then concentrated in vacuo to a residue. The residue is triturated with benzene to give a gray solid which is isolated by filtration, washed with benzene and dried to obtain the product of this step.

B. N- (S-3-Trifluoroacetamido-2-hydroxypropionyloxy) succinimide.

Dissolve 20 mmol of the product of 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 gene and stir the reaction mixture for 3 hours. hours at room temperature. Filtrate to remove the precipitate and concentrate the filtrate to dryness to give the title compound which is dried in high vacuum and used in step D.

C. 61-N-Trifluoroacetylsisomicin.

Dissolve 20 g of sisomicin in 1.2 liters of anhydrous methanol and dropwise add a solution of 6 ml of ethyl thiol trifluoroacetate in 60 ml of methanol over a period of 3 hours with stirring. The reaction is allowed to proceed for 18 hours at room temperature, after which the solvent is removed in vacuo to give a residue of 23.8 g of the product with an approximate purity of 95% and the following physicochemical properties:

Mass spectral data: m / e 543 M +, other crucial peaks at m / e 413, 395, 385, 362, 223 and 126.

NMR (60 MHz, D 2 O) δ 5.37 doublet, J = 2 Hz, H-l'f 5.12 doublet, J = 4 Hz, H1 "; 4.96 wide singlet, H-4 '; 2.57 , singlet, N-CH-.

1.26 singlet, C-CH

10

DK 156070 B

D. 1-N- (S-3-Amino-2-hydroxypropionyl) sisomicin.

Dissolve 814 mg (1.5 mmol) of 6'-N-trifluoroacetyl isomicin in 12 ml of 50% 1 sec aqueous methanol and add dropwise with stirring a solution of 1.5 mmol of the product of step B dissolved in 3 ml of dimethylformamide. . The resulting solution is stirred at room temperature for 18 hours and concentrated in vacuo to a residue. The residue is dissolved in 10 ml of concentrated ammonium hydroxide and allowed to stand for 2 hours (to remove the trifluoroacetyl groups). The solvent is evaporated to give the title compound from a residue. The residue is chromatographed on a silica gel column using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system (2: 1: 1) as eluent. Fractions containing the title compound are combined and concentrated to a residue which is dissolved in water and lyophilized to give an amorphous white solid.

26 o 1-N- (S- * 3-amino-2-hydroxypropionyl) sisomicin, [α] D + 139 (c = 0.3 in water). Analysis: Calculated for C 22 H 42 ° 9 N 6 R 2 H 2 CO 3: C = 47 / 74f H - 7.66, N = 14.85? found: C = 47.39, H = 7.14, N = 15.16.pmr 100 MHz D20: il, 22 (s), C-Me? £ 2.60 (s) N-Me 4.22 (q), J = 4 Hz, 7.5 Hz? £ 5.09 (d), H-1 ", J1If 2n = 4 '° Hz * ^ 5'34 (d)' Π" 1 '' Jl \ 2 '= 2 Hz

Prepared similarly: 1-N- (S-3-amino-2-hydroxypropionyl) gentamicin Cla, pmr 100 MHz, D 2 O: Si, 22, C-Me? 2.59, N-Me? [α] 26 + 115.4 ° (c = 0.3 in water)? 5.09, H-l ", J-Lii / 2 *" = 4 Hz? 5.17, H1 ", J2, 2, = 3.5 Hz. Analysis: calculated for C 22 H 44 ° 9 N 6 'H 2 O / CO 2: C = 45'01' H = 7/39 / N = 13.70? Found: C = 44.98, H = 7.81, N = 13.68%, 1-N- (S-3-amino-2-hydroxypropionyl) gentamicin B, pmr 100 MHz, D20 + DC1: £ 1.18 C -Me (s)? 2.77 N-Me (s)? 4.35 (q), J = 4.5, 8.0, * H-1 ', £ 5.01 (d), Jx, Δ 21 = 4.0 Hz? HI ", 5.43 (d), Jin 2" = 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. 0.297g of t-butoxycarbonylazide is added dropwise with stirring followed by 0.186ml of triethylamine and the resulting solution is stirred for 18 hours. The reaction mixture is evaporated in vacuo to a residue and the residue is chromatographed on 100 g of silica gel using the lower phase of a chloroform: methanol concentrated ammonium hydroxide solvent system (2: 1: 1) as eluent.

n DK 156070B

agent. 2 ml fractions are collected and the column effluent is assayed by thin layer chromatography. Fractions containing the same material are collected (fractions 180-230) and evaporated to give 0.830 g of 6'-Nt-butoxycarbonylgentamicin B with the following physical constants: pmr (60 MHz, D20): <J1, 21 (3H, s, C-CH3 ), 1.42 (9H, s, C (CH 3) 3), 2.53 (3H, s, N-CH 3), 5.2 (1H, d, J = 4.5 HzT H 23 (1H, d 7 "j = 3.0)

Hz, H-1 ') PPM, analysis calculated for C24H46N4 ° i2, CO2, H2O: C = 46'57' H = 7.51, N = 8.69%, found: C = 46.80, H = 7 , 82, N = 8.54%, [α] 20 + 124 ° (c = 1 in methanol).

B. 1-N- (S-4-Amino-2-hydroxybutyryl) -61-N-t-butoxycarbonyl-gentamicin B.

Dissolve 0.582 g of 6'-N-t-butoxycarbonylgentamicin B in 25 ml of methanol: water (1: 3). A solution of 0.334 g of N- (S-4-carbo-benzyloxyamino-2-hydroxybutyryloxy) succinimide in 5 ml of dimethylformamide is added dropwise with stirring. The reaction mixture is stirred at 5 ° C for 5 hours, then evaporated to dryness in vacuo. The residue is chromatographed on 60 g of silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system (2: 1: 1) as eluent. 3 ml fractions are collected, the contents of which are determined by thin-layer chromatography. Fractions 160-235 are combined and evaporated to a residue of 0.280 g, which migrates as a single spot by thin layer chromatography. The residue is dissolved in 8 ml of methanol and 10 ml of water and hydrogenated at 3.7 atmospheres over 60 mg of 5% palladium-on-carbon catalyst. The catalyst is removed by filtration, the filtrate is evaporated to a residue which is chromatographed on 20 g of silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system (1: 1: 1) as eluent. Fractions 79-127 (each of 2 ml) consisting of 1-N- (S-4-amino-2-hydroxybutyryl) -61-N-t-butoxycarbonyl-gentamicin B are combined. Yield 86 mg. pmr: $ 1.21 (3H, s, C-CH3), 1.43 (9H, s, C- (CH3) 3), 2.51 (3H, s, N-CH3), 5.08 (1H, d, J = THz, H1 "), 5.25 (1H, ~ d7 J = 3.5 Hz, H-1 ')" PPM.

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

The product of step B is dissolved in 0.3 ml of trifluoroacetic acid and after 5 minutes 30 ml of ethyl ether is added. A precipitate of the compound of this example is formed in the form of its trifluoroacetate salt and is isolated by filtration. The precipitate is dissolved in water and the solution is passed through a basic hydroxyl ion exchange resin column to transfer the acid addition salt into the free base. colon

12 DK 156070B

the neofluent is lyophilized to give the compound of this example in the form of a white, amorphous solid. Yield 72 mg. pmr D20 100 MHz: δ.14 C-methyl (s), 2.45 (s) N-methyl, 5.04 (d), H1 "= 4/0 Hz; 5.30 (d), H -l ', 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 C

Dissolve 0.84 g of 2 ', 3-di-N-trifluoroacetylgentamicin in 40 ml of tetrahydrofuran and add a solution of 1.19 g of N- (S-3-carbobenzyloxyamino-2-hydroxypropionyloxy) succinimide in 24 ml of tetrahydrofuran dropwise with stirring. The reaction mixture is stirred for 24 hours and then evaporated to a residue. The residue is chromatographed on silica gel using the lower phase of a chloroform: methanolic concentrated ammonium hydroxydivand solvent system (2: 1: 0.2: 0.8) v / v as eluent. The chromatogram is examined by thin layer chromatography, and fractions containing the same product are combined to give the main product of the reaction with the following constants: mp: 125-131 ° C, [.alpha.] @ 6 = + 93 ° (CH2 OH), analysis 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 of step A in 55 ml of methanol and add 25 ml of concentrated ammonium hydroxide with stirring. Stir for 3 days, after which thin layer chromatography on silica gel plates using the lower phase of a chloroform: methanolic concentrated ammonium hydroxide solvent system (1: 1: 1) as mobile phase indicates substantially complete removal of the trifluoroacetyl blocking groups. The solution is evaporated to dryness in vacuo. Yield 0.2 g, mp: 109-112 ° C, [α] D = 6 = + 73 ° (H 2 O).

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

Dissolve 0.153 g of the residue from step B in 8 ml of acetic acid and add 0.05 g of 10% palladium-on-carbon catalyst. The solution is hydrogenated at 4.0 atmospheres and 25 ° C until thin layer chromatography (using the solvent system used in step B) indicates complete reaction to the title compound (i.e., 16 hours to 3 days). The catalyst is removed by filtration and the solution

DK 156070 B

13 is evaporated to a residue in vacuo. The residue is chromatographed on 7 g of silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide mixture (2: 1: 1) as eluent. The column is examined using thin layer chromatography, combining similar materials to give the product of this example. Yield 112 mg, mp: 109-119 ° C [α] D = + 98 ° (E₂O), analysis for the 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 solution (1: 3) and cool to 0-5 ° C. A solution of 3.81 g of N- (S-4-phthalimido-2-hydroxybutyryloxy) succinimide in 20 ml of dimethylformamide is added dropwise with stirring to the antibiotic solution. Stir for an additional 16 hours, then concentrate to a residue in vacuo. The residue is chromatographed on a column of 250 g of silica gel with the lower phase of a chloroform: methanol: concentrated ammonium hydroxide mixture (2: 1: 1) as eluent. The eluate is assayed by thin layer chromatography and fractions containing similar materials are combined. The combined fractions containing the main product are evaporated to give 1-N- (S-4-phthalimido-2-hydroxybutyryl) digestion and an isomer thereof.

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

Dissolve 4.0 g of the product of the previous step in 35 ml of ethanol and add 1.0 g of hydrazine hydrate. The solution is refluxed for 3 hours and then evaporated to dryness in vacuo. The residue is chromatographed over 160 g of silica gel eluting with the lower phase of a chloroform: methanol: concentrated ammonium hydroxide mixture (1: 1: 1) (v / v). The fractions containing the main component of the reaction as shown by thin layer chromatography are combined and evaporated to give the compound of this example as a white amorphous solid.

CMR (25.2 MH, 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.

Similarly prepared: t

14 DK 156070 B

1-N- (S-4-amino-2-hydroxybutyryl) -gentamicin B1 PMR (60 MH, D 2 O) δ 1.28, s, C ~JJ; 1.26, d, CH-CH 2; 1.7-2.3, m, 2xH-3 "and H-2" eq; 2.74, s, 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 mp 128-138 ° C; [a] 2 + + 126.4 (C, 0.28 in water) PMR (100 MH, D 2 O) 61.12, s, C-Me; 1.21, d , CH-Me; 2.48, s, N-Me; 5.08, d, J = 3H, H-1 "? 5r25, d, J = 4Hz, H-1 'ppm and 1-N- (S-3-amino-2-hydroxy-propionyl) -gentamicin PMR (D20): 6 1.08 (3H, d, J = 7 ), 1.28 (3H, s, C-CH 3), 1.38 (1H, q, J = 13.0 Hz, H-2ax,), 1.93 (1H, m, H 2), 2.48 (1H, d, J = 10.5 Hz, H-3 "), 2.5 (3H, s, N-CH-,), 3.93-2eq 4.28 (m , 2H, H-5 "and H-2",) f 5.05 (1H, d, J = 4.0 Hz, H-1 "), 5.25 - (1H, d, J = 3.0 Hz, H-1 ').

CMR (D20), δ (rel TMS, PPM): 179.1, 102.4, 99.1, 89.6, 80.3, 76.1, 75.1, 74.7, 73.5, 73.1 , 72f3, 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-hydroxypropionic acid in 110 ml of dioxane: water (4: 1). 200 mg of palladium-on-carbon catalyst is added and hydrogenated at 3.4 atmospheres for 3 hours. The catalyst is filtered off and the filtrate is evaporated to a residue comprising S-3-amino-2-hydroxypropionic acid. The acid is dissolved in methanol (40 ml) and triethylamine (8.5 ml), the solution is cooled in an ice bath and t-butoxycarbonylazide (4.76 g) is added. The mixture is allowed to stand overnight at room temperature. Most of the methanol is evaporated, diluted with water and acidified with dilute hydrochloric acid to pH 3. The aqueous mixture is extracted several times with ethyl acetate, the ethyl acetate extracts are combined and dried and then evaporated to a residue in vacuo. The residue is recrystallized from ethyl acetate: hexane to give the title compound of this example. Yield 3.5 g, m.p. 92-94 ° C, [α] 26 + 14.9 °, (c = 0.68, 0), analysis calculated for C CH ^NO ^: 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.

2.3 g of S-3-t-butoxycarbonylamino-2-hydroxypropionic acid are dissolved in a mixture of tetrahydrofuran (25 ml) and ethyl acetate (60 ml). With stirring, 1.42 g of N-hydroxysuccinimide is added, followed by a solution of dicyclohexylcarbodiimide (2.51 g) in ethyl acetate (10 ml).

The mixture is stirred for 16 hours, after which the precipitated solid is removed.

, 5 DK 156070 B

filtered. The filtrate is evaporated to give the product of this example. Yield 3.56 g.

2. 2 ', 3-di-N-Trifluoroacetylgentamicin C

A. 2'-N-Trifluoroacetylgentamicin C · ^.

Dissolve 1.7 g of gentamicin in 20 ml of methanol, cool the mixture to 4 ° C and add 0.46 ml (0.563 g) of ethylthioiltrifluoroacetate with stirring. The reaction is allowed to continue for 2 hours and the solution is concentrated to a residue in vacuo. The product is chromatographed on 80 g of silica gel G using the lower phase of a chloroformimethanol: water: ammonium hydroxide mixture in volume ratio of 10: 5: 4: 1 as eluent. The fractions containing the main component are combined and concentrated to give 1.4 g of the title compound, m.p. 108-111 ° C, [α] D = + 128 ° (c = 0.3%, E + O). Analysis for C 23 H 42 N 5 O 8 F 3, H 2 O requires 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. 2f, 3-di-N-Trifluoroacetylgentamicin C

Dissolve 0.66 g of the product of step A in 10 ml of methanol, cool the mixture to 4 ° C and add 0.148 ml (0.182 g) of ethylthiol trifluoroacetate dissolved in 3 ml of methanol. The reaction mixture is stirred for approx. 16 hours and concentrated in vacuo to a residue. Chromatograph the product on 30 g of silica gel as described in step A. The column is assayed by thin layer chromatography, the appropriate fractions are combined and concentrated to give 0.32 g of the title compound, m.p. 121-129 ° C, [α] ρβ = 121 ° (c = 0.3%, H 2 O). Analysis for Ο ^ Η .-, Ν, -ΟοΕ, - requires C = 44.84, H = 6.17, N = 10.46%. Found: C = 44.94, Δο 4i o y b H = 6.35, N = 10.17%.

Example 6 1- N- (S-4-amino-2-hydroxybutyryl) gentamicin C ^ a C A. 1-N- (S-4-Benzyloxycarbonylamino-2-hydroxybutyryl) gentamicin Cla.

Dissolve 2.8 g (4 mmol) of gentamicin C sulfate in 30 ml of water and add 15 ml of methanol. Then 0.56 ml (4 mmol) of triethylamine is added and stirred for 10 minutes. Then, a solution containing 4 mmol of N- (S-4-benzyloxycarbonylamino-2-hydroxybutyryloxy) -succinimide in 20 ml of dry dimethylformamide is added dropwise with stirring to the antibiotic solution. The mixture is stirred overnight (16 hours)

, 6 DK 156070B

at ambient temperature. Thin layer chromatography of the reaction mixture via TLC on silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system (1: 1: 1) shows the presence of smaller amounts of several components and one major component. The reaction mixture is concentrated in vacuo to a residue and the residue is triturated with methanol to give 3.2 g of white solid containing the components previously observed by chromatography.

150 mg of the product is chromatographed on 50 g of silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system (2: 1: 1). The fractions containing the major component are combined and lyophilized to give 1-N- (S-4-benzyloxycarbonylamino-2-hydroxybutyryl) gentamicin C Yield 70 mg. NMR (D 2 O): δ 1.15 (3H, s, C-CH 3)} 2.49 (3H, s, NCH 3); 4.10 (1H, dd, J = 8.0, 4.0 Hz, side chain H-2); 7.36 (5H, m, phenyl) B. B. 1-N- (S-4-Amino-2-hydroxybutyryl) gentamicin Cla ·

The product of Step A is dissolved in a mixture of 12 ml of methanol and 3 ml of water, added to 20 mg of 10% palladium-on-charcoal and hydrogenated at 4 atmospheres and room temperature. After 3 hours, the reaction is essentially complete. The catalyst is removed by filtration and the filtrate lyophilized to give 46 mg of 1-N- (S-4-amino-2-hydroxybutyryl) gentamicin. NMR (D 2 O): δ 1.17 (3H, s, C-CH 3); 2.48 (3H, s, NCH 3); 4.22 (1H, dd, J = 9.5 Hz, 4.0 Hz, side chain CHOH) j 5.04 (2H, m, H-Tr and H1 "). Mass spectral data: (M-H 2 O) m / e 532nd

Example η 1-N- (S-4-Amino-2-hydroxybutyryl) gentamicin B.

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

3.39 g of gentamicin B sulfate are dissolved in 48.4 ml of water and diluted with 23.7 ml of methanol. 0.7 ml of triethylamine is added dropwise with stirring. 1.67 g of N- (S-4-benzyloxycarbonylamino-2-hydroxybutyryl-oxy) succinimide are dissolved in dimethylformamide and the solution is added dropwise with stirring to the antibiotic solution. The resulting solution is stirred at room temperature for 18 hours, then concentrated to a residue in vacuo. The residue is dissolved in water and treated with dilute barium hydroxide solution with stirring until the pH reaches approx.

8.0. The precipitated barium sulfate is removed by filtration using a filter aid. The precipitate is washed with water, the filtrate and the wash water are combined and concentrated to dryness in vacuo. Remaining chromatic

17 DK 156070 B

Charge onto a column containing 600 g of silica gel using the lower phase of a chloroform: methanol: ammonium hydroxide solvent system (1: 1: 1) as the eluant. The material eluted immediately before gentamicin B is combined and the combined fractions are concentrated 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.

The product of step A is dissolved in a mixture of 20 ml of water and 8 ml of methanol. The product is hydrogenated in the presence of € 0 mg of 5% palladium-on-charcoal at 3.4 atmospheres and room temperature for 3 hours. The catalyst is removed by filtration using a filter aid. The filter cake is washed with water and the filtrate and wash water are combined. The combined filtrate and washings are concentrated to dryness in vacuo. The residue is chromatographed on a silica gel column containing 10 g of silica gel using a chloroform: methanol: ammonium hydroxide solution (1: 2: 1) as eluent. The fractions containing the most polar components are combined, concentrated and lyophilized to give 1-N- (S-4-amino-2-hydroxybutyryl) gentamicin B. Yield 12 mg.

Pmr D 2 O 100 MHz:> δ 1.14 c-methyl (S), 2.45 (S), N-methyl, 5.04 (d), H-1 ", Jr.f2" = 4 '° HZ; 5.30 (d) 'H ~ 1' 'Jl' »2 '= 3'5 Hz *

The compounds prepared by the analogous process of the invention are broad-spectrum antibacterial agents that exhibit activity against many organisms resistant to 1-N unsubstituted precursors thereof. Thus, the compounds of the invention can be used alone or in combination with other antibiotic agents to avoid growth or reduce the number of bacteria in different environments. The compounds of the invention are more active against many organisms which inactivate the corresponding antibiotics by acetylation of the 3-amino group and / or by adenylation of the 2 "hydroxyl group.

Thus, the compounds prepared by the analogous process of the invention have the potential to be used as antibacterial agents and can be used for the same purpose as their (parent) non-derived antibiotics, e.g. they can be used as bacteriostatic cleaning agents for hospital glassware, surgical instruments,

, 8 DK 156070B

utensils, bathtubs or for cleaning 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 C1a (U.S. Patent 3,796,699)

Compound 2: 1-N- (S-4-amino-2-hydroxybutyryl) gentamicin C1 (U.S. Patent 3,780,018) b)

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 is given in Table I.

200 ml of Mueller-Hinton medium adjusted to pH 7.2 is sterilized. Transfer 5 ml of the sterile medium to each of the 120 sterile, 16x150 run cotton plug test tubes. The tubes are placed in 10 groups with 12 4 tubes in each. To each tube are added 10 cells of one of the bacterial strains to be tested. To each group is added an aqueous solution of the antibiotic scm to be tested to produce subsequent final concentration per day. tubes: 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). The tubes are incubated for 24 hours at 37 ° C. For each group of tubes, visually the lowest concentration of antibiotic which inhibits bacterial growth and the highest concentration of antibiotic permitting bacterial growth is determined.

The minimum inhibitory concentration for antibiotic testing against each of the bacteria tested is determined by calculating the mean of the two values found.

19 DK 156070 B

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In general, the dose administered of the compounds of the invention depends on the age and weight of the species being treated, the mode of administration, and the type and severity of the bacterial infection to be avoided or reduced. In general, the dose of the derivatives of the 4,6-di- (aminoglycosyl) - 1,3-diaminocyclitols used to control a given bacterial infection will correspond to the dose requirements for the corresponding 1-N unsubstituted 4,6-di- (aminoglycosyl) -1,3-di-aminocyclitols.

The compounds of the invention can be administered orally. They 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 emuction type, or in the form of creams. Pharmaceutical carriers useful in the preparation of such formulations include, e.g. substances such as water, oils, fats, polyesters, polyols and the like.

For oral administration, the compounds of the invention may be formulated in the form of tablets, capsules, elixirs or the like, or may be mixed with animal feed. It is in these dosage forms that the antibacterial agents are most effective in treating bacterial infections of the gastrointestinal tract which infections cause diarrhea.

Generally, the topical compositions contain from ca. 0.1 to approx. 3.0 g of the compounds of the invention per 100 g ointment, cream or lotion. The topical compositions are usually applied gently to lesions of approx. 2 to approx. 5 times a day.

The antibacterial agents of the invention can be used in liquid form, such as solutions, suspensions and the like, for use in the ears and eyes and can also be administered parenterally by intramuscular injection. The injectable solution or suspension will usually be administered in an amount of from ca. 1 mg to approx. 15 mg antibacterial agent per kg body weight per day divided into approx. 2 to approx. 4 doses. The exact dose depends on the stage and severity of the infection, the sensitivity of the infected organism to the antibacterial agent and the individual characteristics of the species of animal being treated.

Claims (2)

22. DK 156070 B 1. Analogy Process for Preparation of Antibiotically Active 1-N-Substituted Derivatives of the 4,6-di- (aminoglycosyl) - 1,3-diaminocyclitolene gentamicin B, gentamicin, gentamicin, gentamicin C., sisomicin, verdamicin, antibiotic G-418 , anti-I 3 biotics 66-40B, antibiotic 66-40D, antibiotic JI-20A, antibiotic JI-20B and antibiotic G-52, wherein the 1-N substituent is designated X and represents S-3-amino-2-hydroxypropionyl or 5-4-amino-2-hydroxybutyryl, with the proviso that in the case of gentamicin C. and gentamicin C, X is S-3-amino-2-hydroxy-propionyl, 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 at any position differently from position 1, is 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 acid, or B) one of the 4,6-di- (aminoglycosyl) -1,3-di-aminocyclitols listed above. partially neutralized by formation of an acid addition salt, treated with an acid of the formula HO - X 'wherein X' is a group as defined for X wherein the amino group and / or hydroxy group may be protected, in the presence of a carbodiimide, or with a reactive derivative of the above acid, which process A) or B) is followed by removal of all protecting groups present in the molecule and isolation of the derivative as such, or as a pharmaceutically acceptable acid addition salt. Process according to claim 1, characterized in that 1-N- (S-3-amino-2-hydrogypropionyl) gentamicin B is prepared and isolated as such, or as a pharmaceutically acceptable acid addition salt.