CS201513B2 - Method of producing novel 6'-n-substituted derivatives of 3-n-formyl-kanamycin - Google Patents

Description

The invention relates to a novel process for the preparation of selectively N-protected kanamycin derivatives as intermediates for the preparation of aminoglycoside antibiotics, in particular 1-N-alkyl-substituted kanamycin derivatives.

Examples of such 1-N-alkyl-substituted kanaimycin derivatives are described in British Patent No. 1,464,401; others are described in Belgian Patent ^sp: the no. 855,709.

If similar compounds are to be prepared from kanamycin, which is a readily available fermentation product, all amino groups of imimo 1-amino groups need to be protected. It is then possible to selectively alkylate the amino group at the 1-position or to simplify the isolation of the final 1-N-substituted product.

In MS. No. 201 512 describes a preparation process. -N-alkyl-substituted kanamycin derivatives of formula I

in which

R is amino or hydroxyl,

R ¹ represents a lower alkyl group having 1 to 6 carbon atoms, wherein any of the carbon atoms, except that which is bound to the nitrogen atom may optionally be substituted by hydroxyl or amino, characterized by alkylating a compound of formula II ·

in which

R ³ is alkanoyl with 2-4 carbon atoms or benzoyl and

R ⁴ represents a hydroxyl group or a group of formula NHR ³ , to give a compound of formula III

wherein R 1, R ³ and R ⁴ are as defined above and after removal of the formyl group and the R ³ group, the desired compound of formula I is isolated.

As used herein, a lower alkyl group refers to a group containing from 1 to 6 carbon atoms and which may be straight or branched. Examples of lower alkyl substituted R ¹ groups include the (S) -4-amino-2-hydroxybutyl group and the 1,3-dihydroxyprop-2-yl group. The protecting group R ³ is preferably an acetyl group.

The above process for the preparation of compounds of formula I is described in U.S. Pat. No. 201,512.

The 6‘-N-substituted-3-N-formyl kanamycin derivatives of formula II are novel compounds. According to the present invention, these novel compounds of formula II can be prepared by a process characterized in that the compounds of formula II A

wherein the substituents R ³ and R ⁴ are as defined above, are formyl and the resulting compounds are selectively hydrolyzed to remove the formyl group from the 1-amino position, while the formyl group at the 3-position remains intact.

The preparation of suitably substituted acylkanamycin derivatives substituted on the amino groups at the 3-position and the 6'-kanamycin A and further on the 2'-amino group of kanamycin B using a C 2 -C 4 alkanoyl or benzoyl group is described in the Belgian patent No. 853,564. For example, the preparation of 3 ', 6 ' -di-N-acetylkanamycin A by performing selective migration of an acetyl group from oxygen to nitrogen is described. Other derivatives can be prepared in a similar manner.

The formylation reaction is. can be performed in a number of ways that are generally known. For example, formylation can be carried out using a mixture of formic anhydride and acetic anhydride, but we have found that formylation is conveniently carried out by treatment with 4-nitrophenyl ester of formic acid. Suitable solvents for this reaction include aqueous dioxane, aqueous tetrahydrofuran or dimethylformamide. The reaction is carried out by adding an excess (e.g., up to 3-fold) of 4-nitrophenyl formate to the kanamycin derivative, usually with a base to facilitate the reaction, and an organic base such as triethanolamine may be used. The athylation reaction is generally complete in 24 hours at room temperature, and the product can then be isolated by conventional means, for example by distilling off the solvent or by precipitation, and if desired, further purified.

Selective hydrolysis is carried out with the 1,3-di-N-formyl derivative of kanamycin dissolved in water or in an aqueous organic solvent, for example aqueous dioxane or tetrahydrofuran. The pH of the solution is carefully adjusted to a range of 12.0 to 12.5 by the addition of an alkali such as dilute sodium or potassium hydroxide solution. The solution is stirred for several days at room temperature and the progress of the reaction is monitored by thin layer chromatography until conversion to the desired 3-N-formyl derivative of general formula II. In this case, the solution is neutralized by the addition of an acid, or preferably by the addition of an ion exchanger in a hydrogen cycle. The use of an ion exchanger has the advantages that inorganic substances do not accumulate in the solution and further that any by-products resulting from the cleavage of both the 1- and 3-formyl groups are adsorbed to the ion exchanger. The product is then isolated by conventional means, for example by evaporating the solvent to a small volume and precipitating it with an organic solvent such as isopropyl alcohol. Further purification, if necessary, is possible by chromatography. Usually, however, the product is sufficiently pure for direct use in the preparation of the compounds of formula (I).

For the preparation of compounds of formula II, 3 ', 6'-di-N-acetylcanamycin A can be used to give a compound of formula II wherein R ³ is acetyl and R ^{4 is} hydroxyl. Similarly, 2 ', 3'-6'-tri-N-acetylkanamycin B can be used to form a compound of formula II wherein R ⁴ is NHR ³ wherein R ³ is acetyl.

The invention is illustrated by the following examples.

Thin layer chromatography was performed on a thin layer of silica gel using the following solvent systems. Detection is carried out by drying the plates by spraying with a 5% solution of tert-butyl hypochlorite in cyiclohexane, drying in a ventilated oven at 100 ° C for 10 minutes, and after cooling by spraying with a solution of sodium iodide with starch.

Thin layer electrophoresis is performed on 20 cm silica gel plates with a potential difference of 900 V for 45 minutes. As the electrolyte, a mixture of formic and acetic acid of pH 2 was used and the detection was carried out as described above.

Example 1

Preparation of 3 ', 6‘-di-N-acetyl-3-N-formylcanamycin A

A) To a suspension of 3 ', 6'-di-N-acetylkanamyeine A (3.9 g) in 21 ml of water and 21 ml of tetrahydrofuran containing 6.91 g of triethylamine was added portionwise over 45 minutes 6.88 g of 4-nitrophenyl ester formic acid. The reaction mixture was stirred at room temperature overnight, then the reaction mixture was concentrated to a small volume · The last traces of water were removed by azeotropic distillation using isopropanol (final volume of reaction mixture 35 ml). 2.42 g of 3 ', 6'-di-N-acetyl-1,3-di-N-forimylcanamycin A are precipitated from the solution, which are filtered off and dried in vacuo at 50 ° C.

In thin-layer electrophoresis (pH = 2) the anti-kanamycin substance has a value of 0.09. When thin layer chromatography using a methanol, ethyl acetate, and ammonia water (40: 40: 1: 30) as mobile phase, the value of R _f 0.42. The NMR spectrum gives signals δ = 7.96 ppm (formyl proton), 8.02 ppm (formyl proton) and 2.0 * 2 ppm (6 protons of acetyl groups).

B) 2.4 g of the product of process A) are dissolved in 72 ml of water, the pH of the solution is adjusted to 12.0 to 12.5 by adding 2.0 ml of 2 N sodium hydroxide solution. The solution is stirred at room temperature for 7 days while maintaining a pH between 12.0 and 12.5, then neutralized by adding 10 ml of Amberlite IR 120 ion exchanger in a hydrogen cycle. The ion exchanger is filtered off, the filtrate is concentrated to a small volume and the last traces of water are removed by refluxing with 5 ml of dimethylformamide. To the dimethylformamide solution was slowly added 30 ml of 2-propanol. The precipitated 3 ‘, 6‘-di-N-acetyl-3-N-formylcanamycin A is filtered off and dried under vacuum at 40 ° C. Yield 1.75 g.

The compound obtained is sufficiently pure for use in the next step of the process. The sample was further purified by chromatography on CM Sephadex C25 in the aimonium form using 0.1 M ammonium hydroxide solution as eluent. 13 C-NMR spectrum is in perfect accord with the desired structure, confirming monoformylaci amino group at position 3. When thin-layer electrophoresis (pH = 2), the substance to ¹ kanamycin A value of 0.43. Thin layer chromatography using methanol, ethyl acetate, ammonia and water (40: 40: 1: 30) as the mobile phase gave the compound an R _f value of 0.23.

Example 1 a d 2

Preparation of 2 ‘, 3,, 6‘-tri-N-acetyl-3-N-formylcanamycin B

A) To a solution of 80.5 g of kanamycin B sulfate and 76 g of sodium carbonate in 400 ^{l of} water was added 128 g of benzyl chloroformate over 10 minutes and the solution was stirred at room temperature for one hour. The precipitate is filtered off, washed with water, dilute hydrochloric acid and again with water, and dried to give 125.7 g of penta-N-benzyloxycarbonylcanamycin B.

B) To a mixture of 189 ml of acetic anhydride, 346 ml of pyridine and 346 ml of dichloromethane is added portionwise over 15 minutes stirring 230.8 g of penta-N-benzyloxycarbonylcanamycin B and the suspension is stirred at room temperature for 48 hours. The solution is then poured into a mixture of 1.5 L of dichloroethane and 2.3 L of water, the organic phase is separated and washed with a dilute hydrochloric acid solution of pH = 4 and then with water. By distilling off the solvent, the volume of the organic phase was adjusted to 0.75 L and this solution was poured into 4 L of diethyl ether. Filter the precipitate of tetra-O-acetyl-penta-N-benzyloxycarbonylcanamycin B and dry under vacuum (yield 244.8 g, 92.6 percent).

C) A solution of 33.0 g of tetra-O-acetyl-penta-N-henzyloxycarbonyl kanamycin B in a mixture of 132 ml of tetrahydrofuran, 66 ml of water and 3.3 ml of acetic acid is hydrogenated in the presence of 3.3 g of 5% palladium on charcoal 7 hours at 50 ° C and 0.35 NPa. The catalyst is filtered off and the filtrate is concentrated to a volume of 25 ml. To this residue was added 81.5 mL of 7 N ammonium hydroxide and the reaction mixture was stirred overnight at room temperature. The solution was evaporated to dryness and the residue was chromatographed on an Amberlite CG-50 ion exchange column in ammonium form (2 L ion exchange resin) using water followed by a 0.01 M aqueous ammonia solution as eluent. The fractions containing the desired product were combined and 9.37 g (61.6%) of 2 ', 3', 6'-tri-N-acetylkanamycin B was isolated after evaporation of the solvent; mp 191-198 ° C (dec.), v _max (potassium bromide) 1650, 1550 µm ^-1 , R f = 0.11 in methanol, diethyl ether, water and 0.88 M aqueous ammonium hydroxide (6: 3) : 1: 2); R _f = 0.11 in a methanol, ethyl acetate, water and 0.88 M aqueous ammonium hydroxide · (40: 40: 30: 1).

D) To a cooled solution of 93 g of 2 ', 3', 6'-tri-N-acetylkanamycin B in a mixture of 465 ml of water, 558 ml of tetrahydrofuran and 154 g of triethylamine is added portionwise over 45 minutes 153.1 g of p-nitroiphenyl ester ant. The reaction mixture was stirred overnight at room temperature and then concentrated to a small volume. The aqueous concentrate was diluted by adding isopropyl alcohol to precipitate the product. Residues of water are removed from the product by azeotropic distillation with isopropyl alcohol. Finally, the product is filtered off, washed with isopropanol and dried under vacuum. 89.2 g of 2 ', 3', 6'-tri-N-acetyl-1,3-di-N-formylkanamycin B are isolated, m.p. 310-318 ° C (dec.), In _max (in bromide dra201513). butyric) 1665, 1545 ^cm-1, Rf = 0.49 in a methanol, diethyl ether, water and 0.88 M aqueous ammonium hydroxide solution (6: 3: 1: 2) _Rf = 0.54 in a methanol, ethyl acetate, water and 0.88 M aqueous ammonium hydroxide (40: 40: 30: 1).

E) A solution of 86.9 g of 2 ', 4', 6'-tri-N-acetyl-1,3-di-N-formylkanamycin B in 2.61 liters of water was adjusted to pH 12.0 by adding 10 N sodium hydroxide. to 12.5. The reaction mixture was left at room temperature for 4 days and adjusted to pH 12 by addition of sodium hydroxide solution if necessary. The solution is then neutralized by addition of an ion exchanger

Amberlite IR 120 in hydrogen cycle. The mixture is filtered and the filtrate is chromatographed on a column containing 5.5 liters of Amberlite 200 ion exchange resin in ammonium form using water followed by 0.1 N aqueous ammonium hydroxide solution as eluent. Concentration of the appropriate fractions isolated 55.4 g of 2 ', 3' -, 6'-tri-N-acetyl-3-N-formylcanamycin B; mp 266-268 ° C (dec.); #max (KBr) 1665, 1555 CIM. ^"1; R _f = 0.49 in a methanol, ethyl acetate, water and 0.88 M aqueous ammonium hydroxide (40: 40: 30: 1). The structure was confirmed by C-13 NMR spectroscopy.

I will invent the object

Claims (7)

I will invent the object A process for the preparation of 6‘-N-substituted-3-N-formylcanamycin derivatives of the general formula II in which: R ³ denotes an alkanoyl group having from 2 to 4 carbon atoms or a benzoyl group and R ⁴ represents OH or NHR · ^3, characterized in that a compound of formula IIA in which R ³ and R ⁴ are as defined above, is formylated and the resulting compound is selectively hydrolyzed to remove the formyl group from the L-amino moieties. 2. The process according to claim 1, wherein 3 ', 6 ' -di-N-acetyl-kanamycin A is used as the starting compound. 3. The process according to claim 1, wherein the starting compound is 2 ', 3 ', 6 ' -tri-N-acetylkanamycin B. 4. A process according to any one of claims 1 to 3 wherein the formylation of the compound of formula IIA is carried out with 4-nitrophenyl formate. 5. The process of claim 4 wherein the formylation is carried out in an aqueous organic solvent in the presence of a base. 6. A process according to any one of claims 1 to 5 wherein the selective hydrolysis is carried out with a dilute sodium or potassium hydroxide solution. 7. The process of claim 6 wherein the selective hydrolysis is carried out at a pH of 12.0 to 12.5. Severography, n. P., Plant 7, Most