NO149635B - PROCEDURE FOR THE PREPARATION OF 1, N- (OMEGA-AMINO-ALFA-HYDROXYALKANOYL) -CANAMYCINE A OR B - Google Patents

Description

The present invention relates to a process for the production of 1-N-[w-amino-α-hydroxyalkanoyl]-kanamycin A or B with the general formula (I):

wherein R is OH or NH 2 and n is an integer from 0 to 2, or a non-toxic, pharmaceutically acceptable acid addition salt thereof.

The kanamycins are well-known antibiotics, such as e.g. is described in the Merck Index, 8th edition, pages 597-598. Numerous derivatives of the kanamycins are known. The structural formulas for kanamycin A and B are given below together with the standard numbering system used in the field. In what follows, the various derivatives of kanamycin, where it will be easily understood, will be referred to as derivatives of kanamycin A or B rather than by the structural formula, so that the need to compare complex structures to determine differences is avoided.

US patent 3,781,268 relates to 1-[L-(-)-γ-amino-α-hydroxybutyryl]-kanamycin A (amikacin) and B, as well as the mono- and di-carbobenzyloxy-protecting derivatives thereof. Regarding lower and higher homologs, reference is made to US patents 3,886,139 and 3,904,597. The compounds are prepared by acylation of a 6'-N-protected kanamycin A or B with an acylating derivative of an N-protected L-(-)-γ-amino-α-hydroxybutyric acid in an aqueous medium, followed by removal of one N -protecting group or both N-protecting groups.

From US patent 3,974,137, a method for the production of 1-[L-(-)-γ-amino-α-hydroxybutyryl]-kanamycin A is known, which comprises reacting 6'-carbobenzyloxykanamycin A with at least 3 mol benzaldehyde, a substituted benzaldehyde or pivaldehyde, to form 6'-N-carbobenzyloxykanamycin A containing Seiffske base groups in the 1,3 and 3" positions, acylate this tetra-protected kanamycin A derivative with the N-hydroxysuccinimide ester of L -(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid and then remove the protecting groups.

From Belgian patent document 828,192, a method for the production of 1-[L-(-)-y-amino-α-hydroxybutyryl]-kanamycin A is known by producing the same tetra-protected kanamycin A derivative as according to US patent document 3,974,137 , acylation with the N-hydroxy-5-norbornene-2,3-dicarboximide ester of L-(-)-y-benzyloxycarbonylamino-α-hydroxybutyric acid and subsequent removal of the protected groups.

The method according to the invention is characterized by

it comprises that kanamycin A or B containing 4-8 trimethylsilyl groups or kanamycin A or B containing 3-7 trimethylsilyl groups and a blocking group different from silyl, in the 6<1->amino group, is acylated with a) an active ester of N-hydroxysuccinimide, N-hydroxy-5-norbonene-2,3-dicarboximide or N-hydroxyphthalimide or b) a mixed acid anhydride of pivalic acid, benzoic acid, isobutylcarboxylic acid or benzylcarboxylic acid and an acid of the general formula (II):

where n is an integer from 0 to 2 and B is an amino blocking group of the formula

where R 1 and R 2 are the same or different and each is H, F, Cl,

Br, NC>2F OH, (lower) alkyl or (lower) alkoxy, and X is Cl,

Br, F or I, and Y is H, Cl, Br, F or I, in a substantially anhydrous organic solvent, and that all blocking groups are then removed.

According to the invention, a commercially attractive method for the production of compounds with the formula (I) has been developed. The use of a polysilylated kanamycin A or B as starting material gives high solubility in the organic solvent system, which enables reaction at high concentrations. Although the reaction is usually carried out in solutions containing 10-20% polysilylated kanamycin starting material, very good results have been obtained at concentrations of approx.

50 g/100 ml solvent.

As with known methods, the method according to the invention gives a mixture of acylated products. The desired 1-N-acylated product is separated from the other products by chromatography, and if desired, the by-products can be hydrolyzed to the starting kanamycin with a view to recycling. By known methods it has been found that any 3"-N-acylated material that was formed caused a loss of approximately a corresponding amount of the desired 1-N-acylated product due to the great difficulty in separating the latter from the former. A a particularly attractive feature of the method according to the invention is the very low amount of unwanted 3"-N-acylated product that is formed (typically none is detected).

In the production of 1-[L-(-)-γ-amino-α-hydroxybutyryl]-kanamycin A, amikacin, by various known methods, the 3"-N-acylated product (BB-K11), the 3-N-acylated product (BB-K29), the 6'-N-acylated product (BB-K6) and polyacylated material together with unreacted kanamycin A. In the commercial production of amikacin by acylation of 6'-N-carbobenzyloxy kanamycin A in an aqueous medium followed by removal of the protecting group, it was thus found that about 10% of the desired amikacin (2.5 kg in a 25 kg batch) was usually lost due to the presence of BB-K11 as a co-product When amikacin is prepared by the method according to the invention, BB-K11 is typically not detected in the reaction mixture.

The blocking groups that can be used for protection

of the 6'-amino group of the anamycin and the amino group of the acyl-

end acid (group B in formula II) are conventional blocking groups for protection of primary amino groups and are well known to those skilled in the art. Suitable blocking groups include "Alkoxy^ carbonyl groups, such as tert-butoxycarbonyl and tert-amyloxycarbonyl, aralkyloxycarbonyl groups such as benzyloxycarbonyl, cycloalkyloxycarbonyl groups such as cyclohexyloxycarbonyl, halogeno-alkyloxycarbonyl groups such as trichloroethoxycarbonyl, acyl groups such as phthaloyl and o-nitrophenoxyacetyl, as well as other known blocking groups, such as the o-nitrophenylthio group, the trityl group, etc.

The acylating acid with the formula (II) can be in the (+)- or (-)-isomeric form or a mixture of the two isomers (d,1-form), so that the corresponding compound with the formula ( I), where the 1-N-[ cu -amino-α-hydroxyalkanoyl ] - group is in its (+)— [or (R)] form or its (-)- [or (S)] form, or a mixture of these. Any such isomeric form and the mixture thereof are within the scope of the invention.

In a preferred embodiment, the acylating acid of formula (II) is in its (-) form. In another preferred embodiment, the acylating acid of the formula (II) is in its (+) form.

In a particularly preferred embodiment, the method relates to the production of 1-N-[L-(-)-γ-amino-α-hydroxybutyryl]-kanamycin A or a non-toxic, - pharmaceutically acceptable acid addition salt thereof, whereby polysilylated kanamycin A is acylated with a mixed acid anhydride of L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid with one of the above-mentioned acids in a largely anhydrous organic solvent.

In another particularly preferred embodiment, the method relates to the production of 1-N-[L-(-)-γ-amino-α-hydroxybutyryl]-kanamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof, whereby polysilylated kanamycin A containing a carbobenzyloxy group of the 6<1->amino group is acylated with a mixed acid anhydride of L-(-)-y-benzyloxycarbonyl lami no-a-hy dr ok sy butyric acid and one of the above-mentioned acids in a largely anhydrous organic solvent, and that one then removes all blocking groups.

In yet another particularly preferred embodiment, the method relates to the production of 1-N-[L-(-)-γ-amino-α-hydroxybutyryl]-kanamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof, whereby polysilylated kanamycin A is acylated with an active ester of L-(-)-y<->benzyloxycarbonylamino-α-hydroxybutyric acid with N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or N-hydroxyphthalimide in a large set anhydrous organic solvent is removed.

In a further particularly preferred embodiment, the method relates to the production of 1-N-[L-(-)-γ-amino-α-hydroxybutyryl]-kanamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof, whereby polysilylated kanamycin A containing a carbobenzyloxy group on the 6<1->amino group is acylated with an active ester of L-(-)-y-benzyloxycarbonyl-amino-α-hydroxybutyric acid with N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or N-hydroxyphthalimide in a largely anhydrous organic solvent.

The term "non-toxic, pharmaceutically acceptable acid addition salt" of a compound of the formula (I) here means a mono-, di-, tri- or tetrasalt formed by reaction between one molecule of a compound of the formula (I ) and 1-4 equivalents of a non-toxic, - pharmaceutically acceptable acid. These acids include acetic acid, hydrochloric acid, sulfuric acid, maleic acid, phosphoric acid, nitric acid, hydrobromic acid, ascorbic acid, malic acid and citric acid, as well as the other acids which are usually used for the production of salts of amine-containing drugs.

Acylation of the polysilylated kanamycin A or B starting material can usually be carried out in an organic solvent

in which the starting material has sufficient solubility. These starting materials are well soluble in most common organic solvents. Suitable solvents are e.g. acetone, diethyl ketone, methyl 1-n-propyl ketone, methyl isobutyl ketone, methyl ethyl ketone, acetonitrile, glyme, diglyme, dioxane, toluene, tetrahydrofuran, cyclohexanone, methylene chloride, chloroform, carbon tetrachloride and mixtures of acetone/butanol or diethyl ketone/butanol. The choice of solvent depends on the particular starting material used. Ketones are usually the preferred solvents. The most advantageous solvent for the particular combination of reaction participants used can easily be determined by routine testing.

Suitable silylating agents for use in manufacturing

of the polysilylated kanamycin starting materials used herein include the silylating agents of the general formulas:

m is an integer of 1 or 2 and X is halogen or

Specific silyl compounds of formulas IV and V are: trimethylchlorosilane, hexamethyldisilazane, methyltrichlorosilane, dimethyldichlorosilane, methyldiethylchlorosilane, dimethylethylchlorosilane, dimethyl-t-butylchlorosilane, etc. Also hexamethylcyclotrisilazanes and octa-methylcyclotetrasilazanes are applicable. Other suitable silylating agents are silylamides (such as methylsilylacetamides and bis-trimethylsilylacetamides), trimethylsilylureas and silyl ureides. Trimethylsilylimidazole can also be used.

Preferred silylating agents for introducing the trimethylsilyl group are hexamethyldisilazane, bis-(trimethylsilyl)-acetamide and trimethylsilylacetamide. Hexamethyldisilazane is most preferred.

When polysilylated kanamycin A or B containing a blocking group other than silyl on the 6'-amino group is used as starting material, this can be produced either by polysilylation of the desired 6'-N-blocked kanamycin A or B, or by introducing of the desired 6'-blocking group in polysilylated kanamycin A or B.

Methods for introducing silyl groups into organic compounds, including certain aminoglycosides, are known in the field. The polysilylated kanamycins (with or without a blocking group other than silyl on the 6<1->amino group) can be prepared by methods known per se or as described in this description.

The term polysilylated kanamycin A or B as used here refers to kanamycin A or B which contains the above stated number of trimethylsilyl groups in the molecule. The term polysilylated kanamycin A or B thus does not include persilylated kanamycin A or B, which would contain 11 silyl groups in the molecule.

The exact number of silyl groups present in the polysilylated kanamycin starting materials (with or without a blocking group other than silyl on the 6'-amino group) or their position is not known. It has been shown that both undersilylation and oversilylation reduce the yield of the desired product and increase the yield of other products. In case of strong under- and over-silylation, little or none of the desired product may be formed. The degree of silylation which will give the greatest yield of the desired product will depend on the particular reaction participants used in the acylation step. The most advantageous degree of silylation using an arbitrary combination of reaction participants can be easily determined by routine experiments.

In the preparation of 1-N-[L-(-)-γ-amino-α-hydroxybutyryl]-kanamycin A by acylation of polysilylated kanamycin A with the N-hydroxysuccinimide ester of L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid in acetone solution, it has been shown that good yields of the desired product are obtained by using polysilylated kanamycin A, which has been prepared by reacting from 4 to 5.5 mol of hexamethyldisilazane per mol kanamycin A. Larger or smaller amounts of hexamethyldisilazane can be used, but the yield of the desired product in the subsequent acylation step is considerably reduced. In the particular method stated above, it is preferred to use from 4.5 to 5.0 mol of hexamethyldisilazane per mol of kanamycin with a view to achieving maximum product yield in the acylation step.

It will be understood that each mole of hexamethyldisilazane is capable of introducing two equivalents of the trimethylsilyl group into kanamycin A or B. Both kanamycin A and B have a total of 11 positions (NH2~ and OH groups) which can be silylated, while kanamycin A and B containing a blocking group different from silyl on the 6'-amino group, has a total of 10 such positions.

5.5 mol hexamethyldisilazane per mol of kanamycin A or B could thus theoretically completely silylate all OH and N^ groups on the kanamycin, while 5.0 mol of hexamethyldisilazane could completely silylate one mol of kanamycin A or B containing a blocking group different from silyl on the 6'- the amino group. However, it is believed that such extensive silylation does not take place with these molar ratios within reasonable reaction times, even if higher degrees of silylation are achieved during a given reaction time when a silylation catalyst is added.

Silylation catalysts greatly accelerate the silylation rate. Suitable silylation catalysts are well known in the field and include, among other things, amine sulphates, e.g. kanamycin sulfate, sulfamic acid, imidazole and trimethylchlorosilane. Silylation catalysts usually promote a higher degree of silylation than is necessary in the process according to the invention. However, oversilylated kanamycin A or B can be used as starting material if it is first treated with a desilylation agent to reduce the degree of silylation before the acylation reaction is carried out.

Good yields of the desired product are obtained when acylating polysilylated kanamycin A prepared using a 5.5:1 molar ratio of hexamethyldisilazane to kanamycin A. However, when kanamycin A silylated with a 7:1 molar ratio of hexamethyldisilazane (or with a 5 .5:1 molar ratio in the presence of a silylation catalyst) was acylated in acetone with the N-hydroxysuccinimide ester of L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid, less than 1% yield of the desired product was obtained. However, when the same "oversilylated" kanamycin A was acylated with the same acylating agent in acetone solution, to which water had been added (21 mol of water per mol of kanamycin, 2.5% water (w/v) as desilylating agent 1 hour before the acylation, a yield of about 40% of the desired product. The same results are obtained if the water is replaced with methanol or another active hydrogen compound capable of effecting desilylation, e.g. ethanol, propanol, butanediol, methyl mercaptan, ethyl mercaptan, phenyl mercaptan or similar.

Although it is common to use dry solvents when working with silylated materials, it has surprisingly been shown that adding water to the reaction solvent before acylation often gives equally good yields and sometimes better yields of the desired product than a dry solvent. In acylation reactions carried out in acetone in the usual concentrations of 10-20% (w/v) of polysilylated kanamycin A, it has been shown that very high yields of 1-N-[ L-( -) -y-amino- α-hydroxybutyryl]-kanamycin A was obtained when up to 28 mol of water per mol polysilylated kanamycin A. At a concentration of 20%, 28 mol per moles approx. 8% water'. With other combinations of reaction participants, even more water can be tolerated or be beneficial. The acylation reaction can be carried out in solvents containing up to approx. 40% water, although with such high water concentrations short acylation times must be used to avoid too strong desilylation of the polysilylated kanamycin A or B starting material. Accordingly, the term "largely anhydrous organic solvent" as used herein is intended to include solvents containing up to approx.

25% water. A preferred interval is up to approx. 20%, a more preferred interval is up to 8% water, and a particularly preferred interval is up to approx. 4% water.

The duration and temperature during the acylation reaction

is not critical. Temperatures in the range from -30 to 100°C

can be used for reaction times in the range from approx. 1 hour and up to a day or more. It has been found that the reaction usually proceeds well at room temperature, and for convenience and economic reasons it is preferred to carry out the reaction at ambient temperature. However, with a view to obtaining maximum yields and selective acylation, it is preferred to carry out the acylation at from 0 to 5°C.

Acylation of the 1-amino group in the polysilylated kanamycin

A or B (with or without a blocking group other than silyl on the 6<1-> amino group) can be carried out with any acylation derivative of the acid of formula II known in the art to be suitable for acylation of a primary amino group. Examples of suitable acylation derivatives of the free acid include the corresponding acid anhydrides, mixed anhydrides, e.g. alkoxyformic anhydrides, acid halides, acid azides, active esters and active thioesters. The free acid can be coupled with the polysilylated kanamycin starting material after the free acid is first reacted with N,N'-

dimethylchloroforminium chloride [see UK Patent 1,008,170

and Novak and Weichet, Experientia XXI, 6, 360 (1065)] or by using an N,N'-carbonyldiimidazole or N,N'-carbonylditriazole (see South African patent 63/2684) or a carbodiimide reagent [especially N,N<1->dicyclohexylcarbodiimide, N,N'-di-isopropylcarbodiimide or N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide; see Sheenan and Hess, J.A.C.S., 77 1967 (1955)] ,

or by an alkynylamine reagent [see R. Buijle and H.G. Viehe,

angew. Chem. International Edition, 3, 582 (1964)] or by an isoxazolium salt reagent [see R.B. Woodward, R.A. Olofson and H. Mayer, J. Amer. Chem. Soc, 83, 1010 (1961)] , or by a ketene-imine reagent [see CL. Stevens and M.E. Munk, J. Amer. Chem. Soc,

80, 4065 (1958)] or of hexachlorocyclotriphosphatetriazine or hexabromocyclotriphosphatetriazine (U.S. Patent 3,651,050) or of diphenylphosphoryl azide [DDPA, J. Amer. Chem. Soc, 94, 6203-6205

(1972)] or by diethylphosphoryl cyanide [DEPC, Tetrahedron Letters

No. 18, pages 1595-1598 (1973)] or of diphenyl phosphite [Tetrahedron Letters No. 49, pages 5047-5050 (1972)]. Another derivative equivalent to the acid is a corresponding azolide, i.e. an amide of the corresponding acid whose amide nitrogen forms part of a quasiaromatic 5-membered ring containing at least two nitrogen atoms, i.e. imidazole, pyrazole, triazoles, benzimidazole, benzotriazole and their substituted derivatives. As those skilled in the art will appreciate, it may occasionally be desirable or necessary to protect the hydroxyl group of the acylated derivative of the acid of formula (II), e.g. when using acylation derivatives such as an acid halide. Protection of the hydroxyl group can be carried out in ways known in the art, e.g. by using a carbobenzyloxy group, by acetylation, by silylation or the like.

After completion of the acylation reaction, all blocking groups are removed in a manner known per se to form the desired product with the formula (I). The silyl groups can e.g. easily removed by hydrolysis with water, preferably at a low pH value. Also, the blocking group B on the acylation derivative of the acid of formula (II) and the blocking group on the 6'-amino group on the polysilylated kanamycin starting material (if present) can be removed by means of known methods. Thus, a tert-butoxycarbonyl group can be removed using formic acid, a carbobenzyloxy group by catalytic hydrogenation, a 2-hydroxy-1-naphthocarbonyl group by acid hydrolysis, a trichloroethoxycarbonyl group by treatment with zinc dust in glacial acetic acid, the phthaloyl group by treatment with hydrazine hydrate in ethanol during warm-up, etc.

Product yields were determined in different ways. After removal of all blocking groups and chromatography on a CG-50 (NH^<+>) column, the yield of amikacin could be determined by isolation of the crystalline solid from the appropriate fractions or by microbiological analysis (turbidimetric or plate) of the appropriate fractions . Another technique used was particularly effective liquid chromatography of the unreduced acylation mixture, i.e. the aqueous solution obtained after hydrolysis of the silyl groups and removal of organic solvent, but before hydrogenolysis to remove the remaining blocking group(s). This assay was not a direct assay for amikacin or BB-K2 9 but for the corresponding mono- or di-N-blocked compounds.

The instrument used was a Waters Associates "ALC/GPC 244" high pressure liquid chromatograph with a Waters Associates model 440 absorption detector and a "u-Bondapak C-18" column that was 30 cm x 3.9 mm ID, under the following conditions:

The curve speed varied but 2 minutes per 2.54 cm was typical. The above conditions produced UV curves with peaks that were easy to measure quantitatively. The results of the above analyzes are referred to in the description as HLPC analyses.

To avoid repetition of complex chemical designations, the following abbreviations are occasionally used in the description: AHBA L-(-)-y-amino-ct-hydroxybutyric acid BHBA N-carbobenzyloxy derivative of AHBA HONB N-hydroxy-5-norbornene-2,3- dicarboximide NAE N-hydroxy-5-norbornene-2,3-dicarboximide-(or BHBA-'ONB') activated ester of BHBA

HONS N-Hydroxysuccinimide

SAE N-hydroxysuccinimide activated ester of (or BHBA-<1>ONS<1>) BHBA

DCC dicyclohexylcarbodiimide

DCU dicyclohexylurea

HMDS hexamethyldisilazane

BSA bis-(trimethylsilyl)-acetamide

MSA trimethylsilylacetamide

TFA trifluoroacetyl

t-BOC tert-butyloxycarbonyl.

"Dicalite" is a diatomaceous earth.

"Amberlite CG-50" is the chromatography grade of a weak

acidic, cationic ion exchange resin of the carboxyl polymethacryl type.

"u-Bondapak" is a series of highly efficient liquid chromatography columns.

The terms "(lower) alkyl" and "(lower) alkoxy" refer to alkyl or alkoxy groups containing from 1 to 6 carbon atoms.

The invention will be described in more detail below

by means of examples illustrating preferred embodiments.

Example 1

Preparation of 1-N-[l-(-)-y-amino-α-hydroxybutyryl]-kanamycin A, amikacin, by selective acylation of poly-(trimethylsilyl) 6<1->N-carbobenzyloxykanamycin A in anhydrous diethyl ketone. 15 g (24.24 mmol) of 6'-N-carbobenzyloxykanamycin A was slurried in 90 ml of dry acetonitrile and heated with reflux under a nitrogen atmosphere. 17.5 g (108.48 mmol) of hexamethyldisilazane was added slowly over 30 minutes, and the resulting solution was refluxed for 24 hours. After removal of the solvent under vacuum (40°C) and complete drying under vacuum (10 mm), 27.9 g of a white, amorphous solid was obtained [90.71% calculated as 6<1->N-carbobenzyloxykanamycin A (silyl)g] .

This solid was dissolved in 150 ml of dry diethyl ketone at 23°C. 11.05 g (2 6.67 mmol) L-(-)-γ-benzyloxycarbonylamino-α-hydroxy-butyric acid-N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) dissolved in 100 ml of dry diethyl ketone at 23°C was added slowly with good stirring over the course of \ hour. The solution was stirred at 23°C for 78 hours. The yellow, clear solution (pH 7.0) was diluted with 100 ml of water. The pH of the mixture was adjusted to 2.8 (3N HCl) and the mixture was stirred vigorously at 23°C for 15 minutes. The aqueous phase was separated, and the organic phase was extracted with .50

ml of water with a pH of 2.8. The combined aqueous fractions were washed with 50 mL of ethyl acetate. The solution was placed in a 500 ml Parr flask along with 5 g of 5% palladium-on-carbon catalyst and reduced at 3.4 atm. H~ for 2 hours at 23°C. The mixture was filtered through a layer of "Dicalite" which was then washed with an additional 30 ml of water. The colorless filtrate was concentrated under vacuum (40-45°C) to 50 ml. The solution was loaded onto a 5 x 100 cm "CG-50" (NH4<+>) ion exchange column. After washing with 1000 ml of water, unreacted kanamycin A, 3-[1-(-)-γ-amino-α-hydroxybutyryl]-kanamycin A (BB-K29) and amikacin were eluted with 0.5 N ammonium hydroxide. Polyacyl material was extracted with 3N ammonium hydroxide. Bioanalysis, thin-layer chromatography and optical rotation were used to record the progress of the elution. The volume and observed optical rotation for each eluate fraction as well as the weight and percentage yield of solid isolated from each fraction by evaporation to dryness are summarized below:

The used diethyl ketone layer was shown by highly efficient liquid chromatography to contain an additional 3-5% amikacin.

The crude amikacin (6.20 g) was dissolved in 20 ml of water and diluted with 20 ml of methanol, and 20 ml of isopropanol was added to induce crystallization. 6.0 g (45.8%) of crystalline amikacin was obtained.

Example 2

Preparation of 1-N-[l-(-)-γ-amino-α-hydroxybutyryl]-kanamycin A, amikacin, by selective acylation of poly-(trimethylsilyl) 6'-N-carbobenzyloxykanamycin A in anhydrous acetone.

103 g (0.081 mol, calculated as 6'-N-carbobenzyloxykanamycin

A (silyl) g) poly-(trimethylsilyl)-6 1-N-carbobenzyloxy kanamycin A prepared as in example 1 was dissolved in 100 ml of dry acetone at 23°C. 35.24 g (0.085 mol) of L-(-)-γ-benzyloxycarbonylamino-o-hydroxybutyric acid-N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) dissolved in 100 ml of dry acetone at 23°C was slowly added with good stirring to the solution of poly-(trimethylsilyl)-6'-N-carbobenzyloxykanamycin A over a period of 15 minutes. The solution was stirred at 23°C for 20 hours under a nitrogen atmosphere. The pale yellow, clear solution (pH 7.2) was diluted with 100 ml of water. The pH of the mixture was adjusted to 2.5 (3N HCl), and stirring was continued at 23°C for 15 minutes. Acetone was removed using steam ejector vacuum at ca. 35°C. The solution was placed in a 500 ml Parr flask along with 10 g of 5% palladium-on-carbon catalyst (Engelhard) and reduced at 2.72 atm. H2 for 2 hours at 23°C. The mixture was filtered through a layer of diatomaceous earth which was then washed with a further 50 ml of water. After concentration to approx. 1/3 volume solution (pH 6.9-7.2) was loaded into a 6 x 110 cm "CG-50" (NH4<+>) ion exchange column and eluted with a stepwise gradient from H20 to 0.6 N ammonium hydroxide for recovery of amikacin. An automatic polarimeter was used to record the progress of the elution. Combinations were made on the basis of thin-layer chromatography assessment. The combined amikacin fractions were concentrated to 25-30% solids. The solution was diluted with an equal volume of methanol, followed by 2 volumes of isopropanol to induce crystallization. 18.2 g (40%) of crystalline amikacin was recovered.

The recovery of 12% kanamycin A, 40% BB-K29 and 5% polyacylated kanamycin gave a material balance of 97%.

Example 3

Preparation of 1-N-[l-(-)-γ-amino-α-hydroxybutyryl]-kanamycin A, amikacin, by selective acylation of poly-(trimethylsilyl)-kanamycin A using in situ blocking.

A. Poly-(trimethylsilyl)-kanamycin A.

Kanamycin A free base (18.g activity, 37.15 mmol) was slurried in 200 ml of dry acetonitrile and heated to reflux. 29.8 g (184.6 mmol) of hexamethyldisilazane was added over 30 minutes, and the mixture was stirred at reflux for 78 hours, yielding a pale yellow, clear solution. Removal of the solvent under vacuum left 43 g (94%, calculated as kanamycin A (silyl)10) of an amorphous solid residue.

B. 1- N- Hl-(-)- y- amino- g- hydroxybutyryl]- kanamycin A.

5.56 g (20.43 mmol) of p-(benzyloxycarbonyloxy)-benzoic acid was slurried in 50 ml of dry acetonitrile at 23°C. 8.4 g (41.37 mmol) of N,0-bis-trimethylsilylacetamide was added with good stirring. The solution was kept at 23°C for 30 minutes and then in

over 3 hours with vigorous stirring added to a solution of 21.5 g (17.83 mmol, calculated as the (silyl)^-compound) of poly-(trimethylsilyl)-kanamycin A in 75 ml of dry acetonitrile at 23°C. The mixture was stirred for 4 hours, the solvent was removed under vacuum (40°C), and the oily residue was dissolved in 50 mL of dry acetone at 23°C.

8.55 g (20.63 mmol) of L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid-N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) in 30 ml of acetone was added to the above solution in the race

of a period of 5 minutes. The mixture was kept at 23°C for 78 hours. The solution was diluted with 100 ml of water and the pH value (7.0) was lowered to 2.5 (6N HCl). The mixture was placed in a

500 ml Parr bottle along with 3 g of 5% palladium-on-carbon catalyst (Engelhard) and reduced at 2.72 atm. H2 for 2 hours at 23°C. The mixture was filtered through a layer of diatomaceous earth which was then washed with 20 ml of water. It was demonstrated by microbiological analysis against E. coli that it combined filtrate and washing water

(168 ml) contained approx. 11,400 mcg/ml (19% yield) amikacin.

Example 4

Preparation of 1-N-[L-(-)-γ-amino-α-hydroxybutyryl]-kanamycin A, amikacin, by selective acylation of poly-(trimethylsilyl)-kanamycin A.

A. Poly-(trimethylsilyl)-kanamycin A.

A suspension of 10 g (20.6 mmol) of kanamycin A in 100 ml of dry acetonitrile and 25 ml (119 mmol) of 1,1,1,3,3,3-hexamethyldisilazane was refluxed for 72 hours. The solution was evaporated to dryness under vacuum at 30-40°C. 21.3 was achieved

g poly-(trimethylsilyl)-kanamycin A as a light, yellow-brown, amorphous powder [J55% yield calculated as kanamycin A (silyl) 1Q] •

B. 1- N- Cl-(-)- y- amino- g- hydroxybutyryl] - kanamycin A.

To a solution of 2.4 g (2.0 mmol) of poly-(trimethylsilyl)-kanamycin A in 30 ml of dry acetone, 2.0 mmol of L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid was slowly added N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) in 10 ml of dry acetone at 0-5°C. The reaction mixture was stirred at 23°C for 1 week and then evaporated to dryness under vacuum at a bath temperature of 30-40°C. 60 ml of water was then added to the residue, followed by 70 ml of methanol to produce a solution. The solution was acidified with 3N HCl to pH 2.0 and then reduced at 3.40 atm. H2 for 2 hours using 500 mg of 5% palladium-on-carbon catalyst. The material was filtered, and it was demonstrated by microbiological analysis against E. coli that the combined filtrate and washing liquid contained amikacin in a yield of 29.4%.

Example 5

Preparation of amikacin by selective N-acylation of polytrimethylsilyl-6'-N-carbobenzoxykanamycin A in anhydrous acetone.

I. Summary.

Silylation of 6<1>N-carbobenzoxykanamycin A in acetonitrile using hexamethyldisilazane (HMDA) gives 6'-N-carbobenzoxykanamycin A (silyl) intermediate Q). This silylated kanamycin A is readily soluble in most organic solvents. Acylation with NAE in anhydrous acetone at 23°C of a 5% molar excess of NAE relative to the 6'-N-Cbz kanamycin A used gave a mixture containing only Cbz derivatives of amikacin and BB-K2 9, somewhat unreacted kanamycin A and some polyacyl material. BB-Kll could not be detected in any of these examinations. Elution of an acetone acylation mixture after reduction and work-up from a "CG-50" (NH4<+>) column using an ammonium hydroxide gradient gave isolated yields of pure amikacin in the range of about 40%.

II. Reaction forms. III. Materials

"CG- 50" (NH4+) - No toxicity data available,

handled with care.

V. Procedure

A. Preparation of 6'-N-carbobenzyloxykanamycin A (silyl)g

J6'-N-Cbz Kana A (silyl) g]

1. 50 g of 6<1->N-carbobenzyloxykanamycin A (KF<4%) is suspended in 300 ml of acetonitrile (KF< 0.01%). The slurry is brought to boiling by reflux (74°C) while maintaining a flow of dry nitrogen through the slurry. 2. Over a period of 30 minutes, slowly add 75.8 ml of hexamethyldisilazane (HMDS). Complete dissolution will occur with the evolution of ammonia gas. 3. Recooling is continued for 18-20 hours under nitrogen purging. 4. The clear, pale yellow solution is concentrated under vacuum (bath temperature 40-50°C) to a foam-like solid. Yield" of the silylyn compound 89-92 g (90-94% of theoretical yield).

NB: For future reference. In other solvent studies, this solid is usually not isolated, but used directly for the acylation.

B. Preparation of N-hydroxy-5-norbornene-2,3-dicarboximide ester of L-(-)-γ-carbobenzyloxyamino-α-hydroxybutyric acid (NAE)

1. 21.5 g of L-(-)-y-carbobenzyloxyamino-α-hydroxybutyric acid (BHBA) are dissolved in 100 ml of dry acetone at 23°C and then 15.2 g of N-hydroxy-5-norbornene-2,3- dicarboximide (HOMB). A complete resolution is achieved. 2. During 30 minutes, a solution of 17.48 g of dicyclohexylcarbodiimide (DCC) in 50 ml of acetone is added with stirring. The temperature will rise to approx. 4 0°C during the addition by precipitation of dicyclohexylurea (DCU). 3. The slurry is stirred for 3-4 hours while the temperature is allowed to equalize to 23-25°C. 4. The urea derivative is removed by filtration. The filter cake is washed with 30 ml of acetone. The filtrate plus the washings are kept for the acylation step below.

C. Acylation of 6'-N-Cbz Kana A (silyl)g.

1. 6'-N-Cbz Kana A (silyl)y isolated during part A, step 4, is dissolved in 100 ml of dry acetone at 23-24°C.

2. Slowly and with good stirring, add it under part

B produced NAE solution over a period of 15 minutes. The temperature will gradually rise to approx. 40°C. The solution is allowed

to reach equilibrium at 23°C and stirring is continued for 18-20 hours under a nitrogen atmosphere.

3. 100 ml of water is added and the pH value (6.9-7.2) is lowered to 2.2-2.5 with 6N hydrochloric acid. It is stirred for 15 minutes at 23°C. (NB: A further layer can be formed. This does not cause any problem during processing). 4. Acetone is removed under vacuum at a bath temperature of 30-35°C. The concentrate is transferred to a suitable hydrogenation vessel (pre-purged with nitrogen). 10 g of 5% palladium-on-carbon catalyst is added, and it is hydrogenated at 2.72 atm. for 2-3 hours. 5. The mixture is filtered through a "Dicalite" layer and the hydrogenation vessel and filter cake are washed with an additional 50 ml of water.

6. The filtrate plus the washing liquid is concentrated to approx. 1/3

of the volume (50 ml) under vacuum at 40-45°C.

7. The pH value is checked. It should be in the range 6.9-7.2. If not set it with IN ammonium hydroxide. The mixture is loaded onto a "CG-50" (NH4<+>) column (6 x 110 cm). 8. The column is washed with 1000 ml of deionized water. It is then eluted with 0.5-0.6 N ammonium hydroxide using an automatic polarimeter to record the progress of the elution. The elution order is as follows:

No BB-Kll was detected in any of the acylation preparations. Polyacyl material, ie the 1,3-diAHBA analogue of kana A, is recovered by washing the column with 3N ammonium hydroxide. 9. The amikacin fractions are combined and concentrated to 25-30% solids. It is diluted with 1 volume of methanol and inoculated with amikacin crystals. 10. Slowly add 2 volumes of isopropanol (IPA) over 2 hours with good stirring, and crystallize at 23°C for 6-8 hours. 11. The solid is filtered off, washed with 50 ml of 1:1:2 water/methanol/IPA mixture and finally with 25 ml of IPA. 12. It is dried under vacuum at 40°C for 12-16 hours. Yield: 17.3-19.0 g (38-42%) amikacin with the following properties:

Thin layer chromatography.

CHCl3-methanol - NH^OH - water (1:4:2:1), 5 x 20 cm silica gel plates from Quantum Industries - one zone as detected with nin-hydrin (RF~ 0.4).

Specific turning.

13. The recovery of BB-K29 in this system was also 39-42%, residual kana A 10-14% and 1,3-diAHBA-kana A approx. 5%, which gives a material balance of over 95%.

Example 6

Preparation of amikacin by selective N-acylation of polytrimethylsilyl kana A in anhydrous acetone.

I. Summary.

Silylation of kana A "base" in acetonitrile using hexamethyldisilazane (HMDA) gave polytrimethylsilyl kana A. The degree of silylation is still uncertain, but is currently believed to be kana A (silyl)1Q. Polysilylated kana A is readily soluble in most organic solvents. Acylation with SAE in anhydrous acetone at 23°C using a 1:1 molar ratio of SAE to cana A used gave a mixture containing Cbz derivatives of amikacin and BB-K29, usually in a ratio of 2-3/1, BB -K6 (5-8%), unreacted kana Å (15-20%) and some polyacyl material (5-10%). As was the case with the previous work on the acylation of polytrimethylsilyl-6'-N-carbobenzoxycan A, no BB-Kll was detected in any of these experiments. Reduction and work-up of an acetone acylation mixture followed by chromatography on a "CG-50"-(NH4<+>) column using 0.5 N ammonium hydroxide afforded isolated crystalline amikacin in the range of 34-39%.

II. Reaction forms. A. III. Materials

IV. Safety

Kana A "base" - Known drug - common

caution is advised.

Kana A (silyl)- No direct information available, approach with caution.

Other materials - See example 5.

V. Procedure

A. Preparation of Kana A (silyl)^q.

1. 50 g kana A "base" (KF 2.5-3.5%) is suspended in 500 ml acetone (KF< 0.01%). The slurry is brought to reflux (74°C) while maintaining a flow of dry nitrogen through the slurry. 2. Over the course of 30 minutes, slowly add 112 ml of hexamethyldisilazane (HMDS). Complete dissolution will occur within 4-5 hours with evolution of ammonia gas. 3. Boiling with recooling is continued for 22-26 hours under nitrogen purging. 4. The clear, slightly yellow solution is concentrated under vacuum (40°C) to a "viscous residue. It is rinsed with an additional 100 mL of acetonitrile and completely dried under high vacuum for 3-6 hours. The yield of whitish, amorphous solid is 109 -115 g (90-95% of the theoretical, calculated as kana A (silyl)-^q) • B. Preparation of N-hydroxysuccinimide ester of L-(-)-cc-carbobenzyloxyamino-g-hydroxybutyric acid ( SAE. 1. 100 g of L-(-)-a-benzyloxycarbonylamino-a-hydroxybutyric acid (BHBA) and 45.38 g of N-hydroxysuccinimide (N-HOS) are dissolved in 1300 ml of ethyl acetate (KF<0.05%) with stirring at 23°C 2. Dissolve 81.29 g of dicyclohexylcarbodiimide (DCC) in 400 ml

ethyl acetate (KF<0.05%) at 23°C. With good stirring, this solution is added over 30 minutes to the solution from step 1. The temperature will rise to 40-42°C during simultaneous precipitation of dicyclohexylurea (DCU).

The slurry is stirred for 3-4 hours whereby the temperature is allowed to equilibrate to 23°C. 3. The DCU is filtered off and the filter cake is washed with 250 ml of ethyl acetate (KF< 0.05%). The DCU cake is discarded while the filtrate and washing liquids are kept.

4. The filtrate plus the washing liquids are concentrated to approx. 500

ml (under vacuum at 30-35°C). A little product will crystallize out.

5. The concentrate is transferred to a suitable container and, with vigorous stirring, 100 ml of heptane is added. If necessary, SAE seed crystals are added. Crystallization will begin almost immediately. The slurry is stirred for 30 minutes at 23°C. 6. In the course of 30 minutes, 400 ml of heptane are added, and the slurry is stirred for 4-5 hours at 23°C. 7. The cake is filtered and washed with 200 ml of 3:1 heptane/ethyl acetate followed by 100 ml of heptane. 8. It is dried in a vacuum oven at 30-35°C for 18-20 hours.

The yield is 110.1-131.4 g (80-95%).

Temp. : 119-120°C with softening at 114°C (corr.) Thin layer chromatography: 4 acetone: 12 benzene:1 CH-jC^H - on display 1% aqueous KMO^.

Rf: 0.7 for SAE, 0.2 BHBA on 2 x 10 cm pre-coated silica gel plates from Analtech Inc.

C. Acylation of Kana A (silyl) -^q.

1. The kana A (silyl) isolated under section A, step 4 is dissolved in 100 ml of dry acetone at 23°C.

2. Quickly and with good stirring, it is added under section

B prepared SAE (35.03 g) as a 10 percent solution in dry acetone over 5-10 minutes. The temperature will rise approx. 5°C. The solution is allowed to equilibrate at 23°C, and stirring is continued for 18-20 hours.

3. The light orange, clear solution is diluted with 400 ml of water,

and the pH value (7.0-7.5) is lowered to 2.2-2.5 with 3N hydrochloric acid.

The clear solution is now stirred at 23°G for 15-30 minutes.

4. Acetone is removed under vacuum at a bath temperature of 30-35°C (a small amount of material may separate at this point, but does not pose a problem). The concentrate is transferred to a suitable hydrogenation container. 10 g of palladium-on-carbon catalyst are added and hydrogenated at 3.40 atm. for 2-3 hours. 5. The mixture is filtered through a "Dicalite" layer, and the hydrogenation vessel and filter cake are washed with additional

2 x 50 ml water.

6. The filtrate plus the washing liquids are concentrated to about a third of the volume (150-165 ml) under vacuum at 40-45°C. 7. The pH value is at this point in the range 6.0-7.0. The mixture is poured into a "CG-50" (NH4<+>) column (6 x 110 cm). 8. The column is washed with 1000 ml of deionized water. It is eluted with 0.5 N ammonium hydroxide using an automatic polarimeter to monitor the progress of the elution. The elution order is-as follows: Rest kana A BB-K6 } BB-K29 amikacin. No BB-Kll was detected in any of the experiments. 9. The amikacin fractions are combined and concentrated to 25-30% dry matter. It is diluted with 1 volume of methanol and inoculated with amikacin crystals. 10. Over the course of 2 hours, 2 volumes of IPA are slowly added with good stirring, and it crystallizes at 23°C for 6-8 hours. 11. The solid is filtered and washed with 35 ml of 1:1:2 water/methanol/IPA and finally with 35 ml of IPA. 12. It is dried in a vacuum oven at 40°C for 16-24 hours.

Yield: 19.91-22.84 g (34-39%). IR, PMR and CMR spectral data as well as specific rotation were in complete <1> agreement with the desired structure.

Thin layer chromatograph system

CHCl3/methanol/NH4OH/water (1:4:2:1) 5 x 20 cm silica gel plates from Quantum Industries, 1 zone amikacin with Rf~ 0.4 (ninhydrin detection).

Example 7

Preparation of amikacin by acylation of poly-(trimethylsilyl)-6 1 -N-Cbz-kana A in tetrahydrofuran with mixed acid anhydride of pivalic acid and BHBA.

A. Preparation of mixed anhydride.

5.066 g (20.0 mmol) of BHBA, 4.068 g (20.0 mmol) of BSA and 2.116 g (22.0 mmol) of ethylamine were dissolved in 200 ml of sieve-dried tetrahydrofuran. The solution was refluxed for 2 1/4 hours and then cooled to -10°C. 2.412 g (20.0 mmol) of pivaloyl chloride was added over 2-3 minutes with stirring, and stirring was continued for 2 hours at -10°C. The temperature was then allowed to rise to 23°C.

B. Acylation of poly-(trimethylsilyl)-6'-N-Cbz kana A.

5.454 g (4.97 mmol, calculated as 6'-N-Cbz kana A (silyl) Q) poly-(trimethylsilyl)-6<1->N-Cbz kana A prepared as in example 1 was dissolved in 50 ml of dry (molecular sieve) tetrahydrofuran at 23°C. Half of the solution prepared in step A above

mixed anhydride (10.0 mmol) was added over 20 min with stirring, and stirring was continued for 7 days.

Then 100 mL of water was added to the reaction mixture and the pH (5.4) was adjusted to 2.0 with 3 M H 2 SO 4 . Stirring was continued for 1 hour and the solution was extracted with ethyl acetate. Polyacylated material began to crystallize out, so the reaction mixture was filtered. After drying over P2°5, the recovered solid weighed 0.702 g. The extraction of the reaction mixture was continued in a total of 4 x 75 ml of ethyl acetate, after which excess ethyl acetate was driven away from the aqueous layer. An aliquot of the aqueous solution was subjected to analysis by HPLC. The resulting curve indicated a 26.4% yield of di-Cbz amikacin.

The aqueous layer was then hydrogenated in a Parr apparatus at 3.40 atm. H 2 ~ pressure for 2 hours using 0.5 g of 10% Pd-on-carbon catalyst. The material was filtered, and it was demonstrated against E. coli that the combined filtrate and washing liquids contained a 31.2% yield of amikacin. The amikacin/BB-K2 9 ratio was approx. 9-10:1. Traces of polyacyl and unreacted kana A were present.

Example 8

Effect of water on the production of amikacin by acylation of poly-(trimethylsilyl)cana A in acetone solution at 23°C.

A. Anhydrous solvent.

2.40 g (2.0 mmol, calculated as kana A (silyl)^q) poly-(trimethylsilyl) kana A prepared as in example 3 was dissolved in 20 ml of acetone which had been dried with a molecular sieve. The solution was stirred at 23°C, and a solution of 0.701 g (2.0 mmol) of SAE in 10 mL of sieve-dried acetone was added over 10 seconds. Stirring was continued at 23°C for 22 hours. 50 ml of water was added,

and pH (7.5) was adjusted to 2.5. The acetone was evaporated under vacuum at 40°C, and the aqueous solution was then reduced at a H 2 ~ pressure of 3.47 atm. at 23°C for 2 hours using 1.0 g of 10% Pd-on-carbon as catalyst. Microbiological analysis showed a 31.24% yield of amikacin.

B. Solvent added to water.

Step A above was repeated, except that 1.0 mL (56 mmol) of water was added to the solution of poly-(trimethylsilyl)cana A,

and it was stirred for 15 min before acylation with SAE. Microbiological analysis showed a yield of 33.80% amikacin.

Example 9

Preparation of amikacin by acylation of poly-(trimethylsilyl)-6 1-N-Cnz kana A in acetone with the mixed anhydride of BHBA and isobutylcarbonic acid.

A. Preparation of mixed anhydride.

1.267 g (5.0 mmol) BHBA and 1.313 g (10.0 mmol) N-trimethylsilylacetamide (MSA) in 20 ml sieve-dried acetone were stirred at 23°C, and 0.70 ml (5.0 mmol) triethylamine (TEA ) was added. The mixture was refluxed under a 2-atmosphere for 2 hours. The mixture was cooled to -20°C and 0.751 g (0.713 mL, 5.50 mmol) of isobutyl chloroformate was added. Triethylamine hydrochloride immediately began to be excreted. The mixture was stirred for 1 hour at -20°C.

B. Acylation.

6.215 g (4.9 mmol, calculated as the (silyl)^-compound) poly-(trimethylsilyl)-6,<0->N-Cbz-cana A prepared as in example 1 was dissolved in 20 ml sieve-dried acetone while stirring at 23°C. The solution was cooled to -20°C and the cold solution of mixed anhydride from step A was slowly added over 30 minutes. The reaction mixture was stirred for an additional 1 hour at -20°C and then for 17 hours at 23°C. The reaction mixture was then poured into 150 mL water at 23°C with stirring, pH (7.75) adjusted to 2.5 with 3N HCl, and stirring continued for 15 minutes. Acetone was then evaporated under vacuum at 40°C. An aliquot of the resulting aqueous solution was subjected to analysis by HPLC. The resulting curve showed a yield of 34.33% di-Cbz-amikacin.

The bulk of the aqueous solution was reduced at 3.40 atm. H 2 pressure at 23°C for 3 1/4 hours using 2.0 g of Pd/C catalyst. The catalyst was removed by filtration, and by microbiological analysis against E. coli it was demonstrated that the combined filtrate and washing liquids contained a yield of 35.0% amikacin.

Example 10

Preparation of amikacin by acylation of poly-(trimethylsilyl)-6'-N-Cbz-cana A in 3-pentanone.

30 g (23.65 mmol, calculated as 6<1->N-Cbz-cana A (silyl)g) poly-(trimethylsilyl)-6<1->N-Cbz-cana prepared as in example 1

and dissolved in 100 mL of sieve-dried 3-pentanone was stirred at 23°C, and NAE (26.02 mmol, 10% excess) was added over 40 minutes. Stirring was continued for 113 hours at 23°C, and the mixture was then added to 250 ml of water with vigorous stirring. The pH (7.3) was adjusted to 2.5 with 3N HCl, after which the mixture was stirred for an additional 30 minutes and 3-pentanone driven off under vacuum at 40°C. The aqueous solution was extracted with 4 x 100 ml of ethyl acetate. An aliquot of the aqueous solution was then subjected to analysis by HPLC. The resulting curve showed a yield of 46.12% di-Cbz-amikacin.

The bulk of the aqueous reaction mixture was reduced at 3.47 atm. t^ pressure at 23°C for 2 hours using 3.0 g of 10% Pd/C catalyst. Microbiological analysis of an aliquot of combined filtrate and washings showed a yield of 40.24% amikacin. The bulk of the reduced aqueous reaction mixture was then concentrated under vacuum at 40°C to approx. 100 ml and fractionated in a "CG-50" (NH^<+>) ion exchange column (approx. 10 cm x approx. 120 cm, containing approx. 10 liters of resin). The aqueous solution was poured into the column which was washed with 5 liters of water, and the material was eluted with 0.5N NH^OH, followed by 3N NH^OH

for elution of polyacylated products. Polarimetry of the fractions showed a yield of 42.7% amikacin, a yield of 12.0% unreacted kanamycin A, a yield of 12.4% polyacylated material and a yield of 23.2% BB-K29.

Example 11

Preparation of amikacin by acylation of poly-(trimethylsilyl)-6 1 -N-Cbz-kana A in anhydrous cyclohexanone for varying periods of time.

A. 2.537 g (2.0 mmol, calculated as 6<1->N-Cbz-cana A (silyl)g) poly-(trimethylsilyl)-6'-N-Cbz-cana A prepared as in example 1

and dissolved in 300 mL of dry cyclohexanone was acylated for 20 h at 23°C with a NAE solution in dry cyclohexanone (10.8 mL of a 0.1944 mmol/mL solution, 2.10 mmol). The reaction mixture was then added to 150 mL of water with stirring, and the pH (5.6) was adjusted to 2.5 with 3N HCl. The cyclohexanone was evaporated under

vacuum at 40°C, and an aliquot of the remaining aqueous phase was removed for analysis by HPLC.

The majority of the aqueous phase was reduced below 3.40 atm. H 2 ~ pressure for 3 hours at 23°C using 1.0 g of 10% Pd/C catalyst. The catalyst was removed by filtration, and the combined filtrate and washings were analyzed microbiologically for amikacin.

B. Reaction A was repeated, except that the acylation was continued for 115 hours instead of 20 hours.

Example 12

Preparation of amikacin by acylation of 'poly-(trimethylsilyl)-6'-N-Cbz-cana A in anhydrous tetrahydrofuran for varying periods of time.

A. Example 11 A was repeated, except that dry tetrahydrofuran was used as solvent instead of dry cyclohexanone.

B. Example 11 B was repeated, except that dry tetrahydrofuran was used as solvent instead of dry cyclohexanone.

Example 13

Preparation of amikacin by acylation of poly-(trimethylsilyl)-6'-N-Cbz-kana A in anhydrous dioxane for varying periods of time.

A. Example 11 A was repeated, except that the acylation was continued for 44 hours using dry dioxane as the solvent.

B. Example 11 B was repeated, except that the acylation 33

was continued for 18% hour using dry dioxane as solvent.

Example 14

Preparation of amikacin by acylation of poly-(trimethylsilyl)-6'-N-Cbz-cana A in anhydrous diethyl ketone at 75°C.

To a stirred solution of 2.537 g (2.0 mmol, calculated as 6'-N-Cbz-cana A (silyl)n) poly-(trimethylsilyl)-6'-N- -Cbz-cana A formed as in example 1, in 32 ml of sieve-dried diethyl ketone at 75 C, a solution of NAE (10.8 ml with 0.1944 mmol/ml diethyl ketone, 2.10 mmol) was added over 15 minutes. Stirring was continued at 75°C for a further 3 hours, after which the mixture was poured into 150 ml of water. The pH was adjusted to 2.8 with 3N HCl, and the diethyl ketone was evaporated under vacuum at 40°C. HPLC analysis of an aliquot of the aqueous phase showed a yield of 39.18% di-Cbz-amikacin.

The majority of the aqueous phase was reduced below 3.39 atm. H 2 ~ pressure for 3 1/4 hours at 23°C using 1.0 g of Pd/c catalyst. The catalyst was removed by filtration, and the combined filtrate and washings were analyzed microbiologically for amikacin. Turbidimetric analysis showed a yield of 27.84%, and plate analysis showed a 28.6% yield.

Example 15

Preparation of amikacin by acylation of poly-(trimethylsilyl) cana A with NAE at 0-5°C after back-hydrolysis with water.

A. Silylation of kanamycin A using HMDS with

TMCS as a catalyst.

10 g of kanamycin A with a purity of 97.6% (20.14 mmol) in 100 ml of sieve-dried acetonitrile was brought to boiling with reflux under a nitrogen atmosphere. A mixture of 22.76 g (141 mmol, 7 mol per mol kanamycin A) of HMDS and 1 ml (0.856 g, 7.88 mmol) of TMCS was added to the cooled reaction mixture over 10 minutes. Refrigeration was continued for 4 3/4 hours after which the mixture was cooled and concentrated under vacuum to a yellow viscous liquid and dried under high vacuum for 2 hours. The yield of product was 23.8 g (97.9%, calculated as kanamycin A (silyl)1Q).

B. Acylation

23.8 g (20.14 mmol) of poly-(trimethylsilyl)-kanamycin A prepared during step A above was dissolved in 250 ml sieve-dried acetone at 23°C and then cooled to 0-5°C. 3.63 mL of water (201.4 mmol, 10 mol per mol of polysilylated kanamycin A) was added with stirring, and the mixture was placed under moderate vacuum for 30 minutes. NAE (19.133 mmol, 0.95 mol per mol polysilylated kanamycin A) in 108.3 mL of acetone was then added in less than 1 minute. The mixture was stirred at 0-5°C for 1 hour,

diluted with water, the pH was adjusted to 2.5 and the acetone then removed under vacuum. The aqueous solution was then reduced at 3.40 atm. H 2 -pressure at 23°C for 2 hours using 2.0 g of 10% Pd/C as catalyst. The reduced reaction mixture was filtered through "Dicalite", concentrated to ca. 100 ml under vacuum at 40°C and then poured into a %CG-50" (NH4<+>)

column (6 liters of resin, 5 x 100 cm). It was washed with water and then eluted with 0.6N-1.0N-3N NH^OH. 60.25% amikacin, 4.37% BB-K6, 4.35% BB-K29, 26.47% kanamycin A and 2.18% polyacyl compounds were obtained.

Example 16

Preparation of amikacin by acylation of poly-(trimethylsilyl)-6'-N-Cbz-cana A with SAE at 0-5°C after back methanolysis.

A. Silylation of 6'-N-Cbz-kanamycin A.

20.0 g (3 2.4 mmol) of 6<1->N-Cbz-kanamycin A in 200 ml of sieve-dried acetonitrile was brought to boiling with reflux under a nitrogen atmosphere. 47.3 mL of HMDS (226.8 mmol, 7 mol per mol 6'-N-Cbz-can A) was added over 10 minutes, and refluxing was continued for 20 hours. The mixture was then cooled, concentrated under vacuum and dried under high vacuum for 2 hours to give 39.1 g of white, amorphous solid (95.4% yield, calculated as 6'-N-Cbz-kana A (silyl) n).

B. Acylation.

39.1 g (32.4 mmol) of poly-(trimethylsilyl)-6<1->N-Cbz-cana A prepared during step A above was dissolved in 400 ml of dry acetone with stirring at 23°C. 6.6 ml of methanol (162 mmol, 5 mol per mol polysilylated 6<1->N-Cbz-cana A) was added, and the mixture was re-

stirred at 23°C for 1 hour under a strong stream of nitrogen. The mixture was cooled to 0-5°C, and a solution of 11.35 g (32.4 mmol) SAE

in 120 ml of dry acetone which had been cooled beforehand was added. The mixture was stirred for a further 3 hours at 0-5°C and then placed in a 4°C cold room for 1 week. 300 ml of water was added and the pH adjusted to 2.0. The mixture was stirred for 1 hour and the acetone was then evaporated under vacuum. The resulting aqueous solution was reduced at 3.67 atm. H₂ pressure for 17 hours at 23°C using 3.0 g of 10% Pd/C as catalyst. It was then filtered through "Dicalite", concentrated under vacuum to 75-100 ml and loaded onto a "CG-50" NH4<+>) column and eluted with water and 0.6N NH4OH. 52.52% amikacin, 14.5% BB-K29, 19.6% kanamycin A and 1.71% polyacyl compounds were obtained.

Example 17

Preparation of amikacin by acylation of poly-(trimethylsilyl) cana A with SAE at 0-5°C after back-hydrolysis with water. <

A. Silylation of kanamycin A with TMCS in acetonitrile below

use of tetramethylguanidine as acid acceptor.

4.88 g (10.07 mmol) of kanamycin A was suspended in 100 ml of sieve-dried acetonitrile with stirring at 23°C. To the stirred suspension was added 16.234 g (140.98 mmol, 14 mol per mol kanamycin A) tetramethylguanidine (TMG). The mixture was heated

to reflux, and 15.32 g (140.98 mmol, 14 mol per mol kanamycin A) of TMCS was added over 15 minutes. A white precipitate of TMG*HCl was formed after approx. half of the TMCS had been added. The mixture was cooled to room temperature, concentrated to a sticky residue and dried under high vacuum for 2 hours. The solid was thoroughly mixed with 100 ml of dry THF, and the insoluble TMG-HCl was decanted and washed with 5 x 20 ml portions of THF. Combined filtrate and washings were concentrated under vacuum at 40°C to a sticky residue and dried under high vacuum for 2 hours. 10.64 g of a light cream-colored, sticky residue was obtained (87.6% yield, calculated as kanamycin A (silyl)^).

B. Acylation

10.64 g (10.07 mmol) of poly-(trimethylsilyl)-kanamycin A prepared during step A above was dissolved in 110 ml sieve-dried acetone with stirring at 23°C, and the solution was cooled to 0-5°C. 1.81 mL (100.7 mmol, 10 mol per mol polysilylated cana A) of water was added and the solution was stirred for 30 minutes

under moderate vacuum. 3.70 g (10.57 mmol, 5% excess) of SAE in 40 mL of pre-cooled acetone was added in less than 1 minute, and the mixture was stirred for 1 hour. The mixture was worked up by the general method in example 16B, whereby approx. 50% amikacin, approx. 10% BB-K29, 5-8% BB-K6, approx. 20% kanamycin A and 5-8% polyacyl compounds.

Example 18

Preparation of poly-(trimethylsilyl)-kanamycin A in pyridine using HMDS.

10.0 g (20.64 mmol) of kanamycin A was suspended in 100 ml of sieve-dried, freshly distilled pyridine at 23°C. Nitrogen flow was started and the suspension was brought to boiling with reflux. 17.33 g (107.32 mmol, 5.2 mol per mol kanamycin A) of HMDS was added over 10 minutes, and the mixture was refluxed for 19 hours. It was then cooled to room temperature, concentrated under vacuum to a light yellow-golden viscous liquid and dried under high vacuum to a white, amorphous solid. 22.1 g were obtained (92.6% yield, calculated as kanamycin A (silyl)1Q).

Example 19

Preparation of poly-(triethylsilyl)-kanamycin A using triethylchlorosilane with triethylamine as acid acceptor.

5.0 g of kanamycin A of 97.6% purity (10.07 mmol) was suspended in 100 ml of sieve-dried acetonitrile at 23°C. 33.8 mL, 24.5 g (241.7 mmol) of triethylamine (TEA) was added and the suspension was brought to reflux. A solution of 23.7 mL, 21.3 g (14 0.98 mmol) of trichloroethylsilane in 25 mL of dry acetonitrile was added over 20 minutes. The reflux was continued for another 7 hours and the mixture was cooled to room temperature after which long fine needles of TEA-HCl separated. The mixture was allowed to stand at room temperature for 16 hours, concentrated under vacuum at 40°C to a sticky solid and dried for 2 hours under high vacuum to a deep orange sticky solid. The solid was mixed thoroughly with 100 ml of dry THF at 23°C, and the insoluble TEA-HCl was filtered off, washed with 5 x 20 ml of THF and dried to give 16.0 g of TEA-HCl. Combined filtrate and washings were concentrated under vacuum to a solid and dried under high vacuum for 2 hours. 19.3 g of poly-(triethylsilyl)-kanamycin A were obtained as a

deep orange, viscous liquid.

Example 20

Preparation of poly-(trimethylsilyl)-kanamycin A using bis-trimethylsilylurea.

10.0 g of kanamycin A of purity 99.7% (20.58 mmol) was suspended in 200 ml of sieve-dried acetonitrile with stirring at 23°C.

To the suspension was added 29.45 g (144.01 mmol, 7 mol per mol kanamycin) of bis-trimethylsilylurea (BSU), and the mixture was brought to reflux under a nitrogen atmosphere. The reflux was continued for 17 hours, after which the reaction mixture was cooled to room temperature. A small amount of insoluble material present was removed by filtration, washed with 3 x 10 mL portions of acetonitrile and dried (1.1381 g). IR analysis showed this to be BSU plus a small amount of unreacted kanamycin A. Combined filtrate and washings were cooled to 4°C for 16 hours. Additional solid separated, removed as above (7.8 g) and was shown by IR analysis to be BSU plus . urea. The pale yellow filtrate and washings were concentrated under vacuum at 40°C and dried under high vacuum, whereby 27.0 g of a white solid was produced which was partly sticky and partly fine, needle-like crystals. The solid was treated with 150 ml of heptane at 23°C. The insoluble portion was removed by filtration, washed with 2 x 50 mL portions of heptane and dried to yield 6.0 g of white needles (shown by IR analysis to be BSU plus urea). Combined filtrate and washings were concentrated under vacuum at 40°C and dried under high vacuum for 2 hours, yielding 20.4 g of white needles whose IR spectrum was typical of polysilylated kanamycin A. Calculations showed that the product contained, on average, 7.22 trimethylsilyl groups.

Example 21

Preparation of amikacin by acylation of per-(trimethylsilyl)-kanamycin A after partial desilylation with 1,3-butanediol.

A. Preparation of per-(trimethylsilyl)-kanamycin A.

10.0 g (20.639 mmol) of kanamycin A was suspended in 100 ml of sieve-dried acetonitrile with stirring at 23°C. The suspension was brought to reflux under a stream of nitrogen, and 23.322 g of HMDS (144.5 mmol, 7 mol per mol kanamycin A) was

added within 10 minutes. Refrigeration was continued for 16 hours after which the mixture was cooled to room temperature, concentrated under vacuum and dried for 2 hours under high vacuum. 24.3 g of a white sticky residue were obtained (92.1% yield, calculated as kanamycin A (silyl)•]_-[_)•

B. Acylation.

24.3 g of per-(trimethylsilyl)-kanamycin A prepared in steps

A above was dissolved in 240 ml sieve-dried acetone with stirring at 23°C. To this solution was added 9.25 ml (103.2 mmol, 5 mol per mol per-(trimethylsilyl)-kanamycin A) 1,3-butanediol. The mixture was stirred at 23°C for 2 hours under a stream of nitrogen and then cooled to 0-5°C. 7.23 g (20.64 mmol) of SAE in 70 ml of pre-cooled acetone was added over ca. 1 minute. The mixture was stirred at 0-5°C for 3 hours and was then placed in a 4°C cold room for approx. 16 hours. 200 mL of water was added, the pH was adjusted to 2.5, and the clear yellow solution was stirred at 23°C

for 30 minutes. The acetone was evaporated under vacuum and the aqueous solution was reduced at 3.74 atm. H 2 ~ pressure at 23°C for 2 hours using 3.0 g of 10% Pd-on-carbon as catalyst. The reduced solution was filtered through "Dicalite" and chromatographed as in Example 16 B, whereby 47.50% amikacin, 5.87% BB-K29, 7.32% BB-K6, 24.26% kanamycin A and 7.41% polyacyl compounds.

Example 22

Preparation of amikacin by acylation of poly-(trimethylsilyl)-kanamycin A prepared in THF using SAE with sulfamic acid catalyst.

To a mixture which was boiled with reflux and which consisted of 5.0 g (10.32 mmol) of kanamycin A in 50 ml of sieve-dried tetrahydrofuran (THF) was added 100 mg of sulfamic acid and 12.32 g (76.33 mmol) of HMDS . The mixture was refluxed for 18 hours, with complete dissolution occurring after 16 hours. The solution was cooled to 23°C, treated with 0.1 ml of water and held at 23°C for 30 minutes. A solution of 3.61 g (10.3 mmol)

SAE in 36 mL of THF was added over 30 minutes. After stirring for 3 hours, the mixture was diluted with 100 ml of water, and pH

was set at 2.2 with 10 percent H-jSO^. The mixture was stirred for 30 minutes at 23°C and then concentrated under vacuum to remove THF. The resulting aqueous solution was reduced at 3.40 atm. H2~ pressure for 2 hours at 23°C using

10% Pd-on-carbon as catalyst. The reduced solution was filtered through "Dicalite" and the solid was washed with water. Microbiological analysis against E. coli showed that combined filtrate and washing liquids (150 ml) contained 1225 mcg/ml (31.5% activity yield) of amikacin.

Example 23

Preparation of amikacin by acylation of poly-(trimethylsilyl)-kanamycin A with the N-hydroxysuccinimide ester of dicarbo-benzyloxy-AHBA.

A. Preparation of dicarbobenzyloxy-L-(-)-α-amino-α-hydroxybutyric acid-N-hydroxysuccinimide ester. 8 g (20.65 mmol) of dicarbobenzyloxy-L-(-)-α-amino-α-hydroxybutyric acid and 2.37 g (20.65 mmol) of N-hydroxysuccinimide were dissolved in 50 ml of dry acetone at 23°C . 4.25 g (20.65 mmol) of dicyclohexylcarbodiimide dissolved in 20 ml of dry acetone was added, and the whole was stirred at 23°C for 2 hours. Dicyclohexylurea was filtered off, the filter cake was washed with 10 ml of dry acetone, and the filtrate and washings were combined.

B. Acylation.

Poly-(trimethylsilyl)-kanamycin A prepared in accordance with the general procedure in Example 21 from 10.0

g (20.639 mmol) of kanamycin A was dissolved in 100 ml of dry acetone. The solution was cooled to 0-5°C, 3.7 ml of deionized water was added, and the solution was stirred at 0-5°C for 30 minutes under moderate vacuum.

The solution prepared in step A was added to this solution

of the di-Cbz-blocked acylating agent added, and the mixture was stirred at 0-5°C for 30 minutes. The mixture was diluted with water, the pH was adjusted to 2.2, and the acetone was removed under vacuum. The aqueous solution was reduced by the general procedure of Example 22 and then filtered through "Dicalite". Chromatography showed 40-45% amikacin, approx. 10% BB-K29, trace amounts

of BB-K6, approx. 30% kanamycin A and a small amount of polyacyl compounds.

Example 24

Preparation of poly-(trimethylsilyl)-kanamycin A using HMDS with imidazole as catalyst. 11 g (22.7 mmol) of kanamycin A and 100 mg of imidazole were heated to reflux in 100 ml of sieve-dried acetonitrile under a stream of nitrogen. 18.48 g (114.5 mmol, 5 mol per mol kanamycin A) of HMDS was added over 30 minutes and the mixture was refluxed for 20 hours. Complete resolution occurred in approx. 2\ hours. The solution was cooled to 23°C, and the solvent was removed under vacuum, leaving 21.6 g of poly-(trimethylsilyl)-kanamycin A as a foamy residue (93.1% yield calculated as kanamycin (silyl) •

Example 25

Preparation of 1-N-[l-(-)-γ-amino-α-hydroxybutyryl]-kanamycin B (BB-K26) by acylation of poly-(trimethylsilyl)-kanamycin B with SAE.

A. Preparation of poly-(trimethylsilyl)-kanamycin B below

application of HMDS with TMCS catalyst.

25 g (51.7 mmol) of kanamycin B in 250 ml of sieve-dried acetonitrile was heated to reflux under a stream of nitrogen. 62.3 g (385.81 mmol, 7.5 mol per mol kanamycin B) of HMDS was added over 30 minutes, followed by 1 mL of TMCS as a catalyst. The mixture was refluxed for 21 hours with complete dissolution after 1 hour. The solvent was removed under vacuum at 60°C, and the oily residue was kept at 60°C under high vacuum for 3 hours. 53.0 g of poly-(trimethylsilyl)-kanamycin B were obtained (85.2% yield calculated as kanamycin B (silyl)^g).

B. Acylation

53.0 g of the poly-(trimethylsilyl)-kanamycin B prepared in step A above was dissolved in 500 ml of dry acetone at 0-5°C, 20.9 ml of methanol was added, and the mixture was stirred under vacuum for 30 minutes at 0-5°C. A solution of 18.1 g (51.67 mmol) SAE

in 200 ml of pre-cooled acetone was added in less than 1 minute and the mixture was stirred for 30 minutes at 0-5°C. The mixture was worked up in accordance with the general procedure of Example 22 and was then loaded into a column of "CG-50" (NH4<+>) (8 x 120 cm). It was eluted with an NH^OH gradient from 0.6N to 3N. 38% BB-K26 was obtained, 5% of the corresponding 6'-N-acylated kanamycin B (BB-K22), 10% of the corresponding 3-N-acylated kanamycin B (BB-K46),

14.63% kanamycin B as well as a small amount of polyacylated kanamycin B.

Example 26

Preparation of poly-(trimethylsilyl)-kanamycin A using HMDS with kanamycin A sulfate as catalyst.

19.5 g (40.24 6 mmol) kanamycin A and 0.5 g (0.8 58 mmol) kanamycin A sulfate (total = 20.0 g, 41.0 mmol) in 200 ml sieve-dried acetonitrile were brought for boiling with cooling. 60.3 mL (287.7 mmol, 7 mol per mol kanamycin A) of HMDS was slowly added and the mixture was refluxed for 28 hours. It was then evaporated to dryness in a rotary evaporator and dried under steam injector vacuum. 47.5 g of poly-(trimethylsilyl)-kanamycin A were obtained as a pale yellow oil (95.82% yield calculated as kanamycin A (silyl)^g).

Example 27

Preparation of amikacin by acylation of poly-(trimethylsilyl)-kanamycin A with N-trifluoroacetyl blocked AHBA-N-hydroxysuccinimide ester.

A. Preparation of N-trifluoroacetyl-AHBA and conversion to

its N-hydroxysuccinimide ester.

To a suspension of 5.0 g (42 mmol) of AHBA in 100 ml of THF, 40 g (191 mmol) of trifluoroacetic anhydride was added with stirring during 10 minutes. The solution was stirred for 18 hours at 23°C and then concentrated to dryness under vacuum at 50°C. The residue was dissolved in 100 ml of aqueous methanol (1:1) and stirred for 1 hour. It was then concentrated to dryness under vacuum and redissolved in 50 mL of I 2 O. The aqueous solution was extracted with 3 x 50 ml portions of MIBK, and the extract, after drying over Na 2 SO 4 , was concentrated to an oil. Traces of solvent were removed by adding and distilling off 4 ml of water. On storage, the oil changed to a waxy, crystalline solid (2.5 g, 28% yield).

2.4 g (11.3 mmol) of N-trifluoroacetyl-AHBA was dissolved in 50 ml of dry acetone, and 1.30 g (11.31 mmol) of N-hydroxysuccinimide was added to the solution. A solution of 2.33 g of dicyclohexylcarbodiimide in 20 ml of dry acetone was slowly added. The reaction mixture was stirred for 2 hours at 23°C, and the precipitated dicyclohexylurea was removed by filtration and washed with a little acetone. Combined filtrate and washings (a solution of the N-hydroxysuccinimide ester of N-trifluoroacetyl-AHBA) were used in the next step without isolation.

B. Acylation

To a solution of poly-(trimethylsilyl)-kanamycin A prepared as in example 26 (11.31 mmol) in 54 ml of acetone was added

2.0 mL (113.4 mmol) of water, and the mixture was stirred under vacuum at 0-5°C for 30 minutes. The N-hydroxysuccinimide ester of N-trifluoroacetyl-AHBA prepared in step A above (11.31 mmol) was added to the mixture which was then kept at 5°C for 1 hour. The pH value was then set to approx. 2.0 with 20 percent H2SO4, the mixture stirred for 30 minutes and the pH then raised to approx. 6.0 with NH^OH. The mixture was then evaporated to dryness in a rotary evaporator, whereby 14.4 g of a sticky, off-white, solid was obtained. The solid was dissolved in 100 ml of water, the pH was raised from 5.5 to 11.0 with 10N NH 2 OH, and the solution was heated in an oil bath at 70°C for 1 hour. The pH (9.5) was lowered to 7.0 with HCl after which the solution was finely filtered to remove a small amount of insoluble constituents, and the filtrate was washed with water. Combined filtrate and washings (188 mL) were fed to a "CG-50" (NH4<+>) column (8 x 90 cm), washed with 2 liters of water <p>g eluted with an NH4OH gradient (0.6N -1,ON-concentrated). 28.9% amikacin, 5.0% BB-K6, 5.7% BB-K29, 43.8% kanamycin A, 3.25% polyacyl compounds and 14.3% of an unknown material that was in the first fraction from the column.

Example 28

Preparation of amikacin by acylation of poly-(trimethylsilyl)-kanamycin A with tert-butyloxycarbonyl-blocked AHBA-N-hydroxysuccinimide ester. A. Preparation of tert-BOC-AHBA and conversion to its N-hydroxysuccinimide ester

A solution of 5.0 g (42 mmol) AHBA in 100 ml water and 20 ml acetone was adjusted to pH 10 with 10N NaOH. In the course of 3-4 minutes, 11.6 g (53 mmol) of di-tert-butyl dicarbonate were added, and the solution was stirred for 35 minutes, the pH value being maintained at 10 by periodic addition of 10N NaOH. The acetone was removed under vacuum and the aqueous phase was washed with 40 ml of ethyl acetate. The pH of the aqueous solution was lowered to 2.0 with 3N HCl, and the solution was then extracted with 3 x 30 mL of MIBK. The combined MIBK extracts were dried over Na 2 SO 4 and concentrated to a clear oily residue (8.2 g, 89%).

4.25 g (19.4 mmol) of tert-BOC-AHBA was dissolved in 50 ml of acetone,

and 2.23 g (19.4 mmol) of N-hydroxysuccinimide was added. One

solution of 4.00 g (19.4 mmol) of dicyclohexylcarbodiimide in 20 ml of acetone was slowly added, and the mixture was stirred for 2 hours at 23°C. The precipitated dicyclohexylurea was removed by filtration and washed with a small amount of acetone. Combined filtrate and washings (a solution of the N-hydroxysuccinimide ester of tert-BOC-AHBA) were used in the next step without isolation.

B. Acylation

To a solution of poly-(trimethylsilyl)-kanamycin A prepared as in Example 26 (41.28 mmol) in 94 ml of acetone was added 3.5 ml (194 mmol) of water, and the mixture was stirred under vacuum at 0-5 °C for 30 minutes. The N-hydroxysuccinimide ester of tert-BOC-AHBA prepared in step A above (19.4 mmol) was added and the mixture was allowed to stand at 5°C for 1 hour. 200 ml of water was added and the pH lowered from 7.0 to 2.0 with 20 percent H 2 SO 4 . After 30 minutes of stirring, the pH was raised to approx. 6.0 with NH 3 OH, and the mixture was evaporated to dryness under vacuum to give 3 6.3 g of a golden oil. The oil was dissolved in 200 ml of trifluoroacetic acid, left for 15 minutes and evaporated to dryness in a rotary evaporator. The oil was washed with water, and the water was rapidly evaporated. Concentrated NH^OH was added to pH 6.0 and rapidly evaporated. The resulting solid was dissolved in water and filtered, and the filter was washed with water. Combined filtrate and washings (259 mL) were loaded into a "CG-50" (NH4<+>) column (8 x 92 cm), washed with 4 liters of water and eluted with an NH4OH gradient (0.6N-1, ON-concentrated). 40.32% amikacin, 4.58% BB-K6, 8.32% BB-K29, 30.50% kanamycin A and 7.43% polyacyl compounds were obtained.

Example 29

The general procedure from Example 1 was repeated, except that the 6<1->N-carbobenzyloxykanamycin A used there was replaced by an equimolar amount by weight of 6'-N-carbobenzyloxykanamycin B, and a 1-N-[l -(-)-γ-amino-α-hydroxybutyryl]-kanamycin B.

Example 3 0

The general procedure from example 1 was repeated, except that instead of the L-(-)-γ-benzyl-oxycarbonylamino-α-hydroxybutyric acid-N-hydroxy-5-norbornene-2,3-dicarboximide ester used there, it was replaced with L-(-)-3-benzyl-oxycarbonylamino-α-hydroxypropionic acid-N-hydroxy-5-norbornene-2,3-dicarboximide ester and L-(-)-6-benzyloxycarbonylamino-α-hydroxyvaleric acid-N-hydroxy-5- norbornene-2,3-dicarboximide ester, and it was thereby prepared respectively

1-N-[L-(-)-8-amino-α-hydroxypropionyl]-kanamycin A and 1-N-[L-(-)-S-amino-α-hydroxyvaleryl]-kanamycin A.

Example 31

It. general procedure from example 25 was repeated, except that instead of the L-(-)-γ-benzyl-oxycarbonylamino-α-hydroxybutyric acid-N-hydroxyester used there, L-(-)-ø-benzyloxycarbonylamino-α- hydroxypropionic acid-N-hydroxysuccinimide ester and L-( - )-6-benzyloxy.carbonylamino-α-hydroxyvaleric acid-N-hydroxysuccinimide ester,

and it was thereby produced respectively

1-N-[L-(-)-ø-amino-α-hydroxypropionyl]-kanamycin B and 1-N-[L-(-)-6-amino-α-hydroxyvaleryl]-kanamycin B.

Example 32

Preparation of BB-K8 by acylation of poly-(trimethylsilyl)-3, 6'-di-N-carbobenzyloxykanamycin A in anhydrous diethyl ketone. A. 3, 6'-di-N-carbobenzyloxykanamycin A.

A suspension of 7.26 g (15 mmol) kanamycin A (free base) and 18.6 g (75 mmol) nickel acetate tetrahydrate in 300 ml dimethylsulfoxide (DMSO) was kept heated at 100°C for approx. 30 minutes with stirring, until a clear, green solution was obtained. After cooling, a solution of 11.8 g (37.6 mmol) of N-carbobenzyloxy-5-norbornene-2,3-dicarboximide in 50 ml of DMSO was added

to the solution. The mixture was stirred at room temperature overnight, treated with 100 ml conc. ammonia water and 1 liter of water, stirred at room temperature for 1 hour and transferred to the top of a "Diaion HP-10" column (300 ml). The column was subjected to stepwise elution, first with 7 n NH^OH, then with methanol-water (1:1)

and finally with methanol-water (10:1) collecting 20 ml fractions and monitoring by thin-layer chromatography on "Merck Silica Gel 60 F-254" plates using chloroform-ethanol-28% ammonium hydroxide (1:2: 1). A portion of the desired product, which crystallized as fine needles from fractions which

contained the product in high concentrations, was filtered off, whereby an analytical sample was obtained. The filtrate and the other fractions containing the desired product (RF 0.42) were combined, and the solution was evaporated under vacuum. The residue was thoroughly mixed with diethyl ether, with which a total of 9.7 g (86%) of the title product was obtained. Temp. 300°C. IR(KBr): uc=o 1690 cm"<1>. NMR (DMS0-dg) + DC1, pD ca. 3): δ 4.76-5.26 ( 611,111.1^ ' ,1^" and CO -OCH2-C6H5 x 2), 7.26 (10H, s , CO-OCH2-CgH5 x 2).

Analysis: Calculated for <C>34<H>48N4015-<H>2<0:> C: 52'98' H: 6'54' N: 7 >21

Found: C: 53.20, H: 6.42, N: 7.04.

.B. Poly-(trimethylsilyl)-3,6'-di-N-carbobenzyloxykanamycin A

A mixture of 1.5 g (2 mmol) of 3,6'-di-N-carbobenzyloxykanamycin A from Step A above and 1.29 g (8 mmol) of hexamethyldisilazane in 15 ml of dry acetonitrile was refluxed for 16 hours. The clear solution was concentrated to dryness under vacuum, and the residue was dissolved in 20 ml of dry diethyl ketone. The solution was used directly in the next step.

C. Acylation of poly-(trimethylsilyl)-3,6'-di-N-carbobenzyloxykanamycin A using an equimolar amount of acylating agent

To the solution from step B above, 700 mg (2 mmol) of L-(-)-α-carbobenzyloxyamino-α-hydroxybutyric acid N-hydroxysuccinimide ester (SAE) was added with stirring. The mixture was stirred at room temperature for 19 hours, then treated with 8 mL of water and 35 mL of tetrahydrofuran (THF), adjusted to pH 3 with aqueous hydrochloric acid, stirred for 30 minutes, and concentrated to dryness under vacuum. The residue was dissolved in a mixture of 300 ml water, 40 ml methanol, 10 ml n-butanol and 40 ml THF and hydrogenated overnight in the presence of 500 mg 10% palladium-on-carbon. The catalyst was removed by filtration, and the filtrate was evaporated under vacuum and lyophilized to give 1.7 g of crude BB-K8. The amorphous powder was redissolved in water and the solution was adjusted to pH

4 with aqueous hydrochloric acid and was chromatographed in a column of "Amberlite CG-50" in the NH4<+->cycle. The column was subjected to stepwise elution with water, 0.1 n NH^OH, 0.3 n NH^OH, 0.5 n NH^OH and 2 n NH^OH, collecting 10 ml fractions and monitoring by thin layer chromatography using "Merck

Silica Gel 60 F-254" plates using chloroform-methanol-28% ammonium hydroxide-water (1:4:2:1). The homogeneous fractions were combined, evaporated and finally lyophilized. Fractions containing BB-K8 and fractions containing recovered kanamycin A were analyzed using K. pneumonia A20680 and B. subtilis PCI 129, respectively.

Hydrogenolysis of the partially deblocked product with Pd-c followed by isolation on a "CG-50" column gave an additional 30 mg (2%) of BB-K8. The total yield of BB-K8 was 846 mg (69%).

D. Acylation of poly-(trimethylsily1-3,6'-di-N-carbobenzyloxykanamycin A using 1.2 equivalents of acylating agent1

Step C above was repeated, except that a 20% excess of acylating agent was used. The following results were obtained:

47 1 49635

Hydrogenolysis of the partially deblocked product with Pd-C followed by isolation on a "CG-50" column gave an additional 21 mg (2%) of BB-K8. The total yield of BB-K8 was 771 mg (62%).

E. Acylation of poly-(trimethylsilyl-3,6'-di-N-carbobenzyloxykanamycin A using 1.5 equivalents of acylating agent1

Step C above was repeated, except that a 50% excess of acylating agent was used. the following results were obtained:

Claims (4)

1. Process for the preparation of 1-N-[o>-amino-a-hydroxyalkanoyl]-kanamycin A or B with the general formula (I): where R is OH or NH2 and n is an integer from 0 to 2, or a non-toxic > pharmaceutically acceptable acid addition salt thereof, characterized in that it comprises that kanamycin A or B containing 4-8 trimethylsilyl groups or kanamycin A or B containing 3-7 trimethylsilyl groups and a blocking group different from silyl, in the 6'-amino group, is acylated with a) an active ester of N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or N- hydroxyphthalimide or b) a mixed acid anhydride of pivalic acid, benzoic acid, isobutylcarboxylic acid or benzylcarboxylic acid and an acid of the general formula (II): where n is an integer from 0 to 2 and B is an amino blocking group of the formula wherein R 1 and R 2 are the same or different and each is H, F, Cl, Br, NO 2 , OH, (lower) alkyl or (lower) alkoxy, and X is Cl, Br, F or I, and Y is H , Cl, Br, F or I, in a largely anhydrous organic solvent, and that all blocking groups are then removed. 2. Method in accordance with claim 1, characterized in that the acylation is carried out with an acylating derivative of the acid in which the amino blocking group is a carbobenzyloxy group. 3. Method in accordance with claim 1 or 2, characterized in that a polysilylated kanamycin A or B containing a carbobenzyloxy group on the 6<1->amino group is used as starting material. 4. Process in accordance with one of claims 1-3 for the production of 1-N-[L-(-)-w-amino-α-hydroxyalkanoyl]-kanamycin A, characterized in that the acylation is carried out with an acylating derivative of the acid with the formula: