SE447259B - PROCEDURE FOR PREPARING A 1-N- / L - (-) - OMEGA-AMINO-ALFA-HYDROXIAL CANOYL / CANAMYCINE A OR B - Google Patents

Description

447 259 2 pglysilylated kanamycin A or B containing another blocking agent group than silyl on the 6'-amino group of a substantially anhydrous technical solvent with an acylating derivative of an acid with meln B-HN-CH2- (CH2) n-ïfi- COOH II OH wherein n is an integer from 0 to 2 and B is an amino blocking group, and then removes all blocking groups.

The kanamycins are well-known antibiotics, such as written in Merck Index, 8th edition, p. 597-8. Numerous derivatives of the kanamycins are also known. The structural formulas of kanamycin A and B is reproduced below along with the standard numbering system used within the technique. In the following, the various kanamycin derivatives will be used. is given as a derivative of kanamycin A or B instead of indicated with structural formulas, so as to avoid the need to compare complex structures in order to identify differences. __6h CB NH 2 2 HO 4 ' HO III Kanamycin A: R = OH Kanamycin B: R = NH2 447 259 3 U.S. Patent No. 3,781,268 discloses and writes 1- (L - (-% - 3'-amino-alpha-hydroxybutryl / kanamycin A) cin) and B as well as their mono- and dicarbobenzyloxy protected derivatives.

Lower and higher homologues thereof are described in U.S. Pat. the writings 3,886,139 and 3,904,597 acylating a 6'-N-protected kanamycin A or B with an acyl- derivative of an N-protected L - (-) - '§'-amino-alpha-hydroxybutyric acid in an aqueous medium, after which one or both N-protecting groups are removed.

U.S. Patent No. 3,974,137 discloses and describes of a process for the preparation of 1- / L - (-) - δ ° amino-alpha-hydrogen oxybutryl / kanamycin A, which process involves reacting 6'-carbobenzyloxycanamycin A with at least 3 moles of benzaldehyde, a sub- substituted benzaldehyde or pivaldehyde for the preparation of 6'-N- -carbobenzyloxycanamycin A, which contains Schiff base groups in 1-, 3- and the 3 "positions, this tetra-protected kanamycin A acylates torn with the N-hydroxysuccinimide ester of L - (-) - @ '- benzyloxycarbonyl- amino-alpha-hydroxybutyric acid and then removes the protecting groups.

Belgian Patent Specification 828,192 discloses and describes a process for the preparation of 1- / L - (-) - α'-amino-alpha-hydroxy- butyryl / kanamycin A by preparing the same tetra-protected can mycine A derivatives as in U.S. Pat. No. 3,974,137, acy- lation with the N-hydroxy-5-norbornene-2,3-dicarboxyimide ester of L - (-) - 3'-benzyloxycarbonylamino-alpha-hydroxybutyric acid and subsequently removal of the protection groups.

The present invention provides an improved and commercially attractive process for the preparation of with formula I above. The use of a polysilylated kanamycin A or B as starting material gives high solubility in the organic solvent system, which thus enables turnover at high concentrations. Although the reaction is usually carried out in solutions such as contains about 10-20% polysilylated kanamycin starting material, has labeled results were obtained at concentrations of about 50 g / 100 ml means. - - Similar to processes known in the art, the process yields according to the invention a mixture of acylated products. The desired the 1-N-acylated product is separated from the other products by chromatography and the by-products can, if desired, hydrolyze 447 259 4 to the kanamycin starting material for recycling. At those within technology known processes it has been shown to possibly formed 3 "-N-acylated material gives a loss which roughly corresponds to amount of the desired 1-N-acylated product, due to the large difficulty the ability to separate the latter product from the former material.

A particularly advantageous feature of the present process is the extremely low amount of undesirable 3 ”'- N-acylated product formed (normally no such product can be detected).

In the preparation of 1- (L - (-) - alpha-amino-alpha-hydroxybutyrate yl / kanamycin A (amikacin) by various methods known in the art The 3 "-N-acylated product (BB-K11) is normally formed on 3-N-acylated product (BB-K29), the 6'-N-acylated product (BB-K6) and polyacylated material together with unreacted kanamycin A. At commercial production of amikacin by acylation of 6'-N-car- bobenzyloxycanamycin A in an aqueous medium, followed by removal of protection groups, it is thus found that about 10% of it desired compound amikacin (2.5 kg in a 25-kg batch) usually goes lost due to the presence of the compound BB-K11 as a by-product duct. In preparing the compound amikacin by means of the present invention procedure, the by-product BB-K11 cannot normally be detected in the reaction. the mixture.

The present invention relates to a process for the preparation of of a 1-N- (N) -amino-alpha-hydroxyalkanoyl / kanamycin A or B with the formula CH NH H0 10 15 20 25 30 35 447 259 wherein R is OH or NH 2 and n is an integer from 0 to 2, or non-toxic, pharmaceutically acceptable acid addition salts thereof, which procedure is characterized by the fact that in and known per se acylates polysilylated kanamycin A or B or polysilylated kanamycin A or B contains containing a blocking group other than silyl of 6 the group, which blocking group has the formula 1 'cxo R o 3 || l H azoc-, czg-c-o-c- C33 112 I s- ' Ü r YZXC @ " IN_ N02 O å 0 y or \ X3C-CH2-0-Q- c / hrs O wherein R 1 and R 2 are the same or different and each represents H, F, Cl, Br, NO 2, OH, lower alkyl or lower alkoxy, X is Cl, Br, F or I and Y is H, Cl, Br, F or I 10 15 20 25 30 35 447 259 and wherein the polysilylated kanamycin derivative contains holds 2-10 silyl groups per molecule, preferably in average 4-8 silyl groups per molecule or 3-7 silyl groups groups per molecule in the case of the polysilylated kanamycin A or the B starting material contains another blocking group than silyl on the 6'-amino group, and the silyl groups are tri- (lower alkyl) silyl groups, with an acylating derivative of an acid in the L - (-) form with the formula B-HN-CH2- (CH2) n ~ CH-COOH (II) OH wherein n is an integer from 0 to 2 and B is a con- conventional amino blocking group, in an organic solvents containing up to 25% water and thereafter in a manner known per se removes all blocking groups.

The acylating acid may be present in its isomeric (+) - or (-) - isomeric form or as a mixture of the two iso- (d, l-form), thus obtaining the counter- corresponding compound of formula I wherein 1-N- / u, -amino-alpha- the hydroxyalkanoyl / group is present in its (+ P-traps (R) í7 form or its (-P- [or (5) i7 form or so one mixture thereof. Each such isomer form and mixture thereof falls within the scope of the present invention.

In a preferred embodiment, the acylating agent is present the acid in its (-) form. In another preferred embodiment, form, the acylating acid is in its (+) - form.

The most preferred blocking agent is carbobenzyloxy. the group.

In a preferred embodiment of the invention it is acylating derivative of the acid an active ester and preferably its active ester with N-hydroxysuccinimide, N-hydroxy-5-nor-bornene-2,3-dicarboximide or N-hydroxy- phthalimide. In another preferred embodiment, it is 10 15 20 25 30 35 447 259 acylating derivative of the acid a mixed acid anhydride and preferably its mixed anhydride with pivalic acid, benzoic acid, isobutyl carbonic acid or benzyl carbonic acid.

An extremely preferred embodiment of the present invention relates to the preparation of 1-N- [I - (-) - α'-amino- alpha-hydroxybutyl glycanamycin A or a non-toxic, a pharmaceutically acceptable acid addition salt thereof, wherein to acylate polysilylated kanamycin A with a mixed acid anhydride of L- (-) - N-benzyloxycarbonylamino-alpha- hydroxybutyric acid (preferably its mixed anhydride with pivalic acid, benzoic acid, isobutylcarboxylic acid or benzyl carbonic acid) in a substantially anhydrous organic solution means and then removes all blocking groups.

Another particularly preferred embodiment of the The invention relates to the preparation of l-N- [L - (-) - α / -amino- alpha-hydroxybutyrylycanamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof by acylating polysilylated kanamycin A, which contains a carbobenzyloxy group on the 6 'amino group, with a mixture acid anhydride of L - (-) - α / benzyloxycarbonylamino-alpha- hydroxybutyric acid (and preferably its mixed anhydride with pivalic acid, benzoic acid, isobutylcarboxylic acid or benzyl carbonic acid) in a substantially anhydrous organic solution means and then removes all blocking groups.

Yet another particularly preferred embodiment of the invention relates to the preparation of 1-N- [i - (-) - à / -amino- alpha-hydroxybutyrylphanamycin A or a non-toxic, pharmaceutical Ceutically acceptable acid addition salt thereof by acylates polysilylated kanamycin A with an active ester of L - (-) - α / benzyloxycarbonylamino-alpha-hydroxybutyric acid (and preferably its active ester with N-hydroxy succinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or N-hydroxyphthalimide) in a substantially anhydrous organic solvent and then remove all blocking agents. groups. 10 15 20 25 30 35 447 259 8 Another particularly preferred embodiment of The invention relates to the preparation of 1-N- [f - (-) - 2''-amino- alpha-hydroxybutyryl] kanamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof by one acylates polysilylated kanamycin A, which contains a carbobenzyloxy group on the 6 'amino group, with an active ester of L - (-) - δ '-benzyloxycarboxylamino-alpha-hydroxy- butyric acid (and preferably its active ester with N-hydroxy- succinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or N-hydroxyphthalimide) in a substantially anhydrous organic solvent and then remove all blocking agents. groups.

In a preferred embodiment, the material is polysilylated kanamycin A or B and preferably polysilylated kanamycin mycin A, which contains on average 4-8 silyl groups and preferably trimethylsilyl groups per molecule. By a another preferred embodiment is the material polysilylated kanamycin A or B and preferably polysilylated kanamycin A, which contains a blocking group other than silyl on the 6 'amino group and contains on average 3-7 silyl groups and preferably trimethylsilyl groups per molecular.

With the expression "non-toxic, pharmaceutically acceptable acid addition salt "of a compound of formula I is referred to in landscape context a mono-, di-, tri- or tetrasalt, which has been formed by reacting a molecule of a compound of formula I having 1-4 equivalents of a non-toxic, pharmaceutically acceptable acid. Such acids include acetic acid, hydrochloric acid, sulfuric acid, maleic acid, phosphoric acid acid, nitric acid, hydrobromic acid, ascorbic acid, malic acid and citric acid and such other acids as usual used for the preparation of amine - 9 pharmaceuticals. 10 15 20 25 30 35 9 447 259 Acylation of polysilylated kanamycin A- or -B- starting material (with or without another blocking group than silyl on the 6'-amino group) can generally be performed in an organic solvent, wherein the starting material has sufficient solubility. These starting materials are soluble in most common organic solvents.

Suitable solvents include, for example, acetone, diethyl ketone, methyl n-propyl ketone, methyl isobutyl ketone, methyl ethyl ketone, acetonitrile, glyme, diglym, dioxane, toluene, tetrahydrofuran, cyclohexanone, methylene chloride, chloroform, carbon tetrachloride and mixtures of acetone / butanol or diethyl ketone / butanol. The choice of solvent is dependent on the special starting material in question. Ketones are in generally the preferred solvents. The most advantageous the solvent for the particular combination of reactants in question used can be easily determined by routine experimentation.

Suitable silylating agents for use in preparation of the polysilylated kanamycin starting materials trimethylchlorosilane, hexamethyldisilazane, triethylchlorosilane, triethyl bromosilane, tri-n-propylchlorosilane, methyldiethyl- chlorosilane, dimethylethylchlorosilane, dimethyl-t-butylchlorosilane, hexaetyldisilazane, tri-n-propylsilylamine, tetraethyldimethyl- disilazane, etc. Other useful silyl compounds are hexa- alkylcyclotrisilazanes and octa-alkylcyclotetrasilazanes as well as silylamides (such as, for example, trialkylsilylacetate mides and bis-trialkylsilylacetamides), silyl ureas (such as, for example, trimethylsilyl urea) and silyl urides.

Trimethylsilylimidazole can also be used.

A preferred silyl group is the trimethylsilyl group and preferred silylating agents for the introduction of trimethyl- the silyl group is hexamethyldisilazane, bis (trimethylsilyl) acetamide and trimethylsilylacetamide. Hexametyldisilazañ ' preferred in particular.

When using polysilylated kanamycin A or B, as contains a blocking group other than silyl on the 6'-amino- the group, as starting material this can be prepared 10 15 20 25 30 10 447 259 either by polysilylation of the desired 6'-N- blocked kanamycin A or B or by insertion of the desired 6'-N-blocking group in polysilylated kanamycin A or B.

Methods for introducing silyl groups into organic compounds compounds comprising certain aminoglycosides are known within the technique. The polysilylated kanamycins (with or without a blocking group other than silyl on the 6'-amino group) can be prepared by methods known in the art or as described in more detail below.

By "polysilylated kanamycin A or B" is meant in the present invention related kanamycin A or B, which contains 2-10 silyl groups in the molecule. The term "polysilylated kanamycin A or B "thus includes non-persilylated kanamycin A or B, which would contain 11 silyl groups in the molecule.

The exact number of silyl groups (or their position) in the polysilylated kanamycin starting materials (with or without a blocking group other than silyl on the 6'-amino- group) is not known. It has been shown that both silylation and oversilylation reduce its yield desired product and increases the yield of other products.

In case of severe under- or oversilylation a small one is formed or no amount of the desired product. The silylation degree that gives the greatest yield of the desired product is dependent on the particular reactants in question used in the acylation step. The most advantageous degree of silylation when using a special combination of reactants can be easily determined by routine experimentation. 447 259 ll In the preparation of 1-N- / L - (-) - '¶'-amino-alpha-hydroxybutyl yryl / kanamycin A by acylation of polysilylated kanamycin A with The N-hydroxysuccinimide ester of L - (-) - E'-benzyloxycarbonylamino-alpha- -hydroxybutyric acid in acetone solution, it has been shown that good yields of the desired product is obtained by using polysilylated kanamycin A, which has been prepared by reacting about 4-5.5 moles hexamethyldisilazane per mole of kanamycin A. Larger or smaller amounts hexamethyldisilazane can be used, but the yield of the desired product in the subsequent acylation step is significantly reduced. At In the aforementioned method, it is preferred to use about 4.5-5.0 moles of hexamethyldisilazane per mole of kanamycin in order to obtain ground yield of the product in the acylation step.

It will be appreciated that each mole of hexamethyldisilazane may be introduced ra two equivalents of the trimethylsilyl group in kanamycin A or B.

Both kanamycin A and B have a total of eleven positions (NH2 and OH groups per), which can be silylated, while kanamycin A and B, which contain a blocking group other than silyl on the 6 'amino group, has a total of ten such positions. Thus, theoretically 5.5 moles of hexamethyldisilate per mole of kanamycin A or B completely silylate all OH- and NH 2 groups in kanamycin, while 5.0 moles of hexamethyldisilazane could constantly silylate 1 mole of kanamycin A or B, which contained 447 259 12 holds a blocking group other than silyl on the 6 'amino group. The however, it is assumed that such extensive silylation does not take place with these molar ratios for a reasonable reaction time, although higher degrees of silylation are achieved during a given reaction time when a silylation ring catalyst is used.

Silylation catalysts greatly accelerate silylation the speed. Suitable silylation catalysts are well known in the art. niken and includes i.a. amine sulfates (eg kanamycin sulfate), sulfamic acid, imidazole and trimethylchlorosilane. Silylation catalyst generally promotes a higher degree of silylation than that obtained required in the process of the present invention. Oversize However, kanamycin A or B can be used as starting material. al if first treated with a desilylating agent for the purpose of reduce the degree of silylation before performing the acylation reaction.

Good yields of the desired product are obtained by acylation of polysilylated kanamycin A, 'which has been prepared in use of a molar ratio of hexamethyldisilazane to kanamycin A of 5.5: 1.

However, if kanamycin A, which has been silylated at a molar ratio of the hexamethyldisilazane to kanamycin A of 7: 1 (or at a molar ratio of 5.5: 1 in the presence of a silylation catalyst) acyl- acetone with the N-hydroxysuccinimide ester of L - (-) - J'-benzyloxy- carbonylamino-alpha-hydroxybutyric acid less than 1% yield is obtained the desired product. However, if the same "oversilylated" kanamy- cin A is acylated with the same acylating agent in acetone solution, to Which water (21 moles of water per mole of kanamycin corresponding to 2.5% (w / v lymph) water) has been added as a desilylating agent 1 hour before the acylation. yield, a yield of about 40% of the desired product is obtained. 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, methylmercaptan, ethylmercaptan, phenyl- mercaptan and the like.

Although it is common to use dry solvents at work with silylated materials, it has surprisingly been found that adding water to the reaction solvent before acylation to and in the absence of "oversilylation" gives equally good yields and sometimes better yields of the desired product than in a dry solution means. In acylation reactions carried out in acetone at usual concentrations of 10-20% (w / v) of polysilylated catalyst 447 259 13 namycin A, it has been found that excellent yields of 1-N- / L - (-) - af - -amino-alpha-hydroxybutyl / kanamycin A is obtained by the addition of to 28 moles of water per mole of polysilylated kanamycin A; at a concentration concentration of 20% is 28 moles of water per mole of polysilylated kanamycin A about 8% water. With other combinations of reactants, t.o.m. even greater amounts of water are tolerated or be favorable. Acyle- the reaction can be carried out in solvents containing up to about 40% water, although at such high water concentrations one must use short acylation times in order to avoid excessive desilylation of the polysilylated kanamycin A or B the walking material. In the present context, the term "substantially anhydrous organic solvent" means a solvent, which contains up to about 25% water. A preferred water content is up to about 20% water, a more preferred water content is up to about 8% water and the most preferred water content is up to about 4% water.

As indicated above, the most suitable degree of silylation for a given combination of acylation reactants is readily determined by routine trials. It is assumed that the preferred average number silyl groups in the starting material are usually between 4 and 8 for kanamycin A or B and between 3 and 7 for kanamycin A or B, containing a blocking group other than silyl on the 6'-amino group, but this is only a theoretical assumption and does not constitute any An essential part of the present invention.

The duration and temperature of the acylation reaction are not critical. Temperatures in the range from about -30 ° C to about 100oC can be used for reaction times, which vary from about 1 hour up tilll-åag or more. It has been shown that the reaction usually runs well at room temperature and from a comfort point of view and for economic reasons, it is preferable to carry out the reaction at the temperature of the giving. For maximum yields and selective acylation however, it is preferred to carry out the acylation at a temperature turn of about 0-50.

Acylation of the 1-amino group in the polysilylated kanamycin net A or B (with or without a blocking group other than silyl on The 6'-amino group) can be performed with any acylating derivative of the acid of formula II above, which derivative is known in the art 447 259 14 to be suitable for acylation of a primary amino group. Examples of suitable acylating derivatives of the free acid include the corresponding the acid anhydrides, mixed anhydrides such as, for example, alkoxy formic acid anhydrides, acid halides, acid azides, active esters and active thio- estrar. The free acid can be coupled with the polysilylated kanamy- cin starting material after the free acid has first been reacted with N, N * - dimethylchloroformiminium chloride (see British patent specification 1 008 170 and Weichet: Experientia XXI, 6 (1965) 360) or by use of an N, N'-carbonyldiimidazole or an N, N'-carbonyldiimidazole triazole (see South African Patent Specification 63/2684), a carbodiimide reagents (especially N, N'-dicyclohexylcarbodiimide, N, N'-diisopropylcarbodiimide carbodiimide or N-cyclohexyl-N '- (2-morpholinoethyl) carbodiimide; see Sheehan and Hess: J.A.C.S. 11 (1955) 1967), an alkynylamine reagent (see R. Buijle and H. G. Viehe: Angew. Chem. International Edition, (1964) 582), an isoxazolium salt reagent (see R. B. Woodward, R.A.

Olofson and H. Mayer: J. Amer. Chem. Soc. §§ (1961) 1010), a tenimine reagents (see C.L. Stevens and M. E. Munk: J. Amer. Chem. Soc.

§Q (1958) 4065), hexachlorocyclo triphosphatriazine or hexabromocyclo- triphosphatriazine (see U.S. Pat. No. 3,651,050), diphenyl- phosphorylation wnPA; see J. mer. Chem. soc. g (1972) 6203-6205), diethylphosphoryl cyanide (DEPC; see Tetrahedron Letters lay (1973) 1595- -1598) or diphenyl phosphite (Tetrahedron Letters 22 (1972) 5047-5050).

Another equivalent of the acid is a corresponding azolide, i.e. and amide of the corresponding acid, the amide nitrogen of which forms part of a quasi-aromatic five-membered ring containing at least two nitrogen atoms, ie imidazole, pyrazole, triazoles, benzimidazole, benzotriazole and their substituents rade derivatives. As will be apparent to those skilled in the art, it is sometimes desirable or necessary to protect the hydroxyl group in it the acylating derivative of the acid of formula II, e.g. when utilizing an acylating derivative such as an acid halide. The hydroxyl group can protect das in a manner known per se, e.g. by using a carbohydrate benzyloxy group, by acetylation, by silylation and the like.

After completion of the acylation reaction, all are removed blocking groups with per se known methods of obtaining of the desired product of formula I. The silyl groups can e.g. easy removed by hydrolysis with water, preferably at low pH.

Blocking group B in the acylating derivative of the acid of formula II and the blocking group on the 6 'amino group of the polysilylated 447 259 15 the kanamycin starting material (if present) may also removed by methods known per se. Thus, a t- -butoxycarbonyl group is removed using formic acid, a car- bobenzyloxy group by catalytic hydrogenation, a 2-hydroxy-1-naphtharbonate onyl group by acid hydrolysis, a trichloroethoxycarbonyl group by treatment with zinc dust in glacial acetic acid, a phthaloyl group by treatment with hydrazine hydrate in ethanol during heating, etc.

Product yields can be determined by various methods. Ef- removal of all blocking groups and chromatography on a column CG-50 (NH 4 +) the yield of amikacin can be determined by isolation of the crystalline solid from the appropriate fractions in question or by microbiological analysis (turbidimetric or on plate) of the appropriate fractions in question. Another tech- technique which can be used is liquid chromatography of the unreduced medium. the acylation mixture, i.e. of the aqueous solution obtained hydrolysis of the silyl groups and removal of organic solution but before hydrogenolysis to remove the remaining ones the blocking groups. This analysis method can not be used for direct analysis of amikacin or the compound BB-K29 but for analysis of corresponding mono- or di-N-blocked compounds.

The instrument that has been used is a high-pressure liquid matographer with the designation Waters Associates ALC / GPC 244 content they an absorbance detector with the designation Waters Associates Model 440 and a column with a length of 30 cm and an inner diameter of 3.9 mm with the designation -Honda ak C-18 column. The anal conditions were P P Y following.

Mobile phase: 25% 2-propanol 4 75% 0.01% Sodium acetate pH 4.0 Flow rate: 1 ml / minute Detector: UV at 254 nm Sensitivity: 0.04 AUFS Diluent: DMSO Injected amount: 5 pl _ _ Concentration: 10 mg / ml The paper speed varied, but 2 minutes / 2.54 cm was used. jades normally. The above conditions gave peaks, which were light to measure quantitatively. The results of the above analysis method are given hereinafter as HLPC analysis. 447 259 16 In order to avoid complicated chemical designations, is sometimes used in the following abbreviations: AHBA L - (-) - Ü'-amino-alpha-hydroxybutyric acid BHBA N-cyclobenzyloxy derivative of AHBA HONB N-Hydroxy-5-norbornene-2,3-dicarboxamide NAE N-hydroxy-5-norbornene-2,3-dicarboxamide-ac- tivered ester of BHBA HONS N-hydroxysuccinimide SAE N-hydroxysuccinimide-activated ester of BHBA (or BHBA-'ONS ') DCC dicyclohexylcarbodiimide DCU dicyclohexylurea HMDS hexamethyldisilazane BSA bis (trimethylsilyl) acetamide MSA trimethylsilylacetamide TFA trifluoroacetyl t-BOC tert-butyloxycarbonyl "Dicalite" is the trade name for diatomaceous earth (Great Lakes Carbon Corporation).

"Amberlite CG-50" is the trade name for the chromatographic the quality of a weakly acidic cation exchange resin of carboxyl polymethacrylic type (Rohm & Haas Co.).

"P-Bondapak" is the trade name for a series of liquid chromatographs high performance fig columns (Waters Associates).

The invention is further illustrated below by the following examples, in which the temperature indications refer to degrees Celsius. With the terms "lower alkyl" and "lower alkoxy" mean alkyl and alkyl, respectively alkoxy groups containing 1-6 carbon atoms.

Example 1 Preparation of 1-N- / L - (-) - 'Z'-amino-alpha-hydroxybutyl / - kanamycin A (amikacin) by selective acylation of noly (trimethylsilicone) yl) -6'-N-carbobenzyloxycanamycin A in anhydrous diethyl ketone 15 g (24.24 mmol) of 6 "-N-carbobenzyloxycanamycin A slurry was dissolved in 90 ml of dry acetonitrile and refluxed under a nitrogen atmosphere. far. 17.5 g (108.48 mmol) of hexamethyldisilazane were slowly added 30 minutes and the resulting solution was refluxed under there 24 hours. After removal of the solvent in vacuo (400) and complete drying in vacuo (10 mm) gave 27.9 g of a white, amorphous solid (90.7l% based on 6'-N-carbobenzyloxycanamycin A 447 259 17 (silyl) 9).

This solid was dissolved in 150 ml of dry diethyl ketone at 230. 11.05 g (26.67 mmol) L - (-) - 3 '- benzyloxycarbonylamino-alpha- -hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboxamide ester (NAE), which had been dissolved in 100 ml of dry diethyl ketone at 230 and for a period of half an hour with good stirring. The mixture was stirred for 78 hours at 230 DEG C. The yellow clear solution (pH 7.0) diluted with 100 ml of water. The pH of the mixture was adjusted to 2.8 (3 N HCl) and the mixture was stirred vigorously for 15 minutes at 230 ° C. The tin phase was separated and the organic phase was extracted with 50 ml water with a pH of 2.8. The combined water fractions are washed with 50 ml of ethyl acetate. The solution was poured into a 500 ml Parr bottle, together with 5 g of a catalyst of 5% palladium on carbon (Engel- hard) and was reduced at a hydrogen pressure of 3.5 kg / cm 2 at 230 2 hours. The mixture was filtered through a pad of Dicalite, which was then after was washed with another 30 ml of water. The colorless filter The mixture was concentrated in vacuo (40-450) to 50 ml. The solution is applied row on a 5 x 100 cm ion exchange column CG-50 (NH4 +). After washing with 1000 ml of water eluted unreacted kanamycin A, 3- / L - (-) - -'X'-amino-alpha-hydroxybutyl / kanamycin A (BB-K29) and amikacin with 0.5 N ammonium hydroxide. Polyacyl material was recovered with 3N ammonium hydroxide. Bioassay, thin layer chromatography and optical rotation was used to follow the elution process. Below are reproduced for each eluate fraction volume and observed optical rotation as well as the amount and the percentage yield of solids isolated from each fraction by evaporation to dryness: Volume Weight Material (ml) oàälâ (gm.)% Yield Kanamycin A 1000 + 0.115 0.989 9.15 BB-K29 1750 +0.24 4.37 32.0 Amikacin 2000 + 0.3l 6.20 47.4 Polyacyls 900 +0.032 0.288 2.0 The spent diethyl ketone layer was found in HLPC analysis contain an additional 3-5% amikacin. 'f The crude amikacin (6.20 g) was dissolved in 20 ml of water and was diluted with 20 ml of methanol, and 20 ml of isopropanol was added to initiate crystallization. 6.0 g (45.8%) of crystalline amine were obtained. kacin. 447 259 18 Example 2 Preparation of 1-N- / L - (-) - 2'-amino-alpha-hydroxybutyl / - U kanamyoin A (amikacin) by selective acylation of poly (trunethylethyl silyl) -6'-N-carbobenzyloxycanamycin A in anhydrous acetone 103 g (0.08l mol, calculated as 6'-N-carbobenzyloxycanamycin) A (silyl) 9) poly (trimethylsilyl) -6'-N-carbobenzyloxy-kanamycin A, SO had been prepared as in Example 1, was dissolved in 100 ml of dry acetone at 230. 35.24 g (0.085 mol) of L - (- J-U'-benzyloxycarbonylamino-alpha- -hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboxamide ester (NAE), which had dissolved in 180 ml of dry acetone at 230 was slowly added good stirring to the solution of poly (trimethylsilyl) -6'-N-carbon benzyloxycanamycin A for a 15-minute period. The solution is stirred for 20 hours at 23 ° under a nitrogen atmosphere. The pale yellow clear solution (pH 7.2) was diluted with 100 ml of water. The pH of the mixture is was set at 2.5 (3N HCl) and stirring was continued for 15 minutes 230. The acetone was removed using a vapor jet vacuum at about 350. The solution was introduced into a 500 ml Parr bottle together with 10 g of a catalyst of 5% palladium on carbon (Engelhard) and reduced was maintained at a hydrogen pressure of 2.8 kg / cm 2 at 230 for 2 hours.

The mixture was filtered through a pad of diatomaceous earth, which was then was washed with an additional 50 ml of water. After concentration to about one third of the original volume, the solution was applied (pH 6.9-7.2) on a 6 x 10 cm ion exchange column CG-50 (NH4 +) and eluting gradient elution with water up to 0.6 was eluted N ammonium hydroxide for the extraction of amikacin. An automatic polarizing meters were used to follow the elution process. Combinations was made on the basis of thin layer chromatography evaluation. The combined the amikacin fractions were concentrated to a solid of 25-30%. The solution was diluted with an equal volume of methanol, followed of two volumes of isopropanol to initiate crystallization. You are- contained l8.2 g (40%) of crystalline amikacin.

The recovery of 12% kanamycin A, 40% of the compound BB-K29 and 5% polyacylated kanamycin gave a material balance of 97%. _ _ Example 3 I Preparation of 1-N- / L - (-) -: f -amino-alpha-hydroxybutyl / - kanamycin A (amikacin) by selective acylation of ooly (trimethyl- silyl) -kanamycin A using in situ blocking A. Poly (trimethylsilyl) -kanamycin A 18 g activity (37, 15 mmol) kanamycin A in the form of the free 447 259 19 the base was slurried in 200 ml of dry acetonitrile and refluxed. 29.8 g (844 mmol) of hexamethyldisilazane were added over 30 minutes and the mixture was refluxed for 78 hours with stirring to obtain of a light yellow clear solution. Removal of the solvent in vacuum gave an amorphous solid residue (43 g, 94%, calculated as kanamy- cin A (silyl) 10).

B. 1-N- / L - (- γ- β-amino-alpha-hydroxybutryl / kanamycin A 5.56 g (20.43 mmol) of p- (benzyloxycarbonyloxy) benzoic acid was slurried in 50 ml of dry acetonitrile at 230. 8.4 g (41, 37 mmol) N, O-bis-trimethylsilylacetamide was added with good stirring. Loose- was held for 30 minutes at 230 and then set for a period of time. period of 3 hours with vigorous stirring to a solution of 21.5 g (10.83 mmol, calculated as the (silyl) 10-compound) in 75 ml of dry aceto- nitrile at 230. The mixture was stirred for 4 hours, the solvent was removed was removed in 40 ml) and the oily residue was dissolved in 50 ml dry acetone at 230. 8.55 g (20.63 mmol) L - (-) - '' 3'-benzyloxycarbonylamino-alpha- Hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboxamide ester (NAE) in 30 ml of acetone was added to the above solution for a period of 5 minutes. The mixture was kept for 78 hours at 230. The solution was diluted with 100 ml of water and the pH (7.0) was lowered to 2.5 (6N HCl).

The mixture was introduced into a 500 ml Parr bottle together with 3 g of a catalyst of 5% palladium on carbon (Engelhard) and was reduced at a hydrogen pressure of 2.80 kg / cm 2 at 230 for 2 hours. Mixture- one was filtered through a pad of diatomaceous earth, which was then washed. was diluted with 20 ml of water. The combination of filtrate and washing liquids (168 ml) was then subjected to microbiological analysis against E.coli and was found to contain approximately ll 400 pg / ml (19% yield) amikacin.

Example 4 Preparation of 1-N- / L - (-) - H '-amino-alpha-hydroxybutyl / - kanamycin A (amikacin) by selective acylation of poly (trimethylsilicone) yl) -kanamycin A A. Poly (trimethylsilyl) -kanamycin A _ 7 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 refluxed for 72 hours. A clear light yellow solution was obtained.

The solution was evaporated to dryness in vacuo at 30-400, stirring obtained 21.3 g of poly (trimethylsilyl) -kanamycin A as a light brown dye. amorphous powder (85% yield calculated as kanamycin A (silyl) 10). 447 259 20 B. 1-N- (L - (-) - '3'-amino-alpha-hydroxybutyryl / kanamycin A To a solution of 2.4 g (2.0 mmol) of poly (trimethylsilyl) Namycin A in 30 ml of dry acetone was slowly added 2.0 mmol of L - (-) - Syloxycarbonylamino-alpha-hydroxymorphic acid-N-hydroxy-5-norbornene-2,3- -dicarboxamide ester (NAE) in 10 ml of dry acetone at 0-50. Reactional the mixture was stirred at 230 for 1 week and then evaporated to dryness in vacuo at a bath temperature of 30-400. 60 ml of water was then added to the residue, followed by 70 ml of methanol to obtain those of a solution. The solution was acidified with 3N HCl to pH 2.0 and was then reduced at a hydrogen pressure of 3.5 kg / cm 2 for 2 hours. using 500 mg of a 5% palladium catalyst on coal. The material was filtered and the combination of filtrate and washings liquids were subjected to microbiological analysis against E. coli, which analysis showed a yield of 29.4% amikacin.

Example 5 Preparation of amikacin by selective N-acylation of poly- trimethylsilyl-6'-N-carbobenzoxy-kanamycin A in anhydrous acetone I. Summary Silylation of 6'-N-carbobenzoxy-kanamycin A in acetonitrile using hexamethyldisilazane (HMDS) gave the intermediate 6'-N-carbobenzoxy-kanamycin A (silyl) 9 <:). This silylated kanamy- cin A was readily soluble in most organic solvents. Acylation with NAE in anhydrous acetone at 230 using a 5% molar excess of NAE relative to the amount of 6 ' -N-carbobenzoxy-kanamycin A gave a mixture containing only the carbobenzoxide derivatives of amikacin and the compound BB-K29, certain amount unreacted kanamycin A and some polyacyl material. You could do not in these investigations demonstrate the association BB-Kll. Elution of an acetone acylation mixture gave after reduction and work-up using a column CG-50 (NH 4 +) with ammonium hydroxide dient elution pure amikacin in a yield of about 40%. 21 II. Reaction scheme 6'-N-Cbz-Kana A c25x42ol3N4 (s1e, ss) + (ena) 3si-ÄNn-si (ca : H3 HMDS (l6l.4) A CH3CN OH R 1 Si (CH 3) 3 Q fi v-N-cbz-xana A cs-.uyng 447 259 447 259 22 OH IN a. cbzna (C112) z-cn-coou + Hon + ncc ' BHBA (253.4) 'HONB (l79.2) Acetoma OH 0 i II DcU + cbzm fl cufz-CH-c-o-N '<2z4, a> <2) NAE (414.6) diCbzamikacin (854) + ® + <2) dicbzaB-Kzs + 5'CbZ ~ 1,3-diBHBA-Kama A H2 + 5% On / C 6'Cbz-Kana A _ J \ __ š; ¿kacin + 35'K29 + 1,3-diAHBA-Kana A + Kan; 3 * (5 ° 5,52> _ <7zz, 1s) (4a4, s) ' cs-so (ux4 +) Anííkacin 447 259 23 III. Material Weight (g) Vol. (Ml) gg; 6'-N-Cbz-Kana A 50 0.081 HMDS 58.9 76.5 0.365 Acetonitrile 300 BHBA 21.5 0.085 HONB 15.2 0.085 DCC 17.48 0.085 Acetone 260 CG-50 (NH4 +) 3000 Methanol As needed IPA As needed IV. Safety instructions 6'-N-Cbz-kana A - Acetonitrile - Hexamethyldisilazane (HMDS) - 6'-N-Cbz-kana A (silyl) n - BHBA _ HONB - DCC - AC QtOIl _ NAE - Methanol - Isogroganol - No direct information available.

Contact with dust should be avoided.

Treated as a cyanide. Inand- vapors must be avoided. can cause skin irritation.

Annoying, handled with care hot.

No direct information available, handled with care.

Toxicity has not been established. Ex- exposure to the solid material should be avoided.

Toxicity unknown. Caution at handling should be observed.

Causes severe skin irritation and eyes. Inhalation of vapors should avoided. Toxic.

Fiery. Inhalation may cause headache, fatigue, anxiety, irritation in the bronchi and in large quantities kos.

No direct information available should always be handled directly as a solution in acetone.

Fiery. Poisoning can occur by oral administration, inhalation or percutaneous äbSOrPti0n- Fiery. Ingestion orally or hauling of large quantities of steam can cause headaches, drowsiness, mental depression, vomiting, anesthesia. 447 259 24 Ammonium hydroxide - Toxic fumes. Breathing mask shall worn and contact with the liquid shall avoided.

CG-50 (NH4 +) -3 No toxicity data available, handled with care.

V. Production procedure A. Preparation of 6'-N-carbobenzyloxycanamycin A (silyl) n J 13'-N-Cbz-Kana A (silyl) 97 1. 50 g of 6 "-N-carbobenzyloxycanamycin A (water content according to Karl Fischer less than 4%) is suspended in 300 ml of acetonitrile (aqueous content according to Karl Fischer less than 0.01%). It all gets reflux- boil (740) while passing a stream of dry nitrogen gas the slurry. 2. Over a 30-minute period, slowly add 75.8 ml hexamethyldisilazane (HMDS). Dissolution of the material takes place during evolution of ammonia gas. 3. The reflux is continued for 18-20 hours during nitrogen purge. 4. The clear light yellow solution is concentrated in vacuo (bath temperature 40-500) to a foamy solid. The yield of sil- The yl9 compound amounts to 89-92 g (90-94% of the theoretical value). the).

/ Note: When carrying out the procedure in other solutions part does not normally insulate this solid material but is used directly for acylation.

B. Preparation of N-hydroxy-5-norbornene-2,3-dicarboximide the ester of L - (-) - γ γ-carbobenzyloxyamino-alpha-hydroxybutyric acid (NAE) 1. 21.5 g of (-) - Qf-carbobenzyloxyamino-alpha-hydroxybutyric acid (BHBA) is dissolved in 10 ml of dry acetone at 23 °, followed by 15.2 g of N- -hydroxy-S-norbornene-2,3-dicarboxyimide (HONB). Full resolution enters. 2. For 30 minutes, add a solution of 17.48 g of dicyclo- hexylcarbodiimide (DCC) in 50 ml of acetone with stirring. The temperature rises to about 400 during the addition during precipitation of dicyclohex ylurea (DCU). 3. The slurry is stirred for 3-4 hours while the temperature is kept at 23-250. 4. The urea derivative is removed by filtration and the cake is washed with 30 ml of acetone. Filtrates plus washing liquids are saved for 447 259 25 the acylation step below.

C. Acylation of 6'-N-Cbz Kana A (Silyl) n. The compound 6'-N-Cbz- isolated under A in step 4 above -Kana A (silyl) 9 is dissolved in 100 ml of dry acetone at 23-240. 2. With good stirring, slowly add it under B above prepared the NAE solution over a 15-minute period. The temperature gradually rises to about 400. The solution may assume a temperature of 230, after which stirring is continued for 18-20 hours under a nitrogen atmosphere. far. 3. Add 100 ml of water and lower the pH (6.9-7.2) to 2.2-2.5 with 6N hydrochloric acid. Stirring takes place for 15 minutes at 230.

(Note: a second layer may form - this does not cause anything reprocessing problems). 4. The acetone is removed in vacuo at a bath temperature of 30- -350. The concentrate is transferred to a suitable hydration vessel (as in pre-flushed with nitrogen). 10 g of a catalyst of 5 g palladium on carbon is added and hydrogenation takes place at 2.8 kg / cm 2 below 2-3 hours. 5. The mixture is filtered through a Dicalite pad, after which the hydration vessel and the filter cake are washed with an additional 50 ml of water. ten. 6. The filtrate plus washing liquid is concentrated to about 1/3 of the initial volume (50 ml) in vacuo at 40-450. 7. The pH value is checked. It should be within the range 6.9-7.2. If not, adjust the pH with 1N ammonium hydride. oxide. The mixture is applied to a 6 x 110 cm column of CG-50 (NH 2). 8. The column is washed with 1000 ml of deionized water. Then elution with 0.5-0.6N ammonium hydroxide using a automatic polarimeter to follow the elution process. Elution the order is as follows: Remaining Kana A --Q) BB-K29 - {> »amikacin.For- no BB-K11 could not be detected in any of the acylation the reprocessings. Polyacyl material, i.e. 1,3-diAHBA-ana- the lodge of Kana A, is extracted by washing the column with 3N ammonium hydroxide; 9. The amikacin fractions are combined and concentrated to one solids content of 25-30%. The whole is diluted with 1 volume of methane. ol and added with amikacing seed crystals. For 2 hours, slowly add 2 volumes of isopropanol 447 259 26 (IPA) with good stirring and crystallization may take place for 6-8 hours. more at 23 °. ll. The solid is filtered, washed with 50 ml of one 1: 1: 2 mixture of water, methanol and IPA and finally with 25 ml IPA. 12. The material is dried in vacuo for 12-16 hours at 400.

The yield amounts to: in 17.3-19, and g (sa-un amikacin with the following properties creates: .TEE CHCl 3 -methanol-NH 4 OH-water (1: 4: 2: 1), 5 x 20 cm silica gel plates from Quantum Industries - a zone for detection with nin- hydrin (RF ^ J 0.4).

Specific rotation [di] sššo + Hig1,6 + O1'ol1rf 9NH4OH + oíåglfšzso4 C = 1.0% 13. The recovery of BB-K29 in this system is also 39-42%, of remaining Kana A 10-14% and of 1,3-di-AHBA-Kana A about 5%, which gives a material balance in excess of 95%.

Example 6 Preparation of amikacin by selective N-acylation of poly- trimethylsilyl-Kana A in anhydrous acetone.

I. Summary Silylation of Kana A base in acetonitrile using hexamethyldisilazane (HMDS) gave polytrimethylsilyl-kana A. Silylation the degree is still uncertain but was assumed to be kana A (silyl) l0. Poly- silylated Kana A was readily soluble in most organic solvents. part. Acylation with SAE in anhydrous acetone at 230 during use of a molar ratio of SAE to Kana A of lzl gave a mixture which contained the Cbz derivatives of amikacin and the compound BB-K29, normally in a ratio of 2-3: 1, the compound BB-K6 (about 5-8%), unreacted Ka- A (15-20%) and a certain amount of polyacyl material (about 5-10%). As at above acylation of polytrimethylsilyl-6'-N-carbobenzoxy-kana-A the compound BB-Klll could not be detected in these experiments. Editors and work-up of an acetone acylation mixture, followed by chromatography CG-50-NH4 + column using 0.5N ammonium hydride. oxide gave isolated crystalline amikacin in a yield of 34-39%. 447 259 27 II. Reaction scheme a, ' Ho ou OK '5 N _32; O * HS ma; M2 Q Kama A- bas clsaušollul; (484, Sl) + (CH3) 3 Si ~ NH-Sl (Cfl3) 3 anus <1s1,4> R 1 Si (CH 3) 3 CD Rana. A (Chile fl lo c48a¿16ollN4sil0 (l2os, 3S) 447 259 for O OH “ I »I / B. cbzna (cx2) 2-ca-coca + ao-N + pcc Base N-Hos (2o6.3) 053.4) i (11s, 9) EtOAc O ox o .ncU + cbzmitczi fl zcx-c-o-N <224.3) 0 (2 SAE (350.33) C.

Cbzähmikacin (720) + Cbz-BB-K29 Acetorw + Q- + @ __í'_å X] cbz-aa-xs + Kana A + q ' 'H2 Polyadyls (mainly 1; 3- šï_ï37E i: _ \\ __ diBHBA-Kana A) ñJç "Ü Amikacin-i-BB-K29 + BB-KG + 1,3-diAHBA-1 (sas, 62) '(722.7s) 'cs-so (Nn4 +) AmikacinA 447 259 29 III. Material Weight (g) Vol. (ml) gg; Kana A- "bas" 50 0.103 HMDS (Sp. Weight 0.774) 86.68 112 0.537 Acetonitrile 600 SAE 35.03 0.10 Acetone 850 cs-so (m4 J ') 3000 Methanol As needed IPA As needed IV. Safety instructions Kana A- "base" _ Known drug - common caution recommended Kana A (Silyl) l0 - No direct information available, handled with care Other materials - See example 5 V. Production procedure A. Preparation of Kana A (Silyl) 10 l. 50 g Kana A-base (water content according to Karl Fischer 2.5-3.5%) suspended in 500 ml of acetonitrile (water content according to Karl Fischer increasing 0.01%). The whole thing gets refluxed (740) while one stream of dry nitrogen gas may pass through the slurry. 2. Over a 30-minute period, slowly add 112 ml hexamethyldisilazane (HMDS). Full resolution occurs within 4-5 hours during evolution of ammonia gas. 3. The reflux is continued for 22-26 hours during nitrogen purge. 4. The clear pale yellow solution is concentrated in vacuo (40 ° L) to a syrupy residue. Evaporation is done with an additional 100 ml acetonitrile and the whole is completely dried in vacuo for 3-6 hours. mar. The yield of a white, amorphous solid is 109-115 g (90-95% of the theoretical value, calculated as Kana A (Silyl) 10).

B. Preparation of the N-hydroxysuccinimide ester of L- (~) -alpha- -carbobenzyloxyamino-alpha-hydroxybutyric acid (SAE). _ 1. 100 g of L - (-) - alpha-benzyloxycarbonylamino-alpha-hydroxy butter acid (BHBA) and 45.38 g of N-hydroxysuccinimide (N-HOS) are dissolved in 1300 ml ethyl acetate (water content according to Karl Fischer less than 0.05%) at 23 ° with stirring. 447 259 30 2. 81.29 Dicyclohexylcarbodiimide (DCC) is dissolved in 400 ml ethyl acetate (water content according to Karl Fischer less than 0.05%) at 230. This solution is added with good stirring to the solution from step l for 30 minutes. The temperature rises to about 40-420 during the same precipitation of dicyclohexylurea (DCU). The slurry is stirred 3-4 hours, whereby the temperature may assume 230. 3. DCU is filtered off and the filter cake is washed with 250 ml of ethyl acetate (water content according to Karl Fischer less than 0.05%). DCU- the cookie is discarded, while the filtrate and washing liquids are saved. 4. The filtrate plus washings are concentrated to a volume of about 500 ml (in vacuo at 30-350). A certain amount of product crystallizes out serar. 5. The concentrate is transferred to a suitable vessel and 100 ml heptane is added with vigorous stirring. Seedlings may be added crystals of SAE. Crystallization occurs practically immediately bart. The slurry is stirred for 30 minutes at 230 ° C. 6. For 30 minutes, add 400 ml of heptane and slurry one is stirred 4-5 hours at 230. 7. The whole is filtered and the filter cake is washed with 200 ml a 3: 1 mixture of heptane and ethyl acetate, followed by 100 ml of heptane. 8. The material is dried in a vacuum oven at 30-350 for 18-20 hours. The yield amounts to 110-1131.4 g (80-95%). Melting point is 119-120 with softening at 114 ° (corr). Thin layer chromatography with a 4: 12: 1 mixture of acetone, benzene and CH3CO2H and ring with a 1% aqueous solution of KMO4 gives an Rf value of 0.7 for SAE and 0.2 for BHBA on 2 x 10 cm pre-prepared silica gel plates from Analtech Inc.

C. Acylation of Kana A (Silyl) 10 l. The compound Kana A isolated under A in step 4 above (Silyl) 10 is dissolved in 500 ml of dry acetone at 230. With good stirring, add it quickly in section B above prepared compound SAE (35.03 g) as a 10% solution in dry acetone for 5-10 minutes. The temperature rises about 50. The solution gets assume 230 and stirring is continued for 18-20 hours. 3. The light orange clear solution is diluted with 400 ml water and the pH (7.0-7.5) is lowered to 2.2-2.5 with 3 N hydrogen chloride acid. The clear solution is now stirred at 230 for 15-30 minutes. 447 259 31 4. Acetone is removed in vacuo at a bath temperature of 30-350 (a small amount of material may precipitate at this stage but causes no problem). The concentrate is transferred to a suitable hydrogen ring vessel. 10 g of a catalyst of 5% palladium on carbon are added and hydrogenation takes place at 3.5 kg / cm 2 for 2-3 hours. 5. The mixture is filtered through a Dicalite pad and hydrated. the ring vessel and the filter cake are washed with an additional 2 x 50 ml of water. 6. The filtrate plus washing liquids is concentrated to approx. a third part of the original volume (150-165 ml) in vacuo at 40-450. 7. The pH at this stage is 6.0-7.0. The mixture is applied on a CG-50 (NH4 +) column (6 x 110 cm). 8. The column is washed with 100 ml of deionized water and eluted with with 0.5 N ammonium hydroxide using an automatic larimeter to follow the elution process. The elution order is following: 'Remaining Kana A --5 BB-K6 --å BB-K29 --å amikacin.

The compound BB-Kll could not be detected in these experiments. 9. The amikacin fractions are combined and concentrated to one solids content of 25-30%. Dilution is done with 1 volume of methanol and seed crystals of amikacin are added. For 2 hours, slowly add 2 volumes of IPA and crisis tallization takes place at 230 for 6-8 hours. ll. The solid is filtered, washed with 35 ml of one 1: 1: 2 mixture of water, methanol and IPA and finally with 35 ml IPA. l2. The material is dried in a vacuum oven at 400 for 16-24 hours. mar. The yield amounts to 19.91-22.84 g (34-39%). IR, PMR and CMR spectral data as well as specific rotation were in complete agreement with the desired structure.

Thin layer chromatography system CHCl 3 / methanol / NH 4 OH / water (1: 4: 2: 1), 5 x 20 cm silica gel plate from Quantum Industries - 1 zone amikacin with Rf about 0.4 (nin- hydrintest).

Example 7 Preparation of amikacin by acylation of poly (trimethylsilicone) yl) -6'-N-Cbz-kana A in tetrahydrofuran with the mixed acid anhydride of pi- valinic acid and BHBA A. Preparation of the mixed anhydride 5.066 g (20.0 mmol) BHBA, 4.068 g (20.0 mmol) BSA and 2.116 g (22.0 mmol) of triethylamine was dissolved in 200 ml of screen dried tetrahydrofuran. 447 259 32 The solution was refluxed for 2.25 hours and then cooled -100. 2.412 g (20.0 mmol) of pivaloyl chloride were added over 2-3 minutes. while stirring and stirring was continued for 2 hours at -100. Tem- the temperature was then allowed to rise to 230.

B. Acylation of poly (trimethylsilyl) -6 ”-N-Cbz-Kana A 5.454 g (4.97 mmol, calculated as 6'-Cbz-Kana A (SiI1) 9) poly- (trimethylsilyl) -6'-N-Cbz-Kana A, which had been prepared as in Example pellet 1, was dissolved in 50 ml of dry (molecular sieve) tetrahydrofuran at 23 °. half of the solution of mixed anhydride prepared in step A above it (10.0 mmol) was added over a period of 20 minutes stirring and stirring was continued for 7 days. 100 ml of water were then added to the reaction mixture and The pH (5.4) was adjusted to 2.0 with 3M H 2 SO 4. Stirring was continued 1 hour and the solution was extracted with ethyl acetate. Polyacylated ma- material began to crystallize, so the reaction mixture was filtered. des. After drying over P 2 O 5, the recovered solid weighed 0.702 g. The extraction of the reaction mixture was continued with total 4 x 75 ml of ethyl acetate, after which the excess ethyl acetate was evaporated off the water layer. An aliquot of the aqueous solution was subjected to HPLC -analysis. The resulting curve indicated a yield of 26.4% di-Cbz-amino kacin.

The aqueous layer was then hydrogenated in a Parr apparatus at one hydrogen pressure of 3.5 kg / cm 2 for 2 hours using 0.5 g of a catalyst of 10% palladium on carbon. The material was filtered and the combination of filtrate and washings was subjected to analysis against E.coli and was found to contain 31.2% amikacin. The relationship amikacin: BB-K29 was about 9-10: 1; traces of polyacyl and unreacted Kana A was present.

Example 8 Effect of water on the production of amikacin by acy- poly (trimethylsilyl) -Kana A in acetone solution at 230 A. Anhydrous solvent 2.40 g (2.0 mmol, calculated as Kana A (Silyl) 10) polyphthriate methylsilyl) -Rana A, which had been prepared according to Example 3, was dissolved in 20 ml of acetone, which had been dried with a molecular sieve. The solution was stirred at 230 and a solution of 0.701 g (2.0 mmol) of SAE in 10 ml of lekyl screen dried acetone was added for 10 seconds. Stirring continued for 22 hours at 230. 50 ml of water were added and the pH (7.5) was set to 2.5. The acetone was evaporated in vacuo at 400 DEG and water. 447 259 33 the tin solution was then reduced at a hydrogen pressure of 3.6 kg / cm 2 at 230 for 2 hours using 1.0 g of a catalyst of 10% palladium on carbon. Microbiological analysis showed an exchange of 31.24% amikacin.

B. Addition of water to the solvent Step A above was repeated except that 1.0 ml (56 mmol) water was added to the poly (trimethylsilyl) -Kana A solution, which was stirred 15 minutes before acylation with SAE. Microbiological analysis they a yield of 33.80% amikacin.

Example 9 Preparation of amikacin by acylation of poly (trimethyl- silyl) -6'-N-Cbz-Kana A in acetone with the mixed anhydride of BHBA and iso- butyl carbonic acid A. Preparation of the mixed anhydride 1.267 g (5.0 mmol) of BHBA and 1.313 g (1.0 mmol) of N-trimethyl- silylacetamide (MSA) in 20 ml of molecular sieve dried acetone was stirred at 230 and 0.70 ml (5.0 mmol) of triethylamine (TEA) were added. Mixture- one was refluxed under a nitrogen atmosphere for 2.5 hours. The mixture cooled to -2 ° and 0.751 g Triethylamine hydrochloride began to precipitate immediately. The mixture is stirred dec 1 hour at -2o °.

B. Acylation 6.215 g (4.9 mmol, calculated as the (silyl) 9 compound) poly- (trimethylsilyl) -6'-N-Cbz-Kana A, which had been prepared according to Example 1, was dissolved in 20 ml of molecular sieve dried acetone at 230 with stirring. ring. The solution was cooled to -200 and the cold mixed anhydride solution one from step A was added slowly over a period of 30 minutes.

The reaction mixture was stirred for an additional 1.5 hours at -200 and then for 17 hours at 23 °. The reaction mixture was poured then in 150 ml of water at 230 with stirring, the pH (7.75) was adjusted to 2.5 with 3N HCl and stirring was continued for 15 minutes. ter. The acetone was then evaporated in vacuo at 400. An aliquot of it obtained aqueous solution was subjected to HPLC analysis. The obtained the curve indicated a yield of 34.33% di-Cbz-amikacin. IN The majority of the aqueous solution was reduced at a hydrogen pressure 2 of 3.5 kg / cm at 230 for 3.25 hours using 2.0 g of a palladium catalyst on carbon. The catalyst was removed by of filtration and the combination of filtrate and washing liquids 447 259 34 microbiological analysis against E.coli and was found to contain a yield of 35.0% amikacin.

Example 10 Preparation of amikacin by acylation of poly (trimethyl- JÉQQQ-6'-N-Cbz-Kana A and 3-pentanone 30 g (23.65 mmol, calculated as 6'-N-Cbz-Kana A (Silyl) 9) poly (trimethylsilyl) -6'-N-Cbz-Kana A, which had been prepared according to Example 1, was dissolved in 100 ml of molecular sieve dried 3-pentanone, mixed with The mixture was stirred at 230 and 26.02 mmol (10% excess) of NAE was added for a period of 40 minutes. Stirring was continued during ll3 hours at 230 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, the mixture was stirred for an additional 30 minutes and the 3-pentanone evaporated in vacuo at 400. The aqueous solution was extracted with 4 x 100 ml of ethyl acetate. An aliquot of the aqueous solution was then subjected HPLC analysis. The resulting curve indicated a yield of 46.12% di- -Cbz-amikacin.

The majority of the aqueous reaction mixture is reduced at a hydrogen pressure of 3.6 kg / cm2 at 230 for 2.5 hours using 3.0 g of a 10% palladium catalyst on carbon.

Microbiological analysis of an aliquot of the combination of filtrates and washing liquids indicated a yield of 40.24% amikacin. The main part of the reduced aqueous reaction mixture was concentrated then in vacuo at 400 to a volume of about 100 ml and fractionating was mounted on an ion exchange column (CG-50 (NH 4 +), 10 x 122 cm, containing about 10 liters of resin). The aqueous solution was applied to the column, column The mixture was washed with 5 liters of water and the material was eluted with 0.5 N NH4OH (followed by 3N NH4OH to elute polyacylated products).

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

Example ll Preparation of amikacin by acylation of poly (trimethyl- silyl) -6'-N-Cbz-Kana A in anhydrous cyclohexanone while varying reaction time A. 2.537 g (2.0 mmol, calculated as 6'-N-Cbz-Kana A (Silyl) 9) - poly (trimethylSilYl) -6'-N-Cbz-kana A, which had been prepared according to Example 1, in 300 ml of dry cyclohexanone was acylated for 20 hours at 447 259 35 23 ° with an NAE solution in dry cyclohexanone (10.8 ml of a solution containing 0.144 mmol / ml = 2.0 mmol). The reaction mixture was then added to 150 ml of water with stirring and the pH (5.6) was set at 2.5 with 3N HCl. The cyclohexanone was evaporated in vacuo at 400 and an aliquot of the remaining aqueous phase was taken for HPLC -analysis.

The majority of the aqueous phase was reduced during a hydrogen pressure of 3.5 kg / cm 2 for 3 hours at 230 using 1.0 g of a catalyst of 10% palladium on carbon. The catalyst is removed filtration and the combination of êV filtrate and washings was analyzed microbiologically for amikacin.

B. Reaction step A above was repeated except that acy- the training was allowed to last 115 hours instead of 20 hours.

Yields ÉÉEÉÉÉEEÄXÉ Microbiological analysis (amikacin) (di-Cbz-amikacin) Turbidimetric Plate Reaction A 49.18% 42.87% 39.16% Reaction B 56.17% 55.39% 38.45% Example 12 Preparation of amikacin by acylation of poly (trimethyl- silyl) -6'-N ~ Cbz-Kana A in anhydrous tetrahydrofuran under variation ..of the reaction time A. Example 11A was repeated except that dry tetra- hydrofuran was used as solvent instead of dry cyclo- hexanone.

B. Example II B was repeated except that dry tetra- hydrofuran was used as solvent instead of dry cyclo- hexanone.

Yields HPLC ~ analysis Microbiological analysis Gm fl æmin) (di-Cbz-amikacin) Turbidimetric Plate Reaction A 29.27% 28.34% _ 328.18% Reaction B 33.39% 21.52% 28.63% 447 259 36 Example 13 Preparation of amikacin by acylation of poly (trimethyl- silyl) -6'-N-Cbz-Kana A in anhydrous dioxane while varying the reaction tion time A. Example 11A was repeated except that the acylation continued for 44 hours using dry dioxane as the solvent. average.

B. Example II B was repeated except for the acylation 18.5 hours using dry dioxane as the solvent. average.

Utbvten HPLC Assay Microbiological Assay (Amikacin) (di-Cbz-amikacin) Turbidimetric Plate Reaction A 39.18% 43.27% 33.36% Reaction B 42.82% 22.55% 33.37% Example 14 Preparation of amikacin by acylation of noly (trimethyl- silyl) -6'-N-Cbz-Kana A in anhydrous diethyl ketone at 750 To a stirred solution of 2.537 g (2.0 mmol, calculated as) 6'-N-Cbz-Kana A (Silyl) 9) poly (trimethylsilyl) 6'-N-Cbz-Kana A, which had been prepared according to Example 1, in 32 ml of molecular sieve dried diethyl ketone at 750 was added a solution of NAE (10.8 ml of a diethyl ketone solution containing 0.144 mmol / ml = 2.10 mmol) for 15 minutes.

Stirring was continued at 750 for another 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 in vacuo at 400. HPLC analysis of an aliquot of the aqueous phase indicated a yield of 39.18% di-Cbz -amikacin.

The majority of the aqueous phase was reduced under a hydrogen pressure of 3.5 kg / cm 2 for 3.25 hours at 230 using 1.0 g of a catalyst of palladium on carbon. The catalyst was removed by filtration and the combination of filtrates and washings microbiologically with respect to amikacin. Turbidimetnic ana- lys showed a yield of 27.84% and plate analysis gave a yield of 28.6%. 447 259 37 Example 15 Preparation of amikacin by acylation of poly (dimethyl- silyl) -Kana A with NAE at 0-50 after hydrolysis with water A. Silylation of kanamycin A using HMDS with TMCS such as catalyst 10 g of kanamycin A (97.6% purity, 20.14 mmol) in 100 ml of molar cold sieve dried acetonitrile was refluxed under a nitrogen atmosphere. One mixture of 22.76 g (141 mmol, 7 moles per mole of kanamycin A) HMDS and 1 ml (0.856 g, 7.88 mmol) of TMCS was added to the refluxing reaction. the mixture for 10 minutes. The reflux was allowed to continue 4.75 hours and the mixture was then cooled, concentrated in vacuo to a yellow viscous syrup and dried in high vacuum for 2 hours.

The product yield was 23.8 g (97.9% calculated as kanamycin A (silyl) 10).

B. Acylation 23.8 g (20.14 mmol) of the compound prepared in step A above no poly (trimethylsilyl) -kanamycin A was dissolved in 250 ml of molecular screen dried acetone at 23 ° and then cooled to 0-50. 3.63 ml (20l, 4 mmol, 10 mol per mol polysilylated kanamycin A) water added was added with stirring and the mixture was allowed to stand for 30 minutes in moderation vacuum. 19.133 mmol (0.95 mol per mol polysilylated kanamycin A) NAE in 108.3 ml of acetone was added over a period of less than 1 ml. nut. The mixture was stirred at 0-50 for 1 hour and then diluted. with water, after which the pH was adjusted to 2.5 and the acetone was removed in vacuo. The aqueous solution was then reduced at one hydrogen pressure of 3.5 kg / cm2 at 230 for 2.5 hours during use of 2.0 g of a catalyst of 10% palladium on carbon. It reduced the reaction mixture was filtered through Dicalite, concentrated to a volume of about 100 ml in vacuo at 400 and then applied to and CG-50 (NH4 +) column (6 liters of resin, 5 x 100 cm). It was washed with water and eluted with 0.6 N-1.0N-3N NH 4 OH. 60.25 were obtained % amikacin, 4.37% BB-K6, 4.35% BB-K29, 26.47% kanamycin A and 2.18% polyacyl material.

Example 16 Preparation of amikacin by acylation of poly (trimethyl- silyl) -6'-N-Cbz-Kana A with SAE at 0-50 after methanolysis A. Silylation of 6'-N-Cbz-kanamycin A 20.0 g (32.4 mmol) of 6'-N-Cbz-kanamycin A in 200 ml of molecular sieve dried acetonitrile was refluxed under a nitrogen atmosphere. 47.3 ml 447 259 38 (226.8 mmol, 7 moles per mole of 6'-N-Cbz-Kana A) HMDS was added below a 10-minute period and refluxing was continued for 20 minutes hours. The mixture was then cooled, concentrated in vacuo and dried in high vacuum for 2 hours to give 39.1 g of a white, amorphous solid (95.4% yield calculated as 6'-N- -Cbz-Kana A (Silyl) q).

B. Acvlation 39.1 g (32.4 mmol) of poly (trimethylsilyl) -6'-N-Cbz-Kana A, which had been prepared according to step A above, was dissolved in 400 ml of dry acetone at 230 while stirring. 6.6 ml (162 mmol, 5 mol per mol polysilyl- 6'-N-Cbz-Kana A) methanol was added and the mixture was stirred at 230 during 1 hour under heavy nitrogen purge. The mixture cooled to 0-50 and a solution of 11.35 g (32.4 mmol) of SAE in 120 ml of pre-cooled, dry acetone was added. The mixture was stirred for an additional 3 hours at 0-5 ° and then placed in a refrigerator. room at a temperature of 4 ° for 1 week. 300 ml of water were added, pH the value was set at 2.0, the mixture was stirred for 1 hour and the acetone was evaporated in vacuo. The resulting aqueous solution was reduced at a hydrogen pressure of 3.8 kg / cm2 for 17 hours at 230 during using 3.0 g of a 10% palladium catalyst on carbon. Loose- The mixture was then filtered through Dicalite, concentrated in vacuo to a volume of 75-100 ml, was applied to a CG-50 (NH 4 +) yolon and eluted with water and 0.6N NH 4 OH. S2.52% of amikacin was obtained, 14.5% BB-K29, 19.6% kanamycin A and 1.71% polyacyl material.

Example 17 Preparation of amikacin by acylation of poly (trimethyl- silyl) -Kana A with SAE at 0-50 after hydrolysis with water A. Silylation of kanamycin A with TMCS in acetonitrile below use of tetramethylguanidine as acid accent 4.88 g (10.07 mmol) of kanamycin A were suspended in 100 ml of molar cold-dried acetonitrile at 230 with stirring. To the stirred To the suspension was added 16234 g (140.98 mmol, 14 moles per mole of kanamycin A) tetramethylguanidine (TMG). The mixture was refluxed at 1.52.32 g (140.98 mol, 14 mol per mol kanamycin A) TMCS was added under 15 minutes. A white precipitate of TMG.HCl was formed after approx half of TMCS had been added. The mixture was cooled to room temperature. concentrated to a sticky residue and dried in high pressure. kuum for 2 hours. The solid was triturated with 100 ml 447 259 39 dry THF and the insoluble precipitate of TMG.HCl were filtered off and was washed with 5 x 20 mL of THF. The combination of filtrate and laundry shoes were concentrated in vacuo at 400 to a sticky residue and dried in high vacuum for 2 hours. 10.64 g of a candle were obtained cream-colored, sticky residue (87.6% yield calculated as kanamycin A (Silyl) 10).

B. Acvlation 10.64 g (IQ07 mmol) of poly (trimethylsilyl) -kanamycin A, which has they were prepared according to step A above, dissolved in 110 ml of molecular sieve dried acetone with stirring at 230 and the solution was then cooled to 0-50. 1.81 ml of water (100.7 mmol, 10 mol per mol of polysilylated Kana A) water was added and the solution was stirred for 30 minutes in moderate vacuum. 3.70 g (10.57 mmol, 5% excess) SAE in 40 ml of pre-cooled, dry acetone was added over a period of less than 1 minute and the mixture was stirred for 1 hour. The mixture is worked up was carried out according to the general procedure described in Example 16B, when about 50% amikacin was obtained, about 10% BB-K29, 5-8% BB-K6, about 20% kanamycin A and 5-8% polyacyl material.

Example 18 Preparation of poly (trimethylsilyl) -kanamycin A in pyridine using HMDS 10.0 g (20.64 mmol) of kanamycin A were suspended in 100 ml of molar cold-dried, freshly distilled pyridine at 230. Nitrogen gas purge began and the suspension was refluxed. l7.33 g (l07.32 mmol, 5.2 moles per mole of kanamycin A) HMDS was added over 10 minutes and the mixture was refluxed for 19 hours. It was then cooled to room temperature, concentrated in vacuo to a light golden color. syrup and dried in a high vacuum to a white, amorphous solid. rial. 22.1 g (92.6% yield calculated as kanamycin A) were obtained. lyl) l0).

Example 19 Preparation of noly (triethylsilyl) -kanamycin A using triethylchlorosilane with triethylamine as acid acceptor, 5.0 g of kanamycin A (97.6% purity, 10.07 mmol) was suspended in 100 ml of viscous-dried acetonitrile at 23 °. 33.8 ml (24, g, 241.7 mmol) of triethylamine (TEA) was added and the suspension was refluxed. kokades. A solution of 23.7 ml (2.1 g, 140.98 mmol) of trichlorethylsilicone lan in 25 ml of dry acetonitrile was added over 20 minutes. Reflux 447 259 40 boiling was continued for another 7 hours and the mixture was cooled. to room temperature, whereupon long fine needles of TEA.HCl precipitated.

The mixture was allowed to stand at room temperature for about 16 hours, concentrated was dried in vacuo at 400 to a sticky solid residue and dried for 2 hours in a high vacuum to a dark orange, sticky solid material. The solid was triturated with 100 mL of dry THF at z3 ° and insoluble -rßzhncl avfiitreraaes, washed with s x zo m1 THF and dried to give 16.0 g of TEA.HCl. The combination of filtrate and washings were concentrated in vacuo to a solid and dried in high vacuum for 2 hours. 19.3 g of powder were obtained. 1y (triethylsilyl) kanamycin A as a dark orange, viscous silica rap.

Example 20 Preparation of poly (trimethylsilyl) -kanamycin A using bis-trimethylsilylurea 10.0 g of kanamycin A (99.7% purity, 20.58 mmol) was suspended in 200 ml of molecular sieve dried acetonitrile with stirring at 230. To to the suspension was added 29.45 g (144.0l mmol, 7 mol per mol kanamycin) - bis-trimethylsilyl urea (BSU) and the mixture was refluxed. there nitrogen atmosphere. Refluxing 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 acetonitrile and dried to give l, l38l g material. Infrared analysis showed that this material was BSU plus a small amount of unreacted kanamycin A. The combination of filtrates and washings were cooled at 40 for 16 hours. Additional fixed ma- material precipitated, and was recovered as described above (7.8 g). Infrared- analysis showed that this material was BSU + urea. The light yellow the filtrate and washings were concentrated in vacuo at 400 ° C and dried. in a high vacuum to give 27.0 g of a white solid. al, which was partly tacky and partly composed of fine, needle-like the crystals. The solid was treated with 150 ml of heptane at 230, the insoluble portion was removed by filtration, sealed with 2 x 50 ml heptane and dried to give 6.0 g of white needles (infrared analysis showed that this material was grounded by BSU plus carbon amid). The combination of filtrate and washings was concentrated in vacuum at 400 and dried in high vacuum for 2 hours to obtain of 20.4 g of white needles, the infrared spectrum of which was typical of lylated kanamycin A. Calculations showed that the product contained in 447 259 41 average 7.22 trimethylsilyl groups.

Example 21 Preparation of amikacin by acylation of per (trimethyl- silyl) -kanamycin A after partial desilylation with 1,3-butanediol A. Preparation of down (trimethylsilyl) -kanamycin A 10.0 g (20.639 mmol) of kanamycin A was suspended in 100 ml of lyophilized acetonitrile at 230 with stirring. The suspension was refluxed under nitrogen purge and 23.322 g (444.5) mmol, 7 mol per mol kanamycin A) HMDS was added over a period of time of 10 minutes. Refluxing was continued for 16 hours and the mixture was then cooled to room temperature, concentrated in vacuum and dried in high vacuum for 2 hours. 24.3 g were obtained of a white, sticky residue (92.1% yield calculated as kanamycin A) (silyl) II).

B.Acylation 24.3 g per (trimethylsilyl) kanamycin A, which had been prepared according to step A above, was dissolved in 240 ml of molecular sieve dried acetone at 230 while stirring. To this solution was added 9.25 mL (10.2 mmol, 5 moles per mole per (trimethylsilyl) -kanamycin A) 1,3-butanediol. Among- The mixture was stirred for 2 hours at 230 with nitrogen permeation and cooling. was then reduced to o-5 °. 7.23 g 00.64 mmol) SAE in 70 ml of off in advance cooled acetone was added over a period of about 1 minute. The mixture was stirred at 0-50 for 3 hours and then allowed to stand in a cold room at 4 ° for about 16 hours. 200 ml of water were added, the pH was adjusted was set at 2.5 and the bright yellow solution was stirred for 30 minutes at 230. The acetone was evaporated in vacuo and the aqueous solution was reduced at a hydrogen pressure of 3.9 kg / cm2 at 230 for 2 hours during use 3.0 g of a 10% palladium catalyst on carbon. The reduced The solution was filtered through Dicalite and chromatographed. as in Example 16 B to give 47.50% amikacin, 5.87% BB-K29, 7.32% BB-K6, 24.26% kanamycin A and 7.41% polyacyl material.

Example 22 Preparation of amikacin by acylation of poly (trimethyl- silyl) -kanamycin A prepared in THF using SAE with sulfur famsyra catalyst To a refluxing mixture of 5.0 g (10.32 mmol) of ca- Namycin A in 50 ml of molecular sieve dried tetrahydrofuran (THF) was added 100 mg sulfamic acid and 12.32 g (76.33 mmol) of HMDS. Mixture reflux 447 259 42 boiled for 18 hours, at which time complete dissolution occurred after 6 hours. The solution was cooled to 230, treated with 0.1 ml water and kept at 230 for 30 minutes. A solution of 3.61 g (10.3 mmol) of SAE in 36 ml of THF was added over a period of 30 ml. nuter. After stirring for 3 hours, the mixture was diluted with 100 ml water and the pH was adjusted to 2.2 with 10% H 2 SO 4. Mixture- one was stirred at 23 ° for / 30 minutes and then concentrated in vacuum to remove THF. The resulting aqueous solution was was produced at a hydrogen pressure of 3.5 kg / cm 2 for 2 hours at 230 using a catalyst of 10% palladium on carbon. The re- the diluted solution was filtered through Dicalite and the solid The wash was washed with water. The combination of filtrate and washing liquids (150 ml) was subjected to microbiological analysis against E. coli and found contain 1225 pg / ml (31.5% activity yield) of amikacin.

Example 23 Preparation of amikacin by acylation of poly (trimethyl- silyl) -canamycin A with the N-hydroxysuccinimide ester of dicarbobenzyl- oxy-AHBA A. Preparation of dicarbobenzyloxy-L - (-) - alpha-amino-alpha- -hydroxybutyric acid-N-hydroxysuccinimide ester 8 g (20.65 mmol) of dicarbobenzyloxy-L - (-) - alpha-amino-alpha-hydro- oxybutyric acid and 2.37 g (20.65 mmol) of N-hydroxysuccinimide were dissolved in so m1 dry acetone at 23 °. 4.25 g (20.6 mmol) of acycionexycarbodi imide, which had been dissolved in 20 ml of dry acetone, was added and whole The mixture was stirred for 2 hours at 230 Dicyclohexylurea. The filter cake was washed with 10 ml of dry acetone and the filtrate the washings were combined.

B. Acvlation Poly (trimethylsilyl) -kanamycin A, which had been prepared according to the general procedure of Example 21 starting from 10.0 g (20.639) mmol) kanamycin A, was dissolved in 100 ml of dry acetone. The solution was cooled to 0-50, 3.7 ml of deionized water was added and the solution was stirred. at 0-50 for 30 minutes under moderate vacuum. _ g To this solution was added the solution prepared in step A. one of the Cbz-blocked acylating agent and the mixture stirred at 0-50 for 30 minutes. The mixture was diluted with water, The pH was adjusted to 2.2 and the acetone was removed in vacuo. Vat- The solution was reduced by the general procedure of Examples 22 and then filtered through Dicalite. Chromatography expelled 447 259 43 40-45% amikacin, about 10% BB-K29, traces of BB-K6, about 30% kanamycin A and a small amount of polyacyl material.

Example 24 Preparation of poly (trimethylsilyl) -kanamycin A under reversal of HMDS with imidazole as catalyst 11 9 (22.7 mmol) kanamycin A and 100 mg imidazole reflux was boiled in 100 ml of molecular sieve-dried acetonitrile under nitrogen blowing. 18.48 g (11.5 mmol, 5 mol per mol kanamycin A) HMDS added was allowed to stand for a period of 30 minutes and the mixture was refluxed. for 20 hours. Full resolution occurred within about 2.5 hours. The solution was cooled to 230 and the solvent was removed in vacuum to give 21.6 g of poly (trimethylsilyl) -kanamycin A so-called as a foamy residue (93.1% yield calculated as kanamycin (silyl) II).

Example 25 Preparation of 1-N- / L - (-) - 7? -amino-alpha-hydroxybutyl / kanamycin B (BB-K26) by acylation of poly (trimethylsilyl) kanamycin cin B with SAE A. Preparation of poly (trimethylsilyl) kanamycin B using reversal of HMDS with TMCS catalyst 25 g (51.7 mmol) of kanamycin B in 250 ml of molecular sieve dried acetone tonitrile was refluxed under a stream of nitrogen. 62.3 g (385.8l mmol, 7.5 moles per mole of kanamycin B) HMDS was added over a period of 30 minutes, followed by 1 ml of TMCS as catalyst. The mixture is 10 ?? Sk0k fi deS for 21 hours with complete dissolution after 1 hour. The solvent was then removed in vacuo at 600 and the oil the residue was kept at 600 in high vacuum for 3 hours. 53.0 were obtained g of poly (trimethylsilyl) -kanamycin B (85.2% yield calculated as mycin B (silyl) 10).

B. Acylation 53.0 g of the compound poly (tri- methylsilyl) -kanamycin B was dissolved in 500 ml of dry acetone at 0-50, 20.9 ml of methanol were added and the mixture was stirred in vacuo at 0 ~ 5 ° for 30 minutes. A solution of 18.1 g (51, 67 mmol) of SAE in 200 ml of pre-cooled, dry acetone was added over a period of less than 1 minute and the mixture was stirred at 0-50 for 30 minutes. ter. The mixture was worked up according to the general procedure in Example 22 and then applied to a column of CG-50 (NH 4 +) (8 x 120 cm). It was eluted with an NH 4 OH gradient from 0.6N to 3N. 447 259 44 38% of BB-K26, 5% of the corresponding 6'-N-acylated kanamycin were obtained B (BB-K22), 10% of the corresponding 3-N-acylated kanamycin B (BB-K46), 14.63% kanamycin B and a small amount of polyacylated kanamycin B.

Example 26 Preparation of poly (trimethylsilyl) -kanamycin A under reversal of HMDS with kanamycin A sulfate as catalyst 19.5 g (40.246 mmol) of kanamycin A and 0.5 g (0.858 mmol) of kanamycin namycin A sulphate (total = 20.0 g, 41.0 mmol) in 200 ml molecular screen dried acetonitrile was refluxed. 60.3 ml (287.7 mmol, 7 mol per mol kanamycin A) HMDS was added slowly and mixed The mixture was refluxed for 28 hours. It was then evaporated to dryness in a rotary evaporator and dried under vacuum. 47.5 g of poly (trimethylsilyl) -kanamycin A were obtained as a pale yellow oil (95.82% yield calculated as kanamycin A (silyl) 10).

Example 27 Preparation of amikacin by acylation of poly (trimethyl- silyl) -cannamicin A with the N-hydroxysuccinimide ester of N-trifluoroacetyl- blocked AHBA A. Preparation of N-trifluoroacetyl-AHBA and conversion thereof of to its N-hydroxysuccinimide ester To a suspension of 5.0 g (42 mmol) of AHBA in 10 ml of THF 40 g (191 mmol) of trifluoroacetic anhydride for a period of 10 minutes while stirring. The solution was stirred at 23 ° for 18 hours. and then concentrated to dryness in vacuo at 500 DEG. The column was dissolved in 100 ml of a 1: 1 mixture of water and methanol and stirred for 1 hour. It was then concentrated to dryness in vacuo and redissolved in 50 ml of water. The aqueous solution is extracted was prepared with 3 x 50 ml MIBK and after drying over sodium sulphate the extract was concentrated to an oil. Traces of solvent were removed by adding and distilling off 4 ml of water. Then the oil was allowed to stand it was converted to a waxy crystalline solid (2.5 g, 28% yield). g 2.4 g (11, 3 mmol) of N-trifluoroacetyl-AHBA were dissolved in 50 ml dry acetone and 1.30 g (11.3 mmol) of N-hydroxysuccinimide were added solution. A solution of 2.33 g of dicyclohexylcarbodiimide in 20 ml of dry acetone was added slowly. The reaction mixture was stirred at 230 ° C. 2 hours and the precipitated dicyclohexylurea was removed by filtration and washed with a small amount of acetone. The combination 447 259 45 of filtrates and washings (a solution of the N-hydroxysuccinimide ester) of N-trifluoroacetyl-AHBA) was used in the following steps without isolation.

B. Acylation To a solution of 11.31 mmol of poly (trimethylsilyl) -kanamycin A, which had been prepared as in Example 26, in 54 ml of acetone was added 2.0 ml (13.4 mmol) of water and the mixture was stirred in vacuo at O-50 for 30 minutes. 11.3 mmol of that prepared in step A above The N-hydroxysuccinimide ester of N-trifluoroacetyl-AHBA was added to the mixture. which was then maintained at 50 for 1 hour. The pH value is set was then added to about 2.0 with 20% sulfuric acid, the mixture was stirred for 30 minutes and the pH was then raised to about 6.0 with NH 4 OH.

The mixture was then evaporated to dryness in a rotary evaporator. to obtain 14.4 g of a sticky, off-white solid.

The solid was dissolved in 100 ml of water, the pH was raised from 5.5 to 11.0 with 10N NH 4 OH and the solution was heated on an oil bath at 700 in 1 hour. The pH (9.5) was then lowered to 7.0 with HCl, the solution was clear filtered to remove a small amount of insoluble matter material and the filter was washed with water. The combination of filtrates and washings (188 ml) were applied to CG-50 (NH 4 +) column (8 x 90 cm), washed with 2 liters of water and eluted with an NH ent (0.6N-1.0N-concentrated). 28.9% of amikacin, 5.0% of BB- -K6, S, 7% BB-K29, 43.8% kanamycin A, 3.25% polyacyl material plus 14.3% of an unknown material, which was found in the first fraction from the column.

Exemgel 28 Preparation of amikacin by acylation of poly (trimethyl- silyl) -kanamycin A with the N-hydroxysuccinimide ester of t-butyloxycarb onyl-blocked AHBA A. Preparation of t-BOC-AHBA and Conversion thereto N-hydroxysuccinimide ester A solution of 5.0 g (42 mmol) of AHBA in 100 ml of water and 20 ml ml of acetone was adjusted to pH 10 with 10N NaOH. During a period of time of For 3-4 minutes, 11.6 g (53 mmol) of di-t-butyl dicarbonate were added, and the solution was stirred for 35 minutes while maintaining the pH at 10 by periodic addition of 10N NaOH. The acetone was removed in vacuo and the aqueous phase was washed with 40 ml of ethyl acetate. PH of the aqueous solution value was reduced to 2.0 with 3N HCl and then extracted with 3 x 30 ml MIKB. The combined MIBK extracts were dried over sodium 447 259 46 sulfate and concentrated to a clear oily residue (8.2 g, 89%), 4.25 g (19.4 mmol) of t-BOC-AHBA were dissolved in 50 ml of acetone and 2.23 g (19.4 mmol) of N-hydroxysuccinimide were added. A solution of 4.00 g (19.4 mmol) of dicyclohexylcarbodiimide in 20 ml of acetone were added slowly and the mixture was stirred for 2 hours at 230. The precipitated di the cyclohexylurea was removed by filtration and washed with a small amount of acetone. The combination of filtrate and washing liquids (a solution of the N-hydroxysuccinimidister of t-BOC-AHBA) was used in the following steps without insulation.

In B. Acylation To a solution of 41.28 mmol of poly (trimethylsilyl) -kanamycin A, which had been prepared according to Example 26, in 94 ml of acetone was added 3.5 ml (194 mmol) of water and the mixture was stirred in vacuo for 30 minutes. at 0-5 °. 19.4 mmol of the N-hydroxy- the succinimide ester of t-BOC-AHBA was added and the mixture was allowed to stand 1 hour at 50. 200 ml of water were added and the pH (7.0) was lowered. to 2.0 with 20% sulfuric acid. After stirring for 30 minutes, the the pH was adjusted to about 6.0 with NH 4 OH and the mixture was evaporated to dryness in vacuo to give 36.3 g of a gold-colored oil. Ol- Jan was dissolved in 200 ml of trifluoroacetic acid, allowed to stand for 15 minutes and evaporated to dryness in a rotary evaporator. The oil was washed with water and the water was evaporated. Concentrated NH 4 OH was added pH 6.0 and evaporated. The resulting oil was dissolved in water and filtered. and the filter was washed with water. The combination of filtrates and washings (259 ml) were applied to a CG-50 (NH 4 +) column (8 x 92 cm), washed with 4 liters of water and eluted with an NH gradient (0.6N-1.0N-concentrated). 40.32% of amikacin were obtained. 4.58% BB-K6, 8.32% BB-K29, 30.50% kanamycin A and 7.43% polyacyl terial- Example 29 The general procedure of Example 1 was repeated except that the compound 6'-N-carbobenzyloxycanicide used therein mycin A was replaced with an equimolar amount of 6'-N-carbobenzyloxycanamic acid. cin B to give i-N- / L- (q- a * -amino-aifa-hyaroxybutyryu- -kanamycin B.

Exemgel 30 The general procedure of Example 1 was repeated with assuming that the L - (-) - Z'-benzyloxycarbonylamino-alpha- 447 259 47 -hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester was added with L - (-) - beta-benzyloxycarbonylamino-alpha-hydroxypropionate acid-N-hydroxy-5-norbornene-2,3-dicarboximide ester, respectively L - (-) - 8'-Benzyloxycarbonylamino-alpha-hydroxyvaleric acid-N-hydro- oxy-5-norbornene-2,3-dicarboximide ester to give 1-N- [L- (-) - beta-amino-alpha-hydroxypropionylphkanamycin A respectively 1-N-ÅL - (-) - 8'-amino-alpha-hydroxyvalerylphanamycin A.

Example 31 The general procedure of Example 25 was repeated except for that the L - (-) - 3β-benzyloxycarbonylamino-alpha- the hydroxybutyric acid N-hydroxy ester was replaced by L - (-) - beta-benzyloxy- carbonylamino-alpha-hydroxypropionic acid N-hydroxysuccinimide ester respectively L - (-) - 6 -benzyloxycarbonylamino-alpha-hydroxyvalerian- acid N-hydroxysuccinimide ester to give 1-N-L - (-) - beta- amino-alpha-hydroxypropionylphkanamycin B and 1-N- [I - (-) - 6-amino-alpha-hydroxyvalerylphanamycin B.

Example 32 Preparation of amikacin by acylation of poly (tri- methylsilyl) -3,6 "-di-N-carbobenzyloxycanamycin A in anhydrous di- ethyl ketone A. 3,6'-Di-N-carbobenzyloxycanamycin A A suspension of 7.26 g (15 mmol) of kanamycin A in the form of the free base and 18.6 g (75 mmol) of nickel acetate tetrahydrate in 300 ml of dimethyl sulfoxide (DMSO) was heated at 1000 for about 30 minutes while stirring until a clear green solution was obtained. ning. After cooling, a solution of 11.8 g (37.6 mmol) was added. N-carbobenzyloxy-5-norbornene-2,3-dicarboximide 50 ml DMSO to solution. The mixture was stirred at room temperature overnight. was treated with 100 ml of concentrated ammonia water and 1 liter water, stirred at room temperature for 1 hour and applied on top of a Diaion HP-10 column (300 ml). The column below stepwise elution was started, starting with 7N NH 4 OH, then after methanol-water in a volume ratio of 1: 1 and finally methanol-water in a volume ratio of 10: 1. 20 ml fractional was elucidated and the elution was monitored by means of layer chromatography on Merck 60 F-254 silica gel plates using a mixture of chloroform, ethanol and 28% ammonium hydroxide in a volume ratio of 1: 2: 1. A share of 447 259 48 the desired product, which crystallized as fine needles from fractions containing the product in high concentrations, was filtered to obtain an analytical sample. The filtrate and other fractions containing the desired product (Rf 0.42) combined and the solution was evaporated in vacuo. The remainder triturated with diethyl ether to give a total of 9.7 g (86%) of the title product with a melting point exceeding 3000. IR (KBr) v¶c = o l690cm_l. NMR (DMSO-d 6 + DC1, pm ca 3) = å '4,76-s, 2e (psn, m, nl', nl "and cø-oggz-cßns x z), 7.26 (10H, s, CO-OCHZ-§6§5 x 2).

Analysis Calculated for C 34 H 48 N 4 O 15 -H 2 O: C 52.98, H 6.54, N 7.27 Found: C 53.20, H 6.42, N 7.04.

B. Poly (trimethylsilyl) -3,6'-di-N-carbobenzyloxycanamycin A A mixture of 1.5 g (2 mmol) of 3,6'-di-N-carbobenzyloxy- kanamycin A from step A above and 1.29 g (8 mmol) of hexamethyldisilate zan in 15 ml of dry acetonitrile was refluxed for 16 hours. The The clear solution was concentrated to dryness in vacuo and the residue it was dissolved in 20 ml of dry diethyl ketone. The solution was used immediately in subsequent steps.

C. Acylation of poly (trimethylsislyl) -3,6'-di-N-carbobenzyl syloxy-kanamycin A using an equimolar amount of the acylating agent To the solution from step B above was added with stirring 700 mg (2 mmol) L - (-) - alpha-carbobenzyloxyamino-alpha-hydroxybutyric acid N-hydroxysuccinimide ester (SAE). The mixture was stirred at room temperature. temperature for 19 hours, then treated with 8 ml of water and 35 ml of tetrahydrofuran (THF), were adjusted to pH 3 with an aqueous solution of hydrochloric acid, stirred for 30 minutes and concentrated to dryness in vacuo. The residue was dissolved in a mixture of 30 ml of water, 40 ml of methanol, 10 ml of n-butanol and 40 ml of THF and was prepared overnight in the presence of 500 mg of 10% palladium on The catalyst was removed by filtration and the filtrate was evaporated. was evaporated in vacuo and lyophilized to give 1.7 g of crude amikacin. The amorphous powder was redissolved in water and the solution was adjusted to pH 4 with an aqueous solution of hydrochloric acid and was matographed on a column of Amberlite CG-50 in the NH 4+ cycle. 447 259 " 49 The column was subjected to stepwise elution with water, 0.1 N NH 4 OH, 0.3 N NH 4 OH, 0.5 N NH 4 OH and 2 N NH 4 OH, taking up 10 ml fractions and monitored the elution using thin layer chromatography using Merck silica gel plates 60 F-254 using a mixture of chloroform, methanol, 28% ammonium hydroxide and water in a volume ratio of 1: 4: 2: 1.

The homogeneous fractions were combined, evaporated and lyophilized. was finally sered. Fractions containing amikacin and fractional containing recovered kanamycin A was analyzed using reversal of K. pneumoniae A20680 resp. B. subtilis PCl 129.

The following results were obtained.

Fraction NH OH Weight Yield Product Rf- No. (3) (%) value 32-39 0.1, 0.3 102 mg partially blocked row product 40-46 0.3 174 mga 7 ° kanamycin A i 0.42 47-59 0.3, 0.5 l06 mg unidentified 0.33 product 60-78 0.5 816 mgb 67 ° amikacin 11.16 89-95 2 70 mg 5 diacylkanamycin A 0.5 (a) 408 pg / mg (b) 956 pg / mg (c) based on bioassay Hydrogenation of the partially unblocked product with Pd-C, followed by isolation on a CG-50 column, gave an additional 30 mg (2%) amikacin. The total yield of amikacin was thus to 846 mg (69%).

D. Acylation of poly (trimethylsilyl) -S fi'- di-N-carbobenzyloxy- kanamycin A using 1.2 equivalents of acylating agent Step C above was repeated except that one was used 20% excess acylating agent. The following results were obtained.

Fraction NH4OH Weight Yield Product Rf- No. (%) value (N) 28-29 0.2 107 mg partially unblocked row product 447 259 50 30-41 0.2 157 mg unidentified 0.35 product 42-52 0.3 120 mg unidentified 0.30 product 53-81 0.3, 0.5 750 mga sob amikacin o, 14 94-116 l, 2,147 mg ll diacylkanamycin A 0.05 (a) 933 pg / mg (b) based on bioassay Hydrolysis of the partially blocked product with Pd-C, followed by isolation on a CG-50 column, gave a further 21 mg (2%) amikacin. The total yield of amikacin was thus to 771 mg (62%).

E. Acylation of polyltrimethylsilyl-3β'-di-N-carbobenzyl- loxy-kanamycin A using 1.5 equivalents of acylation average Step C above was repeated except that one used a 50% excess acylating agent. The following results were obtained.

Fraction NH4OH Weight Yield Product Rf- No. (N) (%) value (double-frame calling) 29-39 0.2 293 mg ”partially blocked row product 40-47 0.3 95 mga e ° kanamycin A 0.67 65-so 0.5 582 mgb 29 ° amikacin 0.33 100-130 l, 2,543 mg 26 diacylkanamycin A 0.09 (a) 860 pg / mg (b) 880 μg / mg (c) based on bioassay

Claims (6)

s: 447 259 PATENT REQUIREMENTS A process for the preparation of a 1-N- [1 - (-)% fl-amino-alpha-hydroxyalkanoyl] kanamycin A or B of the formula C 6 -C 18 N 2 O 2 wherein R is OH or NH and n is an integer from 0 to 2, 2 or a non-toxic pharmaceutically acceptable acid addition salt thereof, characterized in that polysilylated kanamycin A or B or polysilylated kanamycin A or B containing another blocking group is acylated in a manner known per se. the amino group, which blocking group has the formula 10 1. R f: W czzoc-, cna-clz-o-c-, Yzxeå I ca3 2 10 15 20 35 447 259 _ 52 ÖIS: O å. 'Or R or, X C-CH -O-C- 3 2 C, /! H 0 wherein R 1 and R 2 are the same or different and each represents H, F, Cl, Br, NO 2, OH, lower alkyl or lower alkoxy, X is Cl, Br, F or I and Y is H, Cl, Br, F or I and wherein the polysilylated kanamycin derivative contains 2-10 silyl groups per molecule preferably on average 4-8 silyl groups per molecule or 3-7 silyl groups per molecule in case the polysilylated kanamycin A or B starting material contains a blocking group other than silyl of 6 the amino group, and the silyl groups are tri- (lower alkyl) silyl groups, with an acylating derivative of an acid in the L - (-) - form of the formula B-HN-CH 2 - (cnz) n-gn-coo II (II) of : wherein n is an integer from 0 to 2 and B is a conventional amino blocking group, in an organic solvent containing up to 25% water and then in a manner known per se removes all blocking groups. 2. A process according to claim 1, characterized in that the acylating derivative of the acid is its active ester but N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboxyimide or N-hydroxyphthalimide. 10 15 53 447 259 3. A process according to claim 2, characterized in that the amino-blocking group on the acylating derivative of the acid is a carbobenzyloxy group. 4. A process according to claim 1, characterized in that the acylating derivative of the acid is its mixed acid anhydride with pivalic acid, benzoic acid, isobutyl carboxylic acid or benzyl carbonic acid. 5. A process according to any one of claims 1-4, characterized in that the polysilylated kanamycin A or B contains a carbobenzyloxy group on the 6'-amino group. Process according to any one of claims 1-5, characterized in that the silyl groups are trimethylsilyl groups.