SE461148B - POLYSILYLATED CANAMYCIN A OR B DERIVATE - Google Patents

Description

461 148 ' 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 OH. wherein n is an integer from 0 to 2 and B is an amino blocking group, and then removes all blocking groups.

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. given as derivatives of kanamycin A or B instead of indicated by structural formulas, so as to avoid the need to compare complex structures in order to identify differences. 6 ' CEZNRZ 4! HO Kanamycin A: R I OH Kanamycin B: R I NH2 461 148 U.S. Patent No. 3,781,268 discloses and anwaldbbhï fl mmwu fi mwmnmwwyhmwunAhmm- cin) and B as well as their mono- and dicarbobenzyloxy protected derivatives.

Lower and higher homologues thereof are described in U.S. Pat. the articles 3 886 139 and 3 904 597. The associations are produced by acylating a 6'-N-protected kanamyoin A or B with an acyl- derivative of an N-protected L - (-) - zf -amino-alpha-hydroxybutyric acid in an aqueous medium, after which one or both N-protecting groups _perna removed. U.S. Patent No. 974,131 discloses: a description of a process for the preparation of 1- / L - (-) - a4amino-alpha-hydride oxybutryl / kanamycin A, which process involves reacting 6'-carbobenzyloxycanamycin A with at least 3 moles of benzaldehyde, a sub- substituted benzaldehyde or pivaldehyde to produce 6'-N-I -carbobeneyloxycanamycin A, which contains Schiff base groups in 1-, 3- and the 3 "positions, aylate this tetrasodically protected kanamycin A torn with the N-hydroxysucoinimide ether of L - (-) - 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 tetrahedral 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 intermediates for use in an improved and commercially attractive process for the preparation of the compounds of formula I above. The use of a polysilylated kanamycin A or B as starting material gives high solubility in the organic solvent system, thus enabling turnover at high concentrations. Although the reaction is usually carried out in solutions containing about 20% of the polysilylated channel mycine starting material, excellent results have been obtained in concentrating ions of about 50 g / 100 ml of solvent. 461 1481 4 Similar to processes known in the art, said pre-I a mixture of acylated products. The desired 1-N-acy- the separated product is separated from the other products by typography and the by-products can, if desired, be hydrolysed to the can the mycine starting material for recycling. At those known in the art processes, it has been found to possibly form 3 "-N-acylated material gives a loss, which roughly corresponds to the same amount desired 1-N-acylated product, due to the great difficulty of separating the latter product from the former material. A special partial feature of the present process, it is extremely low amount of undesirable 3 "-N-acylated product formed (normally one can do not detect any such product).

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-KG) 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. 461 148 The present invention relates to polysilylated kanamycin A or B or polysilylated kanamycin A or B containing a blocking group other than silyl on the 6'-amino group, which blocking group has the formula R cx o 3 ° §_ -z 3 h. 2 I Q ~ ca,. 461 148 ' wherein R 1 and R 2 here are the same or different and each denotes H, F1, Br, NO, OH, lower alkyl or lower alkoxy, X is Cl, Br, t or I and Y are H, Cl, Br, I or I and wherein the polyethylated kananycin derivative contains 2 - 10 eyyl groups per molecule, average 4 - 8 eyyl groups per molecule or 3 - 7 eyyl groups groups per molecule in the case of the polyylylated kanamy- cin A or B starting material.includes another blocking group other than eilyl on the 6'-amino group and eilyl- the groups are tri (lower alkyl) eilyl groups, for use intermediate product in the preparation of derivatives of mycin A and B down the formula Cllailïl; wherein R is OH or N82 and n is an integer from 0 to 2.

.WE 461 148 The acylating acid of formula II may be present in its iso- more (+) - or (-) - isomeric form or as a mixture of the two the isomers (ml form), thus obtaining the corresponding a compound of formula I wherein the 1-N- / w -amino-alpha-hydroxyalkanoyl / v group exists in its (+) f- [or (~ R) .-] form or its (-) - [or (S) -_ 7 form or as a mixture thereof. In a preferred embodiment the acylating acid of formula II is present in its (-) form. By a In another preferred embodiment, the acylating acid is present in the form flour II in its (+) - form.

The most preferred blocking group for protecting 6 'amino acids the no group in the kanamycin and the amino group of the acylating acid (group B in formula II above) is the carbobenzyloxy group.

In a preferred embodiment, the acylating derivative is of the acid of formula II an active ester and preferably its active ester with N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or clay N-hydroxyphthalimide. In another preferred embodiment, it is the acylating derivative of the acid of formula II a mixed acid anhydride and preferably its mixed acid anhydride with pivalic acid, benzoic acid, iso- butyl carbonic acid or benzyl carbonic acid. ' 461 148 ' A particularly preferred embodiment relates to the production of 1-N- / L - (-) - 2 "-amino-alpha-hydroxybutyl / kanamycin A or an pharmaceutically acceptable acid addition salt thereof, wherein acylates polysilylated kanamycin A with a mixed anhydride of L - (-) - U'-benzyloxycarbonylamino-alpha-hydroxybutyric acid (preferably its mixed anhydride with pivalic acid, benzoic acid, isobutyl carbonic acid or benzyl carbonic acid) in a substantially anhydrous organic solution means and then removes all blocking groups.

Another particularly preferred embodiment relates to 1-N- / L - (-) - 2'-amino-alpha-hydroxybutyl / kanamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof by acylates polysilylated kanamycin A, which contains a carboben- syloxy group on the 6 "amino group, with a mixed acid anhydride of L - (-) - 2- -benzyloxycarbonylamino-alpha-hydroxybutyric acid (and preferably its mixed acid anhydride with pivalic acid, benzoic acid, isobutylcarboxylic acid or benzyl carbonic acid) in a substantially anhydrous organic solvent and then removes all blocking groups. _ ' Yet another particularly preferred embodiment relates preparation of 1-N- / L - (-) - 3'-amino-alpha-hydroxybutyl / kanamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof by acylating polysilylated kanamycin A with an active ester of L - (-) - Z'-benzyloxycarbonylamino-alpha-hydroxybutyric acid (and preferably its active ester with N-hydroxysuccinimide, N-hydroxy-5-nor- bornen-2,3-dicarboximide or N-hydroxyphthalimide) in a substantially aqueous free organic solvent and then remove all blocking groups.

Another particularly preferred embodiment relates to position of 1-N- / L - (-) - Ü "-amino-alpha-hydroxybutyl / kanamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof through acylating polysilylated kanamycin A, which contains a carbohydrate benzyloxy group on the 6'-amino group, with an active ester of L - (-) - ¶'-ben- syloxycarboxylamino-alpha-hydroxybutyric acid (and preferably its active ester with N-hydroxysuccinimide, .N-hydroxy-5-norbornene-2,3-dicarboximide or N-hydroxyphthalimide) in a substantially anhydrous organic solution and then removes all blocking groups. '461 148 According to a preferred embodiment, the present provides invention polysilylated A or B containing another blocking agent group than silyl on the 6 'amino group. In a preferred embodiment, the material polysilylated kanamycin A or B and preferably poly silylated kanamycin A, which contains on average 4-8 silyl groups and preferably trimethylsilyl groups per molecule. At another The preferred embodiment is the material polysilylated kanamycin A or B and preferably polysilylated kanamycin A, which contains another blocking group other than silyl on the 6'-amino group and contains in the 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 herein as a mono-, di-, tri- or tetra-salt, which has been formed by compounding of a molecule of a compound of formula I with 1-4 equivalents of a non-toxic, pharmaceutically acceptable acid. include acetic acid, hydrochloric acid, sulfuric acid, maleic acid, phosphoric acid, nitric acid, hydrobromic acid, ascorbic acid, malic acid and citric acid. acid and other acids commonly used in the manufacture of salts of amine-containing pharmaceuticals. _ Acylation of polysilylated kanamycin A or B starting material material (with or without a blocking group other than silyl on 6'-a- the minogroup) can generally be carried out in an organic solvent, wherein the starting material has sufficient solubility. These starting points terials are readily soluble in most common organic solvents.

Suitable solvents include, for example, acetone, diethylxetone, methyl n-propyl ketone, methyl isobutyl ketone, methyl ethyl ketone, acetonitrile, glyme, diglyme, dioxane, toluene, tatrahydrofuran, cyclohexanone, meth ylen chloride, chloroform, carbon tetrachloride and mixtures of acetone / butanol or diethyl ketone / butanol. the choice of solvent is on the special starting material in question. Ketones are generally the preferred solvents. The most advantageous solvent 10 461 148 ' for the particular combination of reactants in question used can easily determined by routine experimentation.

Suitable silylating agents for use in preparation of the polysilylated kanamycin starting materials include trimethyl- chlorosilane, hexamethyldisilazane, triethylchlorosilane, triethylbromosilane, tri-n-propylchlorosilane, methyl diethylchlorosilane, dimethylethylchlorosilane, dimethyl-t-butylchlorosilane, hexaethyldisilazane, tri-n-propylsilylamine, tetraethyldimethyldisilazane, etc. Other useful silyl compounds are hexaalkylcyclotrisilazanes and octaalkylcyclotetrasilazanes as well silylamides (such as, for example, trialkylsilylacetamides and bis-trial- silylsilyl acetamides), silylureas (such as, for example, trimethylsilylacetamides urea) and silyl urides. Trimethylsilylimidazole can also be used.

A preferred silyl group is the trimethylsilyl group and preferred drawn silylating agents dare introduction of the trimethylsilyl group is hexamethyldisilazane, bis (trimethylsilyl) acetamide and trimethylsilylacetyl amid. Hexamethyldisilazane is particularly preferred. ' When using polysilylated kanamycin A or B, which is contains a blocking group other than silyl on the 6 'amino group, so as a starting material, this can be prepared either by polysilylene the desired 6'-N-blocked kanamycin A or B or by introducing the desired 6'-N-blocking group into the polysilylene kanamycin A or Q.

Methods for introducing silyl groups into organic compounds including certain aminoglycosides are known in the art. The po- lysilylated kanamycins (with or without another blocking agent) group than silyl on the 6 'amino group) can be prepared by means of ll 461 148 known methods or as described in more detail below.

By "polysilylated kanamycin A or B" is meant herein the contexts kanamycin A or B, which contain 2-10 silyl groups in the molecule. The term "polysilylated kanamycin A or B" means thus taking non-persilylated kanamycin A or B, which would contain eleven silyl groups in the molecule.

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

In the case of severe under- or oversilylation, a small or an amount of the desired product. The degree of silylation that gives it greatest yield of the desired product depends on the special reactants used in the acylation step. The advantage- the highest degree of silylation when using a special combination of reactants can be easily determined by routine experimentation.

In the preparation of 1-N- / L - (-) - W'-amino-alpha-hydroxybutyl yryl / kanamycin A by acylation of polysilylated kanamycin A with The N-hydroxysuccinimide ester of L - (-) - '' '- 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 two equivalents of the trimethylsilyl group of 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 12 461 148 ' 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. oversilyle- 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 aoylation 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, S: 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 S, 5: 1 in the presence of a silylation catalyst) acyl- ~ acetone with the N-hydroxysuccinimide ester of L - (-% - ¶'-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 (Z1 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 shown 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 13 461 148 namycin A, it has been found that excellent yields of l-N- / L-4 -) - 3 '- -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 of about -30 ° C to about 100 ° C can be used for reaction times, which vary from about 1 hour up tilll-G89 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-S0. _ 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 14 461 148 ' 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 meg N, N'-dimethylchloroformiminium chloride (see British patent specification 0000 170 and Weichet: Experientia XXI, Q (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. Z1 (1955) 1967), an alkynylamine reagent (see R. Buijle and H. G. Viehe: Angew. Chem. International Edition, 3 (1964) 582), an isoxazolium salt reagent (see R. B. Woodward, R.A.

Olofson and H. Mayer: J. Amer. Chem. Soc. åâ (1961) 1010), ettke- tenimine reagents (see C.L. Stevens and M. E. Munk: J. Amer. Chem. Soc. (1958) 4065), hexachlorocyclo triphosphatriazine or hexabromocyclo- triphosphatriazine (see U.S. Pat. No. 3,651,050), diphenyl- phosphoryl azide (DDPA; see J. Amer. Chem. Soc. gi (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 15 461 148 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 locking 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 P-Bondapak C-18 column. The assay conditions were following.

Mobile phase: 25% 2-propanol - 75% 0,01§ Sodium acetate pH 4.0 s1öaeshast = f 1: nl / 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. 16 461 148 ' In order to avoid complicated chemical designations, is sometimes used in the following abbreviations: AHBA L - (-) - U'-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.

Exemgel 1 e j Preparation of 1-N- / L-Y -) - Cr'-amino-alpha-hydroxybutryl / - kanamycin A (amikacin) by selective acylation of poly (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 (40 °) and complete drying in vacuo (10 mm) gave 27.9 g of a white, amorphous solid (90.7l% based on 6'-N-carbobenzyloxycanamycin A 17 461 148 (silyl) 9).

This solid was dissolved in 150 ml of dry diethyl ketone at 23 °. 11.05 g (26.67 mmol) of L -hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboxamide ester (NAE), which had a maximum of 1 ioo mi tar: ailetiketone via 23 °, was added 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 23 °. Vat- 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 introduced into a S00 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 degrees Celsius 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 concentration was concentrated in vacuo (40-45 °) to 50 ml. The solution is applied on a 5 x 100 cm ion exchange column_CG-50 (NH4 +). After washing with 1000 ml of water eluted unreacted kanamycin A, 3-Ak - (-) - -jr -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 GL Weight Material (ml) âlå (gm.)% Yield Kanamycin A 1000 + 0.1l '0.989 »9.15 aB-xzs 17so + o, 24 '4.37 32.0 'amikacin zoco + 0.31 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.

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. , .. 18 461 1487 Example 2 Preparation of 1-N- / L - (-) - '2'-amino-alpha-hydroxybutyl / kanamycin A (amikacin) by selective vacylation avggoly (trimethyl- 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 - (- M-E'-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 was left 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 steam jet vacuum at about 35 °. 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 23 ° 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%.

Exemgel 3 Preparation of 1-N- / L - (-) - Qf -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 of activity (37, 15 mmol) of kanamycin A in the form of the free 19 461 148 the base was slurried in 200 ml of dry acetonitrile and refluxed. 29.8 g (844 mmol) of hexamethyldisilazane were added over 30 minutes 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 - (-) -? - amino-alpha-hydroxybutyl / kanamycin A 5.56 g (20.43 mmol) of p- (benzyloxycarbonyloxy) benzoic acid slamaaes in so m1 dry acetonitrile at 23 °. 8.4 g (41.37 mmol) N, O-bis-trimethylsilylacetamide was added with good stirring. Loose- The reaction was held for 30 minutes at 23 ° and then set for a period of period of 3 hours with vigorous stirring to a solution of 21.5 g (17.83 mmol, calculated as the (silyl) 10 compound) in 75 ml of dry aceto- nitrile at 23 °. The mixture was stirred for 4 hours, the solvent was removed was removed i.vænmml40 °) 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 23 °. The solution was diluted with 100 ml of water and the pH (7.0) was lowered to 2.5 (6N H61).

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 / omz 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.

Exemgel 4 L Preparation of 1: N- / L - (-) - Tf-amino-alpha-hydroxyQutyryl / - kanamycin A (amikacin) by selective acylation of goly (trimethylsilicone) 11) -kanamycin A A. Poly (trimethylsilyl) -kanamycin A A suspension of 10 g (20.6 mmol) of kanamycin A in 100 ml of dry acetonitrile and 25 ml (19 mmol) of 1,1,1,1,3,3,3-hexamethyldisilazane refluxed for 72 hours. A clear light yellow solution was obtained.

The solution was evaporated to dryness in vacuo at 30-40 °, whereupon obtained 21.3 g of poly (trimethylsilyl) -kanamycin A as a light brown dye. amorphous powder (85% yield calculated as kanamycin A (silyl) 10). 20 461 148 ' B. 1-N- (L - (-) - C 1-4 amino-alpha-hydroxybutyl / 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 moles 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 23 ° 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 23 ° using a 5% molar excess of NAE in relation to the amount invested 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. 461 148 II. Reaction scheme s' m-cbz-xana A cZGHQoBNÅ (e1a, ss) ë + (CH _ 3) 3si-Nx-si (CH 3) 3 HMDS (l6l.4) A casku En - si (aug) 3 CD GWN-Cbz-Kana A (Silyllg csaxxluolazusig (Lamm 22 461 148 O ïn a. cbzNH (cu2) 2-ca-coon + non + Dcc (206) ansa (2s3,4) aonß (179,2) Acaton- ïm fi ocv '+ cbzaN (cu2) 2-ca-c-o-N (zz4,3) O (for now: (41.4, s) c. dicbzargikaçin (854) + CD * GD A °° t ° “dich aa-x "'ššj - få 2 29 + 6'Cb = -1.3-dia fl aa-xana A H + ss rd / c ' 6'Cbz-Kana A _ Jç Qíkacin + §B-K29 + I, 3-diAHBA-K-ana A + kan; Ai 5585: 52) _ (722.16) (4a4.5; cc-so (Nx4 +) Axáikacin III. Material weight (3) 6'-N-Cbz-Kana A 50 HMDS '58.9 Aeetonitrile BHBA 21.5 HONB 15.2 DCC 17.48 Acetone CG-50 (NH4 +) Methanol IPA IV. Safety instructions 6'-N-Cbz-kana A - Acetonitrile - Hexamethyldisilazane (HMDS) - 6'-N-Cbz-kana A (silyl) 9 - BHBA - Methanol - Isogroganol - 461 148 vo1. (m1) gg; 0.081 76.5 0.365 aoo I o, oas 0.085 oh, us 260 3000 Based on needs Based on needs 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 abS0ïPti0 fl- Fiery. Ingestion orally or hauling of large quantities of steam can cause headaches, drowsiness, mental depression, vomiting, anesthesia. 24 461 148 Ammonium hydroxide - 1 Toxic fumes. Breathing mask shall worn and contact with the liquid shall avoided.

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

V. Production procedure ' A. Preparation of 6'-N-carbobenzyloxycanamycin A (silyl), 13'-N-Cbz-Kana A (Chile1) 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. q 4. The clear light yellow solution is concentrated in vacuo (bath temperature 40-50 °) to a foamy solid. The yield of sil- The y19 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-carbobenzyloxyamino-alpha-hydroxybutyric acid (NAB) 1. '21.5 g / l - (-) - β-carbobenzyloxyamino-alpha-hydroxybutyric acid (smaA) is dissolved in 100 ml of dry acetone at 23 °, followed by 15.2 g of -hydroxy-5-norbornene-2,3-dicarboxyimide (HONB). Full resolution enters. 2. For 30 minutes, add a solution of 17.48 g of dicyclo- hexylcarbodinide (DCC) in SO ml acetone with stirring. The temperature rises to about 40 ° during the addition during precipitation of dicyclohex ylurea (DCU). 3. The slurry is stirred for 3-4 hours while the temperature is kept at 23-25 °. 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 25 461 148 the acylation step below.

C- Acylation of 6'-N-Cbz Kana A (Silyl) n. 1. The compound 6'-N-Cbz- isolated under A in step 4 above -xana A (siiyng is dissolved in 100 ml of dry acetone at 23-24 °. 2. With good stirring, slowly add it under B above prepared the NAE solution over a 15-minute period. The temperature rises gradually to about 400. The solution is allowed to assume a temperature of 23 °, after which stirring is continued for 18-20 hours under a nitrogen atmosphere. far. hrs 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 23 °.

(Note: a second layer may form ~ this does not cause anything ~ reprocessing problems). IN 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 40-45 °. 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 (NHÉ). '- 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 solution is as follows: Remaining Kana A --è BB-K29 ---> amikacimFören- no BB-K11 could not be detected in any of the acylation the reprocessings. Bolyacyl material; i.e. 1; 3-diAHBA-ana- the lodge of Rana A, is recovered by washing the column with 3N ammonium hydroxide. IN 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 26 461 148 ' (IPA) with good stirring and crystallization may take place for 6-8 hours. mar 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 12A. _ ' 12. the material is dried in vacuo for 12-16: more at 40 °.

The yield amounts to 17.3-19.0 g (38-42%) of amikacin with the following properties creates: - EEE 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 ^ / 0.4).

Specific rotation 23 ° n o min NH on o in H so 547 sas + eío1, s + 1o1, e ° + 103.52 4 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-Rana 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 Rana A base in acetonitrile using hexamethyldisilazane (HMDS) gave polytrimethylsilyl-kana A. Silylation the degree is still uncertain but was assumed to be kana AÅsilyl) l0. Poly- silylated Rana A was readily soluble in most organic solvents. part. Acylation with SAE in anhydrous acetone at 23 ° during use of a molar ratio of SAE to Kana A of 1: 1 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. Reduction and work-up of an acetone acylation mixture, followed by chromatography on the CG-50-NH2 + column using 0.5N ammonium oxide gave isolated crystalline amikacin in a yield of 34-39%. 27 461 148 II. Reaction schedule 1 .. í ' no Du GK. - e a. 0 /. 2 KZN ca 3 “Ka . Q - Rana A- bas- ° 1sH: s ° L1H4 (434'5l) + (cn3) 3 si-Na-s1 (cu3) 3 aMns' (161.4) a - sL (cH3) w xâníl Ä 28 461 148 0 \ fi * s. cbznz fl cx fi z-CH-cooa + xo-N »+ acc Bum _. N-zzos (20 "6.3J '(zsa, 4_> (nsm Et0Ac '\ - o on o . ncu + cbzmucuzhcx-c-o-N {I 1 <224, s) ' (2 sAB (3so.33) c.

Cbz-Amikacin (720) _ + - cbz-as-éxzs Acetone fl + Q- "'@ 23' 5 cbz-as-xs' '+ _ Kana A + 'H2 ¶Pó1¶eóy1er. (Mainly 1.3 * 5 * Pæc _ v - diam-Karm A) I -_. Ø ^ L ~ _ï Amikacixwßä-Kß + BB-KG + l fi- GLAHBA-Kana A + Rana A ' 585,62) '' 472236) CG-50 (NH +) 29 461 148 III. Material Weight (g) Vol. (ml) gg; Kana A- "bas" 50 0.103 HMDS (Sp. Weight 0.774) 86.68 112 0.537 Acetonitrile 0 600 SAE 35.03 0.10 Acetone 850 cs-so (Naq "'> aooo Methanol As needed IPA As needed IV. Safety instructions Kana A- "base" _ e Known drug - common caution recommended Rana 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 1. 50 g Kana A base (water content according to Karl Fischer 2.5-3.5%) 1 500 ml of acetonitrile (water content according to Karl Fischer increasing 0.01%). The whole is allowed to reflux (74 °) 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 (400) 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) ld).

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-hydroxysuccinunide (N-HOS) are dissolved in 1300 ml ethyl acetate (water content according to Karl Fischer less than 0.05%) at 230 while stirring. IN 30 461 148 ' 2. 81.29 g of dicyclohexylcarhodiimide (DCC) are 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-Ä2 ° during the precipitation of dicyclohexylurea (DCU). The slurry is stirred 3-4 hours, during which the temperature may reach 23 °. 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 30-35 °). A certain amount of product crystallizes out serar.0 I '5 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 30-35 ° for 18-20 hours. The yield amounts to 110.1-113 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 KMO 4 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. I 1 C. Acylation of Kana A (Silyl) lo The compound Rana A isolated under A in step 4 above (suynlo dissolves 1 soo m1 tar: acetone at 23 °. With good stirring, add it quickly in section B above prepared compound SAE (35.03 9) as a 10% solution in dry acetone for 5-10 minutes. The temperature rises about 5 °. The solution gets assume 23 ° 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 23 ° for 15-30 minutes. 31 461 148 4. Acetone is removed in vacuo at a bath temperature of 30-35 ° (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 40-45 °. 7. pix value 1 this stage is e, o-7, o. siananingen applice- on a CG-50 (NH4 +) column (6 x 10 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 the following: ' 'Remaining Kana A --3 BB-K6' - å BB-K29 --9 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 performed with 1 volume of methanol and seed crystals of amikacin are added. J For 2 hours, slowly add 2 volumes of IPA and crisis teilisetion occurs via 23 ° below 6-8 tins. 11. The solid is filtered, washed with 35 ml of one 1: 1: 2 mixture of water, methanol and IBA and finally with 35 ml IPA. in 12. The material is dried in a vacuum oven at 400 for 16-24 hours. down. the yield amounts to 19.91-22.64 g (34-39%). IR-, run- and cun- 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- valinsvra and BHBA A. Preparation of the mixed anhydride 5.066 g (20.0 mmol) of BHBA, 4.068 g (20.0 mmol) of BSA and 2.116 (22.0 mmol) of triethylamine was dissolved in 200 ml of screen dried tetrahydrofuran. 32 461 148 ' 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 -10 °. Tem- the temperature was then allowed to rise to 23 °.

B. Acylation of poly (trimethylsilyl) -6'-N-Cbz-Kana A 5.454 g (4.97 mmol, calculated as 6'-Cbz-Kana A (Silyl) 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 (1.0.0 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 PZOS, 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. 4 Example 8 Effect of water on the production of amikacin by acy- poly (trimethylsilyl) -Kana A in acetone solution at 23 ° A. Anhydrous solvent 2.40 g (2.0 mmol, calculated as Kana A (Silyl) 1 °) poly (tri- methylsilyl) -Kana 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 23 ° 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 23 °. 50 ml of water were added and the pH (7.5) was set to 2.5. The acetone was evaporated in vacuo at 40 ° and aqueous 33 461 148 the tin solution was then reduced at a hydrogen pressure of 3.6 kg / cm 2 at 239 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 23 ° 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 was cooled to 22 ° and 0.751 g (0.713 ml, 50 ml) of the isomtyifomanaa were added.

Triethylamine hydrochloride began to precipitate immediately. The mixture is stirred des l hour at -206.

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 23 ° under 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 -20 ° and then for 17 hours at 230. 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 40 °. 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.

The majority of the aqueous solution was reduced at a hydrogen pressure of 3.5 kg / cm2 at 23 ° under 3.25 tins using 2.0 g of a palladium catalyst on carbon. The catalyst was removed by filtration and the combination of filtrate and washing liquids 34 461 148? microbiological analysis against E.coli and was found to contain a yield of 35.0% amikacin.

Example 10 Preparation of amikacin by acylation of polystrimethyl- jägy fi-6'- N-Cbz-Kana A i 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 23 ° 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 07.3 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 40 °. 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 placed on an ion exchange column (CG-SO (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 NH 4 OH (followed by 3N NH 4 OH to elute polyacylated products).

Polarimetry of the fractions showed a yield of 42.7% amikacin, 2.0% 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-Rana A (Silyl) 9) - poly (trimethylsis fl J-6'-N-Cbz-kana A Example 1, in 300 ml of dry cyclohexanone was acylated for 20 hours at 35 461 148 230 with a NAE solution in dry cyclohexanone (10, 8 ml of a solution containing 0.144 mmol / ml = 2.10 mmol). The reaction mixture was then add 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 40 ° 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 by filtration and the combination of HV filtrate and washings was analyzed microbiologically for amikacin.

B. Reaction step A above was repeated except that acy- the training was allowed to last ll5 hours instead of 20 hours.

Utbxten ÉÉÉÉIÉEÉÅXÉ Microbiological analysis (amikacin) (di-Cbz-amikacin) Turbidimetric Plate Reaction A 49.18% 42.87% 39.16% ReactionOn 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 Gmüæmin) (di-Cbz-amikacin) Turbidimetric Plate Reaction A 29.27% - 28.34% 28.18% Reaction B at 33.39 and 21.52; 28.63 æ 36 461 148 ' 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.

Yields 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 poly (trimethyl- snyn-fr-N-cbz-xana A 1 anhydrous ailetiketone via 1s ° 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 75 °, a solution of NAE (1.0 ml of a diethyl ketone solution containing 0.144 mmol / ml = 2.10 mmol) for 15 minutes.

Stirring was continued at 75 ° for a further 3 hours, after which the mixture was poured into 150 ml of water. The pH was adjusted to 2.8 with 3N HCl and the diethyl ketone was evaporated in vacuo at 40 °. HPLC analysis of an aliquot of the aqueous phase indicated a yield of 39.18% di-Cbz -amikacin. n The majority of the aqueous phase was reduced under a hydrogen pressure of 3.5 kg / cm 2 for 3.25 hours at 23 ° using 1.0 g of a catalyst of palladium on carbon. The catalyst was removed by filtration and the combination of filtrates and washings microbiogly with respect to amikacin. Turbidimetric analysis lys showed a yield of 27.84% and plate analysis gave a yield of 28.6%. 37 461 148 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 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 sieve acetone at 23 ° and then cool to 0 ° C. 3.63 m1 (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 0-5 ° 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 23 ° 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-Rana A with SAE at 0-5 ° after methanolysis IA. Silylation of 6'-N-Cbz-kanamycin A 20.0 9 (32.4 mmol) 6'-N-Cbz-kanamycin A in 200 ml molecular sieve dried acetonitrile was refluxed under a nitrogen atmosphere. 47.3 ml 38 461 148 ' (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. Acylation 39.1 g (32.4 mmol) of P ° iy (trimethylsilyl) -6'-N-Cbz-Kana A, which had been prepared according to step A above, was dissolved in 400 ml of dry acetone at 23 ° with 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 for 1 hour during heavy nitrogen purge. The mixture was cooled to 0-S ° 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 an additional 3: more via o-s ° ee were then introduced into a cooling room at a temperature of 40 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 23 ° 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 +) column and eluted with water and 0.6N NH 4 OH. 52.52% of amikacin were obtained, 14.5% BB-K29, 19.6% kanamycin A and 1.71% polyacyl material.

Exemgel l7 Preparation of amikacin by acylation of poly (trimethyl- silyl) -Rana A with SAE at 0-5 ° 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 16.234 g (140.98 mmol, 14 mol per mol of kanamycin A) tetramethylguanidine (TMG). The mixture was refluxed and 15.32 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 employees. The mixture was cooled to room temperature. concentrated to a sticky residue and dried at high speed. kuum for 2 hours. The solid was triturated with 100 ml 39 461 148 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 40 ° 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 0-5 °. 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. 17.33 g (107.32 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. lyUm).

Example 19 Preparation of noly (triethylsilyl) kanamycin using triethylchlorosilane with triethylamine as the acid acceptor 5.0 g of kanamycin A (97.6% purity, 10.07 mmol) was suspended in 100 ml of molecular sieve dried acetonitrile at 23 °. 33.8 ml (24.5 g, 241.7 mmol) of triethylamine (TEA) was added and the suspension was refluxed. xokaaes. A solution of 23.7 ml (α1, g, 140.98 mmol) of trichioethylene lan in 25 ml of dry acetonitrile was added over 20 minutes. Aterlopps- 40 461 148 ' O 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 23 ° and insoluble TEA.HCl was filtered off, washed with 5 x 20 ml 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. ly (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 (444.0 mmol, 7 mol per mol of 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 4 ° 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 40 ° 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 23 °, the insoluble portion was removed by filtration, washed 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 41 461 148 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 oer (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) mol, 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.Ac1lering 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 z3 ° unasr stirring. from this solution was added 9.25 ml (io3.2 mmsi, 5 moles per mole of peritrimethylsilyl) -kanamycin A) 1,3-butanediol. Among- The mixture was stirred for 2 hours at 23 ° with nitrogen permeation and cooling. was then increased to 0-50. 7.23 g (20.64 mmol) of SAE in 70 ml of 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 40 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 23 °. 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 goly (trimethyl- silyl) -canamycin A prepared in THF using SAE with sulfur famsyra catalyst To a refluxing mixture of 5.0 g (10.32 mmol) of ca- nanycin A and soni mslskyisikssssrkad f-.strahydrofuran (THF) were added 100 mg sulfamic acid and 12.32 g (76.33 mmol) of HMDS. Mixture of reflux 42 461 1487 boiled for hours, with complete dissolution taking place after 6 hours. The solution was cooled to 23 °, treated with 0.1 ml water and kept at 230 for 30 minutes. A solution of 3.61 g (10.3 mmol) SAE in 36 ml 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 230 for 30 minutes and then concentrated in vacuum to remove THF. The resulting aqueous solution was was produced at a wet pressure of 3.5 kg / cm 2 for 2 hours at 23 ° using a catalyst of 10% palladium on carbon. The rv ~ 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 Fg / 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 50 ml of dry acetone at 230. 4.25 g (20.65 mol) of dicyclohexylcarbodic acid 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. Acylation 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 0-5 ° for 30 minutes under moderate vacuum.

To this solution was added the solution prepared in step A. one of the di-Cbz-blocked acylating agent and the mixture was stirred at 0-5 ° 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 43 461 148 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 g (22.7 mmol) of kanamycin A and 100 mg of imidazole reflux was boiled in 100 ml of molecular sieve dried acetonitrile under nitrogen blàening. 16.48 g (ll4, s mmol, s mel per mel kenemyein A) Hnns: lll- 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 23 ° 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 - (- 1H-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 1oypsk0käÖêS for 21 hours with complete dissolution after 1 hour. the solvent was then removed in vacuo at 60 ° and the oily the residue was kept at 60 ° in a medium vacuum for 3 hours. 53.0 was 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 (trñ) prepared in step A above methylsilyl) -kanamycin B was dissolved in 500 ml of dry acetone at 0-SO, 20.9 ml of methanol were added and the mixture was stirred in vacuo at 0-50 for 30 minutes, A solution of 1311 9 (5l, 67 mm fl l) 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 0-5 ° 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. 44 461 148 ' 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-E46), 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 0.59 (0.8S8 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 steam vacuum. 47.5 g of poly (trimethylsilyl) -kanamycin A were obtained as a pale yellow oil (9S, 82% yield calculated as kanamycin A (silyl) 10).

Example 27 Preparation of amikacin by acylation of polytrimethyl- silyl) -kanamycin 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 100 ml of THF satin 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 50 °. Re- 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 SO 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). 2.4 g (11, 3 mol) of N-trifluoroacetyl-ABBA were dissolved in 50 ml dry acetone and 1.30 g (11.3 mol) 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 23 ° C. 2 hours and the precipitated dicyclohexylurea was removed by filtration and washed with a small amount of acetone. The combination 45 '”461 148 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 0-5 ° for 30 minutes. 11.31 mmol of that prepared in step A above The N-hydroxysuccinimide ester of N-trifluoroacetyl-ABBA was added to the mixture. which was then kept at 5 ° 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 1o ° below in tin. the pia value (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 3), 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, 5.7% 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 possible t-BdC-AHBA and conversion thereof to it N-hydroxysuccinimide ester I 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 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 lowered to 2.0 m -1 HCl and then extracted with 3 x 30 ml MIKB. The combined MIBK extracts were dried over sodium 46 461 148 ' 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-ABBA) was used in the following steps without insulation.

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 ° C. 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 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 the filter 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 1-N- / L - (-) - IT -amino-alpha-hydroxybutyl / - -kanamycin B.

Example 30 The general procedure of Example 1 was repeated with assuming that the L - (-) - zr'-benzyloxycarbonylamino-alpha- 47 '461 148 -hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester was added with L - (-) - beta-benzyloxycarbonylamino-alpha-hydroxypropionic acid ra-N-hydroxy-5-norbornene-2,3-dicarboximide ester and I, respectively L - (-) - eel.-benzyloxycarbonylamino-alpha-hydroxyvaleric acid-N-hydro- oxy-5-norbornene-2,3-dicarboximide ester to give 1-N- / L- - (-) - beta-amino-alpha-hydroxypropionyl / kanamycin A respectively 1-N- / L - (-) f Å / raminojalfa-hydroxivaleryl / kanamycin A.

Exemgel 31 The general procedure of Example 25 was repeated except for that the L - (-) - 'of -benzyloxycarbonylamino-alpha-hydride used therein oxybutyric acid N-hydroxy ester was replaced by L - (-) - beta-benzyloxycarbon ylamino-alpha-hydroxypropionic acid N-hydroxysuccinimide ester, respectively L - (-) - 5-Hensyloxycarbonylamino-alpha-hydroxyvaleric acid-N-hydroxy- succinimide ester to give 1-N- / L- (~) -beta-amino-alpha-hydr-r oxypropionyl / kanamycin B and 1-N- / L - (-) - α; -amino-alpha-hydr- oxivaleryl / kanamycin E.

Claims (3)

48 461 148? EBIEHIEBBX Polyeilylated kanamycin A or B or polysilylated kanamycin A or B containing a blocking group other than silyl on the 6'-amino group, which blocking group has the formula 'f * ß fan: H zzoc-, cxa-iz-oc wherein R 1 and R 2 here are the same or different and each represents H, F 1, 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 the case of the polysilylated kanamycin A or the B starting material contains a blocking group other than silyl on the 6 'amino group and the silyl groups are tri (lower alkyl) silyl groups, for use as an intermediate in the preparation of derivatives of kanamycin A ooh B of the formula C fl z fifl z HO wherein R is OH or NH2 and n is an integer from 0 to 2. Polysilylated kanamycin A or B according to claim 1, characterized in that it contains a carbobenzyloxy group on the 6 'amino group. Polysilylated kanamycin A or B according to claim 1 or 50 461 148 '2, characterized in that the silyl groups are trimethylsilyl groups.