GB1598294A - Process for the preparation of kanamycin derivatives - Google Patents

Abstract

1-N-[ omega -amino- alpha -hydroxyalkanoyl] kanamycins A and B of the formula I <IMAGE> in which R represents OH or NH2, are prepared by acylation of polysilylated kanamycin A or B, or polysilylated kanamycin A or B which has a protective group other than silyl on the 6'-amino group, with an acylating derivative of an acid of the formula II <IMAGE> in which n is an integer from 0 to 2 and B is an amino protective group, in an essentially anhydrous organic solvent and by subsequent removal of all blocking groups. The compounds have therapeutic properties.

Description

(54) PROCESS FOR THE PREPARATION OF KANAMYCIN DERIVATIVES (71) We, BRISTOL-MYERS COMPANY, a Corporation organised and existing under the laws of the State of Delaware, United States of America, of 345 Park Avenue, State of New York, 10022, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to an improved process for the preparation of compounds of the formula

wherein R is OH or NH2 and n is 0 or an integer of 1 or 2, or a nontoxic pharmaceutically acceptable acid addition salt thereof which process comprises acylating polysilylated kanamycin A or B or polysilylated kanamycin A or B containing a blocking group other than silyl on the 6'-amino moiety, in a substantially anhydrous organic solvent, with an acylating derivative of an acid of the formula

in which n is 0 or an integer of 1 or 2 and B is an amino-blocking group, and subsequently removing all blocking groups.

The kanamycins are well-known antibiotics, having been described, for example in the Merck Index, 8th edition, pp. 597-8. Numerous derivatives of the kanamycins also are known. The structural formulae of kanamycins A and B are given below, along with the standard numbering system used in the art.

Hereinafter, where readily understandable, the various kanamycin derivatives will be referred to as derivatives of kanamycin A or B rather than by structural formula, so as to avoid the necessity of comparing complex structures to determine differences.

Kanamycin A: R - OH Kanamycin B: R = NH2 U.S. Patent 3,781,268 discloses and claims 1-[L-(-)-y-amino-a- hydroxybutyryl]kanamycin A (amikacin) and B, as well as their mono- and dicarbobenzyloxy protected derivatives. For lower and higher homologs see U.S.

Patents 3,886,139 and 3,904,597. The compounds are prepared by acylating a 6'-Nprotected kanamycin A or B with an acylating derivative of an N - protected L (-) - y - amino - a - hydroxybutyric acid, in an aqueous medium, followed by removal of one or both N-protecting groups.

U.S. Patent 3,974,137 discloses and claims a process for preparing 1 - [L (-)--amino - a - hydroxybutyrylikanamycin A which comprises reacting 6'carbobenzyloxykanamycin A with at least three moles of benzaldehyde, a substituted benzaldehyde or pivaldehyde, to produce 6'-Ncarbobenzyloxykanamycin A containing Schiff base moieties on the 1,3 and 3"positions, acylating this tetra-protected kanamycin A derivative with the Nhydroxysuccinimide ester of L- (-)- - benzyloxycarbonylamino - br- hydroxybutyric acid, and subsequently removing the protecting groups.

Belgian Patent 828,192 discloses and claims a process for preparing I - [L (-) - y - amino - a - hydroxybutyryl]kanamycin A by preparation of the same tetra-protected kanamycin A derivative as in U.S. 3,974,137, acylating with the N hydroxy - 5- norbornene- 2,3 - dicarboximide ester of L - (-) - - benzyloxycarbonylamino - a - hydroxybutyric acid, and subsequently removing the protecting groups.

The present invention provides an improved and commercially attractive process for the preparation of compounds of formula I. The use of a polysilylated anamycin A or B as a starting material gives high solubility in the organic solvent system, thus permitting reaction at high concentrations. Although the reaction is usually conducted in solutions containing about 1020% polysilylated kanamycin starting material, excellent results have been obtained at concentrations of about 50 gms./100 ml. of solvent.

As with prior art processes, the present process gives a mixture of acylated products. The desired l-N-acylated product is separated from the other products by chromatography and, if desired, the by-products may be hydrolyzed to the starting kanamycin for recycling. In prior art processes it was found that any 3"-Nacylated material which was produced caused a loss of about an equal amount of the desired l-N-acylated product, due to the great difficulty of separating the latter from the former. A particularly desirable feature of the present process is the extremely low amount of undesirable 3"-N-acylated product which is produced (typically, none is detected).

In preparing 1 - [L - (-) - a - amino - a - hydroxybutyryl]kanamycin A, amikacin by various prior art procedures, there is typically also produced the 3"-N acylated product (BB-Kl 1), the 3-N-acylated product (BB-K29), the 6'-N-acylated product (BB-K6) and polyacylated material, as well as unreacted kanamycin A.

Thus, in commercial production of amikacin by acylation of 6'-N-carbobenzyloxy kanamycin A in an aqueous medium, followed by removal of the protecting group we found that about 10% of the desired amikacin (2.5 kg. in a 25 kg. batch) usually was lost because of the presence of BB-Kl 1 as a co-product. When preparing amikacin by the present process, BB-Kl 1 typically is not detected in the reaction mixture.

The present invention provides the process for the preparation of a 1 - N [ - amino - a - hydroxyalkanoyl]kanamycin A or B having the formula

in which R' is OH or NH2 and n is 0 or an integer of 1 or 2, or a nontoxic pharmaceutically acceptable acid addition salt thereof, which comprises acylating polysilylated kanamycin A or B or polysilylated kanamycin A or B containing a blocking group other than silyl on the 6'-amino moiety with an acylating derivative of the acid of the formula

in which n is 0 or an integer of 1 or 2 and B is an amino-blocking group, in a substantially anhydrous organic solvent, and subsequently removing all blocking groups.

The blocking groups which may be used to protect the 6'-amino moiety of the kanamycin and the amino group of the acylating acid (group B in Formula II) are conventional blocking groups for the protection of primary amino groups and are well known to those skilled in the art. Suitable blocking groups include alkoxycarbonyl groups such as t-butoxycarbonyl and t-amyloxycarbonyl; aralkoxycarbonyl groups such as benzyloxycarbonyl; cycloalkyloxycarbonyl groups such as cyclohexyloxycarbonyl; haloalkoxycarbonyl groups such as trichloroethoxycarbonyl; acyl groups such as phthaloyl and o-nitrophenoxyacetyl; and other well-known blocking groups such as the o-nitrophenylthio group and the trityl group.

The acylating acid of formula II may be in its (+) or (-) isomeric form or a mixture of the two isomers (the d, I form), thus producing the corresponding compound of formula I in which the 1 - N - [w - amino - a - hydroxyalkanoyl] group is in its (+) [or (R)] form or its (-) [or(S)] form, or a mixture thereof. Each such isomeric form, and the mixture thereof, is included within the scope of this invention. In one preferred embodiment, the acylating acid of formula II is in its (-) form. In another preferred embodiment the acylating acid of formula II is in its (+) form.

In one embodiment of the invention the starting material is polysilylated kanamycin A or B (and preferably polysilylated kanamycin A). In another embodiment the starting material is polysilylated kanamycin A or B (and preferably polysilylated kanamycin A) containing a blocking group other than silyl on the 6'-amino moiety, said blocking group preferably being selected from those of the formulae

wherein R' and R2 are alike or different and each is H, F, Cl, Br, NO2, OH, (lower)alkyl or (lower)alkoxy, and X is Cl Br, F or I, and Y is H, Cl, Br, F or I. The most preferred blocking group is the carbobenzyloxy group. In the case of the last stated blocking group the B-HN grouping then becomes B-N to give phthalimido.

In a preferred embodiment of the invention the acylating derivative of the acid of Formula II is an active ester, and preferably its active ester with Nhydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or Nhydroxyphthalimide. In another preferred embodiment the acylating derivative of the acid of Formula II is a mixed acid anhydride, and preferably its mixed acid anhydride with pivalic acid, benzoic acid, isobutylcarbonic acid or benzylcarbonic acid.

In a most preferred embodiment, this invention relates to the preparation of 1 - N - [L - (-) - y - amino - a - hydroxybutyryl]kanamycin A or a nontoxic pharmaceutically acceptable acid addition salt thereof, which comprises acylating polysilyated kanamycin A with a mixed acid anhydride of L - (-) - y benzyloxycarbonylamino - a - hydroxybutyric acid (and preferably its mixed acid anhydride with pivalic acid, benzoic acid, isobutylcarbonic acid or benzylcarbonic acid) in a substantially anhydrous organic solvent, and subsequently removing all blocking groups.

In another most preferred embodiment, this invention relates to the preparation of 1 - N - [L - (-) - y - amino - a - hydroxybutyryl]kanamycin A or a nontoxic pharmaceutically acceptable acid addition salt thereof, which comprises acylating polysilylated kanamycin A containing a carbobenzyloxy group on the 6' amino moiety with a mixed acid anhydride of L- ()- - - benzyloxycarbonylamino - a - hydroxybutyric acid (and preferably its mixed acid anhydride with pivalic acid, benzoic acid, isobutylcarbonic acid or benzylcarbonic acid) in a substantially anhydrous organic solvent, and subsequently removing all blocking groups.

In another most preferred embodiment, this invention relates to the preparation of I - N - [L - (-) - y - amino - a - hydroxybutyryl]kanamycin A or a nontoxic pharmaceutically acceptable acid addeition salt thereof, which comprises acylating polysilylated kanamycin A with an active ester of L- (-) - - benzyloxycarbonylamino - a - hydroxybutyric acid ( and preferably its active ester with N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or Nhydroxyphthalimide) in a substantially anhydrous organic solvent, and subsequently removing all blocking groups.

In another most preferred embodiment, this invention relates to the preparation of 1 - N - [L - (-) - y - amino - a - hydroxybutyryl]kanamycin A or a nontoxic pharmaceutically acceptable acid addition salt thereof, which comprises acylating polysilylated kanamycin A containing a carbobenzyloxy group on the 6'amino moiety with an active ester of L - (-) - y - benzyloxycarbonylamino - a 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 solvent, and subsequently removing all blocking groups.

In another aspect, the present invention provides polysilylated A or B containing a blocking group other than silyl on the 6'-amino moiety. In a preferred embodiment the material is polysilylated kanamycin A or B (and preferably polysilylated kanamycin A) containing an average number of silyl groups (and preferably trimethylsilyl groups (and preferably trimethylsilyl groups) per molecule of from 4 to 8. In another preferred embodiment the material is polysilylated kanamycin A or B (and preferably polysilylated kanamycin A) containing a blocking group other than silyl on the 6'-amino group and containing an average number of silyl'groups (and preferably trimethylsilyl groups) per moleculae of from 3 to 7.

As used herein and in the claims, the term "nontoxic, pharmaceutically acceptable acid addition salt" of a compound of Formula I means a mono-, di-, trior tetrasalt formed by the interaction of one molecule of a compound of Formula I with 1--4 equivalents of a nontoxic, pharmaceutically acceptable acid. Included among these acids are acetic, hydrochloric, sulfuric, maleic, phosphoric, nitric, hydrobromic, ascorbic, malic and citric acid, and those other acids commonly used to make salts of amine-containing pharmaceuticals.

Acylation of the polysilylated kanamycin A or B starting material (with or without a blocking group other than silyl on the 6'-amino moiety) may, in general be conducted in an organic solvent in which the starting material has sufficient solubility. These starting materials are highly soluble in most common organic solvents. Suitable solvents include for example, acetone, diethyl ketone, methyl npropyl ketone, methyl isobutyl ketone, methyl ethyl ketone, acetonitrile, glyme, diglyme, dioxane, toluene, tetrahydrofuran, cyclohexanone, methylene chloride, chloroform, carbon tetrachloride and mixtures of acetonelbutanol or diethyl ketone/butanol. The choice of solvent is dependent on the particular starting materials employed. Ketones, generally, are the preferred solvents. The most advantageous solvent for the particular combination of reactants being utilized can readily be determined by routine experimentation.

Suitable silylating agents for use in preparing the polysilylated kanamycin starting materials utilized herein include those of the formula

wherein R5, R6 and R7 are selected from hydrogen, halogen, (lower)alkyl, halo(lower)alkyl and phenyl, at least one of the said R5, R6 and R7 groups being other than halogen or hydrogen; R4 is (lower)alkyl, m is an integer of 1 to 2 and X is selected from halogen and

wherein R8 is hydrogen or (lower)alkyl and R9 is hydrogen, (lower)alkyl or

in which R5, R6 and R7 are as defined above.

Specific silyl compounds of Formulas IV and V are: trimethylchlorosilane, hexamethyldisilazane, triethylchlorosilane, methyltrichlorosilane, dimethyldichlorosilane, triethyl bromosilane, tri-n-propylchlorosilane, methyldiethylchlorosilane, dimethylethylchlorosilane, dimethyl-tbutylchlorosilane, phenyldimethylbromosilane, benzylmethylethylchlorosilane, phenylethylmethylchlorosilane, triphenylchlorosilane, triphenylfluorosilane, tri-o-tolylchlorosilane, tri-p-dimethylaminophenylchlorosilane, N-ethyltriethylsilylamine, hexaethyldisilazane, triphenylsilylamine, tri-n-propylsilylamine, tetraethyldimethyldisilazane, hexaphenyldisilazane and hexa-p-tolyldisilazane. Also useful are hexaalkylcyclotrisilazanes and octaalkylcyclotetrasilazanes. Other suitable silylating agents are silylamides (such as trialkylsilylacetamides and bis-trialkylsilylacetamides), silylureas (such as trimethylsilylurea) and silylureides, Trimethylsilylimidazole also may be utilized.

A preferred silyl group is the trimethylsilyl group and preferred silylating agents for introducing the trimethylsilyl group are hexamethyldisilazane, bis(trimethylsilyl)acetamide and trimethylsilylacetamide. Hexamethyldisilazine is most preferred.

When utilizing polysilylated kanamycin A or B containing a blocking group other than silyl on the 6'-amino moiety as a starting material, said starting material may be prepared either by polysilylating the desired 6'-N-blocked kanamycin A or B, or by introducing the desired 6'-N blocking group into polysilyated kanamycin A or B.

Methods for the introduction of silyl groups into organic compounds, including certain aminoglycosides, are known in the art. The polysilylated kanamycins (with or without a blocking group other than silyl on the 6'-amino moiety) may be prepared by methods which are known per se, or as described in this specification.

As used herein, the term polysilylated kanamycin A or B refers to kanamycin A or B containing from two to ten silyl groups in the molecule. Thus, the term polysilylated kanamycin A or B, does not include persilylated kanamycin A or B, which would contain eleven silyl groups in the molecule.

The precise number of silyl groups (or their location) present in the polysilylated kanamycin starting materials (with or without a blocking group other than silyl on the 6'-amino moiety) is not known. We have found that both undersilylation and oversilylation lower the yield of the desired product and increase the yield of other products. In the case of gross under- or oversilylation, little or none of the desired product may be formed. The degree of silylation which will give the greatest yield of desired product will depend on the particular reactants being used in the acylation step. The most advantageous degree of silylation using any combination of reactants can readily be determined by routine experimentation.

When preparing 1 - N - [L - (-) - y - amino - a - hydroxybutyryl]kanamycin A by acylating polysilylated kanamycin A with the N-hydroxysuccinimide ester of L - (-) - p - benzyloxycarbonylamino - a - hydroxybutyric acid in acetone solution, we have found that good yields of the desired product are obtained by utilizing polysilylated kanamycin A which has been prepared by reacting from 4 to 5.5 moles of hexamethyldisilazane per mole of kanamycin A. Greater or lesser amounts of hexamethyldisilazane may be utilized, but the yield of desired product in the subsequent acylation step is lowered significantly. In the specific process set forth above we prefer to utilize from 4.5 to 5.0 moles of hexamethyldisilazane per mole of kanamycin in order to obtain maximum yield of product in the acylation step.

It will be appreciated that each mole of hexamethyldisilazane is capable of introducing two equivalents of the trimethylsilyl group into kanamycin A or B.

Both kanamycin A and B have a total of eleven sites (NH2 and OH groups) which might be silylated, while kanamycin A and B containing a blocking group other than silyl on the 6'-amino moiety have a total of 10 such sites. Thus, 5.5 moles of hexamethyldisilazane per mole of kanamycin A or B could theoretically completely silylate all OH and NH2 moieties of the kanamycin, while 5.0 moles of hexamethyldisilazane could completely silylate one mole of kanamycin A or B containing a blocking group oterh than silyl on the 6'-amino moiety. However, we believe that such extensive silylation does not take place with these molar ratios during reasonable reaction time periods, although higher degrees of silylation are obtained in a given reaction time when a silylation catalyst is added.

Silylation catalysts greatly accelerate the rate of silylation. Suitable silylation catalysts are well known in the art and include inter alia amine sulfates (e.g.

kanamycin sulfate), sulfamic acid, imidazole and trimethylchlorosilane. Silylation catalysts generally promote a higher degree of silylation than is required in the process of this invention. However, oversilylate kanamycin A or B can be used as starting material if it is first treated with a desilylating agent to reduce the degree of silylation before the acylation reaction is carried out.

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

Although it is usual to utilize dry solvents when working with silylated materials, we have suprisingly found that, even in the absence of "oversilylatiori", the addition of water to the reaction solvent prior to acylation often gives equally good yields, and sometimes gives better yields of desired product than in a dry solvent. In acylation reactions conducted in acetone at the usual concentrations of 1020% (W/V) of polysilylated kanamycin A, we have found that excellent yields of 1 - N [L - (-) - y - amino - a - hydroxybutyryl]kanamycin A were obtained when adding up to 28 moles of water permole of polysilylated kanamycin A; at 20% concentration, 28 moles per mole is approximately 8% water. With other combinations of reactants, even more water may be tolerated or be beneficial. The acylation reaction may be conducted in solvents containing up to about 40% water, although at such high water concentrations one must utilize short acylation times in order to avoid excessive desilylation of the polysilylated kanamycin A or B starting material. Accordingly, as used herein and in the claims, the term "substantially anhydrous organic solvent" is intended to include solvents containing up to about 25% water. A preferred range is up to about 20% water, a more preferred range is up to about 8% water, and a most preferred range is up to about 4X water.

As indicated above, the most desirable degree of silylation for any combination of acylation reactants may be readily determined by routine experimentation. It is believed that the preferred average number of silyl groups in the starting material will usually be 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 moiety, but this is only theory and is not considered an essential part of this invention.

The duration and temperature of the acylation reaction are not critical.

Temperatures in the range of -300C to 100 C may be used for reaction times ranging from one hour up to a day or more. We have found that the reaction usually proceeds well at room temperature and, for convenience and economy, prefer to conduct the reaction at ambient temperature. However, for maximum yields and selective acylation, we prefer to conduct the acylation at from 0 to 5 .

Acylation of the 1-amino moiety of the polysilylated kanamycin A or B (with or without a blocking group other than silyl on the 6'-amino moiety) may be conducted with any acylating derivative of the acid of Formula II which is known in the art to be suitable for the acylation of a primary amino group. Examples of suitable acylating derivatives of the free acid include the corresponding acid anhydrides, mixed anhydrides, e.g. alkoxyformic anhydrides, acid halides, acid azides, active esters and active thioesters. The free acid may be coupled with the polysilylated kanamycin starting material after first reacting said free acid with N,N'-dimethylchloroformininium chloride [cf. Great Britain 1,008,170 and Novalk and Welchet, Experientia XXI, 6, 360 (1965)] or by the use of an N,N'-carbonyldiimidazole or an N,N'-carbonylditriazole [cf.

South African Specification 63/2684] or a carbodiimide reagent [especially N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide: cf. Sheenan and Hess, J.A.C.S., 77, 1967 (1955)], or of an alkynylamine reagent [cf. R. Buijle and H. G.

Viehe, Angew. Chem. International Edition, 3, 582, (1964)] or of an isoxazolium salt reagent [cf. R. B. Woodward, R. A. Olofson and H. Mayer, J. Amer. Chem. Soc., 83, 1010 (1961)], or of a ketenimine reagent [cf. C. L. Stevens and M. E. Munk, J.

Amer. Chem. Soc., 80, 4065 (1958)1 or of hexachlorocyclotriphosphatriazine or hexabromocyclotriphosphatriazine (U.S. Pat. No. 3,651,050) or of diphenylphosphoryl azide [DDPA; J. Amer. Chem. Soc., 94, 6203-6205 (1972)1 or of diethylphosphoryl cyanide [DEPC; Tetrahedron Letters No. 18, pp. 1595-1598 (1973)] or of diphenyl phosphite [Tetrahedron Letters No. 49, pp. 5047-5050 (1972)]. Another equivalent of the acid is a corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen is a member of a quasiaromatic five membered ring containing at least two nitrogen atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As will be appreciated by those skilled in the art, it sometimes may be desirable or necessary to protect the hydroxyl group of the acylating derivative of the acid of Formula II, e.g. when utilizing acylating derivatives such as an acid halide. Protection of the hydroxyl group may be accomplished by means known in the art, e.g. by use of a carbobenzyloxy group, by acetylation or by silylation.

After completion of the acylation reaction, all blocking groups are removed by methods known per se to yield the desired product of Formula I. The silyl groups may, for example, readily be removed by hydrolysis with water preferably at low pH. Blocking group B of the acylating derivative of the acid of Formula II, and the blocking group on the 6'-amino moiety of the polysilylated kanamycin starting material (if present) may also be removed by known methods. Thus, a tbutoxycarbonyl group may be removed by the use of formic acid, a carbobenzyloxy group by catalytic hydrogenation, a 2-hydroxy- 1 -naphthcarbonyl group by acid hydrolysis, a trichloroethoxycarbonyl group by treatment with zinc dust in glacial acetic acid, the phthaloyl group by treatment with hydrazine hydrate in ethanol under heating.

Yields of product were determined by various methods. After removal of all blocking groups and chromatography on a CG-50 (NH4+) column, the yield of amikacin could be determined by isolation of the crystalline solid from the appropriate fractions or by microbiological assay (turbidimetric or plate) of the appropriate fractions. Another technique which we utilized was high performance liquid chromatography of the unreduced acylation mixture, i.e. the aqueous solution obtained after hydrolysis of the silyl groups and removal of organic solvent but before hydrogenolysis to remove the remaining blocking group(s). This assay was not a direct assay for amikacin or BB-K29, but for the corresponding mono- or di N-blocked compounds.

The instrument utilized was a Waters Associates ALC/GPC 244 high pressure liquid chromatograph with a Waters Associates Model 440 absorbance detector and a 30 cmx 3,9 mm i.d. ,u-Bondpak C818 column, under the following conditions: Mobile Phase: 25% 2-propanol 75% 0.01M sodium acetate pH 4.0 Flow Rate: 1 ml./minute Detector: UV at 254 nm.

Sensitivity: 0.04 AUFS Diluent: DMSO Injected Amount: 5 5,ul concentration: 10 mg./ml.

Chart speed varied, but 2 minutes/inch was typical. The above conditions gave UV traces with peaks which were easy to measure quantitatively. The results of the above analyses are referred to in the specification as HLPC assays.

In order to avoid the repetition of complex chemical names, the following abbreviations are sometimes utilized in this specification.

AHBA L-()-y-amino-a-hydroxybutyric acid BHBA N-Carbobenzyloxy derivative of AHBA HONB N-hydroxy-5-norbornene-2,3-dicarboximide NAE N-hydroxy-5-norbornene-2,3-dicarboximide (or BHBA-'ONB') activated ester of BHBA HONS N-hydroxysuccinimide SAE N-hydroxysuccinimide activated ester of (or BHBA-'ONS' BHBA DCC dicyclohexylcarbodiimide DCU dicyclohexylurea HMDS hexamethyldisilazane B SA bis)trimethylsilyl)acetamide MSA trimethylsilylacetamide TFA trifluoroacetyl t-BOC tert. butyloxycarbonyl "Dicalite" is a trademark of the Great Lakes Carbon Corporation for diatomaceous ear washing with 1000 ml. of water, unreacted kanamycin A, 3 - [i - (-) - - amino - a - hydroxybutyryl] - kanamycin A (BB-K29) and amikacin were eluted with 0.5N ammonium hydroxide. Polyacyl material was recovered with 3N ammonium hydroxide. Bioassay, thin layer chromatography and optical rotation were used to monitor the progress of elution. The volume and observed optical rotation of each fraction of eluate, as well as the weight and percent yield of solid isolated from each fraction by evaporation to dryness, are summarized below: Volume Weight Material (ml) a578 (gms.) % Yield KanamycinA 1000 +0.115 0.989 9.15 BB-K29 1750 +0.24 4.37 32.0 Amikacin 2000 +0.31 6.20 47.4 Polyacyls 900 +0.032 0.288 2.0 The spent diethyl ketone layer was shown by high performance liquid chromatography to contain an additional 35% amikacin.

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

Example 2 Preparation of 1 -N-[L-(-)-}'-Amino-a-hydroxybutyryl] kanamycin A Amikacin by Selective Acylation of Poly(trimethylsilyl) 6'-N-Carbobenzyloxykanamycin A in Anhydrous Acetone Poly(trimethylsilyl) 6'-N-carbobenzyloxy kana A prepared as in Example 1 (103 g., .081 moles, calculated as 6'-N-Carbobenzyloxykanamycin A (Silyl)9) was dissolved in 100 ml. of dry acetone at 230. L - (-) - y - benzyloxycarbonylamino a - hydroxybutyric acid N - hydroxy - 5 - norbornene - 2,3 - dicaboximide ester (NAE) (35.24 g., .085 moles) dissolved in 180 ml. of dry acetone at 230 was added slowly with good agitation to the solution of poly(trimethylsilyl) 6'-N Carbobenzyloxykanamycin A over a 15 minute period. the solution was stirred at 23 for 20 hours under a nitrogen atmosphere. The pale yellow, clear solution (pH 7.2) was diluted with 100 ml. of water. The pH of the mixture was adjusted to 2.5 (3N HCI) and stirring continued at 23 for 15 minutes. Acetone was removed using steam-ejector vacuum at about 35". The solution was placed in a 500 ml. Parr bottle, together with 10 g. of 5% palladium on carbon catalyst (Engelhard) and reduced at 40 psi H2 for 2 hours at 230. The mixture was filtered through a pad of diatomaceous earth which was then washed with an additional 50 ml. of water.

After concentration to approximately 1/3 volume, the solution (pH 6.9-7.2) was charged on a 6x 110 cm. CG-50 (NH4+) ion exchange column and eluted with a stepwise gradient from H2O to 0.6 N ammonium hydroxide to recover amikacin.

An automatic polarimeter was used to monitor the progress of elution.

Combinations were made on the bases of thin layer chromatography evaluation.

The combined amikacin fractions were concentrated to 25-30% solids. The solution was diluted with an equal volume of methanol, followed by two volumes of isopropanol to induce crystallization. There was recovered 18.2 g. (400) of crystalline amikacin.

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

Example 3 Preparation of l-N-[L-()-y-Amino-a-hydroxybutyryl]kanamycin A Amikacin by Selective Acylation of Poly(trimethylsilyl) Kanamycin A, Using In Situ Blocking A. Poly(trimethylsilyl) Kanamycin A Kanamycin A free base (18 g. activity, 37.15 m. moles) was slurried in 200 ml.

of dry acetonitrile and heated to reflux. Hexamethyldisilazane (29.8 g., 184.6 m.

moles) was added over 30 minutes and the mixture was stirred at reflux for 78 hours to give a light yellow clear solution. Removal of the solvent under vacuum left an amorphous solid residue (43 gm., 94%) [calculated as kanamycin A (silyl)10].

B. 1 -N-[L-(-)--Amino-a-hydroxybutyryl] kanamycin A p-(Benzyloxycarbonyloxy)benzoic acid (5.56 g., 20.43 m. moles) was slurried in 50 ml. of dry acetonitrile at 230. N,O-bis-Trimethylsilyl acetamide (8.4 g., 41.37 m.

mole) was added with good stirring. The solution was held for 30 minutes at 230, and then added over 3 hours with vigorous stirring to a solution of poly(trimethylsilyl)kanamycin A (21.5 g., 17.83 m. mole, calculated as the (silyl)O compound) in 75 ml. of dry acetonitrile at 23". The mix was stirred for 4 hours, the solvent was removed in vacuo (400), and the oily residue was dissolved in 50 ml. of dry acetone at 230C.

L - (-) -- - benzyloxycarbonylamino - a - hydroxybutyric acid Nhydroxy - 5 - norbornene - 2,3 - dicarboximide ester (NAE) (8.55 g., 20.63 m.

moles) in 30 ml. of acetone was added to the above solution over a period of 5 minutes. The mixture was held at 230C for 78 hours. The solution was diluted with 100 ml. of water and the pH (7.0) lowered to 2.5 (6N HCI). The mixture was placed in a 500 ml. Parr bottle together with 3 g. of 5% palladium on carbon catalyst (Engelhard) and reduced at 40 psi H2 for 2 hours at 230 The mixture was filtered through a pad of diatomaceous earth which was then washed with 20 ml. of water.

The combined filtrate and washings (168 ml.) were determined by microbiological assay against E. coli to contain approximately 11,400 mcg/ml. (19% yield) of amikacin.

Example 4 Preparation of 1-N- [L-(-)-y-Amino-a-hydroxybutyryl] kanamycin A Amikacin by Selective Acylation of Poly(trimethylsilyl) Kanamycin A A. Poly(trimethylsilyl) Kanamycin A A suspension of 10 g. (20.6 m. moles) kanamycin A in 100 ml. of dry acetonitrile and 25 ml. (119 m. moles) 1,1,1,3,3,3-hexamethyldisilazane was refluxed for 72 hours. A clear light yellow solution resulted. The solution was stripped to dryness in vacuo at 30--400C. There was obtained 21.3 g. of poly(trimethylsilyl) Kanamycin A as a light tan amorphous powder [85% yield calculated as kanamycin A (silyl)10].

B. 1 -N-[L-(-)-p-Amino-a-hydroxybutyryl]kanamycin A To a solution of 2.4 g. (2.0 m. moles) of poly(trimethylsilyl) Kanamycin A in 30 ml. of dry acetone was added slowly 2.0 m. moles of L- (-) - y- - benzyloxycarbonylamino - a - hydroxybutyric acid N-hydroxy-5-norbornene-2,3dicarboximide ester (NAE) in 10 ml. of dry acetone at 05 C. The reaction mixture was stirred at 230C for a week and then stripped to dryness in vacuo at a bath temperature of 30--40"C. Water (60 ml.) was then added to the residue, followed by 70 ml. of methanol to obtain a solution. The solution was acidified with 3N HCI to pH 2.0 and then reduced at 50 psi H2 for 2 hours, using 500 mg of 5% palladium on carbon catalyst. The material was filtered, and the combined filtrate and washings were determined by microbiological assay against E. coli to contain a 29.4% yield of amikacin.

Example 5 Preparation of Amikacin by Selective N-Acylation of Polytrimethylsilyl 6'-N-Carbobenzoxy Kanamycin A in Anhydrous Acetone I. Summary Silylation of 6'-N-carbobenzoxy Kana A in acetonitrile using hexamethyldisolazne (HMDS) affords the 6'-N-carbobenzoxy Kana A (silyl)9 intermediateO. This silylated Kana A is readily soluble in most organic solvents.

Acylation with NAE in anhydrous acetone at 230 using a 4% molar excess of NAE relative to 6'-N-Cbz Kana A input afforded a mixture containing only Cbz derivatives of amikacin and BB-K29, some unreacted Kana A and some polyacyl material. No BB-Kl 1 was detectable in any of these studies. Elution of an acetone acylation mix, after reduction and workup, from a CG-50 (NH4+) column using an ammonium hydroxide gradient afforded isolated yields of pure amikacin in the 40% range.

II. Equations

A. ii OH OH HO Ooh a2SAo s 0H03 NH2 0 CE 6'-N-Cbz Kana A 4) C26H42013Nq (618.65) + (CE3) 3Si-NE-Si (CH3)3 HMDS (161.4) A 1 CH3CN > z OR OR OR OH 6 "r NH0R C-O Oa 3 +NH 0 I 3CE R = Si (CH3)3 @ 6'-N-Cbz Kana A (Stlyl)9 C33H114O13N4Si9 (1268.3)

OH o B. Cb:NH(CH2)2"CH-COOH + HON 9 + DCC (206) BHBA (253.4) HONB (179.2) Acetone 6 OH O g I II (224.3) DCU + CbzHN 0 G3 NAE (414.6) C.

diCbzamikacin (854) + Acetone 0 + I' > diCbzBB-R29 230 6'Cbz-1,3-diBHBA-Xana A H2 + 5% Pd/C 6'Cb: Kana A C'fbz Kana A I u (585.62) (722.76) (484.5) CG-50(NHqc) Amikacin Aznikac in III. Materials Wgt. g. Vol. ml. Moles 6'-N-Cbz Kana A 50 .081 HMDS 58.9 76.5 .365 Acetonitrile 300 BHBA 21.5 .085 HONB 15.2 .085 DCC 17.48 .085 Acetone 260 CG-50 (NH4+) 3000 Methanol As required IPA As required IV. Safety 6'-N-Cbz Kana A- No direct information available.

Avoid dust contact.

Acetonitrile- Treat as a cyanide. Avoid breathing vapors. May cause skin irritation.

Hexamethyldisilazane- (HNDS) Irritant, handle with care.

6'-N-Cbz Kana A (Silyl)B No direct information available, handle with care.

BHBA- Toxicity is not established.

Avoid exposure to solids.

HONB- Toxicity unknown. Use precaution in handling.

DCC- A severe skin and eye irritant.

Avoid inhalation of mist or vapors. Toxic.

Acetone Flammable, Inhalation may produce headache, fatigue, excitement, bronchial irritation, and, in large amounts, narcosis.

NAE- No direct information available; always handled directly as solution in acetone.

Methanol Flammable. Poisoning may occur from ingestion, inhalation or percutaneous absorption.

Isopropanol- Flammable. Ingestion or inhalation of large quantities of the vapor may cause headache, dizziness, mental depression, vomiting, narcosis.

Ammonium hydroxide Toxic vapors. Wear mask, avoid contact with liquid.

CG-50 (NH4+S No toxicity data available, handle with care.

V. Procedure A. Preparation 6'-N-Carbobenzyloxykanamycin A (silyl)9 [6'-N-Cbz Kana A (Silyl)9] 1. Slurry 50 g. of 6'-N-carbobenzyloxykanamycin A (KF < 4%) in 300 ml. of acetonitrile (KF < 0.01%). Bring to reflux (740) maintaining a stream of dry nitrogen through the slurry.

2. Add slowly over a 30 minute period 75.8 ml. hexamethyldisilazane (HMDS).

Complete solution will occur with evolution of ammonia gas.

3. Continue refluxing for 18-20 hours under a nitrogen purge.

4. Concentrate the clear, light yellow solution under vacuum (bath temp. 40 50 ) to a foamy solid. Yields, of the silyl9 compound 89-92 g. (90940 Theory).

NOTE: For future reference; in other solvent studies this solid is normally not isolated but used directly for the acylation.

B. Preparation of N-hydroxy-5-norbornene-2,3-dicarboximide ester of L-(-)-&alpha;-carbobenzyloxyamino-&alpha;-hydroxybutyric acid (NAE) 1. Dissolve 21.5 g of L-(-)-&gamma;-carbobenzyloxyamino-&alpha;-hydroxybutyric acid (BHBA) in 100 ml. of dry acetone at 23 followed by 15.2 g. of N-hydroxy-5norbornene-2,3-dicarboximide (HONB). A complete solution will result.

2. Over 30 minutes add a solution of 17.48 g. of dicyclohexylcarbodiimide (DCC) in 50 ml. of acetone with agitation. The temperature will rise to approximately 400 during the addition with precipitation of dicyclohexylurea (DCU).

3. Agitate the slurry for 3 hours allowing the temperature to equilibrate to 23250.

4. Remove the urea derivative by filtration; wash the cake with 30 ml.

acetone. Save the filtrate plus washings for the acylation step below.

C. Acylation of 6'-N-Cbz Kana A (Silyl)9 1. Dissolve the 6'-N-Cbz Kana A (silyl)9 isolated in Part A, Step 4 in 100 ml. of dry acetone at 2324 .

2. With good agitation slowly add the NAE solution prepared in Part B over a 15 minute period. The temperature will gradually rise to approximately 40 . Allow the solution to equilibrate to 230 and continue stirring for 18-20 hours under a nitrogen atmosphere.

3. Add 100 ml of water and lower the pH (6.9-7.2) to 2.2-2.5 with 6N hydrochloric acid. Agitate for 15 minutes at 230.

(NOTE: A second layer may form-this does not present a problem in the workup).

4. Remove acetone under vacuum at a bath temperature of 3035 . Transfer the concentrate to a suitable hydrogenation vessel (prepurged with nitrogen). Add 10 g. 5% palladium on carbon catalyst, and hydrogenate at 40 psi for 2-3 hours.

5. Filter the mixture through a Dicalite pad, washing the hydrogenation vessel and cake with an additional 50 ml. water.

6. Concentrate the filtrate plus wash to approximately 1/3 volume (50 ml.) under vacuum at 40---450.

7. Check the pH. It should be in the range 6.9-7.2. If not, adjust with 1N ammonium hydroxide, Charge the mixture on a CG-50 (NH4+) column (6x110 cm.).

8. Wash the column with 1000 ml. of deionized water. Then elute with 0.50.6N ammonium hydroxide using an automatic polarimeter to monitor the progress of elution. The order of elution is as follows: Residual Kana ABB-K29Amikacin. No BB-Kl 1 was detected in any of our acylation workups. Polyacyl material i.e. the l,3-diAHBA analog of Kana A, is recovered by washing the column with 3N ammonium hydroxide.

9. Combine the amikacin fractions and concentrate to 2530% solids. Dilute with 1 volume of methanol, and seed with amikacin crystals.

10. Add slowly over 2 hours 2 volumes of isopropanol (IPA) with good stirring, and crystallize at 23 for 6-8 hours.

11. Filter the solid, wash with 50 ml. of 1:1:2 water/methanoVIPA mixture, and finally with 25 ml. IPA.

12. Dry in a vacuum oven at 400 for 12-16 hours. Yield: 17.3-19.0 g. (3842%) of amikacin having the following properties: TLC CHCl3-methanol-NH4OH-water (1:4:2:1), 5x20 cm. silica gel plates from Quantum Industries-one zone as detected with ninhydrin (RF~0.4).

Specific Rotation 23 H2O 0.lMNH4OH 0.1MH2SO4 [a] 589 +101.6 +101.9 +103.5 C=1.0% 13. The recovery of BB-K29 in this system was also 3942%, residual Kana A 1014 and 1,3-di AHBA-Kana A approximately 5% to give a material balance > 95%.

Example 6 Preparation of Amikacin by Selective N-Acylation of Polytrimethylsilyl Kana A in Anhydrous Acetone I. Summary Silylation of Kana A 'base' in acetonitrile using hexamathyldisilazane (HMDS) yielded polytrimethylsilyl Kana A. The extent of silylation is as yet uncertain, but for the time being is assumed to be Kana A (Silyl)10. Polysilylated Kana A is readily soluble in most organic solvents. Acylation with SAE in anhydrous acetone at 230 using a 1:1 molar ratio of SAE relative to Kana A input afforded a mixture containing Cbz derivatives of amikacin and BB-K29, usually in the ratio 2-3/1; BB-K6 (approximately 58%), unreacted Kana A (15-20%) and zome olyacyl material (approximately 5-10%). Again, as was seen in our previous work on the acylation of polytrimethylsilyl 6'-N-Carbobenzoxy Kana A, no BB-Kl 1 was detected in any of these experiments. Reduction and work-up of an acetone acylation mix, followed by chromatography on a CG-50-(NH4+) column using 0.5N ammonium hydroxide, afforded isolated crystalline amikacin in the 3439% range.

II. Equations

Kana A 'base' C18H36011N4 (484.51) + (CH3) 3 Si-NH-Si(CH3)3

R r Si (CH3) 3 # Kana A (Silyl)10 C48H116O11N4Si10 (1206.35)

OH B. CbzNH(CH2)2-CH-COOH + HO-N + DCC BEBA N-HOS (206.3) (253.4) (115.9) EtOAc I 0 OH O DCU + Cb:NH(CH2)2CH"-C-O (224.3) < > SAE (350.33) C. Cbz Amikacin (720) +. Cb: BB-K29 Acetone | + + + 23e 230 Cb: BB-K6 + Kana Rana A H2 Polyacyls (Primarily 1,3 5% Pd/C $ t diBEBA-Kana A) X . A Amikacin+BB-K29 + BB-K6 + l.3-diAHBA-Kana A + Kana A (585.62) (722.76) G-50(NHq+) Amikacin

III. Materials Wgt. g. Vol., ml. Moles Kana A 'base' 50 .103 HMDS (Sp. gr. 0.774) 86.68 112 .537 Acetonitrile 600 SAE 35.03 .10 Acetone 850 CG-50 (NH4) 3000 Methanol As required IPA As required IV. Safety Kana A 'base' Known drug-usual caution advised.

Kana A (Silyl)10- No direct information available, handle with care.

Other materials- See Example 5 V. Procedure A. Preparation of Kana A (Silyl)10 1. Slurry 50 g. of Kana A 'base' (KF 2.53.5%) in 500 ml. of acetonitrile (KF < 0.01%). Bring to reflux (740) maintaining a stream of dry nitrogen through the slurry.

2. Add slowly over a 30 minute period 112 ml. hexamethyldisilazane (HMDS).

Complete solution will occur within 4--5 hours with evolution of ammonia gas.

3. Continue refluxing for 22-26 hours under a nitrogen purge.

4. Concentrate the clear faint yellow solution under vacuum (40 ) to a syrupy residue. Flush with an additional 100 ml. acetonitrile, and dry completely under high vacuum for 3--6 hours. Yields of whitish amorphous solid are 109-115 g.

(9095% of theory, calculated as Kana A (Silyl)10).

B. Preparation of n-Hydroxysuccinimide ester of L- (~ - a carbobenzyloxyamino - a - hydroxybutyric acid (SAE) 1. Dissolve 100 gl of L-(-)-&alpha;-benzyloxycarbonylamino-&alpha;-hydroxybutyric acid (BHBA) and 45.38 g. of N-hydroxysuccinimide (N-HOS) in 1300 ml. of ethyl acetate (KF < 0.05%) with stirring at 230 C.

2. Dissolve 81.29 g. of dicyclohexylcarbodiimide (DCC) in 400 ml. of ethyl acetate (KF < 0.05%) at 230C. With good agitation add this solution over 30 minutes to step 1 solution. The temperature will rise to 40420C with concurrent precipitation of dicyclohexylurea (DCU). Agitate the slurry 3-4 hours allowing the temperature to equilibrate to 230 C.

3. Filter the DCU; wash the cake with 250 ml. of ethyl acetate (KF < 0.05%).

Discard the DCU cake. Save the filtrate and washes.

4. Concentrate the filtrate plus washes to 500 ml. (in vacuo at 30-35 C).

Some product will crystallize out.

5. Transfer the concentrate to a suitable vessel and add with vigorous agitation 100 ml. of heptane. If necessary, add seed crystals of SAE. Crystallization will begin almost immediately. Agitate the slurry for 30 minutes at 230C.

6. Add, over 30 minutes, 400 ml. of heptane and agitate the slurry 4--5 hours at 23 C.

7. Filter and wash the cake with 200 ml. of 3:1 heptane/ethyl acetate followed by 100 ml. of heptane.

8. Dry in a vacuum oven at 30-35 C for 18-20 hours.

Yield is 110.1-131.4 g. (80-95%).

MP-l l9l200 with softening at 114 (Corr.).

TLC-4 acetone:12 benzene: 1 CH3CO2H- Detection 1% aqueous KMO4.

Rf-0.7 for SAE; 0.2 BHBA on 2x10 cm prescored silica gel plates from Analtech Inc.

C. Acylation of Kana A (Silyl)10 1. Dissolve the Kana A (Silyl),0 isolated in Part A, Step 4 in 500 ml. dry acetone at 230C.

2. With good agitation add rapidly the SAE prepared in Part B (35.03 g.) as a 10% solution in dry acetone over a 5-10 minute period. The temperature will rise approximately 5 . Allow the solution to equilibrate to 230, and continue stirring for 18-20 hours.

3. The light orange, clear solution is diluted with 400 ml. of water, and the pH (7.0-7.5) lowered to 2.2-2.5 with 3N hydrochloric acid. The clear solution is now agitated at 230 for 15-30 minutes.

4. Acetone is removed under vacuum at a bath temperature of 3035 (a small amount of material may separate at this point, but presents no problem).

Transfer the concentrate to a suitable hydrogenation vessel. Add 10 g. 5 , palladium on carbon catalyst, and hydrogenate at 50 psi for 2-3 hours.

5. Filter the mixture through a Dicalite pad, and wash the hydrogenation vessel and cake with an additional 2x50 ml. water.

6. Concentrate the filtrate plug washings to approximately 1/3 volume (150165 ml.) under vacuum at 40--450.

7. The pH at this point is in the range 6.0--7.0. The mixture is charged on CG-50 (NH4+) column (6x 110 cm).

8. Wash the column with 1000 ml. of deionized water. Elute with 0.5N ammonium hydroxide using an automatic polarimeter to monitor the progress of elution. The order of elution is as follows: Residual Kana A~BB-K6oBBK29 < Amikacin. No. BB-Kl 1 was detected in any of our experiments.

9. Combine the amikacin fractions and concentrate to 2530% solids. Dilute with I volume methanol, and seed with amikacin crystals.

10. Add slowly over 2 hours 2 volumes of IPA with good stirring and crystallize at 23 for 6-8 hours.

11. Filter the solid, wash with 35 ml. of 1:1:2 water/methanol/IPA, and finally with 35 ml. IPA.

12. Dry in a vacuum over at 400 for 16-24 hours. Yield: 19.91-22.84 g. (34- 39 XO) IR, PMR and CMR spectral data in addition to specific rotation were completely consistent for the desired structure.

TLC System CHClimethanol/NH4OH/water (1:4:2:1) 5x20 cm. silica gel plates from Quantum Industries-I Zone amikacin having Err0.4 (Ninhydrin Detection).

Example 7 Preparation of Amikacin by Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A in Tetrahydrofuran With the Mixed Acid Anhydride of Pivalic Acid and BHBA A. Preparation of Mixed Anhydride BHBA (5.066 gm., 20.0 m. moles), BSA (4.068 mg., 20.0 m. moles) and triethylamine (2.116 g., 22.0 m. moles) were dissolved in 200 ml. of sieve dried tetrahydrofuran. The solution was refluxed for 2 1/4 hours and then chilled to 10"C. Pivaloyl chloride (2.412 gm., 20.0 m. moles) was added over a period of 23 minutes, with stirring, and stirring was continued for 2 hours at -100C. The temperature was then allowed to climb to 230 C.

B. Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A Poly(trimethylsilyl) 6'-N-Cbz Kana A prepared as in Example 1(5.454 gm., 4.97 m. moles, calculated as 6'-Cbz Kana A (sill)9) was dissolved in 50 ml. dry (molecular sieve) tetrahydrofuran at 230C. One-half of the solution of mixed anhydride prepared in step A, above, (10.0 m. moles) was added over a period of twenty minutes, with stirring, and stirring was continued for 7 days.

Water (100 ml.) was then added to the reaction mixture, and the pH (5.4) was adjusted to 2.0 with 3M H2502. Stirring was continued for 1 hour and the solution was extracted with ethyl acetate. Polyacylated material began to crystallize, so the reaction mixture was filtered. After drying over P2O5, the recovered solids weighed 0.702 gms. The extraction of the reaction mixture was continued for a total of 4x75 ml. of ethyl acetate, after which the excess ethyl acetate was stripped from the aqueous layer. An aliquot of the aqueous solution was subjected to assay by HPLC.

The resulting curve indicated a 26.4% yield of di-Cbz amikacin.

The aqueous layer was then hydrogenated in a Parr apparatus at 50 p.s.i. H2 pressure for two hours, using 0.5 gm. 10% Pd on carbon catalyst. The material was filtered, and the combined filtrate and washings were determined against E. coli to contain a 31.2% yield of amikacin. Amikacin/BB-K29 ratio approximately 9-10/1; traces of polyacyl and unreacted Kana A present.

Example 8 Effect of Water on the Preparation of Amikacin by the Acylation of Poly(trimethylsilyl) Kana A in Acetone Solution at 230C.

A. Anhydrous Solvent Poly(trimethylsilyl) Kana A prepared as in Example 3 (2.40 gm., 2.0 m. moles, calculated as Kana A (silyl)10) was dissolved in 20 ml. of acetone which had been dried with a molecular sieve. The solution was stirred at 230C and a solution of SAE (0.701 gm., 2.0 m. moles) in 10 ml. of sieve dried acetone was added over a period of 10 seconds. Stirring was continued at 230C for 22 hours. Water (50 ml.) was added and the pH (7.5) was adjusted to 2.5. The acetone was stripped in vacuo at 40"C and the aqueous solution was then reduced at 51 p.s.i. H2 pressure at 230C for two hours, utilizing 1.0 gm. of 10% Pd on carbon as catalyst. Microbiological assay showed a 31.24% yield of amikacin.

B. Water Added to Solvent Step A, above, was repeated, except that 1.0 ml. (56 m. moles) of water was added to the poly(trimethylsilyl) Kana A solution, and stirred for 15 minutes, prior to acylation with SAE. Microbiological assay showed a 33.80% yield of amikacin.

Example 9 Preparation of Amikacin by Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A in Acetone with the Mixed Anhydride of BHBA and Isobutylcarbonic Acid A. Preparation of Mixed Anhydride BHBA (1,267 gm., 5.0 m. moles) and N-trimethylsilylacetamide (MSA) (1.313 gm., 10.0 m. moles) in 20 ml. of sieve dried acetone was stirred at 23"C, and triethylamine (TEA) (0.70 ml., 5.0 m moles) were added. The mixture was refluxed under a N2 atmosphere for 2 1/2 hours. The mixture was cooled to -200C and isobutylchloroformate (0.751 gm., 0--713 ml., 5.50 m. moles) was added.

Triethylamine hydrochloride immediately began to separate. The mixture was stirred for 1 hour at --200C.

B. Acylation Poly(trimethylsilyl) 6'-N-Cbz Kana A prepared as in Example 1(6.215 gm., 4.9 m. moles, calculated as the (silyl)9 compound) was dissolved in 20 ml. of sieve dried acetone, with stirring, at 230 C. The solution was cooled to -200C and the cold mixed anhydride solution from step A was slowly added over a period of 30 minutes. The reaction mixture was stirred for an additional 1 1/2 hours at -200C and then for 17 hours at 230C. The reaction mixture was then poured into 150 ml.

of water at 230C with stirring, the pH (7.75) was adjusted to 2.5 with 3N HC1, and stirring was continued for 15 minutes. Acetone was then stripped in vacuo at 400C.

An aliquot of the resulting aqueous solution was subjected to assay by HPLC. The resulting curve indicated a 34.33% yield of di-Cbz ainikacin.

The main portion of the aqueous solution was reduced at 50 p.s.i. H2 pressure at 230C for 3 1/4 hours, utilizing 2.0 gms of Pd/C catalyst. The catalyst was removed by filtration and the combined filtrate and washings were determined by microbiological assay against E. coli to contain a 35% yield of ainikacin.

Example 10 Preparation of Amikacin by Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A in 3-Pentanone Poly(trimethylsilyl) 6'-N-Cbz Kana prepared as in Example 1(30 gm., 23.65 m.

moles, calculated as 6'-N-Cbz Kana A (silyl)9) dissolved in 100 ml. sieve dried 3pentanone was stirred at 230C, and NAE (26.02 m. moles, 10% excess) was added over a period of 40 minutes. Stirring was continued for 113 hours at 23"C and the mixture was then added to 250 ml. water with vigorous stirring. The pH (7.3) was adjusted to 2.5 with 3N HCI, the mixture was stirred for an additional 30 minutes, and the 3-pentanone was stripped in vacuo at 400 C. The aqueous solution was extracted with 4x 100 ml. of ethyl acetate. An aliquot of the aqueous solution was then subjected to assay by HPLC. The resulting curve indicated a 46.12% yield of di-Cbz amikacin The main portion of the aqueous reaction mixture was reduced at 51.0 p.s.i. H2 pressure at 23"C for 2 1/2 hours, utilizing 3.0 gms. of 10% Pd/C catalyst.

Microbiological assay of an aliquot of the combined filtrate and washings indicated a 40.24% yield of amikacin. The main portion of the reduced aqueous reaction mixture was then concentrated in vacuo at 400C to approximately 100 ml. and fractionated on a CG-50 (NH4+) ion exchange column (4 inchesx4 feet, containing approximately 10 liters of resin). The aqueous solution was charged on the column, the column was washed with 5 liters of water, and the material was eluted with 0.5N NH40H (followed by 3N NH40H to elute polyacetylated products). Polarimetry of the fractions showed the presence of a 42.7% yield of amikacin, a 12.0% yield of unreacted kanamycin A, a 12.4% yield of polyacylated material and a 23.20to yield of BB-K29.

Example 11 Preparation of Amikacin by Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A in Anhydrous Cyclohexanone For Varying Times A. Poly(trimethylsilyl) 6'-N-Cbz Kana A prepared as in Example 1(2.537 gm., 2.0 m. moles, calculated as 6'-N-Cbz Kana A (silyl)9) in 300 ml. dry cyclohexanone was acylated for 20 hours at 230C with an NAE solution in dry cyclohexanone (10.8 ml. of 0.1944 m. mole/ml. solution, 2.10 m. mole). The reaction mixture was then added to 150 ml. of water, with stirring, and the pH (5.6) was adjusted to 2.5 with 3N HCI. The cyclohexanone was stripped in vacuo at 400C and an aliquot of the remaining aqueous phase was taken for assay by HPLC. The main portion of the aqueous phase was reduced under 50 p.s.i. H2 pressure for 3 hours at 230C, using 1.0 gm. of 10% Pd/C catalyst. The catalyst was removed by filtration and the combined filtrate and washings were assayed microbiologically for ainikacin.

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

Yields HPLC Assay Microbiological Assay 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(trimethylsilyl) 6'-N-Cbz Kana A in Anhydrous Tetrahydrofuran For Varying Times A. Example 11 A was repeated except that dry tetrahydrofuran was utilized as solvent instead of dry cyclohexanone.

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

Yields HPLC Assay Microbiological Assay Amikacin (di-Cbz Amikacin) Turbidimetric Plate Reaction A 29.27% 28.34% 28.18% Reaction B 33.39% 21.52% 28.63% Example 13 Preparation of Amikacin by Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A in Anhydrous Dioxane For Varying Times A. Example 11 A was repeated except that the acylation was continued for 44 hours utilizing dry dioxane as the solvent.

B. Example 11 B was repeated except that the acylation was continued for 18 1/2 hours utilizing dry dioxane as the solvent.

Yields HPLC Assay Microbiological Assay Amikacin (di-Cbz Amikacin) Turbidimetric Plate Reaction A 39f18% 43.27% 33.36% Reaction B 42.82% 22.55% 33.37% Example 14 Preparation of Amikacin by Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A in Anhydrous Diethyl ketone at 75"C To a stirred solution of poly(trimethylsilyl) 6'-N-Cbz Kana A prepared as in Example 1(2.537 gm., 2.0 m. moles, calculated as 6'-N-Cbz Kana A (silyl)9) in 32 ml. sieve dried diethyl ketone at 750C was added a solution of NAE (10.8 ml. of 0.1944 m. moles/ml. of diethyl ketone, 2.10 m. moles) over a period of 15 minutes.

Stirring was continued at 750C for an additional 3 hours after which the mixture was poured into 150 ml. of water. The pH was adjusted to 2.8 with 3N HCI and the diethyl ketone was stripped in vacuo at 400 C. HPLC assay of an aliquot of the aqueous phase indicated a 39.18% yield of di-Cbz ainikacin.

The main portion of the aqueous phase was reduced under 49.8 p.s.i. H2 pressure for 3 1/4 hours at 23 C, using 1.0 gram of Pd/C catalyst. The catalyst was removed by filtration and the combined filtrate and washings were assayed microbiologically for ainikacin. Turbidometric assay showed 27.84%. yield and Plate assay showed 28.6% yield.

Example 15 Preparation of Amikacin by the Acylation of Poly(triinethylsilyl) Kana A with NAE at 05 After Back Hydrolysis With Water A. Silylation of Kanamycin A Using HMDS With TMCS as Catalyst Kanamycin A (10 gm of 97.6% purity, 20.14 m. moles) in 100 ml. of sieve-dried acetonitrile was brought to reflux under a nitrogen atmosphere. A mixture of HMDS (22.76 gm., 141 m. moles, 7 moles per mole of Kanamycin A) and TMCS (1 ml., 0.856 gm., 7.88 m. moles) was added to the refluxing reaction mixture over a period of 10 minutes. Reflux was continued for 4-3/4 hours and the mixture was then cooled, concentrated in vacuo to a yellow viscous syrup and dried under high vacuum for 2 hours. The yield of product was 23.8 gms. (97.9%, calculated as Kanamycin A (Silyl),O).

B. Acylation Polytrimethylsilyl) kanamycin A (23.8 gms., 20.14 m. moles) prepared in step A above was dissolved in 250 ml. of sieve-dried acetone at 230 and then cooled to 05 Water (3.63 ml, 201.4 m. moles, 10 moles per mole -of polysilylated kanamycin A) was added, with stirring, and the mixture was allowed to stand under moderate vacuum for 30 minutes. NAE (19.133 m. moles, 0.95 moles per mole of polysilylated kanamycin A) in 108.3 ml of acetone was then added over a period of < 1 minute. The mixture was stirred at 05 for 1 hour, diluted with water, the pH adjusted to 2.5, and the acetone was then removed in vacuo. The aqueous solution was then reduced at 50 p.s.i. H2 pressure at 23 for 2-1/2 hours using 2.0 gms of 10% Pd on carbon as a catalyst. The reduced reaction mixture was filtered through Dicalite, concentrated to ca. 100 ml. in vacuo at 400 and then charged on CG50(NH4+) column (6 liters resin, 5x100 cm.). It was washed with water and then eluted with 0.6N-1.0N-3N NH4OH. There was obtained 60.25% ainikacin, 4.37% BB-K6, 4.35% BB-K29, 26.47% kanamycin A and 2.18% polyacyls.

Example 16 Preparation of Amikacin by the Acylation of Poly(trirnethylsilyl) 6'-N-Cbz Kana A with SAE at 05 After Back Methanolysis A. Silylation of 6'-N-Cbz Kanamycin A 6'-N-Cbz kanamycin A (20.0 gm., 32.4 m. moles) in 200 ml. of sieve-dried acetonitrile was brought to reflux under a nitrogen atmosphere. HMDS (47.3 ml., 226.8 m. moles, 7 moles per mole of 6'-N-Cbz kana A) was added over a 10 minute period and reflux was continued for 20 hours. The mixture was then cooled, concentrated in vacuo, and dried under high vacuum for 2 hours to give 39.1 gms. of white amorphous solid (95.4% yield, calculated as 6'-N-Cbz kana A (silyl)9).

B. Acylation Poly(trimethylsilyl) 6'-N-Cbz kana A (39.1 gm., 32.4 m. moles) prepared in step A above was dissolved in 400 ml. of dry acetone, with stirring, at 23 . Methanol (6.6 ml., 162 m. moles, 5 moles per mole of polysilylated 6'-N-Cbz kana A) was added and the mixture was stirred at 230 for 1 hour under a strong nitrogen purge. The mixture was cooled to 0--50 and a solution of SAE (11.35 gm., 32.4 m. moles) in 120 ml. of pre-cooled, dry acetone was added. The mixture was stirred for an additional 3 hours at 05 and then placed in a 40 cold room for 1 week. Water (300 ml) was added, the pH was adjusted to 2.0, the mixture was stirred for 1 hour, and the acetone was then stripped in vacuo. The resultant aqueous solution was reduced at 54.0 p.s.i. H2 pressure for 17 hours at 23 utilizing 3.0 gm. of 10% Pd on carbon as catalyst. It was then filtered through Dicalite, concentrated in vacuo to 75-100 ml., charge on a CG-50(NH4+) column and eluted with water and 0.6N NH4OH. There was obtained 52.52% ainikacin, 14.5% BB-K29, 19.6% kanamycin A and 1.71 polyacyls.

Example 17 Preparation of Amikacin by the Acylation of Poly(trimethylsilyl) Kana A With SAE at 05 After Back Hydrolysis With Water A. Silylation of Kanamycin A with TMCS in Acetonitrile Using Tetramethyl guanidine as Acid Acceptor Kanamycin A (4.88 gm., 10.07 m. mole) was suspended in 100 ml. of sievedried acetonitrile with stirring at 23 . To the stirred suspension was added tetramethylguanidine (TMG) (16.234 gm., 140.98 m. moles, 14 moles per mole of kanamycin A). The mixture was heated to reflux and TMCS (15.32 gm., 140.98 m.

moles, 14 moles per mole of kanamycin A) was added over a 15 minute period. A white precipitate of TMG HCI formed after about one-half of the TMCS had been added. The mixture was cooled to room temperature, concentrated to a tacky residue and dried under high vacuum for 2 hours. The solid was triturated with dry THF (100 ml.), and the insoluble TMG HCI was filtered off and washed with 5x20 ml. portions of THF. The combined filtrate and washings were concentrated in vacuo at 400 to a tacky residue and dried under high vacuum for 2 hours. There was obtained 10.64 gms. of a light cream tacky residue (87.6% yield, calculated as kanamycin A (silyl)10).

B. Acylation Poly(trimethylsilyl) kanamycin A (10.64 gm., 10.07 m. moles) prepared in step A above was dissolved in 110 ml. of sieve-dried acetone, with stirring, at 230 and the solution was cooled to 05 . Water (1.81 ml., 100.7 m. moles, 10 moles per mole of polysilylated kana A) was added and the solution was stirred for 30 minutes under moderate vacuum. SAE (3.70 gm., 10.57 m. moles, 5% excess) in 40 ml. of pre-cooled dry acetone was added over a period of < 1 minute, and the mixture was stirred for one hour. The mixture was worked up by the general procedure in Example 16B to give ca. 50% amikacin, ca. 10% BB-K29, 58% BB-K6, ca. 20% kanamycin A and 5-8 polyacyls.

Example 18 Preparation of Poly(trimethylsilyl) Kanamycin A in Pyridine Using HMDS Kanamycin A (10.0 gms., 20.64 m. moles) was suspended in 100 ml. sieve-dried freshly distilled pyridine at 239. A nitrogen purge was started and the suspension was brought to reflux. HMDS (17.33 gms., 107.32 m. moles, 5.2 moles per mole of kanamycin A) was added over a period of 10 minutes and the mixture was refluxed for 19 hours. It was then cooled to room temperature, concentrated in vacuo to a light yellow-gold syrup, and dried under high vacuum to a white amorphous solid.

There was obtained 22.1 gms. (92.6% yield, calculated as kanamycin A (silyl)10).

Example 19 Preparation of Poly(triethylsilyl) Kanamycin A Using Triethylchlorosilane With Triethylamine as Acid Acceptor Kanamycin A (5.0 gms. of 97.6% purity, 10.07 m. moles) was suspended in 100 ml. of sieve-dried acetonitrile at 230. Triethylamine (TEA) (33.8 ml., 24.5 gm., 241.7 m, moles) was added and the suspension was brought to reflux. A solution of trichloroethylsilane (23.7 ml., 21.3 gm., 140.98 m. moles) in 25 ml. dry acetonitrile was added over a 20 minute period. Reflux was continued for an additional 7 hours and the mixture was cooled to room temperature, whereupon long fine needles of TEA HCI separated out. The mixture was allowed to stand at room temperature for ca. 16 hours, concentrated in vacuo at 400 to a tacky solid and dried for 2 hours under high vacuum to a deep orange tacky solid. The solid was triturated with 100 ml dry THF at 23 and the insoluble TEA HCI was filtered off, washed with 5x20 ml of THF, and dried to give 16.0 gms of TEA HCI. The combined filtrate and washings were concentrated in vacuo to a solid and dried under high vacuum for 2 hours. There was obtained 19.3 gms of poly(triethylsilyl) kanamycin A as a deep orange viscous syrup.

Example 20 Preparation of Poly(trimethylsilyl) Kanamycin A Using bis-Trimethylsilylurea Kanamycin A (10.0 gm. of 99.7% purity, 20.58 m. moles) was suspended in 200 ml. of sieve-dried acetonitrile, with stirring, at 23". To the suspension was added bis-trimethylsilylurea (BSU) (29.45 gms., 144.01 m. moles, 7 moles per mole of kanamycin), and the mixture was brought to reflux under a nitrogen atmosphere.

Reflux was continued for 17 hours and the reaction mixture was then cooled to room temperature. A small amount of insoluble material present was removed by filtration, washed with 3x10 ml. portions of acetonitrile and dried (1.1381 gms.).

Infrared showed this to be BSU plus a small amount of unreacted kanamycin A.

The combined filtrate and washings were cooled at 40 for 16 hours. Additional solid separated, was recovered as above, (7.8 gms.) and was shown by infrared to be BSU plus urea. The light yellow filtrate and washings were concentrated in vacuo at 400 and dried under high vacuum to give 27.0 gm. of a white solid which was partly tacky and partially fine needle-like crystals. The solid was treated with 150 ml. of heptane at 230, the insoluble portion was removed by filtration, washed with 2x50 ml. portions of heptane and dried, to give 6.0 gms. of white needles (shown by infrared to be BSU plus urea). The combined filtrate and washings were concentrated in vacuo at 400 and dried under high vacuum for 2 hours to give 20.4 gms. of white needles, the infrared spectrum of which was typical for polysilylated kanamycin A. Calculations showed the product to contain an average of 7.22 trimethylsilyl groups.

Example 21 Preparation of Amikacin by the Acylation of Per(trimethylsilyl) Kanamycin A After Partial Desilylation With 1,3-Butanediol A. Preparation of Per(trimethylsilyl) kanamycin A Kanamycin A (10.0 gm., 20.639 m. moles) was suspended in 100 ml. of sievedried acetonitrile, with stirring, at 230. The suspension was brought to reflux under a nitrogen purge and HMDS (23.322 gms., 144.5 m. moles, 7 moles per mole of kanamycin A) was added over a period of ten minutes. Reflux was continued for 16 hours and the mixture was then cooled to room temperature, concentrated in vacuo and dried for 2 hours under high vacuum. There was obtained 24.3 gm. of a white, tacky residue (92.1% yield, calculated as kanamycin A (silyl)").

B. Acylation Per(trimethylsilyl) kanamycin A (24.3 gm.) prepared in step A above was dissolved in 240 ml. of sieve-dried acetone, wtih stirring, at 230. To this solution was added 1,3-butanediol (9.25 ml., 103.2 m. mole), 5 moles per mole of per(trimethylsilyl) kanamycin A. The mixture was stirred at 239 for 2 hours under a nitrogen purge and then cooled at 05 . SAE (7.23 gm., 20.64 m. moles) in 70 ml.

of pre-cooled acetone was added over a period of about 1 minute. The mixture was stirred at 05 for 3 hours and then allowed to stand in a 4" cold room for ca. 16 hours. Water (200 ml.) was added, the pH was adjusted to 2.5 and the clear yellow solution was stirred at 230 for 30 minutes. The acetone was stripped in vacuo and the aqueous solution was reduced at 55.0 p.s.i. H2 pressure at 230 for 2 hours using 3.0 gm. of 10% Pd on carbon as catalyst. The reduced solution was filtered through Dicalite and chromatographed as in Example 16B to give 47.50% amikacin, 5.87% BB-K29, 7.32% BB-K6, 24.26% kanamycin A and 7.41% polyacyls.

Example 22 Preparation of Amikacin by the Acylation of Poly(trimethylsilyl) Kanamycin A Prepared in THF Using SAE With Sulfamic Acid Catalyst To a reffuxine mixture of kanamycin A (5.0 gm., 10.32 m. moles) in 50 ml. of sieve-dried tetrahydrofuran (THF) were added sulfamic acid (100 mg.) and HMDS (12.32 gm., 76.33 m. moles). The mixture was refluxed for 18 hours, with complete solution occurring after 6 hours. The solution was cooled to 230, treated with 0.1 ml. of water and held at 230 for 30 minutes. A solution of SAE (3.61 gm., 10.3 m.

moles) in 36 ml of THF was added over a period of 30 minutes. After stirring for 3 hours, the mixture was diluted with 100 ml. of water and the pH was adjusted to 2.2 with 10% H2SO4. It was stirred for 30 minutes at 230 and then concentrated in vacuo to remove THF. The resulting aqueous solution was reduced at 50 p.s.i. H2 pressure for 2 hours at 230 using 10% Pd on carbon as a catalyst. The reduced solution was filtered through Dicalite and the solids were washed with water. The combined filtrate and washings (150 ml.) were determined by microbiological assay against E. coli to contain 1225 mcg./ml. (31.5% activity yield) of amikacin.

Example 23 Preparation of Amikacin by the Acylation of Poly(trimethylsilyl Kanamycin A with the N-Hydroxysuccinimide Ester of Di-Carbobenzyloxy AHBA A. Preparation of Dicarbobenzyloxy L-(-)-a-Ainino-a-hydroxybutyric Acid N-Hydroxysuccinimide Ester Dicarbobenzyloxy L-(-)-cr-amin o-cy-hydroxyb utyric acid (8 gm., 20.65 m.

moles) and N-hydroxysuccinimide (2.37 gm., 20.65 m. moles) were dissolved in 50 ml. of dry acetone at 23". Dicyclohexylcarbodiimide (4.25 gm., 20.65 m. moles) dissolved in 20 ml. of dry acetone was added and the total was agitated at 230 for 2 hours. Dicyclohexylurea was filtered off, the filter cake was washed with 10 ml. of dry acetone, and the filtrate and washings were combined.

B. Acylation Poly(trimethylsilyl) kanamycin A, prepared according to the general procedure of Example 21 from 10.0 gms. (20.639 in. moles) of 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 05 for 30 minutes under moderate vacuum.

To this solution was added the solution of the di-Cbz-blocked acylating agent prepared in step A, and the mixture was stirred at 05 for 30 minutes. The mixture was diluted with water, the pH was adjusted to 2.2 and the acetone was removed in vacua. The aqueous solution was reduced by the general procedure of Example 22 and then filtered through Dicalite. Chromatography showed 4045% ainikacin, ca. 10% BB-K29, a trace of BB-K6, ca. 30% kanamycin A and a small amount of polyacyls.

* Example 24 Preparation of Poly(trimethylsilyl) Kanamycin A Using HMDS with Imidazole as Catalyst Kanamycin A (11 gm., 22.7 m. moles) and 100 mg of imidzole were heated to reflux in 100 ml. of sieve-dried acetonitrile, under a nitrogen purge HMDS (18.48 gm., 114.5 m. moles, 5 moles per mole of kanamycin A) was added over a period of 30 minutes and the mixture was refluxed for 20 hours. Complete solution occurred in ca. 2-1/2 hours. The solution was cooled to 230 and the solvent was removed in vacuo to leave 21.6 gms. of poly(trimethylsilyl) kanamycin A as a foamy residue (93.1% yield, calculated as kanamycin(silyl)ll).

Example 25 Preparation of 1 -N-[L-(-)-y-Amino-a-hydroxybutyryl] kanamycin B (BB-K26) by the Acylation of Poly(trimethylsilyl) Kanamycin B With SAE A. Preparaion of Poly(trimethylsilyl) Kanamycin B Using HMDS With TMCS Catalyst Kanamycin B (25 gm., 51.7 m. moles) in 250 ml. of sieve-dried acetonitrile was heated to reflux under a stream of nitrogen. HMDS (62.3 gm., 385.81 m. moles, 7.5 moles per mole of kanamycin B) was added over a period of 30 minutes followed by 1 ml. of TMCS as catalyst. The mixture was refluxed for 21 hours with complete solution after 1 hour. The solvent was then removed in vacuo at 600 and the oily residue was held at 60 under high vacuum for 3 hours. There was obtained 53.0 gm. of poly(trimethylsilyl) kanamycin B (85.2% yield, calculated as kanamycin B (silyl),o)- B. Acylation The poly(trimethylsilyl) kanamycin B prepared in step A above (53.0 gm.) was dissolved in 500 ml. of dry acetone at 05 , methanol (20.9 ml.) was added, and the mixture was stirred in vacuo for 30 minutes at 05 . A solution of SAE (18.1 gm., 51.67 m. moles) in 200 ml. of pre-cooled dry acetone was added over a period of less than 1 minute and the mixture was stirred for 30 minutes at 05 . The mixture was worked up according to the general procedure of Example 22 and then loaded on a column of CG-50 (NH4+) (8x 120 cm.). It was eluted with an NH40H gradient from 0.6N to 3N. There was obtained 38% of BB-K26, 5% of the corresponding 6' N-acylated kanamycin B (BB-K22), 10% of the corresponding 3-N-acylated kanamycin B (BB-K46) 14.63% kanamycin B and a small amount of polyacylated kanamycin B.

Example 26 Preparation of Poly(trimethylsilyl) kanamycin A Using HMDS With Kanamycin A Sulfate as Catalyst Kanamycin A (19.5 gm., 40.246 m. moles) and kanamycin A sulfate (0.5 gm., 0.858 m. mole) [total=20.0 gm., 41.0 m. moles] in 200 ml. of sieve-dried acetonitrile was brought to reflux. HMDS (60.3 ml., 287.7 m. moles, 7 moles per mole of kanamycin A) was slowly added and the mixture was refluxed for 28 hours. It was then stripped to dryness on a rotary evaporator and dried under steam injector vacuum. There was obtained 47.5 gms. of poly(trimethylsilyl) kanamycin A as a pale yellow oil (95.82% yield, calculated as kanamycin A (silyl)10).

Example 27 Preparation of Amikacin by the Acylation of Poly(trimethylsilyl) Kanamycin A With N-Trifluoroacetyl Blocked AHBA N-Hydroxysuccinimide Ester A. Preparation of N-Trifluoroacetyl AHBA and Conversion to its N-Hydroxysuccinimide Ester To a suspension of AHBA (5.0 gm., 42 m. moles) in 100 ml. THF was added trifluoroacetic anhydride (40 gm., 191 m. moles), with stirring, over a 10 minute period. The solution was stirred for 18 hours at 23C and then concentrated to dryness in vacuo at SOC. The residue was dissolved in 100 ml. of aqueous methanol (1:1) and stirred for 1 hour. It was then concentrated to dryness in vacuo and redissolved in 50 ml. H2O. The aqueous solution was extracted with 3x50 ml.

portions of MIBK and, after drying over Na2SO4, the extract was concentrated to an oil. Traces of solvent were removed by adding and distilling off 4 ml. of water.

On standing the oil changed to a waxy, crystalline solid (2.5 gm., 28% yield.

raised to ca. 6.0 with NH4OH and the mixture was stripped to dryness in vacuo to give 36.3 gms. of a golden oil. The oil was dissolved in 200 ml. of trifluoroacetic acid, allowed to stand for 15 minutes and stripped to dryness in a rotary evaporator.

The oil was washed with water and the water was flashed off. Concentrated NH4OH was added to pH 6.0 and was flashed off. The resulting solid was dissolved in water, filtered, and the filter washed with water. The combined filtrate and washings (259 ml.) were loaded on a CG-50 (NH4+) column (8x92 cm.), washed with 4 liters of water and eluted with an NH4OH gradient (0.6N-1.0N- concentrated). There was obtained 40.32% amikacin, 4.58% BB-K6, 8.32% BB K29, 30.50% kanamycin A and 7.43% polyacyls.

Example 29 The general procedure of Example 1 is repeated, except that the 6'-N carbobenzyloxykanamycin A used therein is replaced by an equimolar weight of 6' N-carbobenzyloxykanamycin B, and there is thereby produced l-N-[L-(-)-y amino-a-hydroxybutyryl] -kanamycin B.

Example 30 The general procedure of Example I is repeated except that the L-(-)-y benzyloxycarbonylamino-&alpha;-hydroxybutyric acid N-hydroxy-5-norbornene-2,3dicarboximide ester used therein is replaced by L-(-)-ss-benzyloxycarbonylamino-&alpha;-hydroxypropionic acid N-hydroxy-5norbornene-2,3-dicarboximide ester and L-(-)-P-benzyloxycarbonyl amino-a-hydroxyyaleric acid N-hydroxy-5-norbornene- 2,3-dicarboximide ester, respectively, and there is thereby produced 1 -N-[L-(-)-p-amino-ct-hydroxypropionyl] kanamycin A and 1-N-[L-(-)-#-amino-&alpha;-hydroxyvaleryl]kanamycin A, respectively.

Example 31 The general procedure of Example 25 is repeated except that the L-(-) y-benzyloxycarbonyl amino-lr-hydroxybutyric acid N-hydroxy-ester used therein is replaced by L-(-)-P-benzyloxycarbonylamino-ct-hydro propionic acid N-hydroxysuccinimide ester and L-(-)-#-benzyloxycarbonylamino-&alpha;-hydroxyvaleric acid N-hydroxysuccinimide ester, respectively and there is thereby produced l-N-[L-(-)-ss-amino--hydroxypropionyl]kanamycin B and 1-N-[L-(-)-&alpha;-amino-&alpha;-hydroxyvaleryl]kanamycin B, respectively.

WHAT WE CLAIM IS: 1. A process for the preparation of a l-N-[w-amino-a- hydroxyalkanoyl]kanamycin A or B having the formula

wherein R is OH or NH2 and n is 0 or an integer of 1 or 2, or a nontoxic pharmaceutically acceptable acid addition salt thereof, which process comprises acylating polysilylated kanamycin A or B or polysilylated kanamycin A or B

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (20)

**WARNING** start of CLMS field may overlap end of DESC **. raised to ca. 6.0 with NH4OH and the mixture was stripped to dryness in vacuo to give 36.3 gms. of a golden oil. The oil was dissolved in 200 ml. of trifluoroacetic acid, allowed to stand for 15 minutes and stripped to dryness in a rotary evaporator. The oil was washed with water and the water was flashed off. Concentrated NH4OH was added to pH 6.0 and was flashed off. The resulting solid was dissolved in water, filtered, and the filter washed with water. The combined filtrate and washings (259 ml.) were loaded on a CG-50 (NH4+) column (8x92 cm.), washed with 4 liters of water and eluted with an NH4OH gradient (0.6N-1.0N- concentrated). There was obtained 40.32% amikacin, 4.58% BB-K6, 8.32% BB K29, 30.50% kanamycin A and 7.43% polyacyls. Example 29 The general procedure of Example 1 is repeated, except that the 6'-N carbobenzyloxykanamycin A used therein is replaced by an equimolar weight of 6' N-carbobenzyloxykanamycin B, and there is thereby produced l-N-[L-(-)-y amino-a-hydroxybutyryl] -kanamycin B. Example 30 The general procedure of Example I is repeated except that the L-(-)-y benzyloxycarbonylamino-&alpha;-hydroxybutyric acid N-hydroxy-5-norbornene-2,3dicarboximide ester used therein is replaced by L-(-)-ss-benzyloxycarbonylamino-&alpha;-hydroxypropionic acid N-hydroxy-5norbornene-2,3-dicarboximide ester and L-(-)-P-benzyloxycarbonyl amino-a-hydroxyyaleric acid N-hydroxy-5-norbornene- 2,3-dicarboximide ester, respectively, and there is thereby produced 1 -N-[L-(-)-p-amino-ct-hydroxypropionyl] kanamycin A and 1-N-[L-(-)-#-amino-&alpha;-hydroxyvaleryl]kanamycin A, respectively. Example 31 The general procedure of Example 25 is repeated except that the L-(-) y-benzyloxycarbonyl amino-lr-hydroxybutyric acid N-hydroxy-ester used therein is replaced by L-(-)-P-benzyloxycarbonylamino-ct-hydro propionic acid N-hydroxysuccinimide ester and L-(-)-#-benzyloxycarbonylamino-&alpha;-hydroxyvaleric acid N-hydroxysuccinimide ester, respectively and there is thereby produced l-N-[L-(-)-ss-amino--hydroxypropionyl]kanamycin B and 1-N-[L-(-)-&alpha;-amino-&alpha;-hydroxyvaleryl]kanamycin B, respectively. WHAT WE CLAIM IS:

1. A process for the preparation of a l-N-[w-amino-a- hydroxyalkanoyl]kanamycin A or B having the formula

wherein R is OH or NH2 and n is 0 or an integer of 1 or 2, or a nontoxic pharmaceutically acceptable acid addition salt thereof, which process comprises acylating polysilylated kanamycin A or B or polysilylated kanamycin A or B

containing a blocking group other than silyl on the 6'-amino moiety with an acylating derivative of the acid of the formula

in which n is 0 or an integer of 1 or 2 and B is an amino-blocking group, in a substantially anhydrous organic solvent, and subsequently removing all blocking groups.

2. A process as claimed in claim 1 wherein the acylating derivative of the acid is an active ester of a mixed acid anhydride.

3. A process as claimed in claim 1 or claim 2 wherein the amino-blocking group B of the acylating derivative of the acid is

wherein R: and R2 are the same or different and each is H, F, Cl, Br, NO2, OH, (lower)alkyl or (lower)alkoxy, and X is Cl, Br, F or I and Y is H, Cl, Br, F or I, and in the case of the last stated blocking group the B-HN grouping then becomes B-N to give rise to phthalimido.

4. A process as claimed in claim 2 or claim 3 wherein the acylating derivative of the acid is its active ester with N-hydroxysuccinimide, N-hydroxy-5-norbornene2, 3-dicarboximide or N-hydroxyphthalimide.

5. A process as claimed in claim 2 or claim 3 wherein the acylating derivative of the acid is its mixed acid anhydride with pivalic acid, benzoic acid, isobutylcarbonic acid or benzylcarbonic acid.

6. A process as claimed in any one of the preceding claims wherein the polysilylated kanamycin A or B contains a carbobenzyloxy group on the 6'-amino moiety.

7. A process as claimed in any one of the preceding claims wherein the polysilylated kanamycin A or B starting material contains an average number of silyl groups per molecule of from 4 to 8.

8. A process as claimed in any one of claims 1 to 6 wherein the polysilylated kanamycin A or B starting material contains a blocking group other than silyl on the 6'-amino moiety and also contains an average number of silyl groups per molecule of from 3 to 7.

9. A process as claimed in any of the preceding claims wherein the silyl groups are trimethylsilyl.

10. A process for the preparation of a I-N[L-(-)-w-amino-cr- hydroxyalkanoyl]kanamycin A as claimed in any one of the preceding claims wherein the acylating derivative of the acid is of the formula

11. A process as claimed in any one of the preceding claims wherein the reaction is conducted in a solution containing 10 to 20% polysilylated kanamycin starting material.

12. A process as claimed in any one of the preceding claims wherein the desired 1 -N-acylated product is separated from the reaction mixture by chromatography.

13. A process as claimed in claim I substantially as hereinbefore described with reference to any one of the Examples.

14. Polysilylated kanamycin A or B, or polysilylated kanamycin A or B containing a blocking group other than silyl on the 6'-amino moiety.

15. A polysilylated kanamycin as claimed in claim 14 in which the silyl groups are trimethylsilyl.

16. Polysilylated kanamycin A as claimed in claim 14 or claim 15 containing an average number of silyl groups per molecule of from 4 to 8.

17. Polysilylated kanamycin A as claimed in claim 14 or claim 15 having a blocking group other than silyl on the 6'-amino moiety and containing an average number of silyl groups per molecule of from 3 to 7.

18. A polysilylated kanamycin A as claimed in any one of claims 14 to 17 in which the blocking group on the 6'-amino moiety is

wherein R1 and R2 are the same or different and each is H, F, Cl, Br, NO2, OH, (lower)alkyl or (lower alkoxy), and X is Cl, Br, F or I and Y is H, Cl, Br, F or I.

19. A polysilylated kanamycin A as claimed in any one of claims 14, 15, 17 or 18, in which the blocking group on the 6'-amino moiety is the carbobenzyloxy group.

20. A 1-N-[#-amino-&alpha;-hydroxyalkanoyl]kanamycin A or B whenever prepared by a process as claimed in any one of claims 1 to 13.