CH639104A5 - Process for the preparation of 1-N-[omega-amino-alpha-hydroxyalkanoyl] kanamycins - Google Patents

Description

The invention relates to an improved and economically useful process for the preparation of compounds of the general formula I. The use of a polysilylated kanamycin A or B as starting material results in a high solubility in the organic solvent system. This allows the reaction to be carried out at high concentrations. Although the reaction is usually carried out in solutions containing approximately 10 to 20% polysilylated kanamycin starting material, excellent t 'is also achieved.

CH_NH-,

HO

25th

30th

35

Results at concentrations of approximately 50 g / 100 ml solvent.

As with the working methods of the prior art, the process according to the invention produces a mixture of acylated products. The desired 1-N-acylated product is separated from the other products by chromatography. If desired, the by-products can be hydrolyzed to the starting kanamycin in order to reuse it. In contrast, it has been found in the prior art procedures that any formed 3 "N-acylated material means loss of approximately an equal amount of the desired 1-N-acylated product due to the great difficulty that A particularly advantageous feature of the process according to the invention can be seen in the fact that only an extremely small amount of undesired product which is N-acylated in the 3 "position is formed (usually the presence of this product cannot be determined at all ).

If one considers the l- [L - (-) - a-amino-a-hydroxybutyryl] kanamy-cin A, d. H. which produces amikacin according to various workings of the prior art, is usually also the 3 "position N-acylated product (BB-Kll), the 3-position N-acylated product (BB-K29), the 6'-position N-acylated product (BB-K6) and polyacylated material are formed and unreacted kanamycin A is also obtained. It has thus been used in the production of amikacin on an industrial scale by acylation of 6'-N-carbobenzyloxykanamycin A in an aqueous medium followed by removal the protecting group, found that approximately 10% of the desired amikacin (2.5 kg with a batch of 25 kg) is usually lost due to the presence of BB-Kll as a by-product, whereas if amikacin is produced by the process according to the invention usually do not even detect the BB-Kll in the reaction mixture.

The invention thus provides a process for the preparation of a 1-N- [co-amino-a-hydroxyalkanoyl] kanamycin A or B of the general formula I:

(I)

wherein R stands for OH or NH2 and n represents an integer from 0 to 2, or non-toxic, pharmaceutically acceptable acid addition salts of these compounds, which is characterized in that polysilylated kanamycin A or B or

65 polysilylated kanamycin A or B, which contains a blocking group other than silyl in the 6'-amino group, with an acylating derivative of the acid of the general formula II:

639 104

b-hn-ch9- (ch9) -ch-c00h

L £ 11 |

Oh

(II)

wherein n is an integer from 0 to 2 and B is an amino-blocking group, acylated in a substantially anhydrous organic solvent and then all blocking groups are removed.

The blocking group used to protect the 6'-amino group of the kanamycin and the amino group of the acylating acid (group B in formula II) can be conventional blocking groups for protecting primary amino groups. These are known to the person skilled in the art. Suitable blocking groups include alkoxycarbonyl groups, for example tert-butoxycarbonyl and tert-amyloxycarbonyl, aralkoxycarbonyl groups, for example benzyloxycarbonyl, cycloalkyloxycarbonyl groups, for example cyclohexyloxycarbonyl, haloalkoxycarbonyl groups, for example tri-chloroethoxycarbonyl, acyl groups, for example phthaloyl and o-nitrophenoxyacetyl groups, known block groups for example, the o-nitrophenylthio groups, the trityl group, and the like.

O ii ch0oc-

5 The acylating acid of the general formula II can be present in its (+) or (-) - isomeric form or as a mixture of the two isomers (d, 1-form). In this way, the corresponding compound of general formula I is formed, in which the IN- [to-amino-a-hydroxyalkanoyl] group in its (+) io [or (R)] form or its (-) [or (S)] form, or in a mixture thereof. Any isomeric form and mixture thereof fall within the scope of the invention. In a preferred embodiment, the acylating acid of the general formula II is in its (-) form. In another preferred embodiment, the acylating acid of the general formula II is in its (+) form.

The starting material for the process according to the invention is polysilylated kanamycin A or B (and preferably polysilylated kanamycin A) or polysilylated kanamycin A 20 or B (and preferably polysilylated kanamycin A) which contains a blocking group on the 6'-amino group other than silyl. This blocking group is preferably selected from the groups of the following formulas:

CH-3 0

I II

ch, -c-ó-c-J I

ck-,

0

y2xcc-

o in which R1 and R2 are identical or different and in each case for H, formula II is an active ester. F, Cl, Br, NO 2, OH (lower) alkyl or (lower) alkoxy are preferably the active ester with N-hydroxysuccinimide, N-hydroxy-5-and X is CI, Br, F or J and Y is H, Cl, Br, norbornene-2,3-dicarboximide or N-hydroxyphthalimide. In egg-F or J owns. The most preferred blocking group is the other preferred embodiment is the carbobenzyloxy group. 65 acylating derivative of the acid of general formula II

Mixture of acid anhydride, preferably the mixture of. In a preferred embodiment of the invention, acid anhydride is pivalic acid, benzoic acid, isobutyl carbon - it is the acylating derivative of the acid of general acid or benzyl carbonic acid.

639 104

In a particularly preferred embodiment, the invention relates to the preparation of IN - [L - (-) - Y-amino-a-hydroxy-butyryl] kanamycin A, or a non-toxic, pharmaceutically acceptable acid addition salt thereof, the process being characterized in this way that acylated polysilylated kanamycin A with a mixed acid anhydride of L - (-) - y-benzyloxycarbonylamino-a-hydroxybutyric acid (preferably its mixed acid anhydride with pivalic acid, benzoic acid, isobutyl carbonic acid or benzyl carbonic acid) in a substantially anhydrous organic solvent and then blocking groups and then blocking groups away.

In a further particularly preferred embodiment, the invention relates to the preparation of 1-N- [L - (-) - y-amino-a-hydroxybutyryl] kanamycin A or non-toxic, pharmaceutically acceptable acid addition salts thereof, the process being characterized in that that polysilylated kanamycin A, which has a carbobenzyloxy group on the 6'-amino group, with a mixed acid anhydride of L - (-) - Y-benzyloxycarbonylamino-a-hydroxybutyric acid (preferably its mixed anhydride with pivalic acid, benzoic acid, isobutylcarbonic acid or benzylcarbonic acid) acylated in an essentially anhydrous organic solvent and then all blocking groups removed.

In a further particularly preferred embodiment, the invention relates to the preparation of IN- [L - (-) - Y-amino- <x-hydroxybutyryl] kanamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof, the process being characterized in that that polysilylated kanamycin A 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) acylated in an essentially anhydrous organic solvent and then all blocking groups removed.

In a further particularly preferred embodiment, the invention relates to the preparation of IN - [L - (-) - Y-amino-a-hydroxybutyryl] kanamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof, the process being characterized in that polysilylated kanamycin A containing a carbobenzyloxy group on the 6'-amino group with an active ester of L - (-) - y-benzyloxycarbonylamino-a-hydroxybutyric acid (and preferably its active ester with N-hydroxysuccinimide, N-hydroxy-xy -5-norbornen-2,3-dicarboximide or N-hydroxyphthalimide) acylated in an essentially anhydrous organic solvent and then all blocking groups removed. . -

R4

Polysilylated kanamycin A or B which contains a blocking group other than silyl on the 6'-amino group can be used as the starting material for the process according to the invention. Preferably polysilylated kanamycin A 5 or B (and preferably polysilylated kanamycin A) containing an average number of 4 to 8 silyl groups (preferably trimethylsilyl groups) per molecule is used. Another preferred starting material is polysilylated kanamycin A or B (preferably polysilylated kanamycin A), which has a blocking group other than silyl on the io 6'-amino group and contains an average number of 3 to 7 silyl groups (preferably trimethylsilyl groups) per molecule.

15

20th

The term “non-toxic, pharmaceutically acceptable acid addition salt” of a compound of the general formula I used here means a mono-, di-, tri- or tetra salt which does not result from the reaction of a molecule of a compound of the general formula I with 1 to 4 equivalents of one -toxic, pharmaceutically acceptable acid is formed. Such acids include acetic acid, hydrochloric acid, sulfuric acid, maleic acid, phosphoric acid, nitric acid, hydrobromic acid, ascorbic acid, malic acid and citric acid, and all other acids commonly used to form salts with amine-containing pharmaceuticals.

The acylation of the polysilylated kanamycin A or B starting material% with or without a blocking group on the 6'-amino group x other than silyl is generally carried out in an organic solvent in which the starting material has sufficient solubility. The starting materials are readily soluble in most organic solvents. Suitable solvents include, for example, acetone, diethyl ketone, methyl n-propyl ketone, methyl isobutyl ketone, methyl ethyl ketone, acetonitrile, Gly-me, diglyme, dioxane, toluene, tetrahydrofuran, cyclohexanone, methylene chloride, chloroform, carbon tetrachloride and mixtures of acetone / butanol or diethyl ketone / Butanol. The choice of solvent depends on the starting material used. In general, ketones are preferred solvents. The cheapest solvent for a particular combination of reactants can easily be found through routine tests.

30th

35

40

45

Suitable silylating agents for use in the preparation of the polysilylated kanamycin starting materials used herein include those of the general formulas IV and V below:

/

Si

R NH

R

4th

Si / \

N-H

■ Si

• R

and m

R "

R -Si-X

R

IV

V

639 104

wherein R5, R6 and R7 can each represent hydrogen, halogen, (lower) alkyl, halogen (lower) alkyl and phenyl, where at least one of the groups R5, R6 and R7 are different from halogen or hydrogen, R4 represents (low) Alkyl is, m represents an integer from 1 to 2 and X has the meanings halogen and o

/ R

~ nc 9 ^ R

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

R-

Si

R '

in which R5, R6 and R7 have the above meanings.

Examples of specific silyl compounds of the formulas IV and V which may be mentioned are: trimethylchlorosilane, hexamethyldi-silazane, triethylchlorosilane, methyltrichlorosilane, dimethyldi-chlorosilane, triethylbromosilane, tri-n-propylchlorosilane, methyldi-ethylchlorosilanesilane, dimethylchlorosilane, dimethylchlorosilane, dimethylchlorosilane, dimethyl Phenyldimethylbromsilan, Benzylmethyläthylchlor silane, Phenyläthylmethylchlorsilan, triphenylchlorosilane, Triphe-nylfluorsilan, tri-o-tolylchlorsilan, tri-p-dimethylaminophe-nylchlorsilan, N-Äthyltriäthylsilylamin, Hexaäthyldisilazan, tri-phenylsilylamin, tri-n-propylsilylamin, Tetraäthyldimethyldisi-lazan, hexaphenyldisilazane, Hexa-p-tolyldisilazane, and the like. Hexaalkylcyclotrisilazanes and octa-alkylcyclotetrasilazanes are also useful. Other suitable silylating agents are silylamides (e.g. trialkylsilylacetamides and bis-trialkylsilylacetamides), silylureas (e.g. trimethylsilylurea) and silylureides. Trimethylsilylimidazole can also be used.

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

If a polysilylated kanamycin A or B is used which contains a blocking group other than silyl on the 6'-amino group, this starting material can either be prepared by polysilylating the desired 6'-position N-blocked kanamycin A or B or introduces the desired 6'-N blocking group into polysilylated kanamycin A or B.

Methods for introducing silyl groups into organic compounds, including certain aminoglycosides, are known. The polysilylated kanamycins (with or without a blocking group on the 6'-amino group other than silyl) can be prepared by methods which are known per se or are described in the present text.

The term “polysilylated kanamycin A or B” used here refers to kanamycin A or B, which has 2 to 10 silyl groups in the molecule. For example, the term "polysilylated kanamycin A or B" does not include a persilylated kanamycin A or B that contains 11 silyl groups in the molecule.

The exact number of silyl groups (or their position) in the polysilylated kanamycin starting material (with or without a blocking group on the 6'-amino group other than silyl) is not known. According to the invention, it was found that undersilylation and oversilylation lower the yield of the desired product and lead to an increase in other products. In the case of severe under- or oversilylation, it can happen that little or no desired product is formed at all. The degree of silylation which leads to the greatest yield of the desired product depends on the particular reactants used in the acylation step. The most advantageous degree of silylation in any combination of reaction partners can easily be determined by routine experimentation.

In the preparation of IN - [L - (-) - y-amino-a-hydroxybutyryljkanamycin A by acylation of polysilylated kanamycin A with the N-hydroxysuccinimide ester of L - (-) - y-benzyl-15 oxycarbonylamino-a- According to the invention, hydroxybutyric acid in acetone solution was found to give good yields of the desired product if polysilylated kanamycin A was used, which was prepared by reacting about 4 to about 5.5 moles of hexamethyldisilazane per molecule of kanamycin A. Larger or smaller amounts of hexamethyldisilazane can also be used, but this significantly reduces the yield of the desired product in the subsequent acylation step. In the above specific procedure, it is preferred to use about 4.5 to about 5.0 moles of 25 hexamethyldisilazane per mole of kanamycin to achieve maximum product yield in the acylation step.

Two equivalents of the trimethylsilyl group in kanamycin A or B can be introduced per mole of hexamethyldisilazane. 30 Both Kanamycin A and Kanamycin B have a total of 11 sites (NH2 and OH groups) that can be silylated, while Kanamycin A and Kanamycin B, which have a blocking group other than silyl on the 6'-amino group, in total 10 such places. Thus, 5.5 35 moles of hexamethyldisilazane per mole of kanamycin A or B would theoretically lead to complete silylation of all OH and NH2 units of the kanamycin, while 5.0 moles of hexamethyldisilazane would result in complete silylation of one mole of kanamycin A or B, which in the 6'-amino group has a blocking group other than silyl 40. However, it is believed according to the invention that such extensive silylation does not occur at reasonable molar ratios during reasonable reaction times, although higher degrees of silylation can be achieved with a given reaction time, 45 if a silylation catalyst is added.

Silylation catalysts result in a significant increase in the rate of silylation. Suitable silylation catalysts are known. These include amine sulfates (for example kanamycin sulfate), sulfamic acid, imida-50 zol and trimethylchlorosilane. Silylation catalysts generally promote a higher degree of silylation than is required in the process according to the invention. However, oversilylated kanamycin A or B can also be used as the starting material in the process according to the invention if it is first treated with a desilylating agent in order to reduce the degree of silylation before the acylation reaction is carried out.

Good yields of the desired product are obtained by acylating polysilylated kanamycin A which has been prepared using a 5.5: 1 molar ratio of hexamethyldisilazane to kanamycin A. However, when kanamycin A with a 7: 1 molar ratio of hexamethyldisilazane (or with a 5.5: 1 molar ratio in the presence of a silylation catalyst) in acetone with the N-hydroxysuccini fi5 mid ester of the L - (-) - y-benzyloxycarbonylamino -A-hydroxybutyric acid acylated, so you get less than a 1% yield of the desired product. However, if you do the same "oversilylated" kanamycin A with the same acylating agent in one

639 104

Acetone solution, to which 1 hour before the acylation reaction water [21 mol water per mol kanamycin / 2.5% water (w / v)] was added as the desilylating agent, yields a yield of approximately 40% of the desired product. The same results are obtained when the water is replaced by methanol or another active hydrogen compound which can lead to desilylation, for example ethanol, propanol, butanediol, methyl mercaptan, ethyl mercaptan, phenyl mercaptan, or the like.

Although dry solvents are usually used when working with silylated materials, it was surprisingly found that even in the absence of "oversilylation", adding water to the reaction solvent before the acylation often gives the same good yield, sometimes even better yields of the desired product than in a dry solvent. In the case of acylation reactions which are carried out in acetone at the customary concentrations of 10 to 20% (w / v) polysilylated kanamycin A, it was found according to the invention that excellent yields of IN- [L - (-) - Y- Amino-a-hydroxybutyryljkanamycin A is obtained by adding up to 28 moles of water per mole of polysilylated kanamycin A. If one works at 20% concentration, 28 moles per mole means approximately 8% water. With other combinations of reactants, the presence of even more water can be tolerated; it can even be beneficial. The acylation reaction can be carried out in solvents containing up to about 40% water, although short acylation times must be chosen at such high water concentrations to avoid excessive desilylation of the polysilylated kanamycin A or B starting material. Accordingly, when the term "essentially anhydrous organic solvent" is used in the present case, this term also includes solvents which contain up to approximately 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 4% water.

As previously stated, the most appropriate level of silylation for any combination of acylation reactants can easily be determined by routine testing. It is believed that the preferred average number of silyl groups in the starting material is usually between 4 and 8 for kanamycin A or B and between 3 and 7 for kanamycin A or B which has a blocking group other than silyl on the 6'-amino group . However, this is a more theoretical consideration and is not necessarily an essential part of the invention.

The duration and temperature of the acylation reaction are not critical. Temperatures in the range of about -30 ° C to about 100 ° C are useful with reaction times ranging from about 1 hour to a day or more. It has been found according to the invention that the reaction usually proceeds well at room temperature, and for reasons of simplicity and economy it is preferred to carry out the reaction at ambient temperature. However, when it comes to maximum yields and selective acylation, it is preferred to carry out the acylation at a temperature from about 0 ° C to about 5 ° C.

Acylation of the 1-amino group of the polysilylated kana-mycin A or B (with or without a blocking group on the 6'-amino group other than silyl) can be carried out with any acylating derivative of the acid of general formula II known to those known is that it is suitable for the acylation of a primary amino group. Examples of suitable acylating derivatives of free acid include the corresponding acid anhydrides, mixed anhydrides, for example alkoxy formic anhydrides, acid halides,

Acid azides, active esters and active thioesters. The free acid can be coupled with the polysilylated kanamycin starting material after first the free acid with N.N'-dimethylchloroformiminium chloride [cf. GB-PS1008170 and Novak and Weichet, Experientia XXI, 6,360 (1965)], or with the aid of an N, N'-carbonyldiimidazole or an N, N'-carbonylditriazole [cf. South African patent 63/2684] or with the aid of a carbodiimide reagent [in particular N, N'-dicyclohexylcarbodiimide, N, N'-diisopropylcarbodiimide or N-cyclohexyl-N '- (2-morpholinoethyl) carbodiimide; see. Sheehan and Hess, J.A.C.S., 77, 1967 (1955)] or using an alkynylamine reagent [cf. R. Buijle and H.G. Viehe, Angew. Chem. International Edition, 3,582 (1964)] or with the aid of an isoxazolium salt reagent [cf. R. B. Woodward, R. A. Olofson, and H. Mayer, J. Amer. Chem. Soc., 83.1010 (1961)] or with the aid of a ketene imine reagent [cf. C.L. Stevens and M.E. Münk, J. Amer. Chem. Soc., 80, 4065 (1958)] or using a hexachlorocyclotriphosphatriazine or Hexabromicyclotriphosphatriazins (US Pat. No. 3,650,050) or using diphenylphosphorylazide [DDPA; J. Amer. Chem. Soc., 94, 6203-6205 (1972)] or with the aid of diethylphosphoryl cyanide [DEPC; Tetrahedron Letters No. 18, pages 1595-1598 (1973)] or with the aid of diphenyl phosphite [Tetrahedron Letters No. 49, pages 5047-5050 (1972)]. Another equivalent of the acid is a corresponding azolide, i.e. H. an amide of the corresponding acid, the amide nitrogen of which is a member of a quasi-aromatic 5-membered ring which contains at least two nitrogen atoms, i.e. H. Imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. It is obvious to the person skilled in the art that it can sometimes be desirable or necessary to protect the hydroxyl group of the acylating derivative of the acid of the general formula II, for example when using acylating derivatives, such as acid halides. The protection of the hydroxyl group can be carried out in a manner known per se, e.g. Using a carbobenzyloxy group, by acetylation, by silylation, and the like.

After the acylation reaction has ended, all blocking groups are removed in a manner known per se in order to provide the desired product of the general formula I.

The silyl groups can be easily removed, for example, by hydrolyzing with water. This is preferably done at low pH. The blocking group B of the acylating derivative of the acid of the general formula II and the blocking group on the 6'-amino group of the polysilylated kanamycin starting material (if present) can also be removed by known methods. For example, a tert-butoxycarbonyl group can be removed by using 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 zinc dust in glacial acetic acid, remove the phthaloyl group by treating hydrazine hydrate in ethanol with heating, and the like.

The yields of product were determined using various methods. After removing all blocking groups and chromatography on a column with CG-50 (NH4 +), the yield of amikacin could be determined by isolating the crystalline solid from the corresponding fractions or by microbiological analysis (turbidimetric method or plate test) of the corresponding fractions. Another technique used in the present invention was high performance liquid chromatography of the unreduced acylation mixture, i.e. with the aid of the aqueous solution obtained after the hydrolysis of the silyl groups and the removal of the organic solvent, but before the hydrolysis to remove the remaining blocking groups.

8th

5

10th

15

20th

25th

30th

35

10th

45

50

55

60

65

639 104

pe (n). This test is not a direct test for amikacin or BB-K29, but for the corresponding mono- or di-N-blocked compounds.

The instrument used was a Waters Associates ALC / GPC 244 high pressure liquid chromatograph with a Waters Associates Model 440 absorption detector and a 30 cm x 3.9 mm (inner diameter) (i-Bondapak C-18 column) under the following conditions:

Mobile phase:

Flow rate:

Detector: Sensitivity: Diluent: Amount injected: Concentration:

25% 2-propanol

75% 0.01 M sodium acetate pH 4.0

1 ml / minute

UV at 254 nm

0.04 on

DMSO

5 ixl

10 mg / ml

The writing speed varied, but 2min / 254 cm were typical. The above conditions gave UV traces with peaks that were easy to quantify. The results of the above analyzes are referred to herein as HLPC tests.

To avoid repetition of complicated chemical names, the following abbreviations are sometimes used in this text:

AHBA BHBA HONB

NAE

(or BHBA-'ONB ')

HONS

SAE

(or BHBA-'ONS ')

DCC

DCU

HMDS

BSA

MSA

TFA

t-BOC

L - (-) - y-Amino-a-hydroxybutyric acid N-carbobenzyloxy derivative of AHBA N-hydroxy-5-norbornene-2,3-dicarboximide

N-Hydroxy-5-norbornene-2,3-dicarboximide activated ester of BHBA N-hydroxysuccinimide N-hydroxysuccinimide activated ester of BHBA

Dicyclohexylcarbodiimide

Dicyclohexylurea

Hxamethyldisilazane

Bis (trimethylsilyl) acetamide

Trimethylsilylacetamide

Trifluoroacetyl tert-butyloxycarbonyl

After removing the solvent in vacuo (40 ° C.) and drying completely under vacuum (10 mm), 27.9 g of a white, amorphous solid are obtained. Yield 90.71%, calculated as 6'-N-carbobenzyloxykanamycin A (silyl) g.

5 This solid is dissolved in 150 ml of dry diethyl ketone at a temperature of 23 ° C. 11.05 g (26.67 mmol) of L - (-) - Y-benzyloxycarbonylamino-a-hydroxybutyric acid re-N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE), dissolved in 100 ml of dry Diethyl ketone slowly at 23 ° C with good io stirring in the course of half an hour. The solution is stirred for 78 hours at 23 ° C. The yellow, clear solution (pH 7.0) is diluted with 100 ml of water. The pH of the mixture is adjusted to 2.8 with 3N HCl and the mixture is stirred vigorously for 15 minutes at a temperature of 23 ° C. The aqueous phase is separated off and the organic phase is extracted with 50 ml of water at pH 2.8 . The combined aqueous fractions are washed with 50 ml of ethyl acetate. The solution is placed in a 500 ml Parr device together with 5 g of 5% palladium on activated carbon catalyst (Engelhard) and reduced for 2 hours at 20 23 ° C. at a pressure of 3.52 kg / cm 2 ( 50 psi) H2. The mixture is filtered through a filter bed of dicalite, which is then washed with an additional 30 ml of water. The colorless filtrate is concentrated in vacuo (40 to 45 ° C.) to 50 ml. The solution is applied to a 5 × 100 cm CG-50 (NH4 +) ion exchange column 25. After washing with 1000 ml of water, unreacted kanamycin A, 3- [L - (-) - Y-amino-a-hydroxybutyryl] kanamycin A (BB-K29) and amikacin are eluted with 0.5N ammonium hydroxide. Polyacyl material is recovered with 3n ammonium hydroxide. To monitor the continuous elution, choose bioassay, thin layer chromatography and optical rotation. Volume, observed optical rotation of each fraction of the eluate and weight and percentage yield of the solid isolated from each fraction by evaporation to dryness are shown below:

30th

material

Volume (ml) a 578 weight (g)% yield

Kanamycin A 1000 BB-K29 1750 40 Amikacin 2000 Polyacyle 900

+ 0.115 0.989

+ 0.24 4.37

+ 0.31 6.20 + 0.032 0.288

9.15 32.0 47.4 2.0

"Dicalite" is a trademark of the Great Lakes Carbon Corporation for a product made from diatomaceous earth.

“Amberlite CG-50” is a trademark of Rohm & Haas Co. for a weakly acidic cation exchange resin of the carboxylic acid polymethacrylic acid type of chromatographic quality.

"H-Bondapak" is a trade name of Waters Associates for a range of high performance liquid chromatography columns.

All temperatures are given in degrees Celsius.

The terms “lower alkyl” and “lower alkoxy” refer to alkyl or alkoxy groups which have 1 to 6 carbon atoms.

example 1

Preparation of l-N- [L (-) - Y-amino-a-hydroxybutyryl] kanamycin-cin A, amikacin, by selective acylation of poly (trimethylsilyl) -6'-N-carbobenzyloxykanamycin A in anhydrous diethyl ketone

15 g (24.24 mmol) of 6'-N-carbobenzyloxykan-mycin A are slurried in 90 ml of dry acetonitrile and heated to reflux under a nitrogen atmosphere. Then 17.5 g (108.48 mmol) of hexamethyldisilazane are slowly added over a period of 30 minutes and the solution obtained is kept under reflux for 24 hours.

High-performance liquid chromatography shows that the used diethyl ketone layer contains a further 3 to 5% amikacin.

The crude amikacin (6.20 g) is dissolved in 20 ml of water and diluted with 20 ml of methanol and then 20 ml of isopropanol is added to initiate crystallization. 6.0 g (45.8%) of crystalline amikacin are obtained.

50

Example 2

Preparation of l-N- [L - (-) - Y-amino-a-hydroxybutyryl] kana-mycin A, amikacin, by selective acylation of poly (trime-55 thylsilyl) -6'-N-carbobenzyloxykanamycin A in anhydrous acetone

103 g [0.081 mol, calculated as 6'-N-carbobenzyl-oxykanamycin A (silyl) g] poly (trimethylsilyl) -6'-N-carbobenzyl-oxykanamycin A, which were prepared as described in Example 1, are dissolved, 60 g, in 100 ml of dry acetone at a temperature of 23 ° C. 35.24 g (0.085 mol) of L - (-) - y-benzyloxycarbonylamino-a-hydroxybutyric acid-N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE), dissolved in 180, are slowly added with good stirring ml of dry acetone at 23 ° C. over the course of 15 min to dissolve the poly (trimethylsilyl) -6'-N-carbobenzyloxykanarny-cins A. The solution is stirred for 20 hours at 23 ° C. under a nitrogen atmosphere. The pale yellow, clear solution (pH 7.2) is diluted with 100 ml of water. The pH of the mixture is measured with

65

639 104

10th

3n HCl set to 2.5. Then the mixture is stirred at 23 ° C. for a further 15 min. The acetone is removed using a steam jet vacuum at approximately 35 ° C. The solution is placed in a 500 ml Parr bottle along with 10 g of 5% palladium on activated carbon catalyst (Engelhard). Reduce at a temperature of 23 ° C for 2 hours at a pressure of 2.81 kg / cm2 (40 psi) H2. The mixture is then filtered through a filter bed of diatomaceous earth, which is then washed with a further 50 ml of water. After concentrating on approximately 'A of the volume, the solution (pH 6.9 to 7.2) is placed on a 6 x 110 cm CG-50 (NH4 +) ion exchange column and eluted with a gradual gradient from H20 to 0.6 n Ammonium hydroxide to obtain the amikacin. An automatic polarimeter is used to monitor the continuous elution. The fractions are pooled based on evaluation by thin layer chromatography. The combined amikacin fractions are concentrated to a solids content of 25 to 30%. The solution is diluted with an equal volume of methanol and then two volumes of isopropanol are added to initiate crystallization. 18.2 g (40%) of crystalline amikacin are obtained.

Recovery of 12% Kanamycin A, 40% BB-K29 and 5% polyacylated Kanamycin gives a material balance of 97%.

de, which is then washed with 20 ml of water. The combined filtrates and washing liquids (168 ml) are tested by E microbiological test. coli found that they contain approximately 11400 mcg / ml amikacin (yield 19%).

Example 4

Preparation of l-N- [L - (-) - y-amino-a-hydroxybutyryl] kana-mycin A, amikacin, by selective acylation of poly (trimethylsilyl) -kanamycin A

15

A) Poly (trimethylsilyl) kanamycin A

A suspension of 10 g (20.6 mmol) of kanamycin A in 100 ml of dry acetonitrile and 25 ml (119 mmol) of 1,1,1,3,3,3-hexamethyldisilazane is refluxed for 72 hours. A clear, light yellow solution is obtained. The solution is evaporated to dryness in vacuo at 30 to 40 ° C. 21.3 g of poly (trimethylsilyl) kanamycin A are obtained in the form of a light ocher-colored, amorphous powder. The yield is 85%, calculated as kanamycin A (silyl) 10.

Example 3

Preparation of l-N- [L - (-) - y-amino-a-hydroxybutyryl] kana-mycin A, amikacin, by selective acylation of poly (trimethylsilyl) -kanamycin A, blocking in situ.

A) Poly (trimethylsilyl) kanamycin A

Kanamycin A is slurried in the form of the free base with 18 g activity (37.15 mmol) in 200 ml dry acetonitrile and heated to reflux. 29.8 g are added over a period of 30 minutes. (184.6 mmol) of hexamethyldisilazane and the mixture is stirred under reflux for 78 h, a light yellow clear solution being obtained. After removing the solvent under vacuum, an amorphous, solid residue remains. The yield is 43 g [94%, calculated as kanamycin A (silyl) wl.

25th

B) l-N- [L - (-) - y-amino-a-hydroxybutyryl] kanamycin A

2.0 mmol L - (-) - y-benzyloxycarbonylamino-a-hydroxybutyric acid-30 N-hydroxy- are slowly added to a solution of 2.4 g (2.0 mmol) of poly (trimethylsilyl) kanamycin in 30 ml of dry acetone. 5-norbornene-2,3-dicarboximide ester (NAE) in 10 ml of dry acetone at a temperature of 0 to 5 ° C. The reaction mixture is stirred at 23 ° C. for 1 week and then evaporated to dryness in vacuo at a bath temperature of 30 to 40 ° C. Then 60 ml of water is added to the residue, followed by 70 ml of methanol to obtain a solution. The solution is acidified to pH 2.0 with 3N HCl and then reduced for 2 hours at a pressure of 3.52 kg / cm2 (50 psi) H2 using 500 mg of 5% palladium on activated carbon catalyst. The material is filtered and combined filtrates and washing liquids are examined with the microbiological test against E. coli. It is found that a yield of 29.4% amikacin was obtained.

45

B) l-N- [L - (-) - y-amino-a-hydroxybutyryl] kanamycin A

5.56 g (20.43 mmol) of p- (benzyloxycarbonyloxy) benzoic acid are slurried in 50 ml of dry acetonitrile at a temperature of 23 ° C. Then 8.4 g (41.37 mmol) of N, 0-bis-trimethylsilylacetamide are added with thorough stirring. The solution is kept at 23 ° C. for 30 minutes and then with vigorous stirring over the course of 3 hours to a solution of 21.5 g [17.83 mmol, calculated as (silyl)] n compound] poly (trimethylsilyl) k- anamycin A in 75 ml dry acetonitrile at 23 ° C added. The mixture is stirred for 4 hours, the solvent is removed in vacuo (40 ° C.) and the oily residue is then dissolved in 50 ml of dry acetone at a temperature of 23 ° C.

8.55 g (20.63 mmol) of L - (-) - y-benzyloxycarbonylamino-a-hydroxybutyric acid-N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) in 30 ml are added in the course of 5 minutes Acetone to the above solution. The mixture is kept at 23 ° C. for 78 h. Then the solution is diluted with 100 ml of water and the pH (7.0) is reduced to 2.5 with 6N HCl. The mixture is placed in a 500 ml Parr bottle together with 3 g of 5% palladium on activated carbon catalyst (Engelhard) and for 2 hours at a temperature of 23 ° C. under a pressure of 2.81 kg / cm 2 (40 psi ) H2 reduced. The mixture is filtered through a filter bed of diatomaceous

Example 5

Production of amikacin by selective N-acylation of polytrimethylsilyl-6'-N-carbobenzoxy-kanamycin A in water-50 free acetone

I. Summary Silylation of 6'-N-carbobenzoxykanamycin A in Ace-55 tonitrile using hexamethyldisilazane (HMDS) leads to 6'-N-carbobenzoxykanamycin A (silyl) 9 intermediate I. This silylated kanamycin A is in easily soluble in most organic solvents. Acylation with NAE in anhydrous acetone at 23 ° C using a 60 5% molar excess of NAE based on the 6'-N-Cbz-kanamycin A used leads to a mixture which only contains Cbz derivatives of amikacin and BB- K29, contains some unreacted kanamycin A and some polyacyl material. In none of these studies can BB-Kll be discovered. Elution 65 of an acetone-acylation mixture after reduction and workup through a CG-50 (NH4 +) column using an ammonium hydroxide gradient gives yields of pure, isolated amikacin in the range of 40%.

11

II. Reaction equations

639 104

H2N

6'-N ~ Cbz Kana A

c26h42 ° 13n4 <618-65>

+ (CH3) 3 "Si-NH-Si (CH3) 3 hmds (161.4)

A

CH3CN

\ /

■ 1 a 2 ~ ° NHR

R «Si (CH3) 3

(D 6'-N-Cbz Kana A (Siigli c53h114 ° 13n4si9 <1268-3>

(Kana = Kanamycin)

639 1Ö4

12

OH

b) CbzNH (CH2) 2-CH-COOH + HON \ (j |

+ DCC (206)

bhba. (253.4)

HONB (179.2)

acetone

OH O '

DCU + CbzEN (CH2) -CE-C-O-N

(224.3)

© NAE (414.6)

c)

q + 0 acetone n.

23 °

H.

5% Pd / C

Q

<3_i -Cbz-Ami kacin (354)

di-Cbz-BB-J <29

* 4 *

6 'Cbz-l, 3-di-B.HBA-Kana A +

6'Cbz Kana A

+ Kana A *

ilcacin. + BB — K2 9 + 1.3 — d.i ~ AHB A-Kaoa A + Kana A (585.62) (722.76) '(484.5)

CG-50 (NH4 +)

Ami kaci n

13

639 104

III. materials

Weight g

Vol. Ml

Mol

6'-N-Cbz-Kanamycin A

50

0.081

HMDS

58.9

76.5

0.365

Acetonitrile

300

BHBA

21.5

0.085

HONB

15.2

0.085

DCC

17.48

0.085

acetone

260

CG-50 (NH4 +)

3000

Methanol

as much as required

IPA

as much as required

IV. Security recommendations

6'-N-Cbz Kanamycin A: No direct information available. Avoid contact with the dust.

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

Hexamethyldisilazane (HMDS): Irritant, handle carefully.

6'-N-Cbz Kanamycin A (silyl) g: No direct information available. Handle with care.

BHBA: The toxicity is not known. Do not touch solids.

HONB: toxicity unknown. To handle with care.

DCC: A powerful irritant to the skin and eyes. Avoid breathing vapors. Toxic.

Acetone: Flammable. Inhalation can lead to headache, weakness, excitement, bronchial complaints and, in large quantities, anesthesia.

NAE: No direct information available, was always handled as a solution in acetone.

Methanol: Flammable, poisoning can occur through ingestion, inhalation or percutaneous absorption.

Isopropanol: Flammable. Ingestion or inhalation of large amounts of the vapor can lead to headache, drowsiness, depression, vomiting and anesthesia.

Ammonium hydroxide: vapors are toxic. Face mask required. Avoid contact with the liquid.

CG-50 (NH4 +): No toxicity data available. To handle with care.

V. How it works

A) Preparation of 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 (74 ° C) while maintaining a dry nitrogen flow through the slurry.

2. 75.8 ml of hexamethyldisilazane (HMDS) are slowly added over the course of 30 minutes. The complete dissolution takes place with the release of ammonia gas.

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

4. The clear, light yellow solution is concentrated under vacuum (bath temperature 40 to 50 ° C.) to a foamy solid. The yield of silylg compound is 89 to 92 g (90 to 94% of theory).

annotation

With other solvents, this solid is usually not isolated, but used directly for acylation.

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

1. Dissolve 21.5 g of L - (-) - Y-carbobenzyloxyamino-a-hydroxy-butyric acid (BHBA) in 100 ml of dry acetone at 23 ° C and then add 15.2 g of N-hydroxy-5-norbornene -2,3-dicarboximide (HONB) too. A complete resolution is obtained.

2. Over 30 minutes, add a solution of 7.48 g dicyclohexylcarbodiimide (DCC) in 50 ml acetone with stirring. During the addition, the temperature rises to approximately 40 ° C, whereby dicyclohexylurea (DCU) fails.

3. Stir the slurry for 3 to 4 hours while the temperature settles at 23 to 25 ° C.

4. The urea derivative is removed by filtration and the cake is washed with 30 ml of acetone. The filtrate and washings are saved for the subsequent acylation step.

C) Acylation of 6'-N-Cbz Kana A (silyl) 9

1. Dissolve the 6'-N-Cbz Kana A (silyl) 9, which was isolated in Part A, Step 4, in 100 ml of dry acetone at 23 to 24 ° C.

2. While stirring well, slowly add the NAE solution prepared in Part B over 15 minutes. The temperature gradually rises to approximately 40 ° C. The temperature of the solution is allowed to settle to 23 ° C. and the mixture is stirred under a nitrogen atmosphere for 18 to 20 h.

3. 100 ml of water are added and the pH (6.9 to 7.2) is reduced to 2.2 to 2.5 with 6N hydrochloric acid. The mixture is stirred at 23 ° C. for 15 min.

(Note): A second layer can form, but this is not a problem during processing.)

4. The acetone is removed under vacuum at a bath temperature of 30 to 35 ° C. The concentrate is transferred to a suitable hydrogenation vessel (which is flushed with nitrogen). 10 g of 5% palladium on activated carbon catalyst are added and the mixture is hydrogenated at 2.81 kg / cm 2 (40 psi) for 2 to 3 hours.

5. The mixture is filtered through a Dicalite filter bed and the hydrogenation vessel and the filter cake are washed with a further 50 ml of water.

6. The filtrate and washes are concentrated to approximately A of their volume (50 ml) under vacuum at 40 to 45 ° C.

7. Monitor the pH. It should be in the range of 6.9 to 7.2. If this is not the case, adjust with In ammonium hydroxide. The mixture is applied to a 6 x 110 cm CG-50 (NH4 +) column.

8. Wash the column with 1000 ml of deionized water. Then elute with 0.5 to 0.6N ammonium hydroxide using an automatic polarimeter to monitor the progress of the elution. The order of the elution is as follows:

Remaining Kanamycin A -> BB-K29 -> Amikacin. BB-Kll is not found in any of the work-up of the acylation products. Polyacyl material, i.e. H. the 1,3-diAHBA analog of kanamycin A is recovered by washing the column with 3N ammonium hydroxide.

9. Combine the amikacin fractions and concentrate to a solids content of 25 to 30%. It is diluted with 1 volume of methanol and inoculated with amikacin crystals.

10. 2 volumes of isopropanol (IPA) are slowly added over the course of 2 h, with thorough stirring, and the mixture is crystallized at 23 ° C. for 6 to 8 h.

11. The solid is filtered, washed with 50 ml of 1: 1: 2 water

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

639104

14

: Methanol: IPA mixture and closes with 25 ml IPA.

12. Dry in vacuo at 40 ° C. for 12 to 16 h. Yield: 17.3 to 19.0 g (38 to 42%) of amikacin with the following properties:

Thin layer chromatography (TLC): CHC13-methanol-NH4OH-water (1: 4: 2: 1), 5 x 20 cm silica gel plates, purchased from Quantum Industries, one zone, determined by ninhydrin (Rr-0.4).

Specific rotation:

[a] 23 ° H20 0.1m NH4OH 0.1mmH2SO4

589 + 101.6 + 101.9 + 103.5

C = 1.0%

13. The recovery of the BB-K29 in this system is also 39 to 42%. Residual kanamycin A is recovered 10 to 14% and 1,3-di-AHBA-kanamycin A is recovered approximately 5%, giving a material balance> 95%.

Example 6

Production of amikacin by selective N-acylation of polytrimethylsilyl] -kanamycin A in anhydrous acetone

I. Summary Silylation of Kanamycin A- "base" in acetonitrile using hexymethyldisilazane (HMDS) leads to polytrimethylsilyl-kanamycin A. The extent of the silylation is still 5 uncertain, but it is currently assumed that the substance is Kanamycin A (silyl)] 0. Polysilylated Kanamycin A is easily soluble in most organic solvents. Acylation with SAE in anhydrous acetone at 23 ° C using a 1: 1 molar ratio of S AE to 1 ° kanamycin A provides a mixture containing Cbz derivatives of amikacin and BB-K29, usually in a ratio of 2 up to 3/1; BB-K6 (approximately 5 to 8%), unreacted kanamycin A (15 to 20%) and some polyacyl material (approximately 5 to 10%) are obtained. In these 15 experiments, too, no BB-Kl 1 was found in the previous work on the acylation of polytrimethylsilyl-6'-N-carbobenzoxy-kanamycin A. Reduction and work-up of an acylation mixture in acetone, followed by chromatography on a CG-50 (NH4 +) column using 0.5N ammo-20nium hydroxide provides isolated crystalline amikacin in the range of 34 to 39%.

IL reaction equations

3 NH2

5, u help

Kana A 'base *

C18H36 ° 1IN4 <484-5D

* (CH3) 3 Si-NH ~ Si (CH3) 3 HMDS (161.4)

A

CH3CN

\ [/

■ 1 D 2; o

5 * NHP "

+ NH.

15

639 104

R - si (CH3) 3 0 channel A (silyl)

C48H116 ° llN4Si10 (1206.35)

OH I -

BÌ CbzNH (CK2) 2-CK-COOK + EO-tf

0 / ■

+ DCC

BHBA (253.4)

N-HOS (206.3) (115.9)

EtOAc

V

. DCU (224.3)

O

T «/ ~ 1

+ CbzNH (CH _) _ CH — C — O — N.

(CH2) 2

© SAE (350.33)

>

C \

acetone

© + © • '23o>

<

H.

5% Pd / C

V

Cb :: Amikacin (720)

Cbz BB-K29 +

Cbz BB-K6 +

Kana A +

Polyacyls (mainly-1,3-diBHBA-Xana A)

639 104

16

f ^

Amikacin * B3-K29 + BB-K6 + 1.3-diAHBA-Kana A + Kana A "

(585.62) (722.76)

CG-50 (NH4 +) r

V

Ami kaci n

III.Materials

Weight g

Vol. Ml

Mol

Kanamycin A- "Base" 50

0.103

HMDS (specific weight 0.774) 86.68

112

0.537

Acetonitrile

600

SAE 35.03

0.10

acetone

850

CG-50 (NH4 +)

3000

Methanol as much as

required

IPA

as much as

required

25th

IV. Security recommendations

Kanamycin A- "Base": Known pharmacon customary precautions recommended.

Kanamycin A (Silyl) 10: No direct information available, handle with care.

Other materials: see example 5.

Vv mode of operation

A) Preparation of Kanamycin A (Silyl) i0

1. 50 g of kanamycin A base (KF 2.5 to 3.5%) are suspended in 500 ml of acetonitrile (KF <0.01%). Bring to reflux (74 ° C) while maintaining a flow of dry nitrogen through the slurry.

2. 112 ml of hexamethyldisilazane (HMDS) are slowly added over a period of 30 minutes. Complete dissolution takes place within 4 to 5 hours with the escape of ammonia gas.

3. The mixture is kept at reflux for a further 22 to 26 hours with nitrogen flushing.

4. Concentrate the clear, pale yellow solution under vacuum (40 ° C) to a syrupy residue. It is rinsed with a further 100 ml of acetonitrile and dried completely under high vacuum for 3 to 6 h. The yield of whitish, amorphous solid is 109 to 115 g [90 to 95% of theory), calculated as kanamycin A (silyl) m].

B) Preparation of the N-hydroxysuccinimide ester of L - (-) - a-carbobenzyloxyamino-a-hydroxybutyric acid (SAE)

1. 100 g of L - (-) - a-benzyloxycarbonylamino-a-hydro-xybutyric acid (BHBA) and 45.38 g of N-hydroxysuccinimide (N-HOS) are dissolved in 1300 ml of ethyl acetate (KF <0.05%) Stir at 23 ° C.

2. 81.29 g of dicyclohexylcarbodiimide (DCC) are dissolved in 400 ml of ethyl acetate (KF <0.05%) at 23 ° C. With good stirring, this solution is added over 30 minutes to the solution according to stage 1. The temperature rises to approximately 40 to 42 ° C., while at the same time dicyclohexylurea (DCU) fails.

The slurry is stirred for 3-4 hours and the temperature is allowed to settle at 23 ° C.

30th

3. The DCU is filtered off, the filter cake is washed with 250 ml of ethyl acetate (KF <0.05%). The DCU filter cake will

15 discarded. The filtrate and washing liquids are saved.

4. Concentrate the filtrate and washes to approximately 500 ml (in vacuo at 30 to 35 ° C). Some product crystallizes out.

5. The concentrate is placed in a suitable vessel and 20 ml of heptane are added with vigorous stirring. If necessary, SAE seed crystals are added. The crystallization takes place almost immediately. The slurry is stirred at 23 ° C for 30 min.

6. 400 ml of heptane are added over 30 minutes and the slurry is stirred at 23 ° C. for 4 to 5 hours.

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

8. Dry in vacuo for 18 to 20 hours at 30 to 35 ° C. The yield is 110.1 to 131.4 g (80 to 95%).

Melting point 119 to 120 ° C with softening at 114 ° C (corrected).

TLC 4 acetone: 12 benzene: 1 CT ^ CC ^ H evolution with 1% aqueous KMn04.

Rf 0.7 for SAE, 0.2 for BHBA on 2 x 10 cm pretreated silica gel plates from Analtech Inc.

35

C) Acylation of Kanamycin A (Silyl) 10

1. Dissolve the Kanamycin A (Silyl) i0 isolated in Part A, Step 4 in 500 ml of dry acetone at 23 ° C.

2. While stirring well, the SAE (35.03 g) prepared in Part B in the form of a 10% strength solution in dry acetone is quickly added over the course of 5 to 10 minutes. The temperature rises approximately by 5 ° C. The temperature of the solution is allowed to settle at 23 ° C. and the mixture is then stirred for a further 18 to 20 h.

3. The light orange, clear solution is diluted with 400 ml of water and the pH (7.0 to 7.5) is lowered to 2.2 to 2.5 with 3N hydrochloric acid. The clear solution is then stirred at 23 ° C. for 15 to 30 minutes.

4. The acetone is removed under vacuum at a bath temperature 50 of 30 to 35 ° C (at this stage a small

Separate the amount of material, but this is not a problem). The concentrate is placed in a suitable hydrogenation vessel. 10 g of 5% palladium on activated carbon catalyst are added and the mixture is hydrogenated at 3.52 kg / cm 2 (50 psi) for 2 to 3 hours.

5. The mixture is filtered through a dicalite filter bed and the hydrogenation vessel and the cake are washed with a further 2 × 50 ml of water.

6. The filtrate and the washing liquids are concentrated to approximately Zz by volume (150 to 165 ml) under vacuum at 40 to 45 ° C.

7. At this stage the pH is in the range of 6.0 to 7.0. Place the mixture on a CG-50 (NH4 +) column (6 x 110 cm)

on.

8. The column is washed with 1000 ml of deionized water. Elute with 0.5N ammonium hydroxide using an automatic polarimeter to monitor the progress of the elution process. The order of the elution is as follows:

40

45

55

60

65

17th

639 104

Remaining Kanamycin A - »BB-K6 - * ■ BB-K29 -> Amikacin.

The presence of BB-Kll was not found in any of the investigations.

9. Combine the amikacin fractions and concentrate to a solids content of 25 to 30%. It is diluted with 1 volume of methanol and inoculated with amikacin crystals.

10. Over the course of 2 h, 2 volumes of IPA are slowly added with thorough stirring and crystallized at 23 ° C. for 6 to 8 h.

11. The solid is filtered, washed with 5 ml 1: 1: 2 water / methanol / IPA and finally with 35 ml IPA.

12. Dry in a vacuum oven at 40 ° C for 16 to 24 hours.

The yield is 19.91 to 22.84 g (34 to 39%). The IR, PMR and CMR spectral data, which were determined in addition to the specific rotation, are in complete agreement with the desired structure.

TLC system: CHCl3 / methanol / NH4OHA water (1: 4: 2: 1), 5 x 20 cm silica gel plates from Quantum Industries; 1 zone amikacin with Rf ~ 0.4 (determination by ninhydrin).

Example 7

Production of amikacin by acylation of poly (trimethylsilyl) -6'-N-Cbz-kanamycin A in tetrahydrofuran with the mixed acid anhydride from pivalic acid and BHBA

A) Preparation of the mixed anhydride

Dissolve 5.066 g (20.0 mmol) of BHBA, 4.068 g (20.0 mmol)

BSA and 2.116 g (22.0 mmol) of triethylamine in 200 ml of tetrahydrofuran, which had previously been dried over a molecular sieve. The solution is refluxed for 2'A h and then cooled to -10 ° C. In the course of 2 to 3 minutes, 2.412 g (20.0 mmol) of pivaloyl chloride are added with stirring and the mixture is then stirred at -10 ° C. for a further 2 hours. Then the temperature is allowed to rise to 23 ° C.

B) Acylation of PoIy (trimethylsiIyI) -6'-N-Cbz-Kanamycin A

5.454 g [4.97 mmol, calculated as 6'-Cbz-kanamycin A (Si

lyl) 9] Poly (trimethylsilyl) -6'-N-Cbz-Kanamycin A, as prepared in Example 1, is dissolved in 50 ml of dry (molecular sieve) tetrahydrofuran at 23 ° C. Half of the solution of the mixed anhydride (10.0 mmol) prepared as described in stage A is added over the course of 20 min with stirring and then stirred for a further 7 days.

Then 100 ml of water are added to the reaction mixture and the pH (5.4) is adjusted to 2.0 with 3 m of H2SO4. The mixture is stirred for a further 1 h and then the solution is extracted with ethyl acetate. The crystallization of polyacylated material begins, so that the reaction mixture is filtered. After drying over P2Os, the solids obtained weigh 0.702 g. The extraction of the reaction mixture is carried out in total with 4 x 75 ml of ethyl acetate. The excess ethyl acetate is then removed from the aqueous layer. A test is performed using HPLC on an aliquot of the aqueous solution. The curve obtained shows a yield of di-Cbz amikacin of 26.4%.

The aqueous layer is then hydrogenated in a Parr device for 2 hours at a H2 pressure of 3.52 kg / cm2 (50 psi) using 0.5 g of 10% Pd on activated carbon catalyst. The material is filtered and the combined filtrate and the washing liquids are determined by testing against E. coli to show that the yield of amikacin is 31.2%. The ratio Amikacin / BB-K29 is approximately 9-10 / 1. There are traces of polyacyl compounds and unreacted kanamycin A.

Example 8

Influence of water on the production of amikacin by acylation of poly (trimethylsilyl) kanamycin A in acetone solution at 23 ° C

A) Anhydrous solvent

2.40 g [2.0 mmol, calculated as Kanamycin A (Silyl) 10] poly (trimethylsilyl) Kanamycin A, which was prepared as described in Example 3, is dissolved in 20 ml of acetone, which has been dried over a molecular sieve was. The solution is stirred at 23 ° C. and a solution of 0.701 g (2.0 mmol) SAE in 10 ml with a molecular sieve-dried acetone is added over the course of 10 seconds. The mixture is stirred at 23 ° C. for a further 22 h. Then 50 ml of water are added and the pH (7.5) is adjusted to 2.5. The acetone is removed in a vacuum at 40 ° C., then the aqueous solution is reduced at a H2 pressure of 3.59 kg / cm 2 (51 psi) for 2 hours at 23 ° C., 1.0 g 10% Pd-on activated carbon used as a catalyst. The microbiological investigation shows a yield of amikacin of 31.24%.

15

B) Add water to the solvent

Step A above is repeated with the exception that 1.0 ml of water (56 mmol) is added to the solution of poly (trimethylsilyl) kanamycin A and stirred for 20 minutes before SAA 15 acylation. The microbiological examination shows a yield of amikacin of 33.80%.

25th

30th

Example 9

Production of amikacin by acylation of poly (trimethylsilyl) -6'-N-Cbz-kanamycin A in acetone with the mixed anhydride from BHBA and isobutyl carbonic acid

A) Preparation of the mixed anhydride

1.267 g (5.0 mmol) of BHBA and 1.313 g (10.0 mmol) of N-trimethylsilylacetamide (MS A) in 20 ml of molecular sieve-dried acetone are stirred at 23 ° C. and 0.70 ml (5.0 mmol) of triethylamine (TEA). The mixture is refluxed for 2/15 h under an N2 atmosphere. The mixture is cooled to -20 ° C and 0.751 g (0.713 ml; 5.50 mmol) of isobutylchloroformate is added. An immediate separation of triethylamine hydrochloride begins. The mixture is stirred at -20 ° C for 1 h.

40

B) acylation

6.215 g [4.9 mmol, calculated as (silyl) 9 compound] poly (trimethylsilyl) -6'-N-Cbz-kanamycin A, which was prepared as described in Example 1, are dissolved in 20 ml of molecular sieve-dried. 45 net acetone with stirring at 23 ° C. The solution is cooled to −20 ° C. and the cold solution of the mixed anhydride from stage A is slowly added over the course of 30 min. The reaction mixture is stirred for a further 114 h at −20 ° C. and then for 17 h at 23 ° C. The reaction mixture is then poured 50 into 150 ml of water at 23 ° C. with stirring, the pH (7.75) is raised to 3 N HCl 2.5 is set, and stirring is continued for 15 min. Then the acetone is removed in vacuo at 40 ° C. An HPLC test is carried out on an aliquot of the aqueous solution obtained. The curve obtained shows a yield of di-Cbz-amikacin of 34.33%.

The majority of the aqueous solution is reduced for 3/4 h at 23 ° C under a H2 pressure of 3.52 kg / cm2 (50 psi), using 2.0 g of Pd / C catalyst. The catalyst is removed by filtration and the combined filtrates and washings are determined by microbiological analysis against E. coli to show a 35.0% yield of amikacin.

Example 10

65 Preparation of Amikacin by Acylation of Poly (trimethylsilyl) -6'-N-Cbz-Kanamycin A in 3-Pentanone

30 g [23.65 mmol, calculated as 6'-N-Cbz-Kanamycin A (Silyl) 9) poly (trimethylsilyl) -6'-N-Cbz-Kanamycin A, as described in

639104

18th

Example 1 was prepared, and which is dissolved in 100 ml molecular sieve-dried 3-pentanone, is stirred at 23 ° C. 26.02 mmol of NAE (10% excess) are added over the course of 40 min. The mixture is stirred for a further 113 h at 23 ° C. and the mixture is then added to 250 ml of water with vigorous stirring. The pH (7.3) is adjusted to 2.5 with 3N HCl, the mixture is stirred for a further 30 min, and the 3-pentanone is removed in vacuo at 40 ° C. The aqueous solution is extracted with 4 x 100 ml of ethyl acetate. Then an aliquot of the aqueous solution is subjected to HPLC analysis. The curve obtained shows a yield of di-Cbz-amikacin of 46.12%.

The main part of the aqueous reaction mixture is reduced for 2 lA h at 23 ° C. under an H2 pressure of 3.59 kg / cm 2 (51.0 psi), using 3.0 g of 10% Pd / C catalyst. The microbiological examination of an aliquot of the combined filtrates and washing liquids shows a yield of amikacin of 40.24%. The major portion of the reduced aqueous reaction mixture is then concentrated in vacuum at 40 ° C to approximately 100 ml and fractionated on a CG-50 (NH4 +) ion exchange column [10.2 cm x 122 cm (4 inches x 4 feet), approximately 101 Resin contains]. The column is loaded with the aqueous solution, washed with 51 water and then the material is eluted with 0.5N NH4OH (followed by 3N NH4OH to elute polyacylated products). Polarimetry of the fractions indicates a 42.7% yield of amikacin, a 12.0% yield of unreacted kanamycin A, a 12.4% yield of polyacylated material and a 23.2% yield of BB-K29.

Exploit:

Reaction A Reaction B

49.18% 56.17%

42.87% 55.39%

39.16% 38.45%

10th

15

Example 12

Production of amikacin by acylation of poly (trimethylsilyl) -6'-N-Cbz-kanamycin A in anhydrous tetrahydrofuran with different reaction times

A) Example 11, A is repeated with the exception that dry tetrahydrofuran is used as the solvent instead of dry cyclohexanone.

B) Example 11, B is repeated, with the exception that dry tetrahydrofuran is used as the solvent instead of dry cyclohexanone.

Exploit:

HPLC test microbiological examination on Amikacin (di-Cbz-Amikacin) turbidimetric plate

Reaction A 20 Reaction B

29.27% 33.39%

28.34% 21.52%

28.18% 28.63%

Example 13

Production of amikacin by acylation of poly (trimethyl-25 silyl) -6'-N-Cbz-kanamycin A in anhydrous dioxane during different reaction times

A) Example 11, A is repeated except that acylation is carried out for 44 hours using dry dioxane as solvent.

30th

B) Example 11, B is repeated with the exception that acylation is carried out using dry dioxane as solvent for 18/2 h.

Example 11

Production of amikacin by acylation of poly (trimethylsilyl) -6'-N-Cbz-kanamycin A in anhydrous cyclohexanone with different reaction times

A) 2.537 g [2.0 mmol, calculated as 6'-N-Cbz-Kanamycin A (Silyl) 9] poly (trimethylsilyl) -6'-N-Cbz-Kanamycin A, which was prepared as described in Example 1, in 300 ml of dry cyclohexanone is acylated for 20 h at 23 ° C. with an NAE solution in dry cyclohexanone (10.8 ml of a 0.1944 mmol / ml solution, 2.10 mmol). Then the reaction mixture is added to 150 ml of water with stirring and the pH (5.6) is adjusted to 2.5 with 3N HCl. The cyclohexanone is removed in vacuo at 40 ° C. and an aliquot of the remaining aqueous phase is used for the analysis by HPLC.

The bulk of the aqueous phase is reduced for 3 hours at 23 ° C under a H2 pressure of 3.52 kg / cm2 (50 psi) using 1.0 g of a 10% Pd / C catalyst. The catalyst is removed by filtration and combined filtrates and washing liquids are examined microbiologically for amikacin.

B) Reaction A above is repeated except that this time the acylation was carried out not for 20 hours but for 115 hours.

35

Exploit:

40

45

HPLC test microbiological analysis

on amikacin

(di-Cbz-Amikacin) turbidimetric

plate

Reaction A

39.18% 43.27%

33.36%

Reaction B

42.82% 22.55%

33.37%

HPLC test microbiological examination on Amikacin (di-Cbz-Amikacin) turbidimetric plate

Example 14

Production of amikacin by acylation of poly (trimethylsilyl) -6 '-N-Cbz-kanamycin A in anhydrous diethyl ketone at 50 75 ° C

To a stirred solution of 2.537 g [2.0 mmol, calculated as 6'-N-Cbz-Kanamycin A (Silyl) 9] poly (trimethylsilyl) -6'-N-Cbz-Kanamycin A, as described in Example 1 was prepared, in 32 ml molecular sieve-dried diethyl ketone at 55 75 ° C., a solution of NAE (10.8 ml with 0.1944 mmol / ml diethyl ketone, 2.10 mmol) was added over the course of 15 min. The mixture is stirred for a further 3 h at 75 ° C. and then the mixture is poured into 150 ml of water. The pH is adjusted to 2.8 with 3N HCl and the diethyl ketone is removed at 40 ° C. in vacuo. HPLC test 60 of an aliquot of the aqueous phase shows a yield of di-Cbz-Amikacin of 39.18%.

The majority of the aqueous phase VA h is reduced at 23 ° C. under an H2 pressure of 3.49 kg / cm 2 (49.8 psi), using 1.0 g of Pd / C catalyst. The catalyst is removed by 65 filtration and combined filtrates and washes are microbiologically examined for amikacin. The turbidimetric test shows a yield of 27.84%, the plate test shows a yield of 28.6%.

19th

639 104

Example 15

Production of amikacin by acylation of poly (trimethylsilyl) kanamycin A with NAE at 0 to 5 ° C after «back» hydrolysis with water

A) Silylation of Kanamycin A using HMDS with TMCS as a catalyst

10 g of kanamycin A (97.6% pure, 20.14 mmol) in 100 ml of molecular sieve-dried acetonitrile are brought to reflux under a nitrogen atmosphere. A mixture of 22.76 g (141 mmol, 7 mol per mol Kanamycin A) HMDS and 1 ml (0.856 g, 7.88 mmol) TMCS is added to the refluxing reaction mixture in the course of 10 min. The mixture is kept under reflux for 4% h and then the mixture is cooled, concentrated in vacuo to a yellow, viscous syrup and dried under high vacuum for 2 h. The yield of product is 23.8 g [97.9%, calculated as kanamycin A (silyl) i0].

B) acylation

23.8 g (20.14 mmol) of poly (trimethylsilyl) kanamycin A, prepared in Stage A, are dissolved in 250 ml of molecular sieve-dried acetone at 23 ° C and then cooled to 0 to 5 ° C. Then 3.63 ml (201.4 mmol, 10 mol per mol polysilylated kanamycin A) of water are added with stirring and the mixture is left under a moderate vacuum for 30 minutes. 19.133 mmol (0.95 mol per mol of polysilylated kanamycin A) of NAE in 108.3 ml of acetone are added in the course of less than 1 min. The mixture is stirred at 0 to 5 ° C. for 1 h, diluted with water, the pH is adjusted to 2.5 and the acetone is then removed in vacuo. Then the aqueous solution is reduced for 2i4 h at 23 ° C at a H2 pressure of 3.52 kg / cm2 (50 psi), using 2.0 g of 10% Pd on activated carbon as a catalyst. The reduced reaction mixture is filtered through dicalite, concentrated to approximately 100 ml in vacuo at 40 ° C and then applied to a CG-50 (NH4 +) column (61 resin, 5 x 100 cm). It is washed with water and then eluted with 0.6 n-1.0 n 3 n NH4OH. 60.25% amikacin, 4.37% BB-K6.4.35% BB-K29.26.47% kanamycin A and 2.18% polyacyl compounds are obtained.

Example 16

Production of amikacin by acylation of poly (trimethylsilyl) -6'-N-Cbz-Kanamycin A with SAE at 0 to 5 ° C after «back» methanolysis

A) Silylation of 6'-N-Cbz-Kanamycin A

20.0 g (32.4 mmol) of 6'-N-Cbz-Kanamycin A in 200 ml of molecular sieve-dried acetonitrile are brought to reflux under a nitrogen atmosphere. In the course of 10 min, 47.3 ml (226.8 mmol, 7 mol per mol of 6'-N-Cbz-Kanamycin A) HMDS are added and the reflux is continued for a further 20 h. The mixture is then cooled, concentrated in vacuo and dried under high vacuum for 2 hours, giving 39.1 g of white amorphous solid. Yield 95.4%, calculated as 6'-N-Cbz-Kana-mycin A (silyl) 9.

B) acylation

39.1 g (32.4 mmol) of poly (trimethylsilyl) -6'-N-Cbz-kanamycin A, which was prepared in step A above, are dissolved in 400 ml of dry acetone with stirring at 23.degree. 6.6 ml (162 mmol, 5 mol per mol of polysilylated 6'-N-Cbz-Kanamy-cin A) of methanol are added and the mixture is stirred for 1 hour under a strong nitrogen purge at 23 ° C. The mixture is brought to 0 to Cooled 5 ° C, and a solution of 11.35 g (32.4 mmol) of SAE in 120 ml of previously cooled, dry acetone is added. The mixture is stirred for a further 3 hours at 0 to 5 ° C and then placed in a cold room kept at 4 ° C for 1 week. Then there

20th

300 ml of water are added, the pH is adjusted to 2.0, the mixture is stirred for 1 h and the acetone is removed in vacuo. The aqueous solution obtained is reduced for 17 h at 23 ° C. under an H2 pressure of 3.80 kg / cm 2 (54.0 psi), using 3.0 g of 10% Pd-on-5 activated carbon as a catalyst. Then it is filtered through dicalite, concentrated in vacuo to 75 to 100 ml, applied to a CG-50 (NH4 +) column and eluted with water and 0.6N NH4OH. 52.52% amikacin, 14.5% BB-K29.19.6% kanamycin A and 1.71% polyacyl compounds are obtained.

10th

Example 17

Production of amikacin by acylation of poly (trimethyl-silyl) -kanamycin A with SAE at 0 to 5 ° C after «back» hydrolysis with water

15

A) Silylation of Kanamycin A with TMCS in acetonitrile using tetramethylguanidine as an acid acceptor

4.88 g (10.07 mmol) of kanamycin A are suspended in 100 ml of molecular sieve-dried acetonitrile with stirring at 23 ° C. 16.234 g (140.98 mmol, 14 mol per mol of kanamycin A) are added to the stirred suspension (tetramethylguanidine ( TMG). The mixture is heated to reflux and 15.32 g (140.98 mmol, 14 mol per mol Kanamycin A) TMCS 25 are added over 15 minutes. Approximately Vi h after the addition of the TMCS, a white precipitate of TMG-HCl is formed. The mixture is cooled to room temperature, concentrated to a sticky residue and dried under high vacuum for 2 hours. The solid is triturated with 100 ml of dry THF and the insoluble TMG-HC1 is filtered off and washed with 5 x 20 ml portions of THF. The combined filtrate and washing liquids are concentrated in vacuo at 40 ° C. to a sticky residue and dried under high vacuum for 2 h. 10.64 g of a light cream-colored, sticky residue are obtained. The yield be-35 is 87.6%, calculated as kanamycin A (silyl) «».

B) acylation

10.64 g (10.07 mmol) of poly (trimethylsilyl) kanamycin are dissolved

40 A, which was prepared in step A above, in 110 ml of molecular sieve-dried acetone with stirring at 23 ° C. and the solution is cooled to 0 to 5 ° C. Then 1.81 ml of water (100.7 mmol , 10 moles per mole of polysilylated kanamycin A) and the solution is stirred for 30 min under moderate vacuum.

45 3.70 g (10.57 mmol, 5% excess) of SAE in 40 ml of pre-chilled dry acetone are added over less than 1 min and the mixture is stirred for 1 h. The mixture is worked according to the general procedure of the example

50 16 B to give about 50% amikacin, about 10% BB-K29.5 to 8% BB-K6, about 20% kanamycin A and 5 to 8% polyacyl compounds.

30th

55

Production of raw materials

A) Preparation of poly (trimethylsilyl) kanamycin A in pyridine using HMDS. 10.0 g (20.64 mmol) of kanamycin A are suspended in 100 ml of 60 molecular sieve-dried, freshly distilled pyridine at 23 ° C. One begins Nitrogen purging and refluxing the suspension. 17.33 g (107.32 mmol, 5.2 mol per mol Kanamycin A) HMDS are added over 10 minutes and the mixture is refluxed for 19 hours. The mixture is then cooled to room temperature, concentrated in vacuo to a light yellow-gold syrup and dried under high vacuum, giving a white, amorphous solid. 22.1 g of product are obtained. Yield 92.6%, calculated as kanamycin A (silyl) | 0.

639 104

20th

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

5.0 g of 97.6% pure Kanamycin A (10.07 mmol) are suspended in 100 ml of molecular sieve-dried acetonitrile at 23 ° C. 33.8 ml (24.5 g, 241.7 mmol) of triethylamine are added and the suspension is brought to reflux. A solution of 23.7 ml (21.3 g 140.87 mmol) of trichloroethylsilane in 25 ml of dry acetonitrile is added over the course of 20 minutes. The mixture is kept under reflux for a further 7 hours and the mixture is cooled to room temperature, after which long, fine needles of TEA-HC1 are separated. The mixture is left to stand at room temperature for about 16 h, concentrated in vacuo at 40 ° C. to a sticky solid and dried under high vacuum to a dark orange, sticky solid for 2 h. The solid is triturated with 100 ml of dry THF at 23 ° C. and the insoluble TEA-HC1 is filtered off, washed with 5 × 20 ml of THF and dried. This gives 16.0 g of TEA-HC1. Combined filtrates and washes are concentrated in vacuo to a solid. Then it is dried for 2 hours under high vacuum. 20 19.3 g of poly (triethylsilyl) kanamycin A are obtained as dark orange, viscous syrup.

25th

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

10.0 g of 99.7% pure kanamycin A (20.58 mmol) are suspended in 200 ml of molecular sieve-dried acetonitrile with stirring at 23 ° C. 29.45 g of bis-trimethylsilylurea (BSU) (144, 01 mmol, 7 mol per mol of Kanamy-30) and brings the mixture to reflux under a nitrogen atmosphere. The mixture is refluxed for a further 17 h and then the reaction mixture is cooled to room temperature. A small amount of insoluble material is removed by filtration, washed with 3 x 10 ml portions of acetonitrile and dried (1,381 35 g). Infrared spectroscopy shows that this is BSU and a small amount of unreacted kanamycin A. Combined filtrates and washing liquids are cooled to 4 ° C for 16 h. Further solid separates, which is obtained as described above. 7.8 g are obtained. Infrared analysis shows that it is BSU and urea. The light yellow filtrate and the washing liquids are concentrated in vacuo at 40 ° C. The mixture is then dried under high vacuum, giving 27.0 g of a white solid which is partly sticky and partly composed of fine, needle-like crystals. The solid is treated with 150 ml of heptane at 23 ° C., the insoluble part is removed by filtration, washed with 2 × 50 ml portions of heptane and dried.

This gives 6.0 g of white needles, which are infrared and BSU and urea. The combined filtrate and washing liquids are concentrated in vacuo at 40 ° C. Then they are dried under high vacuum for 2 hours, giving 20.4 g of white needles. Their infrared spectrum is typical for polysilylated kanamycin A. Calculations show that the product contains an average of 7.22 trimethylsilyl groups.

50

55

Example 18

Production of amikacin by acylation of per (trimethyl-silyl) -kanamycin A after partial desilylation with 1,3-butane-60 diol

A) Preparation of per (trimethylsilyl) kanamycin A

10.0 g (20.639 mmol) of kanamycin A are suspended in 100 ml of molecular sieve-dried acetonitrile with stirring at 23 ° C. 65 The mixture is brought to reflux with nitrogen flushing and then 23.322 g (144.5 mmol 7 moles per mole of kanamycin A) HMDS. You still hold

16 h at reflux and then cool the mixture to room temperature and then concentrate in vacuo. The mixture is then dried under high vacuum for 2 hours. 24.3 g of a white, sticky residue are obtained. The yield is 92.1% when Kanamycin A (silyl) u.

B) acylation

24.3 g of per (trimethylsilyl) -kanamycin A produced in stage A are dissolved in 240 ml of molecular sieve-dried acetone at 23 ° C. with stirring. 9.25 ml of 1,3-butanediol [103.2 mmol, 5 mol per mol of per (trimethylsilyl) -kanamycin A] are added to this solution. The mixture is stirred for 2 hours at 23 ° C. under a nitrogen purge and then cooled to 0 to 5 ° C. In the course of about 1 min, 7.23 g of SAE (20.64 mmol) in 70 ml of pre-cooled acetone are added. The mixture is stirred at 0 to 5 ° C for 3 hours and then left to stand in a cold room kept at 4 ° C for about 16 hours. 200 ml of water are added, the pH is adjusted to 2.5 and the clear, yellow solution is stirred at 23 ° C. for 30 min. The acetone is removed in vacuo and the aqueous solution is stirred at 23 ° C. in an H2 Pressure of 3.87 kg / cm 2 (55.0 psi) was reduced, using 3.0 g of 10% Pd on activated carbon as a catalyst. The reduced solution is then filtered through dicalite and chromatographed as described in Example 16B. 47.50% amikacin, 5.87% BB-K29, 7.32% BB-K6.24.26% kanamycin A and 7.41% polyacyl compounds are obtained.

Example 19

Preparation of amikacin by acylation of poly (trimethylsilyl) kanamycin A, which was prepared in THF using SAE with a sulfamic acid catalyst

100 mg of sulfamic acid and 12.32 g (76.33 mmol) of HMDS are added to a refluxed mixture of 5.0 g (10.32 mmol) of kanamycin A in 50 ml of molecular sieve-dried tetrahydrofuran (THF). The mixture is refluxed for 18 hours, with complete dissolution after 6 hours. The solution is cooled to 23 ° C., treated with 0.1 ml of water and kept at 23 ° C. for 30 minutes. A solution of 3.61 g (10.3 mmol) of SAE in 36 ml of THF is added over the course of 30 minutes. After stirring for 3 hours, the mixture is diluted with 100 ml of water and the pH is adjusted to 2.2 with 10% H2S04. The mixture is stirred at 23 ° C. for 30 minutes and then concentrated in vacuo to remove the THF. The aqueous solution obtained is reduced for 2 hours at 23 ° C at a H2 pressure of 3.52 kg / cm2 (50 psi), using 10% Pd on activated carbon as a catalyst. The reduced solution is filtered through dicalite and the solids are washed with water. Combined filtrates and washing liquids (150 ml) are tested microbiologically against E. coli. It is found to contain 1225 mcg / ml (31.5% yield of active material) amikacin.

Example 20

Preparation of amikacin by acylation of poly (trimethylsilyl) kanamycin A with the N-hydroxysuccinimide ester of di-carbobenzyloxy-AHBA

A) Preparation of the dicarbobenzyloxy-L - (-) - a-amino-a-hydro-xybutyric acid N-hydroxysuccinimide ester

8 g (20.65 mmol) of dicarbobenzyloxy-L - (-) - a-amino-a-hydroxybutyric acid and 2.37 g (20.65 mmol) of N-hydroxysuccinimide are dissolved in 50 ml of dry acetone at 23 ° C. on. 4.25 g (20.65 mmol) of dicyclohexylcarbodiimide, dissolved in 20 ml of dry acetone, are added and the whole is stirred at 23 ° C. for 2 h. The dicyclohexylurea is filtered off, the filter cake is washed with 10 ml of dry acetone and the filtrates and washing liquids are combined.

21st

639 104

B) acylation

Poly (trimethylsilyl) -kanamycin A prepared from 10.0 g (20.639 mmol) of kanamycin A according to the general procedure of Example 21 is dissolved in 100 ml of dry acetone. The solution is cooled to 0 to 5 ° G, 3.7 ml of deionized water are added and the solution is stirred at 0 to 5 ° C for 30 min under a moderate vacuum.

The solution of the di-Cbz-blocked acylating agent prepared in stage A is added to this solution and the mixture is then stirred for 30 minutes at 0 to 5 ° C. The mixture is diluted with water, the pH is adjusted to 2.2 and the Acetone is removed in vacuo. The aqueous solution is reduced according to the general procedure of Example 22 and then filtered through dicalite. Chromatography found 40-45% amikacin, about 10% BB-K29, a trace of BB-K6, about 30% kanamycin A and a small amount of polyacyl compounds.

Example 21

Preparation of poly (trimethylsilyl) kanamycin A using HMDS with imidazole as catalyst

11 g (22.7 mmol) of kanamycin A and 100 mg of imidazole in 100 ml of molecular sieve-dried acetonitrile are heated to reflux with a nitrogen purge. 18.48 g of HMDS (114.5 mmol, 5 mol per mol of kanamycin A) are added in the course of 30 minutes and the mixture is kept under reflux for 20 hours. Complete dissolution occurs after approximately 2Vi h. The solution is cooled to 23 ° C. and the solvent is removed in vacuo, 21.6 g of poly (trimethylsilyl) kanamycin A remaining as a foamy residue. The yield is 93.1%, calculated as kanamycin (silyl) n.

Example 22

Preparation of l-N- [L - (-) - y-amino-a-hydroxybutyryl] kana-mycin B (BB-K26) by acylation of poly (trimethylsilyl) -kanamycin B with SAE

A) Preparation of poly (trimethylsilyl) kanamycin B using HMDS with TMCS catalyst

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

B) acylation

53.0 g of the poly (trimethylsilyl) kanamycin B prepared in step A above are dissolved in 500 ml of dry acetone at 0 to 5 ° C. 20.9 ml of methanol are added and the mixture is stirred at 0 to 5 ° C. in vacuo for 30 min. A solution of 18.1 g (51.67 mmol) of SAE in 200 ml of pre-cooled, dry acetone is added over a period of less than 1 min and the mixture is stirred at 0 to 5 ° C for 30 min. The mixture is worked up according to the general procedure of Example 22 and then applied to a column with CG-50 (NH4 +) (8 x 120 cm). Elution is carried out with an NH4OH gradient from 0.6 n to 3 n. 38% 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.

15

Example 23

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

19.5 g (40.246 mmol) of kanamycin A and 0.5 g (0.858 mmol) of 'kanamycin A sulfate (total = 20.0 g, 41.0 mmol) in 200 ml of molecular sieve-dried acetonitrile are brought to reflux. 60.3 ml of HMDS (287.7 mmol, 7 mol per mol of kanamycin A) are slowly added and the mixture is refluxed for 28 h. The mixture is then evaporated to dryness on a rotary evaporator and then dried under a steam jet vacuum. 47.5 g of poly (trimethylsilyl) kanamycin A are obtained as a pale yellow oil. The yield is 95.82%, calculated as kanamycin A (silyl) ^.

Example 24

Production of amikacin by acylation of poly (trimethylsilyl) kanamycin A with N-trifluoroacetyl-blocked AHB A-N-hydroxysuccinimide ester

20th

A) Preparation of N-trifluoractyl-AHB A and conversion to the N-hydroxysuccinimide ester

40 g (191 mmol) of trifluoroacetic anhydride are added to a suspension of 5.0 g (42 mmol) of AHBA in 100 ml of THF with stirring in the course of 10 minutes. The solution is stirred at 23 ° C. for 18 h and then concentrated to dryness in vacuo at 50 ° C. The residue is dissolved in 100 ml of aqueous methanol (1: 1) and stirred for 1 h. Then concentrated to dryness in vacuo and redissolved in 50 ml of H20. The aqueous solution is extracted with 3 x 50 30 ml portions of MIBK, and after drying over NasS04, the extract is concentrated to an oil. Traces of solvent are removed by adding and distilling off 4 ml of water. When left to stand, the oil turns into a waxy, crystalline solid. 2.5 g of product are obtained, 35% yield 28%.

2.4 g (11.3 mmol) of the N-trifluoroacetyl-AHBA are dissolved in 50 ml of dry acetone and 1.30 g (11.31 mmol) of N-hydroxysuccinimide are added to the solution. A solution of 2.33 g of dicyclohexylcarbodiimide in 20 ml of dry acetone is slowly added. 40 The reaction mixture is stirred at 23 ° C. for 2 hours and then the precipitated dicyclohexylurea is removed by filtration and washed with a small amount of acetone. Combined filtrates and washes (solution of the N-hydroxysuccinimide ester of N-trifluoroacetyl-AHBA) are used in the subsequent step 45 without isolation.

B) acylation

2.0 ml (113.4 mmol) of water are added to a solution of 11.31 mmol of poly (trimethylsilyl) -kanacycin A, which was prepared as described in Example 26, in 50 and 54 ml of acetone, and the mixture is stirred min in vacuo at 0 to 5 ° C. To the mixture are added the N-hydroxysuccinide mid-ester of N-trifluoroacetyl-AHBA (11.31 mmol) prepared in step A above and the mixture is then kept at 5 ° C. for 1 h . The pH is adjusted to approximately 2.0 with 20% H2S04, the mixture is stirred for 30 minutes and then the pH is increased to approximately 6.0 with NH4OH. The mixture is then evaporated to dryness on a rotary evaporator. 14.4 g of a sticky, whitish solid are obtained. The solid is dissolved in 100 ml of water, the pH 60 is raised from 5.5 to 11.0 with 10N NH4OH and the solution is heated in an oil bath at 70 ° C. for 1 h. Then the pH (9.5) is lowered to 7.0 with HCl, the solution is subjected to a clear filtration to remove a small amount of insoluble materials and the filtrate is washed with water. Combined filtrates and 65 washes (188 ml) are applied to a column of CG-50 (NH4 +) (8 x 90 cm), washed with 21 water and with an NH4OH gradient (0.6 n-1.0 n concentrated) eluted. 28.9% amikacin, 5.0% BB-K6.5.7% BB-K29,

639 104 22

43.8% Kanamycin A, 3.25% polyacyl compounds and 14.3% and the mixture is evaporated to dryness in vacuo, an unknown material which in the first fraction from which gives 36.3 g of a gold-colored oil. The oil comes out in the column. 200 ml of trifluoroacetic acid dissolved, left to stand for 15 minutes and then evaporated to dryness on a rotary evaporator. The oil is washed with water and the water is distilled off. Example 25 concentrated HN4OH is added to pH 6.0 and then distilled off. The

Preparation of amikacin by acylation of poly (trimethyl-obtained solid is dissolved in water, filtered, and the filtrate silyl) -kanamycin A blocked with tert-butoxycarbonyl is washed with water. Combined filtrates and

AHBA-N-hydroxysuccinimide ester washes (259 ml) on a CG-50 (NH4 +) column io (8 x 92 cm), washed with 41 water and eluted with an NH4OH-

A) Preparation of tert-BOC-AHBA and conversion in the gradient (0.6 n-1.0 n concentrated). 40.32% Ami-N-hydroxysuccinimide ester kacin, 4.58% BB-K6.8.32% BB-K29.30.50% Kanamycin A are obtained

A solution of 5.0 g (42 mmol) AHBA in 100 ml water and and 7.43% polyacyl compounds.

20 ml of acetone is adjusted to pH 10 with 10N NaOH. in the

The course of 3 to 4 min gives a 6 g (53 mmol) ditert. 15 Example 26

butyl dicarbonate and stir the solution for 35 min while repeating the general procedure of Example 1

maintain the pH at pH 10 by periodically adding 10N NaOH. with the exception that the 6'-N-carbo-

The acetone is removed in vacuo, and the aqueous phase benzyloxy-kanamycin A by an equimolar amount of 6'-N-

is washed with 40 ml of ethyl acetate. The pH of the carbobenzyloxy-kanamycin B is replaced. Here you get the 1-

aqueous solution with 3 N HCl to 2.0 and then extracted with 3 20 N- [L - (-) - y-amino-a-hydroxybutyryl] kanamycin B.

x 30 mlMIBK. The combined MIBK extracts are over

Na2S04 dried and a clear, oily residue Example 27

concentrated. 8.2 g of product are obtained, yield 89%. The general procedure of Example 1 is repeated

4.25 g (19.4 mmol) of the tert-BOC-AHBA are dissolved in 50 ml of 25 with the exception that the L - (-) - y-acetone used there is dissolved, and 2.23 g ( 19.4 mmol) of N-hydroxysucci-benzyloxycarbonylamino-a-hydroxybutyric acid-N-hydroxy-5-nimide. A solution of 4.00 g (19.4 mmol) of dicyclohexylcarnorbornene-2,3-dicarboximide ester by:

bodiimide in 20 ml acetone is slowly added and the L - (-) - ß-benzyloxycarbonylamino-a-hydroxypropionic acid-N-

Mixture is stirred at 23 C for 2 h. The precipitated-hydroxy-5-norbornene-2,3-dicarboximide ester and a dicyclohexylurea are removed by filtration and washed with a small amount of acetone - ^ L - (-) - ö-benzyloxycarbonylamino-a-hydroxyvaleric acid. United filtrates and washing liquid hydroxy-5-norbornene-2,3-dicarboximide ester replaced, whereby ten (solution of the N-hydroxysuccinimide ester of tert-BOC- man

AHBA) in the subsequent stage without isolation, l-N- [L - (-) - ß-amino-a-hydroxypropionyl] kanamycin A, or

used. l-N- [L - (-) - ô-amino-a-hydroxyvaleryl] kanamycin A is obtained.

35

Example 28

B) Acylation The general procedure of Example 25 is repeated to a solution of 41.28 mmol of poly (trimethylsilyl) -kana- with the exception that the L - (-) - y-

mycin A, prepared as described in Example 26, in 94 ml of benzyloxycarbonylamino-a-hydroxybutyric acid-N-hydroxysuc

Acetone is added to 3.5 ml (194 mmol) of water and the solution is stirred through 40 cinimide esters:

30 min in vacuum at 0 to 5 ° C. 19.4 mmol of the L - (-) - ß-Benzyloxycarbonylamino-a-hydroxypropionic acid-N- in the above

Stage A produced N-hydroxysuccinimide ester of tert-hydroxysuccinimide ester, or

BOC-AHB A are added and the mixture is stirred for 1 h at L - (-) - ö-benzyloxycarbonylamino-a-hydroxyvaleric acid-N-

5 ° C. Then 200 ml of water are added and hydroxysuccinimide ester is replaced, whereby the pH (7.0 with 20% H2SO4 is reduced to 2.0. After 30 minutes of 45 l-N- [L - (-) - ß-amino-a-hydroxypropionyl] kanamycin B,

With stirring, the pH is raised to approximately 6.0 with NH4OH, l-N- [L - (-) - ô-amino-a-hydroxyvaleryl] kanamycin B is obtained.

m

Claims (11)

639 104 2 PATENT CLAIMS 1. Process for the preparation of a 1-N- [cu-amino-a-hydroxyalkanoyl] kanamvcins A or B of the general formula I: Ä2, h2 L-C-) (I) wherein R is OH or NH2 and n is an integer from 0 to 2 kanamycin A or B, which on the 6'-amino group is one of silyl, or a non-toxic, pharmaceutically acceptable blocking group, with an acylating Chen acid addition salt thereof, characterized in that the derivative of the acid of the general formula II: polysilylated kanamycin A or B or polysilylated b-hn-ch9- (ch0) -ch-cqoh cvc 'nj -OH (ii) wherein n represents an integer from 0 to 2 and B represents an amino blocking group, acylated in a substantially anhydrous organic solvent and then all blocking groups removed. 2. The method according to claim 1, characterized in that the amino-blocking group of the acylating derivative of the acid corresponds to the following formulas: ch., o i 3 II , ch_ — c-q-c-3 i ch- O ; j y2xcc-, [J 639 104 x3c O R -CH2 "-0 ~ C— and O tl ■ C • c II O 10th wherein R1 and R2 are the same or different and each represent H, F, Cl, Br, NO 2, OH, lower alkyl or lower alkoxy and X represents Cl, Br, F or J and where Y represents H, Cl, Br, F or J stands. 3. The method according to any one of claims 1 and 2, characterized in that the acylating derivative of the acid is the active ester with N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or N-hydroxyphthalimide . 4. The method according to claim 3, characterized in that one uses an acylating derivative of the acid whose amino-blocking group is a carbobenzyloxy group. 5. The method according to claim 2, characterized in that the mixture of the acid anhydride with pivalic acid, benzoic acid, isobutyl carbonic acid or benzyl carbonic acid is used as the acylating derivative of the acid. 6. The method according to any one of claims 1 to 5, characterized in that the polysilylated kanamycin A or B has a carbobenzyloxy group on the 6'-amino group. 7. The method according to any one of claims 1 to 6, characterized in that the polysilylated kanamycin A or B starting material an average number of 4 to 8 Contains 15 silyl groups per molecule. 8. The method according to any one of claims 1 to 6, characterized in that the polysilylated kanamycin A or B starting material which has a blocking group other than silyl on the 6'-amino group has an average Contains 20 che number of 3 to 7 silyl groups per molecule. 9. The method according to any one of claims 1 to 8, characterized in that the silyl groups are trimethylsilyl groups. 10. The method according to any one of claims 1-9 for the production 25 of a 1-N- [L - (-) - co-amino-a-hydroxyalkanoyl] kanamycin A, characterized in that an acylating derivative of the acid of the general formula L - (-) - B-HN-CH2- (CH2) n-CH-C00H OH wherein B represents an amino blocking group. described. There are also numerous derivatives of Kanamycins. 35 The structural formulas of Kanamycins A and B are given below. There is also the commonly used one Standard numbering system. The invention relates to the kanamycin derivatives characterized by the claims as derivatives of the kanamycin A neten subject. or B denotes the need for a comparison Kanamycins are well known antibiotics. They are to be avoided with 40 complex structural formulas to determine differences, for example in the Merck Index, 8th edition, pages 597 to 598. '6'. NK, Kanamycin A: R = OH Kanamycin B: R = NH ^ 639 104 io US Pat. No. 3,781,268 discloses the 1- [L - (-) - Y-amino-a-hydroxybutyryl] kanamycin A (amikacin) and B and their mono- and dicarbobenzyloxy-protected derivatives. Higher and lower homologs are disclosed in U.S. Patent Nos. 3886139 and 3 904597. The compounds are prepared by acylating a 6'-N-protected kanamycin A or B with an acylating derivative of an N-protected L - (-) - y-amino-a-hydroxybutyric acid in an aqueous medium and then one or both N -protective groups removed. U.S. Patent 3,974,137 discloses a process for the preparation of l- [L - (-) - Y-amino-a-hydroxybutyryl] kanamycin A which consists in combining 6'-carbobenzyloxykanamycin A with at least 3 moles of benzaldehyde, one substituted benzaldehyde or pivaldehyde to produce the 6'-N-carbobenzyloxy-15 kanamycin A containing Schiff's base units at the 1,3 and 3 "positions and then this tetrasecured kanamycin A derivative with the N- Hydroxysuccinimide esters of L - (-) - Y-benzyloxycarbonylamino-cx-hydroxybutyric acid are acylated and the protective groups are subsequently removed Belgian patent 828192 discloses a process for the preparation of l- [L - (-) - y-amino-a-hydroxybutyryl] kana-mycin A by producing the same tetra-protected kanamycin A derivative as described in US Pat. No. 3,974,137 , then acylating with the N-hydroxy-5-norbornene-2,3-dicarboximide ester of L - (-) - Y-benzyloxycarbonylamino-no-a-hydroxybutyric acid and finally removing the protective groups.