DK167687B1 - (N)-Hydroxy-amino-alkanoyl-kanamycin derivs. prodn. - by acylating poly:silylated kanamycin, useful as antibiotics - Google Patents

Abstract

Prodn. of kamamycin derivs. of formula (I) and their pharmaceutically acceptable acid additions salts (R = OH, or NH2, n = 0-2). comprises reacting a polysilylated kanamycin A or B (II), or analogous cpd. with the 6'-amino protected by a gp. other than silyl (cpds. (III)), with an acylating deriv. of the acid B.NH.CH2(CH2)nCH(OH).COOH (IV) (B = amino protecting gp). Reaction is carried out in a (nearly)anhydrous organic solvent and then all protecting gps. are removed. Cpds. (II) and (III) are new. Esp. the silyl gp. is trimethylsilyl and (II) contain 4-8, and (III) 3-7 such gps. In (III) the pref. 6'-protecting gp. is carbobenzoxy. Cpds. (I) are antibiotics. (II) and (III) have high solubility so concn. solns. (e.g. 10-20%) can be used and the amt. of 3"-acyl impurity formed is minimal.

Description

in DK 167687 B1

The present invention relates to novel derivatives of kanamycin A or B for use in the preparation of 1N- [w-amino-α: -hydro-xyalkanoyl] kanamycin A or B of the general formula (I) 2 2

Eo / NH2

R

10 7v \ HO / \ Λ / ΛΛ with H0-K? H „OH / c = o VN ^ / / ηο-Ϊη V — ΞΓ ° \ /“ iVn Νη "Γ ^ Τ \ Λ /? H2 15 W HO NH2 wherein R represents OH or NHg, and n is an integer from 0 to 2, or a non-toxic, pharmaceutically acceptable acid addition salt thereof, which derivatives are characterized by having the general formula (Γ) ro- OR ZR ^ -A NHR '25 · \ \? Κ._κηβ., / (1') R'O- ^. CH2OR '/ $ NH S ----- / 30 OR' \ /

ISLAND

wherein Z is -O- or -NH-, R 'is H or a silyl group of the formula R5 R6-Si- \ p wherein R5, R6 and R7 represent hydrogen, halogen, (lower) alkyl, halogen- (lower) alkyl or phenyl, wherein at least one of the groups R5, R6 and R7 does not represent halogen or hydrogen, at least two and at most ten R 'groups are silyl groups and R "is R' or another blocking group other than a silyl group selected from alkoxycarbonyl, aral coxycarbonyl, -cycloalkyl oxy-carbonyl, haloalkoxycarbonyl, halo-methylcarbonyl, acyl, o-nitro-phenylthio and trityl, while R "is not a silyl group when all R'-groups are sylyl groups .

The present derivatives of the general formula (Γ) can be converted to the compounds of the general formula (I) by acylation with an acylating derivative of an acid of the general formula (II) B-HN-CH 2 - (CH 2) n-CH -C00H (II)

OH

Wherein n is an integer of 0 to 2, and B represents an amino blocking group, in a substantially anhydrous organic solvent followed by removal of all blocking groups.

The kanamycins are well known antibiotics such as has been written in the Merck Index, 8th edition, pages 597-598. Numerous derivatives of the kanamycins are known. The structural formulas of kanamycci A and B are listed below together with the standard numbering system used in the art. In the following, the various derivatives of kanamycin, where it will be readily understandable, will be referred to as derivatives of kanamycci A or B rather than by structural formula, so that the need to compare complex structures to detect differences is avoided.

30 35 DK 167687 B1 3 G 'it * CH „NH„ HO _, 2 2 5 HO / „» H0 R 1 HO ^ 5 / W 1 ^ 6 / ^ - NH „HO- ^ CH2 ° h / 2 HO M / III 'O'

Kanamycin A: R = OH

Kanamycin B: R = NH2 US Patent No. 3,781,168 discloses and contains claims directed to 1- [L - (-) -, γ-amino-α-hydroxybutyryl] kanamycin A (amikacin) and B, as well as the 20 no and di-carbobenzyloxy protected derivatives thereof. For lower and higher homologues thereof, reference is made to U.S. Patents Nos. 3,886,139 and 3,904,597. The compounds are prepared by acylating a 6'-N-protected kanamycin A or B with an acylating derivative of an N-protected L - (-) - 7-amino.-o: -hydroxy butyric acid in an aqueous medium followed by removal of 25 one N-protecting group or both N-protecting groups.

U.S. Patent No. 3,974,137 discloses and contains claims directed to a process for the preparation of 1- [L - (-) - 7-amino-α-hydroxybutyryl] kanamycin A, which comprises reacting 6'-carbobene 2 yyloxycanamycin A with at least three moles of benzaldehyde, a substituted benzaldehyde or piv aldehyde to produce 6'-N-carbobenzyloxycanamycin A containing Schiff base groups at the 1.3 and 3 "positions acylates this tetra-protected kanamycin A derivative with the N-hydroxysuccinimide ester of L - (-) - 7-benzyloxycarbonylamino-α-hydroxybutyric acid and then removing the protecting groups.

35 Belgian Patent No. 828,192 discloses and contains claims directed to a process for the preparation of 1- [L - (-) - γ-amino-α-hydroxybutyryl] -canamycin A in the preparation of the same tetra-protected kanamycin A derivative as in the United States. Patent No. 3,974,137, acylation with N-hydroxy-5-norbornene-2,3-dicarboximide ester of L- (-) - γ-benzyloxycarbonylarai-no-hydroxybutyric acid and subsequent removal of the protective groups.

The use of a polysilylated kanamycin A or B as a starting material in the preparation of compounds of formula (I) provides high solubility in the organic solvent system, which enables high concentration reaction. Although the reaction is usually carried out in solutions containing approx. 10-20% polysilylated kanamycin starting material, excellent results have been obtained at concentrations of approx. 50 g / 100 ml solvent.

10 As with known methods, the present process provides a mixture of acylated products. The desired 1-N-acylated product is separated from the other products by chromatography and, if desired, the by-products can be hydrolyzed to the starting kanamycin for recycling. By known methods, it was found that any 3 "N-acylated material produced produced a loss of approximately a corresponding amount of the desired 1-N-acylated product due to the great difficulty of separating A particularly attractive feature of the present process is the extremely low amount of undesirable 3 "N-acylated product produced (typically no 20 is detected).

In the preparation of 1- [L - (-) - γ-amino-α-hydroxybutyryl] kanamycin A, amikacin, by various known methods, the 3 "N-acylated product (BB-K11), the 3 -N-acylated product (BB-K29), the 6'-N-acylated product (BB-K6) and polyacylated material together with unreacted kanamycin A. In commercial production of amikacin by acylation of 6'-N-carbobenzyloxy kanamycin A in an aqueous medium followed by removal of the protecting group, it was found that approximately 10% of the desired amikacin (2.5 kg in a 25 kg charge) was usually lost due to the presence of ΒΒ-ΚΠ When 30 amikacin is prepared by the present process, ΒΒ-ΚΊ1 is typically not detected in the reaction mixture.

The blocking groups which can be used to protect the amino group on the acylating acid (group B of formula II) are conventional blocking groups for protecting 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; aralkoxy-carbonyl groups such as benzyloxycarbonyl; cycloal kyloxycarbonyl groups such as cyclohexyloxycarbonyl; haloalkoxycarbonyl groups such as tri-chloroethoxycarbonyl; 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 the (+) or (-) isomeric form or a mixture of the two isomers (d, 1-form) to produce the corresponding compound of formula (I), wherein the 1-N- [id-aminb-c1: hydroxyalkanoyl] group is in its (+) [or (R)] form or its (-) [or (S)] form, or a mixture thereof. It is preferred that the acylating acid of formula (II) be in its (-) or (+) form.

The blocking group of the 6'-amino group is preferably selected from the groups of the formulas: cHj-c-o-i, y2xcc-, / \ = / CH-3 r2 / 3

. - "Oh

Q— Οχ N ° 2 0 '25

ISLAND

II - · n X-C-CH9-O-C and 3

we you

wherein R and R are the same or different and each represents H, F, Cl,

Br, NOg, OH, (lower) alkyl or (lower) alkoxy, and X represents Cl, Br, F 35 or I, and V represents H, Cl, Br, F or I. The most preferred blocking group is is the carbobenzyloxy group.

In particular, the acylating derivative of the acid of formula (II) is an active ester, preferably an active ester with N-hydroxysuccinimide, N-hydroxy-5- norbornene-2,3-dicarboximide or N-hydroxyphthalimide, or a mixed acid anhydride, preferably a mixed acid anhydride with pivalic acid, benzoic acid, isobutylcarboxylic acid or benzyl carboxylic acid.

It is preferred to prepare 1N- [L - (- γ-amino-α-hydroxybutyryl] catamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof by a process comprising manacylating polysilylated kanamycin A with a mixed acid anhydride of L - (-) - γ-benzyloxycarbonylamino-α-hydroxybutyric acid (and preferably its mixed acid anhydride with pivalic acid, benzoic acid, isobutylcarboxylic acid or benzyl carboxylic acid) in a substantially anhydrous organic solvent and then removes all blocking agents. groups.

It is further preferred to prepare 1N- [L - (-) - γ-amino-α-hydro-xybutyryl3canamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof by a process comprising acylating polysilylated kanamycin A containing a carbobenzyloxy group on the 6'-amino group with a mixed acid anhydride of L - (-) - 7-benzyloxycarbonylamino-α-hydroxybutyric acid (and preferably its mixed acid anhydride with pivalic acid, benzoic acid, isobutyl carboxylic acid or the substantially anhydrous organic solvent and then removes all blocking groups.

In addition, it is preferred to prepare 1N- [L - (-) - γ-amino-α-hydro-xybutyryl] kanamycin A or a nontoxic, pharmaceutically acceptable acid addition salt thereof by a process comprising acylating polysilylated kanamycin A with an active ester of L - (-) - γ-benzyloxy-carbonylamino-α-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 then removes all blocking groups.

Finally, it is preferred to prepare 1N- [L - (-) - 7-amino-α-hydroxybutyryl] kanamycin A or a non-toxic, pharmaceutically acceptable acid addition salt thereof by a process comprising acylating the polysilylated kanamycin A containing a carbobenzyloxy group on the S'-amino group with an active ester of L - (-) - γ-benzyloxycarbonylamino-α-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 then remove all blocking groups.

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

The term "non-toxic, pharmaceutically acceptable acid addition salt" of a compound of formula (I) means herein and in the claims, a mono-, di-, tri- or tetra-salt formed by interaction of a free compound of formula (I) and 1-4 equivalents of a non-toxic, pharmaceutically acceptable acid. These acids include acetic, hydrochloric, sulfuric, maleic, phosphoric, nitric, hydrobromic, ascorbic, malic and citric, and other acids commonly used to form salts of amine-containing drugs.

Acylation of the derivatives of the invention can usually be carried out in an organic solvent in which the starting materials have sufficient solubility. The starting materials are highly soluble in the 20 most-common organic solvents. Suitable solvents include, e.g. acetone, di ethyl ketone, methyl n-propyl ketone, methyl iso-butyl ketone, methyl ethyl ketone, acetonitrile, glyme, diglyme, dioxane, toluene, tetrahydrofuran, cyclohexanone, methylene chloride, chloroform, carbon tetrachloride and mixtures of acetone / butanol or di ethyl ketone / butanol. The choice of solvent depends on the particular starting material used. Ketones are usually the preferred solvents. The most advantageous solvent for the particular combination of reactants used can be readily determined by routine experiments.

Suitable silylating agents for use in the preparation of the derivatives of the invention include the silylating agents of the general form are 35 8 DK Tb / bK / Kl 4 4 R \ / R (\

^ / Si ~ --1IH

"4 NH 5 5 - \ I] ^ Si r4 and R6_si_x R4 // ^ N - Si r7 H ^" R4 J m 10

IV V

Wherein R, R and R are hydrogen, halogen, (lower) alkyl, halogen (lower) alkyl or phenyl, wherein at least one of the groups R, R and R does not represent halogen or hydrogen; R 4 represents (lower) alkyl, m is an integer of 1 to 2, and X represents halogen or the group

-N

20 - - \ g

Ry 8 9 wherein R represents hydrogen or (lower) alkyl and R represents hydrogen, (lower) alkyl or the group R 5 25 h R 6 - Si- 7 7 6 6 30 wherein R, R and R have the meanings defined above .

Specific silyl compounds of formulas IV and V are: trimethylchlorosilane, hexamethyldisilazane, triethylchlorosilane, methyltrichlorosilane, dimethyl dichlorosilane, triethyl bromosilane, tri-n-propylchlorosilane, methyl diethylchlorosilane, dimethylethylchlorosilane t-butylchlorosilane, phenyl dimethyl bromosilane, benzyl methyl ethyl chlorosilane, phenylethyl methylchlorosilane, triphenylchlorosilane, triphenyl fluorosilane, tri-o-tolylchlorosilane, tri-p-dimethylaminophenyl chlorosilane, N-ethyltriethylsilylsilane, N-ethyltriethylsilane , tri-n-propylsilylamine, DK 167687 B1 9 tetraethyldimethyl di si 1 azane, hexaphenyldi si 1 azane, hexa-p-tolyldisilazane, etc. Also hexa-alkylcyclotrisilazanes and octa-alkylcyclotetrasilazanes are useful. Other suitable silylating agents are silylamides (such as trialkylsilylacetamides and bis-trialkylsilylacetamides), silylureas (such as trimethylsilylurea) and silylurides. Also trimethylsilylimidazole can be used.

A preferred silyl group is the trimethylsilyl group, and preferred silylating agents for introducing the trimethylsilyl group are hexamethylsilazane, bis (trimethylsilyl) acetamide and trimethylsilylacetamide. Hexa-10 methyl di silazane is the most preferred.

A polysilylated kanamycin A or B containing a blocking group other than silyl on the 6 'amino group can be prepared either by polysilylation of the desired 6'-N blocked kanamycin A or B or by introducing the desired 6'-N -blocking group in polysilylated kanamycin A or B.

Methods for introducing 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 group) can be prepared by 20 known methods per se or as described herein.

The term polysilylated kanamycin A or B, as used herein, refers to kanamycin A or B containing from 2 to 10 silyl groups in the molecule. Thus, the term polysilylated kanamycin A or B does not include persilylated kanamycin A or B, which would contain 11 silyl groups in the molecule.

The exact number of silyl groups present in the polysilylated kanamycin derivatives (with or without a blocking group other than silyl on the 6 'amino group) (or their position) is unknown. It has been found that both sub-silylation and over-silylation reduce the yield of the desired product and increase the yield of other products. In the case of heavy under- or over-silylation, little or nothing of the desired product can be formed. The degree of silylation which will yield the greatest yield of the desired product will depend on the particular reactants used in the acylation step. The most advantageous degree of silylation using any combination of reactants can be readily ascertained in routine experiments.

In the preparation of 1N- [L - (-) - γ-amino-α-hydroxybutyryl] kanamycin A by acylation of polysilylated kanamycin A with N-hydroxysuccinimide ester of L - (-) - 'Y-benzyloxycarbonylamino α-hydroxybutyric acid in acetone solution has found that good yields of the desired product are obtained using polysilylated kanamycin A, which has been prepared by reacting from ca. 4 to approx. 5.5 moles of hexamethyldisilazane per greater or smaller amounts of hexamethyldisilazane may be used, but the yield of the desired product at the subsequent acylation step is considerably diminished. In the particular method set forth above, it is preferred to use from ca. 4.5 to approx. 5.0 moles of hexamethyldisilazane per mole of kanamycin to obtain maximum product yield at 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 11 positions (NHg and OH groups) which can be silylated while kanamycin A and B containing a blocking group other than silyl on the 6 'amino group have a total of 10 such positions. 5.5 moles of hexamethyldisilazane per Thus, theoretically, moles of kanamycin A or B could completely silylate all OH and IW ^ groups on the kanamycin, while 5.0 moles of hexamethyldisilazane could completely silylate a mole of kanamycin A or B containing a blocking group difference equal to silyl of 6 '. the amino group. However, it is believed that such extensive silylation does not occur with these molar ratios within reasonable reaction times, although higher silylation rates are obtained within a given reaction time when a silylation catalyst is added.

25 Silylation catalysts greatly accelerate the silylation rate. 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 usually promote a higher degree of silylation than is necessary in the present context. However, oversilylated kanamycin A or B can be used as starting material if first treated with a disilylating agent to reduce the degree of silylation before conducting the acylation reaction.

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

Although it is common to use dry solvents when working with silylated materials, it has surprisingly been found that adding water to the reaction solvent prior to acylation 15, even in the absence of "oversilylation", often yields equally good yields and sometimes better yields of desired product than a dry solvent.

In acylation reactions carried out in acetone at the usual concentrations of 10-20% (w / v) of polysilylated kanamycin A, it has been found that excellent yields of 1N- [L - (-) - 7-amino-α-hydroxybu -20 Tyryl'Janamycin A was obtained when up to 28 moles of water per ml. moles of polysilylated kanamycin A were added; at a concentration of 20%, 28 moles per mole about 8% water. In other combinations of reactants, even more water may be tolerated or beneficial. The acylation reaction can be carried out in information agents containing up to approx. 40% water, although at such high 25 water concentrations, short acylation times must be used to avoid excessive disilylation of the polysilylated kanamycin A or B starting material. Accordingly, the term "substantially anhydrous organic solvent", as used herein, is intended to include solvents containing up to ca. 25% water. A preferred interval is up to approx. 20%, a more preferred range is up to 8% water, and a particularly preferred range is up to approx. 4% water.

As noted above, the most desirable degree of silylation for any combination of acylation reactants can be readily determined by routine experiments. 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 β'-amino group, but this is theory only and is not considered an essential part of the invention.

12 UK Ίb / btf / t »l

The duration and temperature of the acylation reaction is not critical. Temperatures in the range of approx. -30 ° C to approx. 100 ° C can be used for reaction times in the range of about 1 hour and up to 1 day or more. It has been found that the reaction usually proceeds well at room temperature, and for convenience and economic reasons it is preferred to carry out the reaction at ambient temperature. However, in order to achieve maximum yields and selective acylation, it is preferable to carry out the acylation by from ca. 0 to 5 °.

Acylation of the 1-amino group in the polysilylated kanamycin A or 10B (with or without a blocking group other than silyl on the 6'-amino group) can be accomplished with any acylation derivative of the formula II acid known in the art. the region as being suitable for acylating a primary amino group. Examples of suitable acylation derivatives of the free acid include the corresponding acid anhydrides, mixed anhydrides, e.g. alkoxy formic anhydrides, acid halides, acid azides, active esters and active thioesters. The free acid can be coupled to the polysilylated kanamycin starting material after the free acid is first reacted with N, Ν'dimethyl chloroformininium chloride [see British Patent Specification No. 1,008,170 and Novak and Weichet, Experientia XXI, 6, 360 20 (1965)] or using an N, N'carbonyl di in mi dazole or N, N'carbonylditriazole (see South African Patent Specification 63/2684) or a carbo-diimide reagent [especially Ν, Ν'-dicyclohexylcarbodiimide, N, N'-diisopropylcarbonyl). diimide or N-cyclohexyl-N, - (2-morpholinoethyl) carbodiimide: see Sheenan and Hess, JACS, 77, 1967 (1955)], or of an alkynylamine reagent [see 25 R. Bui-jle and HG Viehe, Angew. Chem. »International Edition, 3, 582, (1964)] or of an isoxazolium salt reagent [see R.B. Woodward, R.A. Olof-son and H. Mayer, J. Amer. Chem. Soc., 83, 1010 (1961.).], Or by a keteneimine reagent [see. C. L. Stevens and Μ. E. Munk, J. Amer. Chem. Soc., 80, 4065 (1958)] or of hexachlorocyclotriphosphatriazine or hexabromocyclotriphosphatriazine (U.S. Patent No. 3,651,050) or of diphenylphosphoryl azide [DDPA; J. Amer. Chem. Soc., 94, 6203-6205 (1972)] or of di ethyl phosphoryl cyanide [DEPC; Tetrahedron Letters No. 18, pages 1595-1598 (1973)] or of di phenylphosphite [Tetrahedron Letters No. 49, pages 5047-5050 (1972)]. Another acid equivalent derivative is a corresponding azolid, ie. an amide of the corresponding acid whose amide nitrogen is included in a quasi-aromatic 5-membered ring containing at least two nitrogen atoms, i. imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As will be appreciated by those skilled in the art, it may sometimes be desirable or necessary to protect the hydroxyl group on the acylating derivative of the acid of formula (II), e.g. when using acylation derivatives such as an acid halide. Protection of the hydroxyl group may be effected in ways known to the art, e.g. using a carbobenzyloxy group, by acetylation, by silylation or the like.

Upon completion of the acylation reaction, all blocking groups are removed in a manner known per se to produce the desired product of formula (I). The silyl groups may e.g. is easily removed by hydrolysis 10 with water, preferably at low pH. Also, the blocking group B on the acylation derivative of the acid of formula (II) and the blocking group on the 6 'amino group on the polysilylated kanamycin derivative (if present) can be removed by known methods. Thus, a t-butoxycarbonyl group can be removed using formic acid, a carbobenzyloxy group by catalytic hydrogenation, a 2-hydroxy-1-naphthcarbonyl group by acid hydrolysis, a trichloroethoxy-carbonyl group by treatment with zinc dust in glacial acetic acid, a phthaloyl group. by treatment with hydrazine hydrate in ethanol under heating, etc.

Product yields were determined in various ways. After removing all 20 blocking groups and chromatography on a CG-50 (NH4 +) column, the yield of amikacin could be determined by isolating the crystalline solid from the appropriate fractions or by microbial analysis (turbi-dimetric or plate) of Another technique used was HP 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 residual blocking agent (s). group.e (r) This assay was not a direct assay 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 i.d. "μ-Bondapak C-18" column, under the following conditions: 35 Mobile phase: 25% 2-propanol 75% 0.01 M sodium acetate pH 4.0 Flow rate: 1 ml / min.

Detector: UV at 254 nm DK 167687 B1

Sensitivity: 0.04 AUFS

Diluent: DMSO

Injected amount: 5 μ1

Concentration: 10 mg / ml 5

The curve speed varied, but 2 minutes per second. 2254 cm was typical. The above conditions provided UV curves with peaks that were easy to quantitatively measure. The results of the above assays are described in the description as HPLC assays.

10 In order to avoid repetition of complex chemical names, the following abbreviations are occasionally used in the specification.

AHBA L - (-) - 7-amino-α-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

20 (or '~ BHBA- / US /) BHBA

DCC dicyclohexylcarbodiimide DCU dicyclohexylurea HMDS hexamethyldisilazane BSA - bis (trimethylsilyl) acetamide MSA trimethylsilylacetamide TFA tri fluoroacetyl t-BOC tert-butyloxycarbonyl

kana A kanamycin A

30 "Dicalite" is a trademark of Great Lakes Carbon Corporation for diatomaceous earth.

"Amber! Ite CG-50" is a trademark of Rohm and Haas Co. for chromatography in the form of a weakly acidic cationic ion-exchange resin of the carboxyl-polymethacryl type.

35 "μ-Bondapak" is a trademark of Waters Associates for a series of highly effective liquid chromatography columns.

All temperatures herein are listed in ° C.

By the terms 11 (lower) alkyl and "(lower) alkoxy", as used herein, is meant all alkyl or alkoxy groups containing from 1 to 6 carbon atoms.

The following examples illustrate the preparation of the compounds of the invention and their conversion to 1-N- [w-amino-α-hydroxyal-5-canoyl] kanamycin A or B.

Example 1

Preparation of 1- N - [L - (-) - 7-amino-Q? -Hydroxybutyryl] kanam, ycin A, amika-cin, by selective acylation of polyitrimethylsilyD-e'-N-carbobenzyloxy-kanamycin A in anhydrous diethyl ketone 6'- N-carbobenzyloxycanamycin A (15 g, 24.24 m.mol) was slurried in 90 ml of dry acetonitrile and heated to reflux under a nitrogen atmosphere. Hexamethyldisilazane (17.5 g, 108.48 mmol) was added slowly over 15 minutes and the solution obtained was refluxed for 24 hours. After removal of the solvent in vacuo (40 °) and complete drying under vacuum (10 mm), 27.9 g of a white amorphous solid [90.71% calculated as 6'-N-carbobenzyloxycanamycin A (silyl) g] was obtained.

This solid was dissolved in 150 ml of dry diethyl ketone at 23 °. L - (-) - 20 γ-berizyjoxycarbonylamino-α-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) (11.05 g, 26.67 mmol) dissolved in 100 ml dry diethyl ketone at 23 ° was added slowly with good stirring over half an hour. The solution was stirred at 23 ° for 78 hours. The yellow clear solution (pH 7.0) was diluted with 100 ml of water. The pH of the mixture was adjusted to 2.8 (3 N HCl) and stirred vigorously at 23 ° for 15 minutes. The aqueous phase was separated and the organic phase was extracted with 50 ml of water at pH 2.8. The combined aqueous fractions were washed with 50 ml of ethyl acetate. The solution was placed in a 500ml Parr flask with 5 g of 5% palladium-on-carbon catalyst (Engelhard) and reduced at 3.4 atm. H2 for 2 hours at 23 °. The mixture was filtered through a layer of "Dicalite" which was then washed with an additional 30 ml of water. The colorless filtrate was concentrated in vacuo (40-45 °) to 50 ml. The solution was loaded onto a 5 x 100 cm "CG-50" (NH 2+) ion exchange column. After washing with 1000 ml of water, unreacted kanamycin A, 3- [L - (-) - 7-amino-α-35-hydroxybutyryl] -canamycin A (BB-K29) and amikacin were eluted with 0.5 N ammonium hydroxide. Polyacyl material was recovered with 3N ammonium hydroxide. Bioassay, thin layer chromatography and optical rotation were used to record the elution progress. The volume and observed rotation for each eluate fraction, as well as the weight and percent yield of solids isolated from each fraction by evaporation to dryness, are summarized below: 5 volumes

Material (ml) a578 Weight (g)% yield

Kanamycin A 1000 +0.115 0.989 9.15 BB-K29 1750 +0.24 4.37 32.0 10 Amikacin 2000 +0.31 6.20 47.4

Polyacyls 900 +0.032 0.288. 2.0

The spent ethyl ketone ag used was shown by HP liquid chromatography to contain an additional 3-5% amikacin.

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

Example 2 Preparation of 1-N-rL - (-) - 7-amino-O-hydroxybutyrylcanamycin A, amikacin, by selective acylation of poly (trimethylsilyl) -6'-N-carbobenzyloxy-kanamycin A in anhydrous acetone

Poly (trimethylsilyl) -6'-N-carbobenzyloxy kana A prepared as in Example 1 (103 g, 0.081 mol, calculated as 6'-N-carbobenzyloxycanamycin 25 A (silyl) g) was dissolved in 100 ml of dry acetone at 23 °. L - (-) - 7-Benzyloxy-carbonylamino-tt-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) (35.24 g, 0.085 mole) dissolved in 180 ml of dry acetone at 23 ° was added slowly with good stirring to the solution of poly (trimethylsilyl) -6'-N-carbobenzyloxycanamycin A over a period of 15 minutes. 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 (3 N HCl) and stirring was continued at 23 ° for 15 minutes. Acetone was removed using a steam-injector vacuum at ca. 35 °. The solution was placed in a 500 ml Parr flask with 10 g of 5% palladium-on-carbon catalyst (Engelhard) and reduced at 2.72 atm. H2 for 2 hours at 23 °. The mixture was filtered through a layer of diatomaceous earth which was then washed with an additional 50 ml of water. After concentration to about 1/3 volume, the solution DK 167687 B1 17 (pH 6.9-7.2) was charged to a 6 x 110 cm "CG-50" (NH H 2 O to 0.6 N ammonium hydroxide to recover amikacin An automatic polarimeter was used to record the elution progress, combinations were made on the basis of thin layer chromatography assessment, the combined amikacin fractions were concentrated to 25-30% solids. an equal volume of methanol followed by two volumes of isopropanol to induce crystallization, 18.2 g (40%) of crystalline amikacin was recovered.

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

Example 3

Preparation of 1N-rL - (-) - γ-amino-tt-hydroxybutyl, yr.ylkanamycycline A, amicycin, by selective acylation of poly (trimethylsilyl) kanamycin A, using blocking in situ

A. Poly (trimethylsilyl) kanamycin A

Kanamycin A free base (18 g activity, 37.15 m.mol) was slurried in 20 200 ml of dry acetonitrile and heated to reflux. Hexamethyldisilazane (29.8 g, 184.6 mmol) was added over 30 minutes and the mixture was stirred under reflux for 78 hours to give a pale yellow clear solution. Removal of the solvent in vacuo left an amorphous solid residue (43 g, 94%) [calculated as kanamycin A (silyl) jq] -25

B. 1- N- [L - (-) - 7-amino-α-hydroxybutyryl] kanamycin A

p- (Benzyloxycarbonyloxy) benzoic acid (5.56 g, 20.43 m.mol) was slurried in 50 ml of dry acetonitrile at 23 °. Ν, Ο-Bis-trimethylsilylacetamide (8.4 g, 41.37 m.mol) was added with good stirring. The solution was kept at 23 ° for 30 minutes and then stirred vigorously for 3 hours with a solution of poly (trimethylsilyl) kanamycin A (21.5 g, 17.83 m.mol, calculated as the (silyl) compound). ) in 75 ml of dry acetonitrile at 23 ° The mixture was stirred for 4 hours, the solvent was removed in vacuo (40 °) and the oily residue was dissolved in 50 ml of dry acetone at 23 ° C.

L- (-) - γ-Benzyl oxycarbonylamino-α-hydroxy butyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) (8.55 g, 20.63 mmol) in 30 ml of acetone was added to the above solution over a period of 5 minutes. The mixture was kept at 23 ° C for 78 hours. The solution was diluted with 18 UK Ί b / bo7 B1 100 ml water and the pH (7.0) lowered to 2.5 (6 N HCl). The mixture was placed in a 500 ml Parr flask with 3 g of 5% palladium-on-carbon catalyst (Engelhard) and reduced at 2.72 atm. Hg for 2 hours at 23 °. The mixture was filtered through a layer of diatomaceous earth which was then washed with 20 ml of water. The combined filtrate and wash water (168 ml) were determined by microbiological analysis against E. coli to contain about 11.400 mcg / ml (19% yield) of amikacin.

Example 4

Preparation of 1-N- [L - (-) - γ-amino-Q: -hydroxybutyryl Ikanamycin A, amikacin, by selective acylation of poly (trimethylsilyl) kanamycin A

A. Poly (trimethyl silyl) kanam, ycin A

A suspension of 10 g (20.6 mmol) of kanamycin A in 100 ml of dry 1 acetonitrile and 25 ml (119 ml) of 1,1,1,3,3,3-hexamethyldisilazane was refluxed for 72 hours. . This gave a clear pale yellow solution. The solution was evaporated to dryness in vacuo at 30-40 ° C. 21.3 g of poly (trimethylsilyl) kanamycin A was obtained as a light yellow-brown amorphous powder [85% yield calculated as kanamycin A (silyl) jQ].

20

B. 1- N- [L - (-) - γ-amino-α-hydroxybutyryl kanamycin A

To a solution of 2.4 g (2.0 mmol) of poly (trimethylsilyl) kanamycin A in 30 ml of dry acetone was slowly added 2.0 mmol of L - (-) - γ-benzyloxy-carbonylaminoο-ατ-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) in 10 ml of dry acetone at 0-5 ° C. The reaction mixture was stirred at 23 ° C for one week and then evaporated 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 give a solution. The solution was acidified with 3 N HCl to pH 2.0 and then reduced at 3.40 30 atm. H2 for 2 hours using 500 mg of 5% palladium-on-carbon catalyst. The material was filtered and combined filtrate and wash was determined by microbial analysis against E. coli to contain amikacin in a 29.4% yield.

Example 5

Preparation of Amikacin by Selective N-Acylation of Polytrimethylsilyl-6'-N-Carbobenzoxy-Kanamycin A in Anhydrous Acetone DK 167687 B1 19 I. Summary

Silylation of 6'-N-carbobenzoxy-kana A in acetonitrile using hexamethyldisilazane (HMDS) gives the 6'-N-carbobenzoxy-kana A (silyl) 9 intermediate®. This silylated channel A is easily soluble in most 5 organic solvents. Acylation with NAE in anhydrous acetone at 23 ° using a 5% molar excess of NAE relative to the 6'-N-Cbz channel A used yielded a mixture containing only Cbz derivatives of amikacin and BB-K29, somewhat unreacted kana A and some polyacyl material. No BB-K11 was detectable in any of these studies. Elution of an acetone acylation mixture after reduction and reprocessing from a "CG-50" (NH4 +) column using an ammonium hydroxide gradient yielded isolated yields of pure amikacin in the range of about 40 °.

DK 167687 B1 20 II. Reaction Schemes "A.

Λ HO _ OH, 0H

ro / ° h

HO- / OH

-o / ν \ / U / 1 5 '6 NH „V— ^ / Vs n —-o 2 r2n vy ^ -hh2

Channel A "base" C18H36 ° 11N4 (484.51) + (CH3) 3 Si-NH-Si (CH3) 3 HMDS (161.4) ch3cn

V

OR OR

and ro

__ / ° R 0H

RO-5 'CN

/ 6 \\ in ** / NHR

Λ- "7? / RHN ^ i-? JZT 0

RHN or I / rwHR

\ y + nh3 R = Si (CH3) 3 (l) Kana A (silyl) C48B116 ° XlN4Si10 <1206'35 'DK 167687 B1 21

B. CbzNH (CH2) 2-CH-COOH + HON M + DCC

(206)

ISLAND

BHBA (253.4) HONB (179.2)

acetone

V

? H i? DCU + CbzHN (CH9) 2-CH-C-O-N / (224.3)

ISLAND

(2) NAE (414.6) c / [diCbzamikacin (854) + (χ) + (g) Acetone y. diCbzBB-K29 23 ° +

61Cbz-1,3-diBHBÅ-Kana A

H2 Γ "

5% Pd / C Ψ 61Cbz Channel A

_Λ__ (^

Amikacin + BB-K29 + 1,3-diAHBA-Kana A + Kana A

(585.62) (722.76) (484.5) "CG-50 (NH4 +) Ψ

Amikacin DK 167687 B1 22 III. Material is Weight g Vol. ml Mol 5 6'-N-Cbz Channel 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 on demand IPA on demand 15 IV. Security 6'-N-Cbz Kana A - No direct information available. Avoid dust contact.

20

Acetonitrile - Treated as a cyanide.

Avoid inhalation of vapors.

May cause skin irritation.

25 Hexamethyldisilazane - Irritant, (HMDS) is handled with care.

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

BHBA - Toxicity not determined.

Avoid exposure to solids.

35 HONB - Toxicity unknown. Exercise caution during handling.

DK 167687 B1 23 DCC - Severe skin and eye irritant. Avoid inhalation of mist or vapors. Toxic.

5 Acetone - Flammable. Inhalation can cause headaches, fatigue, arousal, bronchial irritation and large amounts of anesthesia.

10 NAE - No direct information available; is always used directly as a solution in acetone.

Methanol - Flammable. Poisoning can occur by administration, inhalation or percutaneous absorption.

Isopropanol - Flammable. Administering or inhaling large quantities of the 20 "vapors can cause headache, dizziness, mental depression, vomiting, anesthesia.

Ammonium hydroxide -. Toxic fumes. Wear mask, avoid 25 contact with liquid.

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

Procedure A. Preparation of 6'-N-carbobenzyloxycanamycin A (silyl) g Γβ'-N-Cbz Kana A (silyl) g] 1. Suspend 50 g of 6'-N-carbobenzyloxycanamycin A (KF <4% ) in 300 ml of acetonitrile (KF <0.01%). Bring the slurry to reflux (74 °) while maintaining a stream of dry nitrogen 9 through the slurry.

2. Add 75.8 ml of hexamethyldisilazane (HMDS) slowly over a period of 30 minutes. Complete solution will occur with the development of ammonia gas.

3. Continue reflux for 18-20 hours during nitrogen flushing.

4. Concentrate the clear, light yellow solution under vacuum 10 (bath temperature 40-50 °) to a foam-like solid. Yields of the silylg compound 89-92 g. (90-94% of theory).

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

B. Preparation of N-hydroxy-5-norbornene-2,3-dicarboximide ester of LM-tt-carbobenzyloxyamino-α-hydroxybutyric acid (NAE) 20 1. Dissolve 21.5 g of L - (-) - γ-carbobenzyloxyamino-α- hydroxybutyric acid (BHBA) in 100 ml of dry acetone at 23 ° and then 15.2 g of N-hydroxy-5-norbornene-2,3-dicarboximide (ΝΒ0ΝΒ). A complete solution is obtained.

2. Add over 30 minutes a solution of 17.48 g of di-cyclohexylcarbodiimide (DCC) in 50 ml of acetone with stirring. The temperature will rise to about 40 ° during the addition of dicyclohexylurea (DCU) precipitation.

3. Stir the slurry for 3-4 hours, allowing the temperature to equalize to 23-25 °. 1

Remove the urea derivative by filtration; wash the cake with 30 ml of acetone. Save the filtrate plus the washings to the acylation steps below.

C. Acylation of 6; -N-Cbz Kana A (silyl) g 25 Dissolve 6'-N-Cbz Kana A (silyl) g isolated under part A, step 4, in 100 ml of dry acetone at 23 -24 °.

2. Slowly add, under good agitation, the NAE solution prepared under Part B over a period of 15 minutes. The temperature will gradually rise to about 40 °. Allow the solution to equilibrate at 23 ° 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. Stir for 15 minutes at 23 °.

15 (Note: An additional layer can be formed. This does not present a problem during reprocessing).

4. Remove acetone under vacuum at a bath temperature of 30-35 °. Transfer the concentrate to an appropriate hydrogenation container (pre-blasted with nitrogen). Add 10 g of 5% palladium-on-carbon catalyst, and hydrogen at 2.72 atm. for 2-3 hours.

5. Filter the mixture through a "Dicalite" layer, wash the hydrogenation vessel and cake with an additional 50 ml of water.

6. Concentrate the filtrate plus the washing liquid to about a three-part volume (50 ml) under vacuum at 40-45 °.

30 7. Check the pH. It should be in the range of 6.9 - 7.2. If not, set with 1 N ammonium hydroxide. Fill the mixture on a "CG-50" (NH4 +) column (6 x 110 cm). 1

Wash the column with 1000 ml of deionized water. Then elute 35 with 0.5-0.6 N ammonium hydroxide using an automatic polarimeter to record the elution progress. The order of elution is as follows: DK 167687 Bl 26

Rest kana A ------> BB-K29 ......> amikacin. No BB-K11 is detected in any of the acylation work-ups. Polyacyl material, i.e. The 1,3-diAHBA analog to channel A is recovered by leaching the column with 3 N ammonium hydroxide.

9. Combine the amikacin fractions and concentrate to 25-30% solids. Dilute with 1 volume of methanol, and pod with amikacrystalline crystal.

10. Slowly add 2 volumes of isopropanol (IPA) over 2 hours with good stirring and crystallize at 23 ° for 6-8 hours.

15 11. Filter off the solid, wash with 50 ml of 1: 1: 2 water / methanol / IPA blend, and finally with 25 ml IPA.

12. Dry under vacuum at 40 ° for 12-16 hours. Yield: 17.3- 19.0 g (38-42%) of amikacin having the following properties: 20

TLC

CHCl 3 - methanol - NH 2 OH - water (1: 4: 2: 1), 5 x 20 cm silica gel pellet veins from Quantum Industries - one zone detected with ninhydrin (RF - 0.4).

25

Specific rotation 23 ° H2 O 0.1 M NH 4 PH 0.1 M H2 SO4 l]] 589 + 101.6 + 101.9 + 103.5 30 C = 1.0% 13. The recovery of BB-K29 in this system was also 39-42%, residual channel A 10-14% and 1,3-diAHBA channel A about 5%, giving a material balance> 95%.

35 DK 167687 B1 27

Example 6

Preparation of Amikacin by Selective N-Acylation of Polytrimethylsilyl Cana A in Anhydrous Acetone 5 I. Summary

Silylation of channel A "base" in acetonitrile using hexa-methyldisilazane (HMDS) gave polytrimethylsilyl-channel A. The degree of silylation is still unknown, but is currently assumed to be channel A (silyl) jQ. Polysilylated channel A is readily soluble in most organic solvents. Acylation with SAE in anhydrous acetone at 23 ° using a 1: 1 molar ratio of SAE to channel A used gave a mixture containing Cbz derivatives of amikacin and BB-K29, usually in the ratio of 2-3 / 1; BB-K6 (about 5-8%), unreacted channel A (15-20%) and some polyacyl material (about 5-10%). As was the case in the previous work on acylation of polytrimethylsilyl-6'-N-carbobenzoxy channel A, no BB-K11 was detected in any of these experiments. Reduction and reprocessing of an acetone acylation mixture followed by chromatography on a "CG-50" (NH 2+) column using 0.5 N ammonium hydroxide yielded isolated crystalline amikacin in the range of 34-39%.

20 28 DK 167687 B1 II. Schemes A.

A HO 1 OH ° H

nn · / oh

H0 - / OH

\ ° l / \ N / · * _ 5 '6 NH „y-rJ X1 5 2 - ~ 0 λ H2N OH l / * 2 Ί-Ν! Ί2

Channel A "base" C18H36 ° 11N4 (484.51) + (CH3) 3 Si-NH-Si (CH3) 3 HMDS (161.4) ck3cn Φ

OR OR

/ OH

V ^ / / ^ \\ / * / G 'NHR

\ ___ f / \ 15 2 ~ j O

RHN> / ^^ Y-nhr u + nh3

R = Si (CH3) 3 (T) Kana A (silyl) ^ Q

C48H116 ° 11N4S: L10 (120o, 35) DK 167687 B1 29 o? H λπ

B. CbzNK (CH9) 9-CH-COOH + HO-N + DCC

BHBA N-HOS (206.3) (253.4) (115.9)

EtOAc N / OH O '$> - I II /

DCU + CbzNH (CH -) - CH-C-O-N Å A

(224.3) f (2) SAE (350.33) / C. Cbz Amikacin (720) +

Cbz BB-K29 + @ + (p-ftcebcne ^^ cbz BB-K6 23 ° I +

Kana A - + H2 Polyacyls (mainly 1.3-5% Pd / C diBHBA-Kana A) Ύ

X V

/ --- A - \

Amikacin + BB-K29 + BB-K6 + 1,3-diAHBA-channel A + channel A

(585.62) (722.76) "CG-50" (NH4 +)

V

Amikacin DK 167687 B1 30 III. Materials Weight g Vol. ml Mol 5 Channel A "base" 50 0.103 HMDS (density 0.774) 86.68 112 0.537

Acetonitrile 600 SAE 35.03 0.10

Acetone 850 "CG-50" (NH ^ 3000)

Methanol on demand IPA on demand IV. Security 15

Kana A "base" - Known drug - usual caution is advised.

Kana A (silyl) ^ - No direct information 20 available, handle with care.

Other Materials - See Example 5.

Process V. Preparation of Kana A (silyl) ^ 1. Suspend 50 g of channel A "base" (KF 2.5 - 3.5%) in 500 ml of acetonitrile (KF <0.01%) . Bring the slurry to reflux (74 °) while maintaining a stream of dry nitrogen through the slurry. 1

Slowly add 112 ml of hexamethyl disilazane (HMDS) slowly over 30 minutes. Complete solution will occur within 4-5 hours of ammonia gas development.

DK 167687 B1 31 3. Continue reflux for 22-26 hours during nitrogen flushing.

4. Concentrate the clear, pale yellow solution under vacuum 5 (40 °) for a syrupy residue. Rinse with an additional 100 ml of acetonitrile and completely dry under high vacuum for 3-6 hours. Yields of whitish amorphous solid are 109-115 g. (90-95% of theory, calculated as channel A (silyl) 10).

B. Preparation of N-hydroxysuccinimide ester of L - (-) - α-carbobenzyloxyamino-α-hydroxybutyric acid (SAE) 1. Dissolve 100 g of L - (-) - γ-benzyloxycarbonylamino-tt-hydroxy acid acid (BHBA) and 45.38 g of N-hydroxysuccinimide (N-HOS) in 1300 ml of ethyl acetate (KF <0.05%) with stirring at 23 ° C.

2. Dissolve 81.29 g of dicyclohexylcarbodiimide (DCC) in 400 ml of ethyl acetate (KF <0.05%) at 23 ° C. Under good stirring, add this solution over 30 minutes to the solution from step 1. The temperature will rise to about 40-42 ° C while simultaneously precipitating dicyclohexylurea (DCU). Stir the slurry 3-4 hours, allowing the temperature to equilibrate to 23 ° C.

3. Filter DCU; wash the cake with 250 ml of ethyl acetate (KF <0.05%). Discard the DCU cake. Save the filtrate and washing fluids.

4. Concentrate the filtrate plus the washings to about 500 ml (in vacuo at 30-35 ° C). Some product will crystallize. 1

Transfer the concentrate to a suitable container and add, with vigorous stirring, 100 ml of heptane. If necessary, add SAE seed crystals. Crystallization will begin almost immediately. Stir the slurry for 30 minutes at 23 ° C.

6. Add 30 ml of heptane over 30 minutes and stir the slurry for 4-5 hours at 23 ° C.

5. 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. The yield is 110.1-131.4 g (80-95%). Mp. 119-120 ° with softening 10 at 114 ° (corr.) TLC - 4 acetone: 12 benzene: 1 CH 2 CO 2 H - detection 1% aqueous kmO4.

Rf - 0.7 for SAE; 0.2 BHBA of 2 x 10 cm-labeled si li keli pi veins from Analtech Inc.

C. Acylation of Kana A (silyl) 1Q

20 "1. Dissolve the channel A (silyl) i0 isolated in section A in step 4 in 500 ml of dry acetone at 23 ° C.

2. Quickly add under good agitation the SAE (35.03 g) prepared as a 10% solution in dry 25 ~ acetone over 5-10 minutes. The temperature will rise about 5 °. Allow the solution to equilibrate to 23 ° and continue stirring for 18-20 hours.

»3. Dilute the light orange clear solution with 400 ml of water, 30 and lower the pH (7.0-7.5) to 2.2-2.5 with 3N hydrochloric acid. The clear solution is now stirred at 23 ° for 15-30 minutes. 1

Acetone is removed under vacuum at a bath temperature of 30-35 ° C (a small amount of material can be separated at this time but presents no problem). Transfer the concentrate to a suitable hydrogenation vessel. Add 10 g of palladium-on-carbon catalyst and hydrogenate at 3.40 atm. for 2-3 hours.

5. Filter the mixture through a "Dicalite" layer and wash the hydrogenation vessel and cake with an additional 2 x 50 ml of water.

6. Concentrate the filtrate plus the washings to about one-third volume (150-165 ml) under vacuum at 40-45 °.

7. The pH value at this point is in the range 6.0-7.0.

The mixture is poured onto a "CG-50" (NH 2 -h) column (6 x 110 cm).

8. Wash the column with 1000 ml of deionized water. Elutes with 0.5 N

15 ammonium hydroxide using an automatic polarimeter to monitor the elution progress. The order of elution is as follows:

Rest channel A ---> BB-K6 ---> BB-K29 ---> amikacin.

20 "- No BB-K11 was detected in any of the experiments.

9. Combine the amikacin fractions and concentrate to 25-30% solids. Dilute with 1 volume of methanol and pod with ami-cocaine crystal clay.

10. Slowly add 2 volumes of IPA under good stirring over 2 hours and crystallize at 23 ° C for 6-8 hours.

11. Filter the solid, wash with 35 ml of 1: 1: 2 water / methanol / - 30 IPA and finally with 35 ml of IPA.

12. Dry in a vacuum oven at 40 ° for 16-24 hours. Yield: 19.91-22.84 g (34-39%) IR, PMR and CMR spectral data, like specific rotation, were completely consistent with the desired structure.

DK 167687 B1 34 TLC system CHCl 3 / methanol / NH 2 OH / water (1: 4: 2: 1) 5 x 20 cm sieve in quartz veins from Quantum Industries - 1 zone amikacin with Rf -0.4 (ninhy -drinpåvisning).

5

Example 7

Preparation of amikacin by acylation of poly (trimethylsilyl) -67-N-Cbz channel A in tetrahydrofuran with the mixed acid anhydride of pi val in acetic acid and BHBA

A. Preparation of mixed anhydride BHBA (5.066 g, 20.0 mmol), BSA (4.068 g, 20.0 mmol) and triethylamine (2.111 g, 22.0 mmol) were dissolved in 200 ml. ml of sieve-dried tetrahydrofuran. The solution was refluxed for 2 1/4 hours and then cooled to -110 ° C.

Pivaloyl chloride (2.412 g, 20.0 mmol) was added over 2-3 minutes with stirring and stirring was continued for 2 hours at -10 ° C. The temperature was then allowed to rise to 23 ° C.

B. Acylation of Poly (trimeth, ylsilyll-B'-N-Cbz channel A

20 '' PDly (trimethylsilyl) -6'-N-Cbz channel A prepared as in Example 1 (5.454 g, 4.97 m.mol, calculated as 6'-Cbz channel A (silyl) g) was dissolved in 50 ml dry (molecular sieve) tetrahydrofuran at 23 ° C. Half of the mixed anhydride (10.0 mmol) solution prepared in step A above was added over 20 minutes with stirring and stirring was continued for 7 days.

Water (100 ml) was then added to the reaction mixture and pH

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

The aqueous layer was then hydrogenated in a Parr apparatus at 3.40 atm H 2 pressure for 2 hours using 0.5 g of 10% Pd-on-carbon catalyst. The material was filtered and combined filtrate and washings were determined against E. coli to contain a 31.2% yield of amikacin.

DK 167687 B1 35

Amikacin / BB-K29 ratios around 9-10 / 1; traces of polyacyl and unreacted channel A present.

Example 8

Effect of Water on the Preparation of Amikacin by Acylation of Poly (trimethylsilyl) Can A in Acetone Solution at 23 ° C

A. Anhydrous solvent

Poly (trimethylsilyl) can A prepared as in Example 3 (2.40 g, 10 2.0 m mol, calculated as channel A (silyl) 2) was dissolved in 20 ml of acetone which had been dried with a molecular sieve. The solution was stirred at 23 ° C and a solution of SAE (0.701 g, 2.0 m.mol) in 10 ml sieve-dried acetone was added over 10 seconds. Stirring was continued at 23 ° C for 22 hours. Water (50 mL) was added and pH (7.5) adjusted to 2.5. The acetone 15 was evaporated in vacuo at 40 ° C and the aqueous solution was then reduced at a Hg pressure of 3.47 atm. at 23 ° C for 2 hours using 1.0 g of 10% Pd-on-carbon as catalyst. Microbiological analysis showed a 31.24% yield of amikacin.

B. Solvent added water

Step A above was repeated except that 1.0 ml (56 mmol) of water was added to the poly (trimethylsilyl) kana A solution and stirred for 15 minutes prior to acylation with SAE. Mi crobi ological analysis showed a 33.80¾ yield of amikacin.

25

Example 9

Preparation of amikacin by acylation of poly (trimethylsilyl) β'-N-Cbz channel A in acetone with the mixed anhydride of BHBA and isobutylcarboxylic acid 30 A. Preparation of mixed anhydride BHBA (1.267 g, 5.0 m.mol ) and N-trimethylsilylacetamide (MSA) (1.313 g, 10.0 mmol) in 20 ml sieve-dried acetone was stirred at 23 ° C, and triethylamine (TEA) (0.70 ml, 5.0 mmol) was added. The mixture was refluxed under Ng atmosphere for 2¼ hours. The mixture was cooled at -20 ° C and isobutyl chloroformate (0.751 g, 0-713 ml, 5.50 m.mol) was added. Triethyl, hydrochloride immediately began to secrete. The mixture was stirred for 1 hour at -20 ° C.

DK 167687 B1 36 B. Acylation

Poly (trimethylsilyl) -6'-N-Cbz channel A prepared as in Example 1 (6.215 g, 4.9 mole, calculated as the (silyl) g compound) was dissolved in 5 ml sieve dried acetone with stirring at 23 ° C. The solution was cooled to -20 ° C and the cold solution of mixed anhydride from Step A was slowly added over 30 minutes. The reaction mixture was stirred for an additional 1% hour at -20 ° C and then for 17 hours at 23 ° C. The reaction mixture was then poured into 150 ml of water at 23 ° C with stirring, pH 10 (7.75) was adjusted to 2.5 with 3 N HCl and stirring was continued for 15 minutes. Acetone was then evaporated in vacuo at 40 ° C. An aliquot of the resulting aqueous solution was subjected to analysis by HPLC. The resulting curve showed a 34.33% yield of di-Cbz amikacin.

Most of the aqueous solution was reduced at 3.40 atm.

15 pressure at 23 ° C for 3 1/4 hours using 2.0 g Pd / C catalyst. The catalyst was removed by filtration and combined filtrate and washings were determined by microbial analysis against E. coli to contain a 35.0% yield of amikacin.

Example 10

Preparation of amikacin by acylation of poly (tri methylsilyl 1-e'-N-Cbz channel A in 3-pentanone

Poly (trimethylsilyl) -6'-N-Cbz kana prepared as in Example 1 (30 g, 23.65 mmol, calculated as 6'-N-Cbz kana A (silyl) g) dissolved in 100 ml 25 sieve dried 3 -pentanone was stirred at 23 ° C and NAE (26.02 mmol, 10% excess) was added over 40 minutes. Stirring was continued for 113 hours at 23 ° C and the mixture was then added to 250 ml of water without vigorous stirring. The pH (7.3) was adjusted to 2.5 with 3N HCl, the mixture was stirred for an additional 30 minutes and the 3-pentanone was evaporated in vacuo at 40 ° C.

The aqueous solution was extracted with 4 x 100 ml of ethyl acetate. An aliquot of the aqueous solution was then subjected to HPLC analysis.

The resulting curve showed a 46.12% yield of di-Cbz amikacin.

Most of the aqueous reaction mixture was reduced by 3.47 atm. H2 ° pressure at 23 ° C for 2 ¼ hours using 3.0 g of 10% Pd / C catalyst. Mi crobi ological analysis of an aliquot of combined filtrate and washings showed a 40.24% yield of amikacin. The majority of the reduced aqueous reaction mixture was then concentrated in vacuo at 40 ° C to about 100 ml and fractionated on a "CG-50" (NH 2+) ion exchange column (about 10 cm x about 120 cm, containing about 10 liters of resin). The aqueous solution was poured onto the column, the column was washed with 5 liters of water, and the material was eluted with 0.5 N NH 2 OH (followed by 3 N NH 4 OH to elute polyacylated products). Polarimetry of the fractions showed a 42.7% yield of amikacin, a 12.0% yield of unreacted quamycin A, a 12.4% yield of polyacylated material and a 23.2% yield of BB-K29.

Example 11 10 Preparation of amikacin by acylation of poly (trimethylsilyl) -6'-N-Cbz channel A in anhydrous cyclohexanone for varying times A. Poly (trimethylsilyl) -6'-N-Cbz channel A prepared as in Example 1 (2.537 g, 2.0 mmol, calculated as 6'-N-Cbz channel A (silyl) g) in 300 ml of dry cyclohexanone was acylated for 20 hours at 23 ° C with a NAE solution in dry cyclohexanone (10 , 8 ml of a 0.1944 m.mol / ml solution, 2.10 m.mol). The reaction mixture was then added to 150 ml of water with stirring and the pH (5.6) adjusted to 2.5 with 3 N HCl. The cyclohexanone was evaporated in vacuo at 40 ° C and an aliquot of the remaining aqueous phase was taken for analysis by HPLC.

The majority of the aqueous phase was reduced below 3.40 atm. H₂ pressure for 3 hours at 23 ° C using 1.0 g of 10% Pd / C catalyst. The catalyst was removed by filtration and combined filtrate and washings were microbiologically analyzed for amikacin.

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

Yields 30 HPLC analysis Mi crobi ological analysis amikacin (di-Cbz amikacin) Turbidimetric PIade

Turnover A 49.18% 42.87% 39.16%

Turnover B 56.17% 55.39% 38.45% 35 38 DK 167687 B1

Example 12

Preparation of amikacin by acylation of polytrimethylsilyl) -6'-N-Cbz channel A in anhydrous tetrahydrofuran for varying periods of 5 A. Example 11A was repeated except that dry tetrahydrofuran was used as a solvent instead of dry cyclohexanone.

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

Yields HPLC analysis Hi crobiological analysis amikacin (di-Cbz amikacin) Turbidimetric Plate 15

Turnover A 29.27% 28.34% 28.18%

Revenue B 33.39% 21.52% 28.63%

Example 13 Preparation of amikacin by acylation of poly (trimethylsily1) 6'-N-Cbz channel A in anhydrous dioxane for varying time A. Example 11A was repeated except that the acylation was continued for 44 hours using dry dioxane as solvent.

Example 11B was repeated except that the acylation was continued for 18 ¼ hours using dry dioxane as the solvent.

Yields 30 HPLC analysis Mi crobi ological analysis amikacin (di-Cbz amikacin) Turbidimetric PIade

Turnover A 39.18% 43.27% 33.36%

Revenue B 42.82% 22.55% 33.37% DK 167687 B1 39

Example 14

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

To a stirred solution of poly (trimethylsilyl) -6'-N-Cbz channel A 5 prepared as in Example 1 (2.537 g, 2.0 m.mol, calculated as 6'-N-Cbz channel A (silyl) g) in 32 ml sieve-dried diethyl ketone at 75 ° C was added a solution of NAE (10.8 ml with 0.1944 m.mol / ml diethyl ketone, 2.10 m.mol) over 15 minutes. Stirring was continued at 75 ° C for an additional 3 hours, after which the mixture was poured into 150 ml of water. The pH was adjusted to 10 to 2.8 with 3N HCl and the ethyl ketone was evaporated in vacuo at 40 ° C. HPLC analysis of an aqueous phase aliquot showed a 39.18% yield of di-Cbz amikacin.

The majority of the aqueous phase was reduced below 3.39 atm. H2 pressure for 3 1/4 hours at 23 ° C using 1.0 g Pd / C catalyst. The catalyst was removed by filtration, and combined filtrate and washings were microbiologically analyzed for amikacin. Turbidimetric analysis showed 27.84% yield and plate analysis showed 28.6% yield.

Example 15 20 Preparation of Amikacin by Acylation of Poly (Tri-methyl Si 1, Y1) -Can A with NAE at 0-5 ° after Back Hydrolyzing with Water A. Silylation of Kanamycin A Using HMDS with THC5 as Catalyst 25 Kanamycin A (10 g of purity 97.6%, 20.14 mmol) in 100 ml of sieve-dried acetonitrile was brought to reflux under nitrogen atmosphere. A mixture of HMDS (22.76 g, 141 moles, 7 moles per mole of kanamycin A) and TMCS (1 mL, 0.856 g, 7.88 m moles) was added to the reflux reaction mixture over 10 minutes. Reflux was continued for 4/4 hours and then the mixture was cooled, concentrated in vacuo to a yellow viscous syrup and dried under high vacuum for 2 hours. The yield of product was 23.8 g (97.9%, calculated as kanamycin A (silyl) io).

B. Acylation 35 Poly (trimethylsilyl) -canamycin A (23.8 g, 20.14 m mol) prepared under step A above was dissolved in 250 ml sieve-dried acetone at 23 ° and then cooled to 0-5 °. Water (3.63 ml, 201.4 mole, 10 moles per mole of polysilylated kanamycin A) was added with stirring and the mixture was left to stand under moderate vacuum for 30 minutes. NAE (19,133 moles, 0.95 moles per mole of polysilylated kanamycin A) in 108.3 ml of acetone was then added in less than 1 minute. The mixture was stirred at 0-5 ° for 1 hour, diluted with water, the pH adjusted to 2.5, and the acetone then removed in vacuo. The aqueous solution was then reduced at 3.40 atm. Expression at 23 ° for 2 ½ hours using 2.0 g of 10% Pd / C as catalyst. The reduced reaction mixture was filtered through "Dicalite", concentrated to ca. 100 ml in vacuo at 40 ° and was then poured onto a "CG-50" (NH 2+) column (6 liters of resin, 5 x 100 cm). It was washed with 10 water and then eluted with 0.6 N - 1.0 N - 3 N NH 2 OH. 60.25% amikacin, 4.37% BB-K6, 4.35% BB-K29, 26.47% kanamycin A and 2.18% polyacyl compound were obtained.

Example 16 Preparation of amikacin by acylation of poly (trimethylsily1) -6'-N-Cbz channel A with SAE at 0-5 ° after back methanolysis.

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

6'-N-Cbz kanamycin A (20.0 g, 32.4 mmol) in 200 ml sieve-dried acetonitrile was brought to reflux under nitrogen atmosphere. HMDS (47.3 mL, 226.8 mmol, 7 mol per mol of 6'-N-Cbz channel A) was added over 10 minutes and reflux continued for 20 hours. The mixture was then cooled, concentrated in vacuo and dried under high vacuum for 2 hours to give 39.1 g of white amorphous solid (95.4% yield, calculated as 25'-N-Cbz channel A (silyl) g).

B. Acylation

Poly (trimethylsilyl) -6'-N-Cbz channel A (39.1 g, 32.4 mmol) prepared under step A above was dissolved in 400 ml of dry acetone with stirring at 23 °. Methanol (6.6 mL, 162 mole mol, 5 mole per mole of polysilylated 6'-N-Cbz channel A) was added and the mixture was stirred at 23 ° for 1 hour under a strong stream of nitrogen. The mixture was cooled to 0-5 ° and a solution of SAE (11.35 g, 32.4 mmol) in 120 ml of precooled dry acetone was added. The mixture was stirred for an additional 3 hours at 0-5 ° and then placed in a 4 ° cold room for 1 week. Water (300 ml) was added, the pH adjusted to 2.0; the mixture was stirred for 1 hour and the acetone was then evaporated in vacuo. The resulting aqueous solution was reduced at 3.67 atm. H2 ~ pressure for 17 hours at 23 ° using 3.0 g of 10% Pd / C as a catalyst. It was then filtered through "Dicalite", concentrated in vacuo to 75-100 ml, loaded on a "CG-50" (NH 4 +) column and eluted with water and 0.6 N NH 4 OH, 52.52% amikacin was obtained. 14.5% BB-K29, 19.6% kanamycin A and 1.71% polyacyl compound ser.

5

Example 17

Preparation of amikacin by acylation of poly (trimethylsilyl) can A with SAE at 0-5 ° after back hydrolysis with water 10 A. Silylation of kanamycin A with TMCS in acetonitrile using tetramethylguanidine as an acid acceptor

Kanamycin A (4.88 g, 10.07 mmol) was suspended in 100 ml screen-dried acetonitrile with stirring at 23 °. To the stirred suspension was added tetramethylguanidine (TMG) (16.234 g, 140.98 m.mol, 14 moles per mole of kanamycin A). The mixture was heated to reflux and TMCS (15.32 g, 140.98 m.mol, 14 moles per mole kanamycin A) was added over 15 minutes.

A white precipitate of TMG.HC1 formed after ca. half of TMCS had been added. The mixture was cooled to room temperature, concentrated to a sticky residue and dried under high vacuum for 2 hours. The solid was triturated with dry THF (100 mL) and the insoluble TMG.HCl was filtered off and washed with 5 x 20 mL portions of THF. Combined filtrate and washings were concentrated in vacuo at 40 ° to a sticky residue and dried. under high vacuum for 2 hours 10.64 g of a light cream sticky residue (87.6% yield, calculated as kanamycin A 25 (silyl) 10) was obtained.

B. Acylation

Poly (trimethylsilyl) -canamycin A (10.64 g, 10.0 µmol) prepared under step A above was dissolved in 110 ml sieve-dried acetone with stirring at 23 ° and the solution cooled to 0-5 °. Water (1.81 ml, 100.7 mole, 10 moles per mole of polysilylated channel A) was added and the solution was stirred for 30 minutes under moderate vacuum. SAE (3.70 g, 10.57 m.mol, 5% excess) in 40 ml precool a dry acetone was added in less than 1 minute and the mixture was stirred for 1 hour. The mixture was worked up by the general procedure of Example 16B to produce ca.

50% amikacin, approx. 10% BB-K29, 5-8% BB-K6, approx. 20% kanamycin A and 5-8% polyacyl compound.

DK 167687 B1 42

Example 18

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

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

Example 19

Preparation of poly (triethylsilyl) -canamycin A using triethylchlorosilane with triethylamine as an acid acceptor

Kanamycin A (5.0 g of 97.6% purity, 10.07 m.mol) was suspended in 100 ml sieve-dried acetonitrile at 23 °. Triethylamine (TEA) (33.8 mL, 24.5 g, 241.7 mmol) was added and the suspension was refluxed.

A solution of trichloroethyl silane (23.7 mL, 21.3 g, 140.98 mmol) in 20 mL of dry acetonitrile was added over 20 minutes. Reflux was continued for another 7 hours and the mixture was cooled to room temperature, after which long fine needles of TEA.HC1 were excreted. The mixture was left at room temperature for 16 hours, concentrated in vacuo at 40 ° to a sticky solid, and dried for 2 hours under high vacuum to a deep orange sticky solid. The solid was triturated with 100 ml of dry THF at 23 ° and the insoluble TEA.HC1 was removed, washed with 5 x 20 ml of THF and dried to give 16.0 g of TEA.HC1. Combined filtrate and washings were concentrated in vacuo to a solid and dried under high vacuum for 2 hours. 19.3 g of poly (triethylsilyl) -canamycin A 30 was obtained as a deep orange viscous syrup.

Example 20

Preparation of poly (trimethylsilyl) kanamycin A using bis-trimethylsilylurea Kanamycin A (10.0 g of 99.7% purity, 20.58 mmol) was suspended in 200 ml sieve-dried acetonitrile with stirring at 23 °. To the suspension was added bis-trimethylsilylurea (BSU) (29.45 g, 144.01 m.mol, 7 moles per mole kanamycin) and the mixture was brought to reflux under nitrogen atmosphere. Reflux was continued for 17 hours and the reaction mixture was then cooled to room temperature. A small amount of insoluble material was removed by filtration, washed with 3 x 10 ml portions of acetonitrile and dried (1.181 g). Infrared analysis showed that this was BSU plus a small amount of unreacted kanamycin A. Combined filtrate and washings were cooled to 4 ° for 16 hours. Additional solid was separated, removed as above (7.8 g) and shown to be BSU plus urea by infrared analysis. The light yellow filtrate and washings were concentrated in vacuo at 40 ° and dried under high vacuum to give 27.0 g of a white solid which was partly sticky and partly fine needle-like crystals. The solid was treated with 150 ml of hepatane at 23 °, the insoluble portion was removed by filtration, washed with 2 x 50 ml portions of heptane and dried to give 6.0 g of white needles (shown to be BSU plus urea by infrared analysis) ). Combined 15 filtrate and washings were concentrated in vacuo at 40 ° and dried under high vacuum for 2 hours to give 20.4 g of white needles whose infrared spectrum was typical of polysilylated kanamycin A. Calculations showed that the product contained, on average, 7.22 trimethylsilyl groups. .

Example 21

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

A. Preparation of Per (trimethylsilyl) -canamycin A

Kanamycin A (10.0 g, 20.639 mmol) was suspended in 100 ml screen-dried acetonitrile with stirring at 23 °. The suspension was refluxed under a stream of nitrogen and HMDS (23.332 g, 144, -5 m mol, 7 moles per mole kanamycin A) was added over 10 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. 24.3 g of a white sticky residue was obtained (92.1% yield calculated as kanamycin A

(Silyl) n).

B. Acylation

Per (trimethylsilyl) -canamycin A (24.3 g) prepared under step A

above was dissolved in 240 ml of screen-dried acetone with stirring at 23 °.

To this solution was added 1,3-butanediol (9.25 ml, 103.2 m mol, 5 moles per mole per (trimethylsilyl) -canamycin A. The mixture was stirred at 23 ° for 2 hours under a stream of nitrogen. and then cooled to 0-5 °. SAE (7.23 g, 20.64 mmol) in 70 ml of precooled acetone was added over about 1 minute. The mixture was stirred at 0-5 ° for 3 hours and was then left to stand in a 4 ° cold room for about 16 hours, water (200 ml) was added, the pH was adjusted to 2.5, and the clear yellow solution was stirred at 23 ° for 30 minutes. The acetone was evaporated in vacuo and The aqueous solution was reduced at 3.74 atm. H 2 "pressure at 23 ° for 2 hours using 3.0 g of 10% Pd-on-carbon as catalyst. The reduced solution was filtered through" Dicalite "and chromatographed as in Example 16 B to produce 47.50% amika-10 cin, 5.87% BB-K29, 7.32% BB-K6, 24.26% kanamycin A and 7.41% polyacyl compound.

Example 22

Preparation of amikacin by acylation of poly (trimethylsily1) kanamycin 15 A prepared in THF using SAE with sulphinic acid catalyst To a reflux mixture of kanamycin A (5.0 g, 10.32 m.mol) in 50 To sieve dried tetrahydrofuran (THF) was added sulfamic acid (100 mg) and HMDS (12.32 g, 76.33 mmol). The mixture was refluxed for 18 hours, with complete dissolution occurring after 6 hours. The solution was cooled to 23 °, treated with 0.1 ml of water and maintained at 23 ° for 30 minutes. A solution of SAE (3.61 g, 10.3 mmol) in 36 ml of THF was added over 30 minutes. After stirring for 3 hours, the mixture was diluted with 100 ml of water and the pH was adjusted to 2.2 with 10% HgSO 4. It was stirred for 30 minutes at 23 ° and then concentrated in vacuo to remove 25 THF. The resulting aqueous solution was reduced at 3.40 atm. W 2 pressure for 2 hours at 23 ° using 10% Pd-on-carbon as catalyst. The reduced solution was filtered through "Dicalite" and the solid washed with water. Combined filtrate and washings (150 ml) were determined by microbiological analysis against E. coli to contain 30 1225 mcg / ml (31.5% activity yield) of amikacin.

Example 23

Preparation of amikacin by acylation of poly (trimethylsilyl) -canamycin A with the N-hydroxysuccinimide ester of di-carbobenzyloxy AHBA 35 A. Preparation of di-carbobenzyloxy L-H-ft-amino-α-hydroxybutyric acid N-hydroxysuccinimide ester

Di-carbobenzyloxy L - (-) - α-amino-α-hydroxybutyric acid (8 g, 20.65 DK 167687 B1 45 m mol) and N-hydroxysuccinimide (2.37 g, 20.65 mmol) were dissolved in 50 ml of dry acetone at 23 °. Dicyclohexylcarbodiimide (4.25 g, 20.65 mmol) dissolved in 20 ml of dry acetone was added and the whole was stirred at 23 ° for 2 hours. Dicyclohexylurea was filtered off, the filter cake washed with 10 ml of dry acetone and the filtrate and washings combined.

B. Acylation

Poly (trimethylsilyl) kanamycin A prepared according to the general procedure of Example 21 from 10.0 g (20.639 m.mol) of 10 kanamycin A was dissolved in 100 ml of dry acetone. The solution was cooled to 0-5 °, 3.7 ml of deionized water was added and the solution was stirred at 0-5 ° for 30 minutes under moderate vacuum.

To this solution was added the solution prepared in step A of the di-Cbz-blocked acylating agent and the mixture was stirred at 0-15 ° for 30 minutes. The mixture was diluted with water, the pH adjusted to 2.2 and the acetone removed in vacuo. The aqueous solution was reduced by the general procedure of Example 22 and then filtered through "Dicalite". Chromatography showed 40-45% amikacin, ca. 10% BB-K29, trace amounts of BB-K6, approx. 30% kanamycin A and a small amount of polyacyl compounds.

Example 24

Preparation of Poly (trimethylsilyl) -kanamycin A using HMDS with imidazole as catalyst 25 Kanamycin A (11 g, 22.7 m.mol) and 100 mg of imidazole were heated to reflux in 100 ml sieve-dried acetonitrile under a stream of nitrogen. HMDS (18.48 g, 114.5 mole, 5 moles per mole of kanamycin A was added over 30 minutes and the mixture was refluxed for 20 hours. Complete solution occurred at about 2 µ h. The solution was cooled to 23 ° and the solvent was removed in vacuo leaving 21.6 g of poly (trimethylsilyl) kanamycin 'A as a foamy residue (93.1% yield calculated as kanamycin (silyl) n).

Example 25

Preparation of 1-N-rL - (-) - γ-amino-o: -hydroxybutyryl 1 kanamycin B (BB-K26) by acylation of polytrimethylsilyl) -canamycin B with SAE

DK 167687 B1 46 A. Preparation of Poly y (Tri Methyl s in Lyl) -Canamycin B Using HMDS with TMCS Catalyst

Kanamycin B (25 g, 51.7 m mol) in 250 ml sieve-dried acetonitrile was heated to reflux under a stream of nitrogen. HMDS (62.3 g, 385.81 mole, 7.5 mole per mole of kanalycin B) was added over 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 removed in vacuo at 60 ° and the oily residue was maintained at 60 ° under high vacuum for 3 hours. 53.0 g of poly (trimethylsilyl) kanamycin B (85.2% yield calculated as kanamycin B (s in lyl) +) were obtained.

B. Acylation

The poly (trimethylsilyl) kanamycin B (53.0 g) prepared in step A above was dissolved in 500 ml dry acetone at 0-5 °, methanol (20.9 15 ml) was added and the mixture was stirred in vacuo for 30 minutes at room temperature. 0-5.

A solution of SAE (18.1 g, 51.67 m.mol) in 200 ml of cold acetone was added over less than 1 minute and the mixture was stirred for 30 minutes at 0-5 °. The mixture was worked up according to the general procedure of Example 22 and then loaded onto a column of "CG-20 50" (TIH4 +) (8 x 120 cm). It was eluted with an NH 1 OH 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 were obtained. -N-acylated kanamycin B (BB-K46) 14.63% kanamycin B and a small amount of polyacylated kanamycin B

Example 26

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

Kanamycin A (19.5 g, 40.246 mmol) and kanamycin Ά sulfate (0.5 g, 0.858 mmol) [total = 20.0 g, 41.0 mmol] in 200 ml sieve-dried acetone-30 trill was brought to reflux. HMDS (60.3ml, 287.7mol, 7mol per mole of kanemycin A) was slowly added and the mixture was refluxed for 28 hours.

It was then evaporated to dryness in a rotary evaporator and dried under steam injector vacuum. 47.5 g of poly (trimethylsilyl) kanamycin A was obtained as a pale yellow oil (95.82% yield calculated as kanamycin A 35 (silyl) 10).

DK 167687 Pg 47

Example 27

Preparation of Amikacin by Acylation of Pol, γ (Trimethylsily1) -Canamycin A with N-Trifluoroacetyl-Blocked AHBA N-h, O-Hydroxysuccinimide Ester 5 A. Preparation of N-Trifluoroacetyl AHBA and Conversion to Its N-Hydroxysuccinimide

To a suspension of AHBA (5.0 g, 42 mmol) in 100 ml of THF was added trifluoroacetic anhydride (40 g, 191 mmol) with stirring over 10 minutes. The solution was stirred for 18 hours at 23 ° and then concentrated to dryness in vacuo at 50 °. 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 of H 2 O. The aqueous solution was extracted with 3 x 50 ml portions of MIBK and the extract was concentrated after drying over Na 2 SO 4 to an oil. Solvent traces were removed by addition and distillation of 4 ml of water. On standing, the oil was changed to a waxy crystalline solid (2.5 g, 28% yield).

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

B. Acylation

To a solution of poly (trimethylsilyl) kanamydn A prepared as in Example 26 (11.31 mmol) in 54 ml of acetone was added 2.0 ml (113.4 mmol) of water and the mixture was stirred in vacuo at 0-5 ° for 30 minutes. The N-30 hydroxysuccinimide ester of N-trifluoroacetyl AHBA prepared under Step A above (11.31 m.mol) was added to the mixture and then maintained at 5 ° for 1 hour. The pH was then adjusted to approx. 2.0 with 20% H 2 SO 4, the mixture was stirred for 30 minutes, and the pH was then raised to ca. 6.0 with NH 3 OH. The mixture was then evaporated to dryness in a rotary evaporator to give 14.4 g of a tacky off-white solid. The solid was dissolved in 100 ml of water, the pH was raised from 5.5 to 11.0 with 10 N NH 2 OH, and the solution was heated in an oil bath at 70 ° for 1 hour. The pH (9.5) was lowered to 7.0 with HCl, the solution fine filtered to remove a small amount of insoluble constituents and the filter washed with water. Combined filtrate and washings (188 ml) were added to a "CG-50" (NH 2+) column (8 x 90 cm), washed with 2 liters of water and eluted with an NH 2 OH gradient (0.6 N - 1 , 0 N - concentrated). 28.9% amikacin, 5.0% BB-K6, 5.7% BB-K29, 43.8% kanamycin A, 3.25% polyacyl compound plus 14.3% of an unknown material were obtained. , which was in the first fraction from the column.

Example 28 Preparation of Amikacin by Acylation of Poly (Trimethylsilyl) -Canamycin A with t-Butyloxycarbonyl-Blocked AHBA N-Hydroxysuccinimide Ester A. Preparation of t-BOC AHBA and Conversion to Its N-Hydroxysuccinimide Ester A solution of AHBA (5.0 g, 42 mMol) in 100 ml of water and 20 ml of ace tone were adjusted to pH 10 with 10 N NaOH. Over 3-4 minutes, 11.6 g (53 mmol) of di-t-butyl dicarbonate) was added and the solution was stirred for 35 minutes keeping the pH of 10 by periodically adding 10 N NaOH. The acetone was removed in vacuo and the aqueous phase was washed with 4'0'ml ethyl acetate. The pH of the aqueous solution was lowered to 2.0 with 3 N HCl and the solution was then extracted with 3 x 30 ml MIBK. The combined MIBK extracts were dried over Na 2 SO 4 and concentrated to a clear oily residue (8.2 g, 89%).

t-BOC-AHBA (4.25 g, 19.4 mmol) was dissolved in 50 ml of acetone and N-hydroxysuccinimide (2.23 g, 19.4 mmol) was added. A solution of dicyclohexylcarbodiimide (4.00 g, 19.4 mmol) in 20 ml of acetone was added slowly and the mixture was stirred for 2 hours at 23 °. The precipitated di-cyclohexylurea was removed by filtration and washed with a small amount of acetone. Combined filtrate and washings (a solution of the N-30 hydroxysuccinimide ester of t-BOC-AHBA) were used in the next step without isolation.

B. Acylation

To a solution of poly (trimethylsilyl) -canamycin A prepared 35 as in Example 26 (41.28 mmol) in 94 ml of acetone was added 3.5 ml (194 mmol) of water and the mixture was stirred in vacuo at 0 DEG. 5 ° for 30 minutes. The N-hydroxysuccinimide ester of t-BOC-AHBA prepared under step A above (19.4 moles) was added and the mixture was allowed to stand at 5 ° for 1 hour. Water (200 DK 167687 B1 49

ml) was added and the pH (7.0) was lowered to 2.0 by 20% 2 SO 2. After stirring for 30 minutes, the pH was raised to approx. 6.0 with NH 2 OH and the mixture was evaporated to dryness in vacuo to give 36.3 g of a golden oil. The oil was dissolved in 200 ml of tri-fluoroacetic acid, allowed to stand for 15 minutes and evaporated to dryness in a rotary evaporator. The oil was washed with water and the water flash evaporated. Concentrated NH 4 OH was added to pH

6.0 and flash evaporated. The resulting solid was dissolved in water, filtered and the filter washed with water. Combined filtrate and washings (259 ml) were charged to a "CG-50" (NH 2+) column (8 x 92 cm), washed with 4 liters of water and eluted with an NH 2 OH gradient (0.6 N - 1.0 N concentrated). 40.32% amikacin, 4.58% BB-K6, 8.32% BB-K29, 30.50% kanamycin A and 7.43% polyacyl compound were obtained.

EXAMPLE 29 The general procedure of Example 1 is repeated except that the 6'-N-carbobenzyloxycanamycin A used therein is replaced by an equivolar amount of 6'-N-carbobenzyloxycanamycin B, thereby producing a 1N- [L- ( -) - γ-amino-Q1-hydroxybutyryl] -canamycin B.

Example 30

The general procedure of Example 1 is repeated except that the L - (-) - γ-benzyloxycarbonylamino-α-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester used is replaced by L - (-) respectively. - N -Benzyloxycarbonylamino-α-hydroxypropionic acid N-hydroxy-5-norbornene-2,3-dicarboximide ester and L - (-) - <$ - benzyloxycarbonylamirro-α-hydroxyvaleric acid N-hydroxy-5-norbornene-2 , 3-dicarboximide ester, thereby producing 1N- [L - (-) - / J-amino-α-hydroxypropionyl] kanamycin A and 1N- [L - (-) - 5-amino-o: -hydroxyvaleryl ] kanamycin A.

Example 31

The general procedure of Example 25 is repeated except that instead of the L - (-) - γ-benzyloxycarbonyl amino-α-hydroxybutyric acid N-hydroxyester used L - (-) - / J-benzyl oxy-carbonylamino, respectively -α-hydroxypropionic acid N-hydroxysuccinimide ester and L - (-) - 5-35 benzyloxycarbonylamino-α-hydroxyvaleric acid N-hydroxysuccinimide ester, thereby producing 1N- [L - (-) - / J-amino-α-hydroxypropionyl] kanamycin respectively B and 1N- [L - (-) - 5-anrino-α-hydroxyvaleryl] kanamycin B.

Claims (6)

A derivative of kanamycin A or B for use in the preparation of 1-N- [a) -amino-α-hydroxyalkanoyl] kanamycin A or B of the general formula (I) CH.NH. EO- ^ i 2 2 / Λ NIS 10. ij ΧΛ__ NH HO- ^ CH, OH / C = 0 xXy * "/ no-in 15 \ ^ - 0X / (έκ.) \ __ \ / l 2 n ^ Wherein R is OH or NH 2 and n is an integer from 0 to 2, or a non-toxic, pharmaceutically acceptable acid addition salt hence, the CHARACTERISTICS of the derivative having the general formula (Γ) 25 \ _ ° \ OR * £ r! '' '' - A. NHR '° oh ^ Vichr · (I1) R' O—. CH.OR ' Wherein Z is -O- or -NH-, R 'is H or a silyl group of formula 35 wherein R5, R6 and R 7 represents hydrogen, halogen, (lower) alkyl, halogen (lower) alkyl or phenyl, wherein at least one of the groups R 5, R 6 and R 7 does not represent halogen or hydrogen, at least two and at most ten R 'groups are silyl groups, R "is R 'or a blocking group other than a silyl group selected from alkoxycarbonyl, aralkoxycarbonyl, cycloalkyloxy-carbonyl, haloalkoxycarbonyl, halomethylcarbonyl, acyl, o-nitr o-phenylthio and trityl, however, R 'is not a silyl group when all R' groups are silyl groups. 2. A derivative according to claim 1, characterized in that the silyl groups are trimethylsilyl. 3. A derivative of kanamycin A according to claim 1 or 2, characterized in that it contains an average number of silyl groups per molecule of 4 15 to 8. 4. A derivative of kanamycin A according to claim 1 or 2, characterized in that it contains a blocking group other than silyl on the 6'-amino group and an average number of silyl groups per molecule of 20. to 7. 5. A derivative of kanamycin A according to any one of the preceding claims, characterized in that the blocking group of the 6 'amino group is selected from groups of the formulas: 25 * CH-, O O Γ \ -4. -3--1 '^) LJ' <X3 30 r2 / O * (Λ- * O'- DK 167687 B1 O in. Li XC-CH ^ -OC- and in 5. c / 1 2 wherein R and R are the same or different and each represents H, F, Cl, Br, NOg, OH, (lower) alkyl or (lower) alkoxy, X represents Cl, Br, F 10 or I, and Y represents H, Cl, Br , F or I. 6. A derivative of kanamycin A according to any one of claims 1, 2, 4 and 5, characterized in that the blocking group of the 6'-amino group is the carbobenzyloxy group. 15 20 1