JPS646200B2 - - Google Patents

Description

Detailed Description of the Invention The present invention relates to a method for producing a polysilylated derivative of kanamycin A or B, and more specifically to a polysilylated kanamycin A or B or a polysilylated kanamycin having a protecting group other than a silyl group on the 6'-amino moiety. Regarding the manufacturing method of A or B. Kanamycin A obtained by the method of the present invention
Or the polysilylated derivative of B has the following formula (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 thereof, as described below. It allows compounds of the above formula to be prepared more efficiently and in higher yields than with other similar intermediates. Kanamycins are well-known antibiotics, as described, for example, in Merck Index, 8th edition, pages 597-8. A number of derivatives of kanamycin are also known. The structural formulas of Kanamycin A and B are shown below along with the standard numbering system used by those skilled in the art. In the main text below, if it can be easily understood,
The various kanamycin derivatives are referred to as derivatives of kanamycin A or B, rather than by their structural formulas, to obviate the need to contrast complex structures to establish differences. U.S. Patent No. 3781268 has 1-[L-(-)-
[gamma]-amino-alpha-hydroxybutyryl] kanamycin A (amikacin) and B and their derivatives with mono- and di-carbobenzyloxy protecting groups have been described and claimed.
For lower and higher congeners, U.S. Pat.
See 3886139 and 3904597.
These compounds are prepared by acylating 6'-protected kanamycin A or B with an acylated derivative of N-protected L-(-)-γ-amino-α-hydroxybutyric acid in aqueous medium, and then converting both or both of the N-protecting groups. It is manufactured by removing one. U.S. Patent No. 3974137 states that 1-[L-(-)
-γ-Amino-α-hydroxybutyryl]Kanamycin A is described and claimed, and this process is applicable to 6'-carbobenzyloxykanamycin A.
containing Schiff base moieties in the 1, 3 and 3″-positions by reacting with at least 3 moles of benzaldehyde, substituted benzaldehyde or pivaldehyde.
6'-N-carbobenzyloxykanamycin A was prepared, and this tetra-protected kanamycin A derivative was acylated with N-hydroxysuccinimide ester of L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid, and then It consists of removing the protecting group. Belgian Patent No. 828192 includes U.S. Patent No.
The same tetra-protected kanamycin A derivative as in No. 3974137 was prepared and acylated with N-hydroxy-5-norbornene-2,3-dicarboximide ester of L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid. A process for the preparation of 1-[L-(-)-γ-amino-α-hydroxybutyryl]kanamycin A by subsequent removal of the protecting group is described and claimed. The present invention provides polysilylated kanamycins A or B useful as starting materials providing an improved and commercially advantageous process for making compounds of formula (), by using the compounds The solubility in organic solvent systems is increased, which allows reactions at high concentrations. The reaction is usually carried out in a solution containing about 10-20% polysilylated kanamycin starting material, but 100 ml of solvent
Excellent results were obtained at a concentration of approximately 50 g per portion. In the prior art process, the 3″-N-acylated product produced results in a loss of approximately equivalent amounts of the desired 1-N-acylated product (with great difficulty in separating the latter from the former). A particularly desirable feature of the method using the compounds contemplated by the method of the present invention is that the undesirable 3″-
The amount of N-acylated product produced is extremely small (in typical examples, it is not detected at all). 1-(L-
(-)-α-amino-α-hydroxybutyl] When producing kanamycin A and amikacin, a typical example is the 3″-N-acylated product (BB-
K11), 3-N-acylated product (BB-K29),
A 6'-N-acylated product (BB-K6) and a polyacylated product as well as unreacted kanamycin A are also produced. Thus, in the commercial preparation of amikacin by acylation of 6'-N-carbobenzyloxykanamycin in aqueous medium and subsequent removal of the protecting group, approximately 10 It has been found that % of the desired amikacin (2.5 Kg in 25 Kg batches) is commonly lost. When amikacin is produced by methods using compounds of the invention, BB-K11 is typically not detected in the reaction mixture. The polysilylated kanamycin A or B obtained by the method of the present invention or the polysilylated kanamycin A or B containing a protecting group other than silyl in the 6'-amino moiety is (where n is an integer from 0 to 2 and B is an amino protecting group) in a substantially anhydrous organic solvent, and then all protecting groups are removed. Compounds of formula can be conveniently obtained by: The protecting groups that can be used to protect the 6'-amino moiety of kanamycin and the amino group of the acylated acid (group B in the formula) are the usual protecting groups for protecting primary amino groups and are known to those skilled in the art. It is well known for. Suitable protecting groups include alkoxycarbonyl groups such as t-butoxycarbonyl and t-
amyloxycarbonyl; aralkoxycarbonyl groups such as benzyloxycarbonyl; cycloalkoxycarbonyl groups such as cyclohexylcarbonyl; haloalkoxycarbonyl groups such as trichloroethoxycarbonyl; acyl groups such as phthaloyl and o-nitrophenoxyacetyl; and other well-known protections Groups such as o-nitrophenylthio, trityl, and the like are included. The acylated acid of formula () may be present in its (+) or (-) isomeric form or in two isomers (d, 1
), so that 1-N-[ω
-amino-α-hydroxyalkanoyl] group in its (+) [i.e. (R)] form or its (-)
Corresponding compounds of formula () in the [i.e. (S)] form or mixtures thereof are produced. In one preferred embodiment, the acylated acid of formula () is in its (-) form. In another preferred embodiment, the acylated acid of formula () is in its (+) form. One embodiment of the target substance of the present invention is polysilylated kanamycin A or B (preferably polysilylated kanamycin A). Another embodiment is a polysilylated kanamycin A or B (preferably polysilylated kanamycin A) containing a protecting group other than silyl on the 6'-amino moiety, said protecting group preferably selected from a group having the formula: be done. [Formula] [Formula] [Formula] [Formula] [Formula] [Formula] and [Formula] In the formula, R ¹ and R ² are the same or different, and are H, F, Cl, Br, NO ₂ , OH, (lower) alkyl or (lower) alkoxy, and
is Cl, Br, F or I, and Y is H,
Cl, Br, F or I. The most preferred protecting group is carbobenzyloxy. In a most preferred embodiment, the polysilylated kanamycin A obtained by the method of the present invention is mixed with a mixed acid anhydride of L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid ( 1-N- [L-(-)-
γ-Amino-α-hydroxybutyryl]kanamycin A or a non-toxic pharmaceutically acceptable acid addition salt thereof is prepared. In another most preferred embodiment, polysilylated kanamycin A containing a carbobenzyloxy group in the 6'-amino moiety obtained by the method of the present invention is prepared in a substantially anhydrous organic solvent with L-(-)-γ.
- acylation with a mixed anhydride of benzyloxycarbonylamino-α-hydroxybutyric acid (preferably its mixed anhydride with pivalic acid, benzoic acid, isobutyl carbonic acid or benzyl carbonic acid) and then removing all protecting groups. Possibly 1-N-
[L-(-)-γ-amino-α-hydroxybutyryl]kanamycin A or a non-toxic pharmaceutically acceptable acid addition salt thereof is produced. In another most preferred embodiment, the active ester of L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid is prepared by preparing the polysilylated kanamycin A contemplated by the method of the invention in a substantially anhydrous organic solvent. (Preferably its N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or N-
1-N-[L-(-)-γ-amino-α-
Hydroxybutyryl]kanamycin A or a non-toxic pharmaceutically acceptable acid addition salt thereof is prepared. In another most preferred embodiment, polysilylated kanamycin A containing a carbobenzyloxy group in the 6'-amino moiety obtained by the method of the present invention is prepared in a substantially anhydrous organic solvent. - active ester of benzyloxycarbonylamino-α-hydroxybutyric acid (preferably its N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or N
1-N-[L-(-)-γ-amino-
α-Hydroxybutyryl]kanamycin A or a non-toxic pharmaceutically acceptable acid addition salt thereof is produced. The present invention provides polysilylated kanamycin A or B containing a protecting group other than silyl in the 6'-amino moiety.
It provides: In a preferred embodiment, the material is polysilylated kanamycin A or B (preferably polysilylated kanamycin A) containing an average number of 4-8 silyl groups (preferably trimethylsilyl groups) per molecule. In another preferred embodiment, the substance contains a polysilylated kanamycin A or B (preferably trimethylsilyl groups) containing a protecting group other than silyl on the 6'-amino group and having an average number of silyl groups (preferably trimethylsilyl groups) of 3-7 per molecule. is polysilylated kanamycin A). As used herein, the term "non-toxic pharmaceutically acceptable acid addition salt" (for a compound of formula) refers to the interaction of one molecule of a compound of formula with 1-4 equivalents of a non-toxic pharmaceutically acceptable acid. means mono-, di-, tri- or tetra-salts formed by action. Among these acids are acetic, hydrochloric, sulfuric, maleic, phosphoric, nitric, hydrobromic, ascorbic, malic, and citric acids, which are commonly used to prepare salts of amine-containing pharmaceuticals. Other acids are included. Acylation of polysilylated kanamycin A or B (with or without a protecting group other than silyl on the 6'-amino moiety) can generally be carried out in an organic solvent with sufficient solubility. These compounds are highly soluble in most common organic solvents. Suitable solvents include, for example, acetone, diethyl ketone, methyl n-propyl ketone, methyl isobutyl ketone, methyl ethyl ketone, acetonitrile, glyme, diglyme, dioxane, toluene, tetrahydrofuran, cyclohexanone, methylene chloride, chloroform, carbon tetrachloride and acetone/butanol or Included are diethyl ketone/butanol mixtures.
The choice of solvent will depend on the particular starting materials used. Generally, ketones are preferred solvents.
The most advantageous solvent for the particular combination of reactants used can be readily determined by routine experimentation. Suitable silylating agents for use in the preparation of the polysilylated kanamycins of the present invention include formulas [Formula] and [Formula] [wherein R ⁵ , R ⁶ and R ⁷ are hydrogen, halogen,
selected from the group consisting of (lower) alkyl, halo(lower) alkyl and phenyl, and the above R ⁵ , R ⁶
and at least one of the R ⁷ groups is halogen or a group other than hydrogen, R ⁴ is (lower) alkyl, m is an integer from 1 to 2, and X is halogen or (R ⁸ is hydrogen or (lower) alkyl,
and R ⁹ is hydrogen, (lower) alkyl or (R ⁵ , R ⁶ and R ⁷ are as defined above)] are included. Special silyl compounds of formulas () and () include trimethylchlorosilane, hexamethyldisilazane, triethylchlorosilane, methyltrichlorosilane, dimethyldichlorosilane, triethylbromosilane, tri-n-propylchlorosilane,
Methyldiethylchlorosilane, dimethylethylchlorosilane, dimethyl-t-butylchlorosilane, phenyldimethylbromosilane, benzylmethylethylchlorosilane, phenylethylmethylchlorosilane, triphenylchlorosilane, triphenylfluorosilane, tri-o-tolylchlorosilane, tri- p-dimethylaminophenylchlorosilane, N-ethyltriethylsilylamine, hexaethyldisilazane, triphenylsilylamine, tri-n-propylsilylamine, tetraethyldimethyldisilazane, hexaphenyldisilazane, hexa-p-tolyldi Silazane, etc. can be mentioned. Hexaalkylcyclotrisilazane and octa-alkylcyclotetrasilazane are also useful. Other suitable silylating agents are silylamides (e.g. tolylalkyl acetamides and bis-trialkylsilylacetamides), silylureas (e.g. trimethylsilylurea).
and silylureido. Trimethylsilylimidazole may also be used. The preferred silyl group is the trimethylsilyl group, and the preferred silylating agents for introducing the trimethylsilyl group are hexamethyldisilazane, bis(trimethylsilyl)acetamide and trimethylsilylacetamide. Most preferred is hexamethyldisilazane. Polysilylated kanamycin A or B containing a protecting group other than silyl in the 6'-amino moiety can be used to
It can be produced by polysilylating 6'-N-protected kanamycin A or B or by introducing a desired 6-N'-protecting group into polysilylated kanamycin A or B. Methods for introducing silyl groups into organic compounds, including certain aminoglycosides, are known in the art. Polysilylated kanamycin A (6′-
The amino moiety with or without a protecting group other than silyl) can be prepared by methods known per se or as described herein. The term "polysilylated kanamycin A or B" as used in this text refers to 2 to 10 silyl groups in the molecule.
(9 to 9 when the 6'-amino moiety has a protecting group other than silyl) is referred to as kanamycin A or B. Thus, the term "polysilylated kanamycins A or B" does not include persilylated kanamycins A or B, which may contain 11 silyl groups in the molecule. We have found that both undersilylation and oversilylation reduce the yield of the desired final product and increase the yield of other products. If the undersilylation or oversilylation is severe, little or no desired product will be formed. The degree of silylation that produces the maximum yield of the desired product depends on the particular reagents used in the acylation step. The most advantageous degree of silylation using any combination of reactants can be easily determined experimentally. 1-N-[L
-(-)-γ-amino-α-hydroxybutyryl] When producing kanamycin A, about 4 to 5.5 hexamethyldisilazane per mole of kanamycin.
It has been found that the desired product can be obtained in good yield by using polysilylated kanamycin A prepared by reacting moles. Higher or lower amounts of hexamethyldisilazane may be used, but the yield of the desired product in the subsequent acylation step will be significantly reduced. In the particular process described above, it is preferred to use about 4.5 to 5.0 moles of hexamethyldisilazane per mole of kanamycin to obtain the highest yield of product in the acylation step. It is clear that 2 equivalents of trimethylsilyl groups can be introduced into kanamycin A or B per mole of hexamethyldisilazane. Kanamycin A and B both have sites that can be silylated (NH ₂ and
Kanamycin A and B, which contain a protecting group other than silyl at the 6'-amino moiety, have a total of 10 such sites. So, 5.5 moles of hexamethyldisilazane per mole of kanamycin A or B could theoretically completely silylate all of the OH and NH _{2 sites of kanamycin, while 5.0 moles of hexamethyldisilazane per mole of kanamycin A or B could theoretically completely silylate all of the OH and NH 2} sites of kanamycin, while 5.0 moles of hexamethyldisilazane per mole of kanamycin A or B could theoretically completely silylate all of the OH and NH 2 sites of kanamycin. Kanamycin A or B1 containing a protecting group other than silyl in
moles can be completely silylated. However, it appears that such extensive silylation cannot occur at such molar ratios during moderate reaction times. However,
If a silylation catalyst is added, a higher degree of silylation is obtained for a given reaction time. The silylation catalyst greatly accelerates the silylation rate. Suitable silylation catalysts are well known in the art and include amine sulfates (eg kanamycin sulfate), sulfumic acids, imidazole and trimethylchlorosilane, among others. The silylation catalyst generally promotes a higher degree of silylation than is required in the process of the present invention. However, hypersilylated kanamycin A or B can be used as a starting material if it is first treated with a desilylating agent to reduce the degree of silylation before carrying out the acylation reaction. The desired product is obtained in good yield when polysilylated kanamycin A prepared using a 5.5:1 molar ratio of hexamethyldisilazane to kanamycin A is acylated. However, kanamycin A was mixed with hexamethyldisilazane in a molar ratio of 7:
1 (or at a 5.5:1 molar ratio in the presence of a silylation catalyst) and acylated with N-hydroxysuccinimide ester of L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid in acetone. If the desired product is 1%
obtained with a yield of less than However, this same "hypersilylated" kanamycin A with the same acylating agent,
One hour before acylation, water was used as a desilylating agent [21 moles of water per mole of kanamycin, 2.5% water (w/
v)] in acetone solution, the desired product was obtained in about 40% yield. The same result is obtained if the water is replaced by methanol or other active hydrogen compounds capable of carrying out the desilylation, such as ethanol, propanol, butanediol, methylmercaptan, ethylmercaptan, phenylmercaptan, etc. Although it is common to use dry solvents when processing silylated materials, we surprisingly found that by adding the reaction solvent to water before acylation, even in the absence of "oversilylation". It has been found that similarly good yields are often obtained, and in some cases better yields of the desired product than with dry solvents. When carrying out the acylation reaction in acetone with a typical concentration of polysilylated kanamycin A of 10-20% (w/v), we have found that less than 28 moles of water per mole of polysilylated kanamycin A (at a concentration of 20%)
%, 28 mol/mol is approximately 8% water), 1-N-[L-(-)-γ-amino-α-hydroxybutyryl]kanamycin A was obtained in excellent yield. I found it. For other reactant combinations, even more water can be tolerated and may be advantageous. The acylation reaction can be carried out in a solvent containing up to 40% water, but at this high water concentration the acylation time must be shortened to avoid excessive desilylation of the polysilylated kanamycin A or B starting material. be. Accordingly, the term "substantially anhydrous organic solvent" herein is intended to include solvents containing up to about 25% water. A preferred range is about 20% water or less, a more preferred range is about 8% water or less, and a most preferred range is about 4% water or less. As indicated in the preamble, the most desirable degree of silylation for any combination of acylating reagents can be readily determined by routine experimentation. The preferred average number of silyl groups in the starting material is usually considered to be 4 to 8 for kanamycin A or B, and 3 to 7 for kanamycin A or B containing a protecting group other than silyl on the 6'-amino moiety. but,
This is just a theory and is not considered an essential component of the invention. The time and temperature of the acylation reaction are not limited.
Temperatures ranging from about -30°C to about 100°C may be used with reaction times ranging from about 1 hour to 1 day or more. It has been found that the reaction normally proceeds satisfactorily at room temperature, and for reasons of convenience and economy it is preferred to carry out the reaction at ambient temperature. However, it is preferred to carry out the acylation at about 0-5°C for highest yields and selective acylation. The art shows that acylation of the 1-amino moiety of polysilylated kanamycin A or B (with or without a protecting group other than silyl on the 6'-amino moiety) is suitable for acylation of the primary amino group. It can be carried out with any acylated derivative of acid of formula () known in the art. Examples of suitable acylated derivatives of free acids include the corresponding acid anhydrides, mixed anhydrides such as alkoxyformic anhydrides, acid halides, acid azides, active esters and active thioesters. The free acid may also be combined with polysilylated kanamycin starting material after first reacting the free acid with N,N'-dimethylchloroforminium chloride.
1008170 and Novak and Weichet, Experientia XXI, 6, 360 (1965)] or the use of N,N'-carbonyldiimidazole or N,N'-carbonylditriazole. [see South African Patent No. 63/2684], or carbodiimide reagents [especially N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or N-cyclohexyl-
N'-(2-morpholinoethyl)carbodiimide: see Sheehan and Hess, JACS, 77, 1967 (1955)] or by the use of alkynylamine reagents [B. Buijle and GH Huie (GH
Angew.Chem.International
Edition), 3,582 (1964)] and the isoxazolium salt reagent [RB Woodward,
(RB, Woodward), RA Olofsson (RA
Olofsoo) and H. Mayer, JACS,
83, 1010 (1961)] and the ketene imine reagent [CL Stevens
and ME Munk (MEMunk), JACS, 80,
4065 (1958)], and by the use of hexachlorocyclotriphosphatriazine or hexabromocyclotriphosphatriazine (U.S. Pat. No. 3,651,050), and by the use of diphenylphosphoryl azide [DDPA: JACS, 94, 6203].
6205 [1972]] and diethylphosphoryl cyanide [DEPC: Tetrahedron Letters, No. 18, 1595].
1598 (1973)] and diphenylphosphite [Tetrahedron Letters, No. 49, 5047-5050
(1972)]. Equivalents of other acids are the corresponding azolides, i.e. quasi-aromatic 5 in which the amide nitrogen contains at least two nitrogen atoms.
Amides of the corresponding acids which are one member of the ring, namely imidazole, pyrazole, triazole, benzimidazole, benzotriazole and substituted derivatives thereof. As is clear to those skilled in the art,
For example, when using acylated derivatives such as acid halides, it may sometimes be desirable or necessary to protect the hydroxy groups of the acylated derivatives of acids of formula (). Protection of hydroxy groups may be achieved by means known in the art, for example by use of carbobenzyloxy groups, by acetylation, by silylation or by similar methods. After completion of the acylation reaction, all protecting groups are removed in a manner known per se to obtain the desired product of formula (). Silyl groups are easily removed, for example by hydrolysis in water, preferably at low pH.
The protecting group B of the acylated derivative of the acid of formula () and the protecting group (if present) of the 6'-amino moiety of the polysilylated kanamycin can also be removed by known methods. Specifically, the t-butoxycarbonyl group was removed by the use of formic acid, the carbobenzyloxy group by catalytic reduction, the 2-hydroxy-1-naphthocarbonyl group by acid hydrolysis, and the trichloroethoxycarbonyl group by the use of zinc powder in glacial acetic acid. By treatment, the phthaloyl group can be removed by treatment with hydrazine hydrate in ethanol under heating. Product yield was determined in various ways. After removal of all protecting groups and chromatography on a CG-50 (NH ₄ ^- ) column, the yield of amikacin was determined by isolation of the crystalline solid from the appropriate fractions or by microbiological assay of the appropriate fractions ( It was measured by turbidimetry or plate). Another technique used was high performance liquid chromatography of the unreduced acylation mixture, an aqueous solution obtained after removal of the silyl group and removal of the organic solvent, before hydrogenolysis to remove residual protecting groups. This assay was not a direct analysis of amikacin or BB-K29, but rather the corresponding mono- or di-N-protecting group compound. The equipment used was tested under Waters under the following conditions:
Waters Associates Model 440
It was a Waters Associates ALC/GPC244 high pressure liquid chromatograph equipped with an absorption detector and a 30 cm x 3.9 mm inner diameter μ-Bondapak C-18 column. Mobile phase 25% 2-propanol 75% 0.01M sodium acetate PH4.0 Flow rate 1ml/min Detector UV.254nm Sensitivity 0.04 AVFS Diluent DMSO Injection volume 5μ Concentration 10mg/ml Chart speed varied, but typical example was 2 minutes/inch. Under the above conditions, a UV trace with peaks that were easy to measure quantitatively was obtained. The results of the above analysis are referred to as the HLPC test in the text. To avoid repetition of complex chemical names, the following abbreviations will be used throughout the text: [Table] Conductor [Table] "Dicalite" is the trade name for diatomaceous earth of the Great Lakes Carbon Corporation. "Amberlite CG-50" is manufactured by Rohm and Haas Company (Rohm
& Haas Co.) is the trade name for a chromatographic grade product of carboxylic acid-polymethacrylic acid based weakly acidic cation exchange resin. "μ-Bondapack" is the trade name of Waters Associates' series of high-performance liquid chromatography columns. All temperatures in this text are expressed in °C. “(lower) alkyl” and “(lower)” used in the text
The term "alkoxy" refers to an alkyl or alkoxy group having 1 to 6 carbon atoms. Example 1 Preparation of poly(trimethylsilyl)6'-N-carbobenzyloxykanamycin A in anhydrous diethyl ketone and its selective acylation of 1-
N-[L-(-)-γ-amino-α-hydroxybutyryl]kanamycin A Production of amikacin 6′-N-carbobenzyloxykanamycin A
[15 g, 24.24 mmol] in 90 ml of dry acetonitrile
The mixture was slurried and heated to reflux under a nitrogen atmosphere.
Hexamethyldisilazane (17.5 g, 108.48 mmol) was added slowly over 30 minutes and the resulting solution was refluxed for 24 hours. Remove the solvent in vacuo (40°) and
Complete drying in vacuum (10 mm) gave 27.9 g of a white amorphous solid. [90.71% calculated as 6'-N-carbobenzyloxykanamycin A (silyl) ₉ ]. This solid was dissolved in 150 ml of dry diethyl ketone at 23°. L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid N-hydroxy-5- dissolved in 100 ml of dry diethyl ketone at 23°
Norbornene-2,3-dicarboximide ester (NAE) (11.05 g, 26.67 mmol) was slowly added over 1/2 hour with good stirring. The solution was stirred at 23° for 78 hours. The clear yellow solution (PH 7.0) was diluted with 100 ml of water. PH of mixture
was adjusted to 2.8 (3N HCl) and stirred vigorously at 23° for 15 minutes. Separate the aqueous phase and remove the organic phase with water at PH2.8.
Extracted with 50ml. The aqueous fractions were combined and washed with 50 ml of ethyl acetate. Add the solution to 5g of 5% palladium-carbon catalyst [Engelhard]
Put it in a 500ml Parr bottle with
Reduced at 50 _psiH2 for an hour. The mixture was passed through a pad of Dicalite and then washed with an additional 30 ml of water. The colorless liquid was concentrated to 50ml in vacuo (40-45°). The solution was loaded onto a 5×100 cm CG-50 (NH ₄ ⁺ ) ion exchange column. After washing with 1000 ml of water, unreacted kanamycin A, 3-[L-(-)-γ-amino-α-hydroxybutyryl] kanamycin A
(BB-K29) and amikacin
Eluted with 0.5N ammonium hydroxide. The polyacyl material was recovered with 3N ammonium hydroxide. Bioassay, thin layer chromatography and optical rotation were used to examine the elution progress. The volume and measured optical rotation of each fraction of eluent and the weight and percent yield of solids isolated from each fraction by evaporation to dryness are summarized below. [Table] The waste diethyl ketone layer was found to further contain 3 to 5% amikacin by high performance liquid chromatography. Dissolve crude amikacin (6.20 g) in 20 ml of water,
Diluted with 20 ml of methanol and added 20 ml of isopropanol to induce crystallization. 6.0 g (45.8%) of crystalline amikacin was obtained. Example 2 Poly(trimethylsilyl) in anhydrous acetone
6′-N-carbobenzyloxykanamycin A
1-N- by production of and its selective acylation
Production of [L-(-)-γ-amino-α-hydroxybutyryl]kanamycin A amikacin Poly(trimethylsilyl)
6'-N-carbobenzyloxycana A (103g,
0.081 mol of 6'-N-carbobenzyloxykanamycin A (silyl) ₉ ) was prepared and dissolved in 100 ml of dry acetone at 23°. L-(-)-γ- dissolved in 180 ml of dry acetone at 23°
Benzyloxycarbonylamino-α-hydroxybutyric acid N-hydroxy-5-norbornene-2,
3-dicarboximide ester (NAE)
(35.24g, 0.085mol) while stirring well.
Poly(trimethylsilyl)6'-N for 15 minutes
- Slowly added to the solution of carbobenzyloxykanamycin A. The solution was heated at 23° under a nitrogen atmosphere for 20
I stirred for hours. The pale yellow clear solution (PH 7.2) was diluted with 100 ml of water. PH of the mixture to 2.5 (3N HCl)
and continued stirring at 23° for 15 minutes. Acetone was removed using a steam-ejector vacuum at 35°. The solution was placed in a 500 ml Par bottle with 10 g of 5% palladium-carbon catalyst (Enzielhard) and reduced with 40 psi H ₂ at 23° for 2 hours. The mixture was passed through a pad of diatomaceous earth and then washed with an additional 50 ml of water. After concentrating to about 1/3 volume, the solution (PH6.9-7.2) was loaded onto a 6 x 110cmCG-50 ( _NH4 ⁺ ) ion exchange column and eluted with 0.6N ammonium hydroxide step concentration from _H2O . Amikacin was collected. An automatic polarimeter was used to check the elution progress. The combination was based on the evaluation of thin layer chromatography. The combined amikacin fraction
Concentrated to 25-30% solids. The solution was diluted with the same volume of methanol and then crystallized with 2 volumes of isopropanol. Crystalline Amikacin 18.2g
(40%) was obtained. Recovery of 12% kanamycin A, 40% BB-K29 and 5% polyacylated kanamycin resulted in a mass balance of 97%. Example 3 Production of poly(trimethylsilyl)kanamycin A and its selective acylation to produce 1-N-[L-
(-)-γ-amino-α-hydroxybutyryl]
Kanamycin A Manufacture of amikacin A Poly(trimethylsilyl)kanamycin A Kanamycin A free base (18g potency, 37.15m
mol) in 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 bright yellow clear solution. Removal of the solvent in vacuo gave an amorphous solid residue (43 g, 94%)
[Calculated as kanamycin A (silyl) ₁₀ ]. B 1-N-[L-(-)-γ-amino-α-hydroxybutyryl]kanamycin A p-(benzyloxycarbonyloxy)benzoic acid (5.56 g, 20.43 mmol) was dissolved in 50 ml of dry acetonitrile at 23°. It turned into slurry. N, O-bis-
Trimethylsilylacetamide (8.4g, 41.37m
mol) was added while stirring well. solution
Hold at 23° for 30 minutes, then dry acetonitrile at 23° for 3 hours with vigorous stirring.
A solution of poly(trimethylsilyl)kanamycin A (21.5 g), 17.83 mmol (calculated as ₁₀ (silyl) compounds) in 75 ml was added. 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°. L-(-)-γ-benzyloxycarbonylamino-α-hydroxybutyric acid N- in 30 ml of acetone.
Hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) (8.55g, 20.63m
mol) was added to the above solution over a period of 5 minutes. The mixture was kept at 23° for 78 hours, the solution was diluted with 100 ml of water and the PH (7.0) was lowered to 2.5 (6H HC1). The mixture was placed in a 500 ml Par bottle with 3 g of 5% palladium-carbon catalyst (Enzielhard) and reduced with 40 psi H ₂ at 23° for 2 hours. The mixture was passed through a pad of diatomaceous earth and washed with 20 ml of water. The liquor and wash liquor were combined (168 ml) and contained approximately 11400 mcg/ml amikacin (19% yield) as determined by microbiological assay for E. coli. Example 4 Production of poly(trimethylsilyl)kanamycin A and its selective acylation to produce 1-N-[L-
(-)-γ-amino-α-hydroxybutyryl]
Kanamycin A Production of amikacin A Poly(trimethylsilyl)kanamycin A 100 ml of dry acetonitrile and 1,1,1,
3,3,3-hexamethyldisilazane 25ml (119
A suspension of 10 g (20.6 mmol) of kanamycin A in 100 mmol) was refluxed for 72 hours. A clear, bright yellow solution was obtained. The solution was dried by vacuum stripping at 30-40°. Poly(trimethylsilyl) kanamycin as a light brown amorphous powder
21.3 g of A (85% yield calculated as Kanamycin A (silyl) ₁₀₎ was obtained. B 1-N-[L-(-)-γ-amino-α-
Hydroxybutyryl]Kanamycin A A solution of 2.4 g (2.0 mmol) of poly(trimethylsilyl)kanamycin A in 30 ml of dry acetone at 0-5° L-(-)-γ- in 10 ml of dry acetone
Benzyloxycarbonylamino-α-hydroxybutyric acid N-hydroxy-5-norbornene-2,
3-dicarboximide ester (NAE) 2.0m
Add moles slowly. reaction mixture for 1 week
Stir at 23° and then vacuum strip dry in a bath temperature of 30-40°. Add water (60ml) to the residue,
Next, 70 ml of methanol was added to obtain a solution.
This solution was acidified to pH 2.0 with 3N HCl for 2 hours.
Reduction was performed using 500 mg of 5% palladium-carbon catalyst at 50 psi H ₂ . The material was filtered and the liquor and washings were combined to contain amikacin in 29.4% yield as determined by microbiological assay against E. coli. Example 5 Preparation of polytrimethylsilyl 6'-N-carbobenzoxykanamycin A and its selective N-acylation to produce amikacin Summary 6'-N-carbobenzoxy in acetonitrile using hexamethyldisilazane (HMDS) Silylation of Kana A provides the 6'-N-carbobenzoxykana A (silyl) ₉ intermediate. This silylated kana A is easily soluble in most organic solvents. Regarding the introduction of 6′-N-Cb _z kana A
Acylation with NAE in anhydrous acetone at 23° using a 5% molar excess of NAE yielded a mixture containing only amikacin and the Cb _z derivative of BB-K29, some terminally reacted kana A, and some polyacyl material. Obtained. In these considerations, BB-
K11 is not detected at all. After reduction and processing,
Pure amikacin was obtained in isolated yields in the range of 40% by elution of the acetone acylation mixture from a CG-50 (NH ₄ ⁺ ) column using ammonium hydroxide step concentrations. equation Materials [Table] Safety [Table] Procedure A Production of 6'-N-carbobenzyloxykanamycin A (silyl) ₉ [6'-N-Cazkana A (silyl) ₉ ] 1 Acetonitrile (KF<0.01%) 300ml 6′-
N-carbobenzyloxykanamycin A (KF
<4%) 50g is slurried. A stream of dry nitrogen is maintained in the slurry to reflux (74°). 2. Slowly add 75.8ml of hexamethyldisilazane (HMDS) over 30 minutes. A complete solution is formed with evolution of ammonia gas. 3 Continue refluxing for 18-20 hours under nitrogen introduction. 4 Pour the transparent bright yellow solution under vacuum (bath temperature 40~
Concentrate under 50°) to obtain a foamy solid. Yield of silyl ₉ compound 89-92 g (90-9.4% of theory). [Note] For reference, when considering other solvents, this solid is usually not isolated and used directly for acylation. B L-(-)-α-carbobenzyloxyamino-α-hydroxybutyric acid N-hydroxy-5
-Production of norbornene-2,3-dicarboximide ester 1 Add L-(-)-γ- to 100 ml of acetone dried at 23°.
Dissolve 21.5 g of carbobenzyloxyamino-α-hydroxybutyric acid (BHBA), then add 15.2 g of N-hydroxy-5-norbornene-2,3-dicarboximide (HONB). A complete solution is obtained. 2. Add a solution of 17.48 g of dicyclohexylcarbodiimide (DCC) in 50 ml of acetone over 30 minutes with stirring. During the addition the temperature rises to about 40° C., accompanied by precipitation of dicyclohexyl urea (DCU). 3 Stir the slurry for 3 to 4 hours and bring the temperature to 23℃.
Allow to equilibrate to ~25°. 4 Remove the urea derivative and add 30ml of acetone to the cake.
wash with Save the liquid + washing liquid for the following acylation step. Acylation of C 6'-N-Cbz kana A (silyl) ₉ 1 Part A, 6'-N-Cbz kana A (silyl) ₉ isolated in step 4, was dried at 23-24° with 100 ml of acetone.
dissolve in 2. While stirring well, slowly add the NAE solution prepared in part B over 15 minutes. The temperature gradually rises to about 40°. Equilibrate the solution to 23° and continue stirring under nitrogen atmosphere for 18-20 hours. 3 Add 100ml of water and adjust the pH (6.9 to 7.2) with 6N hydrochloric acid.
Lower it to 2.2-2.5. Stir at 23° for 15 minutes. (Note: A second layer may be formed. This does not pose a problem during operation.) 4. Remove the acetone under vacuum at a bath temperature of 30-35°.
Transfer the concentrate to a suitable hydrogenation vessel (pre-swept with nitrogen). Add 10 g of 5% palladium-carbon catalyst and hydrogenate at 40 psi for 2-3 hours. 5 Pass the mixture through a Dicalite pad and wash the hydrogenation vessel and cake with an additional 50 ml of water. 6. Concentrate the liquid + washing liquid to about 1/3 volume (50ml) under vacuum at 40-45°. 7 Check the PH. Must be in the range 6.9 to 7.2. If it is not within this range, adjust with 1N ammonium hydroxide. CG-50 mixture
(NH ₄ ⁺ ) column (9-110 cm). 8 Wash the callum with 1000 ml of deionized water. Next, while checking the elution progress using an automatic polarimeter,
Elute with 0.5-0.6N ammonium hydroxide. The elution order is as follows. Residual Kana A → BB-K29 → Amikacin BB-K11 was not detected in any of the acylation procedures. The polyacyl material, the 1,3-diAHBA analog of Kana A, is recovered by washing the column with 3N ammonium hydroxide. 9 Combine Amikacin fraction, then 25
Concentrate to ~30% solids. Dilute with 1 volume of methanol and seed with amikacin crystals. 10 While stirring well, slowly add 2 volumes of isopropanol (IPA) over 2 hours.
Then, crystallize at 23° for 6 to 8 hours. 11 Filter the solids and wash with 50 ml of a 1:1:2 water/methanol/IPA mixture and finally with 25 ml of IPA. 12 Dry in vacuum at 40° for 12 to 16 hours. Amikacin 17.3-19.0g (38-42g) with the following properties:
%) yield. TIC CHCl ₃ - Methanol - NH ₄ OH - Water (1:4:
2:1), 5 x 20 cm Quantum Industries silica gel plate...1 zone (RF~0.4) as detected with ninhydrin. Specific optical rotation [α] 23°589 +H ₂ O 101.6 +0.1MNH ₄ OH 101.9 +0.1MH ₂ SO ₄ 103.5 C = 1.0% 13 The recovery of BB-K29 in this system is 39-42%, and the residual A10-14% and 1,3-di
AHBA-Kana A was about 5% and the material balance was >95%. Example 6 Preparation of polytrimethylsilylkana A and its selective acylation to produce amikacin Summary Polytrimethylsilylkana A was produced by silylation of kana A "base" in acetonitrile using hexamethylene disilazane (HMDS). The degree of silylation is currently unclear, but for the time being it is assumed to be kana A (silyl) ₁₀ .
Polysilylated Kana A is compatible with most organic solvents. By acylation with SAE in anhydrous acetone at 23° using a 1:1 molar ratio of SAE with respect to Kana A, the Cbz derivative of amikacin and BB-K29, BBK6 (2-3/1 ratio is usual) A mixture was obtained containing unreacted Kana A (15-20%) and some polyacyl material (about 5-10%). As seen in the previous experiment with the acylation of polytrimethylsilyl 6'-N-carbobenzoxycana A, BBK11 was not detected in any of these experiments. Reduction and work-up of the acetone acylation mixture followed by chromatography on a CG-50 (NH ₄ ⁺ ) column using 0.5N ammonium hydroxide produced isolated crystalline amikacin in the 34-39% range. equation Materials [Table] Safety Kana A "Base" Known drugs - Usual precautions are recommended Kana A (Cyril) ₁₀ Direct information not available Other materials to be handled with care See Example 5 Operation A Production of Kana A (Cyryl) ₁₀ Slurry 50 g of Kana A "base" (KF 2.5-3.5%) in 500 ml of acetonitrile (KF<0.01%). Reflux (4°) while maintaining a stream of dry nitrogen through the slurry. 2. Slowly add 112 ml of hexamethyldisilazane (HMDS) over 30 minutes. A complete solution forms within 4-5 hours with evolution of ammonia gas. 3 Continue refluxing for 22-26 hours under nitrogen introduction. 4. Concentrate the clear pale yellow solution under vacuum (40°) to a syrupy residue. Add acetonitrile
Wash with 100 ml and dry completely under high vacuum for 3-6 hours. The yield of white amorphous solids is
109-115 g (90-95% of theoretical value, calculated as kana A (silyl) ₁₀ ). B Production of N-hydroxysuccinimide (SAE) of L-(-)-α-carbobenzyloxyamino-α-hydroxybutyric acid 1 Ethyl acetate (KF<
0.05%) Dissolve 100 g of L-(-)-α-benzyloxycarbonylamino-α-hydroxybutyric acid (BHBA) and 45.38 g of N-hydroxysuccinimide (N-HOS) in 1300 ml. 2 Dicyclohexylcarbodiimide (DCC) 81.29 in 400 ml of ethyl acetate (KF<0.05%) at 23°
Dissolve g. Add this solution to the step 1 solution over 30 minutes while stirring well. The temperature increases to 40-42° with simultaneous precipitation of dicyclohexyl urea (DCU). Stir the slurry for 3-4 hours to equilibrate the temperature to 23°. 3 DCU and the cake in ethyl acetate KF
0.05%) Wash with 250ml. Discard DCU cake. Save the solution and washing solution. 4 Concentrate the solution + washing solution to 500ml (vacuum, 30~
35°). Some product crystallizes out. 5 Transfer the concentrate to a suitable container and add 100ml of heptane while stirring vigorously. If necessary, add SAE crystal seeds. Crystallization begins almost immediately. Stir the slurry at 23° for 30 minutes. 6 Add 400ml of heptane over 30 minutes and heat at 23° for 4~
Stir the slurry for 5 hours. 7 Filter and wash the cake with 200 ml of 3:1 heptane/ethyl acetate followed by 100 ml of heptane. 8 Dry in a vacuum oven for 18 to 20 hours at 30 to 35 degrees. Yield 110.1-131.4g (80-95%) MP 119-120°, softened at 114° (corrected) TLC 4acetone: 12benzene: 1CH ₃ CO ₂ H―
1% KMO ₄ aqueous solution - Detection Rf 0.7 for SAE, 0.2 for BHBA, 2 x 10 for Analtech Inc.
cm Pre-recorded silica gel plate C Preparation of Kana A (Syryl) ₁₀ 1 Part A, isolated in step 4 Kana A (Syryl) ₁₀
Dissolve in 500 ml of dry acetone at 23°C. 2 Prepared in part B while stirring well.
Add SAE (35.03 g) rapidly as a 10% dry acetone solution over 5-10 minutes. The temperature rises to about 5°. Allow the solution to equilibrate to 23° and continue stirring for 18-20 hours. 3 Dilute the bright orange clear solution with 400ml of water,
Lower the PH (7.0-7.5) to 2.2-2.5 with 3N hydrochloric acid.
Stir the clear solution for 15-30 minutes at 23°. 4. Remove the acetone under vacuum at a bath temperature of 30-35° (a small amount of material may separate at this point, but this is not a problem). Transfer the concentrate to a suitable hydrogenation vessel. Add 10 g of 5% palladium-carbon catalyst and hydrogenate at 50 _psi for 2-3 hours. 5 Pass the mixture through a dicalite pad, add 2 x 5.0 ml to the hydrogenation vessel and the cake.
Wash with water. 6. Concentrate the liquid + washing liquid under vacuum at 40-45° to about 1/3 volume (150-160 ml). 7 The PH at this point ranges from 6.0 to 7.0. The mixture was loaded into a CG-50 (NH ₄ ⁺ ) column (6 x 110 cm). 8. Wash the column with 1000 ml of deionized water. Using an automatic polarimeter to examine the elution progress
Elute with 0.5N ammonium hydroxide. The elution order is as follows. Residual Kana A → BB-K6 → BB-K29 → Amikacin BB-K11 was not detected in any of the experiments. 9 Combine Amikacin fraction, and 25~
Concentrated to 30% solids. Dilute with 1 volume methanol and seed with amikacin crystals. 10 While stirring well, slowly add 2 volumes of IPA over 2 hours, and crystallize at 23° for 6 to 8 hours. 11 Filter the solid and add 1:1:2 water/methanol/
IPA, and finally wash with 35ml of IPA. 12 Dry in a vacuum oven at 40° for 16 to 24 hours. yield:
19.91-22.84g (34-39%). In addition to the specific rotation, the IR, PMR, and CMR spectral data were also completely consistent with the desired structure. TLC system CHCl ₃ /methanol/NH ₄ OH/water (1:4:
2:1), Quantum Industries’ 5×
20cm silica gel plate...Amikacin (ninhydrin detection) with 1 zone Rf~0.4. Example 7 Preparation of poly(trimethylsilyl) kana A and its acylation to produce amikacin A Silylation of kanamycin A using HMDS with TMCS as catalyst Kanamycin A (10 g of 97.6% purity, 20.14 mmol in 100 ml of sieve-dried acetonitrile) ) was refluxed under nitrogen atmosphere. HMDS (22.76g, 141m
mole, 7 mole/kanamycin A mole) and TMCS
(1 ml, 0.856 g, 7.88 mmol) was added to the refluxing reaction mixture over 10 minutes. Reflux 4-3/4
The mixture was then cooled, concentrated in vacuo to a yellow sticky syrup, and dried under high vacuum for 2 hours. The yield of product was 23.8 g (97.9%, calculated as kanamycin A (silyl) ₁₀ ). B Acylation Poly(trimethylsilyl)kanamycin A (23.8 g, 20.14 mmol) prepared in step A above was dissolved in 250 ml of sieve-dried acetone at 23° and then
Cooled to 5°. While stirring, add water (3.63ml,
201.4 mmol, 10 moles per mole of polysilylated kanamycin A) was added and the mixture was left under medium vacuum for 30 minutes. NAE in 108.3ml of acetone
(19.133 mmol, 0.9 mol per mol of polysilylated kanamycin A) was added in less than 1 minute. Stir the mixture for 1 hour at 0-5°, dilute with water and adjust the pH.
2.5 and then the acetone was removed in vacuo. The aqueous solution was reduced with 2.0 g of 10% Pd-carbon as a catalyst at 50 psiH ₂ at 23° for 21/2 hours. The reduced reaction mixture was passed through Daicalite, concentrated in vacuo at 40° to approximately 100 ml, and then filtered with CG-50
(NH ₄ ⁺ ) column (resin 6, 5 x 100 cm). Wash this with water, then 0.6N - 10N -
Eluted with _3NNH4OH . Amikacin 60.25%, BB
-4.37% of K6, 4.35% of BB-K29, 26.47% of Kanamycin A and 2.18% of polyacyl were obtained. Example 8 Poly(trimethylsilyl)6'-N-Cbz kana A
Preparation of amikacin A by its acylation Silylation of 6'-N-Cbz kanamycin A 6'-N- in 200 ml of sieve-dried acetonitrile
Cbz kanamycin A (20.0 g, 32.4 mmol) was flushed under nitrogen atmosphere. HMDS (47.3 ml, 226.8 mmol, 7 mol per mole of 6'-N-Cbz kanaA) was added once in 10 minutes and refluxed for 20 hours. The mixture was then cooled, concentrated in vacuo, and heated under high vacuum for 2 hours. When dried, 39.1 g of white amorphous solid (yield 95.4%, 6′-N-
Cbz kana A (Cyryl) calculated as ₉ ) was obtained. B Acylation Poly(trimethylsilyl)6'-N-Cbz kana A (39.1 g, 32.4 mmol) produced in step A above was
Dissolved in 400 ml of dry acetone with stirring at 23°. Methanol (6.6 ml, 1.62 mmol, 5 mol per mole of polysilylated 6'-N-Cbz kana A) was added and the mixture was kept under strong nitrogen purge for 1 hour.
It was stirred at 23°. Cool the mixture to 0-5°C,
and SAE in 120 ml of pre-chilled dry acetone.
A solution of (11.35 g, 32.4 mmol) was added. The mixture was stirred for an additional 3 hours at 0-5° and then placed in a cold room at 4° for 1 week. Water (300ml) was added, the pH was adjusted to 2.0, the mixture was stirred for 1 hour and the acetone was stripped in vacuo. The resulting aqueous solution was heated at 23° using 3.0 g of 10% Pd-carbon as a catalyst.
Reduced at 54.0 psiH ₂ pressure for 17 hours. Pass this through Dicalite, concentrate in vacuo to 75-100ml,
Add water and 0.6N NH ₄ OH to CG-50 (NH ₄ ⁺ ).
It was eluted with Amikacin 52.52%, BB-K29
14.5%, 19.6% of kanamycin A and 1.71% of polyacyl were obtained. Example 9 Preparation of poly(trimethylsilyl) kana A and its acylation to produce amikacin A Siluration of kanamycin A with TMCS in acetonitrile using tetramethylguanidine as the acid acceptor Kanamycin A (4.88 g, 10.07 mmol) was added to 23 Dry acetonitrile with sieves while stirring at 100 °C.
ml. Tetramethylguanidine (TMG) (16.234 g, 140.98 mmol, 14 moles per mole of kanamycin A) was added to the stirred suspension.
The mixture was heated to reflux and TMCS (15.32 g, 140.98 m
14 moles per mole of kanamycin A) was added over 15 minutes. After adding about half of the TMCS,
A white precipitate of TMG/HCl was generated. The mixture was cooled to room temperature, concentrated to a viscous residue, and dried under high vacuum for 2 hours. The solid was triturated with dry THF (100 ml) and insoluble TMG.HCl was removed.
Washed with 5 x 20 ml of THF. Combined liquid and washing liquid
Concentrate in vacuo at 40° to a viscous residue and dry under vacuum for 2 hours. 10.64g light cream colored viscous residue
(Yield: 87.6%, calculated as kanamycin A (silyl) ₁₀ ) was obtained. B Acylation Poly(trimethylsilyl)kanamycin A (10.64 g, 10.07 mmol) produced in step A above was
110ml of sieve dried acetone while stirring at 23°
and the solution was cooled to 0-5°. Water (1.81 ml, 100.7 mmol, 10 moles per mole of polysilylated kana A) was added and the solution was stirred under medium vacuum for 30 minutes. 40ml pre-chilled dry acetone
SAE (3.70 g, 10.57 mmol, % excess) was added in less than 1 minute and the mixture was stirred for 1 hour. When the mixture is processed by the general procedure of Example 8B, about 50% of amikacin, about 10% of BB-K29, BB-
5-8% of K6, about 20% of Kanamycin A and 5-8% of polyacyl were obtained. Example 10 Preparation of poly(trimethylsilyl)kanamycin A in pyridine using HMDS Kanamycin A (10.0 g, 20.64 mmol) was suspended in 100 ml of sieve-dried pyridine at 23°. A nitrogen purge was started and the suspension was brought to reflux. HMDS
(17.33 g, 107.32 mmol, 5.2 moles per mole of Kanamycin A) was added over 10 minutes and the mixture was refluxed for 19 hours. It was then cooled to room temperature and concentrated in vacuo to a bright yellow-golden syrup and dried under high vacuum to a white amorphous solid.
22.1g (yield 92.6%, Kanamycin A (silyl) ₁₀
) was obtained. Example 11 Preparation of poly(triethylsilyl)kanamycin A using triethylchlorosilane with triethylamine as acid acceptor Kanamycin A (5.0 g, 97.6% purity, 10.07 mmol) was suspended in 100 ml of sieve-dried acetonitrile at 23°. Triethylamine (TEA) (33.8ml,
24.5 g, 241.7 mmol) were added and the suspension was brought to reflux. A solution of trichloroethylsilane (23.7 ml, 21.3 g, 140.98 mmol) in 25 ml of dry acetonitrile was added over 20 minutes. Refluxing was continued for a further 7 hours and the mixture was cooled to room temperature where it
Long fine needle-like crystals of TEA/HCl precipitated. The mixture was left at room temperature for approximately 16 hours, concentrated in vacuo at 40° to a sticky solid, and dried under high vacuum for 2 hours to a deep orange sticky solid. Dry the solid at 23°
Triturate with 100ml of THF, and insoluble TEA/HCl
Remove, wash with 5 x 20 ml of THF, and dry.
16.0g of TEA/HCl was obtained. The combined liquid and washings were concentrated in vacuo to a solid and dried under high vacuum for 2 hours. 19.3 g of poly(triethylsilyl)kanamycin A was obtained as a deep orange sticky syrup. Example 12 Production of poly(trimethylsilyl)kanamycin A using bistrimethylsilyl urea Kanamycin A (10.0 g, 20.58 m with 99.7% purity)
mol) was suspended in 200 ml of sieve-dried acetonitrile with stirring at 23°. Bis-trimethylsilyl urea (BSU) (29.45 g, 144.01 m
mol, 7 mol per mol of kanamycin),
The mixture was then refluxed under nitrogen atmosphere. Refluxing was continued for 17 hours and the reaction mixture was cooled to room temperature. To remove the small amount of insoluble material present, add acetonitrile 3
× 10 ml aliquots and dried (1.1381
g). Infrared showed this to be BSU plus a small amount of unreacted kanamycin A. The liquid and washing liquid were combined and cooled to 4° for 16 hours. Additional separated material was collected as above (7.8 g) and analyzed by infrared
It turned out to be BSU + urea. The bright yellow liquid and washings were concentrated in vacuo at 40° and dried under high vacuum to yield 27.0 g of a white solid. Part of this solid was viscous and part of it was fine needle-like crystals. The solid was treated with 150 ml of heptane at 23°,
The insoluble portion was removed and washed with 2 x 50 ml portions of heptane to give 6.0 g of white needles (infrared
It turned out to be BSU + urea). The liquid and washing liquid were combined, concentrated under vacuum at 40°, and dried under high vacuum for 2 hours to obtain 20.4 g of white needles. This infrared spectrum was typical of polysilylated kanamycin A. According to calculations, the product is on average
It was found that it has 7.22 trimethylsilyl groups. Example 13 Production of per(trimethylsilyl)kanamycin A by partial desilylation with 1,3-butanediol and production of amikacin by its acylation Production of per(trimethylsilyl)kanamycin A Kanamycin A (10.0 g, 20.639 mmol) ) in sieve-dried acetonitrile while stirring at 23°.
Suspended in 100ml. The suspension was brought to reflux under a nitrogen purge and HMDS (23.322 g, 144.5 mmol, 7 moles per mole of kanamycin A) was added over 10 minutes. Refluxing was continued for 16 hours, and the mixture was cooled to nitrogen temperature, concentrated in vacuo, and dried under high vacuum for 2 hours. 24.3 g of white viscous residue (yield 92.1%, Kanamycin A
(Cyril) calculated as ₁₁ ) was obtained. B Acylation Per(trimethylsilyl)kanamycin A (24.3 g) prepared in step A above was dissolved in 240 ml of sieve-dried acetone with stirring at 23°. Add 1,3-butanediol (9.25ml,
103.2 mmol, 5 mol per mol of per(trimethylsilyl)kanamycin A was added. The mixture was stirred at 23° for 2 hours under a nitrogen purge, then
Cooled to 5°. SAE (7.23 g, 20.64 mmol) was added in 70 ml of pre-chilled acetone in about 1 minute. The mixture was stirred at 0-5° and then left in a cold room at 4° for about 16 hours. Water (200ml) was added, the pH was adjusted to 2.5 and the clear yellow solution was stirred at 23° for 30 minutes. Acetone was stripped in vacuo, and an aqueous solution was used as a catalyst with 3.0 g of 10% Pd-carbon.
Reduced at 55.00 _psiH2 at 23° for an hour. The reduced solution was filtered through Dicalite and chromatographed as in Example 8B to give amikacin 47.50%;
5.87% of BB-K29, 7.32% of BB-K6, 24.26% of kanamycin A and 7.41% of polyacyl were obtained. Example 14 THF using SAE with sulfamic acid catalyst
Production of amikacin by poly(trimethylsilyl)kanamycin A and its acylation in sieve-dried tetrahydrofuran (THF) 50 ml
A refluxing mixture of kanamycin A (5.0 g, 10.32 mmol) in sulfamic acid (100 mg) and
HMDS (12.32g, 76.33mmol) was added. The mixture was refluxed for 18 hours, with complete solution occurring in 6 hours. The solution was cooled to 23°, treated with 0.1 ml of water and held at 23° for 30 minutes. SAE (3.61 g,
A solution of 10.3 mmol) was added over 30 minutes. After stirring for 3 hours, dilute the mixture with 100ml of water and adjust the pH.
Adjusted to _2.2 with 10% _H2SO4 . 30 degrees at 23 degrees
Stir for a minute and then concentrate in vacuo to remove THF. 10% Pd using the resulting aqueous solution as a catalyst.
Reduced with carbon at 23° and 50 psiH ₂ pressure for 2 hours.
The reduced solution was passed through Dicalite and the solids were washed with water. The combined solution and wash solution (150 ml) contained 1225 mcg/ml of amikacin (31.5% active yield) as determined by microbiological assay against E. coli. Example 15 Preparation of poly(trimethylsilyl)kanamycin A using HMDS with imidazole as catalyst Kanamycin A (11 g, 22.7 mmol) and 100 mg of imidazole were heated to reflux in 100 ml of sieve-dried acetonitrile under nitrogen purge. HMDS
(18.48 g, 114.5 mmol, 5 moles per mole of Kanamycin A) was added over 30 minutes and the mixture was refluxed for 20 hours. Complete solution occurred in about 2-1/2 hours. The solution was cooled to 23°, the solvent was removed in vacuo, and the poly(trimethylsilyl)kanamycin
21.6 g of A were obtained as a foamy residue (yield 93.1%, calculated as kanamycin (silyl) ₁₁ ). Example 16 Production of poly(trimethylsilyl)kanamycin B and its acylation to produce 1-N-[L-(-)
Production of γ-amino-α-hydroxybutyryl]kanamycin B A Production of poly(trimethylsilyl)kanamycin B using HMDS with TMCS catalyst Kanamycin B (25 g, 51.7 mmol) in 250 ml of sieve-dried acetonitrile was added under a nitrogen stream. The mixture was heated to reflux. HMDS (62.3 g, 385.81 mmol, 7.5 moles per mole of kanamycin B) was added over 30 minutes, followed by 1 ml of TMCS as catalyst. The mixture was refluxed for 21 hours, with a complete solution occurring after 1 hour. The solvent was then removed in vacuo at 60° to remove the oily residue.
It was held at 60° under high vacuum for 3 hours. Poly(trimethylsilyl)kanamycin B53.0g (yield 85.2%,
Kanamycin B (silyl) (calculated as ₁₀ ) was obtained. B Acylation Dry poly(trimethylsilyl)kanamycin B produced in step A above at 0 to 5° with acetone 500 °C.
ml, methanol (20.9 ml) was added and the mixture was stirred in vacuo at 0-5° for 30 minutes.
SAE (18.1) in 200 ml of pre-chilled dry acetone
g, 51.67 mmol) was added within 1 minute and the mixture was stirred at 0-5° for 30 minutes. Example mixture
14 general operations, then CG-50
(NH ₄ ⁺ ) column (8 x 120 cm).
The column was eluted with stepwise concentrations of _NH4OH from 0.6N to 3N. 38% of BB-K26, equivalent 6′-N-
5% of acylated kanamycin B (BB-K22), corresponding 3-N-acylated kanamycin B (BB-
K46), 14.63% of kanamycin B and a small amount of polyacylated kanamycin B were obtained. Example 17 With Kanamycin A Sulfate as Catalyst
Preparation of poly(trimethylsilyl)kanamycin A using HMDS Kanamycin A (19.5 g, 40.246 mmol) and kanamycin A sulfate (0.5 g, 0.858 mmol) in 200 ml of sieve-dried acetonitrile [total = 20.0
g, 41.0 mmol] was refluxed. HMDS (60.3g,
287.7 mmol, 7 mol per 1 mol of kanamycin A)
was added slowly and the mixture was refluxed for 28 hours.
This was evaporated to dryness on a rotary evaporator and dried under steam syringe vacuum. 47.5 g of poly(trimethylsilyl) kanamycin A was obtained as a pale yellow oil (yield 95.82%, kanamycin A
(Cyril) calculated as ₁₀ ). Example 18 Preparation of BB-K8 Acylation of poly(trimethylsilyl) 3,6'-di-N-carbobenzyloxykanamycin A in anhydrous diethyl ketone 3,6'-di-N-carbobenzylkanamycin A Kanamycin 7.26 g (15 mmol) of A (free base) and 18.6 g (75 mmol) of nickel acetate tetrahydrate were mixed with 300 g of dimethyl sulfoxide (DMSO).
The suspension was heated to 100° C. for 30 minutes with stirring to obtain a clear green solution. After cooling, DMSO50
11.8 g of N-carbobenzyloxy-5-norbornene 2-2,3-dicarboximide (37.6
A solution containing 1 mmol) was added. The mixture was stirred overnight at room temperature, treated with 100 ml of concentrated aqueous ammonia and 1 part water, stirred for 1 hour at room temperature,
It was placed at the top of a "Diaion HP-10" column (300 ml). The column started with 7N NH ₄ OH, followed by methanol-water (1:1) and finally methanol-water (10:1).
20 ml fractions were collected and monitored by thin layer chromatography on Merck silica gel 60F-254 using chloroform-ethanol-28% ammonium hydroxide (1:2:1). A portion of the target portion of fine needle-like crystals was taken from the fraction containing the product at a high concentration and used as an analysis sample. The solution was combined with another fraction containing the target product (RF0.42) and the solution was evaporated in vacuo. The residue was treated with diethyl ether and
9.7 g (86%) of the title product was obtained. Mp＞300℃,
IR (KBr): νc=0 1690cm ^-1 . NMR (DMSO-
d ₆ + DCl, pDCa3): δ4.76−5.26 (6H, m, H ₁ ′,
H″ ₁ and CO―O CH ₂ -C ₆ H ₅ ×2), 7.26 (10H,
s, CO-OCH ₂ - C ₆ H ₅ ×2). Analysis, theoretical value ( _{C34H48N4H15.H2O} ): C, _52.98 : H, 6.54; _N , _7.27 Measured value: C _, 53.20; H, 6.42; N, 7.04. B Poly(trimethylsilyl)3,6'-di-N-
Carbobenzyloxykanamycin A 1.5 g (2 mmol) of 3,6'-di-N-carbobenzyloxykanamycin A from Step A above
and 1.29 g dissolved in 15 ml of dry acetonitrile.
(8 mmol) of hexamethyldisilazane was refluxed for 16 hours. The clear solution was evaporated to dryness under vacuum and the residue was dissolved in 20 ml of dry diethyl ketone. The partially silicified product is syrup-like and has the property of being hydrolyzed during thin layer chromatography. C Acylation of poly(trimethylsilyl) 3,6'-di-N-carbobenzyloxykanamycin A using equimolar acylating agents 700 mg (2 mmol) of L-(- )-carbobenzyloxyamino-α-hydroxybutyric acid N-hydroxysuccinimide ester (SAE) was added. The mixture was stirred at room temperature for 19 hours, treated with 3 ml of water and 35 ml of tetrahydrofuran (THF), and treated with aqueous hydrochloric acid.
The pH was adjusted to 3, stirred for 30 minutes, and dried under vacuum. The residue was dissolved in a mixture of 30 ml of water, 40 ml of methanol, 10 ml of n-butanol and 40 ml of THF and hydrogenated overnight in the presence of 50 mg of 10% palladium on carbon. The catalyst was filtered off and the liquid was evaporated under vacuum and lyophilized.
1.7 g of crude BB-K8 was obtained. The amorphous powder was redissolved in water, brought to pH 4 with aqueous hydrochloric acid, and chromatographed using an Amberlite CG-50 column under NH ₄ ⁺ cycle. The column contains water,
0.1N NH ₄ OH, 0.3N NH ₄ OH, 0.5N NH ₄ OH
and 10 ml of stepwise elution with 2N _NH4OH .
Collect the fractions of chloroform-methanol-28% ammonium hydroxide-water (1:4:2:
1) and monitored by thin layer chromatography using Merck silica gel 60F-254 plates. The homogeneous fractions were combined, evaporated and finally lyophilized. The fraction containing BB-K48 and the fraction containing recovered kanamycin A were combined with K.
pmeumoniae A20680 and B.subtilis PCI129 were used. (a) 408mcg/mg. (b) 956mcg/mg. (c) By bioanalysis. [Table] Hydrogenolysis of the partially deprotected product with Pd-C and CG
-50, BB-K8 is further separated by 30 mg (2
%) obtained. The total yield of BB-K8 is 846 mg (69%)
It was. D. Acylation of poly(trimethylsilyl 3,6'-di-N-carbobenzyloxy-kanamycin A) using 1.2 equivalents of acylating agent. The experiment was carried out under the same conditions as in Step C above, except that a 20% excess of acylating agent was used. , the following results were obtained: (a) 933mc/mg, (b) by bioanalysis. [Table] The partially deprotected product was hydrogenolyzed with Pd-C,
Separation on CG-50 yields an additional 21mg of BB-K8
(2%) was obtained. The total yield of BB-K8 is 771 mg (62
%). E. Acylation of poly(trimethylsilyl 3,6'-di-N-carbobenzyloxykanamycin A) using 1.5 equivalents of acylating agent. Experimented under the same conditions as step C above, except using 50% excess of acylating agent, The following results were obtained: (a) 860mcg/mg, (b) 880mcg/mg, (c) by bioanalysis. [Table]

Claims (1)

[Claims] 1. Kanamycin A or B or in the molecule
Kanamycin A or B in which the 6′-position NH ₂ group is protected with a protecting group other than a silyl group is represented by the formula [Formula] or [Formula] [wherein R ⁵ , R ⁶ and R ⁷ are hydrogen, halogen,
selected from the group consisting of (lower) alkyl, halo(lower) alkyl and phenyl, and the above R ⁵ , R ⁶
and at least one of the R ⁷ groups is halogen or a group other than hydrogen, R ⁴ is (lower) alkyl, m is an integer of 1 to 2, and X is halogen or [Formula] (R ⁸ is and R 9 is hydrogen or (lower) alkyl, and R ⁹ is hydrogen, (lower) alkyl or [Formula] (R ⁵ , R ⁶ and R ⁷ are as defined above) and kanamycin. NH in the molecule of kanamycin A or B by reacting under conditions such that part of the NH ₂ group and/or OH group in the molecule of A or B or 6'-N-protected kanamycin A or B is not silylated. polysilylated kanamycin A or B containing 2 to 10 silyl groups in the molecule as a protecting group for ₂ and (or) OH groups, or at the 6'-position in the molecule of kanamycin A or B.
6′-, in which the NH ₂ group is protected with a protecting group other than the silyl group, and the molecule contains 2 to 9 silyl groups as a protecting group for other NH ₂ groups and/or OH groups;
N-protected polysilylated kanamycin A or B
1. A method for producing a polysilylated derivative of kanamycin A or B, which is useful as an acylation intermediate. 2. The method according to claim 1, wherein the silyl group is trimethylsilyl. 3. The method according to claim 1 or 2, wherein the number of silyl groups in the molecule is 4 to 8. 3. The method according to claim 1 or 2, wherein the NH ₂ group at the 4 6'-position is protected with a protecting group other than silyl, and the number of silyl groups in the molecule is 3 to 7. 5 The protecting group for the NH ₂ group at the 6′-position has the formula [Formula] [Formula] [Formula] [Formula] [Formula] [Formula] and [Formula] (wherein R ¹ and R ² are the same or different, Each is H, F, Cl, Br, NO ₂ , OH, (lower) alkyl or (lower) alkoxy, and X is Cl, Br,
F or I, and Y is H, Cl, Br, F
or I). 5. The method according to any one of claims 1 to 4, wherein the protecting group for the _NH2 group at the 66'-position is a carbobenzyloxy group.