CA1083568A - Process for the preparation of aminoglycoside antibiotics and novel intermediates therefor - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A process for preparing 1-N-substituted kanamycin A
or B derivatives which comprises acylation or alkylation of selectively N-protected intermediate compounds and a process for preparing the novel selectively N-protected intermediate compounds.

Description

356~3 This invention relates to a novel process for the preparation of aminoglycoside antibiotics and with novel inter-mediates for use in such process, and is particularly concerned with a process for ~he preparation of l-N-substituted-kanamycin derivatives and with selectively N-protected kanamycin deriva-tives as intermediates for use in the process.
Examples of such l-N-substituted kanamycin derivatives are described in co-pending Canadian Patent ~PC 5643) Application 238,246 others are known compounds such as l-N-[4-amino-2-hydroxybutyryl]-kanamycin A (BB-K8) described in British Patent Specification Number 1401221. In order to pre-` pare such compounds from the readil~ available fermentation pro-.
` duct kanamycin, it is desirable to protect some, or all, of the amino groups other than the 1-amino group. Substitution may then be effected preferentially on the amino group on the l-position and isolation of the final l-N-substituted product is thereby simplified. It is an object of this present inven-tion to provide a process for the preparation of l-N-substituted ` kanamycin derivatives by providing such selectively N-protected intermediates.
Thus, according to the invention there is provided a process for preparing compounds of the formula:

`~ `
`
, ' .

,.
. . ~ "

.: - . . . -, .
:, -, :. : . , ; ,~ : , . .
, .; ~

s~;~
- ` ~

Ho ~' IH2NH2 H~ ~ , ~'] ~1' a ~ NE12 HO

~HO 1~ ` /
1 ... (I) where R may be an amino or hydroxyl group and Rl may be a lower alkyl or lower aliphatic alkanoyl group either of which may optionally be substituted with hydroxyl and/or amino groups;
which process comprises acylation or alkylation of a compound of the formula:

H

H ~

~EIR
' ~ ~ , HO~
` ' ~H20H /\~H2 . ~ /
R N ~ /

...(II) where R2 is a hydrogen atom or a benæyl group; R3 is a labile amino-blocking acyl group as herein defined; and R~ is a hydr-oxyl group or a group NHR3; to produce a cornpound oE the ~ormula:

: ,.......... .. :
, : -, . ; , 3~

Ho~R3 HO
R4~ 2 C'~

Hn ~ . \
HO ~ / ~ HR
R3N ~ ... (III~

wherein Rl to R are as previously defined; removal of the groups ; R2 ~if benzyl) and R3; and isolation of the compound of Formula (I).
In this specification the term lower alkyl or lower aliphatic alkanoyl indicates that such groups contain from 1 to 6 carbon atoms and may be straight or branched chain. The labile amino-blocking acyl group R3 is an acyl group which may be selectively removed from the compound of Formula (III) by conventional techniques. In the case where Rl is a lower aliphatic alkanoyl group, R3 may be a haloacetyl group, prefer-; ably a trifluoroacetyl group, which may be removed by hydrolysis under mild conditions, for example, with dilute ammonium hydr-oxide solution, such that the alkanoyl group R is not affected.
R may also be a formyl or mono- or dichloro-acetyl group.
When Rl is a lower alkyl group, R3 may additionally comprise an ; acetyl group, a benzoyl group substituted in the aromatic ring with, for example, a nitro group or one or more halogen atoms or a lower alkoxy-carbonyl group, e.g., an etho~ycarbonyl or methoxycarbonyl group. The~e groups require more vigorous _ a~ -,~ . . ....

IV~S~
hydrolysis c~n~itions for their removal and are not ~hereore suitable when Rl i5 a lower alkanoyl group. Preferred labile amino-blocking acyl groups for use in the process of the inven-tion are the acetyl and trifluoroacetyl groups.
This process for the preparation of compounds of Formu~a (I) comprises as an initial step acylation or alkyla-tion of a compound of Formula (II) in order to introduce the substituent Rl onto the amino group at the 1-position. Such a reaction may be performed in a number of ways well known to those skilled in the art. For example, acylation may be achieved using an activated derivative of a lower-aliphatic alkanoic acid, e.g., the N-hydroxysuccinimide ester, or by using the acid chloride or anhydride. Alkylation may also be achieved by con-ventional reactions, for example by reductive alkylation using an appropriate aldehyde or ketone or an aldehyde derivative such (PC 5756) as described in our Canadian SN 272,613 filed Feb. 24, 1~77, or by reduction of the corresponding acylated derivative (e.g., with diborane). Naturally in the case where a compound of Formula (II) is used wherein R2 is a hydrogen atom, reaction will also take place on the 3-N-position bùt if only a slight excess of reagent is used the required l-N-substituted isomer ; can comparatively easily be separated from the 3-N-substituted isomer and from the 1,3-di-N-substituted product by conventional methods, for example, by ion-exchange chromatography. This may 25 be done at this stage of the process or more conveniently after removal of the amino-blocking groups.
The second step of the process comprises removal of the amino-blocking groups R3, from the 2'-amino group, if present, and the 6' and 3"-amino groups and also the benzyl group, if present, from the 3-amino group. In some instances .
:

~ ~ ' ', . .
.
.

wher~ the l-N-substituent itself be~rs an amino substituent group it may be desirable to protect this yroup during -the course of the process and it will then be necessary to remove this amino-blocking group as well in the final step of the process~ There :~ 5 are various conditions for completely removing amino-blocking groups, well known to those skilled in the art, and they will naturally depend on the nature of the protecting group employed and the environment of the protected amine, and will, as already mentioned, need to be chosen having regard for the substituent ; 10 on the N-l position. The medium employed may be anhydrous or aqueous and in particular instances it may be acidic or basic to various strengths. For example, the benzyl group, when present, can be removed by catalytic hydrogenolysis in a conventional manner in the presence of a palladium catalyst. Some acyl groups may be removed by hydrolysis under mild basic conditions, for example, the trifluoroacetyl group may be removed by treatment with l-N ammonium hydroxide at room temperature for 24 hours, while the acetyl, benzoyl and ethoxycarbonyl groups require more vigorous conditions for their removal, e.g., heating with 5N
sodium hydroxide for several hours at 60 - 80C. The product (I) may finally be purified, if desired, by conventional techniques, for example, by crystallization or by chromatography.
The process of the invention is exemplified by the prep-aration of l-N-[(S)-4-amino-2-hydroxybutyryl]-kanamycin A (BB-K8) from 3-N-benzyl-3", 6'-di-N-trifluoroacetyl-kanamycin A. The acylation reaction in this case is conveniently performed using ` the N-hydroxy-succinimide ester of (S)-4-benzyloxycarbonylamino-

2-hydroxy-butyric acid. The reaction is suitably carried out with the reactants dissolved in an inert organic solvent, for example tetrahydrofuran, and is conveniently performed by adding a 501u---6~

, :
,;~ :. , :
.: . . :

..

.33S~
tion of the active ester to a solution of ~he k~namycin deriva-tive at 0C. The reaction can be moni-tored by thin layer chroma-tography and more active ester added if desired to ensure com-plete reaction. The reaction is conveniently allowed to proceed at room temperature and we have found that under these conditions acylation is substantially complete within 48 hours. The product is isolated by evaporation of the solvent and the product may be purified at this stage, if desired, by conventional techniques (e.g., crystallization or chromatography) but is more convenient-ly used in crude form in the next step of the process.
~` Removal of the 3" and 6'-N-trifluoroacetyl groups is achieved by mild base hydrolysis and this may be performed by simply dissolving the product from the first step of the process in lN ammonium hydroxide and allowing the solution to stand for several hours (e.g., overnight) at room temperature. Finally the benzyl and benzyloxycarbonyl groups may be removed together by catalytic hydrogenolysis. This is conveniently performed by dis-solving the product from the previous step in a suitable solvent, e.g., a mixture of methanol, water and acetic acid and subjecting the mixture to a conventional hydrogenation e.g., at 50 p.s.i.
and 40C. in the presence of a palladium catalyst. We have ~ound that under these conditions deprotection is substantially com-plete within 14 hours. The product is isolated, after filtration, by evaporation of the solvent. Purification may then be achieved, if desired, by for example ion-exchange chromatography, to give the required product in pure form.
The process may be performed in an exactly analogous manner, but starting with 311,6'-di-N-trifluoroacetyl-kanamycin A.
In this case the l-N-substituted product is formed, together with the 3~N-substituted derivative and the l,3-N-disubstituted . . . . . :: : ,. : . ~ . .
. .
... .. . , . . . . . ~ -.:.. .. :. - -, . ., : ,.. : .: ~,.... .: . .
.::: .::: :, ,.- , 3S6;~
product. However, the desired l-N-substituted product c~n be readily separated from the other by-products, for example, by the final ion-exchange chromatography step, although naturally the product in this instance is obtained in lower yield.
Si~ilarly 2',3",6'-tri-N-trifluoroacetyl kanamycin B may be used in the process to provide l-N-substituted kanamycin B derivatives.
The process of the invention is also exemplified by the preparation of l-N-~(S)-4-amino-2-hydroxybutyl]-kanamycin A. In this case the protected kanamycin intermediate (II) is first alkylated, for example, by reductive alkylation with an aldehyde [PC5756] derivative, such as described in our Co-pending Patent Applica-tion 272,613 and the N-blocking groups are then removed and the required product isolated. Thus when 3-benzyl-6-[S]-dihydroxy-methyl-tetrahydro-1,3-oxazin-2-one is used in the reductive alkyla-tion with 3",6'-di-N-acetyl kanamycin A, subsequent base hydrolysis to remove the acetyl groups and hydrogenolysis to re-move the benzyl group yields the required compound of Formula (I) in which R is a hydroxy group and Rl is an (S)-4-amino-2-hydroxy-butyl group. The reductive alkylation may conveniently be achieved with the reagents dissolved in a suitable organic solvent, e.g., dimethyiformamide using sodium borohydride and the reactlon is generally complete within several hours at 30CC.
Removal of the acetyl groups is achieved by hydrolysis with 3N
sodium hydroxide at 80 for 4 hours and the benzyl group is remov-ed by catalytic hydrogenation at 60C. and 60 p.s.i. for 16 hours.
The required product is then separated from the co-formed 3-N-substituted isomer by chromatography.
The compounds of Formula (II) are themselves novel com-pounds according to the invention. They may be prepared by a selective 0~ N acyl miyration reaction. Thus ln one process for ' .' ' . , ' - ~Vi5;3~;8 .
their preparation according to the invention an acid addi-tion salt of kanamycin A or B or 3~N-benzyl-kanamycin A is first treated with an excess of acylating agent under acidic conditions such that initially only the hydroxyl groups are acylated.
Secondly, the acid addition salt o~ the C-acylated product, dis-solved in an inert organic solvent is neutralized. Under these conditions intramolecular acyl migration can take place onto any amino group having an acyloxy group on an adjacent ring position, i.e., the 6' and 3" amino groups and the 2' amino group in kanamycin B. The remaining O-acyl groups are then removed in the usual manner e.g., by hydrolysis or alcoholysis and the product may be purified if desired e.g., by chromatography.
This process for the preparation of compounds of Formula (II) has been found to be particularly effective for the prepara-tion of the compounds wherein R4 is a hydroxyl group and R3 is a trifluoroacetyl group. In this case kanamycin A or 3-N-benzyl kanamycin A is dissolved in trifluoroacetic acid and treated at O. with excess trifluoroacetic anhydride. Reaction is substan-tially complste after several hours at OC., (e.g., overnight) and the per-O-trifluoroacetyl-kanamycin derivative as its tri-fluoroacetate salt may be isolated by evaporation of the solvents under vacuum. The product is dissolved in an inert organic solvent, preferably tetrahydrofuran, and neutralized by treating with a base, for example, by stirring the solution with sodium or potassium carbonate. We have found that under these conditions the O-~N acyl migration reaction proceeds rapidly and is substan-` tially complete within 20 minutes at room temperature. The re-maining O-trifluoroacetyl groups are removed in a conventional manner, e.g., by methanolysis, and the 3",6'-di-N-trifluoroacetyl - 30 product may then be i~olated by evaporation of the ~olv~nt and , _g_ .

.
. .. . ~ ~ . ~ , . .. .
- : ,. . :

"... , ., :

: . .. . ~. : , , ~33S6~
puri~ied if desired by conventional colurnn chrom~tography.
As an alternative method of preparation, kanamycin A or

3-N-benzyl ~anamycin A is first treated with a reagent to intro-duce selectively removable amino-bloc~ing groups. Suitable block-ing groups are for example the t-butyloxycarbonyl group or the benzyloxycarbonyl group. The ~ully N-protected product is then O-acylated by known techniques for example by treatment with an acid anhydride or chloride, e.g., acetic anhydride in pyridine or with an alkyl chloroformate e.g., ethyl chloroformate, and the amino-blocking groups are then removed (e.g., the t-butyloxycarb-onyl groups are removed by treatment with trifluoroacetic acid and the benzyloxycarbonyl groups are removed by catalytic hydro-genolysis)0 The O-7N acyl migration can then proceed as before and the remaining O-acyl groups are removed and the product isolated as previously described.
The process may also be applied to kanamycin B, acyl migration in this case additionally proceeding from the 3'-hydroxyl group to the adjacent 2'-amino group to give a tri-N-acylated intermediate.
The compounds of Formula (II) according to the inven-tion, as well as those of Formula ~I) and (III), may exist in ; various conformational forms, and the invention is not lim ted toany one such ~orm thereof. Generally the rings are each in the "chair" form, and each of the substituent groups, is disposed ~5 equatorially with respect to the ring. Furthermore, tha glycosidic linkages between the hexopyranosyl rings and the 2-deoxystrept-arnine ring are more usually X-linkages with respect to the ~ormer.
3-N-Benzyl-kanarnycin A is itself a novel compound. It may be prepared by reductive alkylàtion of kanamycin A with benz-aldehyde under carefully controlled pH conditions. We have dis-. .
.... .. .. : - ......... .
" ' ' ' ' ', ', . :, ' , - .: . . . : ,: ,:
. ., ~ . . . .. .
. . : . - : : .. :

.: -:: .. , : . :
, .:-. , ~ : .

covered that when kanamycin A in aqueous solution i5 subjected to reductive alkylation at room temperature or below, with a slight excess of benzaldehyde in the presence of sodium cyano-borohydride and the pH of the solution is carefully adjusted to 6, then the major product from the reaction is 3-N-benzyl-kanamycin A. Naturally minor amounts of the other N-substituted isomers and poly-substituted products are also produced in the reaction but these may be mainly separated ~y conventional ion-exchange chromatography. The main fraction isolated from the column by elution with ammonium hydroxide is 3-N-benzyl-kanamycin A contaminated with a minor amount of the l-N-benzyl isomer. In practice, this product is sufficiently pure to use directly in ; the process of the invention although naturally the l-N-benzyl isomer present will lead, after acylation or alkylation and deprotection, to the formation of the 3-N-substituted isomer as a minor component together with the required l-N-substituted product of Formula (I). It can, however, then be readily separat-ed by the final chromatography step described.
In the following Examples, Example I describes the preparation of 3-N-benzyl-kanamycin A. Examples 2 to 5 describe the preparation of novel compounds of Formula (II) according to the invention. Examples 6 to 9 illustrates the novel process of the invention for preparing compounds of Formula (I).
Thin layer chromatography was performed on silica plates using the solvent system stated. The spots were visualized after drying the piates by spraying with a 5% solution of t-butyl-hypochlorite in cyclohexane, drying the plates at 100 for 10 minutes in a ventilated oven, cooling and spraying with starch-potassium iodide solution~
~` 30 Temperatures are given in C. "~mberlite" is a Registered - . , . : . . . .
'' ' ' :'' ' , ......... ' ', ~, ', ' .

, .

1~3SÇ;~

Trade Mark.
EX~MPLE 1 Kanamycin A sulphate (24.3 g., 0.03 mole) was dissolved in water (150 ml.) and the pH adjusted to 6 by the dropwise addi-tion of 5-N-hydrochloric acid. Sodium cyanoborohydride (1.95 g., 0.03 mole) was added and the mixture was cooled to OC. and stirred while a solution of benzaldehyde (3.61 g., 0.033 mole) dissolved in methane (15 ml.) was added slowly over the course of 2-1/2 hours. The mixture was allowed to warm to room temperature.
After 16 hours the pH of the solution was adjusted to 5.5 by the addition of l-N-hydrochloric acid and the solution was filtered and added to a column of Amberlite CG-50 ion-exchange resin in the ammonium-ion form. Elution firsk with water and then with a gradient of ammonium hydroxide of increasing concentration from 0 - 0.7N gave as major product 3-N-benzyl-kanamycin A contaminat-ed with some l-N-benzyl derivative (5.0 g., 28%) Rf 0.44 in methanol, chloroform, 17~ ammonium hydroxide 4:1:2. (Kanamycin A gave an Rf value of 0.15).
A sample was converted to the volatile tetra-N-acetyl-hepta-O-trimethylsilyl derivative by treatment with acetic anhydride in methanol at room temperature for 24 hours followed by reaction with a 2:1 mixture of hexamethyldisilazane and tri-methylchlorosilane at room temperature for 24 hours. m/e found C54H106N4l5Si7 requires m/e 1246.
The positlon of substitution was confirmed by the following sequence o reactions: (a) treatment with t-butyloxy-carbonyl aæide gave a compound containing three t-butyloxycarb-onyl groups as well as the benzyl group (from n.m.r.), (b) hydro-genation to remve the benzyl group, (c) acylation with N-[(S)-4-benzyloxycarbonylamino-2-hydroxy-butyryloxy]-suacinimide, and - . , .:: ................. , : .
'''. '' '.' ' ': ', :

B3$~8 (d) ~:emoval of the N-protecting yroups by hydrogenakion ~ollowed by treatment with trifluoroacetic acid gave, as major product, 3-N-[(S)-4-amino-2-hydroxybutyryl]-kanamycin A (BB-K29) identical to a sample prepared according to the procedure of Naito eti al., (J. Antibiotics, 1973, 26, 297).

Trifluoroacetic anhydride (5.0 ml.) was added slowly to a stirred solution of kanamycin ~ (1.0 g.) in trifluoroacetic acid (40 ml.) at O. The solution was allowed to stand at O - 4~. for 20 hours. The solvent was then evaporated under vacuum and the residue treated with toluene (10 ml.) and evaporated to dryness.
The trifluoroacetate salt was taken up in dry tetrahydrofuran and neutralized by slowly adding to a stirred suspension of excess anhydrous potassium carbonate in tetrahydrofuran. The mixture was stirred at room temperature for 20 minutes and the suspension was then filtered and the filtrate evaporated to dryness. The product was taken up in methanol (20 ml.) and kept at room tem-perature for 30 minutes. The solvent was evaporated under reduc-ed pressure and the residue was chromatographed on silica, eluting with a solvent gradient of chloroform, methanol (3:1) to chloro-form, methanol, 17% ammonium hydroxide (8:4:1) to give 3",6'-di-N-trifluoro-acetyl-kanamycin A hydrate (0.52 g.~ dS a white hygroscopic solid. Rf 0.7 in methanol, chloroform, 17%
ammonium hydroxide 4:1:1 (kanamycin A gave an Rf of 0.05).
Vc=o 1665 cm~
A sample was converted to the volatile di-N-acetyl-hepta-O-trimethylsilyl derivative as described in Example 1. m/e found 1264- C47H94N4015F6si7 requires m/e 126~--30 Trifluoroacetic anhydride (0.7 ml., 5 mmole) was added .
" . : . . .. ..
, ~, ; ; , .
: ~ , ~ ~3S~
slow'~y to a solution of 3-N-b~nzyl-kanamycin ~ (0.23 g., 0.4 mmole) in trifluoroacetic acid ~15 ml~) at 0. The solution was kept at -4~. for 20 hours. The solvent was then evaporated and the residue treated with toluene (10 ml.) and evaporated to ~ry-ness. The product was dissolved in tetrahydrofuran (20 ml.) andslowly added to a stirred suspension of excess potassium carbon-ate in tetrahydrofuran. The suspension was stirred at room tem-perature for 30 minutes, filtered and the fil~rate evaporated to dryness under reduced pressure. The residue was taken up in methanol (20 ml.) and allowed to stand at room temperature for 30 minutes. The solvent was then removed under vacuum to yield 3-N-benzyl-3",6'-di-N-trifluoroacetyl-kanamycin A Rf O.S in methanol, chloroform, 8~ ammonium hydroxide, 4:1:0.1 (3-N-benzyl-kanamycin A gave an R~ value of 0.01).

(A) A solution of 1,3,3",6'-tetra-N-benzyloxycarbonyl kanamycin A (Bull. Chem. Soc. Japan, 1965, 38, 1181) (189.4 g.) _ in pyridine (568 ml.) and acetic anhydride (189 ml.) was stirred o~ernight at room temperature and then poured into water (1.9 liters). The aqueous solution was extracted with chloroform (1 x 1.8 liters and 1 x 1.0 liters) and the organic extract was evaporated to dryness under reduced pressure. Trituration of the residue with ether gave penta-0-acetyl-1,3,3",6'-tetra-N-benzyloxycarbonyl kanamycin A (224.8 g.) which was filtered and dried under vacuum. The product had m.p. 223-229; Rf 0.55 in chloroform, industrial methylated spirit (12.1), ~ 1.8 - 2.05 (15 proton multiplet, 5 acetyl groups) and 7.4 (20 proton singlet, 4 phenyl groups).
(B) A solution o~ penta-0-acetyl-1,3,3",6'-tetra-N-benzyl-30- oxycarbonyl kanamycin A (53 g.) in ethyl acetate (260 ml.) con-. .

~...................................... . . .

- ' ~
-~ . . . .
" ~ . ' ' . ,.,., . ' ~ ' ' .
- , , . . :
. .

3s~
.
ta1ning glaclal acetic aci~ (260 ml.) was hydroyenated over 5 palladium on carbon (15 y.) at 60 and 50 p.s.i. for 7 hours.
The solution was filtered and the ~iltrate was evaporated to dryness under reduced pressure. The residue was triturated with ether and the product pe nta-O-acetylkanamycin (32.9 g.~ was collected and dried under vacuum, m.p. 97 - 105, Rf 0.0 in chloroform, industrial methylated spirit tl2:1) compared to an Rf of 0.55 for the starting material. The proton magnetic resonance spectrum showed a complete absence of aromatic protons.
tC~ A solution oE penta-O-acetyl kanamycin A (139.2 g.) in methanol (1.4 liters) saturated with ammonia was allowed to stand overnight at room temperature and then evaporated to dryness under reduced pressure. The residue was dissolved in methanol (140 ml.) and the crude product was precipitated with chloroform (2.5 liters), filtered and dried in vacuum. The crude solid was slurried with industrial methylated spirit (400 ml.~ and the product 3",6'-di-N-acetylkanamycin A (91.9 g.) was collected by filtration, washed with ether and dried under vacuum, m.p. 150 -180, Rf 0.77 in methanol, 0.880 ammonium hydroxide 1:1. It gave a 13C n.m.r. spectrum and proton n.m.r. spectrum in full agreement with the required structure.

Trifluoroacetic anhydride (3.6 mls.) was added slowly ` to a stirred solution of kanamycln B (960 mg., 2 mmole) in tri-- 25 1uoroacetic acid (50 ml.) at 0. The solution was allowed to stand at 0 - 4. for 20 hours. The solvent was then evaporated under reduced pressure and the residue treated with toluene (10 ml.) and evaporated to dryness. The trifluoroacetate salt was dissolved in tetrahydrouran (30 ml.) and added slowly to a ~o stirred solution o excess triethylamine in tetrahydro~uran. The ,~ ~ .,':" ',, ' : ,; :: .
: ; - ' . ' ' . . .
' ~ ' ''' ~' - ' " ' :
.: - .
-,:

~ 3S~;8 soiut.ion was allowed to stand at room temperatur~ ~or 40 minutes and the solvent was then evaporated under reduced pressure. The residue was dissolved in methanol to hydrolyze the remaining O-trifluoroacetyl groups and after 30 minutes at room temperature the solvent was evaporated un~er reduced pressure and the product was chromatographed on silica eluting with a solvent yradient of chloroform, methanol (3:1) to chloroform, methanol, 17% ammonium hydroxide (20:10:1) to give 2',3",6'-tri-N-trifluoroacetyl-kanamycin B (452 mg., 29%~ as a glass. Rf 0.70 in methanol, chloroform, 8% ammonium hydroxide 4:1:0.1 (kanamycin ~ gave an Rf of 0.0~.
The struc~ure was confirmed by the following sequence of reactions: (a3 acetylation with acetic anhydride in methanol for 20 hours at room temperature followed by treatment with lN
ammonium hydroxide for 18 hours to remove the trifluoroacetyl groups gave a product containing two acetyl groups. m/e (field desorption~ found 568, C22H41N5O12 requires M ~ 1 568; (b) Treat-; ment with deuterioacetic anhydride in methanol at room tempera-ture for 24 hours followed by reac~ion with a 2:1 mixture of hexamethyldisilazane and trimethylchlorosilane at room tempera-ture for 24 hours gave the volatile tri-N-deuteroacetyl-di-N-acetyl-hexa-O-trimethylsilyl derivative. m/e Found 1134, C46H86N5O15DgSi6 requires m/e 1134. Diacetylation was shown to have occurred on the 2-deoxystreptamine ring from the fragmenta-tion pattern, thereby confirming that trifluoroacetylation hadinitially taken place on the 2',3" and 6' positions in kanamycin B.

3",6'-Di-N-trifluoroacetyl-kanamycin A (prepared from 1.0 g. kanamycin by the method of Example 2~ in tetrahydrofuran ~n (40 ml.) was treated with N-~S]-4-benzyloxycarbonylamino~2-.

.

, . . . , ~ , ., ~ ~ . . . . . . .
- ., -- ': ' ~153$6;~

hydroxy-butyryloxy) succinimide (1.08 y., 3.1 rnmoles) in tetra-hydrofuran (50 ml.). The solution was allowed to stand at room temperature for 24 hours, then a further 0.54 g. of N-([S]-4-benzyloxycarbonylamino-2-hydroxy-butyryloxy) succinimide was added and the solution was kept at room temperature for a further 24 hours. The solvent was evaporated under vacuum and the residue was dissolved in lN ammonium hydroxide and allowed to stand at room temperature for 20 hours. The solution was concentrated under vacuum and the product taken up in a mixture of dioxan, water and acetic acid (55 ml., 5:5:1) and hydrogenated over 5% palladium on charcoal catalyst at 30. and 50 p.s.i. for 6 hours. The mixture was filtered and the filtrate evaporated.
- The residue was chromatographed on Amberlite CG-50 ion-exchange resin (NH4 ~ form) eluting with a gradient of ammonium hydroxide of increasing concentration from O - 0.5 N, to give BB-K8 (0.11 g., 9O2~ from kanamycin A) identical to a reference sample.

3-N-Benzyl-3"-6'-di-N-trifluoroacetyl-kanamycin A
(prepared from 0.23 g. 3-N-benzyl-kanamycin A as described in Example 3) was treated dlrectly with a solution of N-[S]-4-benzyloxycarb-onylamino-2-hydroxy-butyryloxy) succinimide (.017 g., 0.5 mmole) in tetrahydrofuran (15 ml.) at 0. The solution was allowed to ` stand at room temperature for 24 hours. A further 0.35 g. of the acti~e ester in tetrahydrofuran was then added and the solu-tion kept for a further 20 hours at room temperature. The solu-tion was concentrated under vacuum and the residue taken up in a mixture of methanol, water and acetic acid (30 ml., 10:10:1) and hydrogenated over palladium on charcoal catalyst at 40 and 50 p.s.i. for 13.5 hours. The suspension wa5 filtered and the filtrate evaporated. The product was purified by ion-exchange :
., .

-:, . . .
.
.~. . . .
" , ~
.. . .

5~
chromatography on Amberlite CG-50 lNH4 ~ form) as be~ore to yield BB- K8 (84 mg., 36~ from 3-N-benzyl kanamycin A) identical to a reference sample.

2',3",6'-Tri-N-trifluoroacetyl-kanamycin B is reacted with N-([S]-4-benzyloxycarbonylamino-2-hydroxy-butyryloxy) suc-cinimide in a similar manner to that described in Example 6, to give, after deprotection and purification l-N-([S]-4-amino-2-hydroxybutyryl) kanamycin B (BB-K26~.

.
A solution of 3",6'-di-N-acetylkanamycin A (2.84 g.) and 3-benzyl-6-(S)-dihydroxymethyl-tetrahydro-1,3-oxazin-2-one (1.305 g.) in dimethylformamide (28.4 ml.~ was heated at 60 for one hour and then cooled to 30. Sodium borohydride (0.189 g.) was added and the mixture was stirred for a further one hour.
Water (1.0 ml.~ was added, the mixture was allowed to stand over-night and the sol~ent was then removed under reduced pressure.
The residue was heated with 3N sodium hydroxide solution (28.4 ml.) at 80 for 4 hours and, after cooling, the pH of the reaction mixture was adjusted to 5.7 with concentrated hydro-chloric acid. The crude solution of l-N-[(S)-4-benzylamino-2-hydroxybutyl] kanamycin A and 3-N-[(S)-4-benzylamino-2~hydroxy-butyl] kanamycin A was passed down a column of Amberlite CG-50 ion-exchange resin (NH4 ~ form) eluting first with water 2S to remove inorganics and then with 0.15M ammonia to isolate the crude aminoglycoside mixture. The required column fractions were evaporated and the residue was dissolved in a mixture of methanol (15 ml.), acetic acid (15 ml.), and water (15 ml.) and hydrogenated over 30% palladium on carbon catalyst at ~0 and 3r- 60 p.s.i. fox 16 hours. ~he solution was filtered and the , ,... ., ~
-.. , . ~. .: :
-:- -. . ~ . . . . .
.
. . . . : . : . . .:.

~: . : .

$~;~

solvent removed under reduaed pressure. rrhe product was purified by ion-exchange chromatoyraphy as previously described to yield l-N-[(S)-4-amino-2-hydroxybutyl] kanamycin A identi~al to a reference sample.

~' .. . ... ..

. . ~ ,. .
,: . . .
.

,: . . ., :, . .

Claims (30)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing compounds of the formula:
...(I) where R is an amino or hydroxyl group and R1 is a lower alkyl or lower aliphatic alkanoyl group either of which optionally may be substituted with hydroxyl and/or amino groups;
which process comprises acylation or alkylation of a com-pound of the formula:
...(II) where R2 is a hydrogen atom or a benzyl group; R3 is a labile amino-blocking acyl group; and R4 is a hydroxy-yl group or a group NHR3; to produce a compound of the formula:
...(III) wherein R1 to R4 are as previously defined; removal of the groups R2 (if benzyl) and R3; and isolation of the compound of Formula (I).

2. A process as claimed in claim 1, in which R3 is a tri-fluoroacetyl group.

3. A process as claimed in claim 2, in which the compound of Formula (II) is 3-N-benzyl-3"-6'-di-N-trifluoroacetyl-kana-mycin A.

4. A process as claimed in claim 2, in which the compound of Formula (II) is 3",6'-di-N-trifluoroacetyl-kanamycin A.

5. A process as claimed in claim 2, in which the compound of Formula (II) is 2',3",6'-tri-N-tri-fluoroacetyl-kanamycin B.

6. A process as claimed in claim 1, in which R3 is an acetyl group.

7. A process as claimed in claim 6, in which the compound of Formula (II) is 3",6'-di-N-acetyl kanamycin A.

8. A process as claimed in claim 1, ln which R1 is an (S)-4-amino-2-hydroxybutyryl group.

9. A process as claimed in claim 1, in which R1 is an (S)-4-amino-2-hydroxybutyl group.

10. A process for preparing a compound of the formula:- ...(II) where R2 is a hydrogen atom or a benzyl group; R3 is a labile amino-blocking acyl group; and R4 is a hydroxyl group or a group NHR3, which comprises reacting either an acid addition salt or a fully N-protected derivative of kanamycin A, kanamycin B or 3-N-benzyl kanamycin A with an acylating agent, removing the N-protecting groups if present, neutralizing the O-acylated derivative so formed to effect intramolecular O to N acyl migration, removing any remaining O-acyl groups and isolating the selectively N-protected derivative of formula (II).

11. A process as claimed in claim 10, in which the acylating agent is an acetylating agent.

12. A process as claimed in claim 11, in which the acetylating agent is acetic anhydride.

13. A process as claimed in claim 10, in which the fully N-protected derivative is a benzyloxycarbonyl derivative.

14. A process as claimed in claim 13, in which the fully N-protected derivative is tetra-N-benzyloxycarbonyl-kanamycin A.

15. A process as claimed in claim 13, in which the fully N-protected derivative is penta-N-benzyloxycarbonyl-kanamycin B.

16. A process as claimed in claim 13, in which the benzyloxycarbonyl groups are removed by catalytic hydrogenolysis.

17. A process as claimed in claim 14, in which the tetra N-benzyloxy-carbonyl kanamycin A is acylated with acetic anhydride.

18. A process as claimed in claim 10, in which the trifluoroacetic acid salt of kanamycin A is acylated with trifluoroacetic anhydride.

19. A process as claimed in claim 18, in which the trifluoroacetic acid salt of kanamycin A is acylated with trifluoroacetic anhydride.

20. A process as claimed in claim 18, in which the trifluoroacetic acid salt of kanamycin B is acylated with trifluoroacetic achydride.

21. A process as claimed in claim 18, in which the trifluoroacetic acid salt of 3-N-benzyl kanamycin A is acylated with trifluaroacetic anhydride.

22. A process as claimed in claim 10, in which the fully N-protected derivative is a t-butyloxycarbonyl derivative.

23. A process as claimed in claim 13, in which the acylating agent is ethylchloroformate.

24. A compound of formula (II) when prepared by a process as claimed in claim 10.

25. A compound of formula (II) in which R3 is an acetyl group, when prepared by a process as claimed in claim 12.

26. A compound of formula (II) in which R3 is a trifluoroacetyl group, when prepared by a process as claimed in claim 18.

27. 3",6' -Di-N-trifluoroacetyl-kanamycin A, when prepared by a process as claimed in claim 17.

28. 3",6'-Di-N-trifluoroacetyl-kanamycin A, when prepared by a process as claimed in claim 19.

29. 2',3",6'-Tri-N-trifluoroacetyl-kanamycin B, when preapred by a process as claimed in claim 20.

30. 3-N-benzyl-3",6'-di-N-trifluoroacetyl-kanamycin A, when prepared by a process as claimed in claim 21.