IE45030B1 - Process for the preparation of aminoglycoside antibiotics and novel intermediates therefor - Google Patents

Abstract

The kanamycin derivatives of formula I, in which R is an amino or hydroxyl group and R<1> is an alkyl group optionally substituted by hydroxyl and/or amino groups, are obtained by alkylation of the compounds of formula II, in which R<2> is H or a benzyl group, R<3> is an acyl-protecting group and R<4> is a hydroxyl group or an NHR<3> group, followed by the removal of the R<3> groups and the benzyl group R<2>, where appropriate. The compounds of formula II are new intermediates which are obtained by acylation of a kanamycin A, B or a 3-N-benzylkanamycin A, protected on all its amino functional groups, followed by the removal of the protecting groups and the intramolecular migration of the acyl group from oxygen to nitrogen.

Description

' ’ Thia invention relates to a novel process ior tho prepareticn of aminoglycoside antibiotics end novel intormadiates for use in - ouch process, and is particularly concerned with a process for tho ' preparation of l-H-autotitutcd-kannuycin derivatives end vith oolcc- 5 tivoly H-protectcd kanamycin derivatives es intermediates for use in - ; tho process.

• . .: Examples of such 1-N-eubstitutcd kanamycin derivatives ore ' · described in £aten-USgec±£icat±0a No. others _ ‘ ' ar6“'known compounds^ such as 1 -. N JO £4-araino-?.-hydroxybutyryrj-l£flnaQyeift A (BB-K8) described in ·; ...

Patent Specification No. . In order to prepare such compounds ' from the readily available fermentation product ksnataycin, it is ' desirable to protect seme, or all, of the amino groups other than the 1-amino group. Substitution may then bo effected preferentially on :,.. 15 the amino group on tha 1-position and isolation of the final 1-fi— Substituted product is thereby simplified. It io an object of thia -:· ϊ present invention to provide a process for tha preparation of 1-H— substituted kanamycin derivatives by providing such selectively Mprotectod intermediates. thus, according to the invention there is provided a process for preparing compounds of the formulas t<5θ3ο where ft «ay ha e.n amino or hydroxyl group end ft tiny bo a lower alkyl /either of or lover ; alkanoyl group^ which cay optionally bo nubstituted with hydroxyl and/or imino groups} vhich process comprises acylation or alkylation of a compound of tho ? ‘ 3 where ftB 1» a hydrogen atom or a benzyl group} ft is a labile amino· blocking acyl group as heroin defined} and R^1 in a hydroxyl group or formula (I) “ 3 Case 250C ¢5030 . In this specification the tern/iower alkv? alkanoyl'indicates that such groups cont ain from JI to 6 Carbon atoms may straight or branched chain. The labile aitiino3 blocking acyl group R is an acyl group which may bo selectively ’ removed from tho Compound of formula (III) by conventional techniques. 1 3 In tho case where I! is a lower ·, .ilkanoyl group, R may bo a haloacetyl group, preferably a trifluoroacetyl group, which may bo removed by hydrolysis under mild conditions, for example with dilute ammonium hydroxide solution, such that the alkanoyl group R is not '3 ' : 10 affected. R may also be a formyl or tnonoq; di—or tricliloro-acetyl group.

I ‘ 1 3 When R is a lower alkyl group, fir may additionally comprise a lower alkanoyl .e.g. an acetyl group, groupf a benzoyl group optionally substituted in tha aromatic ring tilth, for example, a nitro group or one or more halogen atoms,or a lower alkoxyearbonyl group^e.g. an ethoxycarbonyl or methoxycarbonyl group. Those groups require more vigorous hydrolysis conditions for their removal and arc wot therefore suitable when R is a lower alkanoyl group.

Preferred labile amino blocking acyl groups for use in the process of the invention are the acetyl and trifluoroacetyl groups.

, This process for tho preparation of compounds of formula (X) comprises as an initial step acylation or alkylation of a compound of formula (ll) in order.to introduce the substituent R* 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 lotzer/ alkanoic acid, e.g. the N-hydroxysuccinimide ester, or a the acid chloride or anhydride. Alkylation may also be achieved · by conventional reactions, for example by reductive alkylation using an appropriate aldehyde or ketone or an aldehyde derivative such as 4^Q3q described in Patent Specification No.. .. or by reduction of the corresponding acylated derivative ic.g. with diboranc). Naturally in the case fc'hero a compound of formula (II) is used wherein R is n hydrogen atosi, reaction will also take place on the 5 3-N- position but if only a slight excess of reagent is used the required 1-N-i‘tibwtituted isomer can comparatively ezisiiy be separated from the J-N-subatituted isomer and from the 3,3-di-N-substituted product by conventional methods, for example·, by ion-exchange chromatography. This may bs done at this stage of the process or more con10 vcniently after removal of the amino-blocking groups. 'fho second step of the process comprises removal of the amino*a blocking groups R, from the S’-emino group, if present, and the 6* and 3,E=-amiitO groups and also the belli;/1, group, if present, from the 3-amino group. Ir. some instances where the i-N-substituent itself bears an amino substituent group it stay co desirable to protect this group during the course cf the process aad ii will then be necessary to remove this amino-blocking group as well in the final step of the process. There are various conditions for completely removing a;ni no-blocking groups, well known to those skilled in tha art, and they will naturally depend on the SO nature of the pro tec-tine croup employed and tho environment of the protected amine, and will, as already mentioned, need tc bo chosen having regard fot> the substituent en the N-i position. The medium employed may be anhydrous or aqueous and in particular irmiunccs it may ba acidic or basic to various strengths, for example^tho benzyl group, whan E5 present, can be- removed by catalytic hydrogenolysis in a conventional manner in thr presence of a palladium catalyst. α ΰ Ο 3 Ο Some acyl groups may be removed by hydrolysis under mild basic conditions, for- example the trifluoroacnty] group may be removed by treatment with 1-N’ ammonium hydroxide at room temperature for 24 hours, while tile acetyl, Jienzoyl and ethoxycarbonyl groups require more vigorous conditions for their removal, e.g. heating with JN sodium hydroxide for several hours at 60 - 80°C. The product (X) may finally be purified, if desired, by conventional techniques, for example·, by crystallisation or by chromatography.

The process of the invention is exemplified by the pre10 paration of 1-N-£(S)-4-amiiio-2-hydroxybutyrylJ -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-hydroxysuccinimide ester of (S)-4-bonzyloxycarbonylamino-2-hydroxy-butyric aeid. 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 solution of tho active ester to a solution of the kanamycin derivative at 0°C. The reaction can be monitored by thin layer chromatography and more active ester added^if desired^ to ensure complete 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 tho solvent and the product may be purified at this stage, if desired, by conventional techniques (e.g. crystallisation or chromatography) but is more conveniently used in crude forir. in the next step of the process. - 6 4Gq3o Removal of tho 3 .ind 6 '-N-trifluoroaccfcyl groups is achieved by mild base hydrolysis and this may ho performed by simply dissolving the product from the first step of tiie process in JN 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 ifl conveniently performed by dissolving the product from the previous step in a suitable solvent., e.g. a mixture of methanol, water ano acetic acidjSnd subjecting the mixture to a conventional hydrogenation^e.g. at JO p.s.i. and 40°C ir< tbs presence of a palladium eataiyflt. Wo have found that under these conditions deprotcction is substantially complete within i-'t hours. The product is isolated, after filtration, by evaporation of the solvent.

Purification may then be achieved, if desired, bv^for example^ionexchange chromatography, to give the required product in pure form.

The process may be performed in an exactly analogous manner, but starting with 3’'56’-di-N-trifluoroaeetyl-kannnycin A. In this case the ί-K-substituted product is formed, together with the 3-Neutetitwted derivative and the i^-Njh'-diBubstituted product. However, the desired 1-N-substituted product can bs readily separated from the other by-products, for example by the final ion-exchange chromatography step, although naturally the product in this instance is j < obtained in lower yield. Similarly 21 ,J,6'-sri-N-trifluoroacetyl— | kan.amycin B may be used in the process to provide 1-N-substituted kanamycin B dcriatives. - 7 α ΰ Ο 3 Ο . ί · The process of tlie invention is also exemplified by the preparation of l~N-£(S)-4-nmino-2-hydroxybutyjJ kanamycin A. In this case the protected lcnnamycin intermediate (II) is first alkylated, for example, by reductive alkylation with an aldehyde derivative, such as described in. Patent Specification No' ' and the N-blocking groups are then removed and tlie required product isolated.

Thus when 3-beir/.yl-6- {sj-dthydroxyructhyl-tctrahydro-l,3-oxazin-2-one is used in the reductive alkylation with 3,6'-di-N-acetyl-kaiinmycih A^ subsequent base hydrolysis to remove the acetyl groups and hydrogenolysis to remove the benzyl group yields the required compound of formula Cl) in which R is a hydroxy group and R1 is an (S)-4-amino-2-hydrcxybutyl group. The reductive alkylation may conveniently be achieved with the reagents dissolved in a suitable organic solvent, e.g. dimethylformamide^ using sodium borohydride and the reaction is generally complete within Several hours ot 30°C. Removal of the acetyl groups is achieved by hydrolysis with 3N sodium hydroxide at 80¾ for 4 hours and tho benzyl group is removed by catalytic hydrogenation at 60°C and 60 p.s.i. for 16 hours. The required product is then separated from the eo-formed 3“JS’ substituted isomer by chromatography.

The compounds of formula (II) are themselves novel compounds according to the invention. They may be prepared by a selective 0 N 'acyl migration reaction. Thus in one process for their preparation according to the invention an acid addition salt of kanamycin A or B Or 3-N-benzyl-kanamycin .'. xs first treated with a.· excess of acylating agent under acidic conditions that initially only the hydroxyl groups are acylated. Secondly., the acid addition salt of the O-acylatod product, ‘ -.: -.«su is neutralised. - 8 10 £0 *J I1* λ - ί® 3 ο Under these conditions intro moleculnr ccyl migration can take piece onto any amino group having nnr.cyloxy group on an adjacent ring position, i.e. the 6' and 3 amino groups and the 3' amino group in kananycin 0. The reoainsng 0-acyl groups are then removed in the usual tannor^e.g. by hydrolysis er alcoholysis^and the product · may bo purifiedjif desired^ e.g. l>y chromatography. this jiroCess for the preparation of compounds of formula (IX) has been found io be particularly effective for the preparation of tho 4 3 compound* wherein S is a hydroxyl group and H is a trifluoroacetyl group. In till;' case kanasnycin A or J-M-'oenayl ksnaBycit: A i.s dissolved in trif ’uo.·.acetic r.eid and fronted at O^with excess trifluoroacetic anhydride. Reaction ia substantially complete afcor severe! hours at ΰ C ie.g. overnight) and the por-O-triiiuoroacotyi-kanfsCsycin derivative at its trifluoroacetate salt «ay be isolated by evaporation of the .solvents undei vaeausa. The product is dissolved in cn inert organic solvent, preferably tetrohydrofuran, end neutralised by treating with a base, for example by stirring the solution with aodiuo or potassium carbonate* Vo have found that under these conditions tha 0 —$>N acyl migration reaction procoedo rapidly snd io substantially complete within £0 minutes at reea temperature* The remaining 0»trifluercace£yi groups are removed in a conventional manner, e.g. hy eethanolysio, end ths 3”jG!T0ih’“trifluorcncetyl product may then be isolated by evaporation of the solvent and p’jrifiedjif desirod^by conventional column ehrematogi aphy.

As an alternative method of preparation, kanaeycinZor 3-K'oensyl-kananiycin A ie first treated with a reagent to introduce selectively removable amino-blocking groups. -9 ¢5030 Suitable blocking groups nre^for example^the t-butyloxycarbonyl group or the benzyloxycarbonyl group. The fully N-protcctod product io then 0acylated by known techniques^for example by treatment v.'ith an acid anhydride or chloride, e.g. acetic anhydride in pyridine,or with an alkyl chioroformate^ o.g. ethyl chloroiurtnato/ and tne amino-blocking groups are then removed (e.g. the t-butyloxycarbonyl groups are removed by treatment with trifluoroacetic acid and the benzyloxycarbonyl groups are removed by catalytic hydrogenolysis) and the solution is neutralised, 'file 0-o-N acyl migration can then proceed as before ahd the remaining 0-acyl groups are removed and tho product isolated as previously described.

Tho process may also be applied to kanamycin Bq acyl migration inthis case additionally proceeding from the 3'-hydroxyl group to the adjacent 2*-amino grown to give a tri-N-acylatcd intermediate.

’ I I ' _ Tne Compounds of lormula (11) according to the invention, as well . 15 as those of formula (I) and (III), may exist in various conformational forms, and the invention is not limited to any one such form thereof.

Generally the rings arfe each in the Chair form, and each of the sub stituent groups^ is disppsed equatorially with respect to the ring.

Furthermore, the glycosidic linkages between the hexopyranosyl rings apd the 2-deoxystreptamine ring are more usually ^-linkages with respect 1 to the former. 3-N-Benzyl-kanamycin A is itself a novel compound. It may be prepared by reductive alkylation of kanamycin A with benzaldehyde under carefully controlled pH conditions. We have discovered that when kanamycin A in aqueous solution is subjected to reductive alkylation at room temperature or below, with a slight excess of benzaldehyde in the presence of sodium cyanoborohydride and the pH of the solution is carefully adjusted to 6, then the major product from the reaction is 3-Nbenzyl-kannmycin A. 3^030 fhitiirally minor amounts of thu othor K-subatitutcd iocaiers and polysubstituted products are Also produced in the reaction but ihoso nay be Bi'ilniy scparntod by conventional ion-exchange cliroisatography. The main fraction isolated from the column by elution with ammonium hydroxide ie J-K-bonzyl-kfiiia.’iyci/; A contaminated v/ith a minor amount of the 1-N-fconzyl isomer. In practice, thisproduct is sufficiently pure to use directly in the process of the invention although natural!) the l-K-fceazyl isomer present will load, after acylation or alkylation and «^protection, to the formation ef the J-K-substituted isomer as a miser component together with the required l-N-euftstitutcd product of forsulh (I). It can, however, then be readily separated by the final Chromatography step described.

In the following Exacts, Example ¢. describes the preparation of J-N-benEvl-Xanainycin A. Example» 2 to 5 describe the preparation of novel compounds of formula (Ii) according to the invention. Examples 6 to 9 illustrate'· tho novel process of the invention for preparing conipounua oi foztiuia (I), 'bin layer chreisitography was performed on silica plates using tho sclvont system etated. The spots were visualised after drying SO the plates by spraying with a pis solution of t-butyl/hypochlorite in cyclohexane, drying the plates fit 1C>%for 10 minutes in a ventilated oven, cooling and spraying with starch-potassium iodide solution.

Temperatures are giver, in °C. Ambarliio ia a Registered Trade Mark. ΛΰΟ3 EXAMPLE 1 Kanamycin A aulptiate {24.3 0, 0.03 mole) was dissolved in water (150 nil) and the pH adjusted to 6 by the dropwise addition of JjN-hydrochloric acid. Sodium cyanoborohydride (l.95 0, 0.03 nolo) was added ahd the mixture was cooled to 0c · and stirred while a solution of benzaldehyde (3.61 g, 0.033 cole) in methanol (15 ral) was . a added slowly over the course of hours. Tho mixture was allowed to warm to room temperature. After l6 hours tlie pH of tho solution was adjusted to 5»5 by the addition of 1-N hydrochloric acid and the solution was filtered and added to a column of Amberlite CG-50 ionexchange resin in the ammonium-ion form. Elution first with water and then with a gradient of ammonium hydroxide of increasing concenI tration from 0 - 0.7N gave as major product 3N~benz>·l-kananr/cln A contaminated with some i-M-benzyl derivative (5.0 g, 28%) Rf 0.44 in methanolt chloroform, 17% ammonium hydroxide 4:1:2. (Kanamycin A gave an Rf value of Ο.13).

A sample was converted te the voletiletetna-N-acety1-heptaO-trinothylsilyl 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 trimethylchlorosilane at room temperature for 24 hours. ra/® found 1246. C54l06H4O ^Si? requires m/e 12460 The position of substitution was confirmed by tho following sequence of reactions: (a) treatment with t-butyloxycarbonyl azide gave a compound containing three t-butyloxyCarbonyl groups 06 well as the benzyl group (from n.m.r.), (b) hydrogenation to remove the benzyl group, (c) acylation viith N-£(S)-4-benzyloxycarbonylnmino-2hydroxy-butyryloxyjsuceinimide, and “ 13 (d) removal of the K-proteeting groups by hydrogenation^foilowed by treatment with trifluoroacetic acid gave, ns major product, 3-N-Qs)4~aii)iijo~2-hydi-oxybutyryllkanamycin Λ ((IN-K29) identical to a sample prepared according to the procedure of Naitc et al. (J. Antibiotics, 1973, 26, 297).

EXAMPLE 2 Trifluoroacetic anhydride (5.0 ml) wz.a added alewly to a stirred solution of tanamyezn A (2.0 g) in trifluoroacetic acid (40 ml) at 0°. The solution was allowed to stand at 0 ·· 4° for 20 Insure.

The solvent van then evaporated undervacuum and the residue treated with toluene (10 ml) and evaporated to dryness. Tlie trifluoroaeetate I salt was taken up in dry totrahydroXuran and neutralised by slowly adding tc a stirred suspension of excess anhydrous potassium carbonate . ir, tetraitydrofuraii. The mixture was stirred at room temperature fetr minutes end the suspension was then filtered and the filtrate evaporated to dryness. The product was taken up in methanol (20 ml) and kept at room temperature for 3θ minutes. The solvent waa evaporated , pressure under rcducad'and the residue was chromatographed on silica, eluting with a solvent gradient of chloroform, methanol (3s l) to chloroform, £0 methanol, ammonium hydroxide (8:4:l) to give 311,61 -di-N-trifluoroacotyl-kanamycin A hydrate (0.52 g) as a white hygroscopic solid.

Did.? in methanol, chloroform, 17% ammonium hydroxide 4: is 1 (kanamycin —1 A gave an Rf of 0.05). D cso ^65 cni · A sample was converted to the volatile di-H-acetyl-liepta-O25 trimethylsilyl derivative as described in Example 1. m/e found 1264.

C47!!94N4Cl5F6Si7 rer*’jires 1264. “ sSO 30 EXAMPLE 3 Trifluoroacetic anhydride (0,7 ml, 5 mmole) was added slowly to a solution of 3-N-benv.yI-kanamycin A (0.23-g, 0.4 mmole) in trifluoroacetic acid (15 ml} al 0°. The solution was kept at 0 - 4° for 20 hours. The solvent Was then evaporated and the residue treated with toluene (l0 ml) nnd evaporated to dryness. The product was dissolved in tetrahydrofuran (20 ml) and slowly added to a stirred . 1 suspension of excess potassium carbonate in tetrahydrofuran. Tho suspension was stirred at room temperature for 30 minutes, filtered nnd the filtrate evaporated to dryness under reduced pressure. The residue was taken up jn methanol (20 ml) and allowed to stand at room temperature for 3θ minutes. The solvent was then removed under vacuum to yield 3-M-benr.yl-3ll,6'-di.-N-tri fluoroacetyl-kananycin A Rf 0.5 in methanol, chloroform, 8% ammonium hydroxide, 4slfO,l (3-Nbetizyl-kanamycin A gave an Rf value of 0.01).

EXAMPLE 4 (Λ) A solution of l,3,3i6'-tetra-ti-benzyloxycarbonyl-kananiycin A (Bull,Chem. See. Japan, 1965, 38, Il8l)(l8g.4 g) in pyridine (568 ml) and acetic anhydride {IS9 ml) was stirred overnight at room temperature and then poured into water (1.9 litres). The aqueous Solution was extracted with chloroform (1 x 1.8 litres and 1 x 1.0 litres) and the organic extract was evaporated to dryness under reduced pressure. Trituration of the residue with ether gave penta-Oacetyl -1,3,3,6'-totra-N-bonzyloxycarbonyl-kanamycin A (224.8 g) which was filtered and dried under vacuum. The product had m.p. 223229°i Rf 0.55 in chloroform, industrial methylated spirit (12.1), i;8 - 2.05 (15 proton multiplet, 5 acetyl groups) nnd 7.4 (20 proton singlet, k phenyl groups). -14*ΰθ3θ (15) ' A solution of penta-0-acetyl-3,3,3s6 ' -tctra-N-bcnzyloxycarbonyl-kanamycin A {53 g) in ethyl acetate {260 nil) containing glacial acetic acid (2ΰ0 ml) was hydrogenated over 5% palladium on carbon (15 g) at 60° and 50 p.s.i. for 7 hours. The solution van filtered and the filtrate was evaporated to dryness under reduced pressure. The residue was triturated with ether and the product Penta-o-acety 1.k.·:ii,-in:vc.in A(32.9 g) was collected and dried under vacuum, m.p. 57 “ Ίθ5°; Rf 0.0 in chioroform, industrial methylated spirit (l2;l) compared to an Rf of 0.55 for the starting material. The proton magnetic resonance spectrum showed a complete absence of aromatic protons, (Ci A solution of ponta-O-aeetyl-kanamycin A (139,2 g) in methanol (3.4 litres) saturated with ammonia v/as allowed to stand overnight at room temperature and then evaporated to dryness under reduced pressure, Tho residue was dissolved in methanol ¢140 nil) and the crude product was precipitated with chloroform (2.5 litres)# filtered and dried in vacuum. The crude solid was slurried v/ith industrial methylated spirit (400 ml) and the product ff^.S’-di-Nccetylkartainycin A (91.9 g) was collected by filtration, washed with £0 ether and dried under vacuum, m.p. 150 - 180°, Ilf O.77,in methanol, 0.880 ammonium hydroxide lsl. It gave a C n.m.r. spectrum and preton n.in.r. spectrum in full agreement with the required structure. “15 EXAMPLE 5 Trifluoroacetic anhydride (3.6 mis) was added slowly to a stirred solution of kanamycin Β (9δ0 my ., 2 mmole) in trifluoroacetic acid (50 ml)-at 0°. Tho soliition was allowed to stand at 0 - 4° for 20 hours. Tlie 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 tetrahydrofuran (30 ml) and added slowly to a stirred solution of excess triethylamine fn tetrahydrofuran. The solution was allowed to stand nt room temperature for 40 minutes and the solvent was then evaporated under reduced pressure. Tlie residue was dissolved in methanol to hydrolyse the remaining 0-trifluoroacetyl groups and after 3θ minutes nt room temperature the solvent was evaporated under reduced pressure and tho product was chromatographed on silica^eluting with-a solvent gradient of chloroform, methanol (3s i) to chloroform, methanol, 17% ammonium hydroxide (20:10:l)sto give 2’ ,3,6'-tri.-N-trifluoroacetylkanamycin D (452 mg, 29%) as a glass. Rf 0.70 in methanol, chloroform, 8% ammonium hydroxide 4sl:0.1 (kanamycin B gave an Rf of 0.0).

The structure was confirmed by the following sequence of reactions: (a) acetylation with acetic anhydride in methanol for hours at room temperature^followed by treatment with IN ammonium hydroxide for 18 hours to remove the trifluoroacetyl groups^ gave a product containing two acetyl groups. m/e (field desorption) found M + 1 pf the di-N-acotyl derivativ: 568, C22H4i^5°i-> recluircs M + 1 5685 (b) Treatment' with deutorioacetic anhydride in methanol at room temperature for 24 hours, followed • > by reaction with a 2:1 mixture of hexamethyldisilazane and trimethylchlorosilane at room temperature for 24 hours^gave the volatile tri-Ndouteroncetyl-di-N-acetyl-hoxa-0-trimethylsilyl derivative. -l6_ ΰ 0 3 0 W/e ioutid Γ-ji, 15^9^6 requires a/a 1134. Dincetylation was shown to have occurred on the 2-deoxystroptamine ring from the fragmentation pattern,thereby confirming that trifluoroacetylation had initially taken place on the 2',3 and 6' positions in kanamycin li.

EXAMPLE 6 3M,6'-Di-N-trifluoroacotyl-kanamycin I, (prepared from 1.0 g kanamycin by tho method of Example 2) in tetrahyd-ofuran (40 ml) was treated with N-ffiO -4-benzyloxycarbonyleiRino-2~hy cS'oxy-butyryl oxy) succinimide (1.08 g5 3.1 mmoles) in tetrahydrofuran (30 ml). The solution was allowed to stand at room tomperaturo for 24 hours, then a further 0,p4 g of N-(£sj-4-berzyloxyearbonylamino-S-hylroxy-butyryloxy) succinimide was added and the solution was kept at room temperature for c further 24 hours. The solvent was evaporated under vacuum and tho residue was dissolved in IH ammonium hydroxide and allowed to stand ei rcais 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 »1» and hydrogenated gver 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-JO ion-exchange resin (ί.Ή^+ form)^ eluting with a gradient of ammonium hydroxide of increasing concentration front 0 0.5 N, to give 3D-K8 (0.11 g, 9.2% from kanamycin A) identical to a reference sample. - 17 EXAMPLE 7 3-N-Bonzyl-J-6 1 -di-ί!-trif] uoroacetyl-kanamycin A (prepared from 0.23 S 3-N-benzyl-kanamycin A as described in Example 3) was treated directly with a solution of N-(fsj-4-benzyloxycarbonylamino5 2-hydroxy-butyryloxy) succinimide (.017 9, 0.5 mmole) in tetrahydrofuran (l5 ml) at 0°. The solution was allowed to stand at room temperature for 24 hours. A further 0.35 9 of the active ester in tetrahydrofuran was then added and the solution kept for a further hours at room temperature. The' solution was concentrated under 10 vacuum and the residue taken,up in a mixture of methanol, water and acetic acid (30 ral, lOilOsi) and hydrogenated over palladium on charcoal catalyst at 4θό and 5θ p.s.i. for 13.5 hours. The suspension was filtered and the filtrate evaporated. Tile product was purified by ion-excliange chromatography on Amberlite CG-JO (NH^+ form) as before to yield BB-K8 (84 mg, 36% from 3-N-benzyl-kanamycin A) identical to a reference sample.

EXAMPLE 8 2r,3”,6'-Tri-N-trifluoroacetyl-kanamycin B is reacted with N-(£s}-4-ber.zyloxycarbonylamino-2-hydroxy-butyryloxy) succinimide in a similar manner to that described in Example 6, to give, after ’deprotection nnd purification^ 1-N- ([s'] -4-amino-2-hydroxybutyryl) kanamycin Β (BB-K26). 18EXAMPLE 9 ί L Ο 3 Q Λ solution of 3,,,C,-di-N-acetyikan.-;iiiycin Λ (2.84 g) and 3-honzyl-C-(S)-diliydroxymethyl-ts:trahydr!)-l,3-oxazin·~3-one (J.30? g) in dimothylformaxnide (28.4 i..l) was heated at 60° for one hour and then cooled to 30°» Sodium borohydride (0.189 g) was added and the mixture was stirred fox· a further one hour. Vater (l.O ml) was added, the mixture was allowed to stand overnight ar.d the solvent was then removed under reduced pressure. Tha residue was heated with 3N sodium hydroxide solution (23.4 ml) at 80° for 4 hours and, after cooling, the pH of the reaction mixture was adjusted to 5-7 with concentrated hydrochloric acid. The crude solutioi: of l-N-^iS)-4-bonzylamino“2hydroxybutyljkanamycin A and 3-N-^(S;-4-benzyl«ruino-2-hydroxybutylJ kanamycin A was passed down a column of Amberlite CG-JO ion-exchange i' -sin (NH^+ forin/j eluting first, with water to remove inorganics and then with 9,55 M ammonia to isolate the crude aminoglycoside mixture· The required column fraotionswere evaporated and the residue was dissolved in a mixture of methane). (15 ml), acetic acid (15 ml), and water (15 ml) and hydrogenated over 305° palladium on carbon catalyst ai 60° and 60 ρ,ε,ϊ. for 16 hours9 The·solution was filtered StO 'and the solvent removed under reduced pressure. The product was purified by .· on-exchange chromatography as previously described to yield l-l'-jjS)-4-amino-2-hydroxybutyfJ kanamycin A (O.J g)s identical to a reference sample.

Claims (4)

1. A process for preparing compounds of tho formula: NHR ---(I) where R may be an amino or hydroxyl group and R may be a lower alkyl or'lower alkanoyl group^either of which may optionally be substituted with hydroxyl and/or amino groups; which process comprises acylation or alkylation of a compouhd of the NIL --- (II)

2. 3 10 where R is a hydrogen atom or a benzyl group; R J is a labile aminoblocking acyl group as herein defined; and R 1 is a hydroxyl group or 3 a group NHR ; to produce a compound of the formula: “20 “ ΐ'ίϊ 0'3O ί 4 2 wherein R to S are as previously defined; removal of the groups R (if benzyl) and Η 7 ; anti isolation of the compound of formula (I), 2, A process as claimed in claim 1 in which R is a trifluoroacetyl group. I

3. , A process as claimed in claim 2 in -which the compound of formula (II) is 3-N~benz.yl-3’j/6'-di-N-trifluoroacetyl-kanawycin A.

4. , A process as claimed in claim 2 in which the compound of formula (II) is 3,G 1 -di-N-trifluoroacotyl-kanamycin 4. J. A process as claimed in claim 2 in which the compound of z-i formula (IX) is 2' ,3,« 1 -tri-N-tri^fluoroacetyl-kanamyein Π. u. A process as claimed m claim 1 in which R is an acetyl group.

7. A process as claimed in claim 6 in which tha compound of formula (II) is 3,6 1 -di-N-acetyl kanamycin A.

8. A process as claimed in any of claims 1 to 5 in which R is an (S)-4-an:iuo-2-hydroxybutyryI group.

9. A process as claimed in any of claims 1 to 7 -( in which R* is an (S)-4-amino-2-hydroxybutyl group.

10. 1-N- £(S)-4 -amino-2-hydroxybutyljkanamycin A when produced by a process ns claimed in claim 9· 4ΰΟ 30 Case 25OC ''11. · Ά process-for the preparation of a compound of the formula (1' substantially as hereinbefore described with reference to any of the Examples 6 to 9.

12. A 1-N-suhstitutod kanamycin Λ or B derivative whenever

5. Produced by a process as'claimed in any of claims 1 to 9 .or 11. -- (II) Ic is a labile amino-blocking acyl group as herein defined; A 3 and R is a hydroxyl group or a group NHR . o3 14, Compounds as claimed in claim 13 in which Ir is a trifluoroacetyl group. 15, Compounds as claimed in claim l3 in which R J is an acetyl group. 15 16, 3,6’-Di-N-trifluoroacetyl-kanamycin A.

17. 3-N-Benzyl-3,6'-di-N-trifluoroacetyl-kannmycin A.

18. 3j6 , -I>i-N-acetyl-kanamycin A.

19. 2’,3' I ,G'-Tri-N-trifluoroacetyl-kanamycin B.

20. A process for preparing compounds of the formula (II), r 20 which comprises either-an acid addition salt of kanamycin A, kanamycin B or 3-N-benzyl t kanamycin A’witn ap. acylating agent - 22 -5 50 3 ... -} oilect intramolecular 0 to N acyl migration, removing any remaining I O-acyl groups and isolating the selectively N-protacted derivative of formula (II). Pi. A process as claimed in claim 20 in which the trifluoro5 acetic acid salt of kanamycin A, kanamycin B or 3-N-benzyl—kanamycin A is acylated with trifluoroacetic anhydride.

22. A process as claimed in claim 20 in which the fully Kprobeeied derivative is a benzyloxycarbonyl derivative.

23. A process as claimed in claim 22 in which the fully ΝΤΟ protected derivative is tetra-N-banzyloxycarbonyl-kanamycin A. 24» A process as claimed in claim 2.3 in which the fully Nprotected derivative is penta-N-bar.zyloxycarbonyl-kanamycin B. 25= A process as claimed in any one of claims 20 or 32 t 0 24 in which the acylating agent is an aeetylatirty agent. 15 i.S, A process as claimed in claim 25 in which the acetylatlng agent io acetic anhydride. 27» A process as e’^is.ed in any one of claims 22 to 26 in which the benzyloxycarbonyl groups are removed by catalytic hydrogenolysis.

23. A process ao claimed in claim 30 in which the fully N20 protected derivative is a t-butyloxycarbonyl derivative. 2.9« A process «as olaimed in any of claims 2 2 to 2 4 in which i I the acylating agent is ethylchloroformate 01, methylchlorofonnote· / 1

30. A process for the preparation of & compound of the formula (XX) substantially as hereinbefore described with reference· to any -s cf the Examples zf to 5.. 2.3 A compound 'of the formula (II) when prepared by a process 31. as claimed in any of claims 20 to 30.