IE48973B1 - The production of a selectively protected n-acylated derivative of an aminoglycosidic antibiotic - Google Patents

Description

The present invention relates to new processes for the product ion of a selectively protected N-acylated derivative of an aminoglycosidic antibiotic in which some amino or alkylamino groups at particular positions of the aminoglycoside molecule have selectively been protected or blocked with an acyl group. The present invention thus relates to new processes for selectively protecting some amino or alkylamino groups at particular positions of the aminoglycosidic antibiotic. The present invention finds its main uses in the production of a selectively protected N-acylated derivative of the aminoglycosidic antibiotic which comprises a deoxystreptamine moiety having a 3-aminoglycosyl group linked with the 6hydroxyl group of the deoxystreptamine moiety in the aminoglycoside molecule. The aminoglycosidic antibiotic to which this invention is applicable may be defined more specifically as an aminoglycosidic antibiotic consisting of a 6-0-(3amino or 3-alkylamino-3-deoxyglycosyl)-2-deoxystreptamine which may optionally have a 4-0-(6’aminoglycosyl) substituent. Typical examples of such aminoglycosidic antibiotics are kanamycins, gentamicins, sisomicin, netilmicin and verdamicin. These new processes may be applied to the production of 1-N(ct-hydroxy-ui-aminoalkanoyl)-amino-glycosidic antibiotics which are known to be useful semi-synthetic antibacterial agents active against drug-resistant bacteria.

Aminoglycosidic antibiotics such as kanamycins are substances containing several amino functions and hydroxyl functions having relatively high aid various degrees of reactivity. Many kinds of semi-synthetic aminoglycosidic antibiotics derived from the parent aminoglycosidic anti5 biotics have been synthetized. In the semi-synthesis of these derivatives, it is often necessary or preferable to ensure that some amino groups and/or some hydroxyl groups in the staring aminoglycosidic antibiotic have selectively been protected or blocked with one or more suitable protective groups.

For selective protection of amino groups and/or hydroxyl groups in the aminoglycosidic antibiotic, various, successful methods have been developed and are available as far as selective protection of hydroxyl group is concerned with.

However, for selective protection of particular some amino groups amongst the existing many amino groups of the aminoglycosidic antibiotic, the presently available methods for this purpose are either difficult to operate or require some complicated operations. This is because all the amino groups in the aminoglycosidic antibiotic have no great difference in their reactivity. As a demonstrative example is provided by 6'-amino group of kanamycin A, however, such an amino or methylamino group which is bound with a certain carbon atom which is, in turn, linking to only one carbon atom in the aminoglycoside molecule exhibits a higher reactivity than that of the amino or methylamino group which is bounded with a certain carbon atom which is linking to two or more carbon 48873 ·' 4 atoms in this aminoglycoside molecule. For this reason the former type of amino or methylamino group is able to much more preferentially react with an acylation reagent having an acyl group to be introduced as the amino-protecting group, as compared to the latter type of amino or methylamino group, whereby the N-protected derivative having the former type of amino or methylamino group preferentially blocked with the acyl group may be produced in a higher yield than the otherwise N-protected derivatives. Several years ago, some of the present inventors found that when amino group and hydroxyl group are neighboring to each other in a pair in the steric configuration of the molecule of the aminoglycosidic antibiotic, these amino and hydroxyl groups can selectively be combined with each other into the form of a cyclic carbamate by treatment with sodium hydride, so that the pair of amino and hydroxyl groups can be blocked simultaneously in the cyclic carbamate without blocking the other amino groups present in the same molecule (see Journal of Antibiotics, 2£, No. 12, 741-742 (1972); U.S. Patent Nos. 3,925,354 and 3,965,089).

In a recent year, Nagabhushan et al have found that when a salt of a divalent transition metal (M++) selected from the group consisting of copper (II), nickel (II), cobalt (II) and cadmium (II) is reacted in an inert organic solvent with an aminoglycosidic antibiotic which belongs to the class of 4-0-(aminoglycosyl)-6-0-(aminoglycosyl)-2-deoxystreptamines represented by kanamycins, gentamicins and sisomicin, this divalent transition metal cation is complexed with a pair of amino and hydroxyl groups which exist at the particular positions of vicinal relationship in the aminoglycoside molecule, whereby the aminoglycosidic antibiotic-transition metal cation complex is formed (see Japanese Patent Application Pre-publication Sho-52-153944 and U.S. Patent Application SN. 697,297 now granted under U.S. Patent No. 4,136,254 issued on January 23, 1979). In this aminoglycosidic antibiotic-transition metal cation complex, the complexed amino group ia being blocked with the divalent transition metal cation. When this complex is subsequently reacted with an acylation reagent having an acyl group, only the non-complexed amino groups in the metal complex which are not blocked by the divalent metal cation can be acylated mainly, so that selective N-protection with the acyl group is achieved.

This is illustrated below with reference to kanamycin A as an example. Thus, when a divalent transition metal cation (M**) chosen from cupper (II), nickel (II), cobalt (II) and cadmium (II) cations is reactd with kanamycin A, complexing reaction of the divalent metal cation (M++) occurrs between 1-amino group and 2-hydroxy group and between 3-amino group and 4-hydroxyl group of kanamycin A molecule, shown by the formula (I) below.

In the above complexing reaction, therefore, it is seen that at least 2 mol of the transition metal salt ie required for 1 mol of kanamycin A. In the resultant metal complex, 1-amino and 3-anino groups are blocked at the same time. When this complex of the formula (I) is treated with an acylation reagent having an acyl group which is available as an amino-protecting group known in the conventional synthesis of polypeptides, the non-complexed 3-amino and 6'10 amino groups only are acylated mainly to give 3,6’-di-Nacylated derivative (see Journal of American Chemical Society IOC, 5253-5254 (1978)).

We have recognized the above fact as reported, but we still have made our further researches on the interaction .. 4 8973 of another, various metal cations with aminoglycosidic antibiotics such as kanamycin A and kanamycin B as well as semisynthetic derivatives of the aminoglycosidic antibiotics.

As a result, we have now found that although divalent zinc cation has behaviours significantly different from those of the above-mentiond divalent, nickel, cobalt, copper and cadmium cations, the zinc cation is ultimately able to strongly complex with and block both 1-amino (or 1-alkylamino) group and 3-amino (or 3-alkylamino) group of an amino10 glycoside compound (such as kanamycin A, B or C) which comprises a deoxystreptamine moiety having a 3-a»inoglycosyl or 3-alkylaminoglycosyl group linked to the 6-hydroxyl group of said deoxystreptamine moiety.

According to Nagabhushan et al, it might be expected that when divalent nickel, cobalt, copper or cadmium cation would be reacted with kanamycin B, for example, there should be formed a kanamycin B-metal salt complex of the following formula (II): 6’ 3 h2nch. nh2 This expectation is supportable by the Nagahhushan et al's disclosure of the aforesaid Journal of American Chemical Society according to which vicinal amino-hydroxyl group pairs should form reversible complexes with the divalent transition metal cations, in view of the fact that kanamycin B contains three pairs of vicinal amino-hydroxyl groups between 1- and 2-positions, between 2'- and 3*-positions and between 2- and 3-positions of the kanamycin B molecule.

However, it has now been found that when kanamycin B is reacted with a divalent metal cation, zinc cation, the kanamycin B-zlnc salt complex actually formed contains free 2'-amino and 3'-hydroxyl groups which are not being blocked by the zinc cation, as be contrary to the Nagahhushan's proposal. Even if complexing reaction of zinc cation with the 2’-amino and 3’-hydroxyl group occurs, the force of complexing is very low, so that substantially 2'-amino and 3'hydroxyl groups are not being blocked in practice. Therefore, when the kanamycin B-zinc cation complex is then acylated by reacting eg. with N-benzyloxycarbonyloxysuccinimido to introduce benzyloxycarbonyl group as the amino-protecting acyl group, tri-3,2’,6'-N-acylated derivative in which three, 3-, 2'- and 6'-amino groups have been acylated is produced, in fact, in a higher yield than the otherwise N-acylated deriva10 tives, but then the 3,6'-di-N-acylated derivative actually cannot be obtained. This experimental fact suggests that zinc cation shows a behaviour different from those of the aforesaid four transition metal cations particularly in that zinc cation does not com15 plex with the vicinal 2'-amino and 3'-hydroxyl group pair.

As a further example, when kanamycin A is reacted with zinc cation followed by acylation with benzyloxycarbonyl group (refer to the formula (I) hereinbefore), the fact is observed that 3,6’-di-N-benzyloxycarbonylkanamycin A is formed as the main acylation product in case zinc cation is provided just in en amount of slightly more than 1 mol. per mol. of kanamycin A. In this case, it must be noticed that this acylation reaction gives formation of 1,3,6’,3tetra-N-benzyloxycarbonyl derivative of kanamycin A and formation of non-acylated, initial kanamycin A simultaneously to some extent but actually brings about formation of tri-N-benzyloxycarbonyl derivative of kanamycin A only in a low yield, in spite of that the Nagabhushan et al's elucidation of the reaction mechanism might expect that the tri-N-benzyloxycarbonyl derivative would be formed in a higher yield than the other N-acylated derivatives. In the specification and particularly claim 4 of U.S. Patent No. 4,136,254, Nagabhushan et al have stated to the effect that a salt of a divalent transition metal such as copper (II), nickel (II), cobalt (II) etc., is necessary to be employed in a total quantity of at least 2 mol. per mol. of kanamycin A for the formation of kanamycin A-transition metal salt complex, as will be seen from the formula (I) given hereinbefore. Our experiment has revealed that,in contrast to the four transition metal cations, zinc cation is able to achieve the effect of block15 ing 1-amino and 3n-amino group of kanamycin A when zinc cation is employed in a total quantity of at least 1 mol per mol of kanamycin A. According to our test, it has been found that when a nickel salt'is reacted in a quantity of slightly more than 1 mol for 1 mol of kanamycin A followed by acylation of the resulting kanamycin A-nickel salt complex with benzyloxycarbonyl group, there is obtained only in a very much low yield 3,6*-di-N-benzyloxycarbonylkanamycin A which would be obtainable in a significant yield when kanamycin Λ-zinc salt complex was acylated. in view of the above-mentioned facts, it is concluded that zinc (II) cation exhibits a mechanism of complexing with an aminoglycoside which is different from the complexing mechanism of nickel (II), cobalt (II), copper (II) and cadmium (II), cation, and that the aminoglycoaidezinc cation complex has a complexing stability which is different from that of the complex of the aminoglycoside with nickel (II), cobalt (II), copper (II) or cadmium (II) cation. For the complexing of zinc cation with the aminoglycosidic antibiotic, zinc cation may be provided in the form of a zinc salt which is advantageously inexpensive and unlikely to be a source of polluting the environment.

In consequence, we,the present inventors, have found that when zinc cation is reacted in an inert organic solvent with an aminoglycosidic antibiotic which contains a deoxystreptamine moiety having a 3-aminoglycosyl or 3-alkylaminoglycosyl group linked to 6-hydroxyl group of the deoxystreptamine moiety and possibly having an aminoglycosyl group linked to 4-hydroxyl group of the deoxystreptamine moiety, zinc cation is complexed with and block amino-hydroxyl pairs locating at particular positions which may vary depending on the nature of the aminoglycosidic antibiotic; and that when the aminoglycosidic antibiotic-zinc cation complex so formed is reacted with an acylation reagent having an acyl group which is used conventionally for introduction of an amino-protecting group in the synthesis of polypeptides, this acylation reagent acylates at least one of such amino groups in the aminoglycosidic antibiotic which are not complexed with and hence not blocked by zinc cation, so that the amino group so acylated is protected; and also that when the resulting acylation product (ie., the aminoglycosidic antibiotic-zinc cation complex having the acylated amino group(s)) is treated with such a suitable reagent which will remove zinc cation from said acylation product, the zinc complex is destroyed, affording a selectively protected N-acylated derivative of the aminoglycosidic antibiotic of which the initially zinc-non-complexed amino group(s) has or have selectively been protected with the io acyl group.

T3 Such partially protected N-acylated derivative of the aminoglycosidic antibiotic consisting of a 6-0-(3amino or 3”-alkylamino-3-deoxyglycosyl)-2-deoxystreptamine with optional 4-0-(aminoglycosyl) group which is to be employed as the starting material in the process of this invention is such one in which derivative the 1-amino and 3-amino or 3-alkylamino groups of the deoxystreptamine moiety are unprotected but all the other amino groups of the aminoglycoside molecule have been protected with an acyl groups. This partially protected N-acylated derivative may be prepared by a method comprising the steps of: (i) reacting a zino salt with the aminoglycosidic antibiotic (as the initial material), in a proportion of more than 1 mol of the zinc salt for 1 mol of said aminoglycosidic antibiotic, at ambient temperature or under heating or under cooling, in an inert organic solvent chosen from dimethylsulfoxide, aqueous dimethylsulfoxide, dimethylformamide, aqueous dimethylformamide, mixture of dimethylsulfoxide ar.d dimethylformamide, tetrahydrofuran, aqueous tetrahydrofuran, methanol, aqueous methanol, ethanol and aqueous ethanol in the presence or absence of eodium acetate added, to form the aminoglycosidic antibiotic-zine cation complex, (ii) reacting said aminoglycosidic antibioticzinc cation complex with an acylating agent which is selected from a carboxylic acid of the following general formula (IVa): (IVa) wherein R^ is hydrogen, an alkyl group, particularly an alkyl group of 1-6 carbon atoms, an aryl group, particularly phenyl, or an aralkyl group, especially benzyl, and these groups being occasionally further substituted, or an acid halide, acid anhydride or active ester of said carboxylic acid (IVa); or a chloroformate of the following general formula (I7b): R5O-CO-CX (IVb) or a p-nitrophenyl carbonate of the following general formula (IVc): R5O-C0-O-C-R--o-NO, (IVc) 0 5 · c or acoive N-hydroxysuccinimide ester of the following formula (IVd): R5O-CO-O-N (IVd) or an azidoformate of the following formula (iVe): where R^ is as defined above; or an alkyl-, aryl- or aralkyl-sulfonic acid of the following general formula (IVf): (IVf) wherein R® is a hydrogen, an alkyl group, especially an alkyl group of 1-6 carbon atoms, an aryl group, particularly phenyl, or an aralkyl group, especially a phenylalkyl group such as benzyl, and these groups being occasionally further substituted, or an acid halide, acid anhydride or active ester of said alkyl-, aryl- or aralkyl-sulfonic acid, at a tenperature of -2O'C to 100'C, to acylate the non-coaplexed amino groups present in said amir.oglyoosic antibiotic-zinc cation complex, and thereby to produce the N-acylated amincglycosidic antibiotic-zinc cation complex, and (iii) reacting said H-acylated aninoglycosidic antibiotic-zinc cation complex with water, or with an aqueous or anhydrous polar organic solvent such as methanol, ethanol, ethylamine and triethylamine or with liquid ammonia, hydrogen sulfide, an alkali metal sulfile or an alkaline metal sulfide or ammonium hydroxide in water or with a cation-exchange resin containing carboxylic or sulfonic aoid functions, or with an anion-exchange resin containing ammonium functions, or with a chelate-exchange resin containing metal-chelating functions, or with chitin or chitosan as the water-insoluble high-colymer containing the metal-combining functions, to remove the zinc cations frcm said complex and thereby to produce the partially protected N-acylated aminoglycosidic antibiotic derivative.

This process is more fully described in Patent Specification No. 2026/79, from which this application is divided.

According to the process as described above (hereinafter sometimes called zinc-complexing process), it is feasible to prepare such a selectively but partially protected N-acylated derivative of the 5 aminoglycosidic antibiotic in which derivative all the ammo groups other than the two, 1-amino and 3-amino (or 3-alkylamino) groups of the aminoglycoside molecule are protected by an acyl group and hence 1amino and 3-amino (or 3-alkylamino) groups are remaining unprotected. Even when this partially protected N-acylated aminoglycosidic antibiotic derivative is reacted with an ai-hydrcxy-W-am.inoalkancic acid or its reactive equivalent for the purpose of effecting the 1-Sacylation as mentioned above, it is actual that there are yielded mixed acylation products comprising (i) the 1-N-acylated product where only 1-am.ino group of the aminoglycoside molecule has been acylated with the 3hydroxy-u.-aminoalkar.oic acid, (ii) the 3-N-acylated product where only 3*-amino (or 3-alkylamino) group has been acylated, (iii) both 1-amino and j-aroino (or 3alkylamino) groups have been acylated, and (iv) the unreacted material where none of 1- and 3-ainino (or 3-alAylarir.o) groups have been acylated. In order to obtain the ultimately desired 1-N-acylation product from the above mixed acylation products, therefore, it is always necessary to carry cut an additional step m which the 1-N-acylation product is isolated therefrom by chromatography or by any other isolation method. As the 1-amino group is fortunately more reactive than the 3-amino (or 3-alkylamino) group, actual yield of the desired 1-N-acylation product usually is about 40% to 60% and exceeds a theoretically maximum yield of 25% which would be calculated with assuming that the reactivity of 1- and 3-amino (or 3-alkylamino) groups should be entirely equal to each other. Nonetheless, even if the reaction conditions for the 1-N-acylation are adjusted to best ones, it is inevitable that the undesirably N-acylated products are by-fcrned, and always it needs an additional step to remove the undesired N-acylated by-products by subjecting the mixed acylated products carefully to a column chromatography.

In order to eliminate this disadvantage, it is obviously required to prepare such a selectively protected N-acylated derivative of the aminoglycosidic antibiotic in which all the amino groups other than 1amino group have been protected. In order to meet this requirement, we have made further research in an attempt to provide a process which is able to selectively protect 3-am.mo (or 3-alkylair.ino) group of the selectively but partially protected N-acylated aminoglycosidic antitictic derivative containing free 1- and 3-amine groups as obtair.ded from the above-described zinc-complexing process, while 1-amino group is remaining unblocked.

As a result, we have now succeeded to find out that when the partially protected N-acylated aminoglycosidic antibiotic derivative as obtained from the zinc-complexing process is reacted with ar. acylating agent selected from formic acid esters, dihalo- or trihalo-alkanoic acid esters, N-formylinidazole, 3amino or 3-alkylamino group can preferentially be acylated for the blocking purpose without acylating 1amino group. This selective 3-N-protecting process may be combined with the above-described zinc-complexing process so that there is produced in a facile and efficient way such a selectively protected N-acylated derivative of the aminoglycosidic antibiotic comprising a 6-0-(325 amino- or 3-alkylamino-3-deoxyglycosyl)-2-deoxystreptamine moiety in which derivative all the amino groups other than 1-amino group of the aminoglycoside molecule have been protected selectively with sarnie or different acyl groups. In the combination of the zinc-complexing process with the selective 3-N-protecting process, an advantage is obtained that the ultimately desired 1-Nur.protected but other N-fully-yrotected derivative of the aminoglycosidic antibiotic can be produced fror. the parent aminoglycosidic antibiotic material in an overall yield of 70% or mere. When this 1-N-unprotected but other N-fully-protected derivative is employed for the 1N-acylation of the aminoglycosidic antibiotic, there is provided a further advantage that the undesirably Nacylated products are substantially not by-formed, so that recovery and purification of the desired 1-Nacylation product is very facilitated.

According to the present invention, therefore, there is provided a process for the production of a selectively protected N-acylated derivative of an aminoglycosidic antibiotic comprising a 6-0-(3-aminoor 3-alkylamino-3-deoxyglycosyl)-2-deoxystreptamine moiety optionally having a 4-3-(aminoglycosyl) group in which derivative 1-amino group of the deoxystreptamine moiety is unprotected but all the other amino groups in the aminoglycoside molecule are protected with same or different acyl groups; the process comprising a step of:(a) reacting an alkanoic acid ester of the formula (VIII):48973 RaC-Rb (VIII) wherein Ra is a hydrogen atom or a dihaloalkyl or trihaloalkyl group of 1-6 carbon atoms, and Rb is an alkyloxy group of 1-6 carbon atoms, an aralkyloxy group, especially benzyloxy group, an aryloxy group, especially phenyloxy group, or an N-formylinidazole as the acylatir.c agent in an inert organic solvent with a partially protected N-acylated derivative of the aminoglycosidic antibiotic in which 1-amino and 3-amino or 3-alkylamino groups are unprotected and all the other amino groups are protected with an acyl group as the amino-protecting group, to effect selective acylation of 3-amino or 3-alkylamino group of the partially protected N-acylated derivative with the acyl group RaC0- of said acylating agent and thereby give the desired 1-N-unprotected and other N-fully-protected derivative of the aminociyccsidic antibiotic.

The acylating agent is preferably reacted with the partially protected N-acylated derivative of: kanamycin A; 6'-N-alkylkanamycin A; 3'-deoxykanamycin A; 6'-N-methy1-3'-deoxykanamycin A; 4’-deoxykanamycin A; J 6'-N-methy1-4'-deoxykanamycin A; 3',4'-dideoxykanamycin ' A; 6-deoxykanamycin A; 4,6-dideoxykanamycin A; kanamycin B; 3'-deoxykanamycin B; 4’-deoxykanamycin B; > 3',4'-dideoxykanamycin B; 3',41-dideoxy-3'-eno-kanamycin B; 6'-N-methyl-3',4'-dideoxykanamycin B; kanamycin C; 3'-deoxykanamycin C; 3',4'-dideoxykanamycin C; gentamicin A; gentamicin B; gentamicin C; verdamicin; sisomicin or netilmicin.

Embodiments of the process according to the present invention are now described more fully.

The partially protected N-acylated aminoglycosidic antibiotic derivative which is to be reacted with the acylating agent of formula (VIII) and of which all the amino groups other than 1-amino and 3-amino (or 3alkylamino) groups in the aminoglycoside molecule are protected may be such one which is produced the afoEsaid zinc-complexing process.

Accordingly, the acyl group originally present in the partially protected N-acylated aminoglycosidic antibiotic derivative 10 is the same as the acyl group (R^co-, R5OCO- or R^O^- group in the formula IVa_g) of the acylation reagent mentioned previously and generally may be an alkanoyl group, an aroyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, an 15 aryloxycarbonyl group, an alkylsulfonyl group, an aralkylsulfonyl group or an arylsulfonyl group known as the conventional amino-protecting group. Moreover, the partially protected N-acylated aminoglycosidic antibiotic derivative employed as the starting material may also be such one which has been prepared by the aforesaid Nagabhushar. et al's method according to U.S. patent No. 4,136,254.

In carrying out the process of the present invention, the partially protected N-acylated amino25 glycosidic antibiotic derivative having the unprotected 1- and 3-amino (or 3-alkylamino) groups is used as the starting material and is dissolved or suspended in an 48873 appropriate inert organic solvent. To the resulting solution or suspension is added an alkanoic acid ester of the fonnula (VIII) or N-formylimidazole as the acylating agent in an amount which is at least equimolar to the starting material used. The inert organic solvent may preferably be such one which shows a high dissolution power for the starting material, for example, dimethylsulfoxide, dimethylformamide and hexamethylphosphoric triamide, but it is possible to use tetrahydrofuran, dioxane, acetonitrile, nitromethane, sulfolane, dimethylacetamide, chloroform,dichloromethane, methanol, ethanol, n-butanol and t-butanol, as well as aqueous ones of these solvents. Benzene, toluene and ethylether, or aqueous mixtures thereof, may be used as the reaction medium solvent, though these are not very suitable as these bring about poorer yield of the desired product. With the acylating agent of the formula (Vlll), Ra may preferably be a dihaloalkyl or trihaloalkyl group, particularly dichloromethyl, trifluoromethyl or trichloromethyl,and Rb may preferably be an alkyloxy group such as methoxy or ethoxy. When Rb is an aryloxy group, it may be phenoxy. Particular examples of the acylating agent (VIII) include methyl formate, ethyl formate, butyl formate, benzyl formate, pbenyl formate, methyl dichloroacetate, ethyldichloroacetate, methyl trichloroacetate, phenyl trichloroacetate, methyl trifluoroacetate, ethyl tri fluoroacetate and phenyl trifluoroacetate. Using this class of the acylating agent, 3-amino group of the starting material can preferentially be formylated, dichloroacetylated, trichloroacetylated or trifluoroacetylated. Trifluoroacetic acid ester, especially ethyl trifluoroacetate is most preferred. This class of the acyl group is advantageous in that it is very easily removable in the subsequent N-deprotecting step by a conventional deprotection method. If the alkanoic acid alkyl ester of the formula (VIII) is not employed as the acylating 1° agent but in stead thereof a corresponding alkanoic acid anhydride or an active ester thereof such as the Nhydroxysuccinimide ester is employed for the acylation process (not in accordance with the invention), the selective acylation of 3-amino group ca.-.not be achieved but there is involved by-formation of 1-N-acylated product and/or formation of mixed acylation products mainly comprising the 1-N-acylated product. It is worthy of attention that the aimed selective acylation of 3-amino group cannot be then achieved when using an acid anhydride or active ester of the same alkanoic acid for the acylating agent.

The acylating agents of the formula (VIII) available in the present invention are different in reactivity and their reactivity are in a wide range of from strong to weak. When an acylating agent of a strong reactivity is employed, the acylating reaction may be conducted for a short reaction time under cooling.

While, when an acylating agent of a weak reactivity is employed, the acylating reaction may be effected either under heating or for a prolonged reaction time. In general, however, the reaction temperature may suitably be in a range of -30’ tc +120°C and the reaction time may appropriately be in a range of 30 minutes to 24 hours or even to 48 hours.

The desired selectively 3--acylated product so obtained may be recovered from the reaction mixture in a known manner, for example, by evaporation of the solvent or by precipitation with addition of water, if necessary, followed by further purification of the product.

The reaction mechanism by which the selective 3N-acylation can be achieved according to the process of the present invention is net yet fully elucidated. A possible interpretation is that the acylating agent of the formula (VIII) acylates at first a hydroxyl group of the starting material to form an ester product intermediately and this O-esterifying acyl 'j 2o group is then shifted or migrated to an amino group r (corresponding the 3-amino or 3-alkylamino group in = the case of the present process) when this amino group | is neighboring to the esterified hydroxyl intermediately formed, whereby the acylation of said amino group is resulted in. If this assumption is followed, it is possible to explain the reason why the 1-amino group which has no neighboring hydroxyl group cannot be acetylated.

Besides, there is a fact that the intermediate ester product cannot be obtained when the trifluoroacetylation or formylation is conducted. Reason why the ester product cannot be recovered upon the trifluoroacetylation or formylation, is probably that the O-trifluoroacetyl group or.O-formyl group is instable and that an amount of the instable O-acyl group which has not undergone the shifting to the amino group (namely, the known Ο * N acyl-migration) is removed from the acylated hydroxyl XO group in the course of recovery and purification of the 3-N-acylation product so as to restore the free hydroxyl group. However, this invention is not limited to the above interpretation of the reaction mechanism involved in the present process. Anyhow, it seems that amongst the compounds which are available as the acylating agent of the formula (VIII) according to the present invention, such ones are more suitable for the purpose of the present invention if they have an acyl group which is likely to give a more instable ester product when this acyl group is transformed into an O-acyl group by reacting with hydroxyl group and thus giving the ester product. Meanwhile, it is very interesting to notice that when the process of the present invention is carried out using in stead of the N-formylimidazol an N-alkanoyl-imidazole such as N-acetyl-imidazole, N-propionyl-imidazole and Nbutyroyl-imidazole, the 3-amino or 3-alkylamino group of the partially protected N-acylated aminoglycosidic antibiotic derivative is not acylated but a hydroxyl group neighboring to said 3-amino or 3-alkylamino group can be esterified by the alkanoyl group of the Nalkanoyl-imidazole employed to give an intermediate 0esterification product. When this O-esterification product or the whole reaction mixture containing this O-esterification product is subsequently treated with an alkaline reagent such as ammonium hydroxide at ambient temperature, the O-esterifying alkanoyl group is caused to shift or migrate to the neighboring 3-amino or 3alkylamino group, resulting in a selective acylation and hence protection of the 3-amino or 3-alkylamino group. Thus, the reaction mixture from the reaction of the partially protected N-acylated aminoglycosidic antibiotic derivative with an N-alkanoyl-imidazole is, at first, not found to contain the desired 3*-N-acylated product, but from said reaction mixture can be recovered the desired 3-N-acylated product only after the reaction mixture has been made alkaline by treating with an alkaline reagent such as aqueous ammonia (see Example 39 given hereinafter) .

As a valuable application of the process of the invention, it is possible to provide a high-yield process for the production of the 1-N-acylated aminoglycosidic antibiotic which is known semi-synthetic antibacterial agent.

A process of producing a 1-N-(α-hydroxy-w-aminoalkanoyl) aminoglyco5 sidic antibiotic starting from the parent aminoglycosidic antibiotic, the process comprising a combination of the step of preparing by the aforesaid zinc-complexing process such a partially protected N-acylated aminoglycosidic antibiotic derivative in which 1-amino and 3-amino or 3-alkylamir.o groups are unprotected and all the other amino groups are protected; the step of preparing the 1-N-unprotected and other N-fully-protected derivative by the selective 3N-acylating process of the present invention, the step of acylating 1-amino group of the 1-N-unprotected and other N-fully protected derivative obtained from the preceding 3-N-acylation step, with an a-hydroxy-ωaminoalkanoic acid, especially 3-amino-2-hydroxypropionic acid (isoserine) or 4-amino-2-hydroxybutyric acid; and finally the step of deprotecting from the 1-N-acylation product so obtained.

More particularly, there is possible an improved process of producing a 1-N-(a-hydroxy-w-aminoalkanoyl) derivative of an aminoglycosidic antibiotic comprising a 6-0-(3-amino or 3-alkylamino-3-deoxyglycosyl)-2-deoxystreptamine moiety having optionally a 4-0-(aminoglycosyl) group, the process comprising the consecutive steps of:(a) reacting zinc cations with the aminoglyco-* sidic antibiotic ir. an inert organic solvent to produce the complex of zinc cations with the aminoglycosidic antibiotic, (b) reacting an acylation reagent having an acy group to be introduced as the amino-protecting group, with the aminoglycosidic antibiotic-zinc cation complex formed in the above step (a) in situ in the inert organic solvent, to produce a complex of zir.c cations with the selectively N-acylated derivative of the aminoglycosidic antibiotic having the initially non-complexed amino groups acylated, (c) reacting the selectively N-acylated amino15 glycosidic antibiotic derivative-zinc cation complex obtained in the above step (b), with a reagent which removes zinc cations from the N-acylated zinc complex, to give a partially and selectively protected N-acylated aminoglycosidic antibiotic derivative which is free 2o from zinc cations and in which 1-amino and 3-amino or 3-alkylamino group are unprotected but all the other amino groups in the aminoglycoside molecule are protected by the acyl group, (d) reacting the partially and selectively 25 protected N-acylated derivative obtained in the above step (c) with an alkanoic acid ester of the formula (VIII) : » 48973 RaC-Rb (VIII) H o wherein Ra is a hydrogen atom or a dihaloalkyl or trihaloalkyl group of 1-6 carbon atoms and Rb is an alkyloxy group of 1-6 carbon atoms, an aralkyloxy group of 1-6 carbon atoms, particularly benzyloxy group or an aryloxy group, particularly phenoxy group, or N-formylimidazole as the acylating agent in an inert organic solvent to selectively acylate the 3-amino or 3-alkylamino group with the acyl group RaCO- of said acylating agent and thereby to give the 1-N-unprotected and other N-fully-acylated-protected derivative of the aminoglycosidic antibiotic in which all the amino groups other than 1-amino group are protected with acyl group, (e) reacting the 1-N-unprotected and other Nfully-protected derivative obtained in the preceding step (d) with an α-hydroxy-u-aminoalkanoic acid of the formula (IX): HOOC-CH(CH-) NH- (IX) ii m i H wherein m is 1 or 2 or an equivalent reactive derivative thereof of which the amino group is either unprotected or protected, to acylate 1-amino group of said 1-Nunprotected derivative, (f) and then removing the residual amino20 protecting groups from the 1-N-acylation product obtained 48873 in the above step (e) by a conventional deprotecting method.

We describe below more fully how to carry out the process described.

The aminoglycosidic antibiotics which are available as the initial material in the first step (a) of the process are the same as these described hereinbefore in respect of the zinc complexing process and the reaction of complexing zinc cations with the aminoglycosidic antibiotic is achieved in the same manner as described hereinbefore, too. The acylation of the aminoglycosidic antibioticzinc cation complex so obtained in the first step (a) may be effected in the second step (b) of the present process in the same way as described hereinbefore.

The removal of zinc cations from the selectively Nacylated aminoglycosidic antibiotic-zinc cation complex so obtained may be conducted in the third step (c) of the present process in various ways as described before, whereby there is obtained a partially and selectively protected N-acylated aminoglycosidic antibiotic derivative which is free from zinc cations and in which 1-amino and 3-amino or 3-alkylamino groups are unprotected but all the other amino groups in the aminoglycoside molecule are blocked with the acyl group of the acylation reagent employed in the step (b).

This partially and selectively protected N-acylated derivative of the aminoglycosidic antibiotic is then reacted with an alkanoic acid ester of the formula (Viii) or N-formylimidazole in the step (d) in the same manner as described hereinbefore in respect of the process of the present invention, to obtain the selective 3-N-acylation of the partially N-protected aminoglycosidic antibiotic derivative without acylation of 1-amino group thereof.

In the fifth step (e), the 1-N-unprotected and other N-fully-protected derivative of the aminoglycosidic antibiotic obtained in the preceding step (d) is reacted with an i-hydroxy-^15 aminoalkanoic acid of the formula (X), particularly 3amino-2-hydroxypropionic acid (as DL-iscserine, Disoserine or L-iscserine) or L-4-amino-2-hydroxybutyric acid to acylate 1-amino group of the aminoglycosidic antibiotic with the 3-amino-2-hydroxypropionyl or 420 amino-2-hydroxyburyryl group. This 1-N-acylation may be conducted generally as described in the specification of U.K. patent No. 1,426,908 or U.S. patent No. 4,001,208 according to any known method of synthesis of amides by reacting the protected aminoglycosidic antibiotic derivative with an isoserine or L-4-amino-2-hydroxybutyric acid, either in its free acid form or in the form of its reactive equivalent such as an active ester, eg. the 48873 dicyclohexylcarbodiinide ester, mixed acid anhydride, acid azide in an inert organic solvent such as dioxane, 1,2-dimethoxyethane, dimethylformamide, tetrahydrofuran or aqueous ones of these solvents. Isoserine and L-45 amino-2-hydroxybutyric acid may be such ones of which amino group has been blocked with an amino-protecting group. Suitable amino-protecting group for this purpose may be the same as or different from that one which was used in the 1-N-unprotected but other N10 fully-protected aminoglycosidic antibiotic derivative to be 1-N-acylated. t-Butoxycarbonyl group is a preferred amino-protecting group, as it is readily removable by treating with a dilute acid such as aqueous trifluoroacetic acid, aqueous acetic acid and diluted hydrochloric acid. Benzyloxycarbonyl group which is removed by conventional catalytic hydrogenolysis over palladium or platinum oxide catalyst, as well as phthaloyl group which is easily removed by hydrolysis with hydrazine are very convenient as the amino20 protecting group to this end.

The acylating reaction in the 1-N-acylation step (e) of the process may preferably be conducted in an aqueous organic solvent using an active ester of the a-hydroxy-w-aminoalkanoic acid (X). The suitable active ester may be Nhydroxysuccinimide ester of isoserine or L-4benzyloxycarbonylamino-2-hydroxybutyric acid, and this active ester may be employed in a quantity of 1 to 2 mol., favorably of 1 to 1.5 mol. per mol. of the aminoglycoside to be 1-N-acylated. The water-miscible organic solvent for use in the reaction medium may preferably be dioxane, 1,2-dimethoxyethane, dimethylformamide, tetrahydrofuran .

Subsequently to the abcve step (e), the Ndeprotection step (f) is carried out to remove all the residual amino-protecting groups from the 1-N-acylation product obtained in the above step (e). The removal of the residual amino-protecting group may be achieved by a conventional N-deprotecting technique. Such a residual amino-protecting group which is of an alkoxycarbonyl type may be removed by hydrolysis with an aqueous solution oi trifluoroacetic acid or acetic acid or with a diluted acid solution such as dilute hydrochloric acid. Such a residual aminoprotecting group which is of an aralkyloxycarbonyl type, for example, benzyloxycarbonyl is readily removed by conventional catalytic hydrogenolysis. When all the residual amino-protecting groups are removed from the 1-N-acylation product of the step (e), the desired 1-N-(2-hydroxy-3-aminopropionyl)or 1-N-(2-hydroxy-4-aminobutyryl)-aminoglycosidic antibiotic is obtained in a high yield.

Examples of the l-N-(a-hydroxy-u-aminoalkanoyl)aminoglycosidic antibiotic which is produced by the process are listed below: (1) 1-N-(L-4-amino-2-hydroxybutyryl)- kanamycin A (2) 1-N-(L-4-amino-2-hydroxybutyryl)-31 - 5 deoxykanamycin A (3) 1-N-(L-4-amino-2-hydroxybutyryl) -3',41-dideoxykanamycin A • (4) 1-N-(L-4-amino-2-hydroxybutyryl)-tobramycin (5) l-N-(L-4-amino-2-hydroxybutyryl)-dibekacin 10 (6) 1-N-(3-amino-2-hydroxypropionyl)-dibekacin.

Another application of the processes of this invention is to produce 1-Nalkyl aminoglycosidic antibiotic from the all Nacylated aminoglycosidic derivatives containing unprotected 1-amino group, and an example of this application is to produce netilmicin or its 1-N-alkylanalogues from sisomicin by alkylation with a lower aliphatic aldehyde and cyanoborohydride.

This invention is further illustrated but not limited by the following Examples Example 1 Production of 3.6'-di-K-ber.zyIoxyoarbenyl-3N-trifluoroacetylkanamycin A A solution of 504 ag of 3,6'-di-.'I-benzyloxycartOnylkanasycin A in 4 mZ of dimethylsulfoxide was admixed with 220 mg of ethyl trifluoroacetate, and the admixture obtained was allowed to stand overnight at ambient temperature. After a small volume of trifluoroacetic acid was added to the reaction mixture, the reaction solution was poured into a large volume of ethyl ether ar.d the resultant oily material deposited was washed well with ethyl ether to afford the solidified material. This material was dried well to obtain 640 ag of the titled compound as a solid substance. Yield 99», [a]^ + 98* (c 1, pyridine) Elemental analysis Calcd.

Found: Exancle 2 for 0,0H47N4Cl6?rCF5CMH C 47.4C; H 5.02s U 5.82% C 47.13} H 5.15: N 5.79/* Production of 5,p'-dl-N-benzylcx.ycarbor.yl-3y-trlfluoroacetylkanaa.ycin A A solution of 20 ag of 3,6'-di-N-ber.zyloxyearbonylkar.amycin A in 0.4 mZ of dimethylsulfoxide was admixed with 6 mg of phenyl trifluoroacetate, and the admixture obtained was allowed to stand overnight at ambient temperature. Subsequently, the reaction mixture was processed in the same manner as in Exanple 1, affording 24.3 mg of the titled croduct which was found identical to that of Example 1 . Yield 97%.

Example 3 Production of 5.6'-dl-N-henzyloxycarbor.yl-3.’.'-triflucroacetylkanamycin A A solution of 10 ag of 3,6'-di-h'-ber.zyloxycarbonylkar.amycir. A ir. 0.3 cZ of hexamethylpr.es choric triamide was admixed wi*h ~ zs; of ethyl trifluoroaceta'e, and the admixture obtained was allowed to stand overnisht at ambient temperature. The reaction solution wa3 admixed with a small volume of trifluoroacetic acid and then poured ir.to a large volume of ethyl ether. The oily material deposited was washed well with ethyl ether and the resultant solid sutstar.-e dried to give 11.7 mg (yield 91%) of the titled product as its mono-trifluoroocetate in the form cf a solid.

Examcle 4 Production of 3,8’-di-N-benzyloxycarbonyl-3S-trlfluorcacetylkanamycln A A sus-ension of 10 mg of 3,6'-di-N-benzyloxycarbonylkar.amycin A ir. C.3 mZ of dimethylformamide was admixed with 7 me of ethyl trifluoroacetate, and the admixture obtained was allowed to stand overnight at ambient temperature.

The homogeneous reaction solution thus obtained was admixed with a small volume of trifluoroacetic acid and then poured into a large volume of ethyl ether. The oily material deposited was washed well with ethyl ether to solidify it and the resultant solid substance dried, affording 11.5 mg (Yield 90») of the titled product as its monotrifluoroacetate ir. the form of a solid.

Example 5 Production of 5,£'—di-N-ben.z,*.,loxycarbonyl-3— N-trif luor pace tylkanamyoir. A A susae-sior. of 10 mg cf T,£'-di-N-benzyloxycarbcnylkan.amycir. A in 0.35 m2 of sulfolane was admixed with 7 mg of ethyl trifluoroacetate, ar.d the admixture was Stirred overnight at ambient temperature. Subsequently, the reaction mixture was processed in the same manner as ln Example 1 , affording 12.0 mg (Yield 94^) of the titled product as the monc-trifluoroacetate in the form of a solid substance.

Example 6 Production of 5.6'-di-N-benzyloxycarbonyl-5N-trifluoroaoetylkanamycln A A suspension of 22 mg of 3,6'-di-N-be.nzyloxycarbonylkar.amyoin A ln 0.5 m2 of tetrahydrofuran was admixed with 10 mg of ethyl trifluoroacetate, and the admixture was stirred for 2 days. The resulting homogeneous reaction solution was admixed with 15 mg of ethyl trifluoroacetate and 8 mg of anhydrous sodium carbonate, stirred overnight and then allowed to stand for 2 days. The resultant reaction solution was concentrated tc a small volume, and the concentrate was washed with water and then dried to give a solid material. The solid material was suspended in a small volume of tetrahydrofuran together with a small amount of trifluoroacetic acid. The admixture so obtained was stirred followed by addition of ethyl ether. The precipitated solid was filtered off, washed with ether and dried, giving 21 mg (Yield 74%) of the titled product mor.o-trifluoroacetate as a solid substance, [a]p^ + gs’ (c 1, Pyridine).

Example 7 Production of 5.6<-dl-N-ber.zyloxycarbonyl-3H-trifluorcacetylkanamycin A A solution of 10 mg of 3,6'-di-N-benzyloxycarbonylkanacycin A in water-tetrahydrofuran (1:1, 0.3 mZ) was admixed with a solution of 5 ng of ethyl trifluoroacetate in 0.1 mZ of tetrahydrofuran, and the resultant admixture was allowed to stand at ambient temperature for one day. Subsequently, a mixture of ethyl trifluoroacetate (10 mg), anhydrous sodium carbonate (4.4 mg) and tetrahydrofuran (0.1 mZ) was added to the resultant solution at 5 hours 20 intervals (four times in all) to effect the 3-N-trifluoroacetylation. The reaction solution was concentrated and then treated in the same manner as in Excmple 6 to give 5.5 mg (Yield 43%) of the titled product mono-trifluoroacetate as a solid substance.

Example 8 Production of 5,6'-dl-N-benzyloxycarbonyl-?N-trlfluoroacetylkar.amycln A A solution of 10 mg of 3,6'-di-N-benzyloxycarbonylkanamycin A in water-ethanol (2:3, 0.6 ml) was admixed with a solution of 5 mg of ethyl trifluoroacetate in 0.1 ml of tetrahydrofuran, and the admixture was allowed to stand at ambient temperature for one day. The reaction solution was thereafter processed in the same way as in Example 6, affording 2.3 mg (Yield 18%) of the titled product monotrifluoroacetate as a solid substance.

Example 9 Production of 3,6'-di-N-t-butoxycarbonyl-3N-trifluoroacetylkanamycin A (a) Preparation of 3,6'-di-N-t-butoxycarbonylkanamycln A 500 mg (1.03 m moles, of kanamycin A (free base) was suspended in 12 ml of dimethylsulfoxide, and 1 g (4.55 m moles) of zinc acetate dihydrate was added to the suspension obtained. The mixture was stirred at room temperature until it formed a homogenous solution, to which was then added 370 mg (2.59 m moles) of t-butoxycarbonyl azide. The resultant mixture was allowed to stand overnight at room temperature and was then poured into ethyl ether and the oil separated was washed several times with further volumes of ethyl ether to afford a thick syrupy material.

The syrup-like material obtained above was dissolved in water-dioxane (1:1) and the solution was passed through a column of Amberlite CG 50 resin (NH4+form) and subjected to linear gradient elution with water-dioxane (1:1) containing 0 to 0.1 N ammonia. No zinc cation was eluted but the desired product was eluted. The fractions of the eluate containing the desired butoxycarbonylation product were concentrated to dryness to afford 590 mg (80%) of a colorless solid of the titled compound, [efJ2® + 89' (c 1, water-dimethylformamide, 1:2). (b) Production of 3,6'-di-N-t-butoxycarbonyl-S11N-trifluoroacetylkanamycln A 3,6'-di-N-t-butoxycarbonylkanamycin A (60 mg) was ,. .4 8973 dissolved in 0.5 mZ of dimethylsulfoxide, and the resulting solution was admixed with 25 ng of ethvl trifluoroacetate, followed by allowing the admixture obtained to stand overnight at ambient temperature. Subsequently, the reaction solution was processed in the same manner as described in Example l, giving 76.8 mg (Yield 98%) of the titled ρς compound trifluoroacetate as a solid. [a]j + 72* (c 1, water-dimethylformamide, 1:2).

Elemental analysis Caled. for CjoH^N^Q^gE^· COOH: C 42.95; H 5.86; N 6.26% Found: C 42.77; H 5.92; N 6.38% Example 10 Production of 3,6'-di-K-(p-methoxybenzyloxycarbonyl)· 3-E-trifluorcacetylksnamycln A A solution of 40 mg of 3,6'-di-N-(p-methoxybenzyloxy· carbonyl)kanamycin A in 0.4 mZ of dimethylsulfoxide was admixed with IS mg of ethyl trifluoroacetate, and the admixture was allowed to stand over20 night at amt lent temperature. Subsequently, the reaction solution was processed in the same manner as in Example 1 , affording 49.3 mg (Yield 98%) of the titled compound as a solid substance. + 75· (c if water-dimethylformamide, 1:2) Elemental analysis Caled. for C38H51N4O18F3’CF3COOH: C 46.97; H 5.12; N 5.48% I Found: C 47.18; H 5.03; N 5.31# Example 11 Production of 3,6'.3-trl-N-trifluoroacetylkanamycin A mg of 3,6'-di-N-trifluoroacetylkanamycin A and triethylamine (12 mg) were admixed with 0.6 mZ of dimethylsulfoxide and then with 35 cc of ethyl trifluoroacetate, and the admixture was stirred overnight to effect the desired 3-N-trifluoroacetylation. The reaction solution was then precessed ir. the same manner as in Example 1 , affording 94.2 mg (Yield 96#) of t're titled oompound as a solid substance, + 76’ (c 1, water-dimethylformamide, 1:2) Elemental analysis Calcd. for C-^jN^^Fg-CFjCOOH C 35.22; H 3.57; N 6.32# Found: C 35.09; H 3.99; N 6.07# Example 12 Production of 3,6t-dl-?J-pkenoxycarbonyi-3N-trlfluoroacetylkar.amycln A A solution of 53 mg of 3,6'-di-N-pher.cxycarbcnylkanamyoin A and triethylamine (9 mg) in 0.5 mZ of dimethylsulfoxide was admixed with 23 mg of methyl trifluoroacetate, and the admixture was then processed in the same manner as ia Example 1 , affording 65 ag (Yield 95#) of the titled compound as a solid material. [α]^5 + 70* (c 1, water-dimethylformamide, 1:2) Elemental analysis Calcd. for Cj^jN^gFj-CFjCOOH C 46.26; H 4.^4: N 5-99% Found: C 45.88; H 4.96; N 5.77% Example 13 Production cf 3,6'.5-tri-3-fcrmylksnaEycin A A mixture of 62 cg of J,ή'-di-Y-forcylkananycin A, mg of ethyl formate and 1 mZ of dimethylsulfoxide was heated at 130*0 for 12 hours in a sealed tube to effect the desired 3-3-formylation. The reaction solution was admixed with a little amount of formic acid, then poured into a large volume of ethyl ether, and processed in the same manner as in Example 1 , affording 6? mg (Yield 98%) of the titled compound as a solid material positive to ninhydrin. [a]p^+ 109* (c 1, water-dimethylformamide, 1:2) Elemental analysis Calcd. for C-^g^O^.HCOOE C 43.00; H 6.23; 3 9.12% Found: C 42.83; H 6.19: 3 9.10% Examcle 14 Production of T.S'-dl-N-te-zyloxycartonyl-S'-Nmethyl-3-N-trlfluoroacetylkar.acycin A A mixture of 68 mg of 3,6'-di-3-benzyloxycarbonyΙό '-N-methylkanamycin A and triethylamine (11 mg), 30 mg of ethyl trifluoroacetate and 0.7 mZ of dimethylsulfoxide was treated in the same manner as in Example 1 , affording 86 mg (Yield 99%) of the titled compound mono-trifluoroacetate as a solid substance. [a)p5 + 65* (e 1, water-dimethylformamide,1:2) Example 15 Production of 3,6l-di-N-benzyloxycarbor.yl-3ldeoxy-5-N-trifluoroacetylkanamycin A A solution of 52 mg of 3,6'-di-N-ter.zyloxycarbonyl3'-deoxykanamycin A and triethyiamine (11 mg) in 0.4 m2 of dimethylsulfoxide was admixed with 21 mg of ethyl trifluoroacetate, and the admixture was allowed to stand overnight at ambient temperature. Subsequently, the reaction solution was processed in the same manner as in Example 1 , affording 64.8 mg (Yield 97%) of the titled compound as a solid material, + 70* (c 1, water-dimethylformamide, 1:2) Elemental analysis Calcd. for CjgH^i^O.-FyCFjCCOH C 48.21; H 5.11; N 5.92% Found: C 47.94; H 5.35; N 5.77% Example 16 Production of 5,6’-di-N-oer.2yloxycarbor.yl-5'deoxy-3-N-formylk5r.amycln A A solution of 78 mg of 3,6'-di-M-ber.zyloxycarbonyl3’-deoxykanamycin A in 0.7 m2 of dimethylsulfoxide was admixed with 65 mg of phenyl formate, and the admixture was heated overnight at 50*C for the 3-N-formylation.

The reaction solution was admixed with a small amount of formic acid, and processed in the same manner as in Example 1 , giving 83 mg (Yield 97%) of the titled compound monoformate as a solid substance. [a]^ + 84' (cl, water5 dimethylformamide, 1:2) Example 17 Production of 5,6'-dl-y-ben2yloxycarbon.vl-5y-dlchlorcacetyl-3'-deoxykanamycin A A solution of 35 mg of 3,6'-di-K-benzyloxyoarbonyl10 3'-deoxykanamycin A in 0,5 tt7 of dimethylsulfoxide was admixed with 12 mg of methyl diohloroacetate, and the admixture was allowed to stand overnight at ambient temperature. The reaction solution was admixed with a small volume of dichlorcaoetio aoid ar.d then treated in the same manner as in Example 1 , giving 44.5 mg (Yield 96%) of the oe titled ccmDound as a solid substance, [al^ + 65* (c 1, water-dimethylformamide, 1:2) Elemental analysis Calcd, for '-36H4gN4Oi5CZ2*CKCZ2CCOH C 46.73; H 5.16; N 5.74; CZ 14.52% Found: C 46.58; H 5.33; S 5.62; CZ 14.28% Example 18 Production of _3,6 '-dl-K-benzyloxyearbor.yl-3N-trlchloroacetyl-3'-deoxykanamycin A A solution of 58 mg of 3,6'-di-N-benzyloxycarbonyl3'-deoxykanamycin A in 0.7 mZ of dimethylsulfoxide was admixed with 25 mg of methyl trichloroacetate, and the admixture was allowed to stand overnight at 50 *C. The reaction solution was admixed with a small volume of trichloroacetic acid and then processed ir. the same manner as in Example l, affording 80.5 mg (Yield 98%) of the titled compound as a solid substance, Ca]^^ + 65· (c q, water-dimethylformamide, 1:2) Elemental analysis Calcd. for C^H^C^CiyCC^CCjH C 43.65: H 4.63; N 5.36; CZ 20.34% Found: C 43.44; H 4.77; N 5.30; CZ 20.19% Example 19 Production of 3.6l-di-.Y-ber.zyIoxycarbonyl-5'deoxy-3-N-trifluoroacetyl-6’-N-methylkanamycin A A solution of 72 mg of 3,6'-di-N-ber.zyloxycarbonyl3'-deoxy-6,-N-cethylkanamyclr. A in 1 mZ of dimethylsulfoxide was admixed with 30 ag of ethyl trifluoroacetate, and the admixture was allowed to stand overnight at ambient temperature. Subsequently, the reaction solution was processed in the 3aae manner as in Example 1 , affording 89.5 ng (Yield 97%) of the titled compound, mono-trlfluoroacetate as a solid substance. [a]p^ + 70’ (c 1, water-dimethylformamide, 1:2) Example 20 Production of 5.6,-di-N-benzyloxycarbonyi-4ldeoxy-3-N-trlfluoroacetylkanamycln A A solution of 71 mg of 3,6'-di-N-benzyloxyearbonyl4’-deoxykanamycin A triethylamine (12 mg) and 30 mg of ethyl trifluoroacetate in 1 mZ of dimethylsulfoxide was processed in the same manner as in Example 1 , giving 90 ng (Yield 99%) of the titled compound mono-trifluoroacetate as a solid substance [a]p5 + 72* (c 1, water-dimethylformamide, 1:2) Example 21 Production of 3,6l-dl-??-'ber.zyloxycarbonyl-?l.4ldidecxy-3”-y-trifluoroacetylkanamycln A A solution of 75 ng of 3,6'-di-II-benzyloxycarbonyl3',4'-dideoxykanamycin A and 30 mg of ethyl trifluoroacetate in 1 mZ of dimethylsulfoxide was treated in the same manner as in Example l , affording 96 ng (Yield 99%) of the titled compound as a solid substance, [a]^ + 72* (c 1, water-dimethylsulfoxlde, 1:2) Elemental analysis Calcd. for C^gH^^N^O^^Ej-CFjCOOH: C 49.03; H 5.20; N 6.02% Found: C 48.83; H 5.46; N 5.87% Example 22 Production of 3.6l-di-y-benzyloxycaΓbor.yl-5l.4,dldeoxy-3-N-formylkanamycln A ng of 3,6'-di-N-tenzyloxycarbony1-3',4'-dideoxykanamycin A and 65 ng of phenyl formate were dissolved in 1 mZ of dimethylsulfoxide and the resultant solution was processed in the same manner as in Example 16 , affording 80 mg (Yield 97%) of the titled compound monoformate aa a solid substance. [a]^ + 80* (c 1, water-dimethylform25 amide, 1:2) Example 23 Production of 3.6,-di-N-benzyloxycarbonyl-3,.4'dideoxy-3-N-dlchloroacetylkanamycln A A solution of 68 mg of 3,6'-di-N-benzyloxycarbonyl31,4’-dideoxykanamycin A in 0.9 m2 of dimethylsulfoxide was admixed with 25 mg of methyl dlchloroacetate, and the admixture was allowed to stand overnight at ambient temperature. The reaction solution was admixed with a small amount of dichloroacetic acid and then processed in the same manner as in Example 1, affording 88 mg (Yield 97#) of the titled compound mono-dichloroacetate as a solid substance, + 67· (c 1, water-dimethylformamide, 1:2) Example 24 Production of 3.2'.6'-trl-?.'-benzyloxycarbonyl-3N-trlfluoroaeetylkanamyeln 3 A solution of 78 mg of 3,2',6’-tri-N-benzyloxycarbonylkanamycin B and triethylamine (11 mg) in 1 m2 of dimethylsulfoxide was admixed with 35 mg of ethyl trifluoroacetate, and the admixture was processed in the same manner aa in Example 1 , affording 92 mg (Yield 95#) of the titled compound monotrlfluoroacetate as a solid substance, [a]^ + 60* (c 1, water-dimethylformamide, 1:2) 8973 Example 25 N-formyltobramycln A solution of 82 mg of 3,2',6'-tri-M-benzyloxycarbonyl-tobramycin and triethylamine (12 mg) in 1.2 mZ of dimethylsulfoxide was admixed with 60 mg of pher.yl formate, and the admixture was processed in the same manner as in Example 16, affording 86 mg (Yield 97%) of the titled compound as a solid subρ C stance, (alp + 71* (c 1, water-dimethylformamide, 1:2) Elemental analysis Caled. for C^K^NjO^-HCCOH C 55.98; H 6.09; N 7.42% Found: C 55.50; H 6.22; N 7.28% Example 26 Production of 3,2',6'-trl-N-ber.zyloxycarbonyl-6lN-methyl-3-N-trifluoroacetyltobramycin A solution of 80 mg of 3,2',6'-tri-N-benzyloxycarbonyl-6'-N-methyltobramyoin ar.d triethylamine (12 mg) in 1.2 mZ of dimethylsulfoxide was admixed with 30 mg of ethyl trifluoroacetate, and the admixture was then processed ir. the same manner as in Example 1, affording 97 mg (Yield 98%) of the titled compound mor.c-trifluoroaeetate as a solid substance. [alp5 + 60’ (c 1, water-dimethylformamide, 1:2) Example 27 Production of 3,2' .C-trl-N-tenmyloxycarbonyl·-?N-trifluoroacetyldibekac in A solution of 82 mg of 3,2',6'-tri-N-benzyloxycarbonyl -dibekacin in 1 m2 of dimethylsulfoxide was admixed with 3C mg of ethyl trifluoroacetate , and the admixture was processed in the same manner as in Example 1, affording 100 mg (Yield 98%) of pC the titled compound as a solid substance. [a]jj + 61* (c 1, water-dimethylformamide, 1:2) Elemental analysis Calcd. for C^H^N^O^FyCFjCOOH C 51.93? H 5.21? N 6.58% Found: C 51.84; H 5.38; N 6.47% Example 28 Production of 3.21,6',3-tetra-N-trifluoroacetyldibekacin A mixture of 71 mg of 3,2',6'-tri-S-trifluoroacetyldibekacin and 30 mg of ethyl trifluorcacetate in 1 m2 of dimethylsulfoxide was allowed to stand overnight at 40*C. Subsequently, the reaction solution was processed in the sane manner as in Example 1, affording 90 mg (Yield 99%) of the titled compound as a solid substance, [a]^ + 70* (c 1, water-dimethylformamide, 1:2) Elemental analysis Calcd. for C 35.42; H 3.61; N 7.38% Found: C 35.40; H 3.89; N 7.17% Example 29 Production of 3.2l.6'-tri-?T-benzyloxycarbonyl3-N-formyldibekacin A mixture of 79 mg of 3,2',6'-tri-N-ber.zyloxycp.rbcnyi-dibekaein and 60 mg of phenyl formate in 1.1 mZ of dimethylsulfoxide was processed in the same manner as in Example 16, affording 84 mg (Yield 98%) of the titled compound monoformate as a solid substance, [a]^^ + 70* (c 1, water-dimethylformamide, 1:2) Example 30 Production of 5,2'.6l-tri-N-benzyloxyearbonyl5°N-dlchloroacetyldibekacin A solution of 84 mg of 3,2',6'-tri-N-benzyloxycarbonyl-iibekacin in 1.2 mZ of dimethylsulfoxide was reacted with 25 mg of methyl dichloroacetate in the same manner as in Example 17, affording 104 mg (Yield 97%) of the titled compound mono-dichloroacetate as a solid substance. + 59· (c water-dimethylformamide, 1:2) Sxamcle 31 Production of 3,2',6'-tri-3-berz.yloxycarbonyl6'-Y-nethyl-5-N-trifluoroacetyldibekacln A solution of 85 mg of 3,2',6'-tri-3-benzyloxycarbonyl-6'-N-methyldibekacin in 1 ai of dimethylsulfoxide was admixed with 30 mg of ethyl trifluoroacetate, and the admixture was processed in the same manner aa in Example 1 , affording 103.5 mg (Yield 98%) of the titled compound mono-trifluoroacetate as a solid substance, + 60’ (c 1, water-dimethylformamide, 1:2) Example 32 Production of 3.2,-dl-N-benzyloxycarbonyl-311N-formylkanamycin C A solution of 81 mg of 3,2'-di-N-benzyloxycarbonylkanamycin C and triethylamine (14 ng) in 1.5 mZ of dimethylsulfoxide was admixed with ?C mg of ethyl formate., and the admixture obtained was treated in the same manner as in Example 16, affording 85.5 ng (Yield 96%) of the titled compound monofornate as a solid substance. [a]p5 + 81* (e 1, water-dimethylformamide, 1:2) Example 33 Production of 3,2'.6'-tri-N-benzyloxyoarbonyl-3N-triflucroacetylsisomicin A solution of S2 mg of 3,2', δ'-tri-E-ber.syloxycarbonyl-sisosicin in 1.5 mZ of dimethylsulfoxide was admixed with 3C mg of ethyl trifluoroaoetate, and the admixture was processed in the same manner as in Example 1 , affording 99 mg (Yield 97%) of the titled compound mono-trifluorcacetate as a solid substance. [a]p^ + 151’ (c 1. water-dicethylfcrmamide, 1:2) Example 34 Production of 3.2',6'-tri-E-benzyloxycarbonyl-5HN-trifluoroacetylnetllnlcin A solution of 85 mg of 3,2' 6'-tri-N-benzyloxy489 73 carbonyl-netilmicin in 1.3 oZ of dimethylsulfoxide waa admixed with 30 mg of ethyl trifluoroacetate, and the admixture was processed in the same manner as in Example 1, affording 103 mg (Yield 98%) of the titled compound monopc trifluoroacetate as a solid substance. + 145’ (e 1, water-dimethylforcamiie, 1:2) Example 35 Production of 5,6'-dl-y-benzyloxycarbonyl-3N-trifluorcacetylgentamlcir. 5 A solution of 72 mg of 3,c'-di-R-benzyloxycarbonylgentamicir. 3 in 1.2 mi of dimethylsulfoxide was admixed with 30 mg of ethyl trifluoroacetace, and the admixture was processed in the same manner as in Example 1, affording 91 mg (Yield 99%) of the titled compound mono-tripc fluoroacetate as a solid substance. + 92* (c 1, water-dicethylformamide, 1:2) Example 36 Production of 3.2' ,6'-tri-”-ber.zyloxycarbonyl-5R-trifluoroacetylger.tamicin and Cj.a mixture A solution of 84 mg of 3,2',6'-tri-R-benzyloxycartonyl-ger.tamirin Cj and 3ia mixture in 1.5 mZ of dimethylsulfoxide was admixed wcth 30 mg of ethyl trifluoroacetate, and the resultant admixture was processed in the same manner as in Example 1 , affording 101 mg of the titled compound mono-trifluoroacetate as a solid substance, [aJp^ + 87’ (c 1, water-dimethylformamide, 1:2) 48873 Example 37 Production of 3.2l.6t-tri-??-ber.zyloxycarbonyl3'.4'-dideoxy-3l-eno-3tl-N-trlfluoroacetylkanamycln B A mixture of 83 mg of 3,2', 6'-tri-N'-benzyloxy5 carbonyl-3',4'-dideoxy-3'-eno-kanamycin B and 35 og of ethyl trifluoroacetate in 1.2 al of dimethylsulfoxide was allowed to stand overnight at ambient temperature. Subsequently, the reaction solution was processed in the same manner as in Example 1 , afford10 ing 99.5 og (Yield 96#) of the titled compound mono-trifluoroacetate as a solid substance, [a]^ + 26* (c 1, water-dimethylformamide, 1:2) Example 38 Production of 3,6l-dl-N-ber.zyloxycarbonyl-5’-decxy15 3'-N-formyIk;:inaaycin A A solution of 90 mg of 3,6'-di-N-benzyloxycarbonyl3'-deoxykanamycin A in 0.8 of of dimethylsulfoxide was admixed with 13 mg of H-formylimidazole, and the admixture was allowed to stand at ambient temperature overnight. 2o The reaction solution was admixed with a little amount of formic acid and then treated with ethyl ether as in Example 1 , affording 94 mg (Yield 95#) of the titled compound monoformate as a solid substance.

Example 39 Production of 3.6'.3-trl-N-aoetylkanamycin A A mixture of 100 ag of 3,6'-di-H-acetylkanamycin A and 20 mg (1.03 molar proportion for 1 mol of the starting material) of N-acetylimidazole in 1 mZ of dimethylsulfoxide was stirred under ice-cooling for 3 hours and then allowed to stand at ambient temperature overnight. The reaction solution was made alkaline by admixing with 0.3 mZ of 28% aqueous ammonia and then allowed to stand at ambient temperature for 3 days. The resultant reaction mixture was treated with ethyl ether to give an ether-insoluble syrup.

The syrup was taken up into water ar.d then passed through a column of CM-Sephadex C-25 (SH4+ -form) (a product of Pharmacia Fine Chemicals Co., Sweden). The resin column was developed with 0.05N aqueous ammonia. The fractions containing the desired product eluted out were combined together and concentrated to dryness. The concentrate was taken up into water, and the aqueous solution was neutralized with acetic acid ar.d again concentrated to dryness, affording 109 mg (Yield 90%) of the titled compound as a solid product. + 98* (c 1, water-dimethylformamide, 1:2) Elemental analysis Calcd. for C24H42N4O14.CH5COOH-H2O C 45.34; H 7.02; N 8.14% Found: C 45.22; H 7.20; N 8.11% The words AMBERLITE and SEPHADEX used herein are Trade Marks.

Claims (5)

CLAIMS;

1. A process for the production of a selectively protected N-acylated derivative of an aminoglycosidic antibiotic comprising a 6-0-(3-amino- or 3-alkylamino-35 deoxyglycosyl)-2-deoxystreptamine moiety in which the 1amino group is unprotected and all the other amino groups in the amino-glycoside molecule are protected, the process comprising a step of reacting an acylating agent which is an alkanoic acid ester of the formula 10 R a C-R b N wherein R a is a hydrogen atom or a dihaloalkyl or trihaloalkyl group of 1-6 carbon atoms, and R b is an alkyloxy group of 1-6 carbon atoms, an aralkyloxy group or an aryloxy group, or an N-formylimidazole with a partially protected N15 acylated derivative of the aminoglycosidic antibiotic ,in which 1-amino and 3-amino or 3-alkylamino groups are unprotected and all the other amino groups are protected with an acyl group, to effect selective acylation of 3-amino or

2. . A process as claimed in Claim 1, in which the acylating agent is reacted with the partially protected N-acylated 5 derivative of kanamycin A; 6'-N-alkylkanaraycin A; 3’deoxykanamyoin A; 6’-N-methyl-3'-deoxykanamycin A; 4’deoxykanamycin A; 6’-N-methyl-4'-deoxykanamycin A; 3',4'dideoxykanamycin A; 6-deoxykanamycin A; 4,6-dideoxykana.-ycin A; kanamycin B; 3’-deoxykanamycin B; «’-decxy10 xar.amycin· B; 3 ' ,4 '-didecxykar.amycin B; 3',4'-dideoxy-3'enc-kar.amycin B; 6 '-N-methyl-3 ' ,u '-didecxykar.amycin B; kanamycin C; 3'-deoxykanamycin C; 3' ,4'-dideexykar.am.ycin C; gentamicin A; gentamicin B; gentamicin C; verdamicin; sisomicin or netilmicin. 15 3 . A process as claimed in either claim 1 or claim 2, in which the acylating agent comprises methyl formate, ethyl formate, butyl formate, benzyl formate, phenyl formate, methyl dichloroacetate, ethyl dichloroacetate, met.nyl trichlorcacetate, phenyl trichloroacetate, methyl trifluoroacetate, 20 ethyl trifluoroacetate or phenyl trifluoroacetate.

3. -alkylamino-3-deoxyglycosyl)-2-deoxystreptamine moiety in which the 1-amino group is unprotected and all the other amino groups in the amino-glycoside molecule are protected, the process comprising the steps of reacting 15 an N-alkanoylimidazole with a partially protected N-acylated derivative of the aminoglycosidic antibiotic, in which 1-amino and 3-amino cr 3-alkylanino groups are unprotected and all the other amino groups are protected with an acyl group, and adding an alkaline reagent to the 20 reaction mixture to effect selective acylation of 3-amino or 3-alkylamino group of the partially protected N-acylated derivative with the alkanoyl group. 8. A process as claimed in claim 7, wherein the N-alkanoylimidazole is N-acetylimidazole. 25 9. A process as claimed in either claim 7 or claim 8, wherein the alkaline reagent is aqueous ammonia. 10. A process for the production of a selectively acylated N-protected derivative of an aminoglycosidic antibiotic as claimed in claim 1 or claim 7 and substantially as hereinbefore described with reference to any one of the Examples. 11. A selectively acylated N-protected derivative 3-alkylamino group of the partially protected N-acylated derivative uith the acyl group R a C0-.

4. . A process as claimed in either claim 1 or claim 2, in which the acylating agent comprises N-formylimiiaocle. 5 . A process as claimed in anyone of the preceding claims, in which the acylating agent is reacted at a temperature 25 of -30°C to +120°C for a time of 30 minutes to 48 hours in an inert organic solvent selected from dimethylsulfoxide, 48873 dimethylformamide, hexamethylphosphoric triamide, tetrahydrofuran, dioxane, acetonitrile, nitromethane, sulfolane, dimethylacetamide, chloroform, dichloromethane, methanol, ethanol, n-butanol, t-butanol, 5 benzene, toluene or ethyl ether. 6. A process as claimed in claim 5, in which the inert organic solvent is mixed with water. 7. A process for the production of a selectively protected N-acylated derivative of an aminoglycosidic 10 antibiotic comprising a 6-0-(3-amino- or

5. Of an aminoglycosidic antibiotic when prepared by a process as claimed in any one of the preceding claims.