CA1105452A - PREPARATION OF 1-N-[.omega.-AMINO-.alpha.- HYDROXYALKANOYL]AMINOGLYCOSIDE ANTIBIOTICS - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

An improved process for the preparation of 1-N-[.omega.-amino-.alpha.-hydroxyalkanoyl] aminoglycoside antibiotics comprises acylating a polysilylated aminoglycoside anti-biotic in a substantially anhydrous organic solvent with an acylating derivative of an .omega.-amino-.alpha.-hydroxyalkanoic acid.

Description

z This invention lelates ~o a process for the preparation of l-N-[~-amino-~-hydroxyalkanoyl]aminoglycoside antibiotics having the formula R O ~

R5 R30 ~ ~ O

HO-CH
(ICH2)n wherein n is an integer of from O to 4; R2 is a substituted hexo-pyranosyl ring as hereinafter defined; R3 is hydrogen or a sub-stituted hexopyranosyl ring as hereinafter defined; R4 is hydrogen, hydroxy or a pentofuranosyl ring as hereinafter defined; and R is hydrogen or hydroxy; provided that, when R3 is other than hydrogen, one of R4 and R5 is hydrogen and the other is hydroxy; and pro-vided that, when R3 is hydrogen, R5 is hydrogen and R4 is a sub-stituted pentofuranosyl ring.
The process involves reacting a polysilylated amino-20 ~lycoside prepared from an aminoglycoside of Formula XIV

R2/~\JNH2 R4 ~ O ~ XIV

optionally containing from 1 to 3 amino blocking groups other than silyl on amino groups other than the C-l amino group, in a sub-stantially anhydrous organic solvent, with an acylating derivative of an acid of the formula B-HN-CH2-(CH2)n-CH-cOoH

OH XIII

- 1 - ~' in which B is an amino-blocking group and n is as described above.
All blocking groups are then removed by conventional means to produce the desired compound of Formula I.
The aminoglycosides are a well-known class of antibiotics and have been widely described in the literature. An excellent review article is that entitled "Structures and Syntheses of Aminoglycoside Antibiotics" by Sumio Umezawa, in Advances in Carbohydrate Chemistry and Biochemistry, 30, 111-182, Academic Press, N.Y. (1974). This review article (and references cited ~ rein) also discusses many known l-N-~acyl)aminoglycoside anti-~iotics such as the l-N-~L~ amino-~-hydroxybutyryl] deriva-tives of kanamycin A, kanamycin B, 3',4'-dideoxykanamycin B, tobramycin, paromomycln I, ribostamycin, 3', 4'-dideoxyribo-stamycin and lividomycin A.
U.S. Patent 4,029,882 discloses l-N-acyl derivatives of ' 1~ Cl~ Cla, C2, C2a and X2, sisomicin, verda-micin, mutamicins 1, 2, 4, 5 and 6, and antibiotics G-418, 66-40B, 66-4QD, JI-20A, JI-20B and G-52, wherein the acyl groups are derived from a straight, branched or cyclic alkyl group containing from 1 to 8 carbon atoms, which may contain an amino or hydroxy substituent, or both an amino and a hydroxy substituent. The compounds are prepared by acylating a partially neutralized acid addition salt of the antibiotic with an acylating derivative of the desired side-chain acid.
U.S. Patent 4,055,715 discloses the l-N-[L-(-)-r amino-~-hydroxybutyryl] derivative of the aminoglycoside XK-62-2, and the process for its preparation by acylating XK-62-2 having its

2'-amino or 2'- and 6'-amino moieties protected by a known amino-protecting group (such as the carbobenzyloxy yroup), with an acylating derivative of L-(-)-r-amino-~-hydroxybutyric acid (such as its N-hydroxysuccinimide ester).

U.K. Patent 1,500,218 discloses the O-, L-, and D, L-forms of l-N-[~-amino-~ hydroxypropionyl]XK-62-2 and its prepara-tion by substantially the same process as described in U.S. Patent 4,055,715.
U.K. Patent 1,499,041 discloses l-N-[L~ y-amino ~-hydroxybutyryl]-6'-N-alkylkanamycin A wherein the 6'-N-alkyl group contains from 1 to 4 carbon atoms. The compounds are prepared inter alia by reacting a 6'-N-alkylkanamycln A (either unprotected or having its 3- or 3"-amino group protected with a conventional amino-blocking group) with an acylating derivative of L-(-)-y-amino-~-hydroxybutyric acid.
U.K. Patent 1,475,481 discloses l-N-acyl derivatives of 6'-N-methyl-3',4'-dideoxykanamycin~B, wherein the acyl groups may be in the L- or D,L-form and have the formula H2N (CH2)n 1 OH

in which n is 1, 2 or 3. The compounds are prepared by acylating the aminoglycoside (having its 6'-amino, and optionally its 2'-amino moiety, protected by a conventional amino-blocking group) with an acylating agent containing the above acyl group, e.g. its N-hydroxysuccinimide ester.
South African Patent 77/1944 discloses inter alia a process for the preparation of l-N(lower)alkanoyl derivatives of kanamycin A and B, in which the alkanoyl groups may be substituted by hydroxy and/or amino. The process involves acylation of kanamycin A or B in which the 3-amino moiety of kanamycin A or B
and the ~'-amino moiety of kanamycin B (and optionally the 6'-amino moiety of each antibiotic) is protected with a conventional amino-blocking group. Acylation is achieved in a conventional manner, such as by use of the N-hydroxysuccinimide ester of the acylating acid.

s~

U.S. Patent 3,974,137 discloses and claims a process ~or preparing l-[L~ amino-a-hydroxy~utyryl]kanamycin A which comprises reacting 6'-carbobenzyloxykanamycin A with at least three moles of benzaldehyde, a substituted benzaldehyde or pivaldehyde, to produce 6'-N-carbobenzyloxykanamycin A containing Schiff base moieties on the 1,3 and 3"-positions, acylating this tetra-protected kanamycin A derivative with the N-hydroxysuccini-mide est~r of L-(-)-~-benzyloxycarbonylamino-~-hydroxybutyric acid, and subsequently removing the protecting groups.
In The Journal of Antibiotics, 26, 790-3 (1973), T.P.
Culbertsoll et al. report the preparation of 5"-amino-5"-deoxy-butirosins A and B from butirosins A and B. The first steps in the synthesis involved:
1) partially N-trifluoroacetylating butirosin base by refluxing in a mixture of methanol and ethyl trifluoroacetate, 2) evaporating to dryness, dissolving the residue in pyridineJ
treating it with hexamethyldisilazane and trimethylchloro-silane, then cooling to <10C and treating it with tri-fluoroacetic anhydride, 0 3) evaporating to dryness and hydrolyzing the residue in a 2:1 mixture of ethanol and 2_ acetic acid at reflux, to give tetra[N-(trifluoroacetyl)]butirosin.
The final products of the synthetic scheme, 5"-amino-5"-deoxy-butirosins A and B, also were reacted according to the above three steps to give penta[N-(trifluoroacetyl)]-5"-amino-5"-deoxy-butirosins A and B. Although this publication discloses the acylation of a trimethylsilylated (and partially acylated) amino-glycoside antibiotic, the result in each instance is complete acylation of all primary amino groups in the molecule (four in the starting butirosin and five in the product). The process of the present invention substantially eliminates polyacylation and provides a high degree of selectivity of acylation in the desired l-N-position.
J.J. Wright et al., in The Journal of Antibiotics, 29, 714-719 (1976), describe a general procedure for the selective l-N-acylation of the gentamicin-sisomicin class of aminoglyco-sides. They report that selectivity in the site of acylation is pH dependent and that the C~l amino group i5 the most reactive toward acylation when the amino groups of the molecule are almost completely protonated. These conditions are achieved by the ad~ition of one equivalent of a tertiary amine base to a solution of the fully neutralized acid addition salt. Although these workers obtained l-N-selectivity in the acylation of gentamicin Cla, sisomicin and verdamicin, they reported that little selec-tivity was observed in the acylation of highly hydroxylated amino-glycosides such as gentamicin B and kanamycin A.
U.K. Patent 1,460,039 discloses a process for the pre-paration of various deoxyaminoglycoside antibiotics by halo-genating a phosphorylated amino~lycoside (one in which the hydroxy group to be removed has been converted to a phosphonoxy group), to produce the corresponding aminoglycoside in which the hydroxy group has been converted to halogen, and reducing the halogen compound to produce the corresponding deoxyaminoglycoside. Before halogenating the phosphorylated amino~lycoside, all of its functional groups are preferably protected by means of silyl or acyl groups.
The present invention provides an improved process fo~
the preparation of l-N-[~-amino-~-hydroxyalkanoyl] aminoglycoside antibiotics. The use of a polysilylated aminoglycoside as a starting material gives high solubility in the organic solvent system, thus permitting reaction at high concentrations. Although the reaction is usually conducted in solutions containing about 5~

10-20% polysilylated aminoglycoside starting material, excellent results have been obtained at concentrations of about 50% W/V
(e.g. 50 gms./100 ml. of solution).
AS with prior art processes, the present process gives a mixture of acylated products, and the desired product is separated from the other products by chromatography. However, the position of substitution is much more selective when utilizing the present invention, thereby giving smaller amounts of undesired products which both increases the yield of desired product and simplifies 1~ chromatographic purification. Thus, in preparing l-[L-(-)-y-amino-~-hydroxybutyryl]kanamycin A amikacin by various prior art procedures, there is typically also produced the 3"-N-acylated product (BB-Kll), the 3-N-acylated product (BB-K29), the 6'-N-acylated product (BB-K6) and polyacylated material, as well as unreacted kanamycin A. In commercial production of amikacin by acylation of 6'-N-carbobenzyloxykanamycin A in an aq~eous medium, followed by removal of the protecting group, we found that about 10% of the desired amikacin (2.5 kg. in a 25 kg. batch) usually was lost because of the presence of BB-Kll as a co-product. Any

3"-N-acylated material which was produced caused a loss of about an equal amount of the desired l-N-acylated product, due to the ~reat difficulty of separating the latter from the former. The selectivity of substitution of the present process is illustrated by the extremely low amount of undesirable 3"-N-acylated product which is produced when preparing BB-K8 by the present process.
Typically, no BB-Kll is detected in the reaction mixture.
The present invention provides a process for the pre-paration of l-N-[~-amino-~-hydroxyalkanoyl] aminoglycoside anti-biotics of the formula I

¢~

R ~ NH2 R ~ ~
H

HO-~H
(CH2)n or a pharmaceutically acceptable acid addition salt thereof, wherein n is an integer of from 0 to 4; R2 is a hexopyranosyl rin~ of the formula CHNHR7 lR6 CHNHR

R8 /~

II III IV

in which R6 is H or CH3, R is H or CH3, R8 is OH or NH21 R9 is H
or OH and R is H or OH;
R3 is H or a hexopyranosyl rin~ of the formula HO ~ ~H
H2N~ Rll \ o HO ~ CHI ~

V VI

HO ~ H3CO ~
HcNH3 or H2~ ~

VII VIII

in which Rll is H or CH3;
R5 is H or OH, and R4 is H, OH or a pentofuranosyl ring of the formula HOCH HOCH

~ or `r OH ~120 OH

IX X

in ~hich R12 is H or a hexopyranosyl ring of the formula Il ~n ~

XI XII

in which R13 is H or ~-D-mannopyranosyl;
provided that, when R3 is other than H, one of R4 and R5 is H and the other is OH; and provided that, when R3 is H, R5 i9 H and R4 is a pentofuranosyl ring of Formula IX or X;
which process comprises reacting a polysilylated aminoglycoside prepared from an aminoglycoside of Formula XIV

R20/~ L
R ~ _ \
\5 R3 ~ NH2 XIV

in which R2, R3, R4, and R5 are as defined above, and which optionally contains from 1 to 3 amino-blocking groups other than silyl on amino groups other than the C-l amino group, in a sub-30 stantially anhydrous organic solvent, with an acylating derivativeof an acid of the formula Div.
SY-1~33s .~
5~

.3 iN CH2 (CH2)n FH COOH XIII
OH

in which B is an amino-blocking group and n is as defined above; and subsequently removing all bloc~ing g~oups.
In the aspects o~ the invention forming the subjec~
matter of this divisional specification provides a process fcr preparing a polysilylated aminoglycoside comprising silylating aminoglycoside of the formula , ~20 ~

~N~E2 XlV

wherein R2 is a hexopyranosyl ring of the formula ~10 ~ ~ or lI III IV

in ~hich R6 is H or CH3, R7 is H or CH3, R is OH or NH2, R
is H or OH and R is H or 0~;

R is EE or a hexopyranosyl ring of the formula - 8(a) -Div .

0~.

2 ~ R ~ ~ __ _-~IO ~ ~ H~l ¦ C~i3 E~O
V VI

K~ ~ - H3CO
C~13 ~ ~ or X

VII . VIII

in which Rll is H or CH3;
5R5 is H or OH; and R4 is H, OH or a pentofuranosyl ring of the formula XCC~

O~ R~ O Oh ~Y X

in which Rl2 is H or a hexopyranosyl ring of the formula C~ ~IY
~~30 ~ o R130 ~ Cll2~d2 a ~a~ or ~ C~

XI XII

- 8(b) -Div .

in which R13 is ~ or ~-D-mannopyranosyl;

provided that, when R3 is other than H, one of the R4 nad R is H or the other is OH; and provided that, when R3 is ~, R is H and R4 is a pentofuranosyl ring of ~he Formula I~ or X;
said aminoglycoside containing ~rom 0 to ~ amino-blocXing groups other thLan silyl on amino groups other than the C-l amino group, said silylating step comprising treating with 8 to 11 equivalents of a silylating agent in the presence of substantially anhydrous solvent, and in the case where the number Oc amino-blocking groups other than silyl is 0 or a number smaller than the number desired, introducing any further desired N-blocking groups into the polysilylated aminoglycoside (after partiaL
desilylation by hydrolysis or solvolysis if necessary).
The amino group of the acylatins acid of Formula XII
above must be protected by an amino-blocking group during ~he acylation reaction. This is normally done by the use of conventional amino-blocking group. These same conventional amino-blocking groups may be utilized to protect amino groups ~0 other than the C-l amino groups of the aminoglycoside. Such conventional blocking groups for the protection of primary amino groups are well known to those skilled in the art.
Suitable blocking aroups include alkoxycarbonyl groups such 3~

- 8(c) -as t-butoxycarbonyl and t-amyloxycarbonyl; aralkoxycarbonyl groups such as benzyloxycarbonyl; cycloalkyloxycarbonyl grcups such as cyclohexyloxycarbony;; haloalkoxycarbony]. groups such as trichioroethoxycarbonyl; acyl groups such as ~hthaloyl ar.d o-~itrophenQ.Yyacetyi; haloacetyl groups such as trifluoro-acetyl: and other well-known blocking groups such as ~he o-nitrophenylthio group, the trityl group, etc.
The acylatln~ acid of Formula XIII cont~ins an asymmetric carbor. atom and may exist in its (+) or (-) form or as a mixtuxe thereof (the d, 1 form), thus producing the corresponding compound of Formula I in which the l-N-[~-amino-~-hydroxyalXanoyl] group is in its (~) [or (R)] form or its [-~ ~or (S~] form or a mixture thereof. Each such opticaily active form, and the mixture thereof, is included within the scope of this invention, but the (-) form is preferred.
~ cylation of the polysilylated amlnoglycoside (~h-ith or without from 1 to 3, amino~blvcklng groups other than siiyl on amino groups other than the C-l amino group) may, in general, be conducted in an organic solvent in which the starting material has su~ficient solubility. These starting materials are highly soluble in most common organic solvents.
Suitabla solvents include for example, acetone, di.ethyi ketone, methyl n-propyl ketone, methyl isobutvl ketone, methyl ethyl ~etone, heptane, glyme, diglyme, dioxane, toluene, tetrahydro-ruran, cyclohexanone, pyridine, methylene chloride, chloroform ~nd carbon tetrachloride. The choice of solvent is dependent on the ~articular starting materials employed Ketones, g~neraily, are the preferred solvents. The most advantageous solvent for the particular combination of reactants being utilized can rea~ily be determined by routine experimentation.

s~

Suitable silylating agents for use in preparing the polysilylated aminoglycoside starting materials utllized herein include those of the formula \si/ _ ~

\ I RlS
/ Si ~ _~ R14 ¦ and R16-1 -Z
\R14 Im xv ~ ~ xv:r - 9(e) --- 9 (a.) --wherein R 5, R 6 and R are selected from the group consisting of hydrogen, halogen, (lower)alkyl, (lower)alkoxy, halo(lower) alkyl and phenyl, at least one of the said Rl5, Rl6 and R17 groups being other than halogen or hydrogen; R14 is (lower)alkyl, m is an integer of 1 to 2 and Z is selected from the group consisting of halogen and wherein Rl8 is hydrogen or (lower)alkyl and Rl9 is hydrogen, (lower)alkyl or R16 Si ----in which R15, R16 and R17 are as defined above.
Specific silyl compounds of Formulas XV and XVI are:
trimethylchlorosilane, hexamethyldisilazane, triethylchlorosilane, methyltrichlorosilane, dimethyldichlorosilane, triethylbromo-silane, tri-n-propylchlorosilane, methyldiethylchlorosilane, dimethylethylchlorosilane, dimethyl-t-butylchlorosilane, phenyl-dimethylbromosilane, benzylmethylethylchlorosilane, phenylethyl-methylchlorosilane, triphenylchlorosilane, triphenyl~luorosilane, tri-o-tolylchlorosilane, tri-p-dimethylaminophenylchlorosilane, N-ethyltriethylsilylamine, hexaethyldisilazane, triphenylsilyl-amine, tri-n-propylsilylamine, tetraethyldimethyldisilazane, hexaphenyldisilazane, hexa-p-tolyldisilazane, etc. Also useful are hexaalkylcyclotrisilazanes and octa-alkylcyclotetrasilazanes.

Other suitable silylating agents are silylamides (such as tri-alkylsilylacetamides and bis-trialkylsilylacetamides), silyl-ureas (such as trimethylsllylurea) and silylureides. Trimethyl-silylimidazole also may be utilized.

A preferred silyl group is the trimethylsilyl group and preferred silylating agents for introducing the trimethylsilyl group are hexamethyldisilazane, bis(trimethylsilyl)acetamide, trimethylsilylacetamide and trimethylchlorosilane. Hexamethyl-disilazane is most preferred.
Polysilylation of aminoglycosides changes the normal order of activity of the amino groups contained therein. Thus, the 6'-amino group of the kanamycins is the most active. If unprotected kanamycin A or B is acylated, the main products are thé 6'-~-acylkanamycins. It is for this reason that prior art procedures for the preparation of l-N-acylkanamycins required protection of the 6'-N-amino moiety (e.g. with carbobenzyloxy) in order to obtain good yields of the l-N-acyl product. However, when acylating the polysilylated kanamycins, the major products are the l-N-acyl kanamycins. It is believed that this is due to steric effects of adjacent (or nearby) silylated hydroxy groups (as well as adjacent glycoside linkages), which hinder acylation at the normally more active amino groups. But this is only a theoretical explanation and does not form a part of the invention.
Kanamycin B has the formula

4 6' ~ CH NH

HO ~ ` ~
~ 1' / 5 7~ ~ ,NH2 4" 6"
HO ~ ~ O

H2N ~ 1" /
HO j /

When kanamycin B having all hydroxy groups silylated is con-sidered in light of the above theory of operation, it is seen that the 3"-amino moiety is sterically hindered by the adjacent 2"- and 4"- silylylated hydroxy groups. It is believed that it is for this reason that no 3"-acylated product is normally detected when acylating polysilylated kanamycin s (or the structually similar polysilylated kanamycins A or C), even though troublesome 3"-N-acylated products are obtained in prior art procedures. Similarly, the 6'-amino moiety is hindered by the nearby 4'- and nearby 3'- silylated hydroxy groups. The 2'-amino moiety is hindered by the adjacent 3'- silylated hydroxy and the adjacent glycoside linkage.
Other aminoglycosides which are structurally related to the kanamycins and which, when polysilylated, give primarily the l-N-acyl product include for example, 3'~deoxykanamycin A, 3'-deoxykanamycin B (tobramycin), the 6'-N-alkylkanamycins A, the 6'-N-alkylkanamycins B, the 3'-deoxy-6'-N-alkylkanamycins A, the 3'-deoxy-6'-N-alkylkanamycins B, gentamicins A, B, Bl, and X2, seldomycin factors 1 and 3 and aminoglycosides NK-1001 and NK-1012-1. Each of these, and other structurally similar amino-glycosides, give primarily the l-N-substituted product when acylated as their polysilylated derivative. Small amounts of 6'-N- and 3-N-substituted products are formed, however, and one or both of these amino moieties may be protected if desired, e.g.
with a carbobenzyloxy group.
Another group of aminoglycosides, although otherwise structurally similar to the kanamycin types described above, does not contain either 3'- or 4'-hydroxy groups (i.e. are 3', 4'-dideoxy compounds). When polysilylated, these do not sterically hinder the 6'-amino moiety (or 2'-amino moiety, if present), and 6'-N-substituted (or 2', 6'-di-N-substituted) compounds are the major products upon acylation. In these amino-glycosides it is necessary to protect the 6'-amino moiety (and 2'-amino moiety, if present) with an amino-blocking group other than silyl (e.g. with carbobenzyloxy) and acylate the poly-silylated 6'-N-blocked (or 2',6'-di-N-blocked) aminoglycoside.
Aminoglycosides which fall into this group include, for example, 3',4'-dideoxykanamycin A, 3',4'-dideoxykanamycin B, the 6'-N-alkyl-3',4'-dideoxykanamycins A, the 6'-N-alkyl-3',4'-dideoxy-kanamycins B, gentamicins Cl, Cla, C2 and C2a, aminoglycoside XK-62-2, aminoglycoside 66-40D, verdamicin and sisomicin.
Another class of aminoglycosides are those wherein the ~lycoside linkage are on the 4- and 5-positions of the deoxy-streptamine ring, rather than on the 4- and 6-positions as in the kanamycin type aminoglycosides described above. These may be illustrated by ribostamycin of the formula 4' 6' HO /~ 2, HO_~ O
~N 11' ~ \ ~
o ~ ~NH2 / 5 ~o

5"
HOCH
4" ~ ~ "

3" ~ 2"
HO OH
In aminoglycosides of the ribostamycin type, polysilylation hinders the desired l-N-amino moiety more than the undesired 3-N-amino moiety (the other amino groups being hindered as described above for the kanamycin types). Thus polysilylated antibiotics of the ribostamycin type will form primarily the 3-N-substituted product upon acylation and it therefore is neces-sary to protect the 3-amino moiety with an amino-b]ocking group such as carbobenzyloxy, in order to ob~ain the l-N-substituted product upon acylation of the polysilylated starting material.
Other aminoglycosides which fall into this class include, for example neomycins B and C, paromomycins I and II, lividomycins A and B, aminoglycoside 2230-C and xylostasin, as well as their 3'-deoxy derivatives. The 6'-N-alkyl and 3'-deoxy-6'-N-alkyl variants of any of the above rlbostamycin type antibiotics which contain a 6'-amino group are also included in this class~ Some of the aminoglycosides in this class contain a 6'-hydroxy group rather than a 6'-amino group.
Another group of aminoglycosides are those of the ribo-stamycin type described above~ but which are 3',4'-dideoxy. As with the 3',4'-dideoxykanamycin type aminoglycosides described above, the 2'- and 6'-amino moieties (of those aminoglycosides in this class which contain a 6'-amino moiety) will not be hindered by polysilylation. Accordingly, with compounds such as 3',4'-dideoxyribostamycin, 3',4'-dideoxyneomycins s and C, and 3',4'-dideoxyxylostasin, as well as their 6'-N-alkyl analogs, it is necessary to protect the 2'-, 3'- and 6'-amino moieties with an amino-blocking group such as carbobenzyloxy, in order that acylation of the polysilylated starting material will pro-duce primarily the l-N-acyl product. In those aminoglycosides of this class which contain a 6'-hydroxy group rather than a

6'-amino group (e.g. 3',4'-dideoxyparomomycins I and II and 4'-deoxylividomycins A or B), it is only necessary to protect the 2'- and 3-amino moieties.
When utilizing as a starting material a polysilylated aminoglycoside containing from 1 to 3 amino-blocking groups other tnan silyl on amino groups other than the C-l amino group, z said starting material may be prepared either by polysilylating the desired N-blocked aminoglycoside or by introducing the desired N-blocking group into the polysilylated aminoglycoside (after partial desilylation by hydrolysis or solvolysis, if necessary).
Methods for the introduction of silyl groups into organic compounds, including certain aminoglycosides, are known in the art. The polysilylated kanamycins (with or without blocking groups other than silyl on amino moieties other than the C-l amino group) may be prepared by methods which are known per se, or as described in this specification.
As used herein, the term polysilylated aminoglycoside does not include a persilylated aminoglycoside. Thus, for example, the term polysilylated kanamycin A includes kanamycin A
containing from 2 to 10 silyl groups in the molecule [there being a total of 11 sites (4 amino groups and 7 hydroxy groups) which may be silylated].
The precise number of silyl groups (or their location) present in the polysilylated aminoglycoside starting materials 2~ (with or without from 1 to 3 amino-bloc]~ing groups other than silyl on amino groups other than the ~--1 amino moiety) is not known. We have found that both undersilylation and oversilyla-tion lower the yield of the desired product and increase the yield of other products. In the case of gross under- or over-silylation, little or none of the desired product may be formed.
The degree of silylation which will give the greatest yield of the desired product will depend on the particular reactants being used in the acylation step. The most advantageous degree of silylation using any combination of reactants can readily be determined by routine experimentation.

~1~5~

It is believed that the preferred average number of silyl groups in the polysilylated aminoglycoside starting material will usually be between a lower limit of 4 and an upper limit which is equal to one more than the total number of hydroxy groups in the aminoglycoside molecule, and that these upper and lower limits are decreased by one for each amino-blocking group present in the aminoglycoside molecule. But this explanation is only theory, and is not considered an essential part of the invention.
Polysilylated aminoglycosides containing the desired number of silyl groups may be prepared ei~her by utilizing an amount of silylating which is only sufficient to add the desired number of silyl groups or by utilizing excess silylating agent to persilylate the aminoglycoside and then partially desilylating by hydrolysis or solvolysis.
Thus, for example, when preparing l-N-[L~ -amino-~-hydroxybutyryl]kanamycin A by acylating polysilylated kanamycin A with the N-hydroxysuccinimide ester of L-(-)-~-benzyloxy-carbonylamino-a-hydroxybutyric acid in acetone solution, we have ~0 found that good yields of the desired product are obtained by utilizing polysilylated kanamycin A which has been prepared by reacting from about 4 to about 5.5 moles of hexamethyldisilazane per mole of kanamycin A. Greater or lesser amounts of hexamethyl-disilazane may be utilized, but the yield of desired product in the subsequent acylation step is lowered significantly. In the specific process set forth above we prefer to utilize from about 4.5 to about 5.0 moles of hexamethyldisilazane per mole of kanamycin in order to obtain maximum yield of product in the acylation step.

It will be appreciated that each mole of hexamethyldi-silazane i5 capable of introducing two equivalents of the tri-methylsilyl group into kanamycin A or B. Kanamycin A or B each have a total of eleven sites (NH2 and OH groups) which might be silylated, while kanamycin A and B containing a blocking group other than silyl on an amino moiety other than the C-l amino group each have a total of 10 such sites. Thus, 5.5 moles of hexamethyldisilazane per mole of kanamycin A or B could theoreti-cally completely silylate all OH and NH2 moieties of the kanamycin, while 5.0 moles of hexamethyldisilazane could com-pletely silylate one mole of kanamycin A or B containing a block-ing group other than silyl on an amino moiety other than the C-l amino group. However, we believe that such extensive silylation does not take place with these molar ratios during reasonable reaction time periods, although higher degrees of silylation are obtained in a given reac-tion time when a silylation catalyst is added.
Silylation catalysts greatly accelerate the rate of silylation. Suitable silylation catalysts are well known in the art and include inter alia amine sulfates (which may be the aminoglycoside sulfate), sulfamic acid, imidazole and trimethyl-chlorosilane. Silylation catalysts generally promote a higher degree of silylakion than is required in the process of this invention. However, oversilylated aminoglycosides can be used as starting material if they are first treated with a desilylat-ing agent to reduce the degree of silylation before the acylation reaction is carried out.
Thus, for example, good yields of desired product are obtained when acylating polysilylated kanamycin A prepared using a 5.5:1 molar ratio of hexamethyldisilazane to kanamycin A.
However, when kanamycin A silylated with a 7:1 molar ratio of 5q~

hexamethyldisilazane (or with a 5.5:1 molar ratio in the presence of a silylation catalyst) was acylated in acetone with the N-hydroxysuccinimide ester of L~ -benzyloxycarbonylamino-~-hydroxybutyric acid, less than a 1% yield of the desired pro-duct was obtained. However, when this same "oversilylated"
kanamycin A was acylated with the same acylating agent in acetone solution to which water [21 moles water per mole of kanamycin; 2.5% water (W/V)] had been added as a desilylating agent 1 hour before acylation, a yield of approximately 40% of the desired product was obtained. The same results are obtained if the water is replaced by methanol or other active hydrogen compound capable of effecting desilylation, e.g. ethanol, pro-panol, butanediol, methyl mercaptan, ethyl mercaptan, phenyl mercaptan, or the like.
Although it is usual to utilize dry solvents when working with silylated materials, we have surprisingly found that, even in the absence of "oversilylation", the addition of water to the reaction solvent prior to acylation often gives equally good yields, and sometimes gives better yields of desired product than in a dry solvent. Thus, for example, in acylation reactions conducted in acetone at the usual concentrations of 10-20% (W/V) of polysilylated kanamycin A, we have found that excellent yields of l-~-[L-(-)-~-amino-~-hydroxybutyryl]kanamycin A were obtained when adding up to 28 moles of water per mole of polysilylated kanamycin A; at 20% concentration, 28 moles per mole is approxi-mately 8% water. With other combinations of reactants and sol vents, even more water may be tolerated or be beneficial. The acylation reaction may be conducted in solvents containing up to about 40% water, although at such high water concentrations one must utili2e short acylation -times in order to avoid excessive desilylation of the polysilylated aminoglycoside starting material. Accordingly, as used herein and in the claims, the term "substantially anhydrous organic solvent" is intended to include solvents containing up to about 40% water. A preferred range is up to about 20% water, a more preferred range is up to about 8% water and the most preferred range is up to about 4%
water.
Except as described above for solvents containing very high water levels, the duration of the acylation reaction is not critical. Temperatures in the range of about -30C to about 100~ may be used for reaction times ranging from about one hour up to a day or more. The reaction usually proceeds well at room temperature and, for convenience, may be conducted at ambient temperature. However, for maximum yields and selective acylation, we prefer to conduct the acylation at from about 0 to 5.
Acylation of the l-amino moiety of the polysilylated aminoglycoside (with or without a blocking group other than silyl on an amino moiety other than the C-l amino group) may be con-ducted with any acylating derivative of the acid of Formula XIII
which is known in the art to be suitable for the acylation of a primary amino group. Examples of sui-table acylating deriva-tives of the free acid include the corresponding acid anhydrides, mixed anhydrides, e.g. alkoxyformic anhydrides, acid halides, acid azides, active esters and active thioesters. The free acid may be coupled with the polysilylated aminoglycoside starting material after first reacting said free acid with N,N'-dimethyl-chloroformiminium chloride [cf. Great Britain 1,008,170 and Novak and Weichet, Experientia XXI, 6, 360 (1965)] or by the use of an N,N'-carbonyldiimidazole or and N,N'-carbonylditriazole [cf. South African Specification 63/2684] or a carbodiimide reagent [especially N,N'-dicyclohexylcarbodiimide, N,N'-diiso-propylcarbodiimide or N-cyclohexyl-N'-(2-morpholinoethyl)-carbodiimide" cf. Sheehan and Hess, J.A.C.S., 77, 1967 (1955)],or of an alkynylamine reagent [cf. R. Buijle and H.G. Viehe, Angew. Chem. International Edition, 3, 582 (1964)] or of an isoxazolium salt reagent [cf. R.B. Woodward, R.A. Olofson and H. Mayer, J. Amer. Chem. Soc., 83, 1010 (1961)], or of a ketenimine reagent [cf. C.L. Stevens and M.E. Munk, J. Amer.
Chem. Soc., 80, 4065 (1958)] or of hexachlorocyclotriphospha triazine or hexabromocyclotriphosphatriazine (U.S. Pat. No.
3,651,050) cr of diphenylphosphoryl azide [DDPA; J. Amer. Chem.
Soc., 94, 6203-6205 (1972)] or of diethylphosphoryl cyanide [DEPC; Tetrahedron Letters No. 18, pp. 1595-1598 (1973)] or of diphenyl phosphite [Tetrahedron Letters No. 49, pp. 5047-5050 (1972)]. Another equivalent of the acid is a corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen is a member of a quasiaromatic five membered ring con-taining at least two nitrogen atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substi-tuted derivatives. As will be appreciated by those skilled in the art, it sometimes may be desirable or necessary to protect the hydroxyl group of the acylating derivative of the acid of Formula XIII, e.g. when utilizing acylating derivatives such as an acid halide. Protection of the hydroxy group may be accom-plished by means known in the art, e.g. by use of a carbobenzyl-oxy group, by acetylation, by silylation~ or the like.
In a preferred embodiment of the invention the acylating derivative of the acid of Formula XIII is an active ester, and preferably its active ester with N-hydroxysuccinimide, N-hydroxy 5-norbornene-2,3-dicarboximide or N-hydroxyphthalimide. In another preferred embodiment the acylating derivative of the acid of Formula XIII is a mixed acid anhydride, and preferably its mixed acid anhydride with pivalic acid, benzoic acid, iso-butylcarbonic acid or benzylcarbonic acid.

After the acylation of the polysilylated aminoglycoside is complete, all blocking groups are removed by methods known per se, to yield the desired product of Formula I. The silyl groups, for example, are readily removed by hydrolysis with water, preferably at low pH. Amino-blocking groups on the amino-glycoside molecule (if present) or on the acyl side-chain may also be removed by known methods. Thus, a t-butoxycarbonyl group may be removed by the use of formic acid, a carbobenzyloxy group by catalytic hydrogenation, a 2-hydroxy-1-naphthcarbonyl group by acid hydrolysis, a trichloroethoxycarbonyl group by t~eatment with zinc dust in glacial acetic acid, the phthaloyl ~roup by treatment with hydrazine hydrate in ethanol under heating, the trifluoroacetyl group by treatment with NH40H, etc.
Preferred amino-blocking groups useful for protecting amino groups in the aminoglycoside molecule as well as the amino group in the acylating acid of Formula XIII are those of the formulae ~ CH OC- , CH3-C-0-C- , Y2XC3-ll ~ cl, X3C-CH2-O-C- and ~/\ lCI /

wherein R20 and R21 are alike or different and each is H, F, Cl, Br, NO2, OH, (lower)alkyl or (lower)alkoxy, X is Cl, Br, F or I, and Y is H, Cl, Br, F or I. A particularly preferred amino-blocking ~roup for use in the aminoglycoside molecule is the carbobenzyloxy group. Particularly preferred amino-blocking ~roups for use in the acylating acid of Formula XIII are the carbobenzyloxy, trifluoroacetyl and t-butyloxycarbonyl groups.
Some of the compounds of Formula I contain a double bond (i.e. where substituent R2 has the structure IV). These are com-pounds derived from aminoglycosides such as sisomicin, verdamicin, G-52, 66-40B and 66-40D. When utilizing such compounds, those skilled in the art will appreciate that any reductive techniques which would reduce the double bond should be avoided. Thus, for example, amino-blocking groups which are removable by hydrolysis or by means of an alkali metal in liquid ammonia should be utilized, so as to avoid reduction of the double bond, as would occur with such techniques as catalytic hydrogenolysis.
Yields oE product were determined by various methods.
After removal of all blocking groups and chromatography on a CG-50 tNH4~) column, the yield could be determined by isolation of the crystalline solid from the appropriate fractions or by micro-biological assay (turbidimetric or plate) of the appropriate fractions. Another technique which we utilized was high perfor-mance liquid chromatography of the unreduced acylation mixture, i.e. the aqueous solution obtained after hydrolysis of the silyl groups and removal of organic solvent but before hydrogenolysis s~

to remove the remaining blocking group(s). This assay was not a direct assay for the final product, but for the corresponding N-blocked compounds.
The instrument utilized was a waters Associates ALC/GPC
244 high pressure liquid chromatograph with a Waters Associates Model 440 absorbance detector and a 30 cm x 3.9 mm i.d. ~-Bondapak C~18 column, under the following conditions:
Mobil Phase: 25% 2-propanol 75% 0.01M sodium acetate pH 4.0 10 Flow Rate: 1 ml./minute Detector: UV at 254 nm.
Sensitivity: 0.04 AUFS
Diluent: DMSO
Injected Amount: 5 ~1 Concentration: 10 mg./ml.
Chart speed varied, but 2 minutes/inch was typical. The above conditions ~ave UV traces with peaks which were easy to measure quantitatively. The results of the above analyses are referred to in the specification as HPLC assays.
In order to avoid the repetition of complex chemical names, the following abbreviations are sometimes utilized in this specification.
AIIBA L-(-)-~-amino-~-hydroxybutyric acid BHBA N-Carbobenzyloxy derivative of AHBA
HON~ ~ N-hydroxy-5-norbornene-2,3-dicarboximide NAE N-hydroxy~5-norbornene-2,3-dicarboximide (or BHBA-'ONB') activated ester of BHBA
HONS N-hydroxysuccinimide SAE N~hydroxysuccinimide activated ester of (or BHBA-'ONS') BHBA
DCC dicyclohexylcarbodiimide DCU dicyclohexylurea HMDS hexamethyldisilazane BSA bis(trimethylsilyl)acetamide MSA trimethylsilylacetamide TFA trifluoroacetyl t-BOC tert. butyloxycarbony]
"Dicalite" is a trademark of the Great Lakes Carbon Corporation for diatomaceous earth.
"Amberlite CG-50" is a Trademark of the Rohm & Haas Co.
for the chromato~raphic grade of a weakly acid cationic exchange resin of the carboxylic-polymethacrylic type.
"~-Bondapak" is a Trademark of Waters Associates for a series of high performance liquid chromatography columns.

All temperatures herein are given in degrees centigrade.

As used herein, the terms "(lower)alkyl" and "(lower)-alkoxy" refer to alkyl or alkoxy groups containing from 1 to six carbon atoms.
As used herein and in the claims, the term "pharmaceuti-cally acceptable acid addition salt" of a compound of Formula I
means a mono-, di , tri-, tetra- (or higher) salt formed by the interaction of one molecule of a compound of Formula I with 1 or more equivalents of a nontoxic, pharmaceutically acceptable acid, d~pending on the particular compound of Formula I. It will be appreciated that an acid addition salt can be formed at each amino group in the molecule, both in the aminoglycoside nucleus and in the acyl side chain. Included among these acids are acetic, hydrochloric, sulfuric, maleic, phosphoric, nitric, hydrobromic, ascorbic, malic and citric acid, and those other acids commonly used to make salts of amine-containing pharmaceu-ticals.
Most of the aminoglycosides used as starting materials in the present invention are known in the art. Any individual amino-glycoside which is not known per se (e.g. a not previously de-scribed 6'-N-methyl derivative of a known aminoglycoside) may readily be prepared by methods well-known in the art for the preparation of analogous compounds.
The compounds of Formula I produced by the present inven-tion are active against Gram-positive and Gram-negative bacteria and are used analogously to other known aminoglycosides. Many of the compounds of Formula I are known ~er se.
In another aspect the present invention provides poly-silylated aminoglycosides of Formula ~IV or polysilylated amino-glycosides of Formula XIV containing from 1 to 3 amino-blocking groups other than silyl on amino moieties other than the C-l amino group.

Description of the Preferred Embodiments Example 1 Preparation of l-N-[L-(-)-y-Amino-a-hydroxybutyryl]kanamycin A
amikac_n by Selective Acylation of Poly(trimethylsilyl) 6'-N-carbobenzyloxykanamycin A in Anhydrous Diethyl Ketone 6'-N-Carbobenzyloxykanamycin A (15 g., 24.24 m. moles) was slurried in 90 ml. of dry acetonitrile and heated to reflux under a nitrogen atmosphere. Hexamethyldisila~ane (17.5 g., 108.48 m.
moles) was added slowly over 30 minutes, and the resulting solu-tion was refluxed for 24 hours. After removal of the solvent ln vacuo (40) and complete drying under vacuum (10 mm), 27.9 g.
of a white, amorphous solid was obtained [90.71% calculated as 6'-N-Carbobenzyloxykanamycin A (Silyl)g].
This solid was dissolved in 150 ml. of dry diethyl ketone at 23. L-(-)-~-benzyloxycarbonylamino-~-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) (11.05 g., 26.67 m. moles) dissolved in 100 ml. of dry diethyl ketone at 23 was added slowly with good agitation over 1/2 hour. The solu-tion was stirred at 23 for 78 hours. The yellow, clear solution(pH 7.0) was diluted with 100 ml. of water. The pH of the mix-ture was adjusted to 2.8 (3N ~Cl) and stirred vigorously at 23 for 15 minutes. The aqueous phase was separated, and the organic phase was extracted with 50 ml. of p~ 2.8 water. The combined aqueous fractions were washed with 50 ml. of ethyl acetate. The solution was placed in a 500 ml. Parr bottle, together with 5 g. of 5% palladium on carbon catalyst (Engelhard) and reduced at 50 psi H2 for 2 hours at 23. The mixture was filtered through a pad of Dicalite which was then washed with an additional 30 ml. of water. The colorless filtrate was concen-trated in vacuo (40-45) to 50 ml. The solution was charged on a 5 x 100 cm CG-50 (NH4+) ion exchange column. After washing with 1000 ml. of water, unreacted kanamycin A, 3-[L-(-)-y-amino-~-hydroxybutyryl]kanamycin A (BB-K29) and amikacin were eluted with 0.5N ammonium hydroxide. Polyacyl material was recovered with 3N ammonium hydroxide. Bioassay, thin layer chromatography and optical rotation were used to monitor the progress of elution. The volume and observed optical rotation of each fraction of eluate, as well as the weight and percent yield of solid isolated from each fraction by evaporation to dryness, are summarized below:

Volume ~ Weight Material (ml) 578 (gms.)~ Yield Kanamycin A 1000 ~0.1150.989 9.15 BB-K29 1750 ~0.24 4.37 32.0 Amikacin 2000 ~0.31 6.20 47.4 Polyacyls 900 ~0.032 0.288 2.0 The spent diethyl ketone layer was shown by high performance liquid chromatography to contain an additional 3~5% amikacin.

s~s~
The crude amikacin (6.20 gms.) was dissolved in 20 ml. of water and diluted with 20 ml. of methanol, and 20 ml. of iso-propanol was added to induce crystallization. There was obtained 6.0 gms. (45.8%) of crystalline amikacin.

Example 2 Preparation of l-N-~L~ -Amino-~-hydroxybutyryl]kanamycin A
Amikacin by Selective Acylation of Poly(trimethylsilyl)Kan mycin A, Using In Situ Blocking A. Poly(trimethylsilyl) Kanamycin A
Kanamycin A free base (18 g. activity, 37.15 m. moles) was slurried in 200 ml. of dry acetonitrile and heated to reflux.
Hexamethyldisilazane (29.8 g., 184.6 m. moles) was added over 30 minutes and the mixture was stirred at reflux for 78 hours to give a light yellow clear solution. Removal of the solvent under vacuum left an amorphous solid residue (43 gm., 94%) [calculated as kanamycin A (silyl)10].
B. l-N-[L-(-~-y-Amino-~-hydroxybutyryl]kanamycin A
p-(Benzyloxycarbonyloxy)benzoic acid (5.56 g., 20.43 m.
moles) was slurried in 50 ml. of dry acetonitrile at 23. N,O-20 bis-Trimethylsilyl acetamide (8.4 g., 41.37 m. mole) was added with good stirring. The solution was held for 30 minutes at 23, and then added over 3 hours with vigorous stirring -to a solution of poly(trimethylsilyl)kanamycin A (21.5 g., 17.83 m. mole, calculated as the (silyl)10 compound) in 75 ml. of dry aceto-nitrile at 23. The mix was stirred for 4 hours, the solvent was removed in vacuo (40 ), and the oily residue was dissolved in 50 ml. of dry acetone at 23C.
L-(-)-y-benzyloxycarbonylamino-~-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester (~AE) (8.55 g., 30 20.63 m. moles) in 30 ml. of acetone was added to the above solution over a period of 5 minutes. The mixture was held at i2 23C for 78 hours. The solution was diluted with 100 ml. of water and the pH (7.0) lowered to 2.5 (6N HCl). The mixture was placed in a 500 ml. Parr bottle together with 3 g. of 5% palladium on carbon catalyst (Engelhard) and reduced at 40 psi H2 for 2 hours at 23. The mixture was filtered through a pad of diato-maceous earth which was then washed with 20 ml. of water. The combined filtrate and washings (168 ml.) were determined by microbiological assay against E. coli to contain approximately 11,400 mcg/ml. (19% yield) of amikacin.

Example 3 Preparation of l-N-[L~ r-Amino-~-hydroxybutyryl]kanamycin A
Amikacin by Selective Acylat-ion of Poly(trimethylsilyl)KanamycinA
~ ~ . _ . _ _ ... . .. . _ . . _ . . .
A. oly(trimethylsilyl) Kanamycin A
A suspension of 10 g. (20.6 m. moles) kanamycin A in 100 ml. of dry acetonitrile and 25 ml. (119 m. moles) 1,1,1,3,3,3-hexamethyldisilazane was refluxed for 72 hours. A clear light yellow solution resulted. The solution was stripped to dryness in vacuo at 30-40C. There was obtained 21.3 g. of poly(tri-methylsilyl) Kanamycin A as a light tan amorphous powder [85%
yield calculated as kanamycin A (silyl)10].
B. l-N-[L-(~ -Amino-~-hydroxybutyryl]kanamycin A
To a solution of 2.4 g. (2.0 m. moles) of poly(trimethyl-silyl) Kanamycin A in 30 ml. of dry acetone was added slowly 2.0 m. moles of L-(-)-y-benzyloxycarbonylamino-~-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester (NAE) in 10 ml. of dry acetone at 0-5C. The reaction mixture was stirred at 23C for a week and then stripped -to dryness in vacuo at a bath temperature of 30-40C. Water (60 ml.) was then added to the residue, followed by 70 ml. of methanol to obtain a solution.

The solution was acidified with 3N HCl to pH 2.0 and then re-duced at 50 psi H2 for 2 hours, using 500 mg of 5% palladium on carbon catalyst. The material was filtered, and the combined filtrate and washings were determined by microbiological assay against E. coli to contain a 29.4~ yield of amikacin.

Example 4 Preparation of Amikacin by Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A in Tetrahydrofuran With the Mixed Acid Anhydride of Pivlaic Acid and BH~A
A. Preparation of Mixed Anhydride BHBA (5.066 gm., 20.0 m moles), BSA ~4.068 gm., 20.0 m moles) and triethylamine (2.116 g, 22.0 m moles) were dissolved in 200 ml. of sieve dried tetrahydrofuran. The solution was refluxed for 2 1/4 hours and then chilled to -10C. Pivaloyl chloride (2.412 gm., 20.0 m moles) was added over a period of 2-3 minutes, with stirring, and stirring was continued for 2 hours at -10C. The temperature was then allowed to climb to ~3C.
B. Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana ~
Poly(trimethylsil~1) 6'-N-Cbz Kana A prepared as in Example I (5.454 gm., 4.97 m moles, calculated as 6'-Cbz Kana A (silyl)g) was dissolved in 50 ml. dry (molecular sieve) tetrahydrofuran at 23C. One-half of the solution of mixed anhydride prepared in step A, above, (10.0 m moles) was added over a period of twenty minutes, with stirring, and stirring was continued for

7 days.
Water (100 ml.) was then added to the reaction mixture, and the pH (5.4) was adjusted to 2.0 with 3M H2SO4. Stirring was continued for 1 hour and the solution was extracted with ethyl acetate. Polyacylated material began to crystallize, so the reaction mixture was filtered. ~fter drying over P2O5, the recovered solids weighed 0.702 gms. The extraction of the reac-tion mixture was continued for a total of 4 X 75 ml. of ethyl acetate, after which the excess ethyl acetate was stripped from the aqueous layer. An aliquot of thP aqueous solution was sub-jected to assay by HPLC. The resulting curve indicated a 26.4%
yield of di-Cbz amikacin.
The aqueous layer was then hydrogenated in a Parr apparatus at 50 p.s.i. H2 pressure for two hours, using 0.5 gm. 10~ Pd on carbon catalyst. The material was filtered, and the combined filtrate and washings were determined against E. coli to contain a 31.2% yield of amikacin. Amikacin/BB-K29 ratio approximately 9-10/1, traces of polyacyl and unreacted Kana A presen~.

Example 5 P paration of Amikacin by Acylation of Poly(trimethylsilyl) 6'-_ N-Cbz Kana A in Acetone with the Mixed Anhydride of BHBA and Isobutylcarbonic Acid A. Preparation of Mixed_Anhydride BHBA (1.267 gm., 5.0m.moles) and N-trimethylsilylacetamide (MSA) (1.313 gm., 10.0 m moles) in 20 ml. of sieve dried acetone was stirred at 23C, and triethylamine (TEA) (0.70 ml., ~.0 m moles) were added. The mixture was refluxed under a N2 atmos-20 phere for 2 1/2 hours. The mixture was cooled to -20C and isobutylchloroformate (0.751 gm., 0-713 ml., S.50 m moles) was added. Triethylamine hydrochloride immediately began to separate.
The mi~ture was stirred for 1 hour at ~20C.
B Acylation .
Poly(trimethylsilyl) 6l-N-Cbz Kana A prepared as in Example 1 (6.215 gm., 4.9 m moles, calculated as the (silyl)g compound) was dissolved in 20 ml. of sieve dried acetone, with stirring, at 23C. The solution was cooled to -20C and the cold mixed anhydride solution from step A was slowly added over a period of 30 minutes. The reaction mixture was s~irred for an additional 1 1/2 hours at -20C and then for 17 hours at 23C.

The reaction mixture was then poured into 150 ml. of water at 23C with stirrlng, the pH (7.75) was adjusted to 2.5 with 3N
HCl, and stirring was continued for 15 minutes. Acetone was then stripped in vacuo at 40C. An aliquot of the resulting aqueous solution was subjected to assay by HPLC. The resulting curve indicated a 34.33% yield of di-Cbz amikacin.
The main portion of the aqueous solution was reduced at 50 p.s.i. H2 pressure at 23C for 3 1/4 hours, utilizing 2.0 gms of Pd/C catalyst. The catalyst was removed by filtration and the combined filtrate and washings were determined by microbiolo~ical assay against E. coli to contain a 35.0% yield of amikacin.

Example 6 Preparation of Amikacin by Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A in Anhydrous Cyclohexanone for Varying Times.
A. Poly(trimethylsilyl) 6'-N-Cbz Kana A prepared as in Example 1 (2.537 gm., 2.0 m moles, calculated as 6'-N-Cbz Kana A
(silyl)g) in 300 ml. dry cyclohexanone was acylated for 20 hours at 23C with an NAE solution in dry cyclohexanone (10.8 ml. of 0.1944 m mole/ml. solution, 2.10 m mole). The reaction mixture was then added to 150 ml. of water, with stirring, and the p~l (5.6) was adjusted to 2.5 with 3N HCl. The cyclohexanone was stripped in vacuo at 40C and an aliquot of the remaining aqueous phase was taken for assay by HPLC. The main portion of the aqueous phase was reduced under 50 p.s.i. H2 pressure for 3 hours at 23C, using 1.0 gm of 10% Pd/C catalyst. The catalyst was removed by filtration and the combined filtrate and washings were assayed microbiologically for amikacin.
B. Reaction A, above, was repeated, except that the acylation was continued for 115 hours instead of 20 hours.

~.~
5~

Yields HPLC Assay Microbiological Assay (Amikacin) (di-Cbz amikacin) Turbidimetric Plate _ Reaction A 4~.18~ 42.87% 39.16%
Reaction B 56.17% 55.39~ 38.45%

Example 7 Preparation of Amikacin by Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A in Anhydrous Tetrahydrofuran ror Varying Times A Example 6 A was repeated except that dry tetrahydrofuran was utilized as solvent instead of dry cyclohexanone.
B. Example 6 B was repeated except that dry tetrahydrofuran was u-tilized as solvent instead of dry cyclohexanone.
Yields HPLC Assay Mic _biological Assay (Amikacin) (di-Cbz amikacin) Turbidimetric Plate Reaction A 29.27% 28.34% 28.18%
Reaction B 33.39% 21.52% 28.63%

Example 8 Preparation of Amikacin by Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A in Anhydrous Dioxane for Varying Times A. Example 6 A was repeated excep-t that the acylation was continued for 44 hours utilizing dry dioxane as the solvent.
B. Example 6 B was repeated except that the acylation was continued for 18 1/2 hours utilizing dry dioxane as the solvent~
Yields HPLC Assay Microbiological Assay (amikacin) (di-Cbz amikacin) Turbidimetric Plate Reaction A 39.18~ 43.27~ 33.36%
Reaction B 42.82% 22.55% 33.37%

3~

Example 9 Preparation of Amikacin by Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A in Anhydrous Diethyl ketone at 75C
To a stirred solution of poly(trimethylsilyl) ~'-N-Cbz Kana A prepared as in Example 1 (2.537 gm., 2.0 m moles, calculated as 6'-N-Cbz Kana A (silyl)g) in 32 ml. sieve dried diethyl ketone at 75C was added a solution of NAE (10.8 ml. of 0.1944 m moles/
ml. of diethyl ketone, 2.10 m moles) over a period of 15 minutes.
Stirring was continued at 75C for an additional 3 hours after which the mixture was poured into 150 ml. of water. The pH was adjusted to 2.8 with 3N HC1 and the diethyl ketone was stripped in vacuo at 40C. HPLC assay of an aliquot of the aqueous phase indicated a 39.18% yield of di-Cbz amikacin.
The main portion of the aqueous phase was reduced under 49.8 p.s.i. H2 pressure for 3 1/4 hours at 23C, using 1.0 gm of Pd/C catalyst. The catal;yst was removed by filtration and -the combined filtrate and washings were assayed microbiologically for amikacin. Turbidimetric assay showed 27.84% yield and Plate assay showed 28.6% yield.

Example 10 Preparation of Amikacin by the Acylation of Pol _trimethylsilyl) Kana A With NAE at 0-5 After Back Hydrolysis With Water . .
A. Silylation of Kanamycin A Using HMDS with TMCS as Catalyst Kanamycin A (10 gm of 97.6% purity, 20.14 m moles) in 100 ml of sieve-dried acetonitrile was brought to reflux under a nitrogen atmosphere. A mixture of HMDS (22076 gm, 141 m moles, 7 moles per mole of kanamycin A) and TMCS (1 ml, 0.856 gm, 7.88 m moles) was added to the refluxing reaction mixture over a period 30 of 10 minutes. Reflux was continued for 4-3/4 hours and the mix-ture was then cooled, concentrated in vacuo to a yellow viscous s~

syrup and dried under high vacuum for 2 hours. The yield of product was 23.8 gms (97.9%, calculated as kanamycin A (silyl)10).
B. Acylation Poly(trimethylsilyl) kanamycin A (23.8 gms, 20.14 m moles) prepared in step A above was dissolved in 250 ml of sieve-dried acetone at 23 and then cooled to 0-5. Water (3.63 ml, 201.4 m moles, 10 moles per mole of polysilylated kanamycin A) was added, with stirring, and the mixture was allowed to stand under moderate vacuum for 30 minutes. NAE (19.133 m moles, 0.95 moles per mole of polysilylated kanamycin A) in 108.3 ml of acetone was then added over a period of <1 minute. The mixture was stirred at 0-5 for 1 hour, diluted with water, the pH adjusted to 2.5, and the acetone was then removed in vacuo. The aqueous solution was then reduced at 50 p.s.i. H2 pressure at 23 for 2-1/2 hours using 2.0 gms of 10% Pd on carbon as a catalyst. The reduced reaction mixture was filtered through Dicalite, concentrated to ca. 100 ml in vacuo at 40 and then charged on CG-50(NH4~) column (6 liters resin, 5 x 100 cm). It was washed with water and then eluted with 0.6N-l.ON-3N NH40H. There was obtained 60.25%
amikacin, 4.37% BB-K6, 4.35% BB-K29, 26.47% kanamycin A and 2.18%
polyacyls.

Example 11 Preparation of Amikacin by the Acylation of Poly(trimethylsilyl) 6'-N-Cbz Kana A with SAE at 0-5 After Back Methanolysis A. Silylation of 6'-N-Cbz Kanamycin A
6'-N-Cbz kanamycin A (20.0 gm, 32.4 m moles) in 200 ml of sieve-dried acetonitrile was brought to reflux under a nitrogen atmosphere. HMDS (47.3 ml, 226.8 m moles, 7 moles per mole of 6'-N-Cbz kana A) was added over a 10 minute period and reflux was continued for 20 hours. The mixture was then cooled, concentrated in vacuo, and dried under high vacuum for 2 hours to give 3901 gms ~S~i2 of white amorphous solid (95.4~ yield, calculated as 6'-N-Cbz kana A (silyl)~).
B. Acylation Poly(trimethylsilyl) 6'-N-Cbz kana A (39.1 gm, 32.4 m moles) prepared in step A above was dissolved in 400 ml of dry acetone, with stirring, at 23. Methanol (6.6 ml, 162 m moles, 5 moles per mole of polysilylated 6'-N-Cbz kana A) was added and the mix-ture was stirred at 23 for 1 hour under a strong nltrogen purge.
The mixture was cooled to 0-5 and a solution of SAE ( 11. 35 gm, 32.4 m moles) in 120 ml of pre-cooled, dry acetone was added.
The mixture was stirred for an additional 3 hours at 0-5 and then placed in a 4 cold room for 1 week. Water (300 ml) was added, the pH was adjusted to 2.0, the mixture was stirred for 1 hour, and the acetone was then stripped in vacuo. The resultant aqueous solution was reduced at 54.0 p.s.i. H2 pressure for 17 hours at 23 utilizing 3.0 gm of 10% Pd on carbon as catalyst.
It was then filtered through Dicalite, concentrated in vacuo to to 75-100 ml, charged on a CG-50(NH4+) column and eluted with water and 0.6N NH40H. There was oktained 52.52% amikacin. 14.5%
BB-K29, 19.6~ kanamycin A and 1.71~ polyacyls.

Example 12 Preparation of Amikacin by the Acylation of Poly(trimethyls_lyl) Kana A With SAE at 0-5 After Back ~ydrolysis With Water A. Silylation of Kanamycin A With TMCS in Acetonitrile Using Tetramethylguanidine as Acid Acceptor Kanamycin A (4.88 gm, 10.07 m mole) was suspended in 100 ml of sieve-dried acetonitrile with stirring at 23. To the stirred suspension was added tetramethylguanidine ~TMG) (16.234 gm, 140.98 m moles, 14 moles per mole of kanamycin A). The mixture was heated to reflux and TMCS (15.32 gm, 140~98 m moles, 14 moles per mole of kanamycin A) was added over a 15 minute period. A

white precipi~ate of TMG-HCl formed after about one-half of the TMCS had been added. The mixture was cooled to room temperature, concentrated to a tacky residue and dried under high vacuum for 2 hours. The solid was triturated with dry THF (100 ml), and the insoluble TMG-HCl was filtered off and washed with 5 x 20 ml portions of THF. The combined filtrate and washings were concen-trated in vacuo at 40 to a tacky residue and dried under high vacuum for 2 hours. There was obtained 10.64 gms of a light cream tacky residue (87.6% yield, calculated as kanamycin A
(silyl)10).
B. Acylation Poly(trimethylsilyl) kanamycin A (10.64 gm, 10.07 m moles) prepared in step A above was dissolved in 110 ml of sieve-dried acetone, with stirring, at 23 and the solution was cooled to 0-5. Water (1.81 ml, 100.7 m moles, 10 moles per mole of poly-silylated kana A) was added and the solution was stirred for 30 minutes under moderate vacuum. SAE (3.70 gm, 10.57 m moles, 5%
excess) in 40 ml of pre-cooled dry acetone was added over a period of <1 minute, and the mixture was stirred for one hour. The mixture was worked up by the general procedure in Example llB
give ca, 50~ amikacin, ca, 10% BB-K29, 5-8% BB-K6, ca. 20%
~anamycin A and 5-8% polyacyls.

Example 13 Preparation of _oly(triethylsilyl) Kanam~cln A Us_ng Triethyl-chlorosilane With Triethylamine as Acid Acceptor Kanamycin A (5.0 gms of 97.6% purity, 10.07 m moles) was suspended in 100 ml of sieve~dried acetonitrile at 23. Triethyl-amine (TEA) (33.8 ml, 24.5 gm, 241.7 m moles) was added and the suspension was brought to reflux. A solution of trichloroethyl-silane (23.7 ml, 21.3 gm, 140.98 m moles) in 25 ml dry aceto-nitrile was added over a 20 minute period. Reflux was continued '5~

for an additional 7 hours and the mixture was cooled to room temperature, whereupon long fine needles of TEA HCl separated out.
The mixture was allowed to stand at room temperature for ca. 16 houxs, concentrated in vacuo at 40 to a tacky solid and dried for 2 hours under high vacuum to a deep orange tacky solid. The solid was triturated with 100 ml dry THF at 23 and the insoluble TEA-HCl was filtered off, washed with 5 x 20 ml of THF, and dried to give 16.0 gms of TEA ~Cl. The combined filtrate and washings were concentrated in vacuo to a solid and dried under high vacuum for 2 hours. There was obtained 19.3 gms of poly(triethylsilyl) kanam~cin A as a deep orange viscous syrup.
Example 14 Preparation of Poly(trimethylsilyl) Kanamycin A Using bis-Trimethylsilylurea Xanamycin A (10.0 gm of 99.7~ purity, 20.58 m moles) was suspended in 200 ml of sieve-dried ace~onitrile, with stirring, at 23. To the suspension was added bis~trimethylsilylurea (BSU) 29.45 gms, 144.01 m moles, 7 moles per mole of kanamycin), and ~he mixture was brought to reflux under a nitrogen atmosphere.
Reflux was continued for 17 hours and the reaction mixture was then cooled to room temperature. A small amount of insoluble material present was removed by filtration, washed with 3 x 10 ml portions of acetonitrile and dried (1.1381 gms). Infrared showed this to be BSU plus a small amount of unreacted kanamycin A. The combined filtrate and washings were cooled at 4 for 16 hours.
Additional solid separated, was recovered as above, (7.8 gms) and was shown by infrared to be BSU plus urea. The light yellow filtrate and washings were concentrated in vacuo at ~0 and dried under high vacuum to give 27.0 gm of a white solid which was 3Q partly tacky and partially fine needle-like crystals. The solid was treated with 150 ml of heptane at 23, the insoluble portion - 37 ~

- `

was removed by filtration, washed with 2 x 50 ml portion of hep-tane and dried, to give 6.0 gms of white needles (shown by infra-red to be BSU plus urea). The combined filtrate and washings were concentrated in vacuo at 40 and dried under high vacuum for 2 hours to give 20.4 gms of white needles, the infrared spectrum of which was typical for polysilylated kanamycin A. Calculations showed the product to contain an average of 7.22 trimethylsilyl groups.
Example 15 Preparation of Amikacin by the Acylation of Perltrimethylsilyl) Kanamycin A After Partial Desilylation With 1,3-Bu~anediol A. Preparation of Per(trimethylsilyl) kanamycin A
Kanamycin A (10.0 gm, 20.639 m moles) was suspended in 100 ml of sieve-dried acetonitrile, with stirring, at 23. The sus-pension was brought to reflux under a nitrogen purge and HMDS
(23.322 gms, 144.5 m moles, 7 moles per mole of kanamycin A) was added over a period of ten minutes. Reflux was continued for 16 hours and the mixture was then cooled to room temperature, con-centrated in vacuo and dried for 2 hours under high vacuum. There was obtained 24.3 gm of a white, tacky residue (92.1~ yield, calculated as kanamycin A (silyl)ll).
B. Acylation Per(trimethylsilyl) kanamycin A (24.3 gm) prepared in step A above was dissolved in 240 ml of sieve-dried acetone, with stirring, at 23. To this solution was added 1,3-butanediol (9.25 ml, 103.2 m mole, 5 moles per mole of per(trimethylsilyl) kanamycin A~ The mixture was stirred at 23 for 2 hours under a nitrogen purge and then cooled at 0-5. SAE (7.23 gm, 20.64 m moles) in 70 ml of pre-cooled acetone was added over a period of about l minute. The mixture was stirred at 0-5 for 3 hours and then allowed to stand in a 4 cold room for ca. 16 hours. Water (200 ml) was added, the pH was adjusted to 2.5 and the clear yellow solution was stirred at 23 for 30 minutes. The acetone was stripped in vacuo and the aqueous solution was reduced at 55.0 p.s.i. H2 pressure at 23 for 2 hours using 3.0 gm of 10 Pd on carbon as catalyst. The reduced solution was filtered through Dicalite and chromatographed as in Example llB to give 47.50% amikacin, 5.87% BB-K29, 7.32~ BB-K6, 24.26% kanamycin A
and 7.41% polyacyls.
Example 16 Preparatlon of Amikacin by the Acylation of Pcly(trimethylsilyl) Kanam~cin A Prepared in THF ~sing SAE With Sulfamic Acid Catalyst To a refluxing mixture of kanamycin A t5. 0 gm., 10.32 m moles) in 50 ml of sieve-dried tetrahydrofuran (THF) were added sulfamic acid (100 mg) and HMDS (12.32 gm, 76.33 m moles). The mixture was refluxed for 18 hours, with complete solution occurring after 6 hours. The solution was cooled to 23, treated with 0.1 ml of water and held at 23 for 30 minutes. A solution of SAE (3.61 gm, 10.3 m moles) in 36 ml of I'HF was added over a period of 30 minutes. After stirring for 3 hours, the mixture 20 was diluted with 100 ml of water and the pH was adjusted to 2.2 with 10~ H2SO4. It was stirred for 30 minutes at 23 and then concentrated in vacuo to remove THF. The resulting aqueous solution was red~lced at 50 p.s.i. H2 pressure for 2 hours at 23 using 10% Pd on carbon as a catalyst. The reduced solution was filtered through Dicalite and the solids were washed with water.
The combined filtrate and washings (150 ml) were determined by microbiological assay against E. coli to contain 1225 mcg/ml ~31.5~ activity yield) of amikacin.

Example 17 Preparation of Amikacin by the Acylation of Poly(trimethylsilyl) Kanamycin A with the N-Hydroxysuccinimide Ester of Di-Carbobenzyl-oxy AHBA
A. Preparation of Dicarbobenzyloxy L-(-)-~-Amino-~-hydrox~-butyric Acid N-Hydroxysuccinimide Ester Dicarbobenzyloxy L-(-)-~-amino-~-hydroxybutyric acid (8 gm, 20.65 m moles) and N-hydroxysuccinimide (2.37 gm, 20.65 m moles) were dissolved in 50 ml of dry acetone at 23. Dicyclohexyl-carbodiimide (4.25 gm, 20.65 m moles) dissolved in 20 ml of dryacetone was added and the total was agitated at 23 for 2 hours.
Dicyclohexylurea was filtered off, the filter cake was washed with 10 ml of dry acetone, and the filtrate and washings were combined.
B~ Acylation Poly(trimethylsilyl) kanamycin A, prepared according to the general procedure of Example 15 from 10.0 gms (20.639 m moles) of kanamycin A, was dissolved in 100 ml of dry acetone. The solution was cooled to 0-5, 3.7 ml of deionized water was added, and the solution was stirred at 0-5 for 30 minutes under moderate vacuum.
To this solution was added the solution oE the di-Cbz-blocked acylating agent prepared in step A, and the mixture was st.irred at 0-5 for 30 minutes. The mixture was diluted with water, the pH was adjusted to 2.2 and the acetone was removed in vacuo. The aqueous solution was reduced by the general pro-cedure of Example 16 and then filtered through Dicalite. Chroma-tography showed 40-45% amikacin, ca. 10~ BB-K29, a trace of BB-K6, ca. 30~ kanamycin A and a small amount of polyacyls.

Example 18 Preparation of Poly(trimeth_lsi~yl) Kana~ycin A Using HMDS with Imidazole as Catalyst Kanamycin A (11 gm, 22.7 m moles) and 100 mg of imidazole were heated to reflux in 100 ml of sieve-dried acetonitrile, under a nitrogen purge. HMDS (18.48 gm, 114.5 m moles, 5 moles per mole of kanamycin A) was added over a period of 30 minutes and the mixture was refluxed for 20 hours. Complete solution occurred in ca. 2-1/2 hours. The solution was cooled to 23 and the solvent was removed in vacuo to leave 21.6 gms of poly(tri~
methylsilyl) kanamycin A as a foamy residue (93.1% yield, calcu-lated as kanamycin (silyl)ll) Example 19 Preparation of l-N-~L~ Amino-~-hydroxybutyryl]kanamycin B
(BB-K26) by the Acylation of Poly(trimethylsilyl) kanamycin B
With SAE
A. Preparation of Poly(trimethylsilyl) Kanamycin B Using HMDS With TMCS Catalyst Kanamycin B (25 gm, 51.7 m moles) in 250 ml of sieve-dried acetonitrile was heated to reflux under a stream of nitrogen.
HMDS (62.3 gm, 385.81 m moles, 7.5 moles per mole of kanamycin B) was added over a period of 30 minutes followed by 1 ml of TMCS
as catalyst. The mixture was refluxed for 21 hours with complete solution af-ter 1 hour. The solvent was then removed in vacuo at 60 and the oily residue was held a-t 60 under high vacuum for 3 hours. There was obtained 53.0 gm of poly(trimethylsilyl) kanamycin B (85.2% yield, calculated as kanamycin B (silyl)10).
B. Acylation The poly(trimethylsilyl) kanamycin B prepared in s-tep A
above (53.0 gm) was dissolved in 500 ml of dry acetone at 0-5, methanol (20.9 ml) was added, and the mixture was stirred in vacuo for 30 minutes at 0-5. A solution of SAE (18.1 gm, 51.67 m moles) in 200 ml of pre-cooled dry acetone was added over a period of less than 1 minute and the mixture was stirred for 30 minutes at 0-5. The mixture was worked up according to the general procedure of Example 16 and then loaded on a column of CG-50 (NH4+) (8 x 120 cm). It was eluted with an NH40H gradient from 0.6N to 3N. There was obtained 38% of BB-K26, 5% of the corresponding 6'-N-acylated kanamycin B (BB-K22), 10~ of the corresponding 3-N-acylated kanamycin B (BB-K46) 14.63~ kanamycin B and a small amount of polyacylated kanamycin B.

Example 20 Preparation of Poly(trimethylsil~l) Kanamycin A Using HMDS With Kanamycin A sul~ate as Catalyst Kanamycin A (19.5 gm, 40.246 m moles) and `kanamycin A sul-fate (0.5 gm, 0.858 m mole) (total = 20.0 gm, 41.0 m moles) in 200 ml of sieve-dried acetonitrile was brought to reflux. HMDS
(60.3 ml, 287.7 m moles, 7 moles per mole of kanamycin A) was slowly added and the mixture was refluxed for 28 hours. It was then stripped to dryness on a rotary evaporator and dried under steam injector vacuum. There was obtained 47.5 gms of poly-(trimethylsilyl) kanamycin A as a pale yellow oil (95.82~ yield, calculated as kanamycin A (silyl)10) Example 21 Preparation of Amikacin by the Acylation of Poly(trimethylsilyl) Kanamycin A W_th N-Trifluoroacetyl Blocked AHBA N-Hydroxy-succinimide Ester A. Preparation of N-Trifluoroacetyl AHBA and Conversion _ to its N-Hydroxysuccinimide Ester To a suspension of A~BA (5.0 gm, 42 m moles) in 100 ml THE' was added trifluoroacetic anhydride (40 gm, 191 m moles), with stirring, over a 10 minute period. The solution was stirred ~or 18 hours at 23 and then concentrated to dryness in vacuo at 50.
The residue was dissolved in 100 ml of aqueous methanol (1:1) and stirred for 1 hour. It was then concentrated to dryness in vacuo and redissolved in 50 ml H2O. The aqueous solution was extracted with 3 x 50 ml portions of MIBK and, after drying over Na2SO4, the extract was concentrated to an oil. Traces of solvent were removed by adding and distilling off 4 ml of water. On standing the oil changed to a waxy, crystalline solid (2.5 gm, 28% yield).
The N-trifluoroacetyl AHBA (2.4 gm, 11.3 m moles) was dis-solved in 50 ml dry acetone and N-hydroxysuccinimide (1.30 gm, 11.31 m moles) was added to the solution. A solution of dicyclo~
hexylcarbodiimide (2.33 gm) in 20 ml of dry acetone was slowly added. The reaction mixture was stirred for 2 hours at 23 and the precipitated dicyclohexylurea was removed by filtration and washed with a small amount of acetone. The combined filtrate and washings (a solution of the N-hydroxysuccinimide ester of N-tri-fluoroacetyl AHBA) was utilized in the next step wlthout isolation.
B. Acylation To a solution of poly(trimethylsilyl) kanamycin A prepared as in Example 20 (11.31 m moles) in 54 ml of acetone was added 2.0 ml (113.4 m moles) of water, and the mixture was s-tirred ln vacuo at 0-5 for 30 minutes. The N-hydroxysuccinimide ester of N-trifluoroacetyl AHBA prepared in step A above (11.31 m moles) was added to the mixture and it was then held at 5 for 1 hour.
The pH was then adjusted to ca. 2.0 with 20% H2SO4, the mixture was stirred for 30 minutes and the pH was then raised to ca. 6.0 with NH40H. The mixture was then stripped to dryness in a rotary evaporator to give 14.4 gm of a tacky off-white solid. The solid was dissolved in 100 ml of water, the pH was raised from 5.5 to 11.0 with 10N NH40H and the solution was heated in an oil bath at 70 for 1 hour. The pH (9.5) was then lowered to 7.0 with HCl, ~ 43 -the solution was polish filtered to remove a small amount of insolubles and the filter was washed with water. The combined filtrate and washings (188 ml) was applied to a CG-50 (NH~+) column (8 x 90 cm), washed with 2 liters o~ water and eluted with a NH40H gradient (0.6N-l.ON-concentrated). There was obtained 28.9% amikacin, 5.0% BB-K6, 5.7~ BB-K29, 43.8% kanamycin A, 3.25~
polyacyls plus 14.3~ of an unknown material which was in the first fraction off the column.

Example 22 Preparation of Amikacin by the Acylation of Poly(trimethylsilyl) Kanamycin A With t-Butyloxycarbonyl Blocked AHBA N-Hydroxy-succinimide Ester A. Preparation of t-BOC AHBA and Conversion to its N-Hydroxysuccinimide Ester . . . :
A solution of AHBA (5.0 gm, 42 m moles) in 100 ml of water and 20 ml of acetone was adjusted to pH 10 with 10N NaOH. Over a period of 3-4 minutes was added 11.6 gm (53 m moles) of di-t-butyl dicarbonate, and the solution was stirxed for 35 minutes while maintaining the pH at 10 by the periodic addition of 10N
NaOH. The acetone was removed in vacuo and the aqueous phase was washed with 40 ml of ethyl acetate. The pH of the aqueous solution was lowered to 2.0 with 3N HCl and it was then extracted with 3 x 30 ml of MIBK. The combined MIBK extracts were dried over Na2SO4 and concentrated to a clear oily residue (8.2 gm, 89%).
The t-BOC-AHBA (4.25 gm, 19.~ m moles) was dissolved in 50 ml of acetone and N-hydroxysuccinimide (2.23 gm, 19.4 m moles) was added. A solution of dicyclohexylcarbodiimide (4.00 gm 19.4 m moles) in 20 ml of acetone was slowly added and the mixture was stirred for 2 hours at 23. The precipitated dicyclohexylurea was removed by filtration and was washed with a small amount of ~s~s~

acetone. he combined filtrate and washings (a solution of the N-hydroxysuccinimide ester of t-BOC-AHBA) was utilized in the next step witho~lt isolation.
B. Acylation To a solution of poly(trimethylsilyl) kanamycin A prepared as in Example 20 (41.28 m moles) in 94 ml of acetone was added 3.5 ml (194 m moles) of water, and the mixture was stirred in vacuo at 0-5 for 30 minutes. The N-hydroxysuccinimide ester of t-BOC-AHBA prepared in step A above (19.4 m moles) was added and the mi~ture was allowed to stand at 5 ~or 1 hour. Water (~00 ml) was added and the pH (7.0) was lowered to 2.0 with 20% H2S04.
After 30 minutes stirring the pH was raised to ca. 6.0 with NH40H
and the mixture was stripped to dryness in vacuo to give 36.3 gms of a golden oil. The oil was dissolved in 200 ml of trifluoro-acetic acid, allowed to stand for 15 minutes and stripped to dryness in a rotary evaporator. The oil was washed with water and the water was flashed off. Concentrated NH40H was added to pH 6.0 and was flashed off. The resulting solid was dissolved in water, filtered, and the filter washed with water. The combined filtrate and washings (259 ml) were loaded on a CG-50 (NH4~) column (8 x 92 cm), washed with 4 liters of water and eluted with an NH40H gradient (0.6N-l.ON-concentrated). There was obtained 40.32% amikacin, 4.58% BB-K6, 8.32% BB-K29, 30.50% kanamycin A
and 7.43% polyacyls.

Example 23 The general procedure of Example 10 is repeated except that the kanamycin A utilized therein is replaced by an equimolar amount of 3'-deoxykanamycin A, 6'-N-methylkanamycin A, 3'-deoxy-6'~N-methylkanamycin A, - ~5 -~s~

kanamycin B, 6'-N-methylkanamycin B, tobramycin (3'-deoxykanamycin B), 6'-N-methyltobramycin, aminoglycoside NR-1001, 3'-deoxy aminoglycoside NK-1001, 6'-N-methyl aminoglycoside NK-1001, 3'-deoxy-S'-N-methyl aminoglycoside NR-1001, gentamicin A, 3'-deoxygentamicin A, gentamicin B, 3'-deoxygentamicin B, 6'-N-methylgentamicin B, 3'-deoxy-6'-N-methylgesltamicin B, gentamicin Bl, 3'-deoxygentamicin Bl, -6'-N-methylgentamicin Bl, 3'-deoxy-6'-N-methylgentamicin Bl, gentamicin X2, seldomycin factor 1 and seldomycin factor 2, respectively, and there is thereby produced l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3'-deoxykanamycin A, l-N-[L-(-)-y-amino-a-hydroxybutyryl]-6'-N-methylkanamyci~ A, L-t-)-y-amino-~-hydroxybutyryl]-3'-deoxy-6'-N-methyl-kanamycin A
l-N-[L-[-)-~-amino-a-hydroxybutyrylJkanamycin B, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6~-N-methylkanamycin B, l-~-[L-(-)-y-amino-~-hydroxybutyryl]tobramycin 1-N-[L-(-)~y-amino-~-hydroxybutyryl~-6'-N-methyltobramycin, l-N-lL-t-)-y-amino-~-hydroxybutyryll aminogly~o~ide NR-1001, l-N-[L-(-)-y-amino~-hydroxybutyryll-3~-deoxy aminoglycoside NK-1001, ., l-N-[L-(-)-y-amino-a-hydroxybutyryl]-6'-N methyl aminoglycoside NK-1001, l-N-~L-(-)-y-amino-~-hydroxybutyryl]-3'-deoxy-6'-N-methyl amino-glycoside NK-1001, l-N-[L-(-)-y-amino-~-hydroxybutyryl]gentamicin A, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3'-deoxygentamicin A, l-N-[L-(-)-y-amino-a-hydroxybutyryl]gentamicin B, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3'-deoxygentamicin B, l-N-[L-(-)-y-amino-~-hydroxybutyryl] 6'-N-methylgentamicin B, 1-N-[L-(-)-y-amino-a-hydroxybutyryl]-3'-deoxy-6'-N-methyl-gentamicin Bl, l-N-[L-(-)-y-amino-~-hydroxybutyryl]gentamicin Bl, l-N-[L-(-)-y-amino-a-hydroxybutyryl]-31-deoxygentamicin Bl, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6'-N-methylgentamicin Bl, l-N-[L-(-)-y-amino-a-hydroxybutyryl]-3'-deoxy-6'-N-methyl-gentamicin Bl, l-N-[L-(-)-y-amino-~-hydroxybutyryl]gentamicin X2, l-N-[L-(-)-y-amino-~-hydroxybutyryl] seldomycin factor 1 and l-N-[L-(-)-y-amino-a-hydroxybutyryl] seldomycin factor 2, respectively.
The reaction of each of the aminoglycoside starting materials listed above in the same manner with L-(-)-~-benzyloxy-carbonylamino-a-hydroxypropionic acid N-hydroxy-5-norbornene-2,3-dicarboximide ester instead of the L-(-)-y-benzyloxycarbonyl-amino-~-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboxi-mide ester produces the corresponding l-N-[L-(-)-~-amino a-hydroxypropionyl] aminoglycosides.
The reaction of each of the aminoglycoside starting materi-als listed above in the same manner with L-(-)-~-benzyloxycar-bonylamino-a-hydroxyvaleric aci.d N-hydroxy-5-norbornene 2,3-di-carboximide ester instead of the L-(-)-y-benzyloxycarbonylamino-~-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester produces the corresponding l-N-[L-(-)-~-amino-~-hydroxy-valeryl] aminoglycosides.

Example 24 The general procedure of Example 10 is repeated except that the L-(-)-~-benzyloxycarbonylamino-~-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester used therein is replaced by L-(-)-~-benzyloxycarbonylamino-~-hydroxypropionic acid N-hydroxy-5-norbornene-2,3-dicarboximide ester and L-(-)-~-benzyloxycarbonylamino-~-hydroxyvaleric acid N-hydroxy-5-norbor-nene-2,3-dicarboximide ester, respectively, and there is thereby produced l-N-[L-(-)-~-amino-~-hydroxypropionyl]kanamycin A and l-N-[L-(-)-~-amino-~- hydroxyvaleryl]kanamycin A, respectively.

Example 25 The general procedure of Example 1 is repeated, except that the 6'-N-carbobenzyloxykanamycin A utilized therein is re-placed by an equimolar amount of 6'-N-carbobenzyloxy-3',4'-dideoxykanamycin A, 6'-N-carbobenzyloxy-3',4'-dideoxy-6'-N-methylkanamycin A, 2',6'-di-(N-carbobenzyloxy)-3',4'-dideoxykanamycin B, 2',6'-di-(N-carbobenzyloxy)-3',4'-dideoxy-6'-N-methylkanamycin s, 2',6'-di-(N-carbobenzyloxy)gentamicin Cl, 2',6'-di-(N-carbobenzyloxy)gentamicin C1a, 2',6'-di-(N-carbobenzyloxy)-6' N-methylgentamicin Cla, 2',6'-di-(N-carbobenzyloxy)gentamicin C2.
2',6'-di-(N-carbobenzyloxy)-6'-N-methylgentamicin C2 and 2',6'-di-(N-carbobenzyloxy)aminoglycoside XK 62-2, respectively, and there is thereby produced l-N-[L-(-)-~-amino-~-hydroxybutyryl]-3',4'-dideoxykanamycin A, l-N-[L-(~ -amino-~-hydroxybutyryl]-3',4'-dideoxy-6'-N-methylkanamycin A, l-N-[L-(-)-~-amino-~-hydroxybutyryl]-3l~4l dideoxykanamycin B, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3',4'-dideoxy-6'-N-methylkanamycin ~, l-N-[L-(-)-~-amino-~-hydroxybutyryl]gentamicin Cl, l-N-[L-(-)-y-amino-~-hydroxybutyryl]gentamicin C1a, l-N-[L-(-)-~-amino-~-hydroxybutyryl]-6'~N-methylgentamicin Cla, l-N-[L-(-)-y-amino-~-hydroxybutyryl]gentamicin C2, l-N-[L-(-)-~-amino-~-hydroxybutyryl]-6'-N-methylgentamicin C2 and l-N-[L-(-)-y-amino-~-hydroxybutyryl] aminoglycoside XK-62-2, respectively The reaction of each of the aminoglycoside starting materials listed above in the same manner with L-(-)-~-benzyloxy-carbonylamino-~-hydroxypropionic acid N-hydroxy-5-norbornene-2,3-dicarboximide ester instead of the L-(-)-~-benzyloxycarbonyl-amino-~-hydroxybutyric acid N-hydroxy-5 norbornene-2,3-dicarbox-imide ester produces the corresponding l-N-[L-(-)-3-amino-~-hydroxypropionyl] aminoglycosides.
The reaction of each of the aminoglycoside startingmaterials listed above in the same manner with L-(-)-~-benzyloxy-carbonylamino-~-hydroxyvaleric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester instead of the L-(-)-~-benzyloxycarbonylamino-~-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester produces the corresponding l-N-[L-(-)-~-amino-~-hydroxy-valeryl] aminoglycosides.
Example 26 2',6'-di-(N-Trifluoroacetyl)sisomicin is slurried in dry acetonitrile and heated to reflux under a nitrogen atmosphere.
Hexamethyldisilazane [4 moles per mole of 2'6'-di-(N-trifluoro-acetyl)sisomicin] is added over a period of 30 minutes and the resulting solutlon is re~luxed for 24 hours. Removal of the solvent in vacuo gives solid polysilylated 2',6'-di-(N~trifluoro-acetyl)sisomicin.
The polysilylated 2',6'-di-(N-trifluoroacetyl)sisomicin is acylated with the N-hydroxysuccinimide ester o~ L-(-)-r-trifluoroacetylamino-~-hydroxybutyric acid according to the general procedure of Example 21B and worked up as in Example 21B
to give l-N-[L-(-)-y-amino-~-hydroxybutyryl]sisomicin.

Example 27 The general procedure of Example 26 is repeated except that the 2',6'-di-(N-trifluoroacetyl)sisomicin utilized therein is replaced by an equimolar amount of 2',6'-di-(N-trifluoroacetyl)-5-episisomicin, 2',6'-di-(N-trifluoroacetyl)-6'-N-methylsisomicin, 2',6'-di-(N-trifluoroacetyl)-6'-N-methyl-S-episisomicin, 2',6'-di-(N-trifluoroacetyl)verdamicin, 2',6'-di-(N-trifluoroacetyl)-5-epiverdamicin, 2',6'-di-(N-trifluoroacetyl)-6'-N-methylverdamicin, 2',6'-di-(N-trifluoroacetyl)-6'-N-methyl-5-epiverdamicin and 2',6'-di-(N-trifluoroacetyl) aminoglycoside 66-40D, respectively, and there is thereby produced.
l-N~[L-(-)-y-amino-~-hydroxybutyryl]-5-episisomicin, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6'-N-methylsisomicin, i-N-[L-(-)-y-amino-~hydroxybutyryl~-6'-N-methyl-5-episisomicin, l-N-[L-(-)-y-amino-~-hydroxybutyryl]verdamicin, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-5-epiverdamicin, l-N-[L-~-)-y-amino-~-hydroxybutyryl]-6'-N-methylverdamicin, l-N-[L-(-)-y-amino-~-hydroxybutyryl~-6'-N-methyl~5-epiverdamicin and l-N-[L-(-)-y-amino-~-hydroxybutyryl] aminoglycoside 66-40D, respectively.

The reaction of each of the 2',6~-di-(N-trifluoroacetyl) aminoglycoside starting materials listed above in the same manner with the N-hydroxysuccinimide ester of L-(-)-~-trifluoro-acetylamino-~-hydroxypropionic acid instead of the N-hydrox-succinimide ester of L-(-)-y trifluoroacetylamino-~-hydroxy-butyric acid produces the corresponding l-N-[L-(-)-~-amino-~-hydroxypropionyl] aminoglycosides.
The reaction of each of the 2',6'-di-(N-trifluoroacetyl) aminoglycoside starting materials listed above in the same manner with the N-hydroxysuccinimide ester of L-(-)~-trifluoroacetyl-amino-~-hydroxyvaleric acid instead of the N-hydroxysuccinimide ester of L-(-)-y-trifluoroacetylamino-~-hydroxybutyric acid produces the corresponding l-N-[L-(-)-~-amino-a-hydroxyvaleryl]
aminoglycosides.

Example 28 The general procedure of ~xample 26 is repeated except that the N-hydroxysuccinimide ester of L-(-)-y-trifluoroacetyl-amino-~-hydroxybutyric acid is replaced by an equimolar amount of the N-hydroxysuccinimide esters of L~ trifluoroacetylamino-~-hydroxypropionic acid and L-(-)-~-trifluoroacetylamino-~-hydroxyvaleric acid, respectively, and there is thereby produced l-N-[L-(-)-~-amino-~-hydroxypropionyl]sisomicin and l-N-[L-(-)-~-amino-~-hydroxyvaleryl]sisomicin, respectively.

Example 29 The general procedure of Example 1 is repeated except that the 6'-N-carbobenzyloxykanamycin A utilized therein is replaced by an equimolar amount of 3-N-carbobenzyloxyribostamycin, 3~N-carbobenzyloxy-3'-deoxyribostamycin, 3-N-carbobenzyloxy-6'-N-methylribostamycin, 3-N-carbobenzyloxy-6'-N-methyl-3'-deoxyribostamycin, 3-N-carbobenzyloxyneomycin B, 3-N-carbobenzyloxy-3'-deoxyneomycin B, 3-N-carbobenzyloxy-6'-N-methylneomycin B, 3-N-carbobenzyloxy-6'-N-methyl-3'-deoxyneomycin B, 3-N-carbobenzyloxyneomycin C, 3-N-carbobenzyloxy-3'-deoxyneomycin C, 3-N-carbobenzyloxy-6'-N-methylneomycin C, 3-N-carbobenzyloxy-6'-N-methyl-3'-deoxyneomycin C, 10 3-N-carbobenzyloxyxylostasin, 3-N-carbobenzyloxy-3'-deoxyxylostasin, 3-N-carbobenzyloxy-6'-N-methylxylostasin, 3-N-carbobenzyloxy-6'-N-methyl-3'-deoxyxylostasin, 3-N-carbobenzyloxyparomomycin I, 3-N-carbobenzyloxy-3'-deoxyparomomycin I, 2',3-di-(N-carbobenzyloxy)-3',4'-dideoxyparomomycin I, 3-N-carbobenzyloxyparomomycin II, 3-N-carbobenzyloxy-3'-deoxyparomomycin II, 2',3-di-(N-carbobenzyloxy)-3',4'-dideoxyparomomycin II, 20 3-N-carbobenzyloxy aminoglycoside 2230-C, 3-N-carbobenzyloxy-3'-deoxy aminoglycoside 2230-C, 3-N-carbobenzyloxylividomycin A and 3-N-carbobenzyloxylividomycin :E~, respectively, and there is thereby produced l-N-[L-(-)-y-amino-c~-hydroxybutyryl]ribostamycin, l-N-[L-(-)-y-amino-c~-hydroxybutyryl]-3'-deoxyribostamycin, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6'-N-methylribostamycin, l-N-[L- (-)-y-amino-c~-hydroxybutyryl]-6'-N-methyl-3'-deoxy-ribos~amycin, 30 l-N-[L-(-)-y-amino-~-hydroxybutyryl]neomycin B, l-N-[L-(-)-y-amino-c~-hydroxybutyryl]3'-deoxyneomycin B, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6'-N-methylneomycin B, l-N-~L-~-)-y-amino-~-hydroxybutyryl]-6'-N-methyl-3'-deoxy-neomycin s, l-N-[L-(-)-y-amino-~.-hydroxybutyryl]neomycin C, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3'-deoxyneomycin C, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6'-N-methylneomycin C, l-N-~L-(-)-y-amino-~-hydroxybutyryl]-6'-N-methyl-3'-deoxy-neomycin C, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-xylostasin, 1-N-[L-(-)-y-amino-~-hydroxybutyryl]-3~-deoxyxylostasin, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6'-N-methylxylostasin, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6'-N-methyl-3'-deoxy-xylostasin, l-N-[L-(-)-y-amino-~-hydroxybutyryl]paromomycin I, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3'-deoxyparomomycin I, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3',4'-dideoxyparomomycin I, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-paromomycin II, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3'-deoxyparomomycin II, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3',4'-dideoxyparomomycin II, l-N-[L-(-)-y-amino-~-hydroxybutyryl]aminoglycoside 2230-C, l~N-[L-(-)-y-amino-~-hydroxybutyryl]-3'-deoxy aminoglycoside 2230-C.
l-N-[L-(-)-y-amino-~-hydroxybutyryl]lividomycin A and l-N-[L-(-)-y-amino-~-hydroxybutyryl]lividomycin B, respectively.
The reaction of each of the carbobenzyloxy-protected aminoglycoside starting matexials listed above in the same manner with L-(-)-~-benzyloxycarbonylamino-~-hydroxypropionic acid N-hydroxy-5-norbornene-2,3-dicarboximide ester instead of the L-(-)-y-benzyloxycarbonylamino-~-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester produces the corresponding l-N-[L-(-)-~-amino-~-hydroxypropionyl] aminoglycosides.

$~

The reaction of each o~ the carbobenzyloxy-protected aminoglycoside starting materials listed above in the same manner with L-(-)-~-benzyloxycarbonylamino-~-hydroxyvaleric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester instead of the L-(-)-y-benzyloxycarbonylamino-~-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester produces the corresponding l-N-[L-(-)-~-amino-~-hydroxyvaleryl] aminoglycosides.

Example 30 The general procedure of Example 1 is repeated except that the 6'-N-carbobenzyloxykanamycin A utilized therein is replaced by an equimolar amount o 2',3,6'-tri-(N-carbobenzyloxy)-3',4'-dideoxyribostamycin, 2',3,6'-tri-(N-carbobenzyloxy)-3',4'-dideoxyneomycin B, 2',3,6'-tri-(N-carbobenzyloxy)-3',4'-dideoxyneomycin C and 3',3,6'-tri-(N-carbobenzyloxy)-3',4'-dideoxylostasin, respectively, and there is thereby produced l-N-~L-(-)-y-amino-~-hydroxybutyryl]-3',4-dideoxyribostamycin, l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3',4'-dideoxyneomycin B, 1-N-[L-(-)-y-amino-~-hydroxybutyryl]-3',4'-dideoxyneomycin C
and l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3',~'-dideoxyxylostasin, respectively, The reaction of each of the 2',3,6'-tri-(N-carbobenzyloxy)-protected aminoglycoside starting materials listed above in the same manner with L-(-)-~-benzyloxycarbonylamino-~-hydroxypropionic acid N-hydroxy-5-norbornene-2,3-dicarboximide ester instead of ~he L-(-)-y-benzyloxycarbonylamino-~-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester produces the corresponding l-N-[L-(-)-~-amino-~-hydroxypropionyl] amino-glycosides.

- 5~ -5~

The reaction of each of the 2',3,6'-tri-(N-carbobenzyloxy)-protected aminoglycoside starting materials listed above in the same manner with L-(-)-~-benæyloxycarbonylamino-a-hydroxyvaleric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester instead of the L-(-)-y-benzyloxycarbonylamino-~-hydroxybutyric acid N-hydroxy-5-norbornene-2,3-dicarboximide ester produces the corres-ponding l-N-[L-(-)-~-amino-~-hydroxyvaleryl] aminoglycosides.

Claims (8)

Div.

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

1. A process for preparing a polysilylated aminoglycoside comprising silylating aminoglycoside of the formula XIV

wherein R2 is a hexopyranosyl ring of the formula , or II III IV

in which R6 is H or CH3, R7 is H or CH3, R8 is OH or NH2, R9 is H or OH and R10 is H or OH;

R3 is H or a hexopyranosyl ring of the formula Div.

V VI
or VII VIII
in which R11 is H or CH3;
R5 is H or OH; and R4 is H, OH or a pentofuranosyl ring of the formula or IX X
in which R12 is H or a hexopyranosyl ring of the formula or XI XII

Div.

in which R13 is H or .alpha.-D-mannopyranosyl;

provided that, when R3 is other than H, one of the R4 and R5 is H and the other is OH; and provided that, when R3 is H, R5 is H and R4 is a pentofuranosyl ring of the Formula IX or X;

said aminoglycoside containing from 0 to 3 amino-blocking groups other than silyl on amino groups other than the C-1 amino group, said silylating step comprising treating with 8 to 11 equivalents of a silylating agent in the presence of substantially anhydrous solvent, and in the case where the number of amino-blocking groups other than silyl is 0 or a number smaller than the number desired, introducing any further desired N-blocking groups into the polysilylated aminoglycoside (after partial desilylation by hydrolysis or solvolysis if necessary).

2. A process as in Claim 1 wherein the polysilylated aminoglycoside contains an average number of silyl groups per molecule of from 3 to 8.

3. A process as in Claim 1 wherein the silyl groups are trimethylsilyl.

4. A process as in Claim 1 wherein the amino-blocking groups are the carbobenzyloxy or trifluoroacetyl group.

5. A polysilylated aminoglycoside prepared from an aminoglycoside of the formula XIV

Div.

wherein R2 is a hexopyranosyl ring of the formula , or II III IV

in which R6 is H or CH3, R7 is H or CH3, R8 is OH or NH2, R9 is H or OH and R10 is H or OH;
R3 is H or a hexopyranosyl ring of the formula , V VI

or VII VIII

in which R11 is H or CH3;
R5 is H or OH; and R4 is H, OH or a pentofuranosyl ring of the formula Div.

or IX X

in which R12 is H or a hexopyranosyl ring of the formula or XI XII

in which R13 is H or .alpha.-D-mannopyranosyl;
provided that, when R3 is other than H, one of R4 and R5 is H and the other is OH; and provided that, when R3 is H, R5 is H and R4 is a pentofuranosyl ring of Formula IX or X;
said polysilylated aminoglycoside optionally containing from 1 to 3 amino-blocking groups other than silyl on amino groups other than the C-1 amino group, whenever prepared or produced by the process of Claim 1 or by an obvious chemical equivalent thereof.

6. A polysilylated aminoglycoside of Claim 5 containing an average number of silyl groups per molecule of from 3 to 8, whenever prepared or produced by the process of Claim 2, or by an obvious chemical equivalent thereof.

Div.

7. A polylsilylated aminoglycoside of Claim 5 wherein the silyl groups are trimethylsilyl, whenever prepared or produced by the process of Claim 3 or by an obvious chemical equivalent thereof.

8. A polylsilylated aminoglycoside as in Claim 5 in which the amino-blocking groups are the carbobenzyloxy or trifluoroacetyl group, whenever prepared or produced by the process of Claim 4 or by an obvious chemical equivalent thereof.