Note: Descriptions are shown in the official language in which they were submitted.
5~L
This invention relates to a process for the preparation
of 1-N-[l~-amino-~-hydroxyalkanoyl]aminoglycoside antibiotics
having the formula
R20
R4~ `
HO-CH
(fH2)n
l H2
NH2
wherein n is an integer of from 0 to 4; R2 is a substituted hexo-
pyranosyl ring as hereinafter defined; R3 is hydrogen or a sub-
stituted hexopyranosyl ring as hereinafter defined; R is hydrogen,
hydroxy or a pentofuranosyl ring as hereinafter defined; and R5 is
hydrogen or hydroxy; provided that, when R3 iS other than hydrogen,
one of R4 and R5 iS hydrogen and the othær 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
R O/~
R4 o \/ 2 XIV
optionally containing from 1 to 3 amino-blocking groups other than
silyl on amino groups o-ther than the C-l amino group, in a sub-
stantially anhydrous organic solvent, with an acylating derivative
of an acid of the formula
3~ B-HN-cH2-(cH2)n~lcH-cooH
OH XI~I
~ - 1- ~'.
~5~
in which B is an amino-blocking group and n is as described above.
~11 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 siochemistry, 30, 111-182, Academic
Press, N.Y. (1974). This review article (and references cited
therein) also discusses many known l-N-(acyl)aminoglycoside anti-
biotics such as the l-N-[L~(-)-~-amino-a-hydroxybutyryl] deriva-
tives of kanamycin A, kanamycin B, 3',4'-dideoxykanamycin B,
tobramycin, paromomycin I, ribostamycin, 3', 4'-dideoxyribo-
stamycin and lividomycin A.
U.S. Patent 4,029,882 discloses l-N-acyl derivatives of
gentamicins A, B, Bl, Cl, Cla, C2, C2a and X2, sisomicin, verda-
micin, mutamicins 1, 2, 4, 5 and 6, and antibiotics G-418, 66-40B,
66-~OD, 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 acylatin~ derivative of
the desired side-chain acid.
U.S. Patent 4,055,715 discloses the l-N-[L~ -amino-
a-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 group), with an
acylating derivative of L-(-)-~ amino-a-hydroxybutyric acid (such
as its N-hydroxysuccinimide ester).
5~
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 1-N-[L~ -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-alkylkanamycin A (either unprotected
or having its 3- or 3"-amino group protected with a conventional
amino-blocking group) with an acylating derivative of L-(-)-~-
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
2 ( 2)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 _ia 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 kanamycln A or B
and the 2'-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 es-ter of the
acylating acid.
.~
5~
U.S. Patent 3,974,137 discloses and claims a process for
preparing l-[L~(-)-~-amino-~-hydroxybutyryl]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 ester of L-(-)-~-benzyloxycarbonylamino-~-hydroxybutyric
acid, and subsequently removing the protecting groups.
In The Journal of Antibiotics, 26, 790-3 (1973), T~P.
Culbertson 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 pyridine,
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 2N acetic acid at reflux, to
give tetra[N-(trifluoroacetyl)]butirosin.
The inal 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
~ s~s~
provides a high degree of selectivity of acylation in the desired
l-N-position.
J.J. Wri~ht 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 is the most reactive
toward acylation when the amino groups of the molecule are almost
completely protonated. These conditions are achieved by the
addition of one e~uivalent 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
Cl , sisomicin and verdamicin, they reported that little selec-
a
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 aminoglycoside (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 aminoglycoside, all of its
functional groups are preferably protected by means of silyl or
acyl groups.
The present invention provides an improved process for
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. ~lthough
the reaction is usually conducted in solutions containing about
5g~
10-20% polysilylated aminoglycoside starting material, excellent
results have been obtained at concentrations of about 50~ W/V
(e.g. 50 ~ms./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 u~ilizing the present
invention, thereby giving smaller amounts of undesired products
which both increases the yield of desired product and simplifies
chromatographic purification. Thus, in preparing l-[L-(-)-~-
amino-a-hydroxy~utyryl]kanamycin A amikacln by various prior art
procedures, there is typically also produced the 3"-~-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 aqueous 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 e~ual amount of the desired l-N-acylated product, due to the
great diEficulty of separating the latter from the :Eormer. 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
R4 ~ _O
HO-CH
2)n
CH2
NH2
or a pharmaceutically acceptable acid addition salt thereof,
wherein n is an integer of from 0 to 4; R2 is a hexopyranosyl
ring of the formula
, CHNHR7 lR6 CHNHR7
9 ~ R10
II III IV
in which R6 is H or CH3, R is H or CH3, R8 is OH or NH2, R9 is H
or OH and R is H or OH;
R3 is H or a hexopyranosyl ring of the formula
HO ~ O
HO ~ CH] ~
V VI
C~13 ;; ~ or ~12
VII VIII
in which R 1 is H or CH3;
RS is H or OHI and
R4 is H, OH or a pentofuranosyl ring of the formula
HOCH~ HOCH
or ~ 7
OH ~120 OH
IX X
J.0 in which R12 is H or a hexopyranosyl ring of the formula
_ R130
H2N
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 is
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~_
R _ ~ \
R R ~ NH2 XIV
in which R~, 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
sq~s~
B-HN-CH2- (CH2) n-CH-COOH
1H XIII
in which B is an amino-blocking group and n is as defined above;
and subsequently removing all blocking groups.
The amino group of the acylating acid of Formula XII
above must be protected by an amino-blocking group during the
acylation reaction. This is normally done by the use of conven~
tional amino-blocking group. These same conventional amino-
blocking groups may be utilized to protect amino groups other
than the C-l amino group of the aminoglycoside. Such con-
ventional blocking groups for the protection of primary amino
groups are well known to those skilled in the art. Suitable
blocking groups include alkoxycarbonyl groups such
- 8a -
s~s~
as t-butoxycarbonyl and t-amyloxycarbonyl; aralkoxycarbonyl
~roups such as benzyloxycarbonyl; cycloalk~lox~carbonyl groups
such as cyclohexyloxycarbonyl; haloalkoxycarbonyl groups such
as triehloroethoxycarbonyl; aeyl groups such as phthaloyl and
o-nitrophenoxyacetyl; haloacetyl groups such as trifluoro-
acetyl; and other well-known blocking groups such as the
o-nitrophenylthio group, the trityl group, etc.
The aeylatin~ acid of Formula XIII contains an
asyn~etric carbon atom and may exist in its (+) or (-) ~orm or
as a mixture thereof (the d, 1 form), thus producing the
corresponding compound of Formula I in which the l-N- L~-amino-
~-hydroxyalkanoyl] group is in its (+) [or (R)] form or its
(-) [or (S)] form or a mixture thereof. Each such optically
active form, and the mixture thereof, is included within the
scope of this invention, but the (-) form is preferred.
Acylation of the polysilylated aminoglycoside (with
or without from 1 to 3, amino-blocking groups other than silyl
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 sufficient solubility. These starting
materials are highly soluble in most common organic solvents.
Suitable solvents include for example, acetone, diethyl ketone,
methyl n-propyl ketone, methyl isobutyl ketone, methyl ethyl
ketone, heptane, glyme, diglyme, dioxane, toluene, te-trahydro-
furan, cyclohexanone, pyridine, methylene chloride, chloroform
and carbon tetrachloride. The choice of sol~ent is dependent
on the particular starting materials employed. Ketones,
generally, are the preferred solvents. The most advantageous
solvent for the particular combination of reactants being
utilized can readily be determined by routine experimentation.
g _
~ ~5~5~
In one aspect the present invention provides a
process for the preparation of l-N-[L-(-)-~-amino-~-
hydroxyalkanoyl]aminoglycoside of the formula
R6
CH-R26
RlO/~
R9 ~ ~ ~
~24 o / \ ~ C-O
R23 ~ / HO~CH L-(-)
R22HN~ H2) n
~ / NH 2
or a pharmaceutically acceptable salt thereof, wherein n is
an integer o rom 0 to 4, R6 is H or CH3, R8 is OH or NH2,
R is H or OH, R is H or OH, R2 ls H or CH3, R 3 is OH or
CH3, R24 is H or OH, R25 is H or CH2OH, and R26 is OH, NH2
or NHCH3; provided that, when R22 is H, R25 is CH2OH, and when
R 2 is CH3~ R25 is H; and provided that, when R is OH, R
is H, and when R23 is CH3, R24 is OH;
which process comprises reacting a polysilylated amino-
glycoside prepared from an aminoglycoside of the formula
- g(a) -
`~
~ ~$~
CH-R
Rl 0
R ~ ~ ~ I ~
R I / HO ~ ~ 7 ~ N~I2
22HN~
HO
o
h R , R , R , R10, R22, R23 R24 R2s 26
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 substantially anhydrous organic
solvent, with an acylating derivative of an acid of the
formula
L-(-) s-HN-CH2-(CH2)n-1CH-COOH XIII
H
in which B is an amino-blocking group and n is as defined
above; and subsequently removing all blocking groups.
In another aspect the invention provides a process
for the preparation of a 1-N-[L-(-)~-amino-~-hydroxyal]canoyl]-
aminoglycoside of the formula
- 9(b) -
R27- HNH-R 8
,~--o NH 2
H2N o HO ~ NH
C=O
/ /HO-CH L-(-)
H3C ~ / (IH2)n
H3CHN ~ / NH2
HO l/ 2
or a pharmaceutically acceptable salt thereof t wherein n is
an integer of from 0 to 4/ R is H or CH3 and R 8 is H or
CH3; which process comprises reacting a polysilylated amino-
glycoside prepared from an aminoglycoside of the formula
R27 -C~NH-R
~ O NH
H2N O HO - - 7~ NH2
OH
~
H3CHnr~ J
in which R27 and R28 are as defined ahove, and which contains
2 or 3 amino-blocking groups other than silyl on amino groups
other than the C-l amino group, in a substantially anhydrous
organic solvent, with an acylating derivative of an acid
of the formula
L-(-) B-HN-CH2-(CH2)n-CH-cOO~
I XIII
OH
-- 9 (c)
5~
in which B is an amino-blocking group and n is as defined
above; and subsequently removing all blocking groups.
In still a further aspect the invention provides a
process for the preparation of a l-N-[L~ ~-amino~-
hydroxyalkanoyl]aminoglycoside of the formula
R10 ~ r~l'6
O / ~ NH
0 / HOC=O
/ HO-CH L-(-)
/ (IH2)n
~/ C~ H22
R2 9 )__(
R30 OH
or a pharmaceutically acceptable salt thereof, wherein n is an
integer of from 0 to 4, R9 is H or OH, R10 iS H or OH, R 6
is OH, NH2 or NHCH3, R 9 is H or OH, and R is H, OH or OR
in which R31 is a hexopyranosyl ring of the formula
~0
R32 ~ r R O
H2N
in which R32 is H or ~-D-mannopyranosyl, and one of R and
R is H and the other is CH2NH2; provided that, when R is
H, R is OH or oR31, and that when R29 is OH, R30 is H;
which process comprises reacting a polysilylated amino-
glycoside prepared from an aminoglycoside of the formula
- 9(d) -
~ .
~ ~5~5~
.
.
R10 ~ ~R26
R ~ \
HO ~ H2
~a
wherein R9, R10, R26, R29 and R30 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 substantially anhydrous organic solvent, with an
acylating derivative of an acid of the formula
L-(-) B-HN-cH2-(cH2)n-f~-cooH XII
OH
in which B is an amino-blocking group and n is as defined
above; and subsequently removing all blocking groups.
Suitable silylating agents for use in preparing the
polysilylated aminoglycoside starting materials utilized herein
include those of the formula
R14 ~R14
R14/ \~ ~ R and R16_1 -Z
\ R m
XV ~ ~ XVI
- 9(e) -
: i .
5~5~
wherein R15, R 6 and R 7 are selected from the group consisting
of hydrogen, halo~en, (lower)alkyl, (lower)alkoxy, halo(lower)
alkyl and phenyl, at least one of the said R15, R16 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
\ Rl 9
~0 wherein R18 is hydrogen or (lower)alkyl and Rl9 is hydrogen,
(lower)alkyl or
R15
R16_ -- Si
117
in which R15, R16 and R17 are as de~ined above.
Specific silyl compounds of Formulas XV and XVI are:
trimethylchlorosilane, hexamethyldisilazane, triethylchlorosilane,
methyltrichlorosilane, dimethyldichlorosilane, trie~hylbromo-
silane, tri-n~propylchlorosilane, methyldiethylchlorosilane,
dimethylethylchlorosilane, dimethyl-t-butylchlorosilane, phenyl-
dimethylbromosilane, benzylmethylethylchlorosilane, phenylethyl-
methylchlorosilane, triphenylchlorosilane, triphenylfluorosilane,
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 trimethylsilylurea) and silylureides. Trimethyl-
silylimidazole also may be utilized.
-- 10 --
A preferred silyl group is the trimethylsilyl group and
preferred silylating agents for introducing the trimethylsi]yl
group are hexamethyldisilazane, bis(trimethylsilyl)acetamide,
trimethylsilylacetamide and trimethylchlorosilane. Hexamethyl-
disilazane is most pre~erred~
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
the 6'-N-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.
20Kanamycin B has the formula
4l 6'
~ CH NH
HO ~ \ ~ ~
~ O / N 2
4,1 6"
HO ~ /5" O
H2N ~ 1" /
HO I /
. . .
s~
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 b~ the adjacent
2"- and 4"- silylylated hydroxy groups. It is believed that i~
is for this reason that no 3"-acylated product is normally
detected when acylating polysilylated kanamycln B (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
a nearby 4'- and nearby 3'- silylated hydroxy groups. The 2'-
amino moiety is hindered by the ad]acent 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 o~
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)
- 12 -
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
10 XK-62-2, aminoglycoside 66-40D, verdamicin and sisomicin.
Another class of aminoglycosides are those wherein the
glycoside 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
O / ~
O `~"NH2
/ 5 HO
5"
HOCH
4" ~
3" ~ 2"
HO OH
In aminoglycosides of the ribostamycin type, polysllylation
hinders the desired l-N-amlno moiety more than -the undesired
3-N-amino moiety (the other amino groups being hindered as
described abo~e for the kanamycin types). Thus polysilylated
antibiotics of the ribostamycin type will form primarily the
- 13 -
s~
3-N-substituted product upon acylation and it ~herefore is neces-
sary to protect the 3-amino moiety with an amino-blocking group
such as carbobenzyloxy, in order to obtain the l-N-substituted
product upon acy~ation 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 s, 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 ribostamycin type antibiotics which
contain a 6'-amino group are also included in this class. Some
of the aminoglycosides in this class contain a 6l-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 B 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 l to 3 amino blocking groups
other than silyl on amino groups other than the C-l amino group,
- 14 -
--
5~
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
tafter partial desilylation by hydrolysis or solvolysis, if
necessary).
Methods ~or 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 o~ silyl groups (or their location)
present in the polysilylated aminoglycoside starting materials
(with or without from 1 to 3 amino-blocking groups other than
silyl on amino groups other than the C-l 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.
- 15 -
.~ . ,
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 par-t of the
invention.
Polysilylated aminoglycosides containing the desired
number of silyl groups may be prepared either 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, ~or example, when preparing 1-N-[L-(-)-y-amino-~-
hydroxybutyryl]kanamycin A by acylating polysilylated kanamycin
A with the N-hydroxysuccinimide ester of L-(-)-y-benzyloxy-
carbonylamino-~-hydroxybutyric acid in acetone solution, we have
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.
- 16 -
s~
It will be appreciated that each mole of he~amethyldi-
silazane is 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, 5O5 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-
in~ 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 reaction 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
amino~lycoside sulfate), sulfamic acid, imidazole and trimethyl-
chlorosilane. Silylation catalysts generally promote a higher
de~ree of silylation 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
;~-
~5~
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-
~-hydroxybu~yric 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 mat~rials, 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-N-[L-(-)-~-amino-~-hydroxybutyryl]kanamycin A were obtained
when adding up to 28 moles of water per mole of polysily]ated
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 utilize short acylation times in order to avoid excessive
desilylation of the polysilylated aminoglycoside starting
- 18 -
. .
~ ~5~
material. Accordingly, as used herein and in the claims, the
term "substantially anhydrous organic solvent" is intended to
inelude 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
eritieal. Temperatures in the range of about -30C to about
100C 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-
dueted with any acylating derivative of the acid of Formula XIII
whieh is known ln the art -to be suitable for the acylation of
a primary amino yroup. Examples of suitable acylating deriva-
tives of the free acid include the corresponding acid anhydrides,
mixed anhydrides, e.g. alkoxyformic anhydrides, acid halides,
aeid azides, active esters and active -thioesters. The free acid
may be eoupled 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
[ef. South African Specifieation 63/2684] or a earbodiimide
reagent [espeeially N,N'-dieyclohexylcarbodiimide, N,N'~diiso-
propylearbodiimide or N-cyclohexyl-Ni-(2-morpholinoethyl)-
19
~5~
carbodiimide" cf. Sheehan and ~Iess, J.~.C.S., 77, 1967 (1955)],or of an alkynylamine reagent [cf. R. Buijle and H.G. Viehe,
Angew. Chem. International Edition, 3, 582 (196~)] or of an
isoxazolium salt reagent ~cf. R.B. Woodward, R.A. Olofson and
H. Mayer, ~. 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) or 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 ~uasiaromatic 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-
o~y 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.
- 20 -
s~
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
treatment with z.inc dust in glacial acetic acid, the phthaloyl
group 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
R2~CH20C- , CH3-C-0-C- , Y2XCC-
CH3
R21
N02
.~
5~5~L
X3C-CH2-O-C- and ~
Il /
wherein R20 and R21 are alike or different and each is H, F, Cl,
Br, NO2, OH, (lower)alkyl or (lower)alkoxy, X ls Cl, sr, F or I,
and Y is H, Cl, Br, F or I. A particularly preferred amino-
blocking group 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 p~2 has the structure IV). These are com-
pounds derived from aminoglycosides such as sisomicin, verdamicin,
G-S2, 66-40s 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 of product were determined by various methods.
After removal of all blocking groups and chromatography on a CG-50
(NH4~) column, the yield could be determined by isolation of the
crystalline solid from the appropriate fractions or by micro-
biological assay (turbidimetxic or plate) of the appropriate
fractions. Another technique which we utilized was hi~h perfor-
mance liquid chromatography of the unreduced acylation mixture,
i.e. the aqueous solution obtained after hydrolysis of the silyl
~roups and removal of organic solvent but before hydrogenolysis
- 22 -
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
24~ high pressure llquid 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./mlA
Chart speed varied, but 2 minutes/inch was typical. The above
conditions gave 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.
AHBA L-(-)-~-amino-~-hydroxybutyric acid
BHBA N-Carbobenzyloxy derivative of AHBA
HONB 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
- 23 -
~Y
~CU dicyclohexylurea
HMDS hexamethyldisilazane
BSA bis(trimethylsilyl)acetamide
MSA trimethylsilylacetamide
TFA trifluoroacetyl
t-BOC tert. butyloxycarbonyl
"Dicalite" is a trademark of the Great Lakes Carbon
Corporation for diatomaceous earth.
"~mberlite CG-50" is a Trademark of the Rohm & Haas Co.
for the chromatographic 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 Formu]a 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,
depending on the particular compound of Formula I. I-t 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 pharmaceuticals.
Most of the aminoglycosides used as star-ting materials in
the present invention are known in the art. ~ny individual amino-
- 24 -
~:~ ,,,
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 per se.
In another aspect the present inventlon provides poly-
silylated aminoglycosides of Formula XIV 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-(-)-r-Amino-~-hydroxybutyryl]kanamycin A
amikacin by Selective Acylation of Polyttrimethylsily1) 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. ~lexamethyldisilazane (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
in 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-(-)-r-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-
~ 25 -
54
tion was stirred at 23 for 78 hours. The yellow, clear solution
(pH 7.0) was diluted with 100 ml. of water. The p~ of the mix-
ture was adjusted to 2.8 (3N ~Cl) and stlrred vigorously at 23
for 15 minutes. The aqueous phase was separated, and the
organic phase was extracted with 50 ml. of pH 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. o~ water. The colorless filtrate was concen-
trated in vacuo (40-45) ~o 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 +0.115 0.989 9.15
BB-K29 1750 10.24 4.37 32.0
Amikacin 2000 ~0.31 6.20 47.4
- ~olyacyls 900 +0.032 0.288 2.0
The spent diethyl ketone layer was shown by high pexformance
liquid chromatography to contain an additional 3-5% amikacin.
- 26 -
~,,
~.
i4~
The erude 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~ y-Amino-~-hydroxybutyryl]kanamycin A
~mikaein b Seleetive Aeylation of Poly(trimethylsilyl)Kanamycin
Y
A, Using In Situ Bloeking
A. Poly(trimethylsilyl) Kanamycin A
Kanamyein A free base (18 g. activity, 37.15 m. moles) was
slurried in 200 ml. of dry aeetonitrile and heated to reflux.
~lexamethyldisilazane (29.8 g., ]84.6 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 vaeuum left an amorphous solid residue (43 gm., 94~)
[ealeulated as kanamyein A (silyl)10].
B. l-N-[L-(-)-y-A-mino-~-hydroxybutyryl]kanamycin A
p-(Benzyloxyearbonyloxy)benzoie acid (5.56 g., 20.43 m.
moles) was slurried in 50 ml. of dry acetonitrile at 23. N,0-
bis-Trimethylsilyl aeetamide (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.~3 m. mole,
ealculated as the (silyl)10 compound) in 75 ml. of dry aeeto-
nitrile at 23. The mix was stirred for ~ hours, the solvent
was removed in vaeuo (40), and the oily residue was dissolved
in 50 ml. of dry acetone at 23C~
L-(-)-~-benzyloxycarbonylamino-~-hydroxybutyric acid N-
hydroxy 5-norbornene-2,3-dicarboximide ester (NAE) (8.55 g.,
20.63 m. moles) in 30 ml. of aeetone was added to the above
solution over a period of 5 minutes. The mixture was held at
.~
~5~
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.
a Example 3
Preparation of l-N-[L-(-)-y-Amino-~-hydroxybu_yryl]kanamyci'n A
Amikacin by Selective Acylation of Poly(trimethylsilyl)KanamycinA
A. Poly(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-(-)-y-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-(-)-~-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 strlpped to dryness ln vacuo at a
bath temperature of 30-40C. Water (60 ml.) was then added to
the residue, followed by 70 ml. of methanol to ob-tain 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
28 -
., ~
54~5~
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 ~ith the Mixed Acid Anhydride
o~ Pivlaic Acid and BHBA
A. Preparation of Mixed Anhydride
BHBA (5.066 gm., 20.0 m moles), BSA (4.068 gm., 20.0 m
.0 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
23C.
B. Acylation of Poly(trimethylsilyl) 6'-N-Cb~ ~ana A
Poly(trimethylsilyl) 6'-N-Cbz Xana 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 ~, 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 mixt~re, 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. After 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
- 29 -
59~
acetate, after which the excess ethyl acetate was stripped from
the aqueous layer. An aliquot of -the 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 present.
Example 5
Preparation of Amikacin by Acylation of Poly(trimethylsilyl) 6'-
N-Cbz Kana A in Acetone with the Mixed Anhydride of ~HBA and
Isobutylcarbonic Acid
A. Preparation of Mixed Anhydride
BHBA (1.267 gm., 5.0m.~olès) and N--trimethylsilylacetamide
(MSA) (1.313 gm., 10.0 m moles) in 20 ml. of ~ieve dried acetone
was stirred at 23C, and triethylamine (TEA) (0.70 ml., 5.0 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., 5.50 m moles) was
added. Triethylamine hydrochloride immediately be~an to separate.
The mixture was stirred for 1 hour at -20C.
B. Acylation
Poly(trimethylsilyl) 6'-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 stirred for an
additional 1 1/2 hours at -20C and then for 17 hours at 23C.
- 30 -
.
The reaction mixture was then poured int~ 150 ml. of water at
23C with stirring, the pH (7.75) was adjusted to 2.5 with 3N
~Cl, 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 solu~ion 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
L0 combined filtrate and washings were determined by microbiological
assay against E. co`li to contain a 35.0% yield of amikacin.
Example 6
Preparation of Amikacin by Acylation of oly(trimethylsily`l) 6'-
N-Cbz Kana A in Anhydrous Cyclohexanone`for Varyin`g 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 23 C 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 pH
(S.6) was adjusted to 2~5 with 3N HCl. The cyclohexanone was
stripped in vacuo at 40C and an aliquot of the remainin~ 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.
- 31 -
3~
S9L
Yields
HPLC Ass'ay 'Microbiologlcal Assay (Amikacin)
(di'-Cbz`amika'cin) Turbi'dimetric Plate
Reaction A 49.18% 42.87~ 39.16%
Reaction B 56.17~ 55.39% 38.45
Example 7
Prepa ation of Amikacin by Acylation o-f Poly(trimethylsilyl) 6'-
N-Cbz Kana A in Anhydrous Tetrahydrofuran for 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 utilized as solvent ins-tead of dry cyclohexanone.
Yields
HPLC Assay Microbiological 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-Cbæ Kana A in Anhydrous DioXane for Vary'ing'Times
A Example 6 A was repeated except that the acylation was
continued for 44 hours utilizing dry dioxane as the solvent.
B. Example 6 B was repeated except that ~he 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~
- 32 -
. ~
~ ~f5
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) 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 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 HCl 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 catalyst 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 Poly(trimethylsilyl)
ICana A With NAE at 0-5 After Back Hydrolysis With Water
A. Silylation of ICanamycin 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 (22.76 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
- 33 -
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
~er 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
Preparatlon 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 39.1 gms
; - 34 -
5~5~
of white amorphous solid (95.4% yield, calculated as 6'-N Cbz
kana A (silyl)g).
B. Acylation
Poly(trimethylsilyl) 6'-N-Cb~ 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 nitrogen 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 addedr
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, concentLated in vacuo to
to 75-100 ml, charged on a CG-50(NH4~) column and eluted with
water and 0.6N NH40H. There was obtained 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(trimethylsilyl)
Kana A With SAE at 0-5 After Back Hydro'lysi's With Wat'er
A. Silylation' of Kanamycin A With TMCS in Acetonit'rile
Using Tetrame*hylguanidine a's'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
- 35 -
white precipitate 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 (lO0 ml), and the
insoluble TMG-HCl was filtered o~f 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
10 (silyl) 10) .
B. Acylation
Poly(trimethylsilyl) kanamycin A (10.64 gm, 10.07 m moles)
prepared in step A above was dissolved in l~0 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, lO 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, 10O57 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%
kanamycin A and 5-8% polyacyls.
Example 13
Preparation of Po~y(triethylsilyl) Kanamycin A Using Triethyl-
chlorosilane With Triethylamine as Acid Acceptor
Kanamycin A (5.0 gms of 97.6% purity, 10.07 m moles) was
suspended in lO0 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. ~eflux was continued
- 36 -
~.
s~
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
hours, concentrated n 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 lO0 ml dry THF at 23 and the insoluble
TEA-HCl was filtered off, washed with 5 x 20 ml of TH~, and dried
to give 16.0 gms of TEA HCl. 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 polyttriethylsilyl)
kanamycin A as a deep orange viscous syrup.
Example 14
Preparation of Poly(trimethylsilyl) Kanamycin A Using bis-
Trimethylsilylurea
Kanamycin A ~10.0 gm of 99.7~ purity, 20.58 m moles) was
suspended in 200 ml of sieve-dried acetonitrile, 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
the 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 40 and dried
under high vacuum to give 27.0 gm of a white solid which was
partly tacky and partially fine needle-like crystals. I'he solid
was treated with 150 ml of heptane at 23, the insoluble portion
S~,
was removed by filtration, washed with 2 x 50 ml por-tion 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 Perttrimethylsilyl)
Kanamycin A After Partial Desilylation With 1,3-Butanediol
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 1 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
~ 38 -
..~,
5~
(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 uslng 3.0 gm of 10%
Pd on carbon as catalyst. The reduced solution was filtered
through Dicalite and chromatographed as in Example lls to give
47.50% amikacinr 5.87% BB-K29, 7.32% BB-K6, 24.26% kanamycin A
and 7.41% polyacyls.
E~ample 16
_
Preparation of Amikacin by the Acylation of Poly(trimethylsilyl)
Kanamycin A Prepared in THF Using SAE With Sulfamic Acid Catalyst
To a refluxing mixture of kanamycin A (5.0 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 THF 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 reduced 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.
- 39 -
Example 17
Preparation of Amikacin by tlle Acylation o~ Poly(trimethylsilyl)
Kanamycin A with the N-Hydroxysuccinimide Ester of Di-Carbobenzyl-
o~ AHBA
A. Preparation of Dicarbobenzyloxy L~ -Amino-~-hydroxy-
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 dry
acetone 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
modera-te vacuum.
To this solution was added the solution of the di-Cbz-
blocked acylating agent prepared in step A, and the mixture was
stirred 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.
- 40 -
5~
Example 18
Preparation of Poly(trimethylsilyl) Kanamycin 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 ni-trogen purge. HMDS (1~.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 after 1 hour. The solvent was then removed in vacuo at
60 and the oily residue was held at 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 step 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 s-tirred in vacuo
- 41 -
. .,
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.
Examp'le 20
Preparation of Poly(trimethyl~silyl) K~anamycin A Using HMDS With
Kanamycin ~ sulfate 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 With 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 AHBA (5.0 gm, 42 m moles) in 100 ml THF
was added trifluoroacetic anhydride (40 gm, 191 m moles), with
stirring, over a 10 minute period. The solution was stirred for
- 42 -
~,
s~
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
alld redissolved in 50 ml H20. The aqueous solution was extracted
with 3 x 50 ml portions of MIBK and, after drying over Na2S04,
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 AHsA (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 preclpitated dicyclohexylurea was removed by filtration and
washed with a small amount of acetone. The combined filtrate and
washings (a solution o~ the N-hydroxysuccinimide ester of N-tri-
fluoroacetyl AHBA) was utili~ed in the next step w~thout 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 stirred ln
acuo at 0-5 for 30 mlnutes. 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% H2S04, the mixture
was stirred for 30 minutes and the pH was then raised to ca. 5.0
with NH40H. The mixture was then stripped to dryness in a rotary
evaporator to give 14.4 gm of a tacky off-white solld. The solid
was dissolved in 100 ml of water, the pH was raised from 5.5 to
11.0 with lON NH40H and the solution was heated in an oil bath
at 70 for 1 hour. The pH (9.5) was th~n lowered to 7.0 with HCl,
- ~3 -
s~
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 (NH4+)
column t8 x 90 cm), washed with 2 liters of water and eluted with
a NH40H gradient (0.6N-l.ON-concentrated). There was obtained
28.9% amikacin, 5.0% BB-X6, 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)
~anamycin A With _-Butyloxycarbonyl Bloc]ce-d AHBA N-Hydroxy-
succinimide Ester
A. Preparation of _-BOC AHBA and Convers-ion 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 lON 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 stirred for 35 minutes
while maintaining the pH at 10 by the periodic addition of lON
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 lowPred 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 Na2S04 and concentrated to a clear oily residue (8.2 gm,
89%).
The t-BOC-AHBA (4.25 gm, 19.4 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 Eiltration and was washed with a small amount of
- 44 -
_
acetone. The combined filtrate and washings (a solution of the
N-hydroxysuccinimide ester of t-BOC-AHB~) was utilized in the
next step without isolation.
B. ~
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 mixture was allowed to stand at 5 for 1 hour. Water (200 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 repeatecl 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,
- 45 -
5~S~
kanamycin B,
6'-N-methylkanamycin B,
tobramycin (3'-deoxykanamycin B),
6'-N-methyltobramycin,
amino~lycoside NK-1001,
3'-deoxy aminoglycoside NK-1001,
6'-N-methyl aminoglycoside NK-1001,
3'-deoxy-6'-N-methyl aminoglycoside NK-1001,
gentamicin A,
3'-deoxygentamicin A,
g~ntamicin B,
3'-deoxygentamicin B,
6'-N-methylgentamicin B,
3'-deoxy-6'-N-methylgentamicin B,
gentamicin Bl,
3'-deoxygentamicin Bl,
6'-N-methylgentamicin Bl,
3'-deoxy-6'-N-methylgentamicin Bl,
gentamicin X~,
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-~-hydroxybutyryl]-6'-N-methylkanamycin A,
l-N-[L-~-)-y-amino-~-hydroxybutyryl]-3'-deoxy-6'-N-methyl-
~anamycin A
l-N-[L-(-)-y-amino-~-hydroxybutyryl]kanamycin B,
l-N-lL-(-)-y-amino-~-hydroxybutyryl]-6'-N-methylkanamycin B,
l-N-[L-(-)-y-amino-~-hydroxybutyryl]tobramycin
1-N-[L-(-)-y-amino-a-hydroxybutyryl]-6'-N-methylto~ramycin,
l-N-[L-(-)-y-amino--hydroxybutyryl] aminoglycoside N~-1001,
l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3'-deoxy amino~lycoside
NK-1001,
- 46 -
5~
l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6'-N-methyl aminoglycoside
NK-1001,
l-N- [L- (-) -y-amino-~-hydroxybutyryl]-3'-deoxy-6'-N-methyl amino-
~lycoside NK-1001,
l-N-[L-(~)-y-amino-~-hydroxybutyryl]gentamicin A,
l-N-[L-(-)-y-amino-~-hydroxybutyryl]-3'-deoxygentamicin A,
l-N- [L- (-) -~-amino-~-hydroxybutyryl]gentamicin B,
l-N- [L- (-) -y-amino-~-hydroxybutyryl]-3'-deoxygentamicin s,
l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6'-N methylgentamicin B,
1-N-[L-(-)-y-amino-~-hydroxybutyryl]-3'-deoxy-6'-N-methyl-
gentamicin sl,
l-N- [L- (- ) -y-amino-~-hydroxybutyryl]gentamici.n Bl,
l-N-[L-(-)-r-amino-~-hydroxybutyryl]-3'-deoxygentamicin Bl,
l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6'-N-methyl~entamicin Bl,
l-N- [L- (-) -y-amino-~-hydroxybutyryl]-3'-deoxy-6'-N-methyl-
gentamicin Rl,
l-N-[L-(-)-y-amino-~-hydroxybutyryl]gentamicin X2,
l-N-[L-(-)-y-amino-~-hydroxybutyryl] seldomycin factor 1 and
l-N-[L- (-)-y-amino-~-hydroxybutyryl] seldomycin factor 2,
respectively.
The reaction of each of the aminoglycoside starting
mate~ials listed above in the same manner with L-(-)-~-benzyloxy~
carbonylamino-~-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-dicarbox.i-
mide ester produces the corresponding l-N-~L-(~ amino-~-
hydroxypropionyl] aminoglycosides.
The reaction of each of the aminoglycoside starting materi-
als listed above in the same manner with L-(-)-~-benzyloxycar-
bonylamino-~-hydroxyvaleric acid N-hydroxy-5-norbornene-2,3-di-
carboximide ester instead of the L~ y-benzyloxycarbonylamino-
- 47 -
N-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 ac.id
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, respec~ively.
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',41-dideoxykanamycin B,
2',61-di-(N-carbobenzyloxy)-3',4'-dideoxy-6'-N-methylkanamycin B,
~',6'-di-(N-carbobenzyloxy)gentamicin Cl,
2',6'-di-(N-carbobenzyloxy)gentamicin Cla,
2',6'-di-(N-carbobenzyloxy)-6'-N-methylgentamicin C1a,
2',6'-di-(N-carbobenzyloxy)gentamicin C2.
2',6'-di-(N-carbobenzyloxy)-6'-N-methylgentamicin C2 and
2',6'-di-(N-carbobenzyloxy)ami.noglycoside XK-62-2, respectively,
and there is thereby produced
- 48 -
~ .
5~
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]-3',4'-dideoxykanamycin s,
l-N-[L-(-)-~-amino-~-hydroxybutyryl]-3',4'-dideoxy-6'-N-
methylkanamycin s,
l-N-[L-(-)-y-amino-~-hydroxybutyryl]gentamicin Cl~
l-N-[L-(-)-~-amino-~-hydroxybutyryl]gentamicin Cla,
l-N-[L-(-)-~-amino-~-hydroxybutyryl]-6'-N-methylgentamicin Cla,
l-N-[L-(-)-y-amino-~-hydroxybutyryl]gentamicin C2,
l-N-[L-(-)-y-amino-~-hydroxybutyryl~-6' N-methylgentamicin C2 and
l-N-[L-(-)-~-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-(~ -amino-~-
hydroxypropionyl] aminoglycosides.
The reaction of each of the aminoglycoside startingmaterials listed above in the same manner with L-(-)-~-benzyloxy-
carb~nylamino-~-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 2'61-di-(N-trifluoro-
- 49 -
acetyl)sisomicin] is added over a pexiod of 30 minutes and the
resulting solution is refluxed 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-trifluoroace-tyl)sisomicin
is acylated with the N-hydroxysuccinimide ester of 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-5-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 66-4OD, respectively,
and there is thereby produced.
l-N-[L-(-)-y-amino-~-hydroxybutyryl]-5-episisomicin,
l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6'-N-methylsisomicin,
l-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-a-hydroxybutyryl]-6'-N-methyl-5-epiverdamicin
and
1-N-~L-(-)-y-amino-~-hydroxybutyryl] aminoglycoside 66-40D,
respectively.
- 50 -
-
?5~S4
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-(-)-~-tri~luoroacetylamino-~-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-(-)-~-trifluoroacetylamino-~-hydroxybutyric acid
produces the corresponding l-N-[L-( )-~-amino-~-hydroxyvaleryl]
aminoglycosides.
Example 28
The general procedure of Example 26 is repeated except
that the N-hydroxysuccinimide ester of L-(-)-~-trifluoroac~tyl-
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 s,
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,
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,
3-N-carbobenzyloxy aminoglycoside 2230-C,
3-N-carbobenzyloxy-3'-deoxy aminoglycoside 2230-C,
3-N-carbobenzyloxylividomycin A and
3-N-carbobenzyloxylividomycin B, respectively,
and there is thereby produced
l-N-[L-(-)-y-amino-~-hydroxybutyryl]ribostamycin,
l-N-[L-(-)-~-amino-~-hydroxybutyryl]-3'-deoxyribostamycin,
l-N-[L-(-)-~-amino-~-hydroxybutyryl]-6'-N-methylribostamycin,
l-N-[L-(-)-y-amino-a-hydroxybutyryl]-6'-N-methyl-3'-deoxy-
ribostamyci~,
l-N-[L-(-)-~-amino-~-hydroxybutyryl]neomycin B,
l-N-[L-(-)-~-amino~-hydroxybutyryl]3'-deoxyneomycin B,
- 52 -
` ~ ~
l-N-[L~ y-amino-a-hydroxybutyryl]-6'-N-methylneomycin B,
l-N-[L-(-)-y-amino-a-hydroxybutyryl]-6'-N-methyl-3'-deoxy-
neomycin B,
l-N-[L-(-)-y-amino-~-hydroxybutyryl]neomycin C,
l-N-[L-(-)-~-amino-~-hydroxybutyryl]-3'-deoxyneomycin C,
l-N-[L-(-)-y-amino-~-hydroxybutyryl]-6'-N-methylneomycin C,
l-N-[L-(-)-y-amino-a-hydroxybutyryl]~6'-N-methyl-3'-deoxy-
neomycin C,
l-N-[L-(-)-y-amino-a-hydroxybutyryl]-xylostasin,
l-N-[L-(-)-y-amino-a-hydroxybutyryl]-3l-deoxyxylostasin~
l-N-[L-(-)-y-amino-a-hydroxybutyrylJ-6'-N-methylxylostasin,
l-N-[L-(-)-y-amino-a-hydroxybutyryl]-6'-N-methyl-3'-deoxy-
xylostasin,
l-N-[L-(-)-y-amino-a-hydroxybutyryl]paromomycin I,
l-N-[L-(-)-y-amino-a-hydroxybutyryl]-3'-deoxyparomomycin I,
l-N-[L-(-)-~-amino-~-hydroxybutyryl]-3',4'-dideoxyparomomycin I,
l-N-[L-(-)-~-amino-a-hydroxybutyryl]-paromomycin II,
l-N-[L-(-)-y-amino-a-hydroxybutyryl]-3'-deoxyparomomycin II,
l-N-[L-(-)-y-amino-a-hydroxybutyryl]-3',4'-dideoxyparomomycin II,
l-N-[L-(-)-y-amino-a-hydroxybutyryl]aminoglycoside 2230-C,
l-N-[L-(-)-y-amino-a-hydroxybutyryl]-3'-deoxy aminoglycoside
2230-C.
l-N-[L-(-)-y-amino-a-hydroxybutyryl]lividomycin A and
l-N-[L-(-)-y-amino-a-hydroxybutyrylJlividomycin B, respectively.
The reaction of each of the 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 the
L-(-)-y-benzyloxycarbonylamino-a-hydroxybutyric acid N-hydroxy-
5-norbornene-2,3-dicarboximide ester produces the corresponding
l-N-[L-(-)-~-amino-~-hydroxypropionyl~ aminoglycosides.
5~
The reaction of each of 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
1-N-[L-(-)-~-amino-~-hydroxyvaleryl] aminoglycosides.
Example 30
The general procedure of Example 1 is repeated except
that the 6'-N-carbobenæyloxykanamycin A utilized therein is
replaced by an equimolar amount of
2',3,6'-tri-(N-carbobenzyloxy)-3',~'-dideoxyribostamycin,
2',3,6'-tri-(N-carbobenzyloxy)-3',4'-dideoxyneomycin s,
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
1-N-[L-(-)-~-amino-~-hydroxybutyryl]-3',4-dideoxyribostamycin,
l-N-[L-(~ -amino ~-hydroxybutyryl]-3',4'-dideoxyneomycin B,
l-N-[L- (-! -~-amino-~-hydroxybutyryl]-3',4'-dideoxyneomycin C
and
l-N-[L-(-)-~-amino-~-hydroxybutyryl]-3',4'-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
the L-(-)-~-benzyloxycarbonylamino-~ hydroxybutyric acid N-
hydroxy-5-norbornene-2,3-dicarboximide ester produces the
corresponding 1-N-[L-(-)~~-amino-~-hydroxypropionyl] amino-
glycosides.
- 54 -
The reaction of each of the 2',3,6'-tri-(N-carbobenzyloxy)-
protected aminoglycoside starting materials listed above in the
same manner with L-(~ -ben~yloxycarbonylamino-~-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.
X
