Note: Descriptions are shown in the official language in which they were submitted.
108169~
BACKGROUND OF THE INVENTION
Field of the invention
This invention relates to new kanamycin C
derivatives active against a wide variety of
kanamycin-resistant bacteria, and a process for the
preparat.ion of these new kanamycin C derivativesO
More particularly, this invention relates to new
and useful l-N-(L-4-amino-2-hydroxybutyryl)
derivatives of kanamycin C, 3'-deoxykanamycin C
and 3',4'-dideoxykanamcyin C, and the preparation
thereof.
Description of the prior art
Butirosin B9 that is, 1-N--(L-4-amino-2-
hydroxybutyryl)-ribostamycin may be produced by a
fermentative method ("Tetrahedron Letters" 28,
2617-2620 (1971)), and 1-N-(L-4-amino-2-hydroxy-
butyryl) derivatives of kanamycin A and kanamycin B
are synthesiæed (UOSo Patent No. 3,781,268 (1973)).
l-N-(L-4-amino-2-hydroxybutyryl) derivatives of
other some aminoglycosidic antibiotics are also
synthesized (UoK~ Patent NoO 1,426,908 (published
in March 1976))o Furthermore~ deoxy derivati~es
of kanamycins are also synthetized on the basis of
the previous findings ~Jhich were obtained by
H. Umezawa et al with respect to the mechanism
of resistance of bacteria to aminoglycosidic
antibiotics owing to various inactivating enzymes
produced by the resistant bacteriaO For instance,
3',4'-dideoxykanamycin B and 3'-deoxykanamycin B
are synthesized which are active against the
- 2 -
10~ 3
resistant bacteria producing aminoglycoside 3'-phosphotransferase
(U. 5. Patent Nos. 3,753,873 and 3,929,762; and H. Umezawa's
"Advances in Carbohydrate Chemistry and Biochemistry" 30, 183
(1974) and "Drug Action and Drug Resistance in Bateria" 2, 211
(1975)). 3',4'-Dideoxykanamycin B has been widely used in thera-
peutic treatment of infections caused by a variety of the resis-
tant bacteria, including Pseudomonas aeruginosa. However, it has
been found that these deoxy derivatives of kanamycin B do not in-
hibit the growth of such resistant bacteria which are capable or
producing the aminoglycoside 6'-acetyltransferase and 2"-nucleo-
tidyltransferase. As a result of a further research, we have
succeeded in synthetically converting the 6'-amino group of kana-
mycin B or its doexy derivatives into hydroxyl group and thereby
producing kanamycin C and its deoxy derivatives which inherently
cannot be inactivated by the 6'-acetyltansferase (see co-pending
Canadian Patent Application S.N. 280,338 filed June 10, 1977).
However, kanamycin C, 3'-deoxykanamycin C and 3',4'-dideoxykana-
mycin C so obtained are not able to inhibit the growth of such
resistant bacteria producing the 2"-nucleotidyltransferase.
SUMMARY OF THE INVENTION
In these circumstances, we have made further research,
and as a result we have succeeded in synthesizing new compounds,
t l-N-(L-4-amino-2-hydroxybutyryl) derivatives of kanamycin C,3'-
.'
:,
.'~
~ 30
. .
,:
3 --
~081~;93
deoxykanamycin C ~nd 3',4'-dideoxykanamycin C which
are active against a wide variety of aminoglycoside-
resistant bacteria, including the 2"-nucleotidyl-
transferase-producing bacteria.
Thus, an object of this invention is to
provide such new kanamycin C derivatives which
inherently cannot be inactivated by the 3'-phospho-
transferase extensively distributed in a variety of
the aminoglycoside-resistant bacteria and also
cannot be inactivated by the 6'-acetyltransferase
and the 2"-nucleotidyltransferase, which is accordingly
active against not only the aminoglycoside-resistant
bacteria producing the 3'-phosphotransferase, but
also the aminoglycoside-resistant bacteria producing
the 6'-acetyltransferase and the aminoglycoside-
resistant bacteria producing the 2"-nucleotidyl-
transferase and which exhibits a very much low
toxicity. Another obJect of this lnvention is to
provide a new process for the preparation of
such new kanamycin C derivatives which can be
operated in a facile way and with a reasonable
efficiencyO Another objects will be seen from the
following descriptionsO
DETAILED DESCRIPTION OF THE INVENTION
According to a first aspect of this invention,
there is provided the novel
l-N-(L-4-amino-2-hydroxybutyryl) derivativeSof
kanamycin C, 3'-deoxykanamycih C or 3',4'-dideoxy-
kanamycin C represented by the general formula:
- 4 -
.
10~1~93
6~
4 l 2' Rl 4
HO - ~ O \ H2N 7 \ R2
H2N ~ 6 OH 14 ~ CH20H
~ ~ ~\5--'-~
HN ~ 3~ MH2 (I)
CO
(L) CHOH
CH2
CH2NH2
wherein Rl and R2 are each hydroxyl, or Rl is hydrogen
and R2 is hydroxyl or Rl and R2 are each hydrogen or
a nontoxic, pharmaceutically acceptable acid-addition
6alt thereof.
The new compounds according to this invention
include l-N-(L-4-amino-2-hydroxybutyryl)-kanamycin
C ~the compound of the formula (I) where both R
and R2 are hydroxyl group); 1-N-(L-4-amino-2-
hydroxybutyry~)-3' deoxykanamycin C ~the compound of
the formula (I) where Rl is hydrogen atom and R2 is
. hydroxyl); and l-N-(L-4-amino-2-hydroxybutyryl~-
3',4'-dideoxykanamycin C ~the compound o~ the formula
(I) where both Rl and R2 are hydrogen atom) as
: well as pharmaceutically acceptable nontoxic acid-addition
~ salts thereofO The chemical, physical and biological
properties of these new compounds are as follows:
l-N-(L-4-Amino-2-hydroxybutyryl)-kanamycin C
is a substance in the form of a colorless powder
having no definite melting point but decomposing
~81t;93
at 167-180C. It shows a specific optical rotation ~~)D +91
(c 1, water). Its elemental analysis is coincident with the
theoretical values of C22H43N5O13.H2O (C 43.77%, H 7.51%, N 11.60%).
This substance gives a single spot positive to ninhydrin at Rf.
0.18 in thin layer chromatography on silica gel (available under
a trade marlc "ART 5721", a product of Merck Co., Germany) deve-
loped with butanol-ethanol-chloroform-17% aqueous ammonia (4:5:2:8
by volume) and at Rf 0.19 in the same thin layer chromatography
developed with chloroform-methanol -28% aqueous ammonia (1:4:2 by
10 volume) as the development solvent, respectively.
l-N-(L-4-Amino-2-hydroxybutyryl)-3'-deoxykanamycin C
is a substance in the form of a colorless powder having no definite
melting point but decomposing at 151-160 C. It shows a specific
optical rotation (~)D +83 (c 1, water). Its elemental analysis
is coincident with the theoretical values of C22H43N5O12.H2O
(C 44.96%, H 7.7296, N 11.92%). This substance gives a single spot
positive to ninhydrin at Rf 0.22 in the above-mentioned silica
gel thin layer chromatography developed with the first-mentioned
development solvent and at Rf 0.24 in the same thin layer chroma-
20 tography developed with the second-mentioned development solvent,
respectively.
L-N-(L-4-Amino-2-hydroxybutyryl)-3',4'-dideoxykanamycin
C is a substance also in the form of a colorless pcwder having no
definite melting
, ,
.
~ - 6 -
~ "~, . ",~ .
~ 081~3
point but decomposing at 142-158Co It shows a
specific optical rotation ~)28 ~76 (c 1, water)D
Its elemental analysis is coincident with the
theoretical values of C22H43N5011 H2
H 7D940~ N 12~25%)o It gives a single spot positive
to ninhydrin at Rf 0O31 in the above-mentioned silica
gelthinlayer chromatogr~phy developed with the
first-mentioned development solvent and at Rf O.30
in the same thin layer chromatography developed
with the second-mentioned development solvent~
The molecular structure of these new kanamycin C
derivatives has been identified by acid hydrolysis
.and by lH and 13C nuclear magnetic resonance
absorption spectraO
The minimum inhibitory concentrations
(mcg/ml.) o~ l-N-(L-4-amino-2-hydroxybutyryl)-
kanamycin C (abbreviated as AHB-KC), l-N-(L-4-
amino-2-hydroxybutyryl)-3'-deoxykanamycin C
(abbreviated as AHB-DKC) and l-N-(L-4-amino-2-
hydroxybutyryl)-3',4'-dideoxykana~ycin C (abbre-
viated as AHB-DDKC) against various microorganisms
were determined according to serial dilution
method on nutrient agar medium at 37C, the
estimation being effected after 18 hours incubationO
For comparison, the minimum inhibitory concentrations
o~ l-N-(L-4-amino-2-hydroxybutyryl)-kanamycin A
(amikacin) was also determined in the same manner
as described aboveD
The antibacterial spectra of these substances
are shown in Table 1 belowO
10~ ;93
.
:~ ~
. 1~ N ~ C-- 1~ ~ ~ ~ U~
O O O O O O O O ~1 0 0 0
~j ~
_
I U~ ~J 11~ U~ U~ U~ U~
~ r-l ~1 ~1 ~1 ~I N N~ N
~q
C)
~1 ~ U~ U~ U~ U~ U~
t~ ~ N U~ N N N N U~ N u~ u~ N r-l
O $ ~0 N ~ L) ~D N ~D ~ N ~t) 1
~ U~ ~ U~ Lr~ U~ U~
$ f q N r1 N U~ N N U~ 1-'\ U~ N I
,Q ~3 ~ ~U~ N ~O N
,~
fi ~ ~ u~
~ ~ o o o
h h
.. o
..
~ ~ ~ o _ = = - = = = = _
~ t>
E~ o r~
O h ~t
t) a) ~:
o ~ C~
.. ~
h
. P1 0
td
':, ~
~O~i~93
O ~D~~D ~~ ~ u~
o o ~~ o ~ ~
o ,,o ~ ~~ ~ o
~, ,, --'
L~
U~ ~ ~.
~oU~ooooo
,, ~ ~ ,1 ~ ,, ~o ~o
OOOOO
, ~ ~ U~ Ln
~U~ooooo
:
o~
o
r.
~, ~ ~ ~ ~ 3 a'
~o ta o
o ~P
o ~, ,.
~, ~ ,
P.
o~
.,1 ~ ~
h = ,1 = o
~ ~,q ,~
.0
P~
_ g _
lV81693
From the above Table, it is seen that the
new compounds of this invention inhibit the growth
of many kinds of bacterial strainsO The new
compounds of this invention further exhibit a
extremely low acute toxicity to animals and men.
It has been estimated that AHB-KC and AHB-DKC
have an LD50 value cf more than 400 mg/kg upon
intravenous injection in miceO Accordingly, the
new compounds of the invention are promising as
chemotherapeutic agents for therapeutic treatment
of infections caused by gram-negative and gram-
positive bacteriaO
l-N-(L-4-Amino-2-hydroxybutyryl)-kanamycin A
(amikacin) and 1-N-(L-4-amino-2-hydroxybutyryl)-
kanamycin B are synthetized by Kawaguchi et al
(U.SO Patent NoO 3,781,268), and these known
compounds are acetylated by a 6'-acetyltransferase
which is obtained from aminoglycoside-resistant
strains, such as Pseudomonas aeruginosa GN315
(see, M~ Yagisawa et al9 J. Antibiot., 28, 486
(1975)) and in this respect they are in contrast
with the new compounds o~ this invention which are
not inactivated by the 6'-acetyltransferaseO
The new compounds of the formula (I)
according to this invention may readily be converted
into the form of a pharmaceutically acceptable, nontoxic
acid-addition salt such as the hydrochloride, sulfate,
phosphate, nitrate 9 acetate, maleate, fumarate,
succinate 9 tartarate, oxalate, citrate, ascorbate,
methanesulfonate, ethanesulfonate and the like by
-- 10 --
1081~93
reacting the free base form of l-N-(L-4-amino-2-
hydroxybutyryl) derivative of kanamycin C, 3'-
deoxykanamycin C or 3'94'-dideoxykanamycin C with
the appropriate acid in ~queous mediumO The
new kanamycin C compounds of this invention and
their pharmaceutically acceptable acid-addition
salt may be administered orally, intraperitoneally,
intravenously, subcutaneously or intramuscularly
using any pharmaceutical form known to the art
for such administration and in a similar manner
to the known kanamycins. For instance, the new
compounds of this invention may be administered
orally using any pharmaceutical form known to the
art for oral administration. Examples of the
pharmaceutical forms for oral administration are
powders, capsules 9 tablets 9 syrup and the like.
A suitable dose of the new compounds of this
invention for effective treatment of bacterial
infections is in a range of 005 to 4 gO per
person a day when it given orallyO It is preferred
that said dose should be orally administered in
three to four aliquots per day. The new compounds
of this invention may also be administered by
intramuscular injection at a dosage of 200 to
2000 mg per person two to four times per dayO
Moreover, the new`compounds of this invention
may be formulated into an ointment for external
application which contains the active compound
at a concentration of 0.5-5% by weight in mixture
with a known ointment base such as polyethylene
-- 11 --
. .
10131f~93
glycol. Furthermore 9 the new compounds of this
invention are useful for sterilization of
surgical instruments~
According to a second aspect of this invention9
therefore, there is provided an antibacterial
composition comprising as the active ingredient
l-N-(L-4-amino-2-hydroxybutyryl)-kanamycin C,
l-N-(L-4-amino-2-hydroxybutyryl)-3'-deoxykanamycin
C or l-N-(L-4-amino-2-hydroxybutyryl)-3',4'-
dideoxykanamycin C or an acid-addition salt thereof
in an antibacterially effective amount to inhibit
the growth of bacteria, in combination with a
carrier or vehicle for the active ingredient
compound.
According to a further aspect of this
invention, there is provided a process for the
preparation of the l-N-(L-4-amino-2-hydroxybutyryl)
derivative of kanamycin C, 3'-deoxykanamycin C
or 3',4'-dideoxykanamycin C represented by the
general formula (I) shown hereinbefore, which
comprises the steps ofo-
acylating the l-amino group of kanamycin
C, 3'-deoxykanamycin C or 3',4'-dideoxykanamycin
C represented by the general formula (II):
1081f~93
6~
4" CH20H O 2' Rl 4~
~z ~ ~ H2N~ ~R2
H2N ~ ~ 6H20H
.. 0~0
H2N ~ / ~ MH2 (II)
wherein Rl and R2 are each hydroxyl, or Rl is hydrogen
and R2 is hydroxyl or Rl and R2 are each hydrogen,or
a nontoxic, pharmaceutically acceptable acid-addition
salt thereof, by reaction with an amino-protected
derivative of L-4-amino-2-hydroxybutyric acid or
a functional equivalent thereof, to produce the
l-N-acylation product of the starting compound (II), .:
and
removing the amino-protecting group from
the resulting 1-N-acylation product to give the
compound of the formula (I). :
The present process may include a further
step of reacting the compound of the formula (I)
so obtained, with a pharmaceutically acceptable
acid to produce the corresponding pharmaceuticaIly
acceptable acid-addition salt of said compound
.~ of the formula (I) 9 if desired.
In one embodiment of the process of this
invention, the process may comprise the following
steps of:-
reacting the starting compound of the
: formula (II) of which amino groupsare not protected,
with an amino-protected derivat.i~e of L-4-amino-2-
. -
1081693
hydroxybutyric acid represented by the formula (III):
(L) / R3
Hooc-cH(oH)-cH2cH2- N~\ (III)
~R4
wherein R3 is a hydrogen atom and R4 is a known
mono-valent amino-protecting group such as alkyloxy-
carbonyl, cycloalkyloxycarbonyl and aralkyloxy-
carbonyl, or R3 and R4 together form a divalent
amino-protecting group such as phthaloyl group,
or a Schiff base group (N=CHR5 in which R5 is a
hydrogen atom, an alkyl group o~ 1-4 carbon atoms
or an aryl group such as phenyl), in a manner
known for the acylation of amino group, to produce
the mixed acylation products comprising a l-N-
(L-4-protected-amino-2-hydroxybutyryl)derivative
of thestartln~ compound of the ~ormula tII).
treating the mixed acylation products in a
known manner to remove the amino-protecting group
therefrom,
and then isolating the desired l-N-(L-4-
amino-2-hydroxybutyryl) derivative of the antibiotic
according to the ~ormula (I) by chromatographic
separation of the acylation products from which
the amino-protecting group has been removed,
to recover the desired compound of the ~ormula (I).
In carrying out the process of this invention,
the starting kanamycin C, 3'-deoxykanamycin C or
3',4'-dideoxykanamycin C o~ the formula (II) of
which amino groups are not prctected, may be used
either in the form o~ the free base or in the form
- 14 -
~081~93
of its acid-addition salt with an appropriate acid
such as hydrochloric acid or sul~uric acidD In
the acylation step of the present process, the
reactionmay be conducted generally as ~escribed
in the specification of U.K. Patent No~ 1,426,908
or U.S. Patent NoO 4,001,208. Preferably, the
starting compound of the formula (II) is dissolved
in water and theresulting aqueous solution is
adjusted to a pH of' 6-8 and more conveniently at
a pH o~ 6.5-7.0 by addition of an ordinary acid
such as hydrochloric acid and sulfuric acid or
an ordinary base such as aqueous sodium hydroxide
and aqueous potassium hydroxideO To this solution
of the above-mentioned partially protonated form of
the startin~ compound is added a solution of an
amino-protected derivative of L-4-amino-2-hydroxy-
butyric acid according to the formula (III) or
a reactive derivative thereof which acts as a
functional equivalent of the aforesaid amino-
protected butyric acid derivative (III). In this
way, the l-amino group of the starting kanamycin
C compound (II) is acylated with the acid (III).
The amino-protecting group available (for
R3 and/or R4) in the amino-protected L-4-amino-2-
hydroxybutyric acld derivative (III) employed
in the acylation step of the present process may
be a known amino-protecting group which is usually
used in the conventional synthesis of peptides.
However, the a~ino-protecting group employed must
be such one of the nature which is removable
.
- 15 -
~081~;93
readily by such a procedure and such reaction
conditions which well not break or damage sub--
stantially the amido linkage present be~ween the
L-4-amino-2-hydroxybutyryl substituent and the
l-amino group of the am,inoglycosidic moiety of the
resulting acylation product when the removal of
the amino-protecting gro~p from the l-N-amino-
alkanoyl substituent of the acylation product is
operatedO Suitable examples of the mono-valent
amino-protecting group which are available (as
the group R4) for the above purpose include an
alkoxycarbonyl group of 2-6 carbon atoms such as
tert-butoxycarbonyl ard tert-amyloxycarbonyl;
,a cycloalkyloxycarbonyl grou~ of 3-7 carbon atoms
such as cyclohexyloxycarbonylg an aralkyloxy-
carbonyl group sv.ch as benzyloxycarbonyl and
p-methoxybenzyloxycarbonyl; and a substituted
alkanoyl gro~p of ~-5 carbon atoms such as tri-
fluoroacet~l and o-nitrophenoxyacetyl~ Preferred
examples of the diva].ent amino-protecting group
available (as the groups R3 and R4 taken together)
for the above-mentioned prupose include phthaloyl
group and a group of ~chiff base type such as
salicylidene~ The-ntroductiQn of the amino-
'~ protecting group into L--4-arnino-~-hydroxybutyric
acid may be achieved by reacting the,latter
with an appropriate reagent for the introduction
of the amino-p.ro-tecting group which is in the
form of an acid halide~ acid azide 9 active ester
or acid anhydrideg in the same manner as described
, - 16 -
1081f~93
e gO in the specifications of UOSo Patent NosO
~,929,762 and 3,939,143 as well as the aforesaid
UoK~ Patent NoO 1,42699080
The acylation of the l-amino gro-up of the
starting kanamycinC compound of the formula (II)
with the amino-protected L-4-amino-2-hydroxy-
butyric acid (III) may be conducted according to
the conventional methods for the synthesis of amides
using the acylating reagent (III) in the form of
active ester such as N-hydroxysuccinimide ester,
mixed acid anhydride or acid azideO As the
starting kanamycin C compound (II) is insoluble
or sparingly soluble in any organic solvent but
soluble in water, it is pre~erred that the acylation
. reaction should be carried out in an aqueous
reaction medium using the acylating agent (III)
in the form of its active esterO For instance, a
solution containing a 1-2 molar proportion of the
N-hydroxysuccinimide ester of L-4-tert-butoxy-
carbonylamino-~-hydroxybutyric acid in a water-
miscible organic solvent such as 1,2-dimethoxyethane
and dimethylformamide may be admixed with an aqueous
solution of 1 molar proportion of the starting
kanamycin C compound (II) at a pH of 6-8 at
ambient temperature under stirring to effect the
desired acylationO The reaction temperature may
be elevated, if desiredO The reaction time may
be for several hours and preferably for 5-6 hoursO
The acylation product so obtained is, in
fact, in the form of the mixed acylation products
;:
~ - 17 -
1081~i93
comprising the desired 1-N-(L-4-protected amino-
2-hydroxybutyryl) derivative as well as the
undesired, otherwise mono-N-acylated, di-N-
acylated and poly-N-acylated products in which
one or more of the l-amino, 3-amino, 2'-amino and
possibly 3"-amino groups of the kanamycin C moiety
has or have been bonded with the L-4!protected
amino-2-hydroxybutyryl substituent. The acylation
product (the mixed acylation products) as such
may be directly treated in the second step of the
present process for the removal of the amino-
protecting group, which is carried out in a manner
known per se in the peptide synthesisO All of
.the amino-protecting groups of .the above-mentioned
nature may be removed readily by weak acid hydro-
lysis using an aqueous solution of trifluoroacetic
acid or acetic acid or dilute hydrochloric acid.
When the amino-protecting group is an aralkyloxy-
carbonyl group9 the latter may be removed also
by an ordinary hydrogenolysisO
The deprotected acylation product obtained
from the second step of the present process is
actually also in the from ofthe mixed products
containing the desired l-N-(L-4-amino-2-hydroxy-
butyryl)-kanamycin C 9 -3'-deoxykanamycin C or
-3~,4'-dideoxykanamycin C of the formula (I)
andthe undesired, otherwise acylated derivatives
of the starting kanamycin C compound, together
with the unreacted starting kanamycin C compound
(II). In order to isolate the desired l-N-
- 18 -
10~16i93
acylation product (I) from the above mixed products,
the latter may be sub~ected to a chromatographic
separation method, such as ion-exchange chromato-
graphy uæing a cation-exchanger containing carbo-
xylic functions, for example, Amberlite CG-50
(a product of Rohm & Haas Co., U.S.A.), CM-sephadex
C-25 (a product of Pharmacia Co., Sweden) and
carboxymethylcellulose; ion-exclusion chromato-
graphy using strong anion-exchange resin, for
example, Dowex l-X2 (a product of Dowex Co.,
U.S.A.); and column chromatography using silica gel.
In th~s way, the isolation and recovery of the
desired product (I) may be achieved with high
efficiencyO Particularly, it is recommendable
that the deprotected mixed acylation product is
chromatographed on Amberlite CG-50 (a weak cation-
exchange resin containing carboxylic functions)
using diluted aqueous ammonia as the development
solvent, because this chromatographic procedure
enables the desired product (I) as well as the
unreacted starting kanamycin C compound (II) to
be recovered efficiently and in pure state.
According to these chromatographic methods, the
desired kanamycin C derivative of the formula (I)
is usually recovered ir, the form of its free
base, hydrate or carbonateO
*
"Sephadex LH-20" is a lyophilic insoluble
molecular-sieve chromatographic medium made by cross-
linking dextran and marketed by Pharmacia, Uppsala,
Sweden. Sephedex LH-20 can be replaced by other
æimilar gel-filtration agents, e.g. Sephadex G25 to
*Trade Marks
1 9
1 ~ 8 1 ~9 ~
G200, Sepharose 4B and 6B (Pharmacia Fine Chemicals AB,
~ppsala, Sweden) and Bio-Gel Al.5 m (Bio Rad Co.). Pre-
ferred gel-filtration agents include the carboxy-
methyl substituted cross-linked dextran gels described in
columns 3 and 4 of U.S. Patent No. 3,819,836.
Dowex l-X2(OH ) is the basic or hydroxlde form
of cholestyramine resin ~hich in its chloride form is a
synthetic, strongly basic anion exchange resin containing
quaternary ammonium functional groups which are attached to
a styrene-divinylbenzene copolymer. Main constituent:
Polystyr~ne trimethylbenzylammonium as Cl anion, also
contains divinylbenzene (about 2%) and water
;. . _ _
. ' . :
... -CH C~2 CH CH2 ^-
CN CN2N+(CN~)~Cl ~ :
,-
typi~ied ~tructure o~ main polymeric group~
(about 43%). Cros~ l~nkage ~ 10.. Particle
size: 50-lOO mesh. Percent volume increa3e, new
to exhausted (Cl to OH ) - 20%. Stable at
temperatures up to 150. Capacity: 3.5 meq/g
: dry, 1.33 meq/ml wet.
*Trade Marks
t~`7 -20-
I
,. .
~(:)8~3
"Amberlite" is a registered trademark o~ the
Rohm and Haas Company, Philadelphia, Pennsylvania.
Amberlite IRC-50 and CG-50 are tradenames for weakly
acidic cation exchange resins of a carboxylic-
polymethacrylic type.
The invention is now illustrated with
', reference to the following ExamplesO
E~ample 1
Synthesis of l-N-(L-4-amino-2-hydroxy-
butyryl)-kanamycin C
A solution of 500 mg (1002 millimole) of
kanamycin C free base (hemihydrate) in 10 ml.
of water was adjusted to pH 6.35 by addition of
,3.2 mlO of lN hydrochloric acid. To this
solution was dropwise added over 5 minutes a
solution of 500 mg (1058 millimole) o~ N-hydroxy-
succinimide ester of L-4-tert-butoxycarbonylamino-
2-hydroxybutyrlc acid ln 5 ml of dimethylformamide
at ambient temperature under stirringO The admixture
so obtained was stirred further for 6 hours to
effect the acylation. The reaction solution
comprising l-N-(L-4-tert-butoxycarbonylamino-2-
hydroxybutyryl)-kana.~ycin C so formed was con-
centrated to dryness under reduced pressure, and
the solid residue ras admixed with 16 ml of an
aqueous solution of 90% trifluoroacetic acidO
The mixture was ~gitated for 1 hour at ambient
temperatureto effect the removal of the amino-
protecting tert-butoxycarbonyl groupO The
reaction solution was concentrated to dryness
'
;, -21-
. .
1C181~;93
under reduced pressure and the residue was washed twice
with 20 ~lof ethylether to give 1u55 g of a colorless
powder comprising a crude product of l-N-(L-4-
amino-2-hy~roxybutyryl)-kanamycin C0
T~.is cru~e product was taken up in 15 ml
Of water and the resulting solution was adjusted
to pH 702 by addition of lN aqueous ammonia and
then rassed through a column (inner diameter 20 mm)
of 190 ml of Amberlite CG-50 (NH4-form) for the
adsorption of the desired productO After the column
was washed with water (660 ml), it was eluted with
1940 ml of O.lN aqueous ammonia and then with
1540 ml of 0.4N aqueous ammonia. The eluate
was collected in 20 ml-fractionsO The fractions
Nos. 75-106 of the eluate obtained using OolN
aqueous ammonia were combined together and con-
centrated to dryness under reduced pressure,
recovering 199 mg of unreacted kanamycin C
(recovery yield,40%). The fractions Nos. 152-175
of the eluate obtained using 004N aqueous ammonia
were combined together and concentrated to dryrless
under reduced pressure to give 214 mg of a colorless
powder containing the desired product~ This
powder was suspended in 2 ml of a mixed solvent
of chloroform-methanol-17% aqueous ammonia
(1:4:2 by volume), and the resulting suspension
was passed through a column (inner diameter 14 mm)
of 50 g of silica gelO The silica gel column
was then developed with the above-mentioned mixed
solvent, and the eluate was collected in 10 ml-frac-
tions. The fractions Nos. 86-114 were combined together
-22-
,
108~6i93
and concentrated to dryness under reduced pressure,and the residue was taken up into water~ The
resulting aqueous solution was chromatographed
in a column of 5 ml of Amberlite CG-50 (NH4-form)
using 0.5N aqueous ammonia as eluent. The eluate
was concentrated to dryness under reduced pressure,
affording 49 mg of pure 1-N-(L-4-amino-2-hydroxy-
butyryl)-kanamycin C in the form of a colorless
powderO Yield 800%o This powder had no definite
melting point and deoomposed at 167-180C.
26
~a)D ~91% (c 1, water).
Example 2
Synthesis of l-N-(L-4-amino-2-hydroxy-
butyryl)-3'-deoxykanamycin C
A solution of 440 mg (0.~2 millimole) o~
3'-deoxykanamycin C free base (hemihydrate) in
808 ml of water was adjusted to pH 6060 by addition
of 2.75 ml of lN hydrochloric acid. To this
resulting solution was dropwise added over
,10 minutes a solution of 449 mg (1.42 millimole)
of N-hydroxysuccinimide ester of L-4-tert-butoxy-
carbonylamino-2-hydroxybutyric acid in 407 ml of
dimethylformamide at ambient temperature under
stirringO The admixture so obtained was stirred
further for 6 hours at ambient temperature to
effect the acylation~ The reaction solution
containing 1-N-~L-4-tert-butoxycarbonylamino-2-
hydroxybutyryl)-3'-deoxykanamycin C so formed was
concentrated to dryness under reduced pressure.
The solid residue was admixed wi~h 11 ml of an
aqueous solution of 90% trifluoroacetic acid, and
-23-
1081~;93
the mixture was agitated at ambient temperature
for 1 hour to effect the removal of the amino-
protecting tert-butoxycarbonyl group. The
reaction solution was concentrated to dryness
under reduced pressure and the residue was washed
twice with 20 ml o~ ethylether to give 2.12 g of
a crude product of l-N-(L-4-amino-2-hydroxybutyryl)-
3'-deoxykanamycin C as colorless powderO
This crude product was taken up into 15 ml
of water, and the resultant aqueous solution was
adjusted to pH 7.8 by addition of lN aqueous ammonia
and then passed through a column (inner diameter
20 mm) of 220 ml of Amberlite CG-50 (NH4-form) to
e~fect the adsorption of the desired product.
After the column was washed with 1040 ml of water,
it was eluted with 1340 ml of 0.2N aqueous ammonia
and then with 1320 ml of 0c5N a~ueous ammoniaO
The eluate was collected in 20 ml-fractionsO
The fractions Nos. 71-~0 of the eluate obtained
using 0.2N a~ueous ammonia were combined together
and concentrated to dryness under reduced pressure,
recovering 149 mg of unreacted 3' deoxykanamy~in C
(recovery yield 34%)0 The fractions Nos. 134-145
of the eluate obtained with 005N aqueous ammonia
were combined together and concentrated to dryness
under reduced pressure to give 155 mg of a colorless
powder containing the desired productO This powder
was suspended in 2 ml of a mixed solvent of chloro-
form-ethanol-17% aqueous ammonia (1:4:2 by volume),
and the suspension so obtained was passed through a
column (inner diameter 10 mm) of 25 g of silica gel,
-24-
'
.
~ ~,
1081~;93
which was then developed with the above-mentioned
mixed solvent. The effluent was collected in 3O2 ml-
fractionsO The combined fractions Nos. 99-140
were concentrated to dryness under reduced pressure,
and the solid residue was taken up into water and
chromatographed in a column of 5 ml of Amberlite
CG-50 (NH4-form) using 0O5N aqueous ammonia as
the eluent The eluate was concentrated to dryness
under reduced pressure. Pure l-N-(L-4-amino-2-
hydroxybutyryl)-3'-deoxykanamycin C was obtained as a
colorless powder. Yield 46 mg (80 5%). This
powder had no definite melting point but decomposed
at 151-160C. (a)D7 +83 (c 1, water)0
Example 3
Synthesis of l-N-(L-4-amino-2-hydroxybutyryl)-
3',4'-dideoxykanamycin C
A solution of 340 mg (0.74 millimole) of
3',4'-dideoxykanamycin C free base (hemihydrate)
in 7.5 ml of water was adjusted to pH 6.70 by
addition of 2.45 ml of lN hydrochloric acid.
To this solution was dropwise added over 5 minutes
a solution of 373 mg (1.18 millimole) of N-hydroxy-
succinimide ester of L-4-tert-butoxycarbonylamino-
2-hydroxybutyric acid in 3.75 ml of dimethylformamide
at ambient temperature under stirring. The admixture
so obtained wa~ stirred further for 6 hours to
effect the acylation. The reaction solution
containing l-N-(L-4-tert-butoxycarbonylamino-2-
hydroxybutyryl)-3',4'-dideoxykanamycin C so formed
was concentrated to dryness under reduced pressure,
and the solid residue was admixed with 8.5 ml
of an aqueous solution of 90% trifluoroacetic acidO
-25-
~081~;93
The mixture was stirred for 1 hour at ambient
temperature to effect the removal of the amino-
protecting tert-butoxycarbonyl groupO The reaction
mixture was then concentrated to dryness under
reduced pressure and the residue was wa~hed twice
with 20 ml of ethylether to give 1.46 g of a crude
product of l-N-(L-4-amino-2-hydroxybutyryl)-
3',4'-dideoxykanamycin C as colorless powder.
This crude product was taken up into 14 ml
of water and the resulting aqueous solution adjusted
to pH 7.8 by addition of lN aqueous ammoniaO
The solution was then passed through a column
(inner diameter 20 mm) of 220 ml of Amberlite
CG-50 (NH4-form) for adsorption of the active
compounds. After the column was washed with water
(1040 ml), ~t was eluted with 1560 ml of 0.2N
aqueous ammonia and then with 1460 ml of 0.5N
aqueous ammonia so that the eluate was collected
in 20 ml-fractions. The fractions Nos. 74-93
of the eluate obtained with 0.2N aqueous ammonia
were combined together and concentrated to dryness
under reduced pressure to recover 153 mg of unreacted
3',4'-dideoxykanamycin C (recovery yield 45%?o
The fractions Nos. 152-168 of the eluate obtained
with 0.5N aqueous ammonia were combined together
and concentrated to dryness under reduced pressure
to give 116 mg of a colorless powder containing
the desired product.
This powder was suspended in 2 ml of a mixed
solvent of chloroform-ethanol-17% aqueous ammonia
(1:4:2 by volume), and the resulting suspension
was placed in a column (inner diameter 10 mm) of
-26-
. .
,
1081f~93
25 g of sllica gel, which was then eluted with
the above-mentioned mixed solvent. The effluent
was collected in 3.2 ml-fractions, and the fractions
Nos. 71-96 were combined together and concentrated
to dryness under reduced pressureO The residue was
dissolved in water and the solution was chromato-
graphed in a column of 5 ml of Amberlite CG-50
(NH4-form) ln the same manner as above using 0.5N
aqueous ammonia as the eluentO The eluate was
concentrated to dryness under reduced pressure to
afford 35 mg of 1-N-(L-4-amino-2-hydroxybutyryl)-
3',4'-dideoxykanamycin C as colorless powder.
Yield 8.3%. This product showed no definite melting
point but decomposed at 142-158C. ~)28 +76
(c 1, water)0
Example 4
The following tests were made to estimate
how the pH of the reaction medium where the acylation
reaction proceeded affected the yield of the
, j .
desired acylation product 1-N-(L-4-amino-2-
hydroxybutyryl)-kanamycin C
Solutions each containing 50 mg (0.10
millimole) of kanamycin C free base (hemihydrate)
in 0.5-1.0 ml of water were prepared and adjusted
to different pH values by addition of varying
volumes of lN hydrochloric acid. To each solution
was added dropwise a solution of 49 mg (0.15 milli-
mole) of N-hydroxysuccinimide ester of L-4-
tert-butoxycarbonylamino-2-hydroxybutyric acid at
ambient te~perature under stirring. The admixture
so obtained was stirred for 6 hours to effect the
acyl~tion. The reaction solution was concentr&ted
-27-
~ 9 3
to dryness under reduced pressure and the solid
residue was taken up into 1.2-1.4 ml of aqueous
90% trifluoroacetic acidO The resultant solution
was stirred for 1 hour at ambient temperature,
and the reaction solution was concentrated to
dryness under reduced pressure. The residue was
washed with ethylether to give a crude productO
This crude product was chromatographed in the
same manner as in Example 1 using a column
(inner diameter 12 mm) of 20 ml of Amberlite
CG-50 (NH4-form), so that the unreacted kanamycin
C was recovered in the pure state and a crude
powder containing l-N-(L-4-amino-2-hydroxybutyryl)-
kanamycin C was obtained~ The contents of l-N-
(L-4-amino-2-hydroxybutyryl)-kanamycin C in the
crude powders so obtained were determined by
measuring the antibacterial potency according to
a standard cup-plate assay method using Bacillus
subtilis PCI Zl9 as the test organismO In Table
2 below, there are shown the yield of unreacted
kanamycin C recovered and the yield of the desired
product formed, together with the pH value of the
aqueous solution of the starting kanamycin C, the
volume of water employed for the dissolution of the
starting kanamycin C and the volume of the lN
hydrochloric acid added for the adjustment of the
pH value~
-28-
.
.. , ~.
-:
1~38169~
Table 2
Volume Volume Yield of Yield of
of of lN kanamycin C desired
water HCl recoverd product
~H (ml) (ml) ) formed
6.30 005 0.32 43 18
6.75 1.0 0.30 38 23
7.40 0.5 0.21 27 19
8.35 1.0 0.10 22 12
3'-Deoxykanamycin C and 3',4'~dideoxykanamycin
C used in Examples 2 and 3 are new compounds
which may be prepared by the following procedure
(see u.S. Patent 4,120,955 issued October 17, 1978):
3'-deoxykanamycin B or 3',4'-dideoxy-
kanamycin B is reacted with tert-butyl chloroformate
or tert-butyl S-4,6-dimethylpyrimid-2-ylthiocarbonate
which is known as a reagent for introducing the
amino-protecting tert-butoxycarbonyl group.
In this way, there is prepared 6'-N-tert-butoxy-
carbonyl-3'-deoxykanamycin B or -3',4'-dideoxy-
kanamycin B. The latter is then reacted with acetic --
anhydride to protect the 1,3,2' and 3"-amino group
of the deoxykanamycin B compound with the another
kind of the amino-protecting group, the acetyl
group, whereby 6'-N-tert-butoxycarbonyl-tetra-
N-acetyl-3'-deoxykanamycin B or -3',4'-dideoxy-
kanamycin B is prepared. When this product is
treated with aqueous 90% trifluoroacetic acid,
the preferential removal of the tert-butoxycarbonyl
group takes place, giving 1,3,2',3"-tetra-N-acetyl-
- 29 -
~ .
1081693
3'-deoxykanamycin B or -3',4'-dideoxykanamycin B.
1,3,2',3"-Tetra-N-acetyl-3'-deoxykanamycin B or
-3',4'-dideoxykanamycin B is dissolved in an
aqueous acetic acid, and the resulting solution
is treated with an aqueous sodium nitrite, when
the 6'-amino group is converted into the 6'-hydroxyl
group, giving 19392',3"-tetra-N-acetyl-3'-deoxy-
kanamycin C or -3'94'-dideoxykanamycin CO When
the latter is treated forthe removal o~ the amino-
protecting acetyl groups 9 ~or example, by alkaline
hydrolysis with 2N aqueous sodium hydroxide, there
is produced 3'-deoxykanamycin C or 3',4'-dideoxy-
kanamycin C.
The preparation of 3'-deoxykanamycin C or
3',4'-dideoxykanamycin C may be achieved in a
similar way to the above-mentioned procedure by
protecting the 6'-amino group of 3'-deoxykanamy~cin
B or 3',4'-dideoxy~anamycin B with benzyloxycarbonyl
group. For this purpose, benzyloxycarbonyl chloride
is employed as the reagent for introducing the
amino-protecting benzyloxycarbonyl groupO In
this case, the removal of the benzyloxycarbonyl
group from 6'-N-benzyloxycarbonyl-tetra-N-acetyl-
3'-deoxykanamycin B or -3' 7 4'-dideoxykanamycin B
which is formed as the intermediate product may
be conducted by corventional hydrogenolysis over
palladium catalyst or platinum catalyst.
The preparation o~ 3'-deoxykanamycin C
and 3',4'-dideoxykanamycin C is now illustrated
below as Examples 5-6.
-~0-
~0~31693
Example 5
Synthesis of 3'-deoxykanamycin ~
(a) A solution o~ 200 g (403 millimole) of
3'-deoxykanamycin B in 40 ml o~ water was admixed
with a solution of 1.03 g (407 millimole) of t-butyl
S-4,6-dimethylpyrimid-2-ylthiocarbonate in 40 ml of
dioxane, and the admixtureso obtained was stirred
for 24 hours at ambie.nt temperature. The reaction
mixture was then concentrated to dryness under
reduced pressure, and the solid residue was taken
up into 32 ml of waterO The resultant aqueous
solution was passed through a column of 160 ml of
a cation-exchange resin, Amberlite CG-50 (ammonium
form) for adsorptionof the formed 6'-N-t-butoxy-
carbonyl-3'-deoxykanamycin Bo The resin column
was washed with 800 ml of water and then eluted
with 800 ml of OolN aqueous ammonia. The elu~te
was collected in 15 ml-fractions,and the fraction
Nos. 26 to 42 were combined together and concentrated
to dryness under reduced pressure to give 1.06 g
o~ a white colored powder comprising 6'-N-t-butoxy
carbonyl-3'-deoxykanamycin BD Yield 44%. The
resin column was further eluted with 0.5N aqueous
ammonia to recover 452 mg of unreacted 3'-deoxy-
kanamycin B.
(b) A solution of 211 mg (0.37 millimole~ of 6l-
N-t-butoxycarbonyl-3'-deoxykanamycin B in 5 ml o~
methanol was admixed with 2~5 ml of acetic anhydride,
and the admixture was agitated ~or 5 hours at ambient
temperature for the acetylation of the remaining
-31-
.
~081693
amino groupsu The reaction solution was admixed
with a volume of water and then concentrated to
dryness under reduced pressure to give a powder
comprising 6'~N-t-butoxycarbonyl-tetra-N-acetyl-
3'-deoxykanamycin Bo Yield 296 mg.
(c) The product (235 mg) obtained in the preceding
step (b) of this Example was dissolved in 2 ml
of an aqueous solution of 90% trifluoroacetic acid
and the resulting mixture was allowed to stand for
45 m$nutes at ambient temperature to effect the
removal of the 6'-butoxycarbonyl groupO The
reaction mixture was concentrated to dryness under
reduced pressure, and the solid residue obtained
was washed with about 2 ml of ethyl ether to give
227 mg of a white colored powder comprising the
tetra-N-acetyl derivative, that is 9 1,3,2',3"-
tetra-N-acetyl3'-deoxykanamycin Bo
(d) The powdery product (193 mg) obtained in
the preceding step (c) of this F,xample was dissolved
in 3.2 ml of an aqueous solution of 33% acetic acid,
and to the resulting solution was added a solution
of 265 mg of sodium nitrite in 302 ml of water
and then 1.6 ml of acetic acid under ice-cooling
and stirringO The mixture so obtained was stirred
for 1 hour under ice-cooling and then for 16 hours
at ambient temperature to effect the reaction where
the 6'-amino group was converted into the 6'-
hydroxyl group. The reaction solution was concen-
trated to dryness under reduced pressure to obtain
240 mg of a solid residueO This solid comprising
-~2-
iO~ i93
1,3,2',3"-tetra-N-acetyl-3'-deoxykanamycin C was
taken up into 4 ml of 2Naqueous sodium hydro~ide,
and the resulting mixture was heated for 7 hours
under reflux to effect the removal of the acetyl
groups.
The reaction solution so obtained was
admixed with 200 ml of water and then passed
through a column (inner diameter i~6 cm) of 50 ml
of a catlon-exchange resin, Amberlite CG-50 (70%
ammonium form) for the absorption of the formed
kanamycin C derivative. The resin column was
washed with 250 ml of water and then eluted with
0 5N aqueous ammoniaO The eluate was collected in
10 ml-fractions, and the fraction Nos. 58 and 59
were combined together and concentrated to dryness
under reduced pressure to give 89 mg of a crude
powder of 3'-deoxykanamycin C. This crude powder
was taken up into 2 ml of water, and the aqueous
solution obtained was again chromatographed using
a column (inner diameter 0075 cm) of 10 ml of
Amberlite CG-50 (ammomium form) in such a manner
that after washing with 30 ml of water, the .
resin column was eluted with 45 ml of OolN aqueous
ammonia, and then with 45 ml of 0~2N aqueous ammoniaO
The eluate was collected in 1 ml-fraction9 and
the fraction NosO 78 to 91 as combined together
were concentrated to dryness under reduced pressureO
A colorless purified powder of 3'-deoxykanamycin C
(54 mg; 0 11 mlllimole) was obtained Yield 45~.
1(~8~693
Example 6
Synthesis of 3'94'-dideoxykanamycin C
(a) To a solution of 13053 g (30 millimole) of
3',4'-dideoxykanamycin B in 135 ml of water was
dropwise added over 1 hour 5061 g (33 millimole)
of benzyloxycarbonyl chloride under ice-cooling
and stirringO After th~ dropwise addition was
completed, the admixture so obtained was stirred
for 1 hour at ambient temperature and the precipitate
which ~ormed was removed by ~iltration. The filtrate
was washed with 135 ml of ethyl ether, and the
aqueous layer was neutralized by addition of
aqueous ammonia ard then concentrated under reduced
pressureO The concentated solution so obtained
was passed through a column of 480 ml of a cation-
exchange resin, Amberlite CG-50 (ammonium form)
for adsorption of the formed 6'-N-benzyloxycarbonyl-
3',4'-dideoxykanamycin Bo The resin column was
washed with 1920 ml of water and then eluted with
O.lN aqueous ammoniaO The first running (960 ml)
of the eluate was discarded and the subsequent
running (780 ml) was collected and concentrated to
dryness under reduced pressure to give 5043 g of
a white colored powder comprising 6'-N-benzyloxy-
carbonyl-3',4'-dideoxykanamycin Bo Yield 31%o
The column was further eluted with 0.5N aqueous
ammonia to recover 2.7 g of unreacted 3',4'-
dideoxykanamycin Bo
(b) 6'-N-Benzyloxycarbonyl-3',4'-dideoxykanamycin
B (1059 g; 2072 milimole) was admixed with 160 ml
of acetic anhydride and 16 g of sodium acetate,
-34-
~01~1~93
and the admixture so obtained was heated for 2 hGurs under reflux
(at 110 C) to effect the acetylation. The reaction mixture was
concentrated to dryness under reduced pressure, and the solid resi-
due was extracted with about 100 ml of acetone. The extract in
acetone was concentrated to dryness under reduced pressure, leaving
a solid (2.5 g). This solid was taken up into 10 ml of chloroform,
and the resulting solution was passed through a column (inner dia-
meter 2.6 cm) of 150 g of silica gel for adsorption of the formed
acetylation product. The silica gel column was washed with 350 ml
of~chloroform and then eluted successively with 900 ml of chloro-
form-methanol (30:1 bY volume), with 900 ml of chloroform-methanol
tl5:1 by volume) and with chloroform-methanol(10:1 by volume).
The eluate was collected in about 14 ml-fractions. The fraction
Nos. 91 to 149 as combined together were concentrated to dryness
under reduced pressure to give 1.80 g of a white colored powder
comprising 6'-N-benzyloxycarbonyl-tetra-N-acetyl-tetra-O-acetyl-3',
4'-dideoxykanamycin B.
(c) The white colored powder (1.18 g) obtained in the above
step (b) of this Example was dissolved in a mixture of 20 ml of
methanol and 5 ml of water, and the resulting solution was subjected
to catalytic reduction for 45 minutes under a stream of hydrogen
over 1.61 g of 5~ palladium-on barium carbonate added to said solu-
tion, so that the benzyloxycarbonyl group was removed. After
removal of the catalyst
, .
:
~ - 35 -
lV~ 9~
by filtration, the reaction mixture was con-
centrated to dryness under reduced pressure, .
affording 942 mg of a white colored powder of the
6'-amino derivative, that is, 1,3,2',3"-tetra-
N-cetyl-5,2",4",6"-tetra-0-acetyl-3',4'-dideoxy-
kanamycin Bo
(d) The white colored powder (942 mg) obtained
in the above step (c) of this Example was dissolved
in 16 ml of a solution of 33% acetic acid in water,
and to the resulting solution were added 16 ml
of a solution of 1.24 g of sodium nitrite and
then 8 ml of acetic acid under ice-cooling and
stirring~ The admixture so obtained was stirred
for 1 hour under ice-cooling and then for 3 hours
at ambient temperature to effect the conversion
of the 6'-amino group into 6'-hydroxyl groupO
The reaction mixture was concentrated to dryness
. under reduced pressure, and the solid residue
was dissolved in 3 ml of chloroform~ The solution
in chloroform was passed through a column (inner
diameter 2 cm) of 100 g of silica gel, which waq
then washed with 210 ml of chloroform and thereafter
eluted successively with 660 ml of chloroform-
methanol (50:1 by volume)~ with 1750 ml o~ chloroform-
methanol (30:1 by volume), with 900 ml of chloroform-
methanol (10:1 by volume) and with 700 ml of
chloroform-methanol (5:1 by volume). The eluate
was collected in about 14 ml-fractionsO The
fraction NosO 220 to 270 as combined together
were concentrated to dryness under reduced pressure,
-36-
1()8i693
giving 587 mg of a white colored powder of tetra-
N-acetyl-tetra-O-acetyl-3',4'-dideoxykanamycin CO
This ~rhite colored powder (234 mg) was
taken up into 4 ml of 2N aqueous sodium hydroxide,
and the resulting solution was heated for 7 hours
under reflux to effect the removal of the acetyl
groupsO The reaction solution was dissolved in
200 ml of water and then passed through a column
(inner diameter 1.6 cm) of 50 ml of a cation-
exchange resin, Amberlite CG-50 (70% ammonium
form) for adsorption of the desired productO
After washing with 250 ml of water, the resin
- column was eluted with 005N aqueous ammonia to
yield 122 mg of a crude powder of 3',4'-dideoxy-
kanamycin CO A solution of this crude powder in
2 ml of water was passed through a column (inner
diameter 0.8 cm) of 14 ml of Amberlite CG-50
(NH4-form) for adsorption of the desired productO
After washing with 45 ml of water, the resin
column was eluted with 40 ml of 0005N aqueous
ammonia, then with 70 ml of OolN aqueous ammonia
and finally with 70 ml of 002N aqueous ammonia~
The eluate was collected in 1 ml-fractions, and
the fractions NosO 119 to 146 were combined together
and concentrated to dryness under reduced pressure
to give gO mg of à colorless purified powder of
3',4'-dideoxykanamycin CO Overall yield 23%o
-~7-
