Note: Descriptions are shown in the official language in which they were submitted.
1 ~S~16
BACKGROUND OF THE INVENTION
Field of the invention
This invention relates to new semi-synthetic
aminoglycosidic antibiotics, including a 1-N-[~-hydroxy-
~-aminoalkanoyl]-5,3',4'-trideoxykanamycin B; l-N-[~-
hydroxy-~-aminoalkanoyl]-5,3',4',6"-tetradeoxykanamycin B
and 5,3',4',6"-tetradeoxykanamycin B as well as a l-N-[~-
hydroxy-~-aminoalkanoyl]-5,3',4'-trideoxy-6'-N-methyl-
kanamycin B and 5,3',4'-trideoxy-6'-N-methylkanamycin B
which are each the new compound useful as antibacterial
agent. This invention also relates to processes for the
production of these new compounds. This invention further
relates to an antibacterial composition comprising one of
these new compounds as the active ingredient.
; 15 Description of the prlor art
Dibekacin, namely 3',4'-dideoxy~anamycin B was
semi-synthetically produced from kanamycin B by the
present inventors (see Japanese patent publication
No. 7595/75; Japanese patent No. 794,612j U.S. patent
No. 3,753,973). Dibekacin has been used extensively in
therapeutic treatment of various bacterial in~ecti.ons as
a chemotherapeutic agent which is active against the
kanamycin-sensitive bacteria and also against various
kanamycin-resistant bacteria. We, the present inventors,
produced semi-synthetlcally habekacin, namely 1-N-(L-4-
amino~2-hydroxybutyryl)-dibekacin which is a
I i75~16
chemotherapeutic agent effective against dibekacin-resistant
bacteria ~see Japanese patent publication No. 33,629/77;
U.S. patent No. 4,107,424). We also produced semi-synthe-
~ tically a l-N-ra-hydroxy-~-amino-alkanoyl~-6'-N-methyldi-
bekacin which has been found to be highly active against
various strains of bacteria (U.K patent No. 1,475,481;
U.S. patent No. 4,147,861~. In our further researches, we
produced synthetically the 6"-deoxy or 4",6"-dideoxy deriva-
tives of dibekacin and habekacin, i.e., l-N-~L-4-amino-2-
hydroxy~utyryl]-dibekacin, respectively. Furthermore, we
have found that these 6" deoxy derivatives and 4",6"-dideoxy
derivatives o~ dibekacin or habekacin exhibit not only a
low o~o-toxicIty but also show an antibacterial activity
as hi~h as that of dibekacin or habekacin tpublished U.K.
patent application 2,058,774 A).
We also produced 5,3',4'-trideoxykanamycin B as
a further deox~ derivative of dibekacin ~Japanese Journa
of Antibiotics, 32, S-178, (1979)). However, it has been
found that 5,3',4'-trideoxykanamycin B is less active than
dibekacin.
It may be added that we further have produced
some deoxy derivatives of l-N-(L-4-amino-2-hydroxybutyryl)-
kanamycin A (i.e. amikacin~, includin~ 3'-deoxyamikacin
mah/ ~ - 2 -
,
~ 175~6
(U~S. patent No. 4,104,372), 3',4'-dideoxyamikacin (publisned
U.R. patent application 2,043,034 A), 6"-deoxyamikacin,
4",6"-di.deoxyamikacin~ 3',4',4",6"-tetradeoxyamikacin and
3',4' r 6 ~'~ trideoxyamikacin, as well as 3l,6"-dideoxyamikacin,
5,3'-dideoxyamikacin and 5,3',6"-trideoxyamikacin, which all
have a low acute toxicity and a lo~ oto-toxicity while
exhibiting a useful antibacterial activity as high as that
of amikacin.
SUMMARY OF THE INVENTION
In further development of our researches on the
. deoxy derivatives of dibekacin or habekacin, we have now
: succeeded to produce a new compound, 5,3',4',6"~tetradeoxy-
kanamycin B which has been found to be significantly act;ve
a~ainst some kinds of hacteria, though the 5,3',4'-triaeoxy-
kanamycin B exhibits no useful antibacterial activity. We
have also succeeded to.produce a new-compound, 5,3',4'-
. trïdeoxy-6'-N-methylkanamycin B which also exhibits an anti-
bacterial actiVity hi.gher than that of the 5,3',4'-trideoxy-
kanamycin B. especially~against some resistant strains. In
an attempt to produce such new
- 3 -
mab/~?~ h
I ~.7~`816
., .
derivatives of 5,3',4'-trideoxykanamycin B, 5,3',4',6"-
tetradeoxykanamycin B or 5,3',4'-trideoxy-6'-N-methyl-
kanamycin B which will have improved antibacterial
activity, we have now synthetized new l-N-[N-hydroxy-~-
aminoalkanoyl] derivatives of these deoxykanamycins B by
acylating the l-amino group of the deoxykanamycin B with
an N-hydroxy-~-aminoalkanoic acid. Then, we have discovered
that the resultant new l-N-[N-hydroxy-~-aminoalkanoyl]
derivatives of 5,3',4'-trideoxykanamycin B, 5,3',4'-
trideoxy-6'-N-methylkanamycin B, and 5,3',4',6"-tetra-
deoxykanamycin B are very much active against kanamycin-
sensitive and kanamycin-resistant bacteria and also exhibit
a high antibacterial activity against a wide range of
bacteria.
According to the first, most generic aspect of this
invention, therefore, there is provided as the new compound
a l-N-[~-hydroxy-~-aminoalkanoyl]-5,3',4'-trideoxy- or
; l-N-[~-hydroxy-~-aminoalkanoyl]-5,3' 5 4'-trideoxy-6'-N-
~ methylkanamycin B or 5,3',4'-trideoxy-6'-N-methylkanamycin
: 20 B or a l-N-[N-hydroxy-~-aminoalkanoyl]-5,3'~4',6"-tetra~
deoxykanamycin B or 5,3',4',6"-tetradeoxykanamycin B
represent0d by the ~ormula (I)
. . .
. ' .
8 1 6
-- 5 --
R \6"
HO ~ ~ O \ H2N ~, ~ '
H N ~ ~~~~~~ ~" ~ ~' ~ (I)
NHR2
RlHN _ ~ H2
wherein R is a hydroxyl group or a hydrogen atom, Rl is
a hydrogen atom or an a-hydroxy-~-aminoalkanoyl group of
formula
: ~ f
; OH
where n is an integer of 1, 2 or 3, and R2 is a hydrogen
atom or a methyl group, provided that R2 is not the methyl
:: group when R is the hydrogen atom, or a pharmaceutically
:
acceptable acid-add~tion salt of said compound.
According to the first preferred embodiment of the
: first aspect invention, there is provided a new compound
which belongs to the new compound o~ the above formula (I)
:
and is a 1-N-[a-hydroxy-~-aminoalkanoyl3-5,3',4'-trideoxy-
: kanamycln B or 1-N~ hydroxy-~-aminoalkanoyl]-5,3',4',6"-
tetradeoxykanamycin B represented by the formula (II)
.
: "~
~ , , . . , :
: ,
:
.
1 17S816
6~
4 ~'H2
(II)
~ NH 2
HN 1 3
CO
CHOH
~CH2)nNH2
wherein R is a hydroxyl group or a hydrogen atom and n is
an integer of 1, 2 or 3 and wherein R is the hydroxyl group
when the compound of the formula (II) is a l-N-[a-hydroxyl-
~-aminoalkanoyl]-5,3',4'-trideoxykanamycin B but R is the
hydrogen atom when the compound of the formula (II) is a
l-N-[a-hydroxy-~-aminoalkanoyl]-5,3',4',6"-tetradeoxy-
kanamycin B, or a pharmaceutically acceptable acid-addition
salt of the compound (II).
Acco.rding to the second preferred embodiment of the
first aspect invention, there is provided a new compound
which belongs to the compound of the formula (I) and is
5,3',4',6'~-tetradeoxykanamycin B represented by the formula
(III)
. .
~ li75~16
-- 7 --
6~ 2
4~ CH3 / o H2N ~ 4,
HO ~ 1" 0 ~ NH2(III)
H2N 1 2 NH2
or a pharmaceutically acceptable acid-addition salt of the
compound (III).
The physico-chemical and biological properties of
the new compounds of the formulae (II) and (III) according
to the first and second preferred embodiments of the first
aspect invention are as follows:-
(1) 1-N-[(S)~4-amino-2-hydroxybutyryl]-5,3',4'-
trideoxykanamycin B monocarbonate monohydrate is a substance
in the form of a colorless powder decomposing at 163-166C
and showing a specific optical rotation [~326 = +87
(c 1, water). Its elemental analysis is coincident with
the theore~ical values of C22H44N609 H2C03 H20 (C 44.80%,
H 7.85%, N 13.63%). This substance gives a single spot
(positive to nlnhydrin) at Rf 0.05 and at Rf 0.0~ ln a thin
layer chromatography on silica gel developed with butanol-
ethanol-chloroform-17% aqueous ammonia (4:5:2:5 by volume)
and with chloroform-methanol-conc. aqueous ammonia-water
(1:4:2:1 by volum~)as the development solvent, respectively.
29 (2) 1-N~[(RS)-3-amino-2 hydroxypropionyl]-5,3l,4'-
trideoxykanamycin B monocarbonate is a substance in the
-` ` 1 17S816
8 -
form of a colorless powder decomposing at 113-116C ~and
showing a specific optical rotatlon Ca]27 = ~120 (c 1,~
water). Its elemental ana~lysis ls coincident with t~he
theoretical value~s ~G21H42N69 ~2C3 (
N 13.63%~. This~substance gives a single spot ;(positive~
to ninhydrin) at~ Rf 0.12~and at~Rf 0.35 in the above~
mentioned thin~layer~chromatography~on sllloa gel developed
with butanol-ethanol-chloroform-171 aqueous;~ammonia~(~4~:~5:2~:;5
by~volume) and with~chloroform-methanol-conc~.~aqueous~
lO ~ ammonia-water~(l 4:2~ by~volume~) as the development
solvent, respect~ively.
(3) 1-N~-~;(S);-4-amino-~2-hydroxybutyr~1]-5~,3~ 4'~,~6~"-t~etra~
deoxykanamyoi;n B~dicarbanate ;i~s~a substance~l~n the~orm~;o~
a~colorless~powder~ decomposlng~at~ 31-1~35C and showine~a~
15~ speolfic opti~cal~rot n ~[~a]27~ +90 (c l,~wat ~ s
elemental~analysi~s~is~coincident~with~t~he~theore~ic l~
values;o~f~C~22H~4~N60 H co3~c~44~7l~%~ H~ 5o%~ N .~0 `-~
This~s~ub~s~tance~g ~ ~ e;apot~(posi~t~i e bo~
at~R~ 0~.~07;:and~a~ Rf~0.;23~in~t~he~abov~-~ ntioned~th
20~ ohromatography an silica gel developedwithbutanol ~ ~ a
ohloro~orm-17g~aqueous ammonla (4:5:2:~5`by volume) and~
wlth chloroform-methanol-conc. aqueous am~on~a-wate~r~
4:2:1 by volumé)~aa the; development solvent, respeotlvely.
(4) 5,3',4',6;~-tetradeoxykanamyoin B dicarb~onate
~monohydrate is~ a sub;s`tance~in the ~orm o~ a colorléss~
powder decompo;sing~at~l28-136C~and~s~howing~a speoi~io
I 175816
optical rotation [~]D3 = +102 (c 1, water). Its elemental
analysis is coincident with the theoretical values of
C18H37N506 2H2C03 H20 (C 42.77%, II 7.72~, N 12-47%)- This
substance gives a single spot (positive to ninhydrin) at
Rf 0.1l2 and at Rf o.56 in the above-mentioned thin layer
chromatography on silica gel developed with butanol-
ethanol-chloroform-17% aqueous ammonia (4:5:2:5 by volume)
and with chloroform-methanol-conc. aqueous ammonia-water
(1:4:2:1 by volume) as the development solvent, respectively.
The minimum inhibitory concentration (mcg/ml) of
[(S)-4-amino-2-hydroxybutyryl]-5,3',4'-trideoxykanamycin
~ B (abbreviated as AHB-trideoxyKMB), l-N-[(RS)-3-amino-2-
; hydroxyprapionyl]-5,3',4'-trideoxykanamycin B (abbreviated
as AHP-trideoxy-KMB) and l-N-[(S)-4-amino-2-hydroxybutyryl~-
5,3',4',6"-tetradeoxykanamycin B (abbreviated as AHB-
tetradeoxyKMB) of the formula (II) according to this
invention as well as of the new compound ~,3',4',6"-
tetradeoxykanamycin B ~abbreviated as tetradeoxyKMB) of
the formula (III) according to this invention against
various microorganisms were determined according to serial
dilution method on a nutrlent agar medium at 37C, the
estimation being made a~ter 18 hours incubation. For
comparison purpose, the minimum inhibitory concentrations
of ~abekacin were also determined in the same manner- as
stated above.
The antibacterial spectra of these new and known
substances are shown in Table l below.
.,
I
,:
' :
~ ~75~16
-- 10 --
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According to the third preferred embodiment of the
first aspect inventlon, there is provided a new compound
which belongs to the compound of the formula (I) and is
a 1-N-[a-hydroxy-~-aminoalkanoyl]-5~3',4'-trideoxy-6'-N-
methylkanamycin B represented by the formula (IV)
HO 6"
~1" H2~ ~,
3...... OH I 6 4 5' HCH3
(IV)
HN ~ NH2
CO
CHOH
(CH2)nNH2
wherein n is an integer of 1, 2 or 3, or a pharmaceutically
acceptable acid-addition salt of the compound (IV).
According to the fourth preferred embodiment of the
first aspect invention, there is further prov1ded a new
compound which belongs to the compound of the formula (I)
and is 5,3'~4'-trideoxy-6'-N-methylkanamycin B represented
by the formula (V)
'
.
~ 1758IB
-- 15 --
6"
HO ~ 2' 4
~ \ X2N~
H2N~llt ~ (V)
o/ ~ NHcH3
H2N --~'~ NH2
or a pharmaceutically acceptable acid-addition salt of
the compound (V).
The physico-chemical and biological properties o~
`the new compounds of the formulae (IV) and (V) according
to the third and fourth preferred embodiment of the first
aspect invention are as follows:-
(5) l~N-[(S)-4-amino-2-hydroxybutyryl]-5,3',4'-
trideoxy-6'-N~methylkanamycin B monocarbonate is a
substance in the form of a colorless powder decomposing
at 162-165C and showing a specific optical rotation
[a]22 = ~88 (c 1, water). Its elemental analysis is
coincident with the theoretical values of C23H46N6O~ H2CO3
(C 47.05%, H 7.~0%, N 13.72%). This substance gives a
single spot (positive to ninhydrin) at Rf 0.05 and at Rf
o . o8 ln a thin layer chromatography on silica gel developed
with butanol-ethanol-chlorof'orm-17% aqueous ammonia
(4:5:2:5 by volume) and with chloroform-methanol-conc.
aqueous ammonia-water (1:4:2:1 by volume) as the develop-
ment solvent, respectively.
. .
1 ~5~1
6 --
(6) 1-N-[(RS)-3-amino-2-hydroxypropionyl]-5,3',4'-
trideoxy-6'-N-methylkanamycin B monocarbonate monohydrate
is a substance in the ~orm of a colorless powder decom-
posing at 162-164C and showing a speci~ic option rotation
[~]D3 = ~80 (c 0.5, water). Its elemental analysis is
coincident with the theoretical values of C22H44N6Og-H2CO3-
H20 (C 44.80%, H 7.85%, N 13.62%). This substance gives a
single spot (positive to ninhydrin) at Rf 0.05 and at Rf
0.14 in the above-mentioned thin layer chromatography on
silica gel developed with butanol-ethanol-chloroform-17%
aqueous amrnonia (4:5:2:5 by volume) and with chloroform-
methanol-conc. aqueous ammonia-water (1:4:2:1 by volume)
as the development solvent, respectively.
(7) 1-N-~(S)-5-amino-2-hydroxyvaleryl]-5~3',4'-
trideoxy-6'-N-methylkanamycin B monocarbonate monohydrate
is a substance in the form of a colorless powder decom-
posing at 163-166C and showing a specific optical rotation
~]D3 = ~84 (c 0.5, water). Its elemental analysis is
coincident with the theoretical values of C24H48N6O9 H2C03
H2O (C 46.57%, H 8.13~, N 13.03%). This substance gives a
single spot (positive to ninhydrin) at Rf 0.03 and at Rf
o . o8 ln the above-mentioned thin layer chromatography on
silica gel developed with butanol-ethanol-chloroform-17%
aqueous ammonia (4:5:2:5 by volume) and with chloroform-
methanol-conc. aqueous ammonla-water (1:4:2:1 by volume)
as the development solvent, respectively.
,. , ~ .. .
~ ~5~1~
- 17
(8) 5,3',4'-Trideoxy-6~-N-methylkanamycin B mono-
carbonate monohydrate is a substance in the form of a
colorless powder decomposing at 137-140C and showing a
specific optical rotation [~]22 - +66 (c 1, water). Its
elemental analysis is coincident with the theoretical
ClgH39N507 H2C03 H20 (C 45.36%, H 8.18%, N 13 23%)
This substance gives a single spot (positive to ninhydrin)
at Rf 0.22 and at Rf 0.49 in the above-mentioned thin layer
chromatography on silica gel developed with butanol-ethanol
chloroform-17% aqueous ammonia (4:5-2:5 by volume) and with
chloroform-methanol-conc. aq~eous ammonia-water (1:4:2:1
by volume) as the development solvent, respectively.
The minimum inhibitory concentrations (mcg/ml) of
l-N-[(S~-4-amino-2-hydroxybutyryl]-5,3',4'-trideoxy-6'-N-
methylkanamycin B (abbreviated as AHB-trideoxyMKMB),
l~N-[(RS)-3-amino-2-hydroxypropionyl]-5,3',47-trideoxy-
6'-N-methylkanamycin B (abbreviated as AHP-trideoxyMKMB)
and l-N-[(S)-5-amino-2-hydroxyvaleryl]-5,3',4'-trldeoxy-
6'-N-methylkanamycin B (abbreviated as AHV-trideoxyMKMB)
of the formula (IV) according to this invention as well as
of the new compound $,3',4'-trideoxy-6'-N-methylkanamycin
B (abbrevlated as trideoxyMKMB) of the ~ormula (V) according
to this invention against various mlcroorganisms were
determined according to serial dilution method on a
nutrient agar medium at 37C, the estimation being made
after 18 hours incubation. For comparison purpose, the
1 ~75~1
- 18 -
minimum inhibitory concentrations of l-N-[(S)-4-amino-2-
hydroxybutyryl~-3',4'-dideoxykanamycin B (ie. habekacin)
were also determined in the same manner as stated above.
The antibacterial spectra of these new and known
compounds are shown in Table 2 below.
, ,
-- 19 --
_ ~
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O U~ J ~ N 1O . ~ . . . . . . . .
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a~ r! O rl ~0 a~ I O ¢
O ~ ~ ~d r-l r~ m ~ LS~
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u~ ~j ¢ r~ 1H ~rl
13 ~ ~ ~ O 1~ I
u~ a) li:~ O c)p~ X~Ir~
~rl o ~ r-l ~ ~;C~ ~i 1--1 1 1 1
td ~ H
bO ~ ? C~C) ~ rl ~3 ~rl
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O U~ rl ~ O~ : ~
c~
u~ c) c~
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E-l C) c~ C) r-l a
o - - o c~
H r-l O ~ rl
P~ C) ~r l H H H
.C~ O~rl r l H H r-l ,Da~
Q~ ~ h~) ~rl~rl ~rl~rl O S
t~ 1 V C)C) c) C)c
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__ _ . ~
~ ~7581B
-- 20 --
L~ L~ Lt~
Lt~Lt~Lt~ Lt~ oLS~ Lr\ ~ o ~ o ~ o o
~ I ~ ~ o ~ ~ ~ o ~ o ~ o Lt~
.
_ ._____
~OtY~ tY~ tr~ Lt~ tY~ tr) t~ Lt~ ~ t~ ~ t~
LS~~1 ~I N r~l ~ I L~ ~--1 Lt~~--1 L~
...............
tY~ ~D tr~ ~D ~ tY~ t~ ~ tr~
tr)~tr~tY~ t~ tY) ~ DtY~ D tYl Lt~
,1,~ ~ ~ ,~ ~ Lt~ Lt~ L~ ~ Lt~ Lt~ Lf~ ~1
.
trl tr) tr~ ~ ~ ~ ~ ~ ~ t~ ~ ~ ~ tY7 ~D
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t~ L~ tY t~ tr~ Lt~ ~o tr~ ~ t
r-~ ~ ~ ~ ~ ~ Lt~ ~ ~ ~ t,~
.
tY~ ~ ~tr) tr~ ~ t~tY~ trl ~D ~ t~ t~ ~ ~
--l 0r--lH H r-lO H Hr-l rl O O ~I r-l
a:) Lt~~o :~
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t,~ XP:; X t~ ~
IO~ O O O ~ ~ O O
r--l~I ~)r--l rl O O O 1:~ 0 t~) r-l
r l ~ ~O
r1 r-lr lr--l ~ ~ 1~ 0 ~ r~l
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Lr~ o V t~
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N ~t~lt~lt~ 1t,~l t~ 1 Lt~ a)
r-lr-lr-l r-lr-l r-l H r~ 0 r l ~1 td
I I I I I I I I ~ rl t,d
rl
O :
r--l rl ~ tl
O ~ O _ t
C~ t~ ~ O
td ~d
~rl ~rl td
H
C) C) r-l t~d
~r-l ~r~ rl
~ t : : ~ rl : r l
tl) a~ u, a)
~0
C) C~ rl
t/~ u~ r--l 5:~
_ .._. _ _ _ _ _ _
~ ~7~16
- 21 -
.. ~ . .
U~
IS~ D L~ ~ O LS~ O O O O O ~ L~
I O ~J OO 1~ 0 0 (~
~1 ~1 ~1 ~1 ~1
A A A
I~ D(Y~co ~f) ~15~
~ .~ . . . -
~D ~Dr I~f) O O ~J O O OL~\ O ~ (~ ~D
LS~ r I L~ OL~
A
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u~
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O ~I L~ ,-1 t'\l r
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r
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D O LD~ J O~1 O O O ~ ~f)
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tr~~D01~ 0 L~
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Lr~ t~J r-l
V
_ . .
r~l ~I
r-l r-l r-t
0~ O~
r ~ ) (~ O O~
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r-l ~D ~d
t~~rl a~ r~ ~D U~
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c/~ ~ ~ 0 3
~r~ D b~
~rl h u~ ~rl ~r/ C~ r~lr-l
a) ~ rl F~
X 1 ~
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r-l O r~ ~ ~ ~O
4~ u~~da l~ ~ a) E3 u~ ~rl rla~
~Ir l~::> h h C~ O ~
~ rlr l t~ L~ O
rl a) (I) ,e
O : -
~rl~rlr~lr-l O O O~ O O(I)
~ h u~
.
~ 175~16
Lr~
L~L~oooooL~o
r~ o o o o ~ o
,,
A ~ A A
~ Lr~ Lf~ ~
Lr~ ~ ~ ~ ~
L~ Lr~ Lr~O ~
J O O
A A
Lf~ ~D ~ L~ ~
L~ L~ Lr~ L~
~D ~ rr~ ~D ~ ~ ~ O ~ O
~1 ~1~I L~ O
Ln ~ ~ L~
.
L~ Lf~ L~ O ~ O
r~ J L~\ O
A
_
rr) ~ ~O
L~ Ln ~ ~ Lr~Lr~ L~ L~
~J H ~1 tr'~ J N O ~ O
~ L~ ~
.
~0 ~
r~ L~ L~ ~ O
rn cr~
~1
r l H ~Z;o~ I H r1~ I r~
v o~ m ~
rl
rl~ rn ~
O O ~rl
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bO ~ o Q~
a~
ra ,1rn rn
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o _ _ : - :- e ~ o o
a) u~
ra orn rn
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~ ~7~81B
- 23 -
~ 'rom Tables 1 and 2, it is seen that the new com-
pounds of this invention according to the formula (I),
including the compounds of the formulae (II), (III), (IV)
and (V), effectively inhibit the growth of many kinds of
bacterial strains. The new compounds of this invention
show a low acute toxicity to animals, including men. It
has been found that the new compounds of the formulae
(II) and (III) according to this invention, such as
l-N-[(S)-4-amino-2-hydroxybutyryl]-5,3',4'-trideoxy-
kanamycin B; l-N-[3-amino-2-hydroxypropionyl]-5,3',4 r _
trideoxykanamycin B; l-N-~(S)-4-amino-2-hydroxybutyryl]-
5,3',4',6l'-tetradeoxykanamycin B; and 5,3',4',6"-tetra-
deoxykanamycin B exhibit an LD50 value of 25 to 50 mg/kg
when their acute toxicity is estimated by intravenous
injection in mice. It has also been found that the new
compounds of the formulae (III) and (IV) according to
this invention, such as l-N-~(S)-4-amino-2-hydroxybutyryl]-
5,3',4'-trideoxy-6'-N-methylkanamycin B; 1-N-(3-amino-2-
hydroxypropionyl)-5,3',4'-trideoxy-6'-N-methylkanamycin B,
1-N-[(S)-5-amino-2-hydroxyvaleryl]-5,3',4'-trideoxy-6'-
N-methylkanamycin B and 5,3',4'-trideoxy-6'-N-methyl-
kanamycin B exhibit an LD50 value of 50 to 100 mg/kg when
their acute toxiclty is estimated by intravenous inJection
in mice.
The new compounds of this lnvention are usually
obtained in the form of its free base or a hydrate or a
~ ~7~6
- 24 -
carbonate thereof. The new compounds of this invention
each may readily be converted into a form of a pharma-
ceutically acceptable acid addition salt thereof~ such as
the hydrochloride, hydrobromide, sulfate, phosphate,
nitrate, acetate, maleate, citrate~ ascorbate, methane-
sulfonate and the like by reacting with the appropriate,
pharmaceutically acceptable inorganic or organic acid in
an aqueous medium.
The new compounds of the formula (I), tII), (III),
(IV) or (V) according to this invention and its pharma~
ceutically 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 pharmaceu-
tical form known to the art for oral administration.
Examples of the pharmaceutical forms for oral administration
are powders, capsules, tablets, syrup and the like. A
suitable dose of the new compounds of this invention for
e~fective treatment of bacterial infections is in a range
of 0.1 to l g. per person a day when it is given orally.
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 admlnistered by intramuscular
inJection at a dosage of 50 to 500 mg per person two to
~ .~758~6
- 25 -
four times per day. Moreoverg the new compounds of this
invention may be formulated into an ointment for external
application which contains the active compound at a con-
centration of 0.5-5% by weight in mixture with a known
ointment base such as polyethylene glycol. Furthermore,
the new compounds of this invention are each useful for
sterilization of surgieal instruments and sanitary
materials.
Aecording to a second aspeet of this invention,
therefore, there is provided an antibacterial composition
comprising as the active ingredient a l-N-[a-hydroxy~
aminoalkanoyl]-5,3',4'-trideoxykanamycin B, a l-N-[~-
hydroxy-~-aminoalkalloyl]-5,3',4',6"-tetradeoxykanamycin B
or a pharmaceutically aceeptable aeid-addition salt thereof
as defined by the formula (II) or 5~3',4',6"-tetradeoxy-
kanamyein B or a pharmaeeutieally aeceptable aeid-addition
salt thereof as defined by the formula (III) in an
antibacterially effeetive amount to inhibit the growth
of baeteria, in eombination with a earrier for the ac~ive
in~redient compound, as well as an antibacterial composi-
tion eomprising as the aetive ingredient a l-N-[~-hydroxy-
~-aminoalkanoyl~-5,3',l~'-trideoxy-6'-N-methylkanamyein B
or a pharmaeeutieally aeeeptable aeid additlon salt thereof
as defined by the formula (IV) or 5,3',l~'-trideoxy 6'-N
methylkanamycin B or a pharmaceutieally acceptable aeid-
addition salt thereof as defined by the formula (V), in
I ~75816
- 26 -
an antibacterially effective amount to inhibit the growth
of bacteria, in combination with a carrier for the active
ingredient compound.
Next, the production of the new compound of the
formula tII), according to this invention is described.
Thus, the new compound of the formula (II) such as
l-N-[(S)-4-amino-2-hydroxybutyryl]-5,3',4'-trideoxy-
kanamycin B and l-N-[(S)-4-amino-2-hydroxybutyryl]-
5,3',4',6"-tetradeoxykanamycin B can be synthetized by
using as the starting compound either the known substance
5,3',4'-trideoxykanamycin B (Japanese Journal of Anti-
biotics, 32, S-178 (1979)) or the new compound 5,3',4'~6"-
tetradeoxykanamycin B as obtained according to this
invention and condensing the l-amino group of the starting
compound with a corresponding ~-hydroxy-~-amino alkanoic
acid or a functional equivalent thereof.
According to the third aspect of this invention,
therefore, there is provided a process for the production
of a 1-N-[~-hydroxy-~-aminoalkanoyl]-5,3',4'-trideoxy~
kanamycin B or l-N-[~-hydroxy-~-aminoalkanoyl]-5,3',4',6'7-
tetradeoxykanamycin B represented by the formula (II)
~ ~7~6
- 27 -
R
HO ~ O \ H2N
H2N \ ~ ~NH2
(II)
HN ~ NH2
CO
CHOH
(CH2)nNH2
wherein R is a hydroxyl group or a hydrogen atom and _ is
an integer of 1, 2 or 3~ which comprises:-
(a) acylating the l-amino group of 5,3',4'-tri-
deoxykanamycin B or 5,3',4',6'r-tetradeoxykanamycin B
represented by the formula (VI)
6"
HO \ H2N ~
H2N ~ OU ~ ~ ~ (VI)
O ~ NH2
H2N~/--\\,~ NH2
whereln R is as de~ined above, or a partially amino-
protected derivative of 5,3',4'-trideoxykanamyci.n B or
5,3',4~,6"-tetradeoxykanamycin B represented by the
~ormula (VI')
..
~ ~7~6
- 28 -
; ~
H2N 1 ~ B
wherein R is a hydroxyl group or a hydrogen atom~ and A
is a hydrogen atom and at least one B is a mono-valent
amino-protecting group but the other B(s) is or are each
a hydrogen atom, or at least one pair of A and B taken
together form a di.-valent amino-protecting group but the
other A and B are each a hydrogen atom, and the amino-
protecting groups represented by A and B may be equal to
each other or dif~erent from each other, by reaction with
an a-hydroxy-~-aminoalkanoic acid or an amino-protected
derivative thereof represented by the formula (VII3
A~
B~ > N(CH2)nlCHCH (VII)
~ H
wherein A' is a hydrogen atom and B' is a hydrogen atom
or a mono-valent am~no-protecting group, or A' and B'
taken together form a di-valent amino protecting group
and n ls an integer of 1, 2 or 3, or a functional
equivalent of the compound (VII), to produce the l-N-
acylated product represented by the formula (II')
31 ~75~16
- 29 -
6"
~1 ~ NH2
~NH2 (II')
CO
CHOH
(CH2)nN - B~
or by the formula (II")
R ~ A
HO ~ ~ \ B ~N ~
A ~ M \ ~ ~ ~ ~ N
N A (II")
CO
CHOH
( CH2 ) nN ~
~ B'
wherein R, A, B, A', B' and n are as def'ined above, and
(b) removing the remaining amino-protecting group(s)
where existts), f'rom the l-N-acylated product of' the f'ormula
(II') or (II") in a known manner to produce the compound of'
the f'ormula (II).
, ' : .
.
1 ~7~16
-- 30 --
The process oF the third aspect of this invention
may include a further step of converting the compound (II)
into a pharmaceutically acceptable acid-addition salt
thereof by reacting with a pharmaceutically acceptable
inorganic or organic acid in a known manner, if desired.
The procedures ~or carrying out the process of the
third aspect of this invention are now described in more
detail.
In carrying out the present process, it is possible
to employ as the starting compound 5,3',41-trideoxy-
kanamycin B or 5~3',4',6T'-tetradeoxykanamycin B (VI) of
which amino groups are not protected at all, in the rorm
of the free base or in the form of an acid-addition salt
with an appropriate acid such as hydrochloric acid or
sul~uric acid. However, it is preferable to employ as
the starting compound such a partially amino~protected
derivative of 5,3',4'-trideoxykanamycin B or 5,3',4',6"-
tetradeoxykanamycin B according to the formula (VI') in
which all or some of the amino groups other than the
l-amino group have been protected with known amino-
protecting group(s) and which may be prepared by
lntroduetion of a known amino-protecting group into the
eompound of the formula (VI) by means of a known amino-
proteeting teehnique previously adopted in the synthesis
of some known deoxy derivatives of kanamycin B. For the
preparation o~ the partially amino-protected 5,3',4'-
~.~7~6
- 31 ~
trideoxykanamycin B or 5,3',4',6"-tetradeoxykanamycin B
of the formula (VI'), it is feasible to utilize the
amino-protecting techniques which were employed, for
instance, in the preparation of the 6'-N-benzyloxycarbonyl
derivative of kanamycin B as described in the specification
of U.S. patent No. 3,781,268 or U.S. patent No. 3,929,762;
the preparation of 2',6'-di-N~tert-butoxycarbonyl-kanamycin
B or 6'-N-benzyloxycarbonyl-kanamycin B, or the mono-N- or
di-N-tert-butoxycarbonyl and even tri-N-tert-butoxycarbonyl
derivative of 6'-N-benzyloxycarbonyl-kanamycin ~, ei.ther
isolated or in mixture thereof~ as described in the
specification of U.~. patent No. 1,426,908 or U.S. patent
No. 3,939,143; or the preparation of 2',3,3",6'-tetra-N-
formyl derivative of kanamycin B as described in the
specification of Belgian patent No. 817,546.
In general, suitable examples of the amino-protecting
group which may be used for the protection of some amino
groups in the partially amino-protected derivative of the
formula (VI') may be an ordinary amino-protecting group,
including an alkoxycarbonyl group such as tert-butoxy-
earbonyl and tert-amyloxycarbonyl; a cycloalkyloxyearbonyl
group sueh as eyelohexyloxyearbonyl; an aralkyloxyearbonyl
group such as benæyloxycarbonyl; an acyl group such as
trifluoroacetyl and o-nltrophenoxyacetyl; a phosphinothioyl
group such as diphenylphosphinothioyl and dimethylphos-
phinothioyl; a phosphinyl group such as diphenylphosphinyl,
~ ~5~6
- 32 -
and the like. Preferred examples of the di-valent amino-
protecting group include phthaloyl group and a group of
Schiff base type such as salicylidene. The introduction
of the amino-protecting group of these kinds may be
conducted by reacting the compound of the formula (VI)
with an appropriate known reagent for introduction of the
amino-protecting group which may be in the form of an
acid halide, acid azide, active ester or acid anhydride
and the like, in the manner known in the conventional
synthesis of peptides. By chosing the quantity of the
reagent for introduction of the amino-protecting group
employed in a proportion of 0.5 to 6 mol. per mol. of the
compound of the formula (VI), it is possible to prepare
a mixture of different 3 partially amino-protected
derivatives (VI') at any ratio, due to the difference
in the reactivity of the respective amino groups of the
compound (VI).
In the process of the third aspect of this inven-
tion, it is practical to employ as the starting compound
such amino-protected 5~3'~4l-trideoxykanamycin B or
5,3',4',6"-tetradeo~ykanamycin B derivative in which all
or some of the amino groups other than the l-amino group
have or has been blocked, for example, a 3,2',6',3"-
tetra~N-protected derivative, a 3,2',6'-tri-N-protected
derivative, a 2',6',3"-tri-N-protected derivative, a
2'~6'-di-N-protected derivative and a 6'-mono-N-protected
~ ~75~1
-- 33 --
derivative. Besides, a mixture of two or more of these
partially N-protected derivatives may, without being
purified, be used for the l-N-acylation step of the
present process.
In order to ensure that the desired product of the
general formula (II) can be produced in a high yield in
accordance with the process of the third aspect invention,
it needs only that just the l-amino group of the compound
of the formula (VI)g namely 5,3',4'-trideoxykanamycin B
or 5,3',4~,6'r-tetradeoxykanamycin B is selectively acylated
with the a-hydroxy-~-aminoalkanoic acid (VII). Accordingly,
: it will be evident that most preferably 9 a 3,2',6',3"-
tetra-N-protected derivat~ve of the compound (VI), that is,
the amino-protected derivative of the compound (VI') in
which all the amino groups other than the l-amino group
have been blocked with the protectlve groups is employed
as the starting compound to be l-N-acylated in the present
process.
To prepare the 3,2',6',3"-tetra-N-protected deriva-
tive of the formula (VI') from the compound of the formula
(VI), the ~ollowing procedure may be used, for instance.
Thus, there can be applied a known method of U.S. patent
No. 4,136,254 o~ Nagabhushan et al by which a 3,2',6'-tri-
N-acylated protected derivative o~ kanamycin B is prepared
by reacting kanamycin B with a di-valent transition metal
cation, for example, cation of copper (II), nickel (II),
~ . , .
~ 1l75~16
cobalt (II), etc. for the formation of a metal complex of
kanamycin B, reacting this kanamycin B-metal complex with
an acylation agent known as the amino~protecting group
introducing reagent for the protection of all the amino
groups other than the l-amino and 3"-amino groups of the
kanamycin B moiety of the kanamycin B-metal complex ~said
1- and 3"-amino groups having been blocked by complexing
with the di--valent metal cation in the kanamycin B-metal
complex), and ~hen removing the di-valent me~al cation from
said complex, eg. by treatment with hydrogen sulfide or by
trea~ment with aqueous ammonia. Or, there can be applied
; - the method of published U.K. patent application 2,036,020 A
or Belgian pa-~ent~No. 879,925, by which a 3,2',6'-tri-N-
acylated protected derivative of kanamycin B is prepared in
a similar way to.the aforesaid known method of Nagabhushan
et al except that zi~nc cation is employed instead of the
d;-valent transition metal cation. In thi~s way, a 3~2',6'-
. tri-N-protected derivative of the formula (VI'). can be
prepared from the compound of the formula ~VI). in a hi.gh
yiel~. The 3"-amino grou~ of this 3,2',6'~.tri~N-protected
der.Lvative (VII~ so prepared can further be protected by
the selective acylation according to a selective. 3"-N-`
acylation method (.see claim 15 of Belgian patent No~ 879,~23
i - 34 -
b.'
mab/~
:: ,
~ ~58~6
for the production of an amino-protected derivative of an
aminoglycosidi.c antibiotic of which all tKe amino groups
other than the l-amino group have been protected selectively,
so that a 3,2',6',3"-tetra-N-protected derivative of the
compound (VI) can be prepared in a high yield. In accor~
dance with the selective 3"-~-acylation method as described
in claim lS of Belgian patent No. 879,923, the above-
mentioned 3j2',6'-tri-N-protected derivative o~ the compound
: (VI) is reacted with.a formic acid alkyl ester, a di-halo
or tri-halo-alkanoic acid alkyl ester, formylimidazole or
an N--alkanoyl-imidazole as the acylation agent, whereby the
3"-amino group can be acylated selectively with the acyl
residue o~ the acylat;on agent employed in a high yield,
wi~th.out involving the acylatïon of the l~amino group of said
3,2l,6'~-tri-N-protected derivatiYe. The 3,2',2~,3"-tetra~
N-acylated derivatlve, for example, 3,2.',6'-tri~N-benzyloxy-
carbonyl-3"-N-trifluoroacetyl derivative, of 5,3',4'-tri-
deoxykanamycin B or 5,3',4',6"-tetradeoxykanamycin B which
may be obtained by applying the above-~mentioned methods of
20. the U,S. patent No. 4,1`36,254 and of the Bel~ian patent No.
8~,923 is a most preferred starting compound to be l-N-
acylated selectiyely with the a-hydroxy-w-aminoa1kanoi~c acid
(~yII) in the l-N-acylation step of the
- ~ ~ - 35 -
mab~7
`
1~7
- 36 -
present process.
In the process of this third aspect invention, the
l-amino group of the compound of the formula (VI) or the
l-amino group of the partially amino-protected derivatives
(VI') thereof, either ~solaked or in mixture of two or
more of them, is acylated with the ~-hydroxy-~-aminoalkanoic
acid of the formula (VII)
A'
B~ / N (C~2)n IHCOOH (VII)
OH
wherein A' and B' are as defined above and _ is an integer
of 1, 2 or 3 of which the amino group is not protected or
has been protected. This ~-hydroxy-~-aminoalkanoiç acid
may be 3-amino-2-hydroxypropionic acid (ie. the compound
of the formula (VII) where _ is 1 and A7 and B' are the
hydrogen atoms), I~-amino-2-hydroxybutyric acid (ie. the
compound of the formula (VII) where n is 2 and A' and B'
are the hydrogen atoms) or 5~amino-2-hydroxyvaleric acid
(ie. the compound of the formula (VII) where is 3 and
A' and B' are the hydrogen atoms). Amongst them, the
(S)-isomer is preferred for use.
In the process of the third aspect invention, the
l-N-acylation with the ~-hydroxy-~-aminoalkanoic acid
(VII) may be conducted according to any of one conventional
methods for the synthesis of peptides, for instance,
according to the known dicyclohexylcarbodiimide method,
I ~ 7
-- 37 --
the known mixed acid anhydride method, the known azide
method or the active ester method and the like, using the
~-hydroxy-~-aminoalkanoic acid as such or in the form of
its reactive derivative (as the ~unctional equivalent
thereof). For the amino-protecting group for protection
of the amino group of the a-hydroxy ~-aminoalkanoic acid
may be employed such an amino-protecting group which is
the same as or different ~rom the one present in the
starting compound (~I'). Particularly, a preferred amino-
protecting group ~or this purpose is tert-butoxycarbonyl
group which is easily removable by treatment with aqueous
trif'luoroacetic acid or acetic acid or with diluted aqueous
hydrochloric acid. Benzyloxycarbonyl group which is
removable by a conventional hydrogenolysis in the presence
of a catalyst such as palladium or platinum oxide is a
convenient N-protecting group.
The l-N-acylation in the present process may pre-
ferably be carried out in an aqueous organic solvent
according to the active ester method using and ~-hydroxy-
~-aminoalkanoic acid in the form o~ its active ester. For
example, N-hydroxysuccinimide ester of L 4-benzyloxy~
carbonylamino-2-hydroxybutyric acid may preferably be used
as the active ester which may be prepared by a conventional
method of prepari.ng the active ester. This active ester
preferably may be used in a proportion of frorn 0.5 to 3
molar equivalents and preferably of from 1 to 1.5 molar
. , ~ .
~ .
,
~75~16
-- 38 --
equivalents per mol of the starting compound (VI) or (VI')
to be l-N-acylated. The aqueous organic solvent used in
the reaction mediurn may be a water-miscible organic solvent
such as dioxane, 1,2-dimethoxyethane, dimekhylformamide,
tetrahydrofuran, and the like. The l-N-acylation may be
effected at ambient temperature or, if desired, at an
elevated temperature of 20-90C and for a reaction time
of several hours and preferably of 5-6 hours.
When the l~N-acylation in the present process is
conducted using as the starting compound such as a partially
amino-protected derivative in which some, but not all, of
the amino groups other than the l-amino group has or have
been protected, for example, the 6'-N-protected derivative
of the starting compound (~I), the acylation products as
formed may partially be purified by a column chromatography,
for example, on silica gel so that the unreacted starting
material is removed, giving a mixture of the desired
; l-N-mono-acylated product with the otherwise N-acylated
products, as the case be in the synthesis of habekacin,
namely 1-N-[(S)-4-amino-2-hydroxybutyryl]-3',4'-dideoxy-
kanamycin B as described in the specification o~ U.S.
patent No. 4,107,424. These mixed acylation products
may~ without being puri~ied and/or isolated, be subjected
immediately to the subsequent de-protecting step of the
2~ present process, followed by the purification and isolation
so that the desired l-N-mono-acylated product is obtained.
.
,
~ ' '
-
~ ~758
-- 39 --
In the second step of the process of this third
aspect invention, the l-N~acylation product (including
the mixed acylation products) as obtained in the l-N-
acylation step of the present process is subjected to the
removal of the amino~protecting groups, if these are still
remaining in the l-N-acylation product. The removal of
the protecting groups is effected by a conventional
deprotecting technique. Thus~ the amino-protecting group
of the alkoxycarbonyl type is removed by weak acid
hydrolysis with an aqueous solution of trifluoroacetic
acid or acetic acid and the like or with a diluted aqueous
solution of an inorganic acid such as hydrochloric acid.
The aralkyloxycarbonyl group such as benzyloxycarbonyl may
be removed by an ordinary catalytic reduction (hydro-
genolysis~-. When-phthaloyl group is present as the amino-
protecting group, it can be removed by heating in a solution
of hydra~ine hydrate in a lower alkanol.
The deprotected acylation product as obtained from
the second, de-protecting step of the present process may
contain the desired l-N-acylation product of the formula
(II) together wi~h some isomers thereo~. The desired
l-N-(a-hydroxy-~-aminoalkanoyl) derivative (II) may be
isolated and puri~ied chromatographically using a cation-
exchanger containing carboxylic ~unctions, such as Amberlite
CG-50 ta product of ~ohm & Haas Co., U.S.A.) or CM-Sephadex
C-25 (a product o~ Pharmacia Co., Sweden) and assaying the
; ,, *trade mark
~ ~758~
antibacterial activity of the fractions of the eluate by
means of a proper ~anamycin-sensitive strain and kanamycin-
resistant s~rain of bacteria.
The 5,3',4',6"-tetradeoxykanamycin B which is
used as the starting compound in the process for the pro-
duction of a l-N-(~-hydroxy-~-aminoalkanoyl)-5,3',4',6"~
tetradeoxykanamycin B in accordance with the third aspect
of this invention may be prepared starting from 3',4',6"-
trideoxykanamycin B already synthesized by the present
in~entor ~this compound was referred to as 6"-deoxydibekacin
în.the specification of published U.K. patent application
2,058~774 A) or an N,O-protected derivative thereof repre-
sented by the formula (VIII-'). .
.
DO ~ o \ B -~N ~
B,,N ~ ~ 1~ (VIII')
B ~ N N `~B
.
wherein A is a hydrogen atom and B is a mono-valent amino-
protecting group, or A and B taken together form a di-valent
amino-protecting group, and D is a hydroxyl-protecting acyl
group.
According to the fourth aspect of this invention,
therefore, there is provided a process for the producti.on
- 40 -
mab/
- 41 ~7~816
of 5,3',4'~6"-tetradeoxykanamycin B as described herein-
before and represented by the formula (III)
CH3 O
HO ~ \ H2N
H N \ ~ ~
2 OH ~ " ~-o NH2 (III)
0~ \
~NH2
H2N~
which comprises the steps of:-
5` (a) protecting the 4"-hydroxyl group of a penta-
N-protected and 2"-0-protected derivative of 3',4',6"-
krideoxykanamycin B represented by the formula (~III)
OH ~ ~ (VIII)
0~0 N~BA
B ~ N i \ N ~-A
wherein A is a hydrogen atom and B is a mono-valent amino-
protecting group, or A and B taken together form a di-valent
amino-proteating group, and D is a hydroxyl-protecting acyl
group, with a mono-valent hydroxyl-protecting acyl g:roup of
the same klnd as the hydroxyl-protecting group (D) present
in the 2"-position of the compound (VIII) to produce the
4"-O-protected compound of the formula (VIII')
' ` ' "
:
. ~
~ ~7~816
- 42 -
t~ N ~ (VIII')
BA ~ N N B
wherein A, B and D are as defined above,
(b) reacting the 41'-O-protected compound of the
formula (VIII') with sulfuryl chloride to replace the
5-hydroxyl group thereof with a chlorine atom to produce
the corresponding 5-chloro derivative,
(c) reducing the 5-chloro deriv,ative to remove the
5-chloro group to produce a protected derivative of
5,3',4',6"-tetradeoxykanamycin B represented by the formula
(IX)
DO ~ \ B ,, N
~N ~ \B (IX)
~ ~0
A " N ~ N B
wherein A, B and D are as deflned above, and
(d) removing the remaining hydroxyl-protectlng
groups and the remaining amino-protecting groups in a
3 17
-- 43 --
known manner from the compound of the formula (IX) to
produce the compound of the formula (III).
In the process of the fourth aspect invention, the
amino-protecting groups present in the penta-N-protected
and 2"-O-protected 3',4'36"-trideoxykanamycin B of the
formula (VIII) employed as the starting material may be
of the same kind as those present in the starting compound
of the formula (VI') which is employed in the process of
the third aspect invention as described hereinbefore. The
preparation of the starting compound of the formula (VIII)
is described hereinafter.
In the first step of the process according to the
fourth aspect invention, the 4"-hydroxyl group of the
starting compound (VIII) is protected with a mono-valent
hydroxyl-protecting group of acyl type which may be a lower
alkanoyl group such as acetyl or an aroyl group such as
benzoyl. Introduction of such hydroxyl-protecting acyl
group into the 4"-hydroxyl group of the starting compound
(VIII) is achieved readily by reacting the starting com-
pound (VIII) with an appropriate acylation reagent in theform of acid anhydride, acid halide or active ester in a
known manner, f`or example, in an organic solvent such as
pyridlne at a temperature of 10-50C, pre~erably at
ambient temperature. Preferred acylation reagent for this
purpose is acetyl chloride or benzoyl chloride. In this
acylation reaction, the 5-hydroxyl group of the starting
~ 17581
-- 44 ~
compound (VIII) is hardly acylated by the acylation reagent
owing to the lower reactivity of the 5-hydroxyl group.
In this wayl there is produced the 1,3,2'6',3"-
penta-N-protec~ed and 2" 9 4"~di-0-protected derivative of
3. ~t ,6"-trideoxykanamycin B of the formula (VIII').
In the second step of the present process, the
protected derivative (VIIII) so obtained is sub~ected to
the deoxygenation at the 5-hydroxyl group thereof in a
known manner as described in the Bulletin of the Chemical
Society of Japan, Vol. 51, page 2354 (1978). Thus~ the
protected derivative (VIII') is reacted with a 1 to 5
-~ . molar proportion of sulfuryl chloride in an organic solvent
such as dry pyridine at a temperature lower than ambient
temperature ror 2 to 20 hours under agitation, af.fording
the 5-chloro derivative.
In the third step of the present process, the 5-
chloro derivative so obtained is redueed to effect the
dehalogenation. This removal of the 5-chloro group may
be achieved by reaction with a metal hydride such as
tributyl tin hydride as described in the above-mentioned
document or by conventional catalytic hydrogenation in
the presence o~ Xaney nickel. In this way, there is
produced the N~0-protected derivative o~ 5,3',4',6"-
tetradeoxykanamycin B o~ the formula (IX).
In the fourth step of the present process, the
N~0-protected 5,3l,4',6"-tetradeoxykanamycin B derivative
*trade mark
:
I ~7~
- 45 -
(IX) is subJected to the deprotection. The mono-valent
hydroxyl-protecting acyl groups (D) present in the 5-
deoxygenated compound (IX) can easily be removed by
alkaline hydrolysis at ambient temperature, for example,
by dissolving the compound (IX) into ammoniac methanol
(ie. a mixture of aqueous ammonia and methanol). The
amino-protecting group present in the starting compound
(VIII) is of the aralkyloxycarbonyl type~ the amino-
protecting group of this nature can be removed concur-
rently to the catalytic hydrogenation to which the 5-chloro
deri~vative is subjected in the third step of the present
proce~s. The amino-protecting groups other than the
aralkyloxycarbonyl group can readily be removed in a known
manner, for example, by hydrolysis wlth weak acid. When
the amino-protecting group is a lower alkoxycarbonyl group
such as ethoxycarbonyl, it can be removed by alkaline
hydrolysls with barium hydroxide.
In the process of the fourth aspect invention, it
is possible to carry out such a modified process in which
3',4',6"-trideoxykanamycin B is used as the intiial
material, its five amino groups are protected and sub-
sequently its two 2"- and li"-hydroxyl groups are protected
at once with the same mono-valent hydroxyl-protecting
groups (D) to prepare the N,0-protected derivative of the
formula (VIII').
The preparation of the penta-N-protected and
', ~ : :
~ J17~816
46 --
2"-O-protected derivative of 3',4',6"-trideoxykanamycin B
of ~he formu].a (VIII~ employed as the starting compound in
the process of the fourth aspect invention may be conducted -
as described in the specification of published U.K. patent application
2,058,774. Thus, a penta-N-protected derivative of
3',4'-dideoxykanamycin B represented by the formula
B (1)
` ~ B - N l 3 N ~ B
wherein A and B are as defined hereinbefore in respect of
the formula (VIII) is used as the initial material, the
: lO two 4"- and 6" hydroxyl groups thereof are protected
s1multaneous1y with a di-valent hydroxyl-protecting group
such as isopropylidene group, the 2"-hydroxyl group thereof
is protected with a mono-valent hydroxyl-protectlng acyl
: group such as benzoyl and acetyl to produce a 2",4",6"-
lS trl-0-protected derivative of the formula
6"
\ C ~ 0D ~ ~ A
: B ~ 0 ~ ~B
. ~ N ~ N B
.~
-
.
~'
- 47 -
wherein A and B are as deflned above, the group
\ C /
y /
is a di-valent hydroxyl-protecting group where X and Y are
each a hydrogen atom, an alkyl group of 1 to 4 carbon, an
aryl group such as phenyl or an alkoxyl group of 1 to 4
carbon atoms, or the group
X
: \ /
~ ~ y/ \
denotes a cyclohexylidene group or a tetrahydropyranylidene
group, and 3 is a mono-valent hydroxyl-protecting acyl
group. The 2",4",6"-tri-0-protected derivative of the
formula (2) so obtained is treated with aqueous acetic
acid to remove the group (y ~ C =) of protecting the 4"-
and 6"-hydroxyl groups the~refrom, and the product so
partially deprotected is reacted with an appropriate
sulfonylation reagent such as p-toluenesulfonyl chloride
in pyridine to preferentially sulfonylate the 6"-hyclroxyl
group thereof and to produce a 6"-0-sulfonylated derivative
of the formula
: ~ . , : . ~ , . - ,, -:
, . - . . , - .
,
~ 8
- 48 -
GS0 ~ A
H0 ~ \ B N ~
A \ ~ O ~ ` ~ / ~ N A
A = N N B
wherein A, B and D are as defined above and ~ is a lower
alkyl group, especially an alkyl group of l to 4 carbon
atoms, an aryl group such as phenyl or p-methylphenyl or
. an aralkyl group such as benzyl. The 6"-O-sulfonylated
derivative obtained is then treated with an alkali metal
iodide or bromide to replace the 6"-sulfonyloxy group
(GSO3-) by the iodo or bromo group and thereby to produce
a corresponding 6"-iodo or 6"-bromo derivative (correspond
ing to such a compound of the formula (3) but where the
group GSO3- has converted into the iodine or chlorine atom),
which is subsequently reduced with hydrogen in the presence
of a known hydrogenation catalyst such as palladium to
effect the dehalogenation, giving the penta-N-protected
and 2"-O-protected 3',4',6"-trideoxykanamycin B derivative
of the f`ormula (VIII) as desired.
~urther7 the production of the new compound of the
~ormula (IV) accord~ng to this invention is described here.
Thus~ the new compound o~ the formula (IV) such as l-N-
[(S)-4~amino-2-hydroxybutyryl~-5,3',4 ~ -trideoxy-6'-N-
I 1i 7 ~
- 49 -
methylkanamycin B, l-N-(3-amino-2-hydroxypropionyl)-
5,3',4'-trideoxy-6'-N-methylkanamycin B and l-N-[(S)-5-
amino-2-hydroxyvaleryl]-5,3',4'-trideoxy-6'-N-methyl-
kanamycin B can be synthetized by using as the starting
compound the new compound 5,3',4'-trideoxy-6'-N-methyl-
kanamycin B as obtained in accordance with this invention
and condensing the 1-amino group of this starting compound
with a corresponding a-hydroxy-~-aminoalkanoic acid or a
functional equivalent thereof.
According to the fifth aspect of this invention,
therefore, there is provided a process for the production
of a l-N-[~-hydroxy-~-aminoalkanoyl]-5,3', L~ ~ ~trideoxy-6'-
N-methylkanamycin B as described hereinbefore and repre-
sented by the formula (IV)
HO
HO ~ O\ H2N ~
H2N ~ ~ ~ ~ NHCH3
~
HN ~ NH2 (IV)
CO
I
CEIOH
(C~I2)nNH2
wherein n is an integer of 1, 2 or 3, which comprises:-
I f~7~16
-- 50 --
(a) acylating the l-amino group of 5,3',4'-trideoxy-
: 6'-N-methylkanamycin B represented by the formula (V)
HO
HO ~ O\ H2N ~
~' ~ H2~ ~ ~ ~ (V) :'
: :OH I ¦ NHCH
~ o 3
o
~` H~2l ~ ~ ~ ~ IB2
or a partially amino-protected derivative of 5,3':,4:'~
trideoxy-6'-N-methy~lkanamycin B represented by the~formula
A ~ ~ ;~ ; B
B OH~ : N-~3( )
whereln A is:~;a hydrogen~at~om~and'at~least one~ B is a~
mono-valent~amino~-p:rote~ct~ing group but th~e other B(s)~
1;0: ~ is or:are ea;ch a~h~drogen atom,~or~at:least one pair of ;
A and B taken together~form a:di-valent~amln'o-protect;ing
group but the other A and B~are eaoh a hydrogen at~om, and
: ~ the amino-protectlng~groups~represented by A and B may be
equal to each other or dlf~erent ~rom each other, by~
reaction with an a-.hydroxy-~-aminoal~anoic acld:or an
: amlno-proteoted derlvative thereo~ represented by the ~ :
, ~ :
~. . .
, ~- - . . ~
~ 775~.~6
- 5I -
formula (VII)
A'
/ N(CH2)nfHCOOH (VII)
B' OK
wherein A' is a hydrogen atom and B' is a hydrogen atom
or a mono-valent amino-protecting group, or A' and B'
taken together form a di-valent amino-protecting group
and _ is an integer of 1, 2 or 3, or a functional equivalent
of the compound (VII), to produce the l-N-acylated product
represented by the formula (IV')
H~ / ~ o ~ ~ N /
NH2 (IV')
HN
CO
CHOH
(CH2)nN ~ B~
or by the formula (IV")
1 1~5~16
- 52 -
HO B /
N ~ OH ~ ~ N
A
7N - B (IV")
CO
CHOH
I,, A
(CH2)nN B~
wherein A~ B, A', B' and n are as defined above3 ancl
(b) removing the remaining amino-protecting group~s)
where exist(s~ ~rom the l-N-acylated product of the formula
(IV') or (IV") in a known manner to produce the compound
of the formula (IV).
The process of the fifth aspect of this invention
may include a further step of converting the compound ~VI)
as produced into a pharmaceutically acceptable acid-addition
salt thereof by reacting with a pharmaceutically acceptable
inorganic or organic acid in a known manner, if desired.
The process of the fifth aspect invention can be
; carried out in the same manner as described hereinbefore
for the process o~ the third aspect of this invention.
In carrying out the process of the ~ifth aspect
invention, it is feasible to employ as the starting com-
pound 5,3',4'-trideoxy-6'-N-methylkanamycln B of the formula
~ `,,'
~ . ~
, - ,,
~ 1~58~6
- 53 -
(V) of which amino groups are not protected at all, in the
form of the free base or in the form of an acid-addition
salt with an appropriate acid such as hydrochloric acid.
However, it is preferred to employ as the starting compound
such a partially amino-protected derivative of 5~3',4'-
trideoxy-6'-N-methylkanamycin B according to the formula
(V') in which all or some of the three amino groups and
methylamino group other than the l-amino group have been
protected with known amino-protecting groups and which may
be prepared by introduction of a known amino-protecting
group into the compound (V) as described detailedly in
respect to the process of the third aspect invention.
In the process of the fifth aspect invention, the
step of 1-N-acylating the starting compound (IV) or (IV')
; 15 and the step of deprotecting the l-N-acylated product (IV')
or (IV") as well as the step of purifying the deprotected
l-N~acylation product are worked out just as described
hereinbefore in respect of the corresponding steps of the
process of the third aspect invention.
The new compound of the formula (V), namely 5,3',4'-
tri-deoxy-6'-N-methylkanamycin B which is used as the
starting compound i.n the process of the aforesaid fifth
aspect invention may be prepared starting from a known
compound, 5,3',4'-trideoxykanamycin B (Japanese Journal of
Antibiotics, Vol. 32, S-178 (1979)) and conducting N-methyl-
ation at the 6'-amino group of said starting compound in
. ~, .
.
~7~6
- 54 -
the same manner as described in U.S. patent No. 3,925,353
or the "Journal of ~ntibiotics" 25, 743 (1972).
According to the sixth aspect of this invention,
5 there is provided a process for the production of 5,3',4'-
trideoxy-6'-N-methylkanamycin B represented by the formula
(V?
HO
HO ~ \ H2N
- H2N ~/~ ~ \~`
: : . OH I ¦ (V)
! ~o NHCH3
H2N ~NH2
.,
which comprises the steps of:-
(a) introducing an alkyloxycarbonyl group, a
cycloalkyloxycarbonyl group or an aralkyloxycarbonyl group
into the 6'-amino group of 5,3',4'-trideoxykanamycin B
: represented bg the formula ~X)
HO
~/ ~ H2N ~
H2N~ \~ (X)
OH I NH2
Io~
H2N ~ MH2
.
~ ~7~
to produce the compound of the formula (XI)
HO
HO ~ O H2
H2N~ r ( XI)
OH \ ~ O NH8R3
H2N~H2
wherein R3 is an alkyl group of 1 to 6 carbon atoms, a
cycloalkyl group of 3 to 6 car~on atoms or an aralkyl
group, particularly a phenyl-(Cl-C4)alkyl group, especially
benzyl, and
(b) reducing the compound of the formula (XI) with
a metal hydride in an anhydrous organic solvent to produce
the compound of the formula (V)
The procedures for carrying ouk the process of the
sixth aspect invention are now described.
In the first step of the present process, an
alkyloxycarbonyl group, a cycloalkyloxycarbonyl group or
aralkyloxycarbonyl group is introduced into the 6'-amino
group of the starting compound (X) and may be the one
which is usually known as the amino-protecting group of
urethane type. Introduction of the alkyloxycarbonyl,
cycloalkyloxycarbonyl or aralkyloxycarbonyl group (-COR3)
into the 6'-amino group of the starting 5,3',4'-trideoxy-
kanamycin ~ (X) is achieved in the same manner as described
'
,
~ ~7581B
- 56 --
hereinbefore for the preparation of the amino-protected
derivative of the starting material (VI') which is employed
in the process of the third aspect invention. The selective
protection of the 6'-amino group can be made because the
6'-amino group is most reactive amongst the amino groups
of the kanamycin B compound (X). The group (-C0~3) to be
., O
intorduced into the 6'-amino group of the starting compound
(X) may preferably be methoxycarbonyl group, ethoxycarbonyl
group or benzyloxycarbonyl group. In this way, the 6'-N-
alkyloxycarbonylated~ 6'-N-cycloalkyloxycarbonylated or
6'~N-aralkyloxycarbonylated product of the formula (XI)
is formed.
The 6' N-alkyloxycarbonyl group, 6'-N-cycloalkyloxy-
carbonyl group or 6'-N-aralkyloxycarbonyl group so intro-
duced is reductively converted into a methyl group to effect
the 6'-N-methylation. This can be achieved by reducing the
compound of the formula tXI) with a metal hydride such as
~; lithium aluminum hydride and diborane in an anhydrous
organic solvent such as tetrahydrofuran. This reduction
may usually be carried out at a temperature o~ 40-90C for
lO hours or longer.
This invention ls illustrated with reference to the
following Examples to which this invention is not limited.
Examples l to 3 are illustrative of the first and third
aspects of this invention, Example 4 illustrative of the
- ~i7~16
- 57 -
first and fourth aspects of this invention, Example 5
illustrative of the sixth aspect of this invention, and
Examples 6-8 illustrative of the fifth aspect of this
invention
Example l
Synthesis of l-N-[(S)-4-amino-2-hydroxybutyryl]-
5,3~,4'-trideoxykanamycin B
(a) 4.36 g (lO mmole) of 5,3',4'-trideoxykanamycin' B
was dissolved in 100 ml of dry dimethylsulfoxide, to which
was then added 10.5 g (48 mmole) of zinc acetate
[Zn(CH3C02)2 2H20]. The resultant mixture was agitated
at ambient temperature for 20 hours to form a complex of
5,3',4'-trideoxykanamycin B with zinc cation. After addi-
kion of 1?.~ g (39.5 mmole) of para-methoxybenzyl S-4,6-
dimethy~pr'yrimid-2-ylthiocarbonate (product of Kokusan
Kagaku X.K., Japan), the resultan~ admixture was agitated
at 50C for 7.5 hours to effect the N-p-methoxybenzyloxy-
.
carbonylation. The reaction solution obtained was then
poured into lO00 ml of water and adjusted to pH ll by
; 20 addition o~ aqueous ammonia to effect the breakdown of the
zinc complex, giving a precipitate. The precipitate was
filtered off, washed with 500 ml of water and dissolved
in 60 ml of a mixed æolvent of chloroform-methanol-17%
aqueous ammonia (50:10:1 by volume). The solution was
subjected to chromatography on a column of 500 g of silica
gel ~Wako-gel C-200~ developing with the same mixed sol~ent
~ I *trade mark
: .
,
~ 1758~
- 58 -
to give 4.1 g (L~4%) of a colorless powder of 3,2',6'-tri-
N-paramethoxybenzyloxycarbonyl-5,3',4'-trideoxykanamycin Bo
3.7 g (4 mmole) of the colorless powder was dissolved
in 50 ml of dimethylsulfoxide and 0.95 ml (8 mmole) of ethyl
trifluoroacetate was added to the solution. The resulting
mixture was stirred at room temperature for 5 hours to
effect the 3"-N-trifluoroacetylatoin and yield 3,2',6'-tri-
N-paramethoxybenzyloxycarbonyl-3"-N-trifluoroacetyl-5,3',4'-
trideoxykanamycin B.
(b) To the reaction solution containing the N-protected
kanamycin B derivative obtained in step (a) above were
added o.6 ml (4.4 mmole) of triethylamine, followed by a
solution of 1.9 g (6 mmole) of N-hydroxysuccinimide ester
of (S)~4-tert-butoxycarbonylamino-2-hydroxybutyric acid in
20 ml of dioxane. The mixture was agitated at room temper-
ature for 19 hours, after which the reaction solution was
poured into 500 ml of water to separate a precipitate. The
precipitate was filtered off and washed with 100 ml of
water to give 10.9 g of a colorless powder. The powder
was dissolved in 20 ml of 90% trifluoroacetic acid and the
solution was allowed to stand at ambient temperature for
45 minutes to perform removal of the amino-protectlng groups.
The solution was then concentrated to dryness and the residue
was taken up in 100 ml of water. The aqueous solution was
adjusted to pH 10.5 by addition of aqueous ammonia and
stirred at ambient temperature for 20 hours to permit
.~
'
.: ~
~ 17581
-- 59 ~
removal of the trifluoroacetyl group. The resultant
reaction solution was concentrated to a volume of about
20 ml, adjusted to pH 7.5 by addition o~ aqueous ammonia,
diluted with 50 ml of water and then passed through a
column of 150 ml of Amberlite CG-50 resin (NH4 form,
product of Rohm & Haas Co., U.S~A.). The column was
washed successively with 750 ml of water and with 500 ml
of 0.5 N aqueous ammonia and then eluted with 0.8 N aqueous
ammonia. ~he eluate fractions containing the desired
product were combined together and concentrated to dryness
to give 1.76 g (72%) of 1-N-~(S)-4-amino-2-hydroxybutyryl~-
5~3Y,4'-trideoxykanamycin B monocarbonate monohydrate as
a colorless powder. Overall yield 32%.
Example 2 -
Synthesis of l-N-(3-amino-2-hydroxypropionyl)-5,3'~4'-
trideoxykanamycin B
- .
(a) 435.5 mg ~1 mmole) of 5,3'~4'-tridecxykanamycin B
was dissolved in 10 ml of dry dimethylsulfoxide, to which
was then added 1.05 g (4.8 mmole) o~ zinc acetate
~Zn(CH3CO2)2 2H2O]. The resulting mixture was agitated
at room temperature ~or 23 hours. Thereafter, a solution
of 937.3 mg (3.9 mmole) of tert-butyl S-4,6-dimethylpyrimid-
2-ylthiocarbonate in 5 ml of dimethylsulfoxide was added
thereto and the resulting admixture was stirred at 50C
~or 24 hours to effect the N-tert-butoxycarbonylatlon.
The reaction solution obtained was admixed with 15 ml of
*trade mark
...... ,~, . . . . . .
l i75816
water~ adjusted to pH 11 with aqueous ammonia~ followed by
addition of 6.4 g of sodium chloride and extraction with
ethyl acetate (2 x 15 ml). The ethyl acetate extracts
were combined together and concentrated to dryness and the
residue was taken up in 6 ml of dimethylsulfoxide. The
solution was admixed with 0.125 ml (1 mmole) of ethyl tri-
fluoroacetate and the admixture was stirred at ambient
temperature for 4 hours to effect the 3"-N-trifluoroacety-
lation.
(b) To the reaction solution containing 3,2',6'-tri-N-
tert-butoxycarbonyl-3"-N-trifluoroacetyl-5,3',4'-trideoxy-
kanamycin B produced in step (a) above were added 0.1 ml
(0.7 mmole) of triethylamine and 384 mg (1.05 mmole) of
N-hydroxysuccinimide ester of paramethoxybenzyl.oxycarbonyl-
isoserine. The resultant mixture was agitated at ambient
temperature for 20 hours, after which the reaction solution
was admixed with 10 ml of water and extracted with ethyl
acetate (2 x 10 ml). The combined extracts were concen-
trated to a small volume, followed by addition of 20 ml
of 3N hydrochloric acid - 50% methanol. The mixture was
stirred at room temperature for 2 hours to perform
simultaneous removal of both the tert-butoxycarbonyl and
para-methoxybenzyloxycarbonyl groups as the amino-protective
~roups. The resulting reaction solution was adjusted to
pH 10 by addition of aqueous ammonia and then stirred at
ambient ternperature for 21 hours to permit removal of the
'
.
:,
~ ~ 7$~16
61 --
trifluoroacetyl group. The reaction solution so obtained
was evaporated to a small volume and diluted with water,
and the resulting aqueous solution was passed through a
column of 25 ml of Diaion WK-lOS (NHLi~ form, product of
Mitsubishi Kasei K.K., Japan). The column was washed with
125 ml of water and then eluted with 0.4 N aqueous ammonia.
The eluate fractions containing the desired product were
combined together and concentrated to dryness to give
257 mg of 1-N-(3-amino-2-hydroxypropionyl)-5,3',4'-
trideoxykanamycin B monocarbonate as a colorless powder.
Qverall yield 42%.
Example 3
Sy_thesis of l-N-[(S?-4-amino-2-hydroxybutyryl]-
5 ? 3 ~ ~ 4'?6"-tetradeo'xykanamycin B
(a) 419.5 mg (0.75 mmole) of 5,3',41,6"-tetradeoxy-
kanamycin B dicarbonate monohydrate was dissoIved in 10 ml
of dry dimethylsul~oxide, to which was then added 1.05 g
(4. 8 mmole~ of zinc acetate ~Zn(CH3C02)2 2H20~ and the
mixture was agitated at room temperature for 18 hours.
Thereafter, a solution of' 1.44 g (6.0 mmole) of tert-butyl
S-4,6-dimethylpyrimid-2-ylthiocarbonate' in 5 ml of'
dimethylsulf`oxide was added thereto and the resulting
admixture was stirred at 50C for 25 hours. The reaction
solution obtained was admixed with 15 ml of water, adjusted
to pH 11 with aqueous ammonia and extracted with ethyl
acetate (2 x 15 ml). ~he aqueous layer was admixed with
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~ ~7~6
- 62 -
6.4 g of sodium chloride and further extracted with 20 ml
of ethyl acetateO The whole ethyl acetate extracts were
combined together and concentrated to dryness and the
residue was taken up in 6 ml of dimethylsulfoxide. The
solution was admixed with 0.125 ml (1 mmole) of ethyl
trifluoroacetate and the admixture was stirred at ambient
temperature for 4 hours.
(b) To the reaction solution containing 3,2',6'-tri-N-
tert-butoxycarbonyl-3"-N-tri~luoroacetyl-5,3',4',6"-tetra-
deoxykanamycin B produced in step (a) above were added0.1 ml (0.7 mmole) of triethylamine and 399.4 mg (1.05~
mmole) of N-hydroxysuccinimide ester of (S)-4-p-methoxy-
benzyloxycarbonylamino-2-hydroxybutyric acid. The resultant
mixture ~as agitated at ambient temperature for 18 hours,
after whlch the reaction solution was admixed with 10 ml
of water and extracted with ethyl acetate ~2 x 10 ml).
The combined extracts were concentrated to a small volume,
followed by addition of 20 ml of 3 N hydrochloric acid -
50% methanol. The mixture was stirred at room temperature
for 2 hours to perform removal of both the tert-butoxy-
carbonyl and para-methoxybenzyloxycarbonyl groups. The
resulting reaction solution was ad~usted to pH 10 by
addition of aqueous ammonia and then stirred at ambient
temperature for 20 hours to permit removal of the tri-
fluoroacetyl group. The reactlon solution so obtained wasevaporated to a small volume and diluted with water, and
. .~
~ ~7~8~1
- 63 ~
the resulting aqueous solution was passed through a column
of 25 ml of Diaion WK-10S (NH4 form). The column was washed
with 125 ml of water and then eluted with 0.32 N aqueous
ammonia. The eluate fractions containing the desired
product were combined together and concentrated to dryness
to give 303 rng of 1-N-[(S)-4-amino-2-hydroxybutyryl)-
5,3',4',6"-tetradeoxykanamycin B dicarbonate as a colorless
powder. Overall yield 63%.
Example 4
Synthesis of 5~3',4',6"-tetradeoxykanamycin B
(a) Preparation of 1,3,2',6',3"-penta-N-ethoxycarbonyl-
; 3~,41-dideoxykanamycin B
4.64 g (10.3 mmole) of 3~,4'-dideoxykanamycin B was
~ disolved in a mixture-of 45 ml. of water and 45 ml of
; 15 methanol, to which was then added 8 g (95.2 mmole) of
sodium bicarbonate, followed by slow addition of 7.2 ml
(75.6 mmole) of ethyl chloroformate ur,der ice-cooline.
The resultant mixture was stirred at room temperatu:re for
18 hours to effect the N-ethoxycarbonylation. The reaction
solution obtained was admixed with 500 ml of water to
æeparate a preoipitate. rrhe latter was filtered off and
washed with water to af~ord 6.49 g (78%) of a colorless
powder of the title compound.
tb) Preparation of 1,3,2',6',3"-penta-N-ethoxycarbonyl-
41l,6l'-0-isopropylidene-3',4~-dideoxykanamycin B
4.83 g (5.95 mmole) of the product obtained in step
~
! ;; *trade mark
"``" 5 ~75~1~
- 64 - ~
(a) above was dissolved in 50 ml of dimethylformamlde, and
to the resulting solution were added 30 mg (0.16 mmole)~of
; p-toluenesulfonic acid and 1.1 ml (9.0 mmole~) of~2,2-
dimethoxypropane. The resulting mixture was agitated at
:
ambient temperature for 25 hours (to effect the 4!l,6ll-o- ;
isopropylidenation), after which the reaction solut-ion~was~
admixed with 1 ml~(7~.~2~ mmole) of triethylamine and then
concentrated to dryness to give~5.1 g (100%) of the title
compound as a pale yellow~powder.
10~ ~ (c)~ Preparatlon of~l,3,2',;6'~,3"-penta-N-ethoxYcarbonyl~
4",6"-0-1sopropylidene-2"-0-benzoyl-3~ 4'-dideoxy~
kanamycin~B~
; 5.1 g (6~.0~mmole) of the~product from step~(b) above
was~dissolved ln~75~ml~o~pyridine,~to~which was~then~added~
15~ ;ml (8.6 mmole);~of benzoyl~ohloride, and~the mlxture~was~
agitated~at~r~oom témp~erature~f~or~5 hours to ef~ect~the~
2"-0-benzoyl`ation. The~react~ion~solution;so obtained~was
;admixed with~10 ~ of~water, stirred;~at~room temp~erature~
;and ~then concentrated~to dryne~ss.~ The residue w~s~takén
2n~ up~ in 250~ml;or c~loroform~and~the~s~lution~was~washed~
successively with`lOO ml~of 0.2 N;hydrochloric acld~and
2 x 100 ml of~sat~urated aqueous~sodium bioarbonate~solutlon~
The chlorofo~r~ lay~èr~was sep~arated,;dried over anhy;droua
sodium sulfate and~concentratéd to dryness to ~ive 5.7 g
(99%) o~ the title compound;~as a pale yellow powder.
':: : `
~ ~758~6
- 65 -
(d) Preparation of 1,3,2 t ~ 6',3" penta-N-ethoxycarbonyl-
2"-0-benzoyl-3',4l-dideoxykanamycin B
5.3 g (5.5 mmole) of the product from step (c) above
was dissolved in 80 ml of a mixture of acetic acid-methanol-
water (2:1:1 by volume) and the resultant solution was
. allowed to stand at ambient temperature for 21 hours and
then concentrated to dryness to yield 4.3 g (97%) of the
title compound as a pale yellow powder.
: (e) Preparation o~ 113,2',6',3~'-penta-N-ethoxycarbonyl-
.
~ 2"-0-be~zoyl-6"-0-tosyl-3',4'-dideoxykanamycin B
. 1.5 g (1.63 mmole) of the product obtained in step
.
. . (d~ above was dissolved in 28 ml of pyridine, to which was
~: then added 374 mg (1.96 mmole) Or paratoluenesulfonyl
- . chloride~ and the mlxture was agitated at room temperature
for 28 hours to effect the 6"-0-tosylation. The resulting
reaction solution was concentrated to dryness, the powdery
residue (2.3 g) was taken up in 12.5 ml of dichlorornethane
and the solution obtained was subjected.to column chromato-
graphy on silica gel (Wako-gel C-200, 200 g). The column
was washed with 1000 ml of dichloromethane-ethanol (60:1)
and then developed with dichloromethane-ethanol (50:1) to
give 1.0 g (60%) of a colorless powder of the title
compound.
(f) Preparation of 1,3,2',6',3"-penta-~-ethoxycarbonyl-
2"--0-benzoyl-6"-iodo-3',4 ~ -dideoxykanamycin B
900 mg (0.84 mmole) of the product from step (e)
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~,
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~ 175~1
-- 66 --
above was dissolved in 18 ml of dimethylformamide, followed
by addition of 12.6 g ~84 mmole) of sodium iodide and
agitation at 95C for two hours to effect the 6~'-iodination.
The reaction solution obtained was poured into 250 ml of
water, and the precipitate deposited was filtered off and
then dissolved in 100 ml of chloroform. The chloroform
; solution was washed with 100 ml of 20% aqueous sodium
thiosulfate and with 100 ml of saturated aqueous sodium
chloride. m e chloroform layer separaked was dried over
anhydrous sodium sulfate and concentrated to dryness ko
- give 817 mg (95%) of khe title compound as a colorless
powder.
(g) Preparation of 1~3,21,6',3'r-penta-N-ethoxycarbonyl-
?"-0-benzoyl-3',4' 9 61'-trideoxykanamycin B
773 mg (0.75 mmole) of the product from step (f)
above was dissolved in 20 ml of dioxane, to which was then
added a small amount of Raney Nickel W-2, and the mixture
was hydrogenated under a hydrogen pressure of 3.6 kg/cm2
~or 23.5 hours using a Parr apparatus. The resultant
reaction solution was filtered to remove the catalyst and
then concentrated to dryness to af~ord 655 mg (97%) of a
colorless powder o~ khe title compound.
th) Preparation o~ 1,3,2',6'~3"-penta-N-ethoxycarbonyl-
21',4'l-dl-0-benzoyl-3',4',6'--trideoxykanamycin B
622 mg ( a . 69 mmole) of the product obtained in step
(g) above was dissolved in 30 ml of pyridine, followed by
' - *trade mark
I 175~s~6
- 67 -
addition of 0.1 ml (o.86 mmole) of benzoyl chloride and
agitation of the mixture at room temperature for 25 hours
to effec-t the 4"-O-benzoylation. The resultant reaction
solution was concentrated to dryness and the residue was
taken up in 50 ml of chloroform. The solution was washed
successively with 10% aqueous potassium bisulfate (2 x
30 ml), saturated aqueous sodium bicarbonate (2 x 30 ml)
and saturated aqueous sodium chloride (l x 30 ml), then
; dried over anhydrous sodium sulfate and concentrated to
dryness to give 663 mg (96%) of the title compound as a
colorless powder.
(i) Preparation of 1,3,2',6',3l'-penta-N-ethoxycarbonyl-
2",4"-di-0-benzoyl-5-chloro-3',4',6"-trideoxy-
: kanamycin B
600 rng (0.6 mmole) of the product from step (h)
above was dissolved in 12 ml of pyridine, to which was
then added 0.1 ml (1.24 mmole) of sulfuryl chloride under
ice-cooling, and the mixture was stirred at ambient temper-
ature for 18 hours to effect the 5-chlorination. The
reaction solution thus obtained was admixed with lO0 ml
of water and extracted with 60 ml of chloroform. The
chloroform extract was washed successively with 60 ml of
10% aqueous potassium bisulfate, 60 ml of saturated aqueous
sodium bicarbonate and60 ml of saturated aqueous sodium
chloride, then dried over anhydrous sodlum sulfate and
concentrated to dryness. The resultant powdery resldue
(572 mg) was taken up in 5 ml of dichloromethane and
8 1
-- 68 --
chromatographed on a column of silica gel (Wako-gel C-200,
50 g). The column was washed with 500 rnl of dichloromethane
and developed with a mixture of dichloromethane-ethanol
~100:1~ to give 356 mg (58%) of a colorless powder of the
title compound.
(;) Preparation of ls3,2',6',3''-penta-N-ethoxycarbonyl-
2",4"-di-0-benzoyl-5,3',4',6~'-tetradeoxykanamycin B
308 mg (0.13 mmole) of the product from step (i)
above was dissolved in 5 ml of dioxane, to which was then
added a small amount of Raney Nickel W-2, and the mixture
was hydrogenated under a hydrogen pressure of 3.6
; kg/cm2 for 23.5 hours using a Parr apparatus to effect the
dechlorination. The resultant reaction solution was
filtered to remove the catalyst and then concentrated to
.
~ 15 dryness to afford-295 mg (100%) of a colorless powder of
~:
the title compound.
k) Preparation of 5,3',4',6"-tetradeoxykanamycin B
120 mg (0.12 mmole) of the product obtained in step
(j~ above was dissolved in a mixture o~ 1 ml of dioxane and
1 ml of water, to which was then added 400 mg (1.27 mmole)
of barium hydroxide [Ba(OH2) 8H20], and the resultant
mixture was subjected tothe deprotection by heating at
110C for 15 hours under re~lux. Thereafter, gaseous
carbon dioxide was passed into the reaction solution to
precipitate barium carbonate, which was filtered off. The
filtrate was admixed with 30 ml of water and passed through
*trade mark
:
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, . .: ~ .
~ ~7~i816
- 69 -
a column of 20 ml of Amberlite CG-50 (NH4 form, product
of Rohm & Haas Co., U.S.A.). The column was washed with
100 ml of water and 75 ml of 0.1 N aqueous ammonia and
then eluted with 0.3 N aqueous ammonia.- The eluate
fractions containing the desired product were combined
together and concentrated to dryness to give 19.5 mg (29%)
o~ 5,3',4l,6~'-tetradeoxykanamycin B dicarbonate monohydrate
as a colorless powder.
Example 5
Synthesis o~ 5,3',4'-trideoxy-6'-N-methylkanamycin B
~a)- 4.0 g (9.18 mmole) of 5,3',4'-trideoxykanamycin B
was dissolved in a mixture of 40 ml of water and 40 ml of
methanol, to which was then added a solution of 5.8 ml
~9.18 mmole) o~ triethylamine and 2.72 g (11.02 mmole) of
2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile
(commercially available under the trade-mark BOC-ON from
Aldrich Co., U.S.A.) in 40 ml of methanol. The resultant
mixture was agitated at room temperature for 3 hours, after
which the reaction solution containing 6'-N-tert-butoxy-
carbonyl-5,3',4'-trideoxykanamycin B formed was admixed
with 2 ml o~ 17% aqueous ammonia and concentrated to
dryness. The residue was taken up ln 100 ml of water and
the solution was ad~usted to pH 7.4 with 6 N hydrochloric
acid, washed with 50 ml of ethyl ether and then passed
through a column (20 mm in inner diameter) of 100 ml of
Amberlite CG-50 (NH4 form, product of Rohm & Haas Co~,
*trade mark
.
~ 1'7~16
- 70 -
U.S.A.). The column was washed with 300 ml of water and
150 ml o~ 0.1 M aqueous ammonia and then eluted with 0.2 M
aqueous ammonia. The eluate was collected in 5 ml-fractions
~ and the fractions No. 11-24 were combined together and; 5 concentrated to dryness to give 2.1 g (42.5%) of 6'-N-tert-
butoxycarbonyl-5,3'~4'-trideoxykanamycin B. Concentration
to dryness of the fractions No. 26-33 combined recovered
1.42 g (35.6%) of unreacted 5~3',4t-trideoxykanamycin B.
(b) 1.86 ~ (3.45 mmole) of the 6'-N-tert-butoxycarbonyl-
553',4t-trideoxykanamycin B obtained just above was
dissolved in 60 ml of dry tetrahydrofuran, followed by
addit~on of 1.31 g (34.5 mmole) of lithium aluminium
hydride and heating at 80C for 20 hours under reflux.
The resulting reaction mixture was added dropwise into
200 ml of water to separate a precipitate, which was
filtered off. The filtrate was evaporated to r-emove the~
tetrahydrofuran and then adjusted to pH 6.5 by addition of
hydrochloric acid. The solution so obtained was passed
through a column (12 mm in inner diameter) Or 30 ml of
Amberlite CG-50 (NH4 ) and the column was washed with
150 ml of water and eluted successively with Q.2 M a~ueous
ammonia (150 ml), 0.3 M aqueous ammonia (150 ml) and 0.4 M
aqueous ammonia. The eluate was collected in 6 ml-fractions
and the ~ractions No. 11-17 were combined together and
concentrated to dryness to recover 740 mg (3g.9%) of
unreacted 6'-N-tert-butoxycarbonyl-5,3',4'-trideoxykanamycin
;~i *trade mark
, ., "~,s~ I .
.
~ ~75~16
- 71 -
B. ~hile~ the ~ractions No. 37-55 were likewise concen-
trated to dryness to a~ford 592 mg (32.4%) o~ the title
compound 5,3',4'-trideoxy-6'-N-methylkanamycin B.
Example 6
Synthesis of l-N-~(S)-4-amino-2-hydroxybutyryl]-5,3'~4'-
trideox~-6'-N-methylkanamycin B
583 mg (1.10 mmole) of the 5~3',4l-trideoxy-6 t -N-
methylkanamycln B prepared as described in ~xample 5 was
dissolved in lQ ml o~ 90% aqueous dimethylsul~oxide, to
which was then added 1.16 g (5.28 mmole) o~ ~inc acetate
~Zn(CH3CO2)2 2H2O]. The mixture was stirred at ambient
temperature ~or 20 hours, followed by addition of a solution
of 1.06 g (4.29 mmole) of 2-(tert~butoxycarbonyloxyimino)-
2-phenylacetonitrile in 5 ml of dimethylsulfoxide and
~urther agitation at 50C for 6 hours to effect the N tert-
butoxycarbonylation. The resulting reaction solution was
admixed with 2 ml of 17% aqueous ammonia and then with
100 ml o~ water and washed with 50 ml of ethyl ether. The
aqueous layer separated was adjusted to pH 11 with 17%
aqueous ammonia and admixed with 2 g o~ sodium chloride,
and the resulting solution was extracted with ethyl acetate
(3 x 100 ml). The ethyl acetate extracts combined were
dried over anhydrous sodium sulfate and concentrated to
dryness. The residue was chromatographed on a column (20
mm in inner diameter) of 50 g of silica gel (Wako-gel C-200
manu~actured by Wako Junyaku K.K., Japan) using as the
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1 6
- 72 -
eluent a mixture of chloroform-methanol-conc. aqueous
ammonia (30:10:1 by volume). The eluate was collected in
10 ml-fractions and the fractions No. 25-50 were combined
together and concentrated ~o dryness to give 607 mg (73.6%)
of 3,2l,6'-tri-N-tert-butoxycarbonyl-5,3',4'-trideoxy-6'-
N-methylkanamycin B.
590 mg (0.787 mmole) of the product obtained just
above was dissolved in 10 ml of dimethylsulfoxide, and to
the solution obtained was added 0.14 ml (1.18 mmole) of
ethyl trifluoroacetate. The admixture was stirred at room
temperature for 1.5 hours to effect the 3'~-N-trifluoro-
acetylation. The resultant reaction solution containing
3,2',6'-tri-N~tert-butoxycarbonyl-3"-N-trifluoroacetyl-
5,3',4'-trideoxy-6'-N-methylkanamycin B produced was
admixed with both 0.16 ml (1.18 mmole) of triethylamine
and 420 mg (1.18 mmole) of N-hydroxysuccinimide ester o~
(S)-4-p-methoxybenzyloxycarbonylamino-2-hydroxybutyric
acid dissolved in 4 ml of tetrahydrofuran and the admixture
was agitated at room temperature for 4 hours to effect the
l-N-acylation. Subsequently, 100 ml of water was added to
the reaction solution, which was then extracted with ethyl
acetate (2 x 100 ml). The extracts combined were dried
over anhydrous sodium sulfate and concentrated to dryness
to afford the l-N-acylated product in the form of the
amino-protected derivative thereof.
This 1-N-aoylated product so obtained was dissolved
. .,
~ ~7581
-- 73 --
in 10 ml of 90% aqueous trifluoroacetic acid and the
solution was agitated at ambien~ temperature for 5 hours
to permit the removal of the tert-butoxycarbonyl and p-
methoxybenzyloxycarbonyl groups and then the reaction
solutîon was concentrated to dryness. The residue was
taken up in 30 ml of water and the solution was adjusted
to pH 10 with 17% aqueous ammonia, followed by stirring
at room temperature for 18 hours to permit the removal of
the trifluoroacetyl group. The reaction solution thus
obtained was adJusted to pH 7.5 by addition of 6 N
hydrochloric acid and passed through a column (13 mm in
~nner diameter) of 20 ml of Amberlite CG-50 (NH4 form).
The column was washed with 100 ml of water and eluted
successively with 160 ml of 0.5 M aqueous ammonia and
100 ml of 0.8 M aqueous ammonia. The eluate was collected
in 4 ml-fractions and the fractions No. 2i-62 were combined
together and concentrated to dryness to yield 346 rn~ (71.8%)
o~ the titled compound, l-N-[(S)-4-amino-2-hydroxybutyryl]-
5,3',4'-trideoxy-6'-N-methylkanamycin B in the form of its
monocarbonate.
Synthesis of l-N-~3-amino-2-hydroxypropiony~-5~3l~4
trideoxy-6'-N~methylkanamycin B
102 mg (0.136 mmole) of the 3,2'~6'-tri-N-tert-
butoxycarbonyl-5,3',4'-trideoxy-6'-N-methylkanamycin B
prepared as in Example 6 was dissolved in 3 ml of
.
*trade mark
I ~75
-- 74 --
dimethylsulfoxide, and to the resultant solution was added
0.02 ml (0.204 mmole) of ethyl trifluoroacetate. The
mixture was stirred at room temperature for one hour. The
resultan-t reaction solution containing 3,2',6'-tri-N-tert-
butoxycarbonyl-3"-N-trifluoroacetyl-5,3',4'-trideoxy-6'-N-
methylkanamycin B produced was admixed with both 0.03 ml
(0.204 rnmole) of triethylamine and 75 mg (0.204 mmole) of
N-hydroxysuccinimide ester of 3-p-methoxybenzyloxycarbonyl-
amino-2-hydroxypropionic acid dissolved in 0.5 ml of tetra-
hydrofuran, and the admixture was agitated at roomtemperature for 7 hours to effect the l-N-acylation.
Subsequently, 10 ml of water was added to the reaction
solution, which was then extracted with ethyl acetate
(2 x 10 ml). The extracts combined were dried over
anhydrous sodium sulfate and concentrated to dryness to
afford 136 mg of the l-N-acylated product in the form of
the amino-protected derivative thereof.
This l-N-acylated product was dissolved in 3 ml of
90% aqueous trifluoroacetic acid and the solution was
agitated at ambient temperature for 2 hours to permit the
removal of the tert--butoxycarbonyl and ~-methoxybenzyloxy-
carbonyl groups, and then the reaction solution was con-
centrated to dryness. ~he residue was taken up in 10 ml
of water and the solution was ad~usted to pH 10 with 17~
aqueous ammonia, ~ollowed by stirring at roorn temperature
for 16 hours to permit the removal of the tri~luoroacetyl
~ 1i7$8~6
-- 75 --
group. The reaction solution thus obtained was ad~usted
to pH 6.4 by addition of 6 N hydrochloric acid and passed
through a column (11 mm in înner diameter) of 10 ml of
Amberlite CG-50 (NH4+ form). The column was washed with
30 ml o~ water and 30 ml of 0.2 M aqueous ammonia and then
eluted with 0.5 M aqueous ammonia. The eluate was collected
in 1 ml fractions and the fractiorls No. 4-7 were combined
together and concentrated to dryness to yield 3801 mg (45.4%)
of the desired l-N-[3-amino-2-hydroxypropionyl]-5,3~94'-
trideoxy-6'-N-methylkanamycin B in the form of its mono-
carbonat e .
Example 8
Synthesis of l-N-[(S)-5-amino-?-hydroxyvaleryi]=5~3',4
trideoxy-6'-N-methylkanamycin B
. ,
The same procedures as described in Example 7 were
- repeated but using 76 mg (0.204 mmolej of N-hydroxysuccin-
imide ester of ~S)-5-p-methoxybenzyloxycarbonylamino-2-
: hydroxyvaleric acid in place of the 3-_-methoxybenæyloxy-
carbonylamino-2-hydroxy-propionic acid N-hydroxysuccinimide
ester to be reacted with the 3,2',6'-tri-N-tert-butoxy-
carbonyl-3l'-N-trifluoroacetyl-5,3',4'-trideoxy-6'-N-
methylkanamycin B.
After the elution of the Amberlite CG-50 column, the
eluate .~ractions No. 11-18 were combined together and
concentrated to dryness to give 47.2 mg (53.8%) of the
title compound (rnonocarbonate).
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