LA 1N-(.alpha.-HYDROXY-.omega.-AMINOALKANOY1)-6'N-METHYL-3',4'-DIDESOXYKANAMYCINE ET SA FABRICATION

1N-(.alpha.-HYDROXY-.omega.-AMINOALKANOYL)-6'N-METHYL-3',4'-DIDEOXYKANAMYCIN B AND THE PRODUCTION THEREOF

Abrégé anglais

1N-(.alpha.-Hydroxy-.omega.-aminoalkanoyl)-6'N-methyl-3',4'-
dideoxykanamycin B and the production thereof
Abstract of the Disclosure
A number of 1N-(.alpha.-hydroxy-.omega.-aminoalkanoyl)-6'N-
methyl-3',4'-dideoxykanamycin B derivatives have been
found to possess excellent antibacterial activity
against most kanamycin susceptible and resistant
organisms. In particular, 1N-(DL-isoseryl)-6'N-
methyl-3',4'-dideoxykanamycin B, 1N-(L-isoseryl)-
6'N-methyl-3',4'-dideoxykanamycin B, 1N-(L-4-amino-2-
hydroxybutyryl)-6'N-methyl-3',4'-dideoxykanamycin B
and 1N-(L-5-amino-2-hydroxyvaleryl)-6'N-methyl-3'-4'-
dideoxykanamycin B, or an acid addition salt thereof
possess these highly desirable attributes.
-1-

Revendications

The embodiments of the invention in which an exclusive property
or privilege is claimed are defined as follows:
1. A process for the production of a 1N-(.alpha.-hydroxy-.omega.-amino-
alkanoyl)-6'N-methyl-3',4'-dideoxykanamycin B of the formula:
<IMG>
wherein n is 1, 2 or 3; which comprises removing the amino-protecting
groups from a 1-N-acylated derivative of 6'N-methyl-3',4'-dideoxykanamycin
B having the formula
<IMG>
wherein R1 is a known mono-valent amino-protecting group and R2 is a
hydrogen atom or a known mono-valent amino-protecting group, R3 is a
-46-

known mono-valent amino-protecting group, R4 is a hydrogen atom, or R3
and R4 taken together form a known di-valent amino-protecting group, and
n is an integer of 1, 2 or 3.
2. The process according to claim 1 in which in the 1-N-acylated
derivative the 1-N side chain is DL-isoseryl group.
3. The process according to claim 1 in which in the 1-N-acylated
derivative the 1-N side chain is L-isoseryl group.
4. The process according to claim 1 in which in the 1-N-acylated
derivative the 1-N side chain is L-4-amino-2-hydroxybutyryl group.
5. The process according to claim 1 in which in the 1-N-acylated
derivative the 1-N side chain is L-5-amino-2-hydroxyvaleryl group.
6. The process according to claim 1, 2 or 3 in which in the
1-N-acylated derivative the amino-protecting groups R1, R2, R3 and R4 are
selected from the groups of a formula -CO-OR6 in which R6 is an alkyl group
of 1-5 carbon atoms, a cycloalkyl group of 3-6 carbon atoms, an aralkyl
group of 1-4 carbon atoms, an aryl group or a heterocyclic group.
7. The process according to claim 1, 2 or 3 in which in the
1-N-acylated derivative the amino-protecting groups R1 and R2 are selected
from the groups of a formula -CO-OR6 in which R6 is an alkyl group of 1-5
carbon atoms, a cycloalkyl group of 3-6 carbon atoms, an aralkyl group of
1-4 carbon atoms an aryl group or a heterocyclic group; and R3 and R4
are, taken together, a di-valent amino-protecting group selected from an
alkylidene or arylidene group of the formula =CHR5 in which R5 is an alkyl
group of 1-6 carbon atoms or an aryl group.
8. The process according to claim 1, 2 or 3 wherein the amino-
protecting groups R1, R2, R3 and R4 are t-butoxycarbonyl group or benzyl-
oxycarbonyl group.
9. The process according to claim 1, 2 or 3 wherein the amino-
protecting groups are an alkyloxycarbonyl group, a cycloalkyloxycarbonyl
group, aryloxycarboxyl group, alkylidene or arylidene group, and the amino-
protecting groups are removed by subjecting the 1-N-acylated derivatives to
a mild hydrolysis treatment with an acid.
-47-

10. The process according to claim 1, 2 or 3 wherein the
amino-protecting groups are an aralkyloxycarbonyl, and the amino-
protecting groups are removed by subjecting the 1-N-acylated derivative
to a hydrogenolysis treatment in the presence of a palladium-carbon or
platinum black catalyst, or by subjecting the 1-N-acylated derivative to a
treatment with hydrogen bromide in acetic acid at low temperature.
11. The process according to claim 1, 2 or 3 wherein the
amino-protecting groups are o-nitrophenoxyacetyl groups which are removed
by a reductive treatment.
12. The process according to claim 1, 2 or 3 wherein the
amino-protecting groups are phthaloyl groups which are removed by treating
the 1-N-acyrlated derivative with hydrazine hydrate in ethanol.
13. The process according to claim 1 wherein the process
further comprises forming a pharmaceutically acceptable, nontoxic acid
addition salt of the 1N-(.alpha.-hydroxy-.omega.-aminoalkanoyl)-6'N-methyl-3',4'-
dideoxykanamycin B.
14. The process according to claim 13 wherein the 1N-(.alpha.-
hydroxy-.omega.-aminoalkanoyl)-6'N-methyl-3',4'-dideoxykanamycin B is reacted
with 1 to 5 moles of a nontoxic, pharmaceutically acceptable acid to form
a mono, di-, tri-, tetra- or penta-salt thereof.
15. The process according to claim 14 wherein the acid is
selected from the group consisting of hydrochloric, hydrobromic, sulfuric,
phosphoric, nitric, carbonic, acetic, maleic, fumaric, succinic, tartaric,
oxalic, methanesulfonic, ethanesulfonic, and ascorbic acids.
16. A process for the production of a 1N-(.alpha.-hydroxy-.omega.-amino-
alkanoyl)-6'N-methyl-3',4'-dideoxykanamycin B of the formula:
-48-

<IMG>
wherein n is 1, 2 or 3, which comprises selectively acylating the 1-amino
group of an amino-protected derivative of 6'N-methyl-3',4'-dideoxykanamycin
B represented by the formula
<IMG>
wherein R1 is a known mono-valent amino-protecting group and R2 is a
hydrogen atom or a known mono-valent amino-protecting group, with an
.alpha.-hydroxy-.omega.-amino acid of the formula
<IMG>
-49-

or its functional equivalent as an acylating agent wherein R3 is a known mono-
valent amino-protecting group and R4 is a hydrogen atom, or R3 and R4
taken together form a known di-valent amino-protecting group, and n is
an integer of 1, 2 or 3, to produce a 1-N-acylated derivative of the formula
<IMG>
wherein R1, R2, R3, R4 and n are as defined above, and then removing the
amino-protecting groups from the 1-N-acylated derivative to produce the
desired compound.
17. A process according to claim 16 in which the amino-
protected derivative of 6'N-methyl 3',4'-dideoxykanamycin B to be acylated
is 2'N,6'-N-di-t-butoxycarbonl-6' N-methyl-3',4'-dideoxykanamycin B or
6'N-t-butoxycarbonyl-6' N-methyl-3',4'-dideoxykanamycin B or a mixture
of them.
18. A process according to claim 16 or 17, in which the
functional equivalent of the .alpha.-hydroxy-.omega.-amino acid is its acid chloride,
mixed acid anhydride, active ester or azide thereof.
19. A process according to claim 16 or 17 in which the
.alpha.-hydroxy-.omega.-amino acid is employed in the form of its N-hydroxysuccinimide
ester.
-50-

20. The process according to claim 16 wherein the process
further comprises forming a pharmaceutically acceptable, nontoxic acid
addition salt of the 1N-(.alpha.-hydroxy-.omega.-aminoalkanoyl)-6'N-methyl-3',4'-
dideoxykanamycin B.
21. The process according to claim 20 wherein the 1N-(.alpha.-
hydroxy-.omega.-aminoalkanoyl)-6'N-methyl-3',4'-dideoxykanamycin B is reacted
with 1 to 5 moles af a nontoxic, pharmaceutically acceptable acid to form
a mono-, di-, tri-, tetra- or penta-salt thereof.
22. The process according to claim 21 wherein the acid is
selected from the group consisting of hydrochloric, hydrobromic, sulfuric,
phosphoric, nitric, carbonic, acetic, maleic, fumaric, succinic, tartaric,
oxalic, methanesulfonic, ethanesulfonic, and ascorbic acids.
23. A process according to claim 17 for the production of
1N-(DL-isoseryl)-6'N-methyl-3',4'-dideoxykanamycin B which comprises
selectively acylating the 1-amino group of an amino-protected derivative
of 6'N-methyl-3',4'-dideoxykanamycin B represented by the formula
<IMG>
wherein R1 is a known mono-valent amino-protecting group and R2 is a
hydrogen atom or a known mono-valent amino-protecting group, with a
DL-.alpha.-hydroxy-.omega.-amino acid of the formula
-51-

<IMG>
or its functional equivalent as an acylating agent wherein R3 is a known
mono-valent amino-protecting group and R4 is a hydrogen atom, or R3 and
R4 taken together form a known di-valent amino-protecting group, and n
is one, to produce a 1-N-acylated derivative of the formula
<IMG>
wherein R1, R2, R3, R4 and n are as defined above, and then removing the
amino-protecting groups from the 1-N-acylated derivative to produce the
desired compound.
24. A process according to claim 23 in which the amino-
protected derivative of 6'N-methyl-3',4'-dideoxykanamycin B to be acylated
is 2'N,6'-N-di-t-butoxycarbonyl-6'N-methyl-3',4'-dideoxykanamycin B
or 6'N-t-butoxycarbonyl-6'N-methyl-3',4'-dideoxykanamycin B or a
mixture of them.
25. A process according to claim 23 or 24 in which the
functional equivalent of the .alpha.-hydroxy-.omega.-amino acid is its acid chloride,
mixed acid anhydride, active ester or azide thereof.
-52-

26. A process according to claim 23 or 24 in which the .alpha.-
hydroxy-.omega.-amino acid is employed in the form of its N-hydroxysuccinimide
ester.
27. A process according to claim 17 for the production of IN-
(L-isoseryl)-6'N-methyl-3',4'-dideoxykanamycin B which comprises
selectively acylating the 1-amino group of an amino-protected derivative
of 6'N-methyl-3',4'-dideoxykanamycin B represented by the formula
<IMG>
wherein R1 is a known mono-valent amino-protecting group and R2 is a
hydrogen atom or a known mono-valent amino-protecting group, with an
L-.alpha.-hydroxy-.omega.-amino acid of the formula
<IMG>
or its functional equivalent as an acylating agent wherein R3 is a known
mono-valent amino-protecting group and R4 is a hydrogen atom, or R3 and
R4 taken together form a known divalent amino-protecting group,and n is
one, to produce a 1-N-acylated derivative of the formula
-53-

<IMG>
wherein R1, R2, R3, R4 and n are as defined above, and then removing the
amino-protecting groups from the 1-N-acylated derivative to produce the
desired compound.
28. A process according to claim 27 in which the amino-
protected derivative of 6'N-methyl-3',4'-dideoxykanamycin B to be acylated
is 2'N,6'-N-di-t-butoxycarbonyl-6'N-methyl-3',4'-dideoxykanamycin B or
6'N-t-butoxycarbonyl-6'N-methyl-3',4'-dideoxykanamycin B or a mixture
of them.
29. A process according to claim 27 or 28, in which the
functional equivalent of the .alpha.-hydroxy-.omega.-amino acid is its acid chloride,
mixed acid anhydride, active ester or azide thereof.
30. A process according to claim 27 or 28 in which the .alpha.-
hydroxy-.omega.-amino acid is employed in the form of its N-hydroxysuccinimide
ester.
31. A process according to claim 17 for the production of
IN-(L-4-amino-2-hydroxybutyryl)-6'N-methyl-3',4'-dideoxykanamycin B
which comprises selectively acylating the 1-amino group of an amino-
protected derivative of 6'N-methyl-3',4'-dideoacykanamycin B represented
by the formula
-54-

<IMG>
wherein R1 is a known mono-valent amino-protecting group and R2 is a
hydrogen atom or a known mono-valent amino-protecting group, with an
L-?-hydroxy-.omega.-amino acid of the formula
<IMG>
or its functional equivalent as an acylating agent wherein R3 is a known
mono-valent amino-protecting group and R4 is a hydrogen atom, or R3 and
R4 taken together form a known di-valent amino-protecting group, and n is
two, to produce a l-N-acylated derivative of the formula
-55-

<IMG>
wherein R1, R2, R3, R4 and n are as defined above, and then removing the
amino-protecting groups from the l-N-acylated derivative to produce the
desired compound.
32. A process according to claim 31 in which the amino-
protected derivative of 6'N-methyl-3',4'-dideoxykanamycin B to be acylated
is 2'N,6'-N-di-t-butoxycarbonyl-6',N-methy1-3',4'-dideoxykanamycin B or
6'N-t butoxycarbonyl-6' N-methyl-3',4'-dideoxykanamycin B or a mixture
of them.
33. A process according to claim 31 or 32, in which the func-
tional equivalent of the ?-hydroxy-.omega.-amino acid is its acid chloride, mixed
acid anhydride, active ester or azide thereof.
34. A process according to claim 31 or 32 in which the ?-
hydroxy-.omega.-amino acid is employed in the form of its N-hydroxysuccinimide
ester.
35. A process according to claim 17 for the production of lN-
(L-5-amino-2-hydroxyvaleryl)-6'N-methyl-3',4'-dideoxykanamycin B which
comprises selectively acylating the l-amino group of an amino-protected
-56-

derivative of 6'N-methyl-3',4'-dideoxykanamycin B represented by the
formula
<IMG>
wherein R1 is a known mono-valent-amino-protecting group and R2 is a
hydrogen atom or a known mono-valent amino-protecting group, with an
L-?-hydroxy-.omega.-amino acid of the formula
<IMG>
or its functional equivalent as an acylating agent wherein R3 is a known mono-
valent amino-protecting group and R4 is a hydrogen atom, or R3 and R4
taken together form a known di-valent amino-protecting group, and n is
three, to produce a l-N-acylated derivative of the formula
-57-

<IMG>
wherein R1, R2, R3, R4 and n are as defined above, and then removing the
amino-protecting groups from the l-N-acylated derivative to produce the
desired compound.
36. A process according to claim 35 in which the amino-
protected derivative of 6'N-methyl-3',4'-dideoxykanamycin B to be acylated
is 2'N,6'-N-di-t-butoxycarbonyl-6' N-methyl-3',4'-dideoxykanamycin B or
6'N-t-butoxycarbonyl-6' N-methyl-3',4'-dideoxykanamycin B or a mixture
of them.
37. A process according to claim 35 or 36, in which the
functional equivalent of the ?-hydroxy-.omega.-amino acid is its acid chloride,
mixed acid anhydride, active ester or azide thereof.
38. A process according to claim 35 or 36 in which the ?-
hydroxy-.omega.-amino acid is employed in the form of its N-hydroxysuccinimide
ester.
39. The process according to claim 16 wherein the acylating
step is carried out in anhydrous dimethylformaldehyde, acetone or tetra-
hydrofuran.
40. The process according to claim 39 wherein the acylating
step is carried out under cooling.
-58-

41. The process according to claim 40 wherein the acylating
step is carried out in the presence of a dehydrating agent.
42. The process according to claim 41 wherein the dehydrating
agent is dicyclohexylcarbodiimide.
43. The process according to claim 16 wherein the starting
compound (IV) is reacted in a 0. 5 to 3 molar proportion of an active ester
of the ?-hydroxy-.omega.-amino acid compound in a reaction medium consisting
of water and an organic solvent.
44. The process according to claim 43 wherein the organic
solvent is dimethoxyethane.
45. The process according to claim 43 or 44 wherein the molar
proportion of the active ester is from 0. 5 to 1. 5.
46. The process according to claim 16 wherein which process
further comprises isolating the desired final product of the formula (I) by
column chromatography.
47. The process according to claim 46 wherein the isolation
step employs silica gel, a cation-exchange resin having carboxylic functions,
a weak cation-exchanger or CM-cellulose.
48. The process according to claim 16, 23 or 27 in which in
the l-N-acylated derivative the amino-protecting groups R1, R2, R3 and R4
are selected from the groups of a formula -CO-OR6 in which R6 is an alkyl
group of 1-5 carbon atoms, a cycloalkyl group of 3-6 carbon atoms, an
aralkyl group of 1-4 carbon atoms, an aryl group, or a heterocyclic group.
49. The process according to claim 16, 23 or 27 in which in the
l-N-acylated derivative the amino-protecting groups R1 and R2 are selected
from the groups of a formula -CO-OR6 in which R6 is an alkyl group of 1-5
carbon atoms, a cycloalkyl group of 3-6 carbon atoms, an aralkyl group of
1-4 carbon atoms, an aryl group or a heterocyclic group; and R3 and R4 are,
taken together, a di-valent amino-protecting group selected from an alkylidene
or arylidene group of the formula =CHR5 in which R5 is an alkyl group of
1-6 carbon atoms or an aryl group.
-59-

50. The proçess according to claim 16, 23 or 27 wherein the
amino-protecting groups R1, R2, R3 and R4 are t-butoxycarbonyl group
or benzyloxycarbonyl group.
51. The process according to claim 16, 23 or 27 wherein the
amino-protecting groups are an alkyloxycarbonyl group, a cycloalkyloxy-
carbonyl group, aryloxycarboxyl group, alkylidene or arylidene group,
and the amino-protecting groups are removed by subjecting the l-N-acylated
derivative to a mild hydrolysis treatment with an acid.
52. The process according to claim 16, 23 or 27 wherein the
amino-protecting groups are an aralkyloxycarbonyl, and the amino-
protecting groups are removed by subjecting the l-N-acylated derivative to
a hydrogenolysis treatment in the presence of a palladium-carbon or
platinum black catalyst, or by subjecting the l-N-acylated derivative to a
treatment with hydrogen bromide in acetic acid at low temperature.
53. The process according to claim 16, 23 or 27 wherein the
amino-protecting groups are o-nitrophenoxyacetyl groups which are removed
by a reductive treatment.
54. The process according to claim 16, 23 or 27 wherein the
amino-protecting groups are phthaloyl groups which are removed by
treating the 1-N-acylated derivative with hydrazine hydrate in ethanol.
55. A 1N-(?-hydroxy-.omega.-aminoalkanoyl)-6'N-methyl-3',4'-
dideoxykanamycin B of the formula:
<IMG>
-60-

wherein n is 1, 2 or 3 whenever prepared by the process of claim 1, 16 or
17 or by an obvious chemical equivalent thereof.
56. A pharmaceutically acceptable acid addition salt of a lN-
(?-hydroxy-.omega.-aminoalkanoyl)-6'N-methyl-3',4'-dideoxykanamycin B of the
formula:
<IMG>
wherein n is 1, 2 or 3 whenever prepared by the process of claim 13 or 20
or by an obvious chemical equivalent thereof.
57. lN-(DL-isoseryl)-6'N-methyl-3',4'-dideoxykanamycin B
whenever prepared by the process of claim 2, 23 or 24 or by an obvious
chemical equivalent thereof.
58. lN-(L-isoseryl)-6'N-methyl-3',4'-dideoxykanamycin B
whenever prepared by the process of claim 3, 27 or 28 or by an obvious
chemical equivalent thereof.
59. lN-(L-4-amino-2-hydroxybutyryl)-6'N-methyl-3',4'-
dideoxykanamycin B whenever prepared by the process of claim 4, 31 or 32
or by an obvious chemical equivalent thereof.
60. lN-(L-5-amino-2-hydroxyvaleryl)-6'N-methyl-3',4'-
dideoxykanamycin B whenever prepared by the process of claim 5, 35 or 36
or by an obvious chemical equivalent thereof.
-61-

Description

~ \
C~537
~Pk~9
L~b ~ o._L~Yo~h~Y~: Thl~ invention
~ relate~ to the preparation o~ new semL~ynthetlc 1-
; sub~tituted derivative~ o~ 6'N-methyl 3l,4'-dideoxy-
kanamycin B, said compound~ being prepared by
~ele¢tlvel~ acylatinig ~he 1-amino~funct~on wlth a
;~ ~-hydroxy-~-aminoalkanoyl moiety,
A) K~inamyc~n B ls a known an~ibiotic de~crlbed
ln Merck Indexj 8th Edition, ppO 597-598. Kanarnycin
B 1~ a compound having the formula
.
6 1
HO - ~ 2
. NO ~
NH2 ~ NH2
r ~ 6--
0H EIO 5 j~
/ ; 2
H N ~ ~ ,
HO
, .
B) 6tN-Meth~l 3~,4'-dideoxykanamycin B i~
- a known compound de~crlbed in British patent 1,384,221
~ and ha~ the ~ormula
., ~ .
~, .
,
: -2-
.
.
. . ..

3L~S~:3537
1~ 1 6 1
~2
\~ 5~ 0
31 /2~ 1~
N~2 1 4 N~2
~"~
` f~E20H HO~1 NH
HO ~,,,~L~ / 2
-, ~ H2N ~
HO \O
C) 31,4~-Dideoxykanamycin B i9 described ln U.S.
Pakent 3,753,973 and has the formula
'. :
:
6 '
~H2-N~2
~15 ' n
31 /~,1'
NH2 1 4 ~2
0~
6FH OH MO ~`~ 2
.', ' ~ ~\~ "
H2N~
.
.
;' "' ' ,, ' . ,: .

~ 3~
D~ IN-(Lr4-Amino-2 hydroxybutyryl~kanamycln
A and lN~(LrJ~-amlno-2-h~droxybutyryl)kanamycin B
are descrlbed in U.S. Patent 3,781~268 and have
; the ~ormula
6~
~0 ~ MH2
',: ~0
HO - ~ 1'
R 4 2
~ ~ 0~,~
2 ~ 1
HO ~ / NH2
H2N~V~
,
,~ :
:~ ,
E) The compoundæ o~ the inætank lnvention
are deæcrlbed in The Journal o~ Antibi~tlcs,
340 (1975),
: :
.: ~ ~ ' :
, . , , - .
.
-4
; . '
. ,, , ., ~ .
:, ' ~ . . .

~1~53
a~ o~ e Ir~Qt~
T~e compound ha~ring the ~ormula
4" ~"
,~ C~OH ~
E211 3~ 3
1~ 10 NH2 ~:
`' CHO~ . . . .
:~ (
NEI2
, . .
,
`ii wherein n iB 1, 2 or 3; or a p~ar~naceut~cal3y
accep~able a¢ld addltion ~alt thereof are valuable - I -
ant~ba~t~rial agerlts u~e~ul in the treatment o~
20 ba~terl~ lOn~ ~n anim~
: ~ .
In one a~pect of thi~ invention there i~ provided a proc~ r>r t~e pro- :
duction of a lN-(t-~ydroxy-llam~ lkalloyl)-6'N-methyl-3' ,4'-dideoxy-
kanamyoin B of th~ tormula:
: 1 ~ ~ , ,
~ 3
30 (~n
~2
5-
'j'

59~
wherein n i~ 1, 2 Ol 3, which compriRe~ removing the amino-protecting
gro~ps from a l-N-acylated derivative of 6'N-methyl-3' ,4'-dideoxykanamycin
B having the formula
~jll
~, R;}IN
~;2N~ln 6 lj~ ~3
; 10 ~ 5 6
HN
CO 1
: 1 3
CHOH ~ R
(CH2 ) nN ~ '
~ ' :
wherein Rl i~ a known mono-valent amino-protecting group and R2 i8 a
hydrogen atom or ~ known mono-valent ami~o-protecting group, R3 i8 a
know~ rnono-valent amino-protectis~g group, R4 i~ a hydrogen atom, or R3
`I and R4 talcen together form a know;l di-valent amino -protecting group, and
n i~ a;l integer of 1, 2 or 3.
~ another a~pect of thi9 inv~ntion there iB provided a process for ~e
production of a lN-(~c(-hydroxy-lJ-arninoalkanoyl)-6~N -methyl-3' , 4' -dideoxy- : .:
kanamycin B of the formula: :
411 6~ . .
0
3~1~ H2N 2~ :
\o ~NI'fCN3
~ o 1 ~ NH2 ,'
d~o~ 3
n
~ I .
511-
', .. '. ' , , , . . ' . ~ . ' , ' ' . . ' ', , ' ' ' , . . . .
., , . , . ,: , , . . " ~ ,: ... .: ... .. . . . . ..

f
~5~53
wherein n i~ 1, 2 or 3, which COmpriBeS ~electively acylating the l-amino
group of an amino-protested derivative of 6~N-methyl-3~ ,4' -dideoxykanamycin
B represented by the formula
6n
49t ICH C)~
~; \ ~IR~
~,2N~ t D
3~ ~
~N ~ N~2
: ' '
wherein Rl i~s a lcn~ rn mono-valent amino-protecting group and R2 i~ a
llydrogen atom or a knovm monD-valen$ am~no-pr~tecting group, with an
hyd~oxy-~-amino acid oi the formula
~,3
:. _
:.' \ N-(CH2~n-CH ~OH)-COOH
R
20 or it~ functional equivalent as an acylating agent wherein R3 i~ a known mono-
valent amino-protecting group and R4 i~ a hydrogen atom, or R3 and R4
taken together form a known di-valent amino-protecting group, and n is
an integer o~ 1, 2 or 3, to produce a l-N-acylated derivative of the formula
'
5b
:
: ,, , , , ~ . , . ~ . . :
, ,

``~ -
3~i5~i37
6~
4" ~2~
~ ~. ,J
~ ~IC0
. - ~o~
',' 10 (C~2)~<R
wherein Rl, R2, R3, R4 and n are as defined above, and then removing the
.~ "
amino-protecting groups ~rom the l-N-acylated derivative to prDduce the
`` desired compound.
. : .
:~ .
, ' '
~ .
~.
,
.. . .
-5c-
,
;~ f .,~, ,~
. ,,"

53~
g~h~ .
ml8 in~rention r~lat~s to new kanamycln B
derivative~ whi~h are prefetably ~elected from the group
~onsl~tine o~ (D~isos~ryl)-6' N-methyl-3',41-
dideoxykanamy~in B" lN~ oseryl) 6' N-me~hyl-
~,4~ dideoxykanamyc~n :3, lN (Ir4-amino-2-
hydroxybutyryl) 6~ N-methgl-~"4~-dideo:~ykanamy~n
:E~ and lN~ 5-amino-2-hydroxy~raleryl)-6 ' N-m~thyl-
~,4~-dideoxykanam~cin :E3 and/or a nontoxic, p~arnaceuti-
call~ acceptable acid add~ tion salt thereo~,
.
Furthermore9 this in~ention al~o relates to a
proce~s Por the semi-synthetic production o~
~ I .
the~e ~ hydroxy-~-~minoa lkanoyl) -6 I N-methyl
derivat.~ves of 3',4' ~dideoxykan,~m~Jcin B.
. .
K~namycins A and B are kno~.~ aTninoglycosidlc
an~1biotlc~ and have been wldely u~ed a~ chemo-
~herapeutic agent~. ~Iowever, many kanamycin-
re~ista~t s~rains Or bact~ria have developed in
re¢ent years. For instance, it ha~ been ~ound
that some R-~actor carrying ~rains o~ the gram-
negatlve b~cteria, such a~ hL~ coll and
oJ~a~ aeru~ino~ have been lsolated ~rom
.j , .. .
patients which are re~istant ~3 the antibacterial
ao~ion o~ the kanam~cins. me mechanism o~
res~3tance of the ~anamycin-resistant bacteria to
~he known amlnoglyco~idlc anttbiotic~ has been
~tudied by H. Umezawa et al., Advance~ in Carbo-
h~drate Cheml~try and Biochemlstry, Vol. 309 pp. -
~,, ...................................................... : '
6--

5 3~
183-225, 1974, Academic Press. It has been
discovered that some kanamycin~resiætant bacteria
produce enzymes capable of phosphorylating the ~'-
hydrox~l grou~ o~ the kanamycins and inactlvate
the kanaT~ycins via these pho~phokrans~erases and
that some kanamycin-resistant bac~eria produce
an enzyme capable o~ nucleotidylating the 2 "-
hydroxyl~group~o~ the kanamycins and thereby
inacti~ate the kanamyclns via a nucleotidyl-
transferase, and that ~ome other kanamycin-
resistant bacteria produce enzymes capable of
., :
acekylating the 6~-amino group o~ the kanamycins
and thereby inactivate the kanamycin~ via these
~; acet~ltran~erases. In this way, the relation-
.
hlp o~ the moleoular structure o~ khe amino-
:gly~osldic antibiot~cs to ~heir antibacterlal
activi~y, a~ well as the biochemical mechanism~
:~ : o~ res~stance o~ the kanamycin-resistant bacteria
to the am~noglycosid~ antibiotics have been
eluc~dated~
: Se~eral ~em~-synthe~ic deriva~ives o~ the
~ anam~cins whlch ~e active against th~ kanamycln-
:~ resl~tant ~acter~a have been ~yn~hetized ~rom the
pare~t kanamycins. m u~ d~deoxykanamycin
(U.S. patent 3,7539973); 6~ N methyl-~,4~-
dideoxykanamycin B ~ rlt~sh patent 1J~j84~221~;
lN~(~4 amino-2-hydroxybu'cyryl)~kanamycin A and
~7
.
., " ,
.
.. . .... ..
- , " : .
,

~5~53~
~kanamycln B (U~S. patent 3~781,268); and a lN-
hydroxy ~-am_noalkanoyl)-~',4'-dideoxykanam~cin
B ~DT-0~ No. 2,350,169) are synthesized, ~or
~; ~ example. mese semi-synthetic kanamycin
: ~ derlvative~ have been found to be active against
: '. a lar~e nu~ber of kana,mycin-resistant bacteria.
We have now per~ormed ~urther research in
' ~ an at~empt to provide new and useful derivatives
o~ ~,4~ dideoxykanamycln B which are e~ective
. .
: not only against.the gram-negative and, ~ram~
positive ba¢teria sen~itive to the kanam~cins
.
: . but al~o agains~-~he kanamycln-resistant bac~eria
We:have now ~ound that selective acylation of the
amino~ group of 6' N-methyl-~"4~-dideoxykanamycin
.
' B~wlth an ~hyd.roxy-~-amino acicl selected ~rom the
' group consistlng o~ lsoserlne, I~-amino-2-h~droxy-
: : .butyrlc aci~ or 5-amino-2-hydroxyvaleric acid9
~,either :in racemi~ form or in the form o~ the L-
omer or in the ~orm o~ the D-isomer9 produces
new:and use~ul ~anamyc~n B derivatives which
exhibit high~an~lbaoterlal activity against the
~gram-neæative and gram-positive bactexia sen~itive
' to ~he kanamycin~, as'well a~ ag~inst the bacteria :
: resi~tant ko the kanamycih~.
An obJeck o~ this invention was to provide new
' and useful kanamycin B~derivatl~es which exhibit
: use~ul antibacterial activlt~ against the kanamycin-
re~i~tant bacteria a`~ well a~ against substantlally
.
-8-
: . , . ~ , . .

: ~5~)537
all o~ the kanamycin~resistant bacteria produclng
the above-mentioned inac~lvatlng enzymes7 Another
ob~ect of this invent~on was to provide a process
~or the production of these new kanamycin B
derivatives :E`~m 6' N-me~hyl-~',4'-dideoxykanamycin
~J which is operable ln a ~aclle way and in a
: Lavorable yield o~ the desiréd product.
.~ .
` A pre~erred embodiment o~ the instant invention
iB t~e~compounds havlng the ~ormula
~: .
4 "
CH20H,
H(~ ~ ~ \ 2 '
~: ~ H2N ~ 1" . H2N--7~ 4 ~
3 OX \ OX lf~ \~C~2 3
CO 1 ~ 2
CHOH 3
( CH2 ) n
: ~ : :whereln~n is 1, 2 or 3; and a pharmaceutically
acceptable acid~-addition ~alt thereof. me a-
- ..
hydroxy-~-amlnoalkanoyl moie~y, that i~J the
i~o~eryl group, 4-amino-2-h~droxybutyryl group
or 5-~mino-2-hydroxyvaleryl ~roup pre~ent in
the:molecule o~ the new oompaund o~ the above
~ormula;(I) ma~ be either in the DL-~orm
(namely, the raoem~¢ ~orm), the Ir~or~ or the
D~orm O
~ ~ ~g_
,
- - . .
, ~

:~5~S~7
Ihe most preferred embodiments are the
compounds:
(1) lN-(DL-isoseryl)~6' N-methyl-3~,4'-
dideoxykanamycin B;
(2) lN-(L~lsoseryl)-61 N-methyl-~',4'-
dideoxykanamycin B;
; (3) lN-(L-4-amino 2-hydroxybutyryl)-6
N-methyl-3',47-dideoxykanamycin B, and
(4) lN-tL-5-amino-2-hydro~yvaleryl)-6' N-
methyl-3',4'-dideoxykanamycin B; or a pharmaceut-
:~ ically acceptable, nontoxlc acid addition salt
thereof.
Examples of the pharmaceutically acceptable
~ . . .
acid addikion sal~ o~ ~he new compounds of
"
: ~ormula (I), according to thi~ inventionj include
the hy~roohloride, hydrobromide, sulfate, phosphate~
L
; ~ nitrate, carbonate, acetate, maleate, fumarate,
~uccinate, tartarate~J oxalate, cltrateg methane-
~ulfonate, ethanesulfonate, ascorbate salts and
the like~ whi¢h ma~ be a mono-, di-, tri-, tetra-
or penta-~alt formed by the interaction of one
-
molecule of the new oompound of the formula (I)
wlth 1-5 mole~ or a nontoxic, pharmaceuticall~y
acceptable acid. ~he pharrnaceutically acceptable
acld inaludes hydrochloric, h~drobromic, ~ulfuric,
pho~phoric~ nitric, carbonic, acetic, malelc,
-10~ '
.

~S~53~
fUmariC9 8UCC~niC~ tartarlc, oxalic, methane
sul~onic, ethanesul~onic, ascorbic acld and
the llke~
The new compound~ according to thl~
invention have the ~ollowing ~hysioal, chemical
and biological propertie~:
a~ La~Is a sub~tanc~ ~n the ~or~m of a
colorle~s cr~sital~line powder with a decomposition
point of 165 169 C., [d~24 _ ~96~ (c 1,175,
water)~. It~ elemental analysis ls con~istent
with the theoYetical values C2~H44N601o (C,
47.81%9 XJ 8.0~%~ N, 15.21%)~ ~is substance
glve~ a single spot (positive to the ninhydrin
rea~tion) at R~ 0.51 by thin layer chlormatography
on ~ilica gel (available under a trade name l'AR~
5721", a~product o~ Merck Co. J Germany) when
dev~loped wlth n-butanol-ethanol-chloroform-28%
:aqueous a~nonia (4:5:2:8 by volume~
~e,~ 6'_,,
a substanee in the ~orm o~ a
colorle3~ crystalline powder with a decompo~ltion
polnt o~ 162-166 C., [~24 _ ~80 (c, 1,02, water),
Iks elemen~al analysls ls con~istent wlth the
theoretical values o~ C22Hl~4N601o (C, 47081%,
H, ~.03%, N, 15.21%)~ Thi~ su~atance gives a
single spok (posltlve to the ninhydrin reaction)
at R~ 0.51 by the above-mentioned silica gel thin
.. . .
.

S;3~7
layer chrom~tography,
ls a ~ub~tance
in the ~orm of a colorle~s crystalline powder
;~ with a decompositlon point o~ 158-161 C.,
325 _ ~71 (c~ 0.8, water). It~ elemental
analy~ls i8 con~tent with the theorekical
,
alue~ of C2~H~N601o (C, ~8~75~ 8-1~%~
N, 14.83%). Thls substance gives a single ~pot
. ~ .:
~: (po6itive to the ninhydrin reaction) at R~ 0.38
by khe above-men~ioned thln lay~r chromatography
on silioa gel, ~
s a substance in the
orm o~ a colorless ory~talllne powd~r wlth a
deoomposi:tion polnt o~ 152-155~ C., [a]24 _ ~79O
(c , 0.71, w~ter3. Its elemental analysis is
,.
conslstent~ wlth the theoretical value~ of C24H48N601o
, 49.64~H, 8.~3~j N, 14,47~). This ~ubstance give~
a ~ingle spot (po~ltive to the ninhydrin reactlon)
- : .
. at R~ V~9 by the above mentioned ~illca gel thin
la~e~ ohromatography.
~ he new compound~ o~ ~ormula (I), a~oordlng
to thiq invention, are characterlzed in that they
,
are no~ susceptible to all the enæy~atic, inactlvatlng ~ -
reaotions w~th the above-menkioned enzyme~ which
...
, ,
-~2- :
.
. . . . .
, . . .
., - . .. . ..
. ' ' , ; ~ ~

`~\
- ~ ~ 5~ 53 ~
inactivake the kanamycin A and kanamycin B. m us,
-~. the new co~pound~ of this invention are neither
~us¢eptible to inactivation by the 6~-acetyltrans~era$e
becau~e the 6~-amlno group of the new compound o~
thls lnvention has be~n methylated, nor are the~
.~ .
~u~cepti~le to lnactivation by the 2 ~-nucleotidyl-
t~ansferase and the ~-phQ~photran~erase because
the l-amino group of the new co!npound of this
.
~ ~ inventlon has been acylated:with ~he a-hydroxy ~-
; ~ amlnoacyl group,- nor are they su~ceptible to
, . ~
iP~et~vation by the other type o~ the 3'-phoæ-
photrans~era$e because the 3'- and 41-hydroxyl
group t~leh are present in kanamycin B have been
removed iP the new compounds of this invent~on.
a~ordingly~ the new compounds of this lnvention
ar~ remarkedly advan~ageous in khat they exhibit
h~gh~antibacterial acti~ity not only against
. j ~ .
v~r:ious kind9 o~ gram-ne~atlve and gram-positive
bac~er~a which are sensitive to the kanamy~ins,
but alæo against the kanamycin-~e istant strainæ
of the~e bacteri~, particularly the kanamycin-
reæi~tank strain~ of ~g9kg~iQh~ and :
~Q~Qm~ ~L.
The mlnlmum ~nhlbitory concentrations (mcg./ml.)
o~ lN-(DL-~soseryl)~6~ N-methyl-3~94~-dldeoxy-
kanamy¢ln R (abbreviated as DL-IS-MDKB); lN-(Ir
~s~seryl)-6~ N-methyl-3~,4~-dideoxykanamyoin B
-13~
.
,

~ ~ 5~ ~3 ~
(abbre~lated as L-IS-MDKB); lN~ 4-amino-3-
hydro~butyryl)-6' N-methyl~,4'-dideoxykanamycin
(abbrevlated a~ AHB-MDKB); and lN-(L-5-amino-2-
hydroxyvaleryl)-6' N-methyl~3',4~dideoxykanam~cin
~ tabbreviated as AH~-MDBK) against various
or~anisms were determined accordlng to serial
dilution me~hod u~ing nutrient agar medium at
37 0~, the readings being made after 18 hours
inc~batlon~ For the comparison purpose, the
minimum inhlbitory concen~ration3 (mcg./ml.~ o~
} N~ 4-amino-~-hydroxybutyryl)-kanamycin A
(abbreviated as,AHB~KMA) and lN-tL-4-amino-2
hydroxybutyryl~-kanamyoln B (abbrevlated as
AHB-KMB) which are known ~rom U.S. Patent
,
: 3,781,268 were al~o determined in the sa~e
manner as stated above.
:. ~
The antibacterial spectra o~ the concerned
oompound are shown in Table 1 below.
. ..
.
,'~ : ' ....
.
.,: , . ,, ., , ,: , ., :
.
.. . . . . . . . . .

53'7
~ o o~ o ~ c~ o o co co o ~ r~ ~O O~ co CO ~ ~O
_ , C~ ~ N ~I C\l C~l Cl ~ ~ N ~1 ~I L~ 1~ ~ t~ ~ L~
~ ~ O O O ~ O O O O O O ~ 1~ ~ O O O O
~ CC V V VVV V
_ ~
O C~ O O O O ~ CO ~ ~ ~ CO CO 00 CO rr~
C~J ~ N ~ l N N L~ CU ~ ~1
O l O O O N O O O ~i ~ O ~ ~ 1~\ O O 0. 0 i'~
~ ~ V V ~ VVV
O ~ O CO O ~ O O O ~ ~ O rr\ ~ L~ ~ ~O ~O C
C~ ~: N ~ N rJ N N N ~ r~l C~.l r i --I CU -1 ~ U~ ~ ~1
P ~ O O O ~ O O O 1~ 1~ 0 1~ i 0 1
~': O 3; V V VVV
q_
, ~ .
H
~ o O O ~ O O O ~O CO 0~ ~ ~O ~ ~ 0~ CO CO U~
E I N cU CU ~ CU CU CU IS~
~rl I O O O ~ O O O ~ O O ~ ~i ~\ O O O O r-i
. . l ~/Vv Vvv
' . r~l
:', ' O
`~" ~! .
I O CO O ~ O O O ~ O ~ ~O ~O
v~ CU C-- CU N ~ CU CU ~I ri ~\ N -1 ~1 In ~1 IS~ L~ ~1
.. O O O ~O O O O ~ ~ C~ ~O ~ r~ ~1 ~ ~ ~1
~: V V V`~/ V
:, .
.
.1
CO
O O
, C~ ~ CO 'C~ 'I 'I O
- :, N '~ 1~ V a~ cO ~1 ~0 o
CU O E~ ~ Z
- C~ I O ~I cC ~1 ~1 ~0 CO H
q ~ cc~ ) N
(u O ~1 ~ 4 CU
1 ~ 0 0 ¢ R ~ ~ ~rI q ~1
.', ~ ~ o ~ ~ O '~
O O O
O C~ O . ~ ~q ~q ~q ~ ~ ~ ~ ~ ~1 ~1 ~ ~
P~ h h ~ M a~ lD
O O O a) b~ Ul
a, O ~ ~ o bO bO ~ n
, o C~ i O a~
~ ~ CO~ ~ ~ s S ~
-15-
.~. . .... . . . . .
.

~5~537
I
~ a) ) a~ ~ ~ ~ ~ o~ ~ O~ O ~O ~ ~ ~ U~
t~ ~ A ~ A ~
, P~ o o o o o o ~1 o o o o ~ ~ ~ o ~
~ CC V O
O ~ O O ~1 0 ~1 ~ O O O O r~ ) CU
h
c~ : ~ W 0
~1 0 ~ ~ O ~ ~J O O O O
o : ~
:
H ~ : ~D 0 ~
- ~3 ~ . , , t-- U~ ~I N
,,_ ~ r-l O, ~ ~ O O O ~1 ~; 1~ ~D L~\ It`\
,! 1 cu ~1 N
~ ~ :
- :~
' tl~ ~ , o
rl )~1 ~1 ~ ~ ~1 ~ 1~ 0 t;~ r~ ~ CU N
~ ~ ~ ..
.
:
' ' ~ " CO ,
~;
0~ O O O N
C!J ~ r~i r-l ~1 ~ L~
0 ~ . r 1 1--I
N ~J N N cu CU o~ a 0 ~a
~ ~ ~J rl ~1 --I ~ ~ C!J N ~ ~5) 0 0 0 0 0
b~ O O O Q~
o o V o , o C) O O C) o o , o ta
a ~ a ~ a ca ~a
~ ~rJ rl ~ ~ ~ ~ ~ ~r( ~ ~ ~ ~ ~(1 ~ t~ t~t t,n
~ ~ ~ ~ C ~ S 5 ~ L~ ~o ~0 ~ o ~o ~0 S3
t;~ ~ h h h h h ~ h h O O O O
S
c~ o c) o ~ o v c~ t~ O
--16-
.

5~537
.
The new compounds of this invention are of
low toxicity to animals, including man, as they
show LD50 values of more than 100 mgO/kg. upon
intravenous ih~ection of the compounds in mice.
In additlon, the new compounds o~ thi~ invention
exhibit high antibacterial activity agalnst
various gram-negatlve and gram-positive bacteria
sensitive to kanamycins, as well as a~ainst t~e
kanamycin-re~istant strains thereof as stated
:"
hereinberore, so that the new compounds of this
invention may be useful in therapeutic treatment
o~ infections by varlous gram-nlegative and gra~-
positive bacteria.
e compounds of this invention may be
admlnistered orally, intraperltoneally, intravenou~ly~
subcutaneously o~ intramu~cularly using any
,
pharmaceutlcal form known to the art ~or such
administration and in a similar manner ~o kanamycins.
For lnstance, the compounds of the ~ormula (I) Or
this invention may be adminlstered oraIly using
.
any pharmaceutical form known to the art for such
oral administration. Examples of pharmaceutical
forms for oral administration are powder~, capsules3
tablets~ syrup and the like. Suitable dose of the
compound for the e~fective treatment of bacterlal
in3eotions is in a range o~ 0.25-2 g. per person
a day when i~ is glven orally. It is preferred
-17-
, . . , , . ~. .
.
;, .;

~L~)51D537
that said dose s~ould be orall~ admini~tered in
three to ~our aliquots per day. ~he compounds
o~ this invention may al~o be adminlstered by
intramuscular in~ectlon at a dosage of 50-500 mg.
per do~e two to four time~ per day. Moreover,
the ~W oomPounds o~ the invention may be
~ormulated into an ointment ~or external application
hich contains a compound of thi~ invention at a
,
cono~ntrakion o~ 0.5-5% by welght in mixture with
:a known oin~ment ba~e such as polye~hylene glycol.
Moreover, the compound~ o~ this :invention are
u8e~ul to sterillze surg~cal instruments when the
sterilization i8 accompanied by adequate mechanical
Gle~nsing.
; In prlnciple, the new campounds o~ the ~ormula
(I),;according to ~his lnvention, may be prepared
rom~a known compou~d, 6I N-methyl-3~ dldeoxy-
kanamycln B, o~ the formula
. ,: -
:6" :~
H0 ~ ~ 2'
H2N ~ \ ~ ~ ~I2N ~
5'~--- 2~ 5
. : 3 ~ :
: ~ - :'
; . ~ i . ~II)
.
. .
.

5~i3~7
by ~electively acylating the l-amino group of
'~ 6~N-methyl-31,41-dideoxykanamy~ln B with an
a-hydroxy-~-amino acid o~ the ~ormula
~' '
' H2N-(cH2~n-cH(o~)-cooH (III)
;,
wherein n is an integer of l, 2 and 3, in a
manner known per se in the prior art, consi~tent
with the acylation of an amino group in the
synthesis o~ peptides. The 6~N-methyl-3~94'-
dideo~ykanamycin B of ~ormula II contains four
:,
: ~ ~.ree primary amino groups (l-, 3-, 2'- and ~"-
., ,
amino groups) and one secondary methylamino
group at khe 6'-po3ition, ~o produce the
'~ ~ compounds o~ ~ormula (I),-according to thi
,:~ invention3 it is required that the l-amino
,
'' group o~ 61N-methyl-3',4~-dideoxykanamycin B
be~ele~otively acylated with the a-hydroxy-~-
,
amino ac~d o~ the ~ormula ~IIX)~without.
cau31ng ~he acylation o~ the other three
amino group~ and the 6~-me~hylamino group. Ihe
new compound3 o~ the ~ormula (I) are obtained in
the be3t, yJ,eld when ~he a-hydroxy-~-amino acid
reaotant o~ th~ rormula (XII) -;s reacted with an
amino-protected derivative o~ compound II in
i~hlch the 61-methylamino group and the free
amino ~roups (that 3.~,~the 3-, 2i- and ~"-
amino group3) oth,er than the l-amino group have
'' -19-
. . .

been blocked by a-known amino~protectin~ group with
only khe l-amino group remainlng fre~, The
prepara~ion of the amlno-protected derivative is
dl~icult but po~ible and a number o~ reaction
steps are requ~red ~or the preparation. It i~
.~ .
preferred ln~ead to prepare 3uch an amino-
protectecl derivat~ve of compound II in whiçh
. ~ ~ only the 6tmethylamino ~roup and optionally
the 29-amino group have been blocked by the
amino ~rot~cting group while the other amino
,
group~ are ret~ined in the ~ree state, Prepar-
. : atlon~o~ the latber type of the mono and di-amino-
protected der~vative of ~:ompound II iB reIatively
~ ~ ~ eas1er to prepare due to the ~act that the 6'-
`~ m~thylamino and the 2'-amlno groups are more
.;,. . , ~ . .
reac~ve than the other amino groups o~ oompound
a~d h~nce can~be bloaked preferentially by
the;amino-proteot~ng group while keeping the
othe~amino groups unblo¢ked~ As the reaot~vity
or th~ 2~ amino group is a llttle lower than that
o~ the 6~-methylam1no group but l~ higher than that
or th~ 3- and 3"-am~no groups, the 2'-amlno
group may also be blocked, if desired.
When the amino-protected derlvativ0 prepared
~rom oompound II in whi¢h tho 6t-methylamino group
and optionally the 21-amino group have been blocked
is reaobed w1th the ~-hydroxy-~-amino acld (I~I)
ln wh1ch the ~-amlno group may pre~erably be
.
o~O

S3t7
blocked by an amino-protecting group3 there is
formed a reactlon product comprising mainly the
desired lN-mono-acylated derivative together
with lesser quantitles of the mono- and poly
N-acylated derivati~es in which one or more of
the amino groups other than the l-amlno group
and occasionally the 2'-amino group have been
acylaked with the a-hydroxy-~-amino acid (III)/
re~pectively. Thus, the acylatlon product
resultlng from the above reaction is actuall~
obtained in the ~orm o~ a mlx~ure o~ di~erentl~
N-acylated derlvatives including the desired
N-mono-acylated derivative. It is possible
to isolate the de~ired lN~mono-acylated
derivative ~rom the mixed N-acylated derivatives
by subjectlng the mlxture to a ohromatographlc
separation. However, the m~ed N-ac~lated
derivatives may be directly treated to remove
the amino-protecting groups therefrom. mis
produces a mixture o~ the desired lN-mono-
acyla~ed product o~ the ~ormula (I) wlth the
otherwlse mono- and poly-N-acylatedJ undesired
by-produot,s derived ~rom the selectively amino-
protected startlng compound. The desired product
~I) may,be isolated ~rom the unde~ired by-product~
by sub3ecting the m~xture o~ them to a chromatographic
separation,
'
-21~
.. . . .
. . , . , . :
. .

~6~5~D~i3~7
According to the ~ond ob~ec~ o~ thi~
imTention, there 18 provided a proce~ ~or the
produetion o~ the lN- (a-h~droxy-~-aminoalkanoyl)-
6i N-methyl 3~,4~-dideoxykanamyc~n :3 compound3 o~
the a~ore~aid ~ormula (I)~, whlch comprise3
~electively acylating the l-amino group o~ an
am~no-protected deriva'c~ve o~ 6~ N-methyl-3~94~-
dldeoxykanamycin B represented b~ the ~ormula
' ~.''
,~, 6n
. ~,.0 ~ 27
'~2~ 6 ' ~ :: ::
~ Cli2N~
7~2N ~ ~2 `
~ .
wherein Rl i8 a known mono-valent amino-protectlng
group and ~ i~ a hydrog~n atom or a known mono- .
;
valent amino-protec~lng group, wl~h an a-hydro~y~
am~no acld Or ~he rormula
3 ~ .
~0 N ,(C~ ~n-CH(OH~-COOH ~V) ~'
R
or itB runctional equivalent as an aoylating agen~, ~
herein R3 1~ a known mono-valent amino~protect~ng ~,
group and R~ i~ a hydro~en Qtom, or R3 and R4 taken
j.,: '..
;,."';.. ,,.;.1 ~ . .
. ,, , ,", .. ,
.. . . . . . .
. .
,, . .: . .... .
.,, ,: . .

537
together form a kno~Yn di-valenk amin~protectirlg
group* and n ls an lnteger o~ 1, 2 and ~) to produce
a l-N-acylated compound of the ~orm~la
,
:~ 6n
HO ~ CHz~ O
~ \ 21l \ R2~ --~S
: ~: ~ k ~ ~
HN ~
c~o~'. gV:E)
;~ ) N< 3
}a4
.,
...
Rl, R~,, R~ 4 and n are as de~ined above
and then removing the amino-pro~ectlng groups ~rom
,
aid l-N~a¢ylat~d: compound ko produce the deslred
~.
compound of the ,~ormula tI) . ~iB proceB~ may be
~ollowed by the additional step of' i~ola~ing the
desired compound o~ the ~ormula (I) ~rom any
unde~i~?ed N~aoylated b~r-product~.
To prepare the compound o~ the ~ormula (IV)
havlng the blocked 6~-me~hylamino group and khe
optionally blocked 2'-amlno group which i~
.
i
--23-
: . . , . . : ,; ' ' . : . , ' , ....
, .
,, , ., , ; ,,

~ ` -
~S~3537
employed as the start~ng material in the process of
this inventlon~ the sompound of the formula (II) is
treated with a reagent whieh is commonly used in
the conventional synthesis of peptldes for the
purpose o~ introducing a known amino-protecting
group into the amino acld employed. Accordingly,
the amino-protecting group available in the proce~s
o~ thiæ inventlon ma~ be any o~ the known amino- -
proteeting groups which are commonly used in the
~ynthesis o~ peptides, as long as it is reaclily
removable from the acylated derivative VI as
produced in the acylation step of the present
process. When the acylated derivative having the
blocked amino groups is treated in a manner known
.,
for the removal o~ the amino-protecting group, the
amino-protecting groups used must be removed
readily without sub~tantlally af~ectin~ the a~ide
: linkage which has been formed between the a-hydroxy-
~-aminoalkanoyl radical and the l-amino group in the
said acylated derivative VI.
As sultable examples of the mono-valent amino-
protec~ing groups for Rl, R2, R3 and R4 ~n thlæ
in~ention, there may be mentioned an al~yloxy-
carbonyl group o~ 2-6 carbon atoms suah as ethoxy-
carbonyl, t-butoxycarbonyl and t-amyloxyparbonyl; a
~ycloal~yloxycarbonyl group of 4-7 carbon atoms
~uch aæ cyclop~ntyloxycarbonyl and cyclohexyloxycarbonyl;
-21~--
~ , . .
:~ . . . . . . . .

~ \ -
~5~537
an aralkyloxysarbonyl group such as benzyloxy
carbonyl and p-nltrobenzyloxycarbonyl; an aryloxy-
carbon~l group such as phenoxycarbonyl; and
~urfuryloxycarbonyl and an acyl group such as o~
nltrophenoxyacetyl and the llke. When a pair o~
the groups R3 and ~4 taken together ~orms a k~own
dl-valent amino-protecting group, th~ 3 di-valent
amino-protecting group may be a phkhaloyl group
or a sali.cylldene group, and generally an alkylidene
or:arylldene group o~ the ~ormula _CHR5 ln which R5
i8 an alkyl group of 1-6 carbon atoms ~uch as
methyl, ethyl, propyl, isopropyl, butyl or pentyl
or an aryl group such as phenyl, tolyl, p-me~hoxy-
phenyi~or o-hydroxyphenyl.
. . .: ~ ;
Suoh known mono-valent amino-protecting groups
as~a~lkyloxycar~onyl~ aralk~loxycarbonyl or aryloxy-
carbonyl group may be shown by a ~ormula -CO-0~6 ln
whioh R6 ls an;alkyl group of 1-5 carbon atoms such
as methyl5 ethyl~ t-butyl and t-amyl or a cycloalkyl
group o~ 3-6 càrbon atoms such as cyclopentyl and ~ -
oyclohexyl; an aralkyl group such as phenyl-alkyl
group containing the alkyl o~ carbon atom~ ~or
example, benzyl and p-nitrobenzyl; an aryl group
~uch as phenyl o~ a heterocy¢lic group such as
fur~uryl.
~or the prepara tlon o~ the amlno~protected
.
, .
: ~ -25-
':
, ,~ ~ , . . . .
,,:,

~50~37
.
6 ~ N-me thy 1-3 1 9 4 ~ -d ide oxykanamyc ln B der iva t ive
~ (IV) o~ such type in which the 6S-methylamlno
:~ group and the 21-amino group have been blocked
by a mono-valent amino protectlng group of
the ~ormula GO-OR6, 6 9 N-me~hyl-3',4'-
dideoxykanam~cin B (II) ma~ be reacted wlth
:
2-3 molar prop.or~ions of a ohloroformate of
the formula
Cl-CO-OR6 (VII );
.
or a p-nitrophenyl carbonate of the f'ormula
p-N~2-c6~s~0-co OR6 (V~
.
~ o r ~an N-h~droxysuccinimlde e~ter of the formula
: . : .
5
N O~CO-OR6 (VII ~ t );
'' .`' `. ~
", ' ' ` ` ~ 0
or an azldo~orma~e o~ the :f ormula
N~-aO-OR6 ~ (VI~
or an ,s 4, 6-dim~thylpyrlmid-2-ylthlocarbonate
; Or ~he ~ormula H C
. ~ 3 ~
~N
S-CO-OR6 (VII~ ");
~ ~ :
E3C
~6
.. . : , . . :
. .

S3~
wherein R6 is as defined above, in a suitable
solvent ~uch as water~ ethanol3 acetone, dimethoxy-
ethane or a mixture thereof under neutral or
basiç conditlons in a manner known in the prior
ar~ ~or the synthesis of peptides. me reaction
products so obtained usually consIst o~ a
mlxture o~ various amlno-protected derivatives of
the compound (II),the main compound of which is an
amino-prQtected der~ati~e in which the 6'-met;hyl-
amino group and the 2t-amino group have co~pletely
been b~locked by the prokective group -CO-OR6, and
a mlnor proportion o~ an amino-protected derlvatlve
in whiah only ~he 69-methylam:tno group has been
blocked~ plu8 small amounts of Imdesired amino-
protected derlvatives in which t;he l-amino group
. ~ .
: has been blocked.together with the blocked amino
group~ the co~npound (II) ls reacted with the
~: acylating rea~ent (VII), (VII~3, (VIIt ~3 or (VII~
.
in ~ubstantially equimolar proportior.s, the
proportion o~ the~amino-protected derlvatlve in
which only the 6~ methyla~ino group has been blocked
will be increased.
The most preferred amino-protectlng group~
are the t butoxycarbonyl group and benzyloxycarbonyl
group, as the~e are capable of rea¢ting preferentially
with the 6~-mekhylamino group and occaslonally with
.
-27-

i37
the 2' amino group of the 6' N-methyl-3',4'-
dideoxykanamycin B compound (II)~ and then being
readily removable ~rom the ao~lated derlvatives
VI which are produced in the acylation step o~
the present process.
For In~tance,2tN~6lN-di t-buto~ycarbon~l~6
N-me~hyl-3',4'-dldeoxykanamycin B~ a pre~era~le
~tartln~ rnaterial ~or the present process, may
be prepared ln a high ~ield by reacting 6IN-methyl-
794~dideoxykanamycin B in solution in a m~xtureo~pyridineJ water and triethylamine wlth a 2-3
molar proportion of t-butoxyca~bonyl az~de added
dropwise thereto wlth agitation, stirrin~the
resulting admixture at amblent temperature over-
night,~concentratin~ the reacti.on mlxture to dryness
, . .
~ C~l~ and then puri~ying the solid residue by
; :
,'colu,mn chroma~o~raphy, or alternative~ly b~ reactin~
6'N-mekhyl-3~,4~-d1deoxykana.nycin B in the ~orm o~
g aqueous so1ution with a 2-3 molar proport~on of
t-buk~l S-4 ,6 -d~methylpyrim1d-2-ylt11iocarbonate,
' . whlch 1~ added thereto under agitation,~stirring the
' ~ ~ resultin~ admixture at ambient temperature overnight,
' concentrat~ng the reac~lon m~xture to dryness in Y~~ :
and then puri~ing the ~olid residue by a column
chrolnatography. me puri~1cati.on o~ said solid
: residue by column chromatoæraphy ma~ be ¢onduct~d
:~ u~ing a cat10n-exchange resin having carboxylic
~unc~ion~, ~or e~ample, a copolymer of mèthacyclic
.
_~0
;, : '. . ; : .

~S~i3~7
acld with divlnylbenzene9 e.g., a product known as
"Amberllte" C~ 50 (ammonlum rorm; ~ommercfally
avallabl~ fro~ Rohm & Haa~3 U~S~Ao)~ me solld
residue obtained in the above proce~ure comprlse~
es~entlally the de~ired 2~N~6~N-di-t-butoxycarbo~yl-
6~N-methyl-3l,4~-dideoxykanamyc~n and 6~N-mono-t-
; butoxycarbonyl 6~N-methyl-3~,4~-dideoxykanamy~ln B .
and may directly be used a~ the raw mater~al ~or the
acylatio~ step o~ the pre~ent proces~
Wlth re~pect to the a-hydrQxy - ~-amlno acid (V)
which i~ employed as the acylat~ng agent ~n the
present pro~ess, the amlno-protecting
R3 group ~ay be those whlch are commonly used in the
: conventional ~ynthe~s o~ peptlde~ It i5 preferred
khat the am~no-protecting group present in the
acylatln~ agenk compound o~ the formula (V) be the
same as that present in the ~tarting amlno-
` protected 6~N-methyl-~,4'-dideoxykanamycin B
der~vative (I~. Preparation o~ the a-hydro~y~ amlno
acid reactant (V) of whioh the ~-amino ~roup ha~ been
blocked by a mono-valent amino-protecting group may ~ -
be ¢arried out in the ~ame manner a s ln the
preparation o~ the am~no-protected 6 'N-methyl-~ ', 4 '
dideoxykanamyoin B derivatlve (IV),. Where the group~
R~ and R~ taken to~ther form a di-valent amino-
prot~tlng group~ thi3 di-valent amino-protect~ng
group may preferably be a phthaloyl or ~allcylidene
group and may generally be an alkylldene or arylidene
* Trademark.
J -29-
.. ,.;.,.,.......................................................... ~
:-, , . . - . , , : . .. : .

537
group of the ~ormula -CHR~ in which R5 is an alkyl
group o~ 1 6 carbon atoms such a 8 methyl, ethyl,
propgl, lsopropyl, butyl or pentyl or an aryl group
such as phenyl~ tolylJ p-met~oxyphenyl or o-hydroxy-
phen~l. Preparation o~ the a-hydroxy-~-amino acid
reaçtant (V) in wh~ch the w-amino group h~s been blo~ked
by a di-valent amino-prokecting group o~ the ~ormula
_~HR5 rnay be carried out by alkylidenating or aryl-
idenating the W-amino group by reactlng the a-
hydroxy~ r.lino acld reactant V) with a substantially
equimolar proportion o~ an aldehyde of the formula
: :
,:
OHC-Rs (VIII3
~whereln R5 is as deflned above,, in a manner l~own
~n the preparation of Schi~f b~ses. Suitable
: ~ ~ aldehydes ~or this purpose include acetaldehydeJ
:
-. anisaldehyde, tolualdehyde, p-nitrobenzaldehyde
and ~alicylaldehyde.-
he a-hydroxy-~-ami~o acid oompound (V~ employed
: ~ ln khe present process~may either be in the ~orm o~
racemic m~xture or ln an optic~lly act~ve ~orm; the
,
L-iso~er and the D-isomer~ I~ is pre~erred, how-
ever, that a~hydroxy ~-aminobutyric acid which is
a compound o~ the ~ormula (I3I) where n is 2~ and
:a-hydroxy-~-alrlinovaleric:acid which is a conpound
- o~ the ro.rmula (III) where n is 3 should be in the
~orm Or the opkically active L-isomer, as the ~inal
product derived therefrom exhibiks a higher anti
. '
. . ~
.. . . .. .

S 37
bacterial activity than the ~inal product derived
~rom the D-isomer.
In the a~ylation step of the proGess according
to this lnvention, the amino-protectsd 6'N methyl-
3',4'-dideoxyka~amycin B derivative (IV) i~ reacted
with ~he orhydroxy~-amino acid reactant tv) ~n a
manner;known in the conventional prep~ration o~
amldes. ThUSJ the starting compound (IV) may be
reaoted wlth the acylating reagent (V) in solution
in anhydrous dimethyIformamide~ a~etone or tetra
hydrofuran under ice-cooIing and in the presence of
a dehydrating agent such as dic,~clohexylcarbodiimide.
Of cour~e, the~a-hydroxy~-amino acid reactant (V)
.
. ~ .. may ~ o be employed in the ~orrn of its functionally
equivaIent,..reactive derivati~e such as the acid
~ : :¢hlorid`e, the mixed acid anhydride, the active ester8:. ~ : . or the azide d.er~vatlve thereo~. For instance, the
a~hydroxy-~-amino-acid reactant tv) may ~irst be
reaoted wlth N-hydroxysucciimide in the presence of
dlcyclohexyl-carbodiimide as the dehydrating a~ent
t o prepare its active ester o~ the forrnula ~ -
, ~ , : :
COC-CE(CH)~(cH2)nN \
O ' 4
. .
V')
. ~hi¢h is, in turn, reacted wlth the starting compound
~ ~ ,
,
- .
~ ~; 1~ '
,.: : ': . ,. : . . . ; ' . ' :
' ' ' .. ' ''' ' ',' . ~., ,' : . .

.
3!L~5~3S3~
~I~) for khe N-acylation of the latter compound.
It is pre~erred that the starting compound (IV)
should be reacted in a 0.5 to 3 molar proportion
and preferably in a 0.5 to 1.5 molar proportion
of the actlve eæter form o~ the a-hydroxy-amino
acid compound (V') ln a reactlon medium con~isting
of water and an organic solvent such as dlmethoxy-
ethane,
.~ ,
In the acylating step of the present proce~s,
there is usually produced a mlxture of mixed N-
acylated derivatives of the starting compound (IV).
The mixture generally consists of a mixture of the
deslred lN-mono-acylated derivative and other
undesired mono-N-acylated derivatlves and undeslred
poly-N-acylated derivatives. m e mixture so
produced ~a~ then directly be tr~ated so as to
remove any amino-protecting groups therefrom: that
is to say, to convert the remaining amino~protecting
groups into hydrogen atoms, respectively.
The removal o~ the amino-protecting groups from
the above-mentioned mixed N-acylated derivatives
which are produced by the acglation step of the
pre~ent process ~ay be ef~ected in the ~ollowing
dif~'erent ways known per se, m us~ when the
amino-protecting group ts an alkyloxycarbonyl group,
æuch a~ t-butoxycarbonyl, an cy¢loalkyloxycarbonyl
-32-
, ''~ ' ' ' , ',' ' ,

37
group, aryloxycarbonyl group, alkylidene or
arylidene group, the removal of this kind o~
amino-prokecting group may be ef~ected by
sub~ecting the mixed N-acylated derivatives to
a mil~ hydrolysis treatment wikh an acid such
as aqueous tri~luoroacetic acid, aqueous acetic
acid and diluted hydrochloric acid. When the
amino-prokecting groups is an aralkyloxycarbonyl
group such as benzyloxycarbGnyl~ the removal of
thi~ type of amino-protecting group may be
ef~ected by sub~ecting the mixed N-acy~ated
der~vatives ko a hydrogenolysis treakment in the
presence o~ a palladium-carbon or platinum black
catalyst or to a treatment wit;h hydrogen hromlde
in acetic acid at low temperature, The o-nitro-
phenoxyacetyl amino-protecting group may be
removed by a reductive treatment. When the amino-
protecting group is phthaloyl group~ khe removal
o~ the phthaloyl group may be achieved by treaking
the mixed N-acylated derlvatives wikh hydrazine
hydrate in ethanol, When the N-acylated derivatives
con~aln di~erent kinds o~ amino-protecting groups?
~he N-acylated derlvatives ma~ be sub~ected to
simultaneous or successive kreatments to remove the
di~erent amino-protecting groups there~rom.
'
~33-

S~537
.
~ he removal o~ the remairling amlno-prote~tlnE;
group~ ~ive~ a mixture of the dif~erently N-acylated
product~ derlved from 61N msthyl-3'34'-dldeoxy- :
kanam~oln B. me mixture i~ Qompri~ed o~ the
d~slred :~ina l produc t 9 l~- (aohydroxy-~-amlnoa lk~noyl) -
6IN-methyl-5~,4l-dideoxykanaTnycin B, its positlon-
omer~ and the poly-N-acylated product~, together
with ~ome unreacted 6lN-methyl-3l~4~-dideoxykanam~rcln
}3. ma i~olatlon of` the des~red final product o~
the f'ormula (I3 may e~iciently be achieved by
~ubJectin~ said mixture t;o coluTnrl chromatograp~y
using9 ~or exampleJ silica gel or a ca~on-exchange
resin havlng carboxylic runctlon~, such.as
Amberlit~IRC 50 or Amberlite*CG 50 (a produot o~ -
Rohm & Haa~7 Co~, U.S.A~,), a w ;ak cation-exchanger
uch a~ CM Sephadex*C-25 (a produ¢k o~ Pha~nac~a
Co., Sweden) or CM-cellulo~e. me eluate ~rorn the
~h~omato~raphic proce~a iæ collected in ~ractions, ; :
and the antlbacterial actiYlty Or these fraction~ is - :
.. . . .
detected uslng sensitlve and resl~tant bacteria
as the tes~ mlcroorganism~. Through the detectlon
~ of the ankibacterial actlvity of each ~raction,
,A~ it 18 relatively ~lmple to determlne the active
~ractlon~ containing the de31red compound o~ the ~.
~ormula (I). A portion o~ these actlve ~ractions
were subJected to a thin layer ohromatography with
~ilica gel using~ for example, a ~olvent system
* Trademark~. .
~34~

537
of butanol-ethanol-chloroform-17% aqueous ammonia.
In this way, it was possible to de~ermine the
fractions wh~ch give a single 9pot at the speci~ic
R~ value of the desired 1 N-(a-hydroxy-~ amino-
alkanoyl)-6~ N-methyl ~34~-dldeoxykanam~cin B
o~ the ~ormula (I) and hence it contains ~olely
the desired product (I). Such fractlons may b0
oombined together and concentrated to dryness
under reduced pressure to recover the desired
.~ .
~ compound (I).
-
The new compounds of the formula (I) accordlng
to th~s invention are use~ul to therapeutically
treat bacterlal ln~ections as stated hereinbe~ore.
According to a third aspect of this invention, ~here-
ore, the~e i5 provided a pharmaceutical composition
f~or treatlng bacterial infections in livlng anlmals,
lnoluding man, which oomprises administering a
therapeut~cally e~ec~lve dose o~ a l N~ hydroxy-~-
aminoalkanoyl) 6i~ N-methyl-~',4~-dideoxykanamycin
B o~ the aforesàld formula (I); or a pharmaceutlcall~
acceptable acid-addition salt thereo~ as the active
.
ingred~ent in combination wlth a pharmaceuticall~
acceptable carrier ~or the active ingredlent.
"
, ~ ' :
, ~ ~ . ' ,
, .
:. .
~ ~~5~
. ` .
~ :, , .,, ., : . , ... . . . :

~L~5~53~7
This invention is now illustrated with references
~o the ~ollowing examples to which this invention i~
not llmited in any way,
Example 1
the
fo~
(a) To a solution o~ 930 mg. (2 mlllimoles) of
6lN-methy~ 4l-dideoxykanaTnycin B in 5 ml. of water
was added a solution o~ 960 mg. (4 millimoles) o~
t~butyl S 4,6-dimethylpyrimid-2-ylthiocarbonate in
5 ml~ o~ dioxane. The mixture was stirred overnigh~
at ambient temperature to effect the t-butoxycarbonyl-
ation, me reaction mixture was then concentrated
to dryness under reduced pressure ko give a solid
residue. m is solid was taken up into 40 ml. o~
waker and the insoluble matter was ~llt,ered o~.
The solutlon (the f'~ltrake) was passed through a
column (20 by 290 m~ of 100 ml, of a cation-
.. . .
exchange re~ln, Amberlite CG 50 (NH4 form) to ef`~ectthe adsorption of' the t-butoxycarbonylation products
by the resin. The resin column was washed with
water (50~ ml.) and then was eluted with O~lN aqu~ous
ammonia, Such ~ractions o~ the eluate whicll were
positive to the ninhydrin reactlon and to the
Rydon-Smith reactiorl and whlch also gave a mal~ ~pot
-~6-
,
,
.

~ 5~537
at RF o.60 by thin layer chromatography on silica
gel using butanol ekhanol-chloroform~17% ammon~a
(4 5:2:3 by volume) as a developing solvent were
co~bined together and concentrated to dryness
under reduced pressure, affording 538 mg. of a
colorless powder mainly comprising 2'N,6'N-di-t-
butoxycarbonyl-6'N-methyl-3',4'-dideoxykanaTnycin
B. Yield 41%.
(b) m is colorless powder (100 mg.) mainly
comprising ~'N,6'N-di-t-butoxycarbonyl-6'N-methyl-
3'J4'-dideoxykanamycin B (0.15 millimole) was
dissolved in a mixture of 1 ml, of water and 1 mlO
of dimethoxyethane, and to the resulting solution
was added a solution of 54 mg. (0.17 milli~ole)
of N-hydroxysuccinimide ester of L-4 t-butoxy-
carbonylamido-2-hydroxybutyric acid in Z ml. o~
: .
dimethoxyme~hane. me mixture ~las stirred for
22 hours at am~lent temperature to effect the
aoylation of the am~no-protected 6'N-methyl-~,4~-
dldeoxykanamycin B makerial. The reactlon mixture
wa~ then conoentrated to dryness under reduoed
pressure to a~ord a solid re~idue comprising the
mlxed N-acylated derlvative~ of the N-protscted
6IN-methyl-3~,4~-dideoxykanamycin B materlal.
(c) The solid residue ~rom step b wa~
di~301ved in 2.4 ml. o~ aqueous 9 ~ trifluoro-
.
.
-37~
. , .

~`:
537
acetic acid and the solution was allowed to stand
for 1 hour at ambient temperature to e~ect the
removal o~ the t-bukoxycarbonyl group. me
reaction mlxture was concentrated ko dryness under
reduced pressure~ and the residue was taken up into
4 ml. of water. The ~olution was ad~usted to pE 8
by the addition o~ concentrated aqueous ammonia
and then passed through a colu~n (8 by 400 mm) of
20 ml. of a cation-exchange resln, Amberllte CG 50
(NH4 ~orm) to effect the adsorption o~ the mixed
,
N-acylated 6IN-methyl-3~,4l-dideoxykanamycin B
products by ~he resin. A~ter the resin column
~ .
was washed successively with 100 ml. of water,
with 100 ml. Or 0.3 N aqueous ammonia and with
0.5 N aqueous ammonia, the resirl column was eluted
with 0.75 M aqueous ammon~a. The eluate was
,
~ collected ln 2 ml. ~rac~ions, and ever~ ~raction
.... .
was~tested accordlng to a u~ual plate method ~or
the~r antibacterial activlty against the kanamycln-
"
sensitive straln a~g~Jah~i sub~til~s PCI 219 and
kanamycln-resiskant strain _o~3~h~ia coli
JR66/W677. Those ~ractlons which showed~hi~h
~antibacterial activity agaln~t both the above-
mentioned ~train3 were combined kogether (to a
volume o~ 26 ml.) and then concentrated to dryness
.
to g~ve 39 mg. o~ a colorless powder mainly com-
prising the desired productl 1 N-(L-I~-amino-2-
hydroxybutyryl)-6'N-methyl-3~,41-dideoxykanamycin B.
.
-38-
.
. . .
, , : , -,

3~
For ~urther puri~lcatlon, this colorless powder was
dissolved ln 0,5 ml. o~ methanol-chloro~orm-17
aqueous ammonia (4-1:2 by volume ) ~ and the
resultlng solutlon was subjected to column chroma-
tography on ~ gO of silica gel using methanol-
ohloro~orm-17~ aqueous ammonla (4:1:2 by volume)
as the e:Luant. The eluate was collected in 1 ml.
fractions, and ~raction Nos. 46-78 were ~ound to
contain solely 1 N-(L-4 amino-2-hydroxybutyryl)-6'N-
methyl-3',4'-dideoxykanamycin B which ga~e a single
spot of R~ 0.38 in a thin layer chromatography on
sil1ca gel ("ART" 5721) usin~ butanol-ethanol-
chloro~orm-28% aqueous ammonia (4:5:2:8 by volume)
., .
as eluant. These fraotions were combined together
and concentrated to dryness to glve 15 mg. of pure
1 N-(Ir4-amino-2-hydroxybutyryl)-6'N~methyl-3',4'-
dideox~kanamycin B as a colorless powder,
.
Decomposltion point; 158-161 C.
~X~m~3,Q~
~LQf ~ (p~Ir~isos-eryl)-6-~N-meth~
. , .
~ _h~yæ~ L~
- (a) The colorless powder (403 mg.) mainly
comprising 2'N,6'N-di-t-~utoxycarbonylw6'Nwmethyl-
3',4'-d1dsoxykanamycin B (o.6 milllmole) which was
obtained in ~xample l(a) was dissolved in a mi~ture
o~ 4 ml. o~ water and 4 ml~ o~ dimethoxyethane.
.~ .
39~
": ,. . .. . ... . .
: . ~ , . . . .. .. .. .
., ,~ . .. . . . . . . . .
. .

~S~i3~
The solution ~o obtained was admixed with a
solution o~ 221 mg. (o,66 millimole) of N-
hydroxysuccinlmide ester o~ N-t-butoxycarbonyl-
D~ oserine in 8 ml. of dimethoxyethane. The
admixture was stirred ror 2~,5 hours at ambient
temperature to ef~ect the acylation. The reaction
mixture was then concentrated to dryness under
reduced pressure to g~ve a solid residue malnly
comprising the mixed N-acylated derivatlves of
2~N,6~N-di~t-butoxycarbon~J1~6'N~methyl-3~,4'-
dldeoxykanamycin Bo
(b) Thls solid residue product was dissolved
in 7~.5 ml. of aqueous 90~ trl~luoroace~ic acid,
and the solution was allowed to stand ~or 1 hour
at amkient ~emperature to e~ect the removal
of the t-butoxycarbonyl group. me reaotion
mixture was concentrated to drynes~ under reduced
: ~ -
pressure, and the residue was dissolved in 16 ml.
of water. The~aqueous solution so obtalned wa~
ad lsted to PE 8 by aùdition of cOnoentrated
aqueous ammonia and wa ~then passed through a
¢olumn ~10 4y 560 mm) o~ 43 ml. of a oation-
exchange resln, Amberlite CG 50 (NH4 ~orm) to
e~re¢t the adsorpt~on o~ the mixed N-acylated
product~. The re~in coJ.umn was washed wlth 200
ml, o~ water and then with 400 ml. of 0.3N aqueous
`
~ -40-
: . ~ .. . . . . .

37
ammon~a and was subsequently eluted with 0.5 N
aqueous ammonia~ The eluate was collected in
4 ml. ~raction~i9 and every ~raction was tested
according to a usual plate method ~or their
antibacterial activity against ~ L kLLi subtl~
PGI 219 and jE~ richia coli JR66~W677. The fractions
which showed a high antibacterial activity against
, .
these two strains were combined together (to a
volume o~ lO0 ml.) and then concentrated to
dryness to give 135 mg. of a colorless powder
; mainly comprislng the desired product, l N-(DL-
. . -
isoseryl~ 6IN-methyl-3~49-dideoxykanamycin B.
:
~ur ~urther puri~ication, khls colorless powder
was dissolved in 2.6 ml. of methanol-chloroform-
; 17% agueous ammonia (4:1:2 by ~olume), and theresulting solution was subjected ko a column
chromatograph~ on sllica gel (8 g.) using
~ , .
methanol~ohloroform-17% aqueous ammonia
(4:1:2 by volume) as eluant. The eluant was
. ~
collected in 2 ml. ~ractions, and fraction Nos.
; 20-27 ~ were ~ound ~o oontain solely the desired
product which gave a single spok o~ 0.51 in a
. ,
thin layer chromatography on silica gel ("ART"
5721) using bukanol-ekhanol-chloro~orm-28~ aqueous
ammonia (4:5:2:8 by volume) as eluank. These
frac'G~on~ were combined togethe~ and concentrated
to dryness to give ~8 mg. o~ pure lN~(DIrisoseryl)-
6lN-methyl-3~4~dideoxykanam~cin B as a colorless
~ 1
.,; i . . . . .

L)f i37
powder Decompo~itlon poin.t; 165-169 C.
B~maL~
where n ls 1
(a) ~Butoxycarbonyl azide (465 mg.; ~.~
millimoles) wa~ added to a ~olution o~ 500 mg.
l.l millimoles) o~ 6'N-meth~ ',4~-dideoxy-
kanamyoin B in a mixture o~ 21 ml. of pyridlne,
.~ 21 ml. o~ triethylamine and 12.6 ml. o~ water
. . . .
~ The mixture wa~ stirred overnight at ambient
: .
temperature to ef~ect the t-butoxycarbonylation~
:me reaction mixture was concent;rated to dryne~s
~ ~ ; u~der reduced pressure, to a~for-d 7Z7 mg. of a
colorle~s powder ~alnly compri ing a.m~xture o~
N,~'N-di-t-butox~carbonyl-6'N-methyl-3',4'-
:dideo~ykanamycin B and 61N-t-butoxycarbonyl-6
meth~1~3~,4'-dideoxykanam~cin 3.
(b) The above colorles~ powder (510 mg ~
mainl~compri~ing a mixture o~ the partly~amino-
~:protected derivatives or~6~N-methyl-3~,4'~dideoxy~
kanamyo~n ~ was, without purification thereo~,
;di~olved in a mlxture o~ 5 Tnl. o~ water and
: 5 ml. of dtmetho~yethane. To the re~ulting
~a~ution wa~ added a 301ution o~ 254 mg. (o.84
millimole):o~ N-hydrox~succinimida e~tar of N-t-
butoxycarbon~l-L-iso~e.rlne in 10 ml. o~
,~ .
.,
.
.

~ 51~ i37
dimethoxyethane. The mixture was stlrred ~or 19
hours at ambient temperature to e~ect the
acylation, The reaction mixture was concentrated
to dryness under reduced pressure to give 794 mg.
o~ a solid residue comprislng the mixed N-acylated
derlvatlves of 2'N,6'N-di~t-butoxycarbonyl- and
6'N-t-~utoxycarbonyl-6lN-methyl-3',4'-dldeoxy-
kanamyoin B.
(c) The solid residue product was treated
.
with aqueous 90% tri~luoroacetic acid for the
: removal o:~ the t-butoxycarbonyl group, and was
then sub~ected to the puri~ication by column
:~ chromatography with Amberlite CG 50 and ~ub-
- sequently to purl~ication by column chromatography
on silica gel in the same manner as in ~xample
~i 2(b)~ Pure IN~(1risoser,~1)~6.'N-methyl~3~,4~-
- ~ dideoxykanamycin B was obtained as a colorless
powder~ ~ield 34 mg. Decomposition point;
162-166 C0
~ a~E~Q-~
1,
~e
(a) The colorle~s powder ~510 mg,) mainly
comprising a mixture o~ 2~N~6'N-di-t-butoxycarbonyl- .
6IN-methyl-3',4~-dideoxykanamycin B and 61N-t-
butoxyc~rbonyl-6~N~methyl-3',4'-dideoxykanamycin B
-43
, . , , . , , . . ~
,-, " ,,,, , . ' :,, ' . ,, ~ . ,'. ' ,
. .

53'7
which was prepared in the same manner as ln
Example 3(a) wa~g without purlflcation thereof,
di~solved ln a m~xture of 5 ml, of water and
5 ml. of dimethoxyethane. To the resulting
solutlon was added a solution of 278 mg.
(0.84 milllmole) o~ N-hydroxysuccinimide ester
o~ I~5-t-~utoxycarbonylamido-2-hydroxyvalerlc
acid in 10 ml. o~ dimethoxyethane. The mixture
was stirred ~or 18 hours at ambient temperature
to~efPect the acylationO The reaction mixture
was concentrated to dryness under reduced pres~ure
to gi~e 834 mg. o~ the solld residue comprising
the mixed N-acylated derivatives o~ the 21N,6'N-
di~t-butoxycarbonyl- and 6~N-t-butoxycarbonyl-6lN-
methyl~ dldeoxykanamyclns B.
(b) The solid re~idue product was dissolved
in 8 ml. of a~ueous 90~ trifluoroacetic acid9 and
the solution was allowed to stand for 1 hour at
ambient temperature to e~fect the removal of the
t-butoxycarbonyl group. The reaction m~xture was
concentrated to dryness under reduced pressur~
an~ the resldue ~o obtained was taken up into
16 ml~ o~ water. The resultant aqueous solution
wa3 ad~usted to pH 8.4 by addltion of concentrated,
aqueous ammonia and was then adsorbed on column
(10 by 560 mm) o~ 40 ml. of Amberlite CG-50 resin
'
-4J~-
. .

l~S3~
(NH4 form). A~ter the reæin column was washed
successivel~ with 200 ml. o~ water, with 250 ml.
o~ 0,3 N aqueous ammonia and with 240 ml. of
0~5 N aqueouæ ammonia3 the res~n column was
eluted with 0.75 N aqueous ammonla. The eluate
was collected ln 4 ml. fractions, and such
fractions whlch ~howed a hlgh antibacterial
activity against Bacil,lus ~Y~ PCI 219 and
E~Q~lchi~ ~oli JR66/W677 were dekected,
combined together (to a volume o~ 60 ml.),
concentrated to dryness and then chromakographed
in a slllca ~el column ~n khe same man~er as ln
Example l(c)~ ,Pure lN-(L-5-amino-2-hydroxyl- :
va1eryl)-6~'N-methyl-3'~4~-dldeoxykanam~cin B
was obtained as a colorless powderO Yield 29 mg.
Decomposition point; 152-155 C0
~ ,
. , .
., ~ .
:
' , . .
., .
-45-
,, " ",; ,... ; .. , ... , ;,, .: , . . , .. , ,.,.. .. , :, . ..