6"-DESOXYKANAMICIDE ET 6"-DESOXYTOBRAMYCINE

6"-DEOXYKANAMYCIN B AND 6"-DEOXYTOBRAMYCIN

Abrégé anglais

6"-DEOXYKANAMYCIN B AND 6"-DEOXYTOBRAMYCIN
Abstract of the Disclosure
There are disclosed two new antibiotics,
6"-deoxykanamycin B and 6"-deoxytobramycin. These
compounds are highly active, broad spectrum antibiotics.

Revendications

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of 6"-deoxykanamycin B and
6"-deoxytobramycin from kanamycin B which comprises the
following sequential steps:
(A) treating kanamycin B with a large molar excess of
an acetylating agent selected from the group consisting of
acetic anhydride in a lower alkanol or acetyl chloride in
conjunction with a base to take up free HCl to form penta-
N-acetylkanamycin B;
(B) treating said penta-N-acetylkanamycin B with p-
toluenesulfonylchloride in the presence of a tertiary amine
selected from the group consisting of pyridine, triethylamine
or dimethylaniline to form the 3', 6"-ditosylate derivative
thereof;
(C) reacting said ditosylate derivative with a member
selected from the group consisting of sodium iodide, lithium
iodide, sodium bromide or lithium bromide in a solvent system
comprising
i) a compound selected from the group consisting
of N,N-dimethylformamide or a ketone having the formula
<IMG> wherein R' and R" are alike or different and each
is an alkyl group containing up to 6 carbon atoms; or
ii) hexamethylphosphortriamide in an aromatic hydro-
carbon selected from the group consisting of toluene, benzene,
xylene or mixtures thereof, at reflux temperature;
(D) hydrogenating the 3', 6"-di-iodo or 3', 6"-di-bromo
derivative obtained in step C) in the presence of a hydro-
genation catalyst; and
(E) hydrolyzing the product obtained in step D) to remove
the acetyl groups and to produce 6"-deoxykanamycin B and
16

6"-deoxytobramycin.
2. A process as defined in claim 1 wherein said
base used to take up free HCl in step A is triethylamine
or pyridine.
3. A process as defined in claim 1 wherein said
lower alkanol in step A) is selected from the group consist-
ing of methanol, ethanol, n-propylalcohol, isopropyl alcohol,
n-butyl alcohol, sec-butyl alcohol, iso-butyl alcohol and
t-butyl alcohol.
4. A process as defined in claim 1 wherein said
ketone in step C) is a member selected from the group con-
sisting of acetone, methyl isobutyl ketone or methyl ethyl
ketone.
5. A process as defined in claim 1 wherein said
hexamethylphosphortriamide in step C) is present in an
amount of about 1.1 mole per mole or sodium iodide, lithium
iodide, sodium bromide or lithium bromide.
6. A process as defined in claim 1 wherein said
3', 6"-di-iodide or 3', 6"-di-bromide obtained in step C)
is isolated in the form of an aqueous solution.
7. A process as defined in claim 1 wherein said
hydrogenation catalyst is a member selected from the group
consisting of Raney nickel, palladium, platinum, rhodium
or ruthenium.
8. A compound having the formula
<IMG>
17

in which R is hydrogen or OH; or a nontoxic pharmaceutically
acceptable acid addition salt thereof, whenever prepared or
produced by the process of Claim 1, or by an obvious chemical
equivalent thereof.
9. A compound having the formula
<IMG>
in which R is hydrogen or OH; or a nontoxic pharmaceutically
acceptable acid addition salt thereof, whenever prepared or
produced by the process of Claim 2, 3 or 4, or by an obvious
chemical equivalent thereof.
10. A compound having the formula
<IMG>
in which R is hydrogen or OH; or a nontoxic pharmaceutically
acceptable acid addition salt thereof, whenever prepared or
18

produced by the process of Claim 5, 6 or 7, or by an obvious
chemical equivalent thereof.
19

Description

sackqrouna o~ the In~ention
~ b~ .er ~ This invention relatesto
two novel semi-synthetic derivatives of kanamycin B, i.e.,
6"-deoxykanamycin B and 6"-deoxytobramycin These compounds
are prepared by dehydroxylation of the 6"- and 3',6"-positions
of Kanamycin B.
2) Description of the Prior Art: Kanamycin B is
a known antibiotic described in Merck Index, 8th Edition,
pp. 597-598. Kanamycin B is a compound having the
formula
~H2~H2
Ei ~
H ~ \ ~H2
NH2 ~ ~ ~
HO ~- ~ \
\ ~ ~^2
CH20~ /
H ~ ~ ~ 1
H2N ~ j!
_l ~ .

~053666
Tobramycin is also a known antibiotic which
chemically is known as 3'-deoxykanamycin B. This
compound is described by K. F. Koch and 3. A. Rhoades
in Antimicrobial A~ents ~nd Chemotherapy, Pages
309-313 (1970) as having the formula
~2NH2
H0
~ I

10536f~6
Summary of the Invention
The compounds having the formula
H~NH2
O Ny2
~\0/ ~ ~ N~2
HO ~ O
~12N ~"C/
in which R is hydrogen or OH or the nontoxic pharmaceutically
acceptable acid addition salt thereof are valuable antibac-
terial agents.
This invention relates to semi-synthetic deriva-
tives of kanamycin B, said compounds being known as 6n-deoxy-
kanamycin B and 6n-deoxytobramycin and having the formula
6'
CH2NH2
HO ~ 4 NH
O //~_ NH2
6"
HO
NH ~
HO 1 V
-3-

lQ53666
wherein R is hydrogen or OH; or a nontoxic pharma-
ceutically acceptable acid addition salt thereof.
For thè purpose of this disclosure, the term
"nontoxic pharmaceutically acceptable acid addition
salt" shall mean a mono-, di-, tri-, tetra- or pentasalt
by the interaction of 1 molecule of 6"-deoxykanamycin B
or 6"-deoxytobramycin with 1-5 moles of a nontoxic,
pharmaceutically acceptable acid. Included among
these acids are acetic, hydrochloric, sulfuric, maleic,
phosphoric, nitric, hydrobromic, ascorbic, malic and
citric acid, and those o~her acids co~monly used to
make salts of amine containing pharmaceuticals.
T.he compounds of the present invention are
prepared by the following diagrammatic scheme:
HO ~ CH2NH2
H ~ ~
; ~ 2
(1) / acetic anhvdride~
/
/
N~2 ~ ~ /
~kanamycin B]
--4--

1053666
HO \~ r~ \ NHAc
Ac ~ ~ ~
H ~ \
/ ~ HAc
CH2H
H~ ~__O
~_-~ / 2
AcNH ~ \J
(2) Compound 2 p-toluenesulfonyl>
chloride
CH2NHAc
~(f~ /
H3C~S02~
Ac ~ NHAc
`
~ Ac
H3C ~ ~ CH2
HO ~ ~ 3
Acl~ HO
(3) Compound 3 ~odium~
iodide
_5_

1053666
CH2NHAC
HO ~~
--~Q\
AcNH`1, ~
~iO ~ . . \
/ ~--Na~c
..
\CH2 ¦
H~ ~
~ ~~ 1 4
Ac~ Hl \~
(4) Compound 4 H2/Ni
~c, ~ ,
Ac~
NHAc
SH3
I
AcN H \~
(5 ) Compound 5 Ba (OH) ,. . 8H20

~053666
ÇH2NH2
HC~ o
\~\ NH2
NH2 ~
HO~_ 7~NH2
~H3 / 6
H~
~ I
AcN~ OH
and
HO~ f H2NH2
\~
~\
~H2
~H2
CH3
H ~ /
N~2 O

105~ i6
It will be noted that step 5 in the diagrammatic
scheme shows the production of 6"-deoxykanamycin B as
well as 6"-deoxytobramycin. The exact mechanism by which
6"-kanamycin B is obtained is not known since one would
expect from the preceding steps to obtain only 6"-deoxy-
tobramycin. However, it is hypothesized that in
step 2, some 6"-monotosylate of penta-N-acetylkanamycin
B may be obtained, in which case, some 6"-iodopenta-N-
acetylkanamycin B would be obtained in step 3 and, in
step 4, some penta-N-acetyl-6"-deoxy~anamycin B would
be obtained. However, the analytical data does not
confirm the presence of such derivatives in these steps.
The objective~ of the present invention have been
achieved, by the provision according to the present
invention of the process for the preparation of 6"-
deoxytobramycin and 6"-deoxykanamycin B: or a nontoxic
pharmaceutically acceptable acid addition salt thereof;
which process comprises the following consecutive steps:
(A) Kanamycin B is treated with a large molar
excess of an acetylating agent, e.g., acetic anhydride
in a lower alkanol orwith acetyl chloride in conjunction with
a base to take up free HCl, such as triethylamine or pyridine.
By "lower alkanol" is meant an alkanol having up to 4 carbon
atoms, e.g., methanol, ethanol, N-propyl alcohol, isopropyl
alcohol, N-butyl alcohol, sec-butyl alcohol, iso-but~l
alcohol and t-butyl alcohol.

-
1053666
Preferably, at least 5.1 moles of acetylating agent are used
per mole of kanamycin B and the reaction is conducted at
a temperature below 25C, and, more preferably, at about
room temperature. The product obtained by the treatment
of kanamycin B with the acetylating agent is penta-N-
acetylkanamycin B.
(B) Penta-N-acetylkanamycin B is treated with
p-toluenesulfonyl chloride in the presence of a tertiary
amine chosen from the group consisting of pyridine, triethyl-
amine and dimethylaniline. Preferably, at least 2.0 molesof p-toluenesulfonyl chloride per mole of penta-N-acetyl-
kanamycin B are employed and the reaction is conducted at a
temperature below 25C. and, more preferably, at about room
temperature.
(C) The tosylate derivative product obtained in
step (B) is reacted with a halide selected from the group
consisting of sodium iodide, lithium iodide, sodium bromide
or lithium bromide in an appropriate solvent system. The
solvent may be a compound selected from the group consisting
of N,N-dimethylformamide, or a ketone having the formula
R'-~-R" wherein R' and R" are alike or different and each is
an alkyl group containing up to 6 carbon atoms. Examples of
such ketones include acetone, methyl isobutyl ketone and
methyl ethyl ketonR. An alternative solvent system is
hexamethylphosphortriamide in an aromatic hydrocaxbon selected
from the group consisting of toluene, benzene, xylene or
mixtures thereof, at reflux
_ g _
~,~

1053666
temperature. About 1.1 mole of hexamethylphosphortriamide
should preferably be used in this system per mole of sodium
iodide, lithium iodide, sodium bromide or lithium bromide.
Preferably, at least 6 moles of halide per mole of tosylate
derivative are used and the reaction is conducted at a
temperature above 80C., and, more preferably, at a temperature
of from 100 to 125C.
(D) An aqueous solution of the 3',6"-di-iodo or
3',6"-di-bromo product obtained in step C is hydrogenated in
the presence of a known hydrogenation catalyst, e.g. Raney
nickel, palladium, platinum, rhodium or ruthenium. The hydro-
genation reaction is preferably conducted at about room
temperature.
(E) Hydrolyzing an aqueous solution of the product
obtained in step B, preferably by heating in the presence of a
strong base, e.g. sodium hydroxide, lithium hydroxide,
potassium hydroxide, or barium hydroxide, to remove the acetyl
groups and to yield a mixture of 6"-deoxykanamycin B and
6"-deoxytobramycin.
The compounds of this invention are valuable as anti-
bacterial agents, nutritional supplements in animal feeds,
therapeutic agents in poultry and animals including man, and
are especially valuable in the treatment of infectious diseases
caused by Gram-negative bacteria. They are effective in the
treatment of systemic bacterial infections when administered
parenterally in the dosage range of about 250 mg. to about
3000 mg. per day in divided doses three or four times a da~y.
Generally, the compounds are effective when administered at a
dosage of about 5.0 to 7.5 mg./kg. of body weight every 12 hours.
-- 10 --

1053666
EXAMPLES
Example
Penta-N-acetvlkanamYcin B (2)
To a stirred suspension of 40.0 g. (0.0827 mole~
of kanamycin B in 800 mL. of methanol was added dropwise
117.2 ml (1.24 moles) of acetic anhydride. Before all
of the acetic anhydride was added, a clear solution was
obtained but almo~t immediately thereafter a precipitate
began to form. Stirring was continued for 16 hours after
completing the addition of acetic anhydride. During this
time, the reaction mixture solidified and several hundred
ml. of methanol were added in order to obtain a filterable
mixture. ~he damp solids were suspended in 800 ml. of
fre~h methanol and the suspen~ion was stirred for one hour.
~he solids were collected by filtration and were then stirred
with 800 ml. of methylene chloride for 1.5 hours. The
product wa~ then collected by filtration, rinsed with
methylene chloride and thoroughly dried to give 44.4 g.
(77.4% yield) of 2 in the form of a crystalline solid
having a melting point of 340-343C~
Anal. Calcd. for C28H47Nsl5
N, 10.09.
Found: [Corrected for 1~37Yo H20 fou~d):
C, 48.62; H, 6.83; N, 10.13.
The infrared and NMR spectra were consiste-_ with
the structure 2.

~053666
Example 2
Penta-N-acetylkanamycln B-3',6"-ditosylate (3 ?
A mixture of 1.000 g. of penta-N-acetylkanamycin B
(1.442 mmole) and 1.645 g. of p-toluenesulfonylchloride
~8,649 mmole) in 10 ml. of anhydrous pyridine was stirred at
room temperature for 23 1/4 hrs. The reactants dissolved
slowly and were completely dissolved in 5 hours. After 5
hours, the solution was orange in color and the color did not
change during the remainder of the reaction. The reaction
mixture was poured into 200 ml. of methylene chloride and
the resultant precipitated creamy-white product was dried
thoroughly. It was then suspended in 30 ml. of ~ethylene
chloride and heated at reflux fbr two minutes. The insoluble
solid was recovered from the hot solvent by filtration and
thoroughly dried over P2O5 to give 1.043 g. (72.2% yield) of
3. The infrared spectrum showed typical tosylate bands at
1180 and 1220 cm.-l. A sample of the product was subjected
to liquid chromatography by injecting 35 microliters of a
suspension of 1 ml. of the product per ml. of a solution con-
sisting of 30~ water and 70~ methanol onto a tandem ~roup
of four 1/8 in. x 2 ft. columns. The columns were packed
with C18/CORASIL I, a monomolecular layer of octadecyl-tri-
chloro-silane chemically bond~d to a porous silica layer on
a solid glass bead core. It was found that the product
consisted of a very minor oomponent with a retention time of
16 1/2 minutes and a major component with a retention time
of 19 3/4 minutes.
Anal. Calcd. for C42HsgNsOlgS2: C, 50.34; H, 5.93;
N, 6.99; S, 6.40.

~053666
Found (corrected for 2.66% moisture found):
C, 48.67; H, 5.64; N, 6.83; S, 5.87.
Example 3
Penta-N-acetYl-3',6"-diiodokanamYcin B (4)
A mixture of 834 mg. of the product obtained in
Example 2 (0.832 mmole) which had been dried in vacuo over
P2O5 at room temperature, and 1.998 g. of sodium iodide 113.3
mmole) which had been air dried in an oven at 110C. for 20
hours, in 1.5 ml. of N,N-dimeth~lformamide was placed in a
60 ml. heavy-walled glass vial which was purged with N2 and
heated at 100C for 23 hours. The vial was then opened,
cooled and filled with ether. After sever~l hours, the ether
was decanted and the residue was heated on a steam bath until
a thick syrup was formed. The vial was then filled with
dioxane, heated to 90C. and the inso~uble solids, which
included the desired product, were recovered by filtration,
rinsed with ether and dried over P2O5 in vacuo. The recovered
solids were de-salted by dispersing them in 15 ml. of water
and then removing the water insoluble solids by filtration
over a bed of diatomaceous earth. The filtrate was passed
over an 800 ml. column of Sephadex G10, a cross-linked dex-
tran having a molecular weight retention of up to about 700.
The column was pumped at 1 ml./min. and 27 ml. fractions
were collected. The product was determined to be in tubes
11-21. The components of these bubes were combined, reduced
to a volume of 20 ml. by evaporation at 50C. and the product,
504 mg. of 4 (66.3% yield), was recovered therefrom by
lyophyllization. This product was used directly in the next
example without further purification.

~053666
Example_ 4
Penta-N-a~y~ _-deoxytobramycin ~5)
An aqueous solution of 388 mg. of the product
obtained in Example 3 (0.425 mmole) in 60 ml. of water was
hydrogenated for 21 hours in the presence of about 4 gm. of
Raney nickel catalyst at room temperature and at a hydrogen
pressure of 44 psi. The catalyst was then filtered off and
washed thoroughly with water. The combined filtrates were
evaporated to dryness at 50C. and dried over P2O5 in vacuo
to give 180 mg. (63.1% yield) of 5.
Example 5
6''-Deoxytobramycin (6) and 6"-deoxykanamycin B (7)
A mixture of 180 mg. of the product obtained in
Example 4 (0.272 mmole), 2.55 g. of Ba(OH)2.8H2O (8.08 mmole)
and 10.2 ml. of water was heated at reflux for 17 hours.
The reaction mixture was then diluted to 35 ml. by the addi-
tion of water and solid carbon dioxide was added until the
pH fell to 7Ø The resultant BaCO3 was removed by filtra-
tion over fine filter paper and washed with a few ml. of
water. The combined filtrates were flashed down to 10 ml.
and then lyophyllized. The crude product weighed 170 mg.
The product was dissolved in 2.00 ml. of water for analysis
by thin layer chromatography on a column of silica gel SllO

1053666
using the solvent system H20:CH30H: conc. NH40H:CHC13
(1:4:2:1). The analysis showed that kanamycin B had an
Rf - 0.52, while the two major ninhydrin-positive components
of the product mixture had Rf = 0.47 and Rf = 0.61. When
a duplicate plate was overlaid with an agar plate seeded with
Bacillus subtilis at a pH of 8 and incubated overnight at
37C., large zones of inhibition were found at Rf = 0.47
and Rf = 0.61. A 0.100 ml. aliquot of the 2.00 ml. solution
of the crude product was diluted to 5.00 ml. with 0.1 N phos-
phate buffer having a pH of 8.0 and submitted for a differen-
tial bioassay for tobramycin-like and kanamycin B-like ac-
tivities. The yield of 6"-deoxytobramycin is determined
from the bioassay data to be about 7.1% and the yield of
6"-deoxykanamycin B is calculated from the bioassay data
to be about 1.5~.
-15-