CA1053666A - 6"-deoxykanamycin b and 6"-deoxytobramycin - Google Patents

Abstract

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.

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 ~ 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' HO ~ 4 NH

O //~_ NH2 6"
HO

NH ~

-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]

HO \~ r~ \ NHAc Ac ~ ~ ~
H ~ \
/ ~ HAc 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_ HO ~~

--~Q\
AcNH`1, ~

~iO ~ . . \
/ ~--Na~c ..

\CH2 ¦
H~ ~
~ ~~ 1 4 Ac~ Hl \~

(4) Compound 4 H2/Ni ~c, ~ , Ac~

NHAc 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 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.

-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 _ ~,~

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.

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

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~.

Claims (10)

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 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 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 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 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 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 5, 6 or 7, or by an obvious chemical equivalent thereof.