CA1087612A - 6'-n-methyl-xk-88-5 and process for the production thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A new semi-synthetic antibiotic, 6'-N-methyl-XK-88-5, is produced by chemically modifying the antibiotic XK-88-5.

Description

lZ

BACKGROUND OF THE INVENTION
The present invention relates generally to a deri-vative of the antibiotic XK-88-5 and more specifically to a new semisynthetic antibiotic, 6'-N-methyl-XK-88-5 and a method for the production thereof.
XK-88-5 is one factor of a series of related antibiotic compounds produced by culturing a microorganism such as Strepto-myces hofuensis, ATCC 21970 under suitable conditions. XK-88-5 and the fermentative production thereof is described in United States Patent No. 3,939,043 issued February 17, 1976. The XK-88 series of antibiotics are also known as the Seldomycins.
Although XK-88-5 (Seldomycin Factor-5) exhibits good antibacterial activity, certain bacterial have the ability to enzymatically inactivate the base compound. It has now been found that a particular derivative of XK-88-5 is not suscep-tible to such enzymatic inactivation.
SUMMARY OF THE INVENTION
The present invention relates to a new derivative of the antibiotic XK-88-5, that is, 6'-N-methyl-XK-88-5 represented by the formula:

.1 ~ \ NH2 HO - ~ \ ~ \
NH2 ~ ~ ~ NH2 '. '~' ' O
~` '~

H2N ~
NE~ 2 ph ~rs^~ :

1~761;~ :

/ 6~-N-methyl-xK-88-5 is a new compound, which exhibits excellent road spectrum antibacterial activity and is also effective f against those bacteria having a resistance to ~K-88-5.
Included in the composition of matter aspect of the s inventiOn are the pharmaceutically acceptable, non-toxic acid ~ ~ addition salts of 6'-N-methyl-XK-88-5. Suitable acid addition `~ 7 salts are prepared in known manner by, for example, reacting one 3 molecule of 6'-N-methyl-XK-88-5 with one to six molecules of 9 acid. The pharmaceutically acceptable non-toxic acids include ~10 various inorganic acids such as hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, phosphoric acid, carbonic 7 acid, etc., and various organic acids such as acetic acid, fumaric ~1~ acid, malic acid, citric acid, mandelic acid, tartaric acid, .:~ ' .. .....
succinic acid, ascorbic acid, etc.
The process of the present invention is that of produc-ing 6'-N-methyl-XK-88-5 which comprises reacting the antibiotic XX-88-5 represented by the formula:
1~18 ~19 r NH2 i~ ~80 ~
`~ ~0 NH2 ;~24 2s 0 ~2- ~ C~3 `29 NH2 ~3 ' ~'.

76~LZ
-;i with an agent capable of introducing into the amino group at the / 6'-position, a group represented by the general formula R-O-C-, / . wherein R is an alkyl group having 1 to 5 carbon atoms, an aralkyl '1~ . group having 7 to 13 carbon atoms, or a substituted or unsubsti-tuted aryl group having 6 to 12 carbon atoms, 6 . .. : ^ .............. in a suitable solvent to prepare 7 an intermediate compound represented by the general formula:

9 . ~ NH-C-OR

. 10 ~, o\ N~2 ,' HO ~ ~

13 HO _ ~ 2 ¦ ~2N ~ OCH~
18 ` NiI2 9 wherein R has the same significance as defined above; and thereafter reducing the intermediate compound to prepare the 21 ~ 6'-N-methylated.end product.

¦ 23 ` ~ DETAILED DESCRIPTION OF THE INVENTON

2~ In accordance with the present invention 6'-N-methyl- :
XK-88-5 is prepared by reacting the antibiotic XK-88-5 with ~6 an agent. ~ . to introduce a R-O-~- group into the 27 amino group at the 6'-position of XK-88-5 Thereafter, the 28 intermediate compound is reduced to produce the desired compound. ~; .
For example, one mole of XK-88-5 dissolved in an appropriate solvent is reacted with 0.1 to 3.0 moles, preferably 0.3 to 1.5 ¦31 moles of an agent to introduce a R-O-C-group into the amino ! group at the 6' -position of XK-88-5 at a temperature of about -20C
_ 5 --' .:
. . .
. : ~ - , , :

¦ / to 50~C, preferably, -10C to 30C. The reaction is usually / complete in 0.5 - 48 hours.
/ , As an agent to introduce a R-O-C-group into the amino -~ group at the 6' -position of XK-88-5 any introducible R-O-C-group into the amino group at the 6'-position of XK-88-5 may be 6 used. Examples of preferred R-O-C- groups and corresponding 7 reagents are shown below.

R2 CH2-0-1l- Rl3~--CE2-0-C-O-

3~--CH2-o-C-X

~13 ' 1 l5 R 3~CH 2-o-C-o~

3~--CE~ -o-c-o~ C
9 R2 Cl Cl t1 C(CH3)-O-cO- tC(CH3)3-O-CQN3]
t2 CH2-O-CO- (CH3-O-COX) t3 C H -O-CO- (C2H5-0-COX) t4 X-CH2-0-CO- X-C~2-0-COX
~2s lRl and R2 in the above formulae may be the same or different ~
~;26 and are H, Cl, Br, I, alkyl groups having 1 to 5 carbon atoms --n or alkoxy groups ha~ing 1 to 5 carbon atoms, and X is Cl, Br ~ 28 or Il ; 29 Other specific examples of agents for carbamation are set forth in ~Protective Groups in Organic Chemistry", pages 56 31 60 (Plenum Press 1973).
. ', ~ ~ ~
- 6 - ~ ~
. ~' :

10~761;~ 1 Solvents suitable for this reaction are selected from the group consisting of dioxane, tetrahydrofuran, ethylene glycol dimethylether, N,N-dimethylformamide, dimethylsulfoxide, methanol, I
~4 -ethanol, propanol, water, acetone, acetonitrile, pyridine, and ¦
the like. A mixed solvent of water and organic solvent (1 : ¦
6 0.1 - 10 by volume) is especially preferred. j 7 ; The intermediate product is isolated and purified from 8 the thus obtained reaction mixture by column chromatography using 9 adsorbents such as ion exchange resins, silica gel, alumina and 1~ cellulose, or thin layer chromatography using silica gel, alumina or cellulose.
' The thus obtained intermediate compound is reduced by 3 dissolving the compound in a solvent having no active hydrogen ; 14 atom such as tetrahydrofuran, dioxane, trioxane and the like.
. .-Then, 1 - 60 moles of lithium-aluminum hydride is added to the }16 solution and the mixture is heated under reflux for 12-72 hours.
17 After completion of the reaction, excess lithium-aluminum hydride 18 iS decomposed by adding a solvent such as ethyl acetate, ethanol . 19 or water. The solvent is removed by distillation and water is then added to the residue. Insoluble inorganics are removed and~
21 the solution is subjected to chromatography using ion exchange ~ 22 ~ resin, silica gel or the like to isolate the desired 6'-N-methyl-`:~23 XK-88-5.
~ The reduction of a urethane group such as a benæyloxy-carboxyl group using lithium aluminum hydride is a known process 26 described in "Journal of Organic Chemistry" Vol. 20, page 92 2~ ~(1955) by R. L. Danley et al.
~29 6'-N-methyl-XK-88-5 of the present invention exhibits 29 excellent broad spectrum antibacterial activity and is character-ized by a strong antibacterial activity against Serratia marcescens ~31 strains which show a resistance to known aminoglycoside antibiotics.
. ~.
: - 7- '~

; ' - . .

~/ 108q61Z

/ The following Table 1 illustrates minimum inhibitory I
' / concentrations (y/ml) of ~'-N-methyl-XK-88-5 against various ¦
~ Gram-positive and Gram-negative bacteria determined by agar ,~ dilution method.
~ Table 1 .,~ . ~
~ .
6'-N-Methyl-3 Microorganisms XK-88-5 XK-88-5 ¦
~,3 ;, 5 '~ Vibrio percolans KY41741.25 1.25 Pseudomonas alkaligenes KY4656 0.64 0.16 Erwinia aroideae ~Y32410.32 0.16 ~1~ Staphylococcus aureusKY4279 0.16 0.04 -'~~3 Escherichia coli KY42710.32 0.32 Bacillus subtilis KY42730.08 0.04 ~l5 ' Proteus vulgaris KY4277 0.64 0.64 ~sl5 Shigella sonnei RY42811.25 0.32 !~ 7 Salmonella typhosa K~4278 0.32 0.32 Klebsiella pneumoniaeKY4275 0.16 0.08 ~19 Serratia marcescensPOE1065 12.5 >100 .
~1- ; - ..

'i2l Serratia marcescens POE1065 produces acetyl-transferaset of kanamycin.

j 23 ~ As illustrated above, the compound of the present

4 invention is useful as an-antibacterial agent to clean and disin-fect laboratory glassware and surgical instruments, and may also 6 be used for pharmaceutical and sanitation purpose in cleaning and ~27 sanitizing hospital rooms and areas.

The acute toxicity (LD50) of 6'-N-methyl-XK-88-5 is 620 mg/kg ~i.v.).
Practice of certain specific embodiments of the present invention is illustrated by the following representative examples and figures.

~!
,. .. .. . . . . . . ... . . . . . .. . . .

1~37612 Wherein Fig 1 illustrates the C13 nuclear magnetic resonance spectrum of 6'-N-benzyloxycarbonyl-XK-88-5 measured in D20 at pD 10; and Fig. 2 shows C13 nuclear magne~ic resonance spectrum of free base of 6'-N-methyl-XK-88-5 measured in D20 by using dioxane as an internal standard compound, - 8a -dc/

, ' ~ i 376~Z :

Example 1. Preparation of 6'-N-benzyloxycarbonyl-XK-88-5.
In this example, five grams of the free base of XK-88-5 is dissolved in a mixture o~ 190 ml of water and 160 ml ,~ of ethyleneglycol-dimethylether and the solution is stirred in an Js ice bath. To the solution, a solution of 3.09 g of N-benzyl-16 oxycarbonyloxysuccinimide dissolved in 30 ml of ethyleneglycol-7 dimethylether is added by drops. The resulting solution is ¦B stirred for 3 hours in the ice bath and then allowed to stand at 5C for 15 hours in a refrigerator.
1~ A small amount of precipitate is removed by filtration;
1l and the filtrate is adjusted to pH 4.4 with hydrochloric acid -In~ ~ ~ and then passed through a column packed with 500 ml of Amberlite s . - :.
~3 CG50 (NH4 form) tRohm and Haas Co., U.S.A.).
The column is washed with 1500 ml of water and 400 ml ~5 of O.lN aqueous ammonia. Then, elution is carried out with O.lN
~16 a~ueous ammonia checking the components of the eluate by a thin ~!? layer chromatography. The eluate is fractionated into 22 ml 3 . fractions and fraction Nos. 45 - 87 containiny 6'-N-benzyloxy-19 carbonyl-XK-88-5 are combined and concentrated to dryness under ' reduced pressure to obtain 4.1 g of the crude product.
~I To purify the 6'-N-benzyloxycarbonyl-XK-88-5, 250 g of ¦ 2 silica gel is packed into a column and 4.1 g of the powder obtained ~'3 in the above step is charged. Then, elution is carried out with ~'~ a solvent comprising n-butanol~ ethanol, chloro~orm and concen-trated aqueous ammonia (4 : 5 : 2 : 1 by volume). The eluate is 26 fractionated into 22 ml fractions and each of the fractions is 27 checked by a thin layer chromatography. Fraction Nos. 82 - 162 ~9 containing only the desired product are combined and concentrated 23 to dryness under reduced pressure to obtain 1~64 g of pure 6'-N-~;~ benzyloxycarbonyl-XK-88-5 as a white amorphous powder.
3~ The physicochemic~l p~operties of the intermediate ! g ~.

'l/[IIE~761Z

compound are as follows:
Melting point: 170 - 172C
Specific rotation: [a]l6= +115 (C = 0 300, w~ter) Elementary analysis (~):
Calculated for C26H44N69 2H2 3 H - 7.38, N = 13.65 Found: C = 51.11, H = 7.26, N = 13.22 The Rf value is 0.62 on silica geI thin layer chromatography (Developer;:~N~tanol :~ethanol : chloroform : concentrated aqueous ammonia = 4 : 5 : 2 : 5 by volume).
Figure 1 illustrates the C13 nuclear magnetic resonance spectrum of 6'-N-benzyloxycarbonyl-XK-88-5 measured in D2O at pD 10. The chemical shift of each signal is shown in p.p.m. from TMS.
158.8, 137.1, 129.5, 129.3, 128.9, 102.7. 100.1, 89.1, 86.6, 80.1, 74.9, 69.5, 68.8, 67.7, 67.4 (standard compound), 60.8, 5~.8, 57.7, 56.1, 54.8, 51.1, 50.2, 45.4, 36.9, 36.3.
EXample 2. Production of 6'-N-methyl-XK-88-5.
To produce the desired end product, 0.81 g of 6l-N-benzyloxycarbonyl-XK-88-5 is suspended in 400 ml of tetra-hydrofuran. To this suspension, 2.43 g of lithium aluminum hydride is added, and the mixture is heated under reflux for 23 hours with vigorous stirring.
An additional 1.30 g of lithium aluminum hydride is then added and the mixture is heated under reflux for a further 19 hours. After cooling the mixture to room temperature,

5 ml of ethyl acetate is added slowly thereto to decompose the excess lithium aluminum hydride and then 20 ml of water is added in drops to insure complete decomposition of excess lithium aluminum hydride.
- The solvent is then removed by distillation under reduced pressure,- and 150 ml of water is added to the residue.
The mixture is stirred at room temperature for 1 hour and then subjected tb centrifugation to obtain a supernatant solution.
dC/~b , - 10 -.

1~7612 .~
To the resulting precipitate, 150 ml of water is added, and the mixture is stirred at room temperature for 1 hour. This mixture is also subjected to centrifugation to obtain a supernatant solu-; tion. The supernatant solutions are combined and the mixture is concentrated to 30 ml under reduced pressure. The concentrate is

6 adjusted to pH 4.50 with hydrochloric acid. A small amount of

7 precipitate is removed by filtration.

8 The filtrate is passed through a column of 77 ml of

9 ~ Amberlite CG50 (NH4 form) which is then washed with 360 ml of o water. Then, elution is carried out with 456 ml of O.lN aqueous ~1 ammonia and 456 ml of 0.2N aqueous ammonia while checking the 12 components of the eluate by a thin layer chromatography.
13 The eluate is fractionated into 12 ml fractions and 4 fraction Nos. 25 - 30 containing unreacted 6'-N-benzyloxy-carbonyl-XK-88-5 are combined and concentrated to dryness under reduced 16 pressure to obtain 0.32 g of the intermediate product. Fra~tion 17 Nos. 45 -~ 55 containing the des~red compound are combined and 18 concentrated to dryness under reduced pressure to obtain 0.35 g ;.
9 of a crude powder of 6'-N-methyl-XK-88-5.
To purify the 6'-N-methyl-XK-88-5, 25 g of silica gel 21 is packed into a column and 0.35 g of the powder obtained in the 22 above ste~ is charged therein. Then, elution is carried out with 23 a solvent comprising chloroform, methanol and concentrated aqueous ~--. . . .
24 ammonia ~1~5 : 1 : 1 by volume). The eluate is fractionated into ., . -5 ml fractions and each of the fractions is checked on the compo-26 nents of the eluate by a thin layer chromatography. Fraction 27 Nos. 25 - 32 are combined and concentrated to dryness under 28 reduced pressure to obtain 90 mg of pure 6'-N-methyl-XK-88-5 as a 29 white amorphous powder.
The phys chemica~ properties of this end product are ~ 31 i3S follOWS:

-: - .. ..
. : .: : :

', 108761Z

~elting Point: 173 - 175C `I
Specific rotation: [~]20= +100 (C = 0.342, water) Elementary analysis (~): i~
; Calculated for ClgH40N6O7.H2 3 ;
!5 ; H = 15.96 ~ Found: C = 46.50; H = 7.12; N = 16.38 ~-li Molecular weight decided by high resolution mass 8 spectrum: 464.3001 19 ~ The Rf value is 1.19 on silica gel thin layer chromato-graphy (Developer : n-butanol : ethanol : chloroform : concen-trated aqueous ammonia = 4 : 5 : 2 : 5 by volume) when the Rf 7 ' value of the antibiotic XK-88-5 is 1.00.
i3 i Figure 2 shows C13 nuclear magnetic resonance ;~
14 spectrum of free base of 6'-N-methyl-XK-88-5 measured in D20 ;by using dioxane as an internal standard compound. The chemical shift of each signal is shown below in p.p.m. from TMS.
l? lOl.9, 99.9, 87.6, 86.8, 80.2, 75.1, 68.9, 67.4, (standard l~ compound), 60.8, 58.7, 57.6, 56.2, 55.3, 54.8, 51.1, 50.2, 37.5, 19 ~ 36.6, 3s.8.
: 7 Example 3.
21 In this example, 100 my of 6'-N-methyl-XK-88-5 is ..
22 dissolved in 0.5 ml of water and the solution is adjusted to pH
3 2.0 with sulfuric acid. To the solution 20 ml of acetone is added in small portions with stirring. The precipitate is separated by filtration, washed with 50 ml of acetone and dried 26 in vacuo to yield 138 mg of sulfate of 6'-N-methyl-XK-88-5 as a 27 white amorphous powder characterized as follows:
28 ~elting point: 210 - 235C (decomposition) 29 Specific rotation: E~] 20= +91. 2 (c = o. 4I2, water) ~ Elementary analysis ~%):
;31 Calculated for ClgH40~67-3H2S4 2 .. ..
.

$~761Z

H = 6. 24; N = 10. 82; S = 12. 38 2 Found: C = 29. 00 ~ = 6.18; W = lO. Sl 5 = 12 01 l : .

g ', :
- :
11 ': ' I?

:

. ~ .

,. .
. 3~ .
.. . :
-- 1 3 ~
'' ' ~ :'' "... , :: . ' . -. , , . . ' ' : ~ . .: ,," ' - " . ' ' ' . . : : . . ~ ,: : .: ' ' ' ~ -

Claims (2)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for producing 6'-N-methyl-XK-88-5 which comprises reacting the compound XK-88-5 with an agent capable of introducing into the amino group at the 6'-position of XK-88-5, a group represented by the formula:

wherein R is an alkyl group having 1 to 5 carbon atoms, an aralkyl group having 7 to 13 carbon atoms, or a substituted or unsubstituted aryl group having 6 to 12 carbon atoms to prepare an intermediate compound represented by the formula:

and thereafter reducing the intermediate compound to prepare 6'-N-methyl-XK-88-5.

2. 6'-N-methyl-XK-88-5, having the formula:

whenever prepared according to the process of Claim 1, or by an obvious chemical equivalent.