Aminocyclitol Antibiotics - American Chemical Society

11 The Effect of O-Methylation on the Activity of

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Aminoglycosides J. Β. McALPINE, R. Ε. CARNEY, R. L. DΕVAULT, A. C. SINCLAIR, R. S. EGAN, M. CIROVIC, R. STANASZEK, and S. MUELLER Abbott Laboratories, Division of Antibiotics and Natural Products, North Chicago, IL 60064

Following the discovery by Hamao Umezawa (1) and co-workers that certain aminoglycoside resistant organisms of clinical origin owed their resistance to an R-factor mediated ability to phosphorylate kanamycin and neomycin at the 3'-hydroxyl group chemical modification of these antibiotics was directed at possible ways of overcoming this inactivation. The 3'-O-methyl and 3'-deoxy derivatives of kanamycin A were prepared by glycosidation of a suitably protected pseudodisaccharide with appropriately substituted glucopyranosyl chlorides (2). The 3'O-methylkanamycin 1 obtained was a 1:1 mixture of anomers at the 1' position and was found to be essentially devoid of anti bacterial activity whereas the 3'-deoxy derivative, obtained as the α-glycoside was found to have strong antibacterial activity against strains of Escherichia coli and Pseudomonas aeruginosa resistant to the parent antibiotic. Similar preparations of 3'O-methylneamine 2 and 4'-O-methylneamine 3 (3) by the same group led to the same disappointing lack of antibacterial activity. Subsequently the wide variety of chemical modifications of aminoglycoside antibiotics carried out in many laboratories have tended to avoid O-alkylations and to emphasize deoxygenation. I t was therefore somewhat s u r p r i s i n g when two d i f f e r e n t f a m i l i e s of aminoglycosides discovered under a j o i n t research p r o j e c t between Abbott Laboratories and the Kyowa Hakko Kogyo Company should have h i g h l y a c t i v e components each of which c o n t a i n an Oj-methyl group i n t h e i r s t r u c t u r e s . F o r t i m i c i n A 4, i s the most a c t i v e of a group of a t y p i c a l pseudodisaccharides (4) many of the members of which c a r r y an (^-methyl s u b s t i t u t e n t on the c y c l i t o l . Seldomycin f a c t o r 5 ^ (5) i s the most a c t i v e of a f a m i l y of aminoglycosides produced by Streptomyces hofunensis and possesses an (^-methyl group at the 4" p o s i t i o n . This a n t i b i o t i c i s immediately recognized as a close r e l a t i v e of the kanamycin-gentamicin group of amino g l y c o s i d e s and i t was decided to i n v e s t i g a t e the e f f e c t of a 4"-0-methyl group on the a c t i v i t y of t h i s f a m i l y of a n t i b i o t i c s .

0-8412-0554-X/80/47-125-183$05.00/0 © 1980 American Chemical Society

Rinehart and Suami; Aminocyclitol Antibiotics ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

184

AMINOCYCLITOL ANTIBIOTICS


11.

MCALPINE ET A L .

O-Methylation of Aminoglycosides

185

The gentamicins ffâÂfi and sagamicin (y^ were a p a r t i c u l a r l y a t t r a c t i v e subgroup f o r O^methylation a t t h i s p o s i t i o n . They possess two secondary h y d r o x y l groups a t C-5 and C-2" and a t e r t i a r y h y d r o x y l a t the C-4" p o s i t i o n a l b e i t i n the epimeric stereochemistry t o that of the methoxy group i n seldomycin f a c t o r 5 ^. The g e n e r a l approach shown i n Scheme 1 was adopted. The amino groups and the two secondary h y d r o x y l were t o be p r o t e c t e d and the product t r e a t e d w i t h the Goldman-Albright o x i d a t i o n r e agent t o convert the t e r t i a r y h y d r o x y l i n t o a methylthiomethyl ether (6). D e s u l f u r i z a t i o n of t h i s would r e s u l t i n the d e s i r e d 4"-0_-methyl d e r i v a t i v e which on d e p r o t e c t i o n would g i v e the r e q u i r e d product. This scheme was executed u s i n g gentamicin C l a ^ as s u b s t r a t e and a c e t y l as the 0_ and N_ p r o t e c t i n g group. S e l e c t i v i t y i n preparing the r e q u i r e d h e p t a - a c e t y l d e r i v a t i v e was l e s s than expected. The t e r t i a r y h y d r o x y l was r e l a t i v e l y e a s i l y a c e t y l a t e d and an o c t a - a c e t y l and a non-acetyl product, the o t h e r , i n w h i r l the 6 -amine had been converted i n t o an imide n i t r o g e n bearing two a c e t y l groups, were major contaminants i n the i n i t i a l product. These were separated by s i l i c a g e l chromatography. The h e p t a - a c e t y l gentamicin C l a was converted i n good y i e l d s t o 4"-0^methylgentamicin C l a by the steps o u t l i n e d . The d e p r o t e c t i o n was by a simple barium hydroxide h y d r o l y s i s . The 4"-0-methylgentamicin C l a was c h a r a c t e r i z e d by an O C H 3 s i n g l e t a t 63.67 i n the proton magnetic spectrum. PMR spectrum determined i n deuterium oxide w i t h t e t r a m e t h y l s i l a n e as e x t e r n a l reference quoted unadjusted. The C-5 h y d r o x y l groups of 4 , 6 - d i - 0 - s u b s t i t u t e d 2-deoxystreptamine a n t i b i o t i c s i s known t o be s t e r i c a l l y hindered and under m i l d e r c o n d i t i o n s of a c e t y l a t i o n i t was p o s s i b l e t o p r e pare a h e x a - a c e t y l d e r i v a t i v e . F o l l o w i n g the same r e a c t i o n s l e d to the sequence i n Scheme 2. This s u b s t r a t e gave the 5oxo compound which was s t e r e o s e l e c t i v e l y reduced during the Raney N i c k e l r e d u c t i o n t o give a 5-epi-hydroxy 4"-0-methyl d e r i v a t i v e . Barium hydroxide h y d r o l y s i s of t h i s l e d t o 5 - e p i 4"-0-methylgentamicin C l a . The chemical s h i f t s from the carbon magnetic resonance s p e c t r a of these compounds and t h e i r parent a n t i b i o t i c are shown i n Table 1 . The resonances assigned to the carbons of the purpurosamine r i n g are v i r t u a l l y i d e n t i c a l f o r a l l three compounds as a r e the resonances of the carbons of the 2-deoxystreptamine r i n g of gentamicin C l a and the 4"-0-methyl d e r i v a t i v e . The three carbons, C-3", C-5", and the C-methyl carbon show the expected u p f i e l d 3 s h i f t f o l l o w i n g a l k y l a t i o n of the 4"hydroxyl w h i l e the 4"-carbons hows a downfield 3 s h i f t . The methoxyl carbon resonance occurs a t 49.7 ppm d o w n f i e l d from t e t r a m e t h y s i l a n e . The resonances of the carbons of the garosamine r i n g of 4"-0-methylgentamicin C l a are i n d i s t i n g u i s h e d from those of i t s 5-epimer. The carbon resonances f o r the 2-deoxystreptamine r i n g o f 5-epi-4"-0-methylgentamicin C l a 1


186

AMINOCYCLITOL ANTIBIOTICS

TABLE 1 Carbon Magnetic Resonance Spectra

Gentamicin C l a 1

4"-O-Me thy1gentamicin C l a

5-Epi-4"-O-methy1gentamicin C l a

C-l C-2 C-3 C-4 C-5' C-6

102.2 51.0 27.1 28.5 71.5 46.1

101.9 50.8 26.9 28.3 71.2 45.9

96.2 50.3 27.2 28.3 70.7 45.7

C-l C-2 C-3 C-4 C-5 C-6

51.7 36.7 50.6 88.3 75.4 87.8

51.5 36.6 50.4 87.9 75.3 86.6

48.2 36.7 47.5 85.8 68.7 79.6

C-l" C-2" C-3" C-4" C-5" CCH NCH OCH

101.3 70.2 64.4 73.3 68.7 23.0 38.0

101.0 70.0 62.2 77.7 65.3 17.4 38.0 49.7

102.3 70.1 62.0 77.7 65.1 17.3 38.0 49.7

1

f

f

f

3

3

3


11.

MCALPiNE

ET AL.



187

188

AMINOCYCLITOL


ANTIBIOTICS

11.

MCALPINE

189

O-Methyhtion of Aminoglycosides

ET AL.

show an u p f i e l d s h i f t from those of other normal isomer as would be a s s o c i a t e d w i t h i n v e r s i o n from an e q u a t o r i a l to an a x i a l s u b s t i t u e n t . In an attempt to improve the s e l e c t i v i t y of the O - p r o t e c t i o n s t e p , carboethoxy groups were chosen as the N - p r o t e c t i n g group i n the s y n t h e s i s s t a r t i n g w i t h sagam i c i n 6d. The per-N-carboethoxy d e r i v a t i v e was prepared q u a n t i t a t i v e l y and ()-acetylated t o give the d e s i r e d 5,2"-di-0acetyl-penta-N-carboethoxysagamicin. This was converted i n good y i e l d t o the 4 -0-methyl d e r i v a t i v e by treatment w i t h d i m e t h y l s u l f o x i d e / a c e t i c anhydride followed by Raney N i c k e l d e s u l f u r i z a t i o n . Barium hydroxide removal of the p r o t e c t i n g groups gave the d e s i r e d 4"-0-methylsagamicin and a major byproduct 4"-0-methylsagamicin-lN,3N-cyclic ureide ^7. The carbon magnetic resonance s p e c t r a l data of these compounds and the parent a n t i b i o t i c are shown i n Table 2. Again no s i g n i f i c a n t d i f f e r e n c e occurs i n the resonances assigned t o the N-methylpurpurosamine r i n g , and the resonances of the carbons of the garosamine r i n g of 4"-0-methylsagamicin shows the s h i f t s from t h e i r p o s i t i o n i n the spectrum of the parent which would be expected from 4 " - 0 - a l k y l a t i o n . The spectrum of the 2-deoxystreptamine r i n g of the c y c l i c ureide shows a cons i d e r a b l e u p f i e l d s h i f t of a l l carbons i n comparison t o t h e i r p o s i t i o n i n the spectrum of the parent. This t r a n s f o r m a t i o n has f l i p p e d the c h a i r form of t h i s r i n g from that conformation w i t h a l l f i v e s u b s t i t u e n t s e q u a t o r i a l to that w i t h a l l f i v e a x i a l . The m u l t i p l i c i t y of the C-2 resonance a t 18.2 ppm was confirmed as a t r i p l e t by ORSFD experiments. The a n t i b a c t e r i a l a c t i v i t y of these compounds i s shown i n Table 3. This assay i s run under c o n d i t i o n s , ( s e n s i t i v e organisms, low n u t r i e n t s , and h i g h pH) which maximize the apparent a c t i v i t y o f the a n t i b i o t i c s . The data show that the 4"-0-methyl d e r i v a t i v e s o f gentamicin C l a and sagamicin are from 2 t o 4 f o l d l e s s a c t i v e than the parent a n t i b i o t i c s . E p i m e r i z a t i o n a t the 5 - p o s i t i o n , as has been shown i n the case of s i s o m i c i n ( 7 ) , i s b e n e f i c i a l . This change r e s t o r e d a c t i v i t y l o s t by 4"-0-methylation and 5-epi-4"-0-methylgentamicin C l a i s as a c t i v e as the parent a n t i b i o t i c . Thus, m e t h y l a t i o n of a 4 " - a x i a l h y d r o x y l group of the gentamicins i s d e t r i m e n t a l t o a c t i v i t y a l b e i t much l e s s so than m e t h y l a t i o n of the 2 and 3' e q u a t o r i a l h y d r o x y l groups of neamine and kanamycin. I t i s i n t e r e s t i n g t o note that deoxygenation of gentamicin CI fa at the 4" p o s i t i o n has been reported by Mallams and co-workers (8>) t o give a compound i n a c t i v e except f o r some very weak a c t i v i t y against s e n s i t i v e Gram p o s i t i v e s t r a i n s . Thus a t t h i s p a r t i c u l a r p o s i t i o n , O-methylation would appear t o be much l e s s d e t r i m e n t a l than deoxygenation. The 4 " - e q u a t o r i a l methoxy group of seldomycin f a c t o r 5 fa was removed by the Monneret r e a c t i o n (9) u s i n g l i t h i u m i n ethylamine t o give O-demethylseldomycin f a c t o r 5. The same r e a c t i o n was c a r r i e d out on 3 -deoxyseldomycin f a c t o r 5 fa, a semisynthetic ,,

f

f


ο

CO

I

3

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H

r

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11.

MCALPiNE E T A L .

191


TABLE 2 Carbon Magnetic Resonance Spectra

Sagamicin

4"-0-Methylsagamicin

4 ' ' -O-Methylsagamicin lN,3N-cyclic

101· 2 50· 5 26·5 28· 5 68.1 55· 1 35.3

101· 3 50.5 26·5 28.6 68.3 55.1 35.3

100.4 50.3 27.0 28.5 68.2 54.9 35.2

C-l c-2 C-3 C-4 C-5 C-6 C-0

51.5 36.5 50.3 87.7 75.4 86.9

51.5 36.5 50.3 87.6 75.3 87.0

47.9 18.2 45.9 78.7 69.3 77.9 159.2

C-l" C-2" C-3" C-4" C-5" CCH NCH OCH

101.2 70.1 64.2 73.1 68.5 22.5 37· 7

101.0 69.9 62.0 77.7 65.2 17.3 37.9 49.7

98.8 70.5 62.1 77.5 65.6 17.4 38.3 49.7

f

C-l C-2 C-3 C-4 C-5 C-6 NCH f

1

f

1

1

3

3

3

3



coli

ATCC 10031

0.01

0.02

Salmonella t y p h i ATCC 9992

K l e b s i e l l a pneumonia

0.04

S h i g e l l a sonnei ATCC 9290

0.2 0.04

BMH #1

3

Proteus v u l g a r i s ATCC 6897

Pseudomonas aeruginosa

Escherichia

0.01

Escherichia c o l i

ATCC 26

20

1.25 10

0.31

Proteus v u l g a r i s ATCC 6897

Providencia s t u a r t i i 164

0.04

K l e b s i e l l a pneumoniae ATCC 10031

19

1 6

5

3

0.31 >20 >20 2.5 2.5 0.63 1.6

10

Streptococcus f a e c a l i s ATCC 10541

ATCC-26

0.04

Staphylococcus aureus ATCC 6538P

Escherichia c o l i R R R R (AAC-3-I) 76-2 NR-79

20

,

3 -H-4"OCH^

0.63 1.25 >20

2.5 20

0.31

0.04

0.16 2.5 0.8 0.4 0.16 0.16 0.16

>20

0.02

Aminocyclitol Antibiotics - American Chemical Society

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