Aminocyclitol Antibiotics - American Chemical Society

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12 The Synthesis and Biological Properties of 3'- and 4'-Thiodeoxyneamines and 4'-Thiodeoxykanamycin Β

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THOMAS W. KU, ROBERT D. SITRIN , DAVID J. COOPER , JOHN R. E. HOOVER, and JERRY A. WEISBACH 3

Research & Development Division, Smith Kline & French Laboratories, Philadelphia, PA 19101

The aminoglycosides are a clinically important class of antibiotics with broad activity against many strains of gram-neg­ ative bacteria. Concurrent with the extensive use of aminoglyco­ sides, resistant organisms, many of which contain transferable R-factors, have become more prevalent. The plasmids in the resistant strains code for enzymes which inactivate various aminoglycosides by phosphorylation, acetylation or adenylation (1,2). The antibiotics can be rendered resistant to inactivation through appropiate structual modifications (1,2). For our purposes the pseudodisaccharide neamine (1), a component of

neomycin and kanamycin B, provided a model substrate for carry­ ing out modifications with this objective. Although 1 is less active against bacteria than typical pseudotrisaccharides such as kanamycin or gentamicin, it is also less toxic (2). Such a 1

Author to whom correspondence is to be addressed

2

Current Address: Orlando Regional Medical Center Orlando, Florida 32806

3

Current Address: Warner-Lambert/Parke Davis Pharmaceutical Research Division, Ann Arbor, Michigan 48105 0-8412-0554-X/80/47-125-197$05.00/0 © 1980 American Chemical Society

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

198

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ANTIBIOTICS

pseudodisaccharide, suitably modified, might have useful activity of itself, or it might serve as a basis for the construction of appropriate pseudotrisaccharides. The principal mechanism for neamine inactivation is phosphorylation of its 3'-hydroxyl group, a reaction which has an important role in the development of resistance to neomycin and kanamycin. I t has been demonstrated that removal o f the oxygen f u n c t i o n a t the 3 ' - p o s i t i o n r e s u l t s i n a n t i b i o t i c s w i t h enhanced a c t i v i t y a g a i n s t such r e s i s t a n t organ­ isms (1 _2) . Along these l i n e s , we have synthesized the 3'- and 4'-thio-3'-and 4'-deoxy- analogs of neamine and 4'-thio-4'-deoxykanamycin Β as p a r t o f a more general program of modifying amino­ g l y c o s i d e a n t i b i o t i c s . These analogs were obtained by nucleop h i l i c opening o f the a p p r o p r i a t e epoxy precursors u s i n g b e n z y l mercaptide. 5

P r e p a r a t i o n of Epoxide

Intermediates

S e l e c t i v e t o s y l a t i o n (3) of the 3'-hydroxyl group of 5,6-0cyclohexylidine-tetracarbomethoxy neamine, 2 (4), f o l l o w e d by treatment w i t h sodium methoxide y i e l d e d the p r e v i o u s l y described (5) c r y s t a l l i n e allo-eipoxlde 4 (Figure 1) . Although the i s o m e r i c galacto-epoxide 7 (Figure 2) could be obtained by methoxide t r e a t ­ ment of the 4 ' - t o s y l a t e , which i n t u r n was i s o l a t e d as a minor product from the t o s y l a t i o n o f 2 ( 5 ) , a more e f f i c i e n t route was needed f o r i t s l a r g e s c a l e p r e p a r a t i o n . Reaction of 2 w i t h excess benzoyl c h l o r i d e i n p y r i d i n e a t low temperature y i e l d e d , along w i t h some dibenzoate, the 3'-mono-benzoate 5 : y i e l d , 68%; [ a ] p +67.4° (c 1, CHC1 ). The monoester was r e a d i l y separated from the more s o l u b l e d i e s t e r by p r e c i p i t a t i o n from ether-petroleum ether. Since only one monoester could be detected i n the product, any 4'-monobenzoate formed i n the r e a c t i o n mixture must have been benzoylated t o the d i e s t e r . M e s y l a t i o n of 5 u s i n g methanesulfonyl c h l o r i d e and t r i e t h y l a m i n e i n methylene c h l o r i d e a t -10° gave 6+: y i e l d , 86%; [ a ] ^ +32.5° (c 1, CHC1 ); nmr, 7.2-8.2 ppm (5H, m, a r o m a t i c ) , 2.9 ppm (3H, s, mesylate). On treatment w i t h sodium methoxide, 6 gave galaoto-epoxide 7^: y i e l d , 81%; [ a ] ^ +2.5° (c 1, 0Η01 )Γ Epoxide 7 was r e a d i l y d i s t i n g u i s h a b l e from the isomer 4 by t i c ( s i l i c a , a c e t o n i t r i l e - e t h e r , 1:1) and HPLC (Microporasil®, CHCl -MeOH, 95:5). Although none of these i n t e r ­ mediates were c r y s t a l l i n e , product 7, obtained by p r e c i p i t a t i o n , was chromatographically homogeneous and i t s p r e p a r a t i o n was amenable t o l a r g e s c a l e work. T

5

3

5

3

5

3

3

Opening of Epoxides w i t h Benzyl Mercaptan Galacto-epoxlde 7 (Figure 3) was t r e a t e d under n i t r o g e n f o r 3 h r w i t h two e q u i v a l e n t s of b e n z y l mercaptide i n r e f l u x i n g t

S a t i s f a c t o r y combustion a n a l y s e s , nmr and i r s p e c t r a were obtained f o r these compounds.

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

12.

Thiodeoxyneamines and Thiodeoxykanamycin Β

KU ET AL.

199

4* r—NHCOnMe

NHC0 Me 2

HO-

Pyr

P\X

Me0 CNH] 2

M e 0 H

T s o \ ^ ^ S

NHCOoMe

Me0 CHN I 0-R

Me0 CNH I 0-R

2

2

NHC0 Me 2

NHCOoMe

Figure 1.

H O - V T - T

HO

' % ·

Me0 CNH| 0-R 2

"faZST 200

200

200

Strain

Kleb. pneumoniae SK&F 4200

6.3

50

200

50

Sal. paratyphi ATCC 12176

12.5

200

200

100

Shigella paradysenteriae

25

200

>200

100

Ps. aeruginosa HH 63

12.5

>200

25

25

Ser. marcescens ATCC 13880

12.5

>200

>200

200

Proteus morgani 179

12.5

100

200

100

Enterobacter aerogenes

12.5

100

200

100

Agar dilution, pH 8.0

Table III.

In vitro activities against resistant organisms, (^g/ml)

Strain

Enzyme System

—

15

Neamine

4-Thioneamine

3-Thioneamine

A -Thioneamine disulfide

X = OH Y = OH

X = OH Y = SH

X = SH Y = OH

X = OH Y = disulfide

1

16

125

500

63

APH(3')-I

>1000

2000

500

2000

E. coli K802N(pJR214)

APH(3')-I + ANT(2")

>1000

2000

500

2000

E. coli K802N(pJR67)

APH(3')-II

>1000

1000

500

500

250

1000

2000

500 125

E. coli K802N E. coli K802N(pR6)

E. coli K802N(pR5)

AAC(6)

E. coli K802N(pJR88)

AAC (3)-I

Ps. aeruginosa HH63

-

Ps. aeruginosa PSI -1 Prov. s p. 64

AAC(3)-II AAC (2)

31

250

1000

250

63

63

16

1000

125

125

63

>1000

>2000

>2000

>2000

Broth dilution, pH 8.0

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

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206

ANTIBIOTICS

Table I V .

OR In vitro Antimicrobial Activities (^g/ml)

1 Strain

Neamine X = OH R=H

£§,

Kanamycin Β X = OH R = 3AG

4-ThioKanamycin Β

4^ 4-Thioneamine X = SH

X = SH R = 3AG

R=Η

Staph, aureus HH127

25

1.6

100

E. coli SK&F 12140

12.5

1.6

>200

6.3

0.4

50

12.5

Sal. paratyphi ATCC 12176

12.5

0.8

200

12.5

Shigella paradysenteriae

25

3.1

200

25

>200

12.5

25

25

Ser. marcescens ATCC 13880

12.5

3.1

>200

50

Proteus morgani 179

12.5

0.8

100

25

Enterobacter aerogenes

12.5

1.6

100

25

Kleb. pneumoniae SK&F 4200

Ps. aeruginosa HH 63

6.3 25

Agar dilution, pH 8.0

+

(1660 c i r r i ) and f i e l d d e s o r p t i o n mass s p e c t r a (m/e 902, (MfH) ) . Apparently, the two benzyl groups f l a n k i n g the amide i n h i b i t i t s h y d r o y s i s . Therefore, 22 (Figure 7) was debenzylated w i t h sodium i n l i q u i d ammonia, r e - a l k y l a t e d on the mercaptan w i t h benzyl c h l o r i d e i n methanol ( 8 ) , and the product was i s o l a t e d as i t s peracetate,23+: y i e l d , 64%; [a]£ +84.7° (c 0.5, CHC1 ). H y d r o l ­ y s i s w i t h Ba(0H) (15% w/v i n 1:1 MeOH-water, r e f l u x overnight) f o l l o w e d by treatment w i t h η-butyl amine a t 150° overnight i n a sealed bomb t o remove the N - l , N-3 c y c l i c urea (9, 10) y i e l d e d the deblocked b e n z y l t h i o e t h e r which showed two α-anomeric protons i n i t s nmr spectrum: y i e l d 55%; MS, m/e 590 (Mt-H) ; nmr (D 0) , 66.1 (1H, d, J=4 Hz), 5.2 (1H, d, J=4 Hz), and 7.5 ppm (5H, s, aromatic). Thus, the product i s the d e s i r e d α-glycoside presum­ ably attached t o the 0-6 hydroxyl as described e a r l i e r (11, 12). F i n a l l y , r e d u c t i o n w i t h sodium i n l i q u i d ammonia y i e l d e d 4 - t h i o 4-deoxykanamycin Β (24), i s o l a t e d as i t s s u l f a t e s a l t : y i e l d , 73%; [α]£ +65.6° (c 0.2, H 0 ) ; SH, 60% o f theory based on MW 744 ( I t i t r a t i o n ) . On t e s t i n g in vitro (Table IV), the a n t i c i p a t e d improvement i n a c t i v i t y over pseudodisaccharide 15 was observed against most of the s t r a i n s o f b a c t e r i a . However, the a c t i v i t y against Pseudomonas aeruginosa remained the same and was, i n f a c t , weaker than that o f kanamycin Β (25). 5

3

2

+

2

5

2

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

2

12.

κυ E T A L .

Thiodeoxyneamines and Thiodeoxykanamycin Β

Figure 7.

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

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AMINOCYCLITOL

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ANTIBIOTICS

Acknowledgements The authors wish t o acknowledge the a s s i s t a n c e of Mr. N. H a l l Ms. J . Rosenbloom, Dr. G. Wellman and Dr. F. P f e i f f e r f o r the p r e p a r a t i o n o f i n t e r m e d i a t e s , Ms. E. Reich f o r performing elemen­ t a l analyses and o p t i c a l r o t a t i o n s , Mr. G. Roberts f o r o b t a i n i n g the mass s p e c t r a l data, and Dr. J . U r i , Dr. S. Grappel and Mr. J . G u a r i n i f o r s u p p l y i n g the b i o l o g i c a l data. The amino­ g l y c o s i d e r e s i s t a n t s t r a i n s were constructed by Mr. L. Fare.

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November 15, 1979.

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