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