Aminocyclitol Antibiotics - American Chemical Society

8 Spectinomycin Modification W. ROSENBROOK, JR. and RONALD E. CARNEY

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: August 4, 1980 | doi: 10.1021/bk-1980-0125.ch008

Abbott Laboratories, Division of Antibiotics and Natural Products, North Chicago, IL 60064

Spectinomycin is an aminocyclitol antibiotic unique both in structure (1,2) and in biological activity. The antibiotic (Figure 1) is a pseudodisaccharide in which a non-aminosugar moiety, actinospectose, is fused to an aminocyclitol, actinamine or Ν,Ν'-dimethy1-2-epi-streptamine by both a β-glycosidic bond and a hemiketal linkage. The bioactivity of spectinomycin resembles that of other aminocyclitol antibiotics only in that its antibacterial spectrum is broad and its mode of action is the inhibition of protein synthesis by an interaction with the 30s ribosomal subunit (3). Spectinomycin's action is generally bacteriostatic rather than bacteriocidal and, although its antibacterial spectrum is described as broad, in vitro potency is generally low. In vivo potency is, however, considerably better than one would predict on the basis of in vitro activity. Most importantly, spectinomycin is devoid of the ototoxic and nephrotoxic properties normally associated with the amino -glycosidic aminocyclitol class of antibiotics (4). In our hands, we have been unable to demonstrate ototoxicity in the rat at a dose of 820 mg/kg/day for 14 days or nephrotoxicity in the rat at a dose of 810 mg/kg/day for 14 days. This lack of toxicity makes spectinomycin an appealing substrate for chemical modification. Our major concern has been the enhancement of antibiotic potency and bacteriocidal action. The r a t i o n a l e of our approach to the m o d i f i c a t i o n of spect inomycin i s based on the o b s e r v a t i o n that a 2-deoxystreptamine or streptamine moiety, w h i l e not s u f f i c i e n t , i s g e n e r a l l y necessary f o r a n t i b i o t i c a c t i v i t y among the a m i n o c y c l i t o l a n t i b i o t i c s (Figure 2) Ç 5 ) . The stereochemistry a t the 2 - p o s i t i o n of the a m i n o c y c l i t o l moiety i s a l s o important. The semisynthetic neomycin analogs, hybrimycins and B 2 of Rinehart and co-workers ( 6 ) , i n which 2-epi-streptamine has been i n c o r p o r a t e d , e x h i b i t g r e a t l y reduced a n t i b i o t i c a c t i v i t y . Spectinomycin has, t h e r e f o r e , been modified at the 7 - p o s i t i o n , which corresponds to the 2 - p o s i t i o n of streptamine, i n an e f f o r t to enhance potency. I n a d d i t i o n , the R - f a c t o r mediated i n a c t i v a t i o n of spectinomycin by 0-8412-0554-X/80/47-125-133$05.00/0 © 1980 American Chemical Society

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


AMINOCYCLITOL ANTIBIOTICS



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a d e n y l y l a t i o r i of the 9-hydroxy group (7,8) prompted m o d i f i c a t i o n s at the 9 - p o s i t i o n . The l a b i l i t y of the spectinomycin molecule s e v e r e l y l i m i t s d i r e c t chemical m o d i f i c a t i o n and a s u i t a b l y blocked d e r i v a t i v e was r e q u i r e d to withstand the r i g o r s of deoxygenation and e p i merization. The most unstable f e a t u r e of the molecule i s the alpha keto hemi k e t a l f u n c t i o n a l i t y at 4a. In b a s i c s o l u t i o n the i n c i p i e n t 1,2-diketone undergoes a b e n z i l i c a c i d rearrangement to the r i n g opened a c t i n o s p e c t i n o i c a c i d ( F i g u r e 3), while i n a c i d , both the hemi k e t a l and g l y c o s i d i c bridges are cleaved to give the component a c t i n o s p e c t o s e and actinamine. Although the C-4 carbonyl of spectinomycin normally e x i s t s as a gem d i o l , the usual ketone d e r i v a t i v e can be formed and s t a b i l i z e the molecule. Our attempts to regenerate the ketone f u n c t i o n were, however, unsuccessful. As reported by Wiley, e t . a l . (1,9), spectinomycin can be reduced to an epimeric mixture of 4-dihydrospectinomycins. In our hands, r e d u c t i o n of spectinomycin d i h y d r o c h l o r i d e with borohydride i n methanol proceeded s t e r e o s p e c i f i c a l l y to the 4 ( S ) or e q u a t o r i a l epimer of dihydrospectinomycin (Figure 4). Hydrog e n t a t i o n , u s i n g a rhodium on carbon c a t a l y s t on the other hand, gave predominately (up to 95%) the 4(R)- or a x i a l dihydrospectinomycin (10). The stereochemistry of the two epimers was e s t a b l i s h e d by a n a l y s i s of t h e i r PMR s p e c t r a and a knowledge of spectinomycin stereochemistry as e s t a b l i s h e d by the x-ray study of Chochran and co-workers (2)· 4(R)-Dihydrospectinomycin i s a convenient s t a r t i n g m a t e r i a l , c o n s i d e r a b l y more s t a b l e than spectinomycin, and i s amenable to p r o t e c t i o n of i t s alpha hydroxy hemi k e t a l f u n c t i o n a l i t y v i a the 4,4a acetonide. A l s o , the a x i a l hydroxyl group of a s u i t a b l y blocked d e r i v a t i v e undergoes s e l e c t i v e o x i d a t i o n back to the 4-oxo or spectinomycin analog. The amino b l o c k i n g group should be one which can be removed under c o n d i t i o n s which maintain the i n t e g r i t y of the spectinomycin molecule. Reaction of an epimeric mixture of d i h y d r o s p e c t inomycins with 2,2-dimethoxy propane gives only the expected c i s product, 4(R)-dihydrospectinomycin 4,4a-acetonide. Both the 4(R)- and 4(S)-dihydrospectinomycins, although subs t a n t i a l l y l e s s a c t i v e than spectinomycin, e x h i b i t both In v i t r o and i n v i v o a n t i b i o t i c a c t i v i t y , with the R-epimer being somewhat more a c t i v e than the S. While the dihydrospectinomycin a n t i b a c t e r i a l s p e c t r a are d i f f e r e n t than that of spectinomycin, organisms r e s i s t a n t to spectinomycin are a l s o r e s i s t a n t to the dihydrospectinomycins. E x t r a p o l a t i o n of s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s from the d i h y d r o - s e r i e s to the 4-oxo or spectinomyc i n s e r i e s i s at best u n c e r t a i n . E p i m e r i z a t i o n at C-7 was achieved v i a r e d u c t i o n of a 3 diketone (Figure 5). Oxidation of the N,N -dicarbobenzoxy-4(R)dihydrospectinomycin-4,4a-acetonide 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 f o r 16 hours at room temperature gave 7,9-dioxo-di-Zacetonide. The crude diketone was immediately t r e a t e d with sodium f


Figure 5.




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borohydride to provide a s t e r e o s p e c i f i c r e d u c t i o n a t both s i t e s to give the d e s i r e d 7-epi_-di-Z-4(R)-dihydrospectinomycin-4,4aacetonide (11)* Removal of the acetonide block f o l l o w e d by o x i d a t i o n with a mixture of DMSO and a c e t i c anhydride f o r a short p e r i o d of time gave the 4-oxo-analog, 7-epi-di-Z-spectinomycin (Figure 6 ) . Removal of the carbobenzoxy groups by c a t a l y t i c hydrogénation t o provide 7-epi-spectinomycin was achieved only a f t e r exhaustive purification. C a t a l y s t poisons, apparently d e r i v e d from the DMSO o x i d a t i o n step, were f i n a l l y separated by p a r t i t i o n chromatography. Evidence f o r the C-7 stereochemistry was obtained from an a n a l y s i s of the 270 MHz PMR spectrum of 7-epi-4(R)-dihydrospectinomycin. Only the C-3 e q u a t o r i a l and C-4 protons e x h i b i t small c o u p l i n g constants, which r e q u i r e s that a l l the protons i n the actinamine d e r i v e d r i n g are now a x i a l . A n a l y s i s of the PMR s p e c t r a of carbobenzoxy-blocked intermediates c o u l d not be achieved due to the presence of rotamers causing broad and i l l defined resonances. Extensive chemical s h i f t overlap of r i n g protons precluded a n a l y s i s of the spectrum of 7-epi-spectinomycin. Di-Z-4(R)-Dihydrospectinomycin-4,4a-acetonide was s e l e c t i v e l y o x i d i z e d i n h i g h y i e l d by the P f i t z n e r - M o f f a t t technique (12) to give the 9-oxo-analog (Figure 7) (13). Complete c h a r a c t e r i z a t i o n of t h i s ketone was precluded by the ready e l i m i n a t i o n of water during s i l i c a g e l column chromatography to give an a , 3 unsaturated ketone. This intermediate was i d e n t i f i e d as the Δ7,8-9-oxo-di-Z-4(R)-dihydrospectinomycin-4,4a-acetonide. T r e a t ment of t h i s unsaturated ketone with sodium borohydride provided a s t e r e o s p e c i f i c r e d u c t i o n with r e g e n e r a t i o n of the n a t u r a l stereochemistry a t both C-8 and C-9 to g i v e 7-deoxy-di-Z-4(R)dihydrospectinomycin-4,4a-acetonide. Removal of the carbobenzoxy groups (Figure 8) y i e l d e d 7deoxy-4(R)-dihydrospectinomycin-4,4a-acetonide, an intermediate whose s t r u c t u r e and stereochemistry c o u l d be assigned from an a n a l y s i s of i t s 270 MHz PMR spectrum. The acetonide block was then removed from 7-deoxy-di-Z-4(R)dihydrospectinomycin-4,4a-acetonide to provide 7-deoxy-di-Z-4(R)dihydrospectinomycin, which was f u r t h e r deblocked t o 7-deoxy4(R)-dihydroxpectinomycin. S e l e c t i v e o x i d a t i o n of the C-4 a x i a l hydroxyl group of 7deoxy-di-Z-4(R)-dihydrospectinomycin by DMSO-acetic anhydride again gave the d e s i r e d 4-oxo-analog, 7-deoxy-N,N -dicarbobenzoxyspectinomycin. A l l atempts to remove the carbobenoxy groups by c a t a l y t i c hydrogénation, however, f a i l e d to give the d e s i r e d 7deoxyspectinomycin. N,N -9-0-Triacetyl-4(R)-dihydrospectinomycin-4,4a-acetonide was d e r i v e d by s e l e c t i v e a c e t y l a t i o n of the 4 ( R ) - d i h y d r o s p e c t i n omycin-4,4a-acetonide (Figure 9) (10). The amino groups i n t h i s compound are p r o t e c t e d from o x i d a t i o n and the 9-hydroxyl f u n c t i o n i s converted to a l e a v i n g group. Treatment of t h i s t r i a c e t a t e ,

f




138

Figure 7.


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with a modified C o l l i n s reagent (14) gave another ot,3-unsaturated ketone, which was i d e n t i f i e d as the A8,9-7-oxo-N,N -diacetyl-4(R)dihydrospectinomycin-4,4a-acetonide on the b a s i s of i t s PMR spectrum. C a t a l y t i c hydrogénation s e l e c t i v e l y reduced the Δ 8 , 9 double bond w i t h concomitant r e g e n e r a t i o n of the n a t u r a l s t e r e o chemistry at C-8 to give 7-oxo-9-deoxy-N,N -diacetyl-4(R)-dihydrospectinomycin-4,4a-acetonide. Sodium borohydride r e d u c t i o n again proceeded i n a s t e r e o s p e c i f i c manner to give only the unnatural epimer at C-7, 7-epi-9-deoxy,N-N'-diacetyl-4(R)-dihydrospectinomycin-4,4a-acetonide. The a c e t y l blocks were removed with base and the acetonide block with d i l u t e a c i d to provide 7 - e p i - 9 deoxy-4(R)-dihydrospectinomycin s u l f a t e . The s t r u c t u r e and stereochemistry were assigned from a complete a n a l y s i s of the 270 MHz PMR spectrum i n p y r i d i n e ^ s o l u t i o n at 1 1 0 ° . A l l of the spectinomycin analogs d e s c r i b e d (Figure 1 0 ) , 7epi-spectinomycin and 7-epi-4(R)-dihydrospectinomycin, 7-deoxy4(R)-dihydrospectinomycin and 7-epi-9-deoxy-4(R)-dihydrospectinomycin, are devoid of a n t i b i o t i c a c t i v i t y as measured by the agar d i l u t i o n method on pH 8 n u t r i e n t agar at 500 ug/ml versus a v a r i e t y of organisms. At t h i s p o i n t , i t i s s u f f i c e to say that our m o d i f i c a t i o n r a t i o n a l e , d e r i v e d from the various a m i n o c y c l i t o l a n t i b i o t i c s , i s not a p p l i c a b l e to spectinomycin. Both the stereochemistry and the presence of the C-7 hydroxyl group i s important to a n t i b i o t i c activity. The question of m o d i f i c a t i o n at C - 9 , however, was unanswered s i n c e the 7-epi-analogs proved to be i n a c t i v e . In an e f f o r t to deoxygenate the 9 - p o s i t i o n , the 9 - 0 - t o l u e n e s u l f o n a t e e s t e r of N,N -di-Z-4(R)-dihydrospectinomycin-4,4aacetonide (Figure 1 1 ) was prepared by standard procedures. Also prepared were the 9-mesylate and the 7,9-dimesylate as w e l l as the corresponding t r i f l a t e s . We were unable to introduce a t o s y l group i n t o the 7 - p o s i t i o n . None of these s u l f o n a t e e s t e r s c o u l d be d i s p l a c e d or r e d u c t i v e l y cleaved by a v a r i e t y of reagents. A f t e r removal of the carbobenzoxy groups, however, the 9 - t o s y l a t e r e a d i l y c y c l i s e d (15) to give the 8,9-epimino-4(R)-dihydrospectinomycin-4,4a-acetonide. High pressure hydrogénation of t h i s epimine, as w e l l as i t s 6-acetyl d e r i v a t i v e , under a v a r i e t y of c o n d i t i o n s (16), f a i l e d to give a r i n g opened analog. The r i n g was r e a d i l y opened with N H 4 C I and with H C 1 (17) to g i v e , r e s p e c t i v e l y , and a f t e r removal of the acetonide block, 9 - c h l o r o 9-deoxy-4(R)-dihydrospectinomycin and 8-epi-chloro-8-des-(methylamino)-9-epi-(methylamino)-9-deoxy-4(R)dihydrospectinomycin. 8,9-Epimino-4(R)-dihydrospectinomycin i t s e l f was obtained by removal of the acetonide block with s u l f u r i c a c i d . 9-Epi-chloro-9-deoxy-4(R)-dihydrospectinomycin and 9 - e p i chloro-9-deoxyspectinomycin were prepared d i r e c t l y (Figure 1 2 ) from s u i t a b l y blocked intermediates by c h l o r i n a t i o n , with i n v e r s i o n , using the N-chlorosuccinimide-triphenylphosphine method of Hanessian, e t . a l . (18). f


f

f


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OH

HNCH

O 3

H

OH

7-Deoxy-4(R)-Dihyrospectinomycin

HNCH3

° OH H

7-Epi-9-Deoxy-4(R)-Dihydrospectinomycin Figure 10.

Figure 11.


142

AMINOCYCLITOL

OH

CI

1

^yVVV" H O - ^ V ^ O ^ T ^ ι OH" ZNCH Ο

ANTIBIOTICS

1.NCS-(C H ) P

Η

2.H /Pd-BaS0

ΗΟ^^ν^Ο^'Γ^ Τ OH» HNCH Ο

6

5

3

2

4

€

^ ^ Ο ^ Ο γ Ο Η

3


3

Ν

Figure 13.


3

8. ROSENBROOK AND CARNEY


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Again, a l l of these analogs f a i l e d to e x h i b i t a n t i b i o t i c a c t i v i t y and the e f f e c t of C-9 deoxygenation and e p i m e r i z a t i o n remained unknown. Recently, however, F o l e y , L i n , and Weigle of HoffmannLa Roche (19,20) prepared the 9-deoxy- and 9-epi-analogs of both spectinomycin and 4(R)-dihydrospectinomycin ( F i g u r e 13). A l l four compounds were found to be devoid of a n t i b i o t i c a c t i v i t y . A l l of the m o d i f i c a t i o n s thus f a r made i n the c y c l i t o l p o r t i o n of spectinomycin and 4(R)-dihydrospectinomycin, i n c l u d i n g such changes as N-demethylation, N - a l k y l a t i o n , N - a c y l a t i o n , and 9-0-acylation (21), r e s u l t i n the complete l o s s of a n t i b i o t i c activity. We have managed to confirm that spectinomycin i s indeed an a t y p i c a l a m i n o c y c l i t o l a n t i b i o t i c . Acknowledgement We wish to acknowledge the c o n t r i b u t i o n s of M. C i r o v i c , D. A. Dunnigan, R. S. Egan, R. J . M a u r i t z , S. L. M u e l l e r and R. S. Stanaszek of Abbott L a b o r a t o r i e s and K. Mochida, Y. Mori and T. Nishinaga of the Tokyo Research Laboratory, Kyowa Hakko Kogyo Company, L t d .

Literature Cited 1.

Wiley, P. F., Argoudelis, A. D., and Hoeksema, H., J. Amer. Chem. Soc. (1963) 85, 2652~2659.

2.

Cochran, T. G., Abraham, D. J., and Martin, L. L., J.C.S. Chem. Comm. (1972) 494~495.

3.

Gale, E. F., Cundliffe, Ε., Reynolds, P. Ε., Richmond, Μ. Η., and Waring, M. J., in "The Molecular Basis of Antibiotic Action", pp. 306~307, John Wiley and Sons, London, 1972.

4.

Novak, E., Schlagel, C. Α., LeZotte, L. Α., and Pfeifer, R. T., J. Clin. Pharm. (1974) 442~447.

5.

Davies, J., Antimicro. Agents and Chemoth. (1968) 1967, 297~303.

6.

Shier, W. T., Rinehart, K. L., and Gottlieb, D., Proc. Natl. Acad. Sci. U.S. (1969) 63, 198~204.

7.

Benveniste, R., Yamade, T., and Davies, J., Infect. Immun. (1970) 1, 109~119.

8.

Kawabe, Η., Inoue, Μ., and Mitsuhashi, S. Antimicro. Agents and Chemoth. (1974) 5, 553~557.




144

9.

Knight, J. C. and Hoeksema, Η., J. Antibiot. (1975) 28, 136~142.

10.

Rosenbrook, Jr., W. and Carney, R. Ε., J. Antibiot. (1975) 28, 953~959.

11.

Rosenbrook, Jr., W. Carney, R. E., Egan, R. S. Stanaszek, R. S., Cirovic, Μ., Nishinaga, T., Mochida, Κ., and Mori, Y., J. Antibiot. (1975) 28, 960~964.

12.

Pfitzner, Κ. E. and Moffatt, J. G., J. Amer. Chem. Soc. (1965) 87, 5670~5678.

13.

Rosenbrook, Jr., W. Carney, R. Ε., Egan, R. S., Stanaszek, R. S., Cirovic, Μ., Nishinaga, T., Mochida, K, and Mori, Y., J. Antibiot. (1978) 31, 451~455.

14.

Ratcliffe, R. and Rodehorst, R., J. Org. Chem. (1970) 35, 4000~002.

15.

Carney, R. E. and Rosenbrook, Jr., W. J. Antibiot. (1977) 30, 960~64.

16.

Daniels, P.J.L., Weinstein, J., Tkach. R. W., and Morton, J., J. Antibiot. (1974) 27, 150~154.

17.

Ali, Y., Richardson, A. C., Gibbs, C. F., and Hough, L., Carbohyd. Res. (1968) 7, 225~271.

18.

Hanessian, S., Ponpipom, Μ. Μ., and Levallee, P., Carbohyd. Res. (1972) 24·, 45~56.

19.

Foley, L., Lin, J.T.S., and Weigle, Μ., J. Antibiot. (1978) 31, 979~984.

20.

Foley, L., Lin, J.T.S., and Weigle, Μ., J. Antibiot. (1978) 31, 985~990.

21.

Rosenbrook, Jr., W., Carney, R. E., Nishimaga, T., Mochida, K, and Mori, Y., unpublished work.

RECEIVED

November 15, 1979.


Aminocyclitol Antibiotics - American Chemical Society

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