Aminocyclitol Antibiotics - American Chemical Society

10 Chemical Modification of Aminoglycosides: A Novel Synthesis of 6-Deoxyaminoglycosides

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BARNEY J. MAGERLEIN

The Upjohn Company, Kalamazoo, MI 49001

As part of a program to study structure-activity relationships among the semisynthetic aminoglycoside antibiotics, we elected to prepare a family of 6-deoxyaminoglycosides. 6-Deoxyneomycin and related compounds have been described in the literature (2, 3, 4, 5). The starting point in our synthesis was neamine which is readily obtained by methanolysis of neomycin. As shown in Figure 1 neamine (1) was blocked on nitrogen by the trifluoroacetyl group giving tetrakis-amide (2) in high yield. The trifluoroacetyl blocking group proved to be quite desirable in this situation since it not only could be readily removed with dilute alkali, but also conferred good solvent solubility on the intermediates. Then too, the trifluoroacetyl intermediates were sufficiently volatile to permit satisfactory vpc-mass spectrum evaluation. The trifluoracetyl derivative (2) when treated with 2,2-dimethoxypropane under mild conditions gave a high yield of monoketal (3) with varying amounts of diketal (4). This diketal was readily converted to monoketal (3) in the presence of dilute acid. Carbon-13 nuclear magnetic resonance definitely established that the ketal group in monoketal (3) was at 0-5,6 as shown. This is in agreement with the findings of ketalization of neamines blocked by other groups on nitrogen. The next two steps proceeded smoothly and i n high y i e l d (Figure 2). The hydroxyls a t C-3 and 4 were a c y l a t e d e i t h e r w i t h a c e t y l , o r i n cases where a UV v i s i b l e group was d e s i r e d f o r TLC r e f e r e n c i n g , w i t h p - n i t r o b e n z o y l , t o form (5a) o r (5b). M i l d a c i d h y d r o l y s i s gave blocked neamine d e r i v a t i v e s (6a) o r (6b) i n almost q u a n t i t a t i v e y i e l d . The question now was which of these two e q u a t o r i a l hydroxyls would be more r e a c t i v e . The pioneering work of Umezawa i n the s y n t h e s i s of the kanamycins i n d i c a t e d that the 6-hydroxyl would be more a v a i l a b l e f o r g l y c o s y l a t i o n by the Koenigs-Knorr r e a c t i o n than the 5-hydroxyl (6). He pointed out that t h i s s e l e c t i v i t y was i n keeping w i t h the observation that hydroxyls adjacent t o a g l y c o s i d i c bond show diminished This i s the second p a r t of a s e r i e s on the m o d i f i c a t i o n o f aminoglycosides. See Réf. JL f o r P a r t I . f

f

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0-8412-0554-X/80/47-125-169$05.00/0 © 1980 American Chemical Society

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

170

AMINOCYCLITOL ANTIBIOTICS


10.

MAGERLEiN

6~Deoxyaminoglycosides

171

r e a c t i v i t y . This observation has been repeatedly confirmed by other i n v e s t i g a t o r s (7, 8_, 9). We have examined t h e g l y c o s y l a t i o n o f t h i s d i o l using both the Koenigs-Knorr and the g l y c a l g l y c o s y l a t i o n procedures. S u b s t i t u t i o n occurred s e l e c t i v e l y a t 0-6. I n no case d i d we i s o l a t e a pure f r a c t i o n which could be assigned an 0-5 s u b s t i t u ted s t r u c t u r e . Thus 5-0-substituted-neamines are not r e a d i l y a v a i l a b l e from t h i s intermediate. One f a c e t of our program, however, was the p r e p a r a t i o n o f j u s t such analogs. One p o s s i b l e way t o prepare such compounds from the a v a i l a b l e intermediates would be t o remove the more r e a c t i v e 6-hydroxyl, l e a v i n g only t h e 5- hydroxyl as a s i t e f o r g l y c o s y l a t i o n . From some of our r e l a t e d work, as w e l l as r e p o r t s i n the l i t e r a t u r e , we know that the v a r i o u s hydroxyls on the neamine moiety c o n t r i b u t e l i t t l e t o in vitro a n t i b a c t e r i a l potency (10, 11). A 5-0-substituted 6-deoxy-neamine (7) may be expected t o be as potent as a 5-0-substituted-neamine (8) but l e s s d i f f i c u l t t o prepare (Figure 3 ) . The removal of the 6-hydroxyl was t h e r e f o r e of i n t e r e s t and was accomplished by what we b e l i e v e t o be an unique r e a c t i o n . T o s y l a t i o n o f d i o l (6a) was q u i t e s e l e c t i v e even i n the presence of a l a r g e excess of t o s y l c h l o r i d e t o g i v e 6- t o s y l a t e (9) (Figure 4 ) . I n a d d i t i o n t o the 6 - t o s y l a t e , about 5% o f t h e 5 - t o s y l a t e could be i s o l a t e d . CMR c l e a r l y i n d i c a t e d t h a t the major product was the expected 6-0-tosylate (9). When t r e a t e d w i t h potassium i o d i d e i n DMF, t h e r e p l a c e ment o f i o d i d e f o r t o s y l was not i n evidence, but ketone (10) was i s o l a t e d i n 60% y i e l d . A c l u e as t o how t h i s transformation takes p l a c e may be gained i n n o t i n g that treatment of t o s y l a t e (9) w i t h L i C l i n DMF r e s u l t e d i n f a c i l e displacement of c h l o r i d e f o r t o s y l . This suggests that the f i r s t step i n ketone formation i s replacement t o i o d i d e (11) which l o s e s HI i n the presence o f DMF t o y i e l d ketone (10). I n a d d i t i o n t o the major product of t h i s r e a c t i o n , s e v e r a l minor products were a l s o i s o l a t e d . One o f these products, formed by the l o s s o f a t r i f l u o r o a c e t a m i d o group, i s unsaturated ketone (12) (Figure 5 ) . When the ketone forming step was c a r r i e d out under more vigorous c o n d i t i o n s o r i n hexamethylphosphoramide, the major r e a c t i o n product was s u b s t i t u t e d c a t e c h o l (13). Reduction o f ketone (10) w i t h sodium cyanoborohydride gave c h i e f l y t h e 5 - e q u a t o r i a l a l c o h o l (14) as would be p r e d i c t e d by Barton's r u l e (Figure 6). I n a d d i t i o n , a few percent of the isomeric 5 - a x i a l a l c o h o l (15) was a l s o i s o l a t e d . Degradation o f 5-alcohol (14) w i t h concentrated hydrobromic a c i d followed by chromatography over an i o n exchange r e s i n r e s u l t e d i n the i s o l a t i o n o f 2,6-dideoxy-D-streptamine (16). This m a t e r i a l proved t o be i d e n t i c a l w i t h a known sample prepared by independent s y n t h e s i s and obtained from Dr. S. D. Gero of the I n s t i t u t de Chimie des Substances N a t u r e l l e s ( 2 ) . Thus, the c o n f i g u r a t i o n o f the 5-hydroxyl i n (14) i s u n e q u i v o c a l l y e q u a t o r i a l o r 3 as shown. Hydrogénation of ketone


172


Figure 4.


MAGERLEiN

6-Deoxy'aminoglycosides


174


(10) over platinum i n methanol s o l u t i o n gave p r i m a r i l y the a x i a l a l c o h o l (15). T h i s hydrogénation was h i g h l y s t e r e o s p e c i f i c though t r a c e amounts of the 5-3-alcohol were i s o l a t e d on chromatography. Degradation of a x i a l a l c o h o l (15) w i t h hydrobromic a c i d a f f o r d e d a d i a m i n o - t e t r a d e o x y i n o s i t o l (17) which was s i m i l a r , but d i f f e r e n t , from i t s isomer (16). The b l o c k i n g groups on oxygen and n i t r o g e n of compounds (14) and (15) were r e a d i l y removed w i t h a l k a l i to g i v e the epimeric p a i r of 6-deoxyneamines (18) and (19) (Figure 7 ) . The a n t i b a c t e r i a l spectrum and potency of these two neamine d e r i v a t i v e s were almost i d e n t i c a l w i t h that of neamine. T h i s i n d i c a t e d that the presence of the 6-hydroxyl i s not c r i t i c a l to a n t i b a c t e r i a l a c t i v i t y , nor must the c o n f i g u r a t i o n of the hydroxy at C-5 be e q u a t o r i a l as i n neamine. Maximum potency of neamine analogs i s only r e a l i z e d when s u b s t i t u t i o n i s present at e i t h e r 0-5 or 0-6 (11). Therefore, the 5 - e q u a t o r i a l or n a t u r a l a l c o h o l (14) was g l y c o s y l a t e d w i t h 2 , 3 , 5 - t r i - 0 - a c e t y l - D - r i b o f u r a n o s y l bromide i n the Koenigs-Knorr r e a c t i o n (Figure 8 ) . The 3 epimer (3 at 1") (203) was i s o l a t e d i n 35% y i e l d , w h i l e only 8% of the l e s s d e s i r a b l e α epimer (20a) was obtained. The b l o c k i n g groups were r e a d i l y removed w i t h d i l u t e a l k a l i to a f f o r d 6-deoxyribostamycin (213) and i t s epimer (21a) (Figure 9 ) . A s i m i l a r p a i r of epimeric aminoglycosides (223) and (22a) were prepared from the u n n a t u r a l 5 - a x i a l a l c o h o l (15) by g l y c o s y l a t i o n f o l l o w e d by h y d r o l y s i s . Thus we have the 4-isomeric 6-deoxyribostamycins to evaluate by in vitro a n t i b a c t e r i a l assay. The in vitro a n t i b a c t e r i a l t e s t i n g data f o r these amino g l y c o s i d e s i s t a b u l a t e d i n F i g u r e 10. In the f i r s t column are l i s t e d the b a c t e r i a a g a i n s t which the compounds were t e s t e d . This spectrum c o n t a i n s a few Gram-positive b a c t e r i a , but i t i s weighted i n f a v o r of the more d i f f i c u l t gram-negative b a c t e r i a . The r e s u l t s of the assay are expressed as MIC v a l u e s , the minimum c o n c e n t r a t i o n of the drug expressed i n micrograms per ml which w i l l completely i n h i b i t growth of the given b a c t e r i a under c o n d i t i o n s of the assay. In g e n e r a l , 6-deoxyribostamycin was the most potent. I t s 5-epimeric analog, r a t h e r s u r p r i s i n g l y , a l s o showed s i g n i f i c a n t a c t i v i t y . The l - a - e p i m e r s , given i n the two columns on the r i g h t , were g e n e r a l l y l e s s a c t i v e . This d i f f e r e n c e i s l e s s i n the C-5 u n n a t u r a l s e r i e s than i n the C-53 or n a t u r a l s e r i e s . One of our main o b j e c t i v e s of t h i s program was the prep a r a t i o n of aminoglycosides which possess u s e f u l a c t i v i t y versus pseudomonads. While 6-deoxyribostamycin has s i g n i f i c a n t a n t i b a c t e r i a l potency, i t s MIC vs Pseudomonas aeruginosa i s something l e s s than s a t i s f a c t o r y . The i n e f f e c t i v e n e s s of many aminoglycocosides to pseudomonads i s due to the p r o d u c t i o n of i n a c t i v a t i n g enzymes by these b a c t e r i a (10). The 3'-hydroxyl group i s one of the prime s i t e s f o r a t t a c k by phosphorylating enzymes. Amino g l y c o s i d e s which l a c k a 3'-hydroxyl or i n which the 3'-hydroxyl M


10.

MAGERLEiN

6-Deoxyaminoglycosides


175

176


Organism S. aureus UC 76 S. pyogenes UC 152 S. faecalis UC 694 S. pneumoniae UC 41 £. coli UC 45 K. pneumoniae UC 58 S. schottmuelleri UC 126 Ps. aeruginosa UC 95 P. vulgaris UC 93 P. mirabilis A-63 S. marcescens UC 131 S. f/exner/ UC 143 S. fyp/i/ TG-3 Figure 10.

6-Deoxy- 6Deoxy-5ribosta- epi-ribostamycin mycin 31.2 3.9 500 15.6 31.2 2.0 7.8 >500 31.2 125 62.5 31.2 15.6

250 31.2 1000 31.2 125 15.6 62.5 1000 62.5 500 62.5 125 31.2

6-Deoxy-aribostamycin

6Deoxy-5epi-a-ribostamycin

125 31.2 >500 125 250 15.6 125 >500 500 >500 500 500 62.5

31.2 31.2 1000 31.2 31.2 15.6 31.2 1000 500 1000 31.2 125 31.2

Antibacterial activities of 6-deoxyribostamycins (minimum inhibitory concentration mcg/mL)


10.

MAGERLEiN

6-Deoxy amino glycosides

177

has been c h e m i c a l l y removed, possess much g r e a t e r potency t o pseudomonads than those c o n t a i n i n g t h i s group. Therefore, removal of the 3'-hydroxyl, o r more simply the removal of both the 3'- and 4'-hydroxyls, was an a t t r a c t i v e means t o i n c r e a s e potency vs pseudomonads (11). The steps f o r removal of the 3',4'-hydroxyl u s i n g well-documented chemistry are o u t l i n e d i n F i g u r e 11 (12, 13). D i o l (23), whose p r e p a r a t i o n was d e s c r i b e d e a r l i e r , was converted t o dimesylate (24) i n high y i e l d . When t r e a t e d w i t h potassium i o d i d e and z i n c i n DMF, unsaturated compound (25) was obtained. T h i s m a t e r i a l was c a t a l y t i c a l l y hydrogenated t o form d i o l (26). In the manner p r e v i o u s l y d e s c r i b e d , t h i s d i o l was s e l e c t i v e l y monotosylated t o the 6t o s y l a t e (27), ( F i g u r e 12). Conversion t o ketone (28) and r e d u c t i o n proceeded smoothly, though the r e d u c t i o n was l e s s s t e r e o s p e c i f i c than i n the 3',4'-diacetoxy s e r i e s . The i s o m e r i c a l c o h o l s (283) and (29a) were then g l y c o s y l a t e d and s a p o n i f i e d t o complete the p r e p a r a t i o n of the two p a i r s o f epimeric 3',4',6t r i d e o x y r i b o s t a m y c i n s shown i n F i g u r e 13. In vitro a n t i b a c t e r i a l t e s t i n g data f o r these compounds u s i n g the same spectrum of organisms as shown before a r e o u t l i n e d i n F i g u r e 14. Once again the most a c t i v e i s the 53-l"3 or n a t u r a l isomer, 3',4',6t r i d e o x y r i b o s t a m y c i n . Note p a r t i c u l a r l y the MIC v a l u e VS Pseudomonas aeruginosa which i s 2 meg/ml compared w i t h a value o f g r e a t e r than 500 i n the 3',4'-dihydroxy s e r i e s . A comparison o f in vitro a c t i v i t y of 3',4',6-trideoxyribostamycin and 6-deoxyr i b o s t a m y c i n w i t h r i b o s t a m y c i n and kanamycin i s shown i n F i g u r e 15. Note that MIC values f o r 3',4',6-trideoxyribostamycin a r e c o m p e t i t i v e w i t h those f o r r i b o s t a m y c i n and kanamycin. Against one s t r a i n of Pseudomonas aeruginosa shown here, 3 ' , 4 ' , 6 - t r i deoxyribostamycin i s much more potent than the o l d e r a n t i b i o t i c s . The MIC values f o r 3',4',6-trideoxyribostamycin Vs a group of r e s i s t a n t pseudomonads and Staphylococcus aureus c l i n i c a l i s o l a t e s are shown i n F i g u r e 16. 3',4',6-Trideoxyribostamycin i s more e f f e c t i v e Vs v a r i o u s pseudomonads and some s t a p h y l o c o c c i than kanamycin, but l e s s e f f e c t i v e than gentamicin. In vivo t e s t i n g data f o r 3',4',6-trideoxyribostamycin a g a i n s t s e v e r a l organisms when administered subcutaneously i n the mouse i s given i n F i g u r e 17. Each v a l u e i s a CD59, the dose of compound, expressed i n mg/mg, which p r o t e c t s 50% of the mice from a l e t h a l i n f e c t i o n of the given organism. Whereas 3 ' , 4 ' , 6 - t r i deoxyribostamycin was somewhat l e s s e f f e c t i v e than r i b o s t a m y c i n vs K. pneumoniae i t was much more potent vs Ps. aeruginosa. T r i d e o x y r i b o s t a m y c i n was only about 1/4 o r 1/6 as potent as gentamicin a g a i n s t Ps. aeruginosa i n t h i s assay and a l s o l e s s e f f e c t i v e a g a i n s t E. coli. However, as shown i n F i g u r e 18, 3',4',6-trideoxyribostamycin possesses only about 1/5 the acute t o x i c i t y of gentamicin when assayed i n the mouse. Thus the t h e r a p e u t i c index f o r 3',4',6-trideoxyribostamycin a g a i n s t


178



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MAGERLEiN

HO

OH

HO OH

31ft

3lg

Figure 13.

Organism S. aureus UC 76 S. pyogenes UC 152 S. faecalis UC 694 S. pneumoniae UC 41 E. co// UC 45 K. pneumoniae UC 58 S. schottmuelleri UC 126 Ps. aeruginosa UC 95 P. vulgaris UC 93 P. mirabilis A-63 S. marcescens UC 131 S. f/exner/ UC 143 S. typ/i/ TG-3 Figure 14.

179


Isomeric 3'',4''fi'-trideoxyribostamycins

3\4\6-Trideoxyribostamycin

3',4',6-Trideoxy-5-epi ribostamycin

3',4',6Trideoxy-α- ribostamycin

3\4\6-Tri. deoxy-5-ep/a ribostamycin

15.6 2.0 250 15.6 31.2 2.0 3.9 2.0 7.8 125 7.8 31.2 7.8

31.2 15.6 >500 125 125 15.6 62.5 31.2 62.5 >500 62.5 250 125

7.8 7.8 >500 31.2 125 15.6 125 62.5 125 500 31.2 125 62.5

62.5 3.9 >250 >250 15.6 62.5 31.2 125 >250 62.5 125 31.2

Antibacterial activities of 3'4'6-trideoxyribostamycins (minimum in hibitory concentration mcg/mL)


180


3',4',6-Trideoxyribo- 6Deoxyribostamycin stamycin

Organism S. aureus UC 76 S. pyogenes UC 152 S. faecalis UC 694 S. pneumoniae UC 51 E. co/i UC 45 K. pneumoniae UC 58 S. schottmuelleri UC 126 Ps. aeruginosa UC 95 P. vulgaris UC 93 P. mirabilis A-63 S. marcescens UC 131 S. f/exner/ UC 143 S. typ/i/ TG-3

15.6 2.0 250 15.6 31.2 2.0 3.9 2.0 7.8 125 7.8 31.2 7.8

31.2 3.9 500 15.6 31.2 2.0 7.8 500 31.2 125 62.5 31.2 15.6

Ribo stamycin

Kanamycin

15.6 7.8 250 7.8 7.8 1.0 3.9 500 15.6 62.5 31.2 15.6 7.8

7.8 15.6 250 125 3.9 1.0 2.0 62.5 15.6 62.5 7.8 15.6 2.0

Figure 15. In vitro comparison of 3',4',6-trideoxyribostamycins and 6-deoxyri bostamycin with ribostamycin and kanamycin (minimum inhibitory concentration mcg/mL )

Organism (Resistant) Ps. aeruginosa 6436 6437 3680 3681 3682 3683 S. aureus 6686 6687 6688 6691 6695 Figure 16.

3\4\6·Τπ· Gentamicin Kanamycin deoxyribostamycin >250 15.6 31.2 15.6 62.5 15.6

31.2 250 1.0 2.0 3.9 2.0 250 2.0 31.2 >250 >250

125 0.25 3.9 3.9 3.9

>250 >250 250 >250 >250

In vitro testing vs. clinical isolates (minimum inhibitory concentration mcg/mL )


10.

MAGERLEiN


Compound 3',4',6-Trideoxyribostamycin Sulfate

381 mg/kg

Gentamicin Sulfate Figure 17.

85 mg/kg

In vitro antibacterial testing (mouse protection assay (CD SQ, mice))

50

3,4',6-Trideoxyribostamycin Sulfate

Ribostamycin Sulfate

Gentamycin Sulfate

K. pneumoniae UC 58 28.3 (19.6-40.7)

10.7 (8.2-14.0)

-

82 (54-136) 121 (79-187)

-

19(13-27)

23 (16.5-32)

-

0.51 (0.35-0.72)

21 (14-32) 20 (15-26)

-

2.5 1.09 (0.88-1.34)

Organism

Ps. aeruginosa UC 231

S. aureus UC 76

E. Coii UC 311 UC 45

Figure 18.

Acute toxicity (LD , TV-mouse) 50


mg/kg

182


Ps. aeruginosa i n the mouse i s not too f a r removed from that of gentamicin. The s y n t h e s i s o f a f a m i l y of 6-deoxyribostamycins has been described and in vitro and in vivo a n t i b a c t e r i a l t e s t i n g data have been reviewed. The key step i n the p r e p a r a t i o n of these semisynthetic a n t i b i o t i c s i s the unique p r e p a r a t i o n of 3\W-dl0-acetyl-5,6-dideoxy-5-oxo-l,2 ,3,6 -tetrakis-N-(trifluoro a c e t y l ) neamine (10) from 3 ' , 4 - d i - 0 - a c e t y l - 6 - 0 - t o s y l - l , 2 , 3 , 6 t e t r a k i s - N - ( t r i f l u o r o a c e t y l ) n e a m i n e ( 9 ) . This intermediate has p o t e n t i a l use f o r the p r e p a r a t i o n of other 5 - s u b s t i t u t e d - 6 deoxy-neamines. f

1

1

?

f

Acknowledgements The author i s indebted to G. E. Zurenko f o r in vitro a n t i b a c t e r i a l t e s t i n g , to K. F. Stern f o r mouse p r o t e c t i o n assays, to Dr. J . E. Gray f o r t o x i c i t y determinations and t o R. J . Reid f o r t e c h n i c a l a s s i s t a n c e .

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Reid, R. J., Mizsak, S. Α., Reineke, L. Μ., Zurenko, G. Ε., and Magerlein, B. J., Abstracts of Papers, 176th ACS National Meeting, Miami Beach, Florida, September 11-14, 1978, Medi 18, Cleophax, J., Gero, S. D., Leboul, J., Akhtar, Μ., Barnett, J. Ε. G., and Pearce, C. J., J. Amer. Chem. Soc. (1976) 98, 7110. Suami, T., Nishiyama, S., Ishikana, Y., and Katsura, S., Carbohyd. Res. (1977) 53, 239. Cleophax, J., Delaumeny, J. M. Gero, S. D., Rolland, Α., and Rolland, Ν., J. Chem. Soc., Chem. Commun. (1978) 773. Hayashi, T., Iwaoka, T., Takeda, Ν., and Ohki, Ε., Chem. Pharm. Bull. (1978) 26, 1786. Umezawa, S., Tsuchiya, T., Jihihara, T., and Umezawa, Η., J. Antibiot. (1971) 24, 711. Hasegawa, Α., Nishimura, D., and Nakajima, Μ., Carbohyd. Res. (1973) 30, 319. Suami, T., Nishiyama, S., Ishikawa, Y., and Katsura, S., Carbohydr. Res. (1976) 52, 187. Tanabe, M., Yasuda, D. Μ., and Detre, G., Tetrahedron Lett. (1977) 3607. Benveniste, R., and Davies, J., Antimicrobial Agents and Chem. (1973) 4, 402. Price, Κ. Ε., Godfrey, J. C., and Kauaguchi, in D. Perlman, Ed., "Structure-Activity Relationships Among the Semisynthetic Antibiotics," pp. 239-395, Academic Press, New York (1977). Jikihara, T., Tsuchiya, T., Umezawa, S., and Umezawa, Η., Bull. Chem. Soc., Japan (1973) 46, 3507. Umezawa, S., Umezawa, H., Okazaki, T., and Tsuchiya, T., Bull. Chem. Soc., Japan (1972) 45, 3624.

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November 15, 1979. Rinehart and Suami; Aminocyclitol Antibiotics ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Aminocyclitol Antibiotics - American Chemical Society

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