Fluorinated Analogs of Cell-Surface Carbohydrates as Potential

Chapter 11

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Fluorinated Analogs of Cell-Surface Carbohydrates as Potential Chemotherapeutic Agents Moheswar Sharma, Ralph J. Bernacki, and Walter Korytnyk Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263 The rationale for designing fluorinated analogs of cell surface carbohydrates as anticancer agents is described. Using diethylaminosulfur trifluoride, a general and a convenient method for synthesis of 6-deoxy-6-fluoro-hexosamines and 9-deoxy-9-fluoro-N-acetylneuraminic acid has been developed. This re-agent has also been used to introduce geminal fluorine both in the 6-position and 4-position (6,6-difluoro-D-galactose and 4,4-difluoro-N-acetyl-D-glucosamine). Another method for incorporation of fluorine is the addition of fluorine to the double bond in the acetylated glycals using XeF2 in the presence of BF3. This process is shown to be a convenient route to synthesize 1,2-dideoxy-1,2-difluoro sugars and 2-deoxy-2,3-di-fluoro-N-acetyl-D-neuraminic acid. Conventional methods of fluorination are used to synthesize 6-deoxy-6-fluoro-D-galactosamine and 4-deoxy-4-fluoro-D-galactosamine. The effects of these analogs on the growth of tumor cells (in vitro) and inhibition of macromolecular biosynthesis, as well as their therapeutic activity on syngenic mice with ascites L1210 leukemia (in vivo) are reported. As p a r t o f our program on a n t i t u m o r plasma-membrane m o d i f i e r s and i n h i b i t o r s , we have s y n t h e s i z e d s e v e r a l c a r b o h y d r a t e anal o g s , s u b s t i t u t i n g a f l u o r i n e f o r a hydroxyl group. These a n a l o g s were d e s i g n e d t o a c t as c a r b o h y d r a t e c h a i n t e r m i n a t o r s o f c e l l s u r f a c e g l y c o c o n j u g a t e s o r as i n h i b i t o r s o f v a r i o u s enzymes involved i n g l y c o p r o t e i n metabolism. A l t e r a t i o n o f the c e l l s u r f a c e c a r b o h y d r a t e s c o u l d be a c h i e v e d e i t h e r by i n h i b i t i o n o f N O T E : This chapter is dedicated to the late Walter Korytnyk.

0097-6156/88/0374-0191$06.00/0 • 1988 American Chemical Society

TAYLOR; Fluorinated Carbohydrates ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

t h e i r b i o s y n t h e s i s o r by i n c o r p o r a t i o n o f m o d i f i e d derivatives. T h i s c o u l d r e s u l t i n s i g n i f i c a n t changes i n the membrane s t r u c t u r e . The c h e m i c a l s y n t h e s i s o f t h e f l u o r i n a t e d d e r i v a t i v e s o f c e l l surface carbohydrates, namely galactose, L-fucose, galactosamine, g l u c o s a m i n e , mannosamine and M - a c e t y l - D - n e u r a m i n i c a c i d as w e l l as t h e i r b i o l o g i c a l e f f e c t s w i l l be reviewed i n t h e c o n t e x t o f ongoing research in t h i s area. There has been considerable interest to pursue approaches d i r e c t e d towards the m o d i f i c a t i o n and i n h i b i t i o n o f c e l l - s u r f a c e glycoprotein. Such m o d i f i c a t i o n may change the immunogenicity, t u m o r o g e n i c i t y and/or m e t a s t a t i c p o t e n t i a l o f cancer c e l l s . The p r i m a r y t a r g e t o f such m o d i f i c a t i o n a r e membrane s i a l i c a c i d , amino s u g a r s , and the o t h e r c e l l - s u r f a c e n e u t r a l s u g a r s . In a d d i t i o n , some c a r b o h y d r a t e a n a l o g s a r e e x p e c t e d t o modify the n u c l e o t i d e pool s i z e s and hence have p o t e n t i a l use i n c o m b i n a t i o n chemotherapy with n u c l e o s i d e a n a l o g s . Plasma-membrane components a r e i n v o l v e d i n c e l l g r o w t h , d i v i s i o n , movement, c o m m u n i c a t i o n , differentiation and a n t i g e n i c i t y . F x t e n s i v e i n v e s t i g a t i o n has shown t h a t changes occur i n these c e l l - s u r f a c e p r o p e r t i e s f o l l o w i n g oncogenic t r a n s f o r m a t i o n by v i r u s e s o r c a r c i n o g e n s . One o f the major components o f t h e c e l l s u r f a c e found t o be a l t e r e d f o l l o w i n g t r a n s f o r mation are the glycoconjugates. The g l y c o c o n j u g a t e s comprise glycoproteins and g l y c o l i p i d s t h a t a r e a t t a c h e d by t h e i r hydrophobic region to the l i p i d b i l a y e r . The c a r b o h y d r a t e c h a i n s o f these m o l e c u l e s a r e found on the o u t e r s u r f a c e o f t h e plasma membrane and c o n s i s t of fa) amino sugars, M-acetyl-P-glucosamine (GlcMAc), N-acetyl-D-galactosamine (GalMAc) and N - a c e t y l - D - n e u r a m i n i c a c i d "(MAMA); (b) n e u t r a l s u g a r s l i k e L - f u c o s e , D-mannose and D-galactose. The attachment t o membrane p r o t e i n o c c u r s e i t h e r as GlcNAc-asparagine l i n k e d N - g l y c o s i d i c a l l y o r as G a l N A c - s e r i n e ( o r t h r e o n i n e ) which a r e l i n k e d £ - g l y c o s i d i c a l l y . L-Fucose and s i a l i c a c i d s g e n e r a l l y o c c u r as t h e t e r m i n a l sugar (4). T h i s c o m p l e x i t y c o n f e r s a good deal o f i n f o r m a t i o n a l p o t e n t i a l t o t h e s u r f a c e c a r bohydrate p r e s e n t i n the v a r i o u s branched o l i g o s a c c h a r i d e structures (5). The r a t i o n a l e t o pursue s t u d i e s on the s y n t h e s i s and b i o c h e m i s t r y o f f l u o r o s u g a r s i s based on the o b s e r v a t i o n s t h a t : I. Biochemical d i f f e r e n c e s e x i s t i n the c e l l s u r f a c e complex carbohyd r a t e s o f tumor c e l l s . II. Sialic a c i d s may mask cell-surface a n t i g e n ; i t s removal enhances c e l l u l a r i m m u n o g e n i c i t y . I I I . Carboh y d r a t e s a r e i n v o l v e d i n the s o c i a l b e h a v i o r o f c e l l s such as c e l l to c e l l communications r e s u l t i n g i n c o n t a c t i n h i b i t i o n . I V . Changes in c e l l s u r f a c e c a r b o h y d r a t e s a r e found t o e f f e c t t h e uptake o f drugs and n u t r i e n t s . V . D i f f e r e n c e s a r e found t o d e v e l o p i n the m e t a b o l i s m o f membrane sugar f o l l o w i n g o n c o g e n i c t r a n s f o r m a t i o n . V I . Some membrane sugar and t h e i r a n a l o g s a r e c y t o t o x i c t o tumor cells (£). V I I . Uptake and a c c u m u l a t i o n o f some membrane sugars results in the lowering of specific intracellular nucleotide pools. V I I I . Some membrane sugars and t h e i r a n a l o g s have been shown t o i n h i b i t e n v e l o p e DNA and RMA v i r u s p r o l i f e r a t i o n . IX. Membrane s i g n a l t r a n s d u c t i o n i s a l t e r e d by oncogene p r o d u c t s following t r a n s f o r m a t i o n . The membrane sugar a n a l o g s under development i n t h i s program, may a c t on tumor o r v i r a l l y i n f e c t e d c e l l s i n t h e f o l l o w i n g manner:


11.

SHARMAET AL.

Fluorinated Analogs ofCell-Surface Carbohydrates

(a) In c o m p e t i t i o n as an a n t i m e t a b o l i t e w i t h the n a t u r a l sugar subs t r a t e l e a d i n g t o the i n h i b i t i o n o f membrane g l y c o c o n j u g a t e form a t i o n , (b) V i a i n c o r p o r a t i o n o f s t r u c t u r a l l y s i m i l a r a n a l o g s i n t o g l y c o n j u g a t e l e a d i n g t o c h a i n t e r m i n a t i o n , (c) In c o n j u g a t i o n with s p e c i f i c n u c l e o t i d e s l e a d i n g t o an a l t e r a t i o n i n n u c l e o t i d e pool s i z e s ( p o t e n t i a l use i n c o m b i n a t i o n chemotherapy), (d) Through i n t r o d u c t i o n o f changes i n c e l l s u r f a c e a n t i g e n i c i t y and immunogenicity. (e) By m o d i f i c a t i o n o f the s o c i a l b e h a v i o r o f c e l l s l e a d i n g to d e c r e a s e d tumor growth and m e t a s t a s i s , (f) A n t i v i r a l a c t i v i t y . S y n t h e s i s o f F l u o r i n a t e d Membrane Sugar A n a l o g s F u l l y a c e t y l a t e d membrane sugars and t h e i r a n a l o g s a r e found to be more c y t o t o x i c than t h e i r corresponding unacetylated derivatives. These d i f f e r e n c e s may have been the r e s u l t o f an i n c r e a s e i n the a g e n t s c e l l u l a r p e r m e a b i l i t y and p a s s i v e u p t a k e , s i n c e it has been found t h a t f u l l y a c e t y l a t e d glucosamine (e-PAGlcNAc) has a h i g h e r o c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t ( 0 . 4 2 ) than the p a r e n t compound, N - a c e t y l - D - g l u c o s a m i n e (0.17). After intracellular upt a k e , PA-GTcMAc was O - d e a c e t y l a t e d , p h o s p h o r y l a t e d , and a c t i v a t e d t o form UDP-GlcNAc, r e s u l t i n g i n a s i g n i f i c a n t l o w e r i n g o f c e l l u l a r UTP and PTP p o o l s ( 6 ) . A general and c o n v e n i e n t method f o r the s y n t h e s i s o f 6 - d e o x y - 6 f l u o r o h e x o s e s has been d e v e l o p e d u s i n g the f l u o r i n a t i n g agent N_,Ndiethylaminosulfur trifluoride (DAST) (7h This reagent permits the use o f the a c e t y l as the p r o t e c t i n g g r o u p ; however, the r e a c t i o n a l s o runs smoothly when the p r o t e c t i n g groups a r e b e n z y l o r methyl e t h e r s as w e l l ( 8 ) . DAST a l s o d i s p e n s e s w i t h the n e c e s s i t y o f i n t r o d u c i n g a l e a v i n g group as the h y d r o x y l group i s being directly r e p l a c e d with f l u o r i n e . The f o l l o w i n g 6-deoxy-6-fluoro hexopyranoses have been prepared using this reagent from the 6-hydroxy-acetylated hexoses: D-glucose, N-acetyl-D-glucosamine, M - a c e t y l - D - g a l a c t o s a m i n e and M-acetyl-D-mannosamine. In t h e p r e p a r a t i o n o f 6 - d e o x y - 6 - f l u o r o - D - m a n n o s a m i n e from 6 - d e o x y - 6 - f l u o r o - D glucosamine by base e p i m e r i z a t i o n o f the M - a c e t y l - d e r i v a t i v e , the s e p a r a t i o n was v e r y d i f f i c u l t for large-scale preparations, even though the e p i m e r i c m i x t u r e c o n t a i n e d about 20% o f the D-mannoepimer. We r e s o r t e d , t h e r e f o r e , t o the use o f N-acetyl-D-mannosamine ( 9 J . The usual p r o c e d u r e f o r the p r e p a r a t i o n o T the 6 - h y d r o x y t e t r a a c e t y l - d e r i v a t i v e c o u l d not be a p p l i e d w i t h N-acetyl-D-mannosamine as t h e i n i t i a l t r i t y l a t i o n s t e p i n v o l v e d p a r t i a l epimerizat i o n to N-acetyl-P-glucosamine i n the p r e s e n c e o f p y r i d i n e . The s t a r t i n g m a t e r i a l was 2 - a c e t a m i d o - l , 3 - d i - 0 - a c e t y l - 2 - d e o x y - 4 , 6 - C i s o p r o p y l idene-D-manno-pyranose (1) (10F. The a c e t a l group was opened up w i t h aqueous a c e t i c a c i d (60%T t o g i v e (2) ( F i g u r e 1) and the p r i m a r y h y d r o x y l group was t r i t y l a t e d i n the usual way t o g i v e ( 3 ) . The h y d r o x y l group i n the 4 - p o s i t i o n was a c e t y l a t e d and the t r i t y l group was removed t o y i e l d (4) which was then f l u o r i n a t e d w i t h DAST i n anhydrous diglyme t o g i v e ( 5 ) . Anhydrous diglyme was a l s o used as the s o l v e n t i n the p r e p a r a t i o n o f 6 - d e o x y - 6 - f l u o r o - D - g a l a c t o s e (11) and 6 - d e o x y - 6 , 6 d i f l u o r o - D - g a l a c t o s e (8) ( L e e , H . H . ; Hodgson, P . G . ; B e r n a c k i , R . J . ; K o r y t n y k , W.; Sharma, M. Carbohydr. Res., in press) (34) from 1,2:3,4-di-0-isopropyl idene-a-D-galactose and 1,2:3,4-dl-Cpsopropylidene-a-D-oalactohexo-dialdo-1,5-pyranose (6) respectively, by t r e a t m e n t w i t h DAST. D e b l o c k i n g o f the i n t e r m e d i a t e d i f l u o r o d e r i -


193

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v a t i v e (7) t o g i v e t h e t a r g e t compound (8) was a c c o m p l i s h e d by c o n ventional a c i d h y d r o l y s i s (Figure 2 ) . S i m i l a r l y , the preparation o f the 6 - d e o x y - 6 - f l u o r o - L - g a l a c t o s e o r 6 - f l u o r o - L - f u c o s e , from 1 , 2 : 3,4-di-O-isopropylidene-L-galactopyranose (12) has been reported (13). F l u o r i n a t i o n o f N - a c e t y l - D - g a l a c t o s a m i n e by t r e a t m e n t o f 1 , 3 , 4 t r i - O - a c e t y l - N - a c e t y l - D - g a l a c t o s a m i n e w i t h DAST gave low y i e l d s o f the H e s i red p r o d u c t . An a l t e r n a t i v e p r o c e d u r e was adopted t o p r e pare 6 - d e o x y - 6 - f l u o r o - N - a c e t y l - D - g a l a c t o s a m i n e (11) ( 1 4 ) . Treatment o f benzyl 2-acetamldo-2-deoxy-3,4-0-isopropylidene3"-0-mesyl-aD - g a l a c t o p y r a n o s i d e (9) ( F i g u r e 3) witFT cesium f l u o r i d e Th b o i l i n g ethanediol gave (10) whicfT was d e b l o c k e d by c a t a l y t i c hydrogenol y s i s t o (11) and then a c e t y l a t e d t o ( 1 2 ) . —

F l u o r i n a t i o n o f the anomeric carbon atom a l o n g w i t h the a d j a c e n t c a r b o n atom, o c c u r s by t h e a c t i o n o f f l u o r i n e on a c e t y l a t e d g l y c a l s (15). A n o t h e r method o f f l u o r i n a t i o n o f a c e t y l a t e d g l y c a l s i s t h e use o f t r i f l u o r o m e t h y l hypofluorite (CF3OF) ( 1 6 , 1 7 ) , which r e s u l t s i n a s i d e product o f t r i f l u o r o m e t h y l g l y c o s i d e . We a r e u s i n g xenon d i f l u o r i d e ( 1 8 ) , a r e a g e n t t h a t can be handled w i t h o u t spec i a l care or apparatus. The c o n v e n i e n c e o f t h i s r e a g e n t a l l o w s t h e preparation of F - l a b e l l e d compounds and hence i s o f potential use i n p o s i t r o n emmission tomography. Boron trifluoride-etherate was c a r e f u l l y used as a m i l d a c i d c a t a l y s t t o e f f e c t t h e f l u o r i n a t i o n and b e n z e n e - e t h e r m i x t u r e as a s o l v e n t t o a v o i d d i m e r i c and polymeric products. Thus, 3,4,6-tri-0-acetyl-2-deoxy-2-fluoro-a-Dglucopyranosyl f l u o r i d e (14), 3 , 4 , 6 - t r i - 0 - a c e t y l - 2 - d e o x y - 2 - f l u o r o - e D - g l u c o p y r a n o s y l f l u o r i d e (15) and 3 , 4 , 6 - t r i - 0 - a c e t y l - 2 - d e o x y - 2 f l u o r o - e - D - m a n n o p y r a n o s y l f l u o r i d e (16) were a l l o b t a i n e d by f l u o r i nation o f 3 , 4 , 6 - t r i - O - a c e t y l - D - g l u c a l (13) w i t h xenon difluoride ( F i g u r e 4) through both c i s and t r a n s a d d i t i o n o f f l u o r i n e ( 1 9 ) . The same mode o f r e a c t i o n was o b s e r v e d w i t h t r i - 0 - a c e t y l g a l a c t a T T 1 8

The composition of products resulting from cis and trans a d d i t i o n o f f l u o r i n e was found t o be the same when t h e r e a c t i o n was c a r r i e d o u t i n an oxygen o r n i t r o g e n atmosphere t o a v o i d any f r e e r a d i c a l r e a c t i o n mechanism. This r e a c t i o n with 3 , 4 - d i - 0 - a c e t y l L - f u c a l (17) (20) gave a l m o s t e x c l u s i v e l y t h e s i n g l e a d d i t i o n p r o duct 3,4-di-(Tacetyl-l,2-dideoxy-1,2-difluoro-L-fucose (18) ( 1 9 ) , which on a c i c T h y d r o l y s i s gave 2 - d e o x y - L - f u c o s e (19) ( F i g u r e T ) . From o u r e x p e r i m e n t a l data i t appears t h a t the r e a c t i o n proceeds as shown i n F i g u r e 6 . The r e a c t i o n o f u n s a t u r a t e d s i a l i c a c i d w i t h xenon d i f l u o r i d e was a l s o s t u d i e d i n t h e s i m i l a r f a s h i o n ( F i g u r e 7 ) . Methyl 4 , 7 , 8 , 9 t e t r a - 0 - a c e t y l - 2 , 3 - d e h y d r o - 2 - d e o x y - N - a c e t y l n e u r a m i n a t e (20)(21) r e a c t e d with xenon d i f l u o r i d e i n methylene c h l o r i d e i n t h e p r e s e n c e of boron t r i f l u o r i d e e t h e r a t e i n an oxygen atmosphere t o g i v e t h e 2,3-difluoro-derivative (21). In t h e absence o f oxygen the r e a c t i o n was v e r y s l u g g i s h and c o n s i d e r a b l e d e c o m p o s i t i o n o c c u r r e d . The a c e t y l a t e d 2 , 3 - d i f l u o r o - m e t h y l e s t e r (21) was d e a c e t y l a t e d t o the c o r r e s p o n d i n g d i f l u o r o a c i d (22) a t pH 7 w i t h o u t l o s s o f f l u o r ine. The l a t t e r compound on a c i d h y d r o l y s i s gave t h e 3 - f l u o r o - N a c e t y l n e u r a m i n i c a c i d (23) w i t h the h y d r o l y t i c removal o f anomeric fluorine. T h i s p r o c e s s s i g n i f i c a n t l y improved t h e y i e l d o f 3 f l u o r o - N - a c e t y l n e u r a m i n i c a c i d o v e r the p r e v i o u s l y r e p o r t e d p r o c e -


11. SHARMAETAL.


(I)

(2)

,(3)

(5)

(4)

F i g u r e 1. S y n t h e s i s o f t e t r a - 0 - a c e t y l - 6 - d e o x y - 6 - f l u o r o - D mannosamine (5). (a) AeOH-^O, 50%, (b) tritylation, A c 0 / P y . ( c ) Pd/c-AcOH, [ H ] ( d ) DAST. ?

2

F i g u r e 3 . Scheme f o r s y n t h e s i s o f N - a c e t y l - 3 , 4 - d i - 0 - a c e t y 1 - 6 deoxv-6-fluoro-Dgalactosamine (11). (a) CsF/ethyTene-alycol (200 C ) , (b) Pd/C i n AcOH, [ H ] , ( c ) A c 0 / P y . 2



F i g u r e 5. Synthesis of 2-deoxy-?-fluoro-L-fucose (19). (a) X e F , B F - e t h e r a t e , (b) 0 . 5 M HC1. 2

3


SHARMA ET AL.

Fluorinated Analogs of Cell-Surface Carbohydrates

Figure 6. Possible mechanism of addition of f l u o r i n e to glycals with XeF . (a) E l e c t r o p h i l i c addition of F from XeF -BF3 complex, (b) addition of F on carboxonium i o n , (c) anomerization of pyranoxylfluoride with BF3. 2

2

Figure 7. Synthesis of 3-deoxy-3-fluoro-NANA (23). (a) XeF , BF -etherate, (b) NaOMe-MEOH, (c) 0.5 N HC1. 2

3


198


dure (22). The F-NKR data were used t o d e t e r m i n e the conf i g u r a t i o n o f t h e f l u o r i n e atom o f methyl 4 , 7 , 8 , 9 - t e t r a - 0 - a c e t y l - 2 deoxy-2,3-difluoro-N-acetylneuraminic acid (21). The magnitude o f the J p _ 2 , F - 3 (I -5" Hz) indicates an a x i a l - e q u i t o r i a l arrangement. The c o u p l i n g c o n s t a n t J p _ H-3 d * Hz) i n d i c a t e s a d i a x i a l o r i e n t a t i o n o f the anomeric' f l u o r i n e and t h e H-3. These c o u p l i n g c o n s t a n t s and the absence o f any long range c o u p l i n g b e tween f l u o r i n e and hydrogen s u g g e s t t h e r i n g c o n f o r m a t i o n t o be C 5 (D) w i t h F-2 a x i a l and F-3 e q u i t o r i a l . 19

9

9

5

2

2

The 9 - p o s i t i o n o f N - a c e t y l n e u r a m i n i c a c i d (NANA) was s u b s t i t u t e d f o l l o w i n g two r o u t e s : Figure 8a, 2-Acetamido-2,6-dideoxy-6fluoro-D-glucopyranose (24) (8^,23) was condensed with potassium di-tert-butyloxaloacetate (Sharma, M.; Korytnyk, W., Carbohydr. R e s . , i n p r e s s ) and s u b s e q u e n t l y h y d r o l y z e d to 9 - d e o x y - 9 - f l u o r o NANA. Figure 8b, Methyl 2-0-methyl-4,7,8-tri-0-benzyl-N-acetylneuraminate (30) was o b t a i n e d by b e n z y l a t i o n o f meThyl 2 - 0 - m e t h y l 9-0-trityl-N-acetylneuraminate (28) and f o l l o w e d by d e t r i t y l a t i o n . Treatment o7 the d e t r i t y l a t e d p r o d u c t (30) w i t h DAST, f o l l o w e d by hydrogenolysis and hydrolysis, gave the 9-deoxy-9-fluoro-NANA (27). The d e t r i t y l a t e d i n t e r m e d i a t e p r o d u c t (30) was o x i d i z e d a t the 9 - p o s i t i o n t o t h e c o r r e s p o n d i n g 9 - a l d e h y d e d e r i v a t i v e (31) and then c o n v e r t e d to the 9 - d e o x y - 9 - d i f l u o r o - N - a c e t y l n e u r a m i n i c acid derivative (32). The C MMR spectrum o f 9-deoxy-9-fluoro-NANA (27) showed t h a t t h e i n t r o d u c t i o n o f f l u o r i n e a t the 9 - p o s i t i o n d e s h i e l d e d C-9 from 663.5 p . p . m . t o 8 1 . 4 (as a d o u b l e t Jrj_p 164.9 Hz). The l - F NMR spectrum o f 9 - d e o x y - 9 - f l u o r o - N A N A gave a c l e a r sextet (-235.4 p . p . m . , J _ g , 4 6 . 8 Hz; J H-8> -° ) ind i c a t i n g t h e absence o f a n y ' a n o m e r i c i s o m e r . 1 3

2

F

j

H

F

6

H z

>

S u b s t i t u t i o n o f the secondary h y d r o x y l group o f c a r b o h y d r a t e s with f l u o r i n e o c c a s s i o n a l l y g i v e s r i s e t o t h e problem o f i n v e r s i o n , both i n a c o n v e n t i o n a l d i s p l a c e m e n t r e a c t i o n o r r e a c t i o n w i t h DAST. However, t h e use o f t h e s e methods i n t h e f l u o r i n a t i o n r e a c t i o n a r e w o r k a b l e , i n such c a s e s , with a p r e c e e d i n g s t e p o f i n v e r s i o n when n e c e s s a r y , t o o b t a i n the r e q u i r e d e p i m e r i c f l u o r i n a t e d p r o d u c t ( 2 4 , 25). We were i n t e r e s t e d i n i n t r o d u c i n g f l u o r i n e i n the C-4 p o s i t i o n o f 2 - a c e t a m i d o - 2 - d e o x y - D - g l u c o p y r a n o s e and 2 - a c e t a m i d o - 2 deoxy-D-galactopyranose s i n c e these molecules occur i n the core r e g i o n o f the g l y c o p r o t e i n s . Our f i r s t attempt was t h e opening o f the 3 , 4 - e p o x i d e w i t h the f l u o r i d e n u c l e o p h i l e . Benzyl 2 - a c e t a m i d o 3,4-anhydro-2-deoxy-6-0-trityl-a-D-glucopyranoside (33) was t r e a t e d w i t h tetrabutylammonium f l u o r i d e i n a c e t o n i t r i l e t o g i v e the c o r r e s p o n d i n g D-gulosamine d e r i v a t i v e (26) ( F i g u r e 9 ) . Inversion of the h y d r o x y l group o f (34) a t C-3 co~UTd not be a c h i e v e d t o o b t a i n the D - g a l a c t o s a m i n e d e r i v a t i v e . However, 2 - a c e t a m i d o - l , 3 , 6 - t r i - 0 acetyl-2,4-dideoxy-4-fluoro-D-gulose (35) was i s o l a t e d and showed e n c o u r a g i n g i n h i b i t o r y a c t i v i t y on t h e growth o f L1210 leukemia cells. 2-Acetamido-2,4-dideoxy-4-fluoro-D-galactopyranose (38) and 2-acetamido-2,4,6-trideoxy-4,6-difluoro-D-galactopyranose (43) were s y n t h e s i z e d from b e n z y l 2-acetamido-3,4-di-0-benzyl-2-deoxy-4O - m e s y l - a - D - g l u c o p y r a n o s i d e (36) and b e n z y l 2 - a c e t a m i d o - 3 - 0 - b e n z y l 2-deoxy-4,6-di-P-mesyl-a-D-glucopyranoside (41) (Figure 10), res p e c t i v e l y , by d i s p l a c e m e n t o f the methanesulphonyloxy groups with tetrabutylammonium f l u o r i d e . These f l u o r o sugar d e r i v a t i v e s were


FluorinatedAnalogs of Cell-Surface Carbohydrates

SHARMA ET AL.

F i g u r e 8. T o p , s y n t h e s i s o f 9 - d e o x y - 9 - f l u o r o - N A N A from 6-deoxy6 - f l u o r o - N - a c e t y l - D - g l u c o s a m i n e ( 2 7 ) . (a) Potassium d i - t e r t b u t y l - o x a l o a c e t a t e - M E O H ( e p i m e r i z a t i o n ) , (b) c o n d e n s a t i o n and h y d r o l y s i s . Bottom, s y n t h e s i s o f 9 - d e o x y - 9 - f l u o r o - N A N A from NANA ( 2 7 ) . (a) Benzyl b r o m i d e - B a ( 0 H ) - D M F , (b) AcOH-H 0 (70%), 90 ° C , (c) DAST-CHoClo, NaOH-HoO, 0.05 N HC1, (d) Py-CrOo-CHoCl (e) D A S T - C H C 1 . 2

2

2

2


199

200


0-\-^0CH Ph 2

NHAc

\

/nr^PK

HO HNAc

(33)

(34)

F i g u r e 9. S y n t h e s i s fluoro-D-gulosamine [ H ] , Py/AcOH.

\

/ OAc HNAc (35)

of N-acetyl-1,3,6-tri-0-acetyl 4-deoxy-4( 3 D " , ( a ) BU4NF-CH3CN, (b) Pd/C-AcOH

HNAC

HNAc

(39)

(38)

HNAc (44)

HNAc (40)

HNAc (45)

F i g u r e 1 0 . Scheme f o r s y n t h e s i s o f ^ - a c e t y l - 4 - d e o x y - 4 - f l u o r o and N-acetyl-4,6-dideoxy-4,6-difluoro-D-galactosamine (39) and R 5 ) . (a) BU4NF-CH3CN, (b) [ H ] , Pd/c-AcOH, (c) Pt/C-H 0-0 , (d) Ac 0/Py, (e) BU4NF-CH3CM, (f) [H], Pd/C-AcOH, ( g ) P t / C - H 0 - 0 , (h) A c 0 - P y . 2

2

2

2

2

2


11.

SHARMAETAL.


a l s o o b t a i n e d by t r e a t i n g b e n z y l 2 - a c e t a m i d o - 3 , 6 - d i - 0 - b e n z y l - 2 d e o x y - a - D - g l u c o p y r a n o s i d e and b e n z y l 2-acetamido-3-(P>enzyl-a-Dglucopyranoside (27-29) w i t h DAST i n methylene c T i l o r i d e , but in much l o w e r y i e l d . Tfie" i n t e r m e d i a t e s were d e p r o t e c t e d by hydrogenol y s i s t o g i v e the f l u o r i n a t e d sugars (38) and ( 4 3 ) . The a l d o n o l a c tones (40) and (44) d e r i v e d from t h e s e s u g a r s by o x i d a t i o n w i t h m o l e c u l a r oxygen i n the p r e s e n c e o f p l a t i n u m on a c t i v a t e d c h a r c o a l were found t o be s t r o n g i n h i b i t o r s ( K i 27.2 uM and 41.4 nM, r e s p e c t i v e l y ) of hexosaminidases ( 2 5 , 3 2 ) . The a c e t y l a t e d d e r i v a t i v e s o f t h e s e s u g a r s (39) and (45) were shown t o be very good a n t i t u m o r agents in t i s s u e c u l t u r e (Table I ) . In the F NMR spectrum of the 4,6-dideoxy-4,6-difluoro-galactosamine derivative (42), it appeared from the v i c i n a l c o u p l i n g c o n s t a n t s ( J p . ^ H-5 ) t h a t t h e f a v o r e d rotamer about the C - 5 - C - 6 has F-6 a ' n t i - p e r i p l a n a r to C - 4 - C - 5 . 1 9

1

2

H

z

The b i o l o g i c a l a c t i v i t y o f these f l u o r o s u g a r s suggested the p r e p a r a t i o n o f the 4 - d e o x y - 4 , 4 - d i f l u o r o (48) and 3 - d e o x y - 3 - f l u o r o M-acetyl-P-glucosamine d e r i v a t i v e s . Benzyl-2-acetamido-3,6-di-0"5enzyl-2-deoxy-a-D-xylo-4-uloside (46) ( F i g u r e 11) was t r e a t e d with DAST to g i v e the 4 - d e o x y - 4 , 4 - d i f l u o r o - D - g l u c o s a m i n e d e r i v a t i v e (47) i n v e r y good y i e l d . A f t e r d e p r o t e c t i o n and a c e t y l a t i o n t h e d i f l u o r o compound (48) showed v e r y good i n h i b i t o r y a c t i v i t y o f c e l l growth (L1210) i n v i t r o ( T a b l e I ) . 2 - A c e t a m i d o - l , 3 , 6 - t r i - 0 - a c e t y l 2,4-dideoxy-4-fTuoro-P-glucosamine (52) was s y n t h e s i z e d by double i n v e r s i o n a t the 4 - p o s i t i o n f o l l o w i n g the scheme as shown i n F i g u r e 12. Compound (49) was o b t a i n e d from (36) by t r e a t m e n t w i t h l i t h i u m benzoate i n hot N , M - d i m e t h y l f o r m a m i d e , f o l l o w e d by d e b e n z o y l a t i o n with Amberlyst A-26-(0H) r e s i n i n methanol. Treatment o f (49) w i t h DAST and subsequent h y d r o g e n o l y s i s gave 2 - a c e t a m i d o - 2 , 4 - d i d e o x y - 4 f l u o r o - D - g l u c o s a m i n e (51) which was a c e t y l a t e d t o ( 5 2 ) . B i o l o g i c a l A c t i v i t y o f F l u o r i n a t e d Carbohydrates In view o f the p h y s i c o - c h e m i c a l p r o p e r t i e s o f the CF b o n d , which by s i z e f a l l s between the CH and C-OH b o n d , i t s i n t r o d u c t i o n i n t o carbohydrates should r e s u l t in b i o l o g i c a l l y analogous molecules. Numerous b i o c h e m i c a l a p p l i c a t i o n s o f f l u o r o s u g a r s have appeared i n the e a r l i e r literature (30,31), and t h e s e studies prompted the development o f s e v e r a l f l u o r o s u g a r s w i t h i n t h i s program. A number o f t h e s e sugars have been t e s t e d f o r t h e i r e f f e c t on tumor c e l l growth iji v i t r o , t h e i r e f f e c t s on m a c r o m o l e c u l a r b i o s y n t h e s i s and t h e i r antitumor a c t i v i t y in mice. These p r e l i m i n a r y s t u d i e s demons t r a t e t h a t the f l u o r o s u g a r s as a group a r e b i o l o g i c a l l y active. Their addition at concentrations ranging from 10~ M to 10~ M ( T a b l e I) to L1210 leukemia c u l t u r e s r e s u l t e d i n c e l l growth i n hibition. G e n e r a l l y , the f u l l y a c e t y l a t e d compounds h a v i n g g r e a t e r lipophilicity were more c y t o t o x i c to c e l l s . The nonacetylated a g e n t s , 6 - F - D - g a l a c t o s e and 9-F-MANA demonstrated c e l l growth i n h i b i t i o n at higher concentration, 1.0 and 0 . 8 mM, respectively. These two agents in addition demonstrated an a b i l i t y to alter c e l l u l a r s i a l i c acid metabolism. Thus, 6-F-D-galactose treatment resulted in d e c r e a s e d L1210 leukemia cell ectosialyltransferase a c t i v i t y (34) w h i l e 9-F-NANA was found t o a c t as a s u b s t r a t e f o r CMP-MAMA synthetase (Petrie, C.R.; Sharma, M.; Simmons, O.D.; Korytnyk, W., unpublished results 1987) and thus serves as a p r e c u r s o r f o r membrane i n c o r p o r a t i o n . 3


6

201

202


The f l u o r i n a t e d n e u t r a l s u g a r s a l s o mimicked t h e i r p a r e n t sugars by competing w i t h them f o r i n c o r p o r a t i o n i n t o m a c r o m o l e c u l a r components ( T a b l e I I ) . 6 - F - P - G a l a c t o s e and 6 - F - L - f u c o s e specifically d e c r e a s e d t h e i n c o r p o r a t i o n o f t h e i r c o r r e s p o n d i n g p a r e n t sugar t o 39 and 9%, r e s p e c t i v e l y , i n murine P288 lymphoma c e l l s grown i n tissue culture. L i t t l e s p e c i f i c e f f e c t was noted on leukemia c e l l i n c o r p o r a t i o n o f glcNAc o r l e u c i n e ( l e u ) w i t h t h e s e two a g e n t s , w h i l e f u l l y a c e t y l a t e d sugars reduced t h e i n c o r p o r a t i o n o f a l l p r e c u r s o r s , n o n s e l e c t i v e l y (Table I I ) . The a d m i n i s t r a t i o n o f f l u o r o sugars t o mice w i t h L1210 leukemia r e s u l t e d , i n a l l c a s e s , with small i n c r e a s e s i n l i f e span (% I L S ) , r a n g i n g from 11 t o 68% ( T a b l e III). The most a c t i v e a g e n t , 4 - F - g a l N A c - t r i - 0 - a c e t a t e or f l u g a l (68% ILS) i s c u r r e n t l y b e i n g e v a l u a t e d i n mice w i t h v a r i o u s o t h e r tumors t o more f u l l y a s s e s s i t s a n t i t u m o r p o t e n t i a l .

Table

I.

Fluoro

F f f e c t s o f F l u o r o - S u g a r A n a l o g s on Murine L1210 Leukemia C e l l Growth in v i t r o I C f ) 9 M* LeuK,emia L1210 5

Sugar

6-F-G1cNAc-tri-O-acetate 6-F-ManNAc-tri-U-acetate 6-F-GalNAc-tri-U-acetate 6-F-Gul NAc-tri-TJ-acetate 6-F-4-ene-lyxNAc-di-0-acetate 6-F-4-ene-lyx-tri-0-acetate 6 - F - G a l (34) 6-F-Mann-tetra-0-acetate 6-F-Glc-tetra-O-acetate 4-F-GalNAc-tri-O-acetate (flugal) 4,4-Di-F-GlcNAc-tri-O-acetate 4,6-Di-F-GalMAc-di-0-acetate 4-F-G1cNAc-Tri-O-acetate 9-F-MAMA 9-F-MAMA

5.0 1.0 1.7 6.0 1.0 3.2 1.0 5.5 5.0 3.0 3.4 2.0 3.0 3.6 8.0

X X X X X X X X X X X X X X X

10-5 10-4 10

1

10-5 io-; 10-6 10-3 10-5 lO10-5 10-5 10-5 10-5 lO-* l O " (TA3) 5

4

*The c o n c e n t r a t i o n o f f l u o r o s u g a r which i n h i b i t e d murine L1210 l e u kemia c e l l growth i n c u l t u r e by 50% o v e r a two-day p e r i o d ( 3 3 ) . C u l t u r e s were i n o c u l a t e d w i t h 5 x 1 0 c e l l s / m L i n RPMI 1640 medium c o n t a i n i n g 10% f e t a l b o v i n e serum. Sugar a n a l o g s were added and growth a l l o w e d f o r 24 h . C e l l number was measured and % c o n trol (no sugar a n a l o g added) growth was c a l c u l a t e d . The IC50 (50% growth i n h i b i t o r y c o n c e n t r a t i o n ) was d e t e r m i n e d from the dose response c u r v e . A l l a s s a y s were performed i n d u p l i c a t e on a t l e a s t two s e p a r a t e o c c a s s i o n s . TA3:Mouse mammary adenocarcinoma. 4


SHARMA ET AL.

Fluorinated Analogs of Cell-Surface Carbohydrates

(46)

(47)

(48)

F i g u r e 11. Synthesis of M-acetyl-l,3,6-tri-0-acetyl-4-deoxy4 , 4 - d i f l u o r o glucosamine T48). (a) DAST-CH2CT2, (b) [ H ] , Pd/C-ACOH, Ac^O-Py.

NHAc

(52)

(51)

F i g u r e 12. Synthesis of N-acetyl-l,3,6-tri-0-acetyl-4-deoxy4-fluoro-D-glucosamine fB2). (a) DAST-CH2CI2, (b) [H], Pd/C-AcOH, ( c ) A c 0 - P y . 2


204 Table

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS I I . E f f e c t s of Fluoro-Sugars P288 Lymphoma C e l l s

Fluoro-Sugars,

Cone.

6 - F - G a l (14) 6 - F - L - F u c o s e (14) 6-F-Glc-B-tri-flT

~T0=^ 10-

on M a c r o m o l e c u l a r B i o s y n t h e s i s

P288 Lymphoma Incorporation, and Leu % Control**

[M]

L-Fuc* 9

3

lO-

acetate, 6-F-ManNAc-tri-0acetate, 4-F-GalNAc-tri-Oacetate, 6-F-Mann

lOIPlO"

Gal* 39

3

3

5

GlcNac* 89 88

in

o f [ H ] Sugars 3

Leu* 84

-

46

51

-

55

71

88

86

3

**P288 lymphoma c e l l s (10 cells/mL) were i n o c u l a t e d i n medium RPMI 1640 c o n t a i n i n g 10% FBS (fetal bovine serum) with the i n d i c a t e d [ H ] r a d i o l a b e l e d p r e c u r s o r * (1 n C i / m L ) . A f t e r 5 h the amount o f i n c o r p o r a t e d r a d i o a c t i v i t y was d e t e r m i n e d and compared with c o n t r o l . A l l a s s a y s were performed i n d u p l i c a t e on t h r e e s e p a r a t e o c c a s s i o n s and the r e s u l t s l i s t e d a r e means. 5

3

Table

III. Effects vivo *

of Fluoro-Sugars

Fluoro-Sugar

on L1210 Leukemia i n Mice

Tumor

6-F-GlcNAc-tri-P-acetate 6-F-Mann-tri-0-acetate 4,6-Pi-F-GalNAc-tri-0-acetate 4-F-G1cNAc-tri-O-acetate 6-F-Mann 6-F-Mann-tetra-O-acetate 6-F Gal 4-F-GalNAc-tri-O-acetate ( F l u g a l ) (30) ~ 4-F-GulNAc^Tri-0-acetate 9-F-NANA 6-F-G1c-tetra-O-acetate 1,2-Pi-F-Glc-tri-O-acetate

Dose(mg/kg)

in

%ILS

L1210 L1210 L1210 L1210 L1210 L1210 L1210

6.25 25.0 10.0 100.0 200.0 200.0 25.0

12.2 11.5 11.4 33.0 17.8 27.4 32.0

L1210 L1210 L1210 L1210 L1210

50.0 50.0 50.0 6.25 400.0

68.0 12.2 19.0 21.0 35.0

l a t e d i . p . w i t h ID* L1210 leukemia c e l l s on day z e r o . Starting on day one mice were g i v e n v a r i o u s dosages (5 mice/dosage l e v e l ) o f the t e s t agent i . p . , once p e r day through day 5 . L i f e span was checked d a i l y . The % measure i n l i f e s p a n (% ILS) was c a l c u l a t e d as compared t o t h e c o n t r o l g r o u p , c o n s i s t i n g o f 8-10 a n i m a l s . The optimum % ILS i s l i s t e d . 5


11. SHARMA ETAL.


C o n c l u s i o n s and F u t u r e S t u d i e s T h i s program has attempted t o demonstrate t h a t , by i n t e r f e r i n g o r m o d i f y i n g t h e b i o s y n t h e s i s o f components o f tumor c e l l surface glycoconjugates, development o f chemotherapeutic agents may r e sult. In p u r s u i n g t h i s avenue several analogs of cell-surface carbohydrates were s y n t h e s i z e d and t e s t e d both i_n v i t r o and i j i vivo as a n t i t u m o r a g e n t s . C o m b i n a t i o n chemotherapy w i t h nucleos i d e a n a l o g s a l s o may improve t h e t h e r a p e u t i c r e s p o n s e as noted with P-glucosamine and p e n t a - O - a c e t y l - B - D - g l u c o s a m i n e (B-PAG1CNAC) in combination with a z a r i b i n e (330. I t appears from t h e p r e liminary experimental data i n our l a b o r a t o r y and o t h e r s that f l u o r i n a t e d s u g a r a n a l o g s a r e membrane-active a g e n t s and a r e worth f u r t h e r s y n t h e t i c and b i o c h e m i c a l investigation. Acknowledgments We would l i k e t o thank P r . E . M i h i c h f o r h i s a c t i v e e n c o u r a g e ment t h r o u g h o u t t h e program. We would a l s o l i k e t o thank D r . B. Paul and M r . N . J . A n g e l i n o f o r t h e i r a c t i v e support i n the program. T h i s s t u d y was s u p p o r t e d by CA24538, CA130?8 and CA42898 from t h e N a t i o n a l Cancer I n s t i t u t e .

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January 18, 1988


Fluorinated Analogs of Cell-Surface Carbohydrates as Potential

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