Biologically Active Synthetic Anionic Polymers - American Chemical

1 Biologically Active Synthetic Anionic Polymers DAVID S. BRESLOW

Downloaded by UNIV OF ALABAMA BIRMINGHAM on May 1, 2013 | http://pubs.acs.org Publication Date: August 12, 1982 | doi: 10.1021/bk-1982-0195.ch001

Hercules Research Center, Wilmington, D E 19899

Maleic anhydride and vinyl ether undergo a cyclic alternating copolymerization in a 2:1 ratio. 13 NMR has shown the polymer to contain a mixture of tetrahydrofuran and tetrahydropyran rings in an approximately 0.8:1 ratio. The copolymer shows a remarkable variety of biological activities. It is an antitumor agent; i t induces the formation of interferon; i t has antiviral, antibacterial, and antifungal activity; i t is an anticoagulant and an anti-inflammatory agent; and it aids in the removal of plutonium from the liver. The copolymer is an immunostimulant, and appears to act by stimulating macrophages. A study of the effect of molecular weight and molecular weight distribution on toxicity and biological activity has led to the synthesis of an active copolymer with low toxicity, which is being investigated clinically as an antitumor agent. C

Maleic anhydride and vinyl ether undergo a radical-catalyzed cyclic alternating copolymerization in a 2:1 ratio. This copolymer, frequently designated in the literature as DIVEMA or as pyran copolymer, has shown unusual and exciting biological activity, and has therefore undergone intensive investigation. Structure The 2:1 copolymer, first reported in 1958 (1), was assumed to have the tetrahydropyran structure, I in Scheme 1 (2). Although this structure had been widely accepted (3), there was no convincing evidence for i t . In fact, a number of reports in the literature suggested that the kinetically controlled route leading to a tetrahydrofuran ring, II in Scheme 1, would be more likely. Thus, i t was shown by ESR that the 5-hexenyl radical, formed by photolysis of 6-heptenoyl peroxide, cyclizes 0097-6156/82/0195-0001 $06.00/0 © 1982 American Chemical Society

In Cyclopolymerization and Polymers with Chain-Ring Structures; Butler, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

POLYMERS WITH CHAIN-RING


2

STRUCTURES

LU LU Χ Ο CO



1.

BRESLOW

Biologically Active Synthetic Anionic Polymers

e x c l u s i v e l y t o the cyclopentylmethylene r a d i c a l ( 4 ) , whereas hexenyl r a d i c a l s s t a b i l i z e d by carbonyl or n i t r i l e groups c y c l i z e to cyclopentane and/or cyclohexane d e r i v a t i v e s , depending on the s u b s t i t u t i o n pattern ( 5 ) . S i m i l a r l y , m e t h y l d i a l l y l a m i n e undergoes c y c l o p o l y m e r i z a t i o n e x c l u s i v e l y t o form a polymeric p y r r o l i d i n e , whereas methyl-substituted d e r i v a t i v e s give polymers c o n t a i n i n g both f i v e - and six-merabered rings (6). Since i t was q u i t e apparent from the l i t e r a t u r e that the s t r u c t u r e o f the maleic a n h y d r i d e - v i n y l ether copolymer could not be p r e d i c t e d , we s e t out to determine the s t r u c t u r e by l ^ C NMR, using model compounds to a i d i n a s s i g n i n g the resonances ( 7 ) . F i g u r e 1 shows the l^C NMR spectrum o f the polymer, prepared i n benzene with carbon t e t r a c h l o r i d e as a c h a i n t r a n s f e r agent, a f t e r h y d r o l y s i s i n D 2 O . I t c o n s i s t s of seven s e t s o f peaks, l a b e l e d A-H. By a s i n g l e frequency off-resonance decoupling experiment, i t was p o s s i b l e to show that peaks A and Β are t r i p l e t s due to methylene carbons, peaks C-F are doublets due to methine carbons, and peaks G and H are due to carbonyls· D e f i n i t i v e assignments were made by comparison with the spectra of the model compounds. 2,6-Dimethyltetrahydropyran-3,4d i c a r b o x y l i c a c i d was prepared, as shown i n Scheme 2, as the six-membered r i n g model; only one isomer, i d e n t i f i e d as a l l - t r a n s by proton NMR, was i s o l a t e d .

Scheme 2 2,5-Dimethyltetrahydrofuran-3,4-dicarboxylic a c i d was prepared, as shown i n Scheme 3, as the five-membered r i n g model; three isomers were i s o l a t e d , but the stereochemistry could not be assigned u n e q u i v o c a l l y . Inspection o f the NMR s p e c t r a o f the model compounds, plus that of commercially a v a i l a b l e


3

4

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STRUCTURES

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Scheme 3 d i m e t h y l s u c c i n i c a c i d (meso plus racemic), showed only one unique carbon atom, at 34.6 ppm (using dioxane a t 67.0 ppm as an i n t e r n a l r e f e r e n c e ) , a s s i g n a b l e to the methylene carbon o f the tetrahydropyran r i n g . T h i s corresponds to peak Β i n the polymer spectrum. However, peak areas (from suppressed Ν0Ε experiments) showed that the polymer could not c o n s i s t s o l e l y of t e t r a hydropyran r i n g s ; c a l c u l a t i o n s show the r a t i o o f f i v e - t o six-membered rings to be approximately 0.8 to 1. We f e e l there i s i n s u f f i c i e n t evidence a t the present time t o make d e f i n i t e assignments of stereochemistry to the polymer Ο ) · Kunitake and Tsukino (8) a l s o used l^C NMR to determine r i n g s i z e i n the copolymer, but they c a l c u l a t e d chemical s h i f t s by e x t r a p o l a t i n g published data on simpler c y c l i c compounds. By t h i s method they concluded that a copolymer prepared i n c h l o r o form contained only t e t r a h y d r o f u r a n r i n g s . However, t h e i r published spectrum of the copolymer contained the same peak Β as ours, and t h e r e f o r e the copolymer must c o n s i s t o f both f i v e - and six-membered r i n g s . The spectrum of a copolymer prepared i n acetone-carbon d i s u l f i d e was too p o o r l y resolved t o show peak B, but Kunitake and Tsukino estimated the polymer to c o n t a i n about 90% tetrahydropyran r i n g s . Biological Activity The 2:1 maleic a n h y d r i d e - v i n y l ether copolymer shows a b e w i l d e r i n g v a r i e t y o f b i o l o g i c a l a c t i v i t i e s (9). I t was f i r s t shown to have antitumor a c t i v i t y against a number o f s o l i d tumors (10). I t induces the formation o f i n t e r f e r o n ( V I ) , and therefore shows a n t i v i r a l a c t i v i t y . I t has been shown t o be a c t i v e a g a i n s t over 20 v i r u s e s , i n c l u d i n g a number o f cancerinducing v i r u s e s and herpes simplex (2.*1I)· Qui surprisingly, i t has a l s o been shown t o have a n t i b a c t e r i a l a c t i v i t y a g a i n s t both gram-positive and gram-negative organisms (JL3). I t a l s o shows a n t i f u n g a l a c t i v i t y (14) and acts as an a n t i c o a g u l a n t (_15). I t i n h i b i t s adjuvant d i s e a s e i n r a t s (16), a syndrome b e l i e v e d to be r e l a t e d t o rheumatoid a r t h r i t i s i n humans. In conjunction with c e r t a i n c h e l a t i n g agents, i t has been shown to be a c t i v e i n removing polymeric plutonium from the l i v e r (17). t e



6

POLYMERS

WITH

CHAIN-RING

STRUCTURES

T h i s broad spectrum of a c t i v i t y q u i t e o b v i o u s l y l e d to considerable a c t i v i t y by b i o l o g i s t s and p h y s i c i a n s to e x p l a i n the polymer's mode of a c t i o n . One of the f i r s t d i s c o v e r i e s showed t h a t , under c e r t a i n c o n d i t i o n s , i t g r e a t l y a c c e l e r a t e d the r a t e of phagocytosis, i . e . , the rate of removal of f o r e i g n bodies from the blood stream (18). Quite s u r p r i s i n g l y , i t showed the same a c t i v i t y i n immunosuppressed mice as i n normal mice (19). I t was shown to i n h i b i t reverse t r a n s c r i p t a s e i n b i r d s , r e p t i l e s , and mammals (20). A c o n s i d e r a b l e amount of work i n a number of l a b o r a t o r i e s f i n a l l y reached the c o n c l u s i o n that the major mode of a c t i o n i n v o l v e s the a c t i v a t i o n of macrophages (21, 22), l a r g e white blood c e l l s which have the f u n c t i o n of removing f o r e i g n bodies from the blood stream. T h i s hypothesis i s g e n e r a l l y accepted, but by no means proven (9)· Narrow Molecular Weight D i s t r i b u t i o n Polymer The i n i t i a l polymer i n v e s t i g a t e d was prepared u s i n g benzoyl peroxide i n a nonsolvent f o r the polymer, and the polymeri z a t i o n was c a r r i e d to a very high conversion. As a r e s u l t , the polymer had a very broad molecular weight d i s t r i b u t i o n . This polymer underwent Phase I c l i n i c a l e v a l u a t i o n as a chemotherapeutic agent against cancer. U n f o r t u n a t e l y , i t showed a number of u n d e s i r a b l e s i d e e f f e c t s (23), and i t was decided i n t h i s l a b o r a t o r y to see i f we could decrease the t o x i c i t y without l o s i n g the antitumor a c t i v i t y . Degradation of the polymer showed that decreasing the molecular weight d i d indeed decrease the t o x i c i t y of the polymer (24). However, before going f u r t h e r we had to develop a means f o r c h a r a c t e r i z i n g the polymer. T h i s was done by c o n v e r t i n g i t to i t s methyl e s t e r and running s i z e e x c l u s i o n chromatography (SEC) on a p r e p a r a t i v e s c a l e . These f r a c t i o n s , which had reasonably narrow molecular weight d i s t r i b u t i o n s on an a n a l y t i c a l instrument, were then compared with polystyrene standards and shown to f i t the u n i v e r s a l c a l i b r a t i o n curve i n the usual f a s h i o n . In order to o b t a i n s u f f i c i e n t l y l a r g e q u a n t i t i e s of polymer f o r both c h a r a c t e r i z a t i o n and b i o l o g i c a l e v a l u a t i o n , a s o l u t i o n p o l y m e r i z a t i o n with l i m i t e d conversion was devised; a comparison of the two polymers by SEC i s shown i n Figure 2; the o r i g i n a l polymer i s NSC 46015 and the new polymer MVE-2. B i o l o g i c a l t e s t i n g showed that t h i s new polymer d i d indeed have low t o x i c i t y and maintained i t s antitumor a c t i v i t y (24). Meanwhile, however, research done at the N a t i o n a l Cancer I n s t i t u t e , as w e l l as a t other l a b o r a t o r i e s , demonstrated that the most d e s i r a b l e way of using t h i s m a t e r i a l was as an immunoadjuvant i n c o n j u n c t i o n with other treatments (25, 26). The goal i s to reduce the tumor burden by some means - surgery, r a d i a t i o n , chemotherapy - and


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Biologically Active Synthetic Anionic Polymers - American Chemical

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