bk-1980-0140.ch010

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Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on December 7, 2014 | http://pubs.acs.org Publication Date: December 22, 1980 | doi: 10.1021/bk-1980-0140.ch010

The Potential of Ruthenium in Anticancer Pharmaceuticals MICHAEL J. C L A R K E Department of Chemistry, Boston College, Chestnut Hill, M A 02167

Pertinent aspects of the chemistry of Ru(II) and Ru(III) complexes are briefly outlined with regard to the utility of these ions i n anticancer pharmaceuticals. Of particular interest i s that Ru(lII) ammine complexes containing hard ligands may be activated by reduction in vivo to lose the acido groups and subsequently add a nitrogen base, which can then be firmly coordinated to the metal in either oxidation state. Work with nucleosides and mucleic acids indicates that the coordination of Ru(II) to such ligands is similar to that of the Pt(II) anticancer drugs, so that analogous effects might be exerted on nucleic acid metabolism. Ammineruthenium(III) ions, on the other hand, form unusual cytosinato and aderosinato species. Collaborative studies demonstrate that a number of Ru compounds serve as bacterial mutagens, and so indicate that at least some Ru complexes are capable of damaging genetic material. The in vivo production of the more easily substituted Ru(II) aquoammine species from the Ru(III) prodrug should be favored in the relatively reducing and hypoxic environment provided by the interior of many tumors. In v i t r o experiments u t i l i z i n g subcellular components as electron-transfer catalysts provide support for t h i s . Screening studies on a series of Ru compounds show that many complexes, which would be thought to function by an activation-by-reduction mechanism, do exhibit a n t i tumor a c t i v i t y . Tissue distribution studies by other workers reveal significant concentrations of ruthenium, injected as cis-[Cl (CNH ) Ru]Cl , i n tumor tissue. The potential of ruthenium isotopes for incorporation into radiodiagnostic pharmaceuticals for imaging tumors is also b r i e f l y discussed. 2

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0-8412-0588-4/80/47-140-157$06.00/0 © 1980 American Chemical Society In Inorganic Chemistry in Biology and Medicine; Martell, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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P o s s i b l e a p p l i c a t i o n s of ruthenium complexes i n the treatment of cancer have been recognized by workers i n d i v e r s e areas and the u t i l i z a t i o n of t h i s metal has been approached from widely d i f f e r e n t p e r s p e c t i v e s . E a r l y i n t e r e s t centered on the t h e r a p e u t i c p r o p e r t i e s o f ruthenium complexes with aromatic c h e l a t e s (1) and then, f o l l o w i n g a dormancy, e f f o r t s became focused on complexes bearing analogies to c i s - C l 2 ( N H ) P t (2,3). S l i g h t l y l a t e r , the suggestion was made that 97R could provide the b a s i s f o r a f a m i l y of r a d i o d i a g n o s t i c agents f o r organ imaging ( 4 ) . T h i s suggestion holds the promise that tumors may be s p e c i f i c a l l y imaged, located and diagnosed w i t h the help o f t u m o r - l o c a l i z i n g Ruc o n t a i n i n g radiopharmaceuticals (5,6). In a d d i t i o n t o r a d i o s c i n t i g r a p h i c agents of t h i s type, t u m o r - s p e c i f i c i t y i s a d e s i r e d (but not a r e q u i r e d ) property f o r chemotherapeutic pharmaceuticals. A f i n a l category of a n t i c a n c e r drugs, which has not been addressed w i t h ruthenium-containing compounds, i s that o f r a d i o t h e r a p e u t i c pharmaceuticals, which would provide a dose o f short range r a d i a t i o n d i r e c t l y at the tumor s i t e . These might be p o s s i b l e with the g-emitting r a d i o n u c l i d e s , *03R or ^06R , provided that a high degree o f t u m o r - l o c a l i z a t i o n could be obtained. 3

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Approaches t o the i n c o r p o r a t i o n of 97R o r 103R i n t o r a d i o s c i n t i g r a p h i c agents i n c l u d e : 1) The use of ruthenium-red (7)a U^oxo t r i m e r which i s known t o bind p r e f e r e n t i a l l y t o a c i d i c animal mucopolysaccharides. The stroma of many neoplasms are high i n these m a t e r i a l s and so could conceivably cause the ruthenium dye t o concentrate i n some tumors. 2) A range o f ruthenocene complexes i n which the c y c l o p e n t a d i e n y l m o i e t i e s serve as d e r i v a t i z a b l e u n i t s f o r the attachment of organ s p e c i f i c molecules (8-10). 3) Complexes with biomolecules which are known to concentrate i n tumors. P a r t i c u l a r i n t e r e s t centers on the bleomycins, a group o f tumor s p e c i f i c a n t i b i o t i c s which induce n u c l e i c a c i d cleavage. Glucose, n u c l e i c a c i d and p r o t e i n p r e c u r s o r s , DNA and some p r o t e i n s a l s o tend to concentrate i n some types of tumor c e l l s . With many of these biochemicals the r e l a t i v e l y high a f f i n i t y of Ru(II) and Ru(III) ions f o r n i t r o g e n l i g a n d s can be taken to advantage. 4) The use o f Ru(III) complexes as prodrugs, which can be transformed by the body i n t o more a c t i v e s p e c i e s which, i n t u r n , should behave s i m i l a r l y to the platinum chemotherapeutic agents (11). The l a s t approach depends upon p a r t i c u l a r aspects of ruthenium i n the I I and I I I o x i d a t i o n s t a t e s as w e l l as c e r t a i n d i f f e r e n c e s between tumor and normal t i s s u e metabolism. I t i s , i n concept, a p p l i c a b l e t o each of the major c a t e g o r i e s of a n t i c a n c e r pharmaceuticals mentioned above and provides u

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In Inorganic Chemistry in Biology and Medicine; Martell, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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a r a t i o n a l b a s i s f o r drug design. The remainder of t h i s r e p o r t w i l l focus upon the chemical and b i o l o g i c a l hypotheses i n v o l v e d i n t h i s approach and w i l l present the r e s u l t s of experiments which these have guided. Ruthenium-Nucleoside I n t e r a c t i o n s . The i n i t i a l phases of t h i s work were c a r r i e d out i n the l a b o r a t o r y of Henry Taube at Stanford and were i n i t i a t e d to explore the e f f e c t s of a r e l a t i v e l y h a r d " metal i o n , and a r e l a t i v e l y " s o f t " , pi-donor metal i o n , on nucleosides and n u c l e o t i d e s (12-14). Because of t h e i r s i m p l i c i t y , high a f f i n i t y f o r n i t r o g e n h e t e r o c y c l e s , and ease of switching o x i d a t i o n s t a t e s , the pentammineruthenium ( I I - I I I ) ions were chosen f o r study. A primary focus of these s t u d i e s , which were begun before i t was widely known that the a c t i v i t y of the platinum anticancer drugs probably depended upon n u c l e i c a c i d b i n d i n g , was to examine novel modes of metalpurine c o o r d i n a t i o n , p a r t i c u l a r l y carbenoid b i n d i n g (14,15) Nucleosides o f f e r a number and a v a r i e t y of metal c o o r d i n a t i o n s i t e s , but many of these can be e l i m i n a t e d by j u d i c i o u s a l k y l a t i o n of the h e t e r o c y c l e . T h i s coupled with unique s p e c t r a l patterns a r i s i n g from l i g a n d - t o - n e t a l charge t r a n s f e r (LMCT) t r a n s i t i o n s and p r e d i c t a b l e e f f e c t s of the metal i o n on the i o n i z i n g a b i l i t y of r i n g protons u s u a l l y e l i m i n a t e s the need f o r s t r u c t u r a l assignments by x-ray c r y s t a l l o g r a p h y . Indeed, the cautious i n t e r p r e t a t i o n of chemical and s p e c t r o s c o p i c data has provided the c o r r e c t assignment of the many p o s s i b l e linkage isomers i n every case i n v o l v i n g ammineruthenium(III) ions that have subsequently been v e r i f i e d by x-ray methods. Moreover, s o l u t i o n c o n d i t i o n s can be chosen so that only a s i n g l e species e x i s t s and, owing to the r e l a t i v e i n e r t n e s s to s u b s t i t u t i o n of Ru(II) and Ru(III) i o n s , these complexes p e r s i s t f o r periods of time s u f f i c i e n t f o r chemical and biochemical study. The dominant mode of pentaammineruthenium c o o r d i n a t i o n to purine nucleosides with a keto group at the 6 - p o s i t i o n i s at the N(7) s i t e on the imidazole r i n g (12-14,16). F i g u r e 1 i l l u s t r a t e s t h i s mode of c o o r d i n a t i o n and the hydrogen bonds, which f u r t h e r add s t a b i l i t y to the complex, that form between coordinated ammines and 0(6). A l k y l a t i o n at N(9), as occurs i n n u c l e o s i d e s , prevents metal binding at both the N(9) and N(3) s i t e s . At low pH protonation i s p r e f e r r e d over m e t a l l a t i o n at N ( l ) and no ruthenium complexes i n v o l v i n g N ( l ) c o o r d i n a t i o n to t h i s type of nucleoside have been i s o l a t e d or c h a r a c t e r i z e d . Attachment at the N(7) of deoxyguanosine, which i s thought to be the i n i t i a l p o i n t of attack of the platinum pharmaceuticals on DNA, has been shown to occur f o r ammineruthenium(II and I I I ) ions (13,17), F i g u r e 2 i n d i c a t e s that N(9) c o o r d i n a t i o n i s indeed


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Figure 2.

Structure of 9-[(Hyp)(NH ) Ru(III)] 3 5

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p o s s i b l e , when t h i s s i t e i s a v a i l a b l e (18). The c o r r e sponding N(3)-bound complex has a l s o been prepared pure i n s o l u t i o n (16), but has not been f u l l y c h a r a c t e r i z e d as a s o l i d . A novel mode of purine b i n d i n g which, among true t r a n s i t i o n metal i o n s , has only been observed f o r Ru(II and III) i s shown i n F i g u r e 3 (14,15). In t h i s case, the c a f f e i n e l i g a n d i s bound v i a the C(8) p o s i t i o n . Purines other than xanthines have not y i e l d e d to attempts to form y l i d e n e or carbenoid complexes of t h i s type. The (NR3X5RUCIII) group i s a l s o the only s p e c i e s known to form s t a b l e menodentate complexes w i t h c y t i d i n e , adenosine and r e l a t e d nucleosides v i a c o o r d i n a t i o n to the e x o c y c l i c n i t r o g e n (Figure 4) (19). The Ru-N(4) bond i n the c y t i d i n e complex i s approximately 0.13 & s h o r t e r than that expected f o r a Ru(III)-N s i n g l e bond (20) and i n d i c a t e s that the p a r t i a l l y f i l l e d d ^ - o r b i t a l on the metal i s accepting some degree of e l e c t r o n d e n s i t y from a p ^ - o r b i t a l on the n i t r o g e n . While complexes of t h i s type are s t a b l e over a wide range of pK, they are most r e a d i l y formed by redox c a t a l y s i s at n e u t r a l pH. C a t a l y t i c s y n t h e s i s i n the presence of a small amount of Ru(II) suggests that i n i t i a l attack probably occurs by the metal i n t h i s o x i d a t i o n s t a t e on the a v a i l a b l e pyrimidine r i n g n i t r o g e n (19). Oxidation of the metal ion to Ru(III) should f a c i l i t a t e deprotonation of the e x o c y c l i c amine, thus allowing f o r a subsequent and f a i r l y r a p i d r i n g - t o exocyclic nitrogen linkage isomerization. Spectral studies suggest that at low pH, r e p r o t o n a t i o n occurs at the adjacent r i n g n i t r o g e n r a t h e r than on the exo-N (19). Since adenine occurs i n a v a r i e t y of water s o l u b l e coenzymes, these can a l s o coordinate Ru(III) v i a the exo-N s i t e (21). Other n u c l e o s i d e s (or near nucleosides) such as r i b o f l a v i n are capable of forming s t a b l e ruthenium adducts and such c o o r d i n a t i o n may s e v e r e l y a f f e c t the coenzymic a c t i v i t y (22), Figure 5 i l l u s t r a t e s the mode of metal b i n d i n g and the s t r u c t u r a l bending induced i n a f l a v i n . The p i r e t r o d a t i v e bonding i n these species i s intense and, i n t e r e s t i n g l y , the Ru(IT)-N(5) bond length (1.979 A) i s q u i t e c l o s e to that of the Ru(III)-N(4)bond i n the c y t o s i n a t o complex (1.983 F i g u r e 4 ) . The s u r p r i s i n g l y s i m i l a r geometries around the l i g a n d n i t r o g e n i n both of these complexes suggest a near equivalency i n the mode of b i n d i n g , even though one would normally be considered to be a Ru(II) complex and the other R u ( I I I ) . In f a c t , the formulation of the f l a v i n complexes of R u ( I I ) - F l does not always appear to be appropriate and the c a n o n i c a l form R u ( I I I ) - F l may be p r e f e r r e d f o r some purposes (23). T


In Inorganic Chemistry in Biology and Medicine; Martell, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980. 2

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Ffeiire 3. Structure of 8-[(1,3,7Xan)(Cl (NH ) Ru(III))]

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Structure of 4,5-[(10MelAlo)NH ), Ru\ + 3

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The s p e c t r a of ( N K ^ ^ R u ( I I I ) - n u c l e o s i d e complexes i n v a r i a b l y e x h i b i t f a i r l y intense LMCT t r a n s i t i o n s , which are w e l l r e s o l v e d from the i n t r a l i g a n d bands and provide a convenient probe i n t o the nature of these metal-nucleoside i n t e r a c t i o n s (12-14,16,19). Reference to F i g u r e 6 r e v e a l s that these absorptions are u s u a l l y l o c a l i z e d i n two d i s t i n c t regions of the spectrum, one i n the near UV and the other i n the v i s i b l e . The energy of these bands i s p r i m a r i l y dependent upon the p a r t i c u l a r purine or p y r i m i d i n e i n v o l v e d , i t s p r o t o n a t i o n s t a t e and s i t e of p r o t o n a t i o n or deprotonation. T h e i r i n t e n s i t i e s , on the other hand, are l a r g e l y a f u n c t i o n of the metal binding s i t e , but a l s o depend somewhat on the nature of the l i g a n d (16). Bands of t h i s type have not been reported f o r any other metal-nucleoside adducts and c o n t r i b u t e to making the present system one of the most convenient f o r study. A p p l i c a t i o n of the maxim that the c l o s e r a hard metal i o n i s to a deprotonation s i t e , the g r e a t e r the i n c r e a s e i n a c i d i t y of that s i t e ( c f . F i g u r e 7) f a c i l i t a t e s assignments of the metal c o o r d i n a t i o n p o s i t i o n and s e p a r a t i o n of the v a r i o u s l i n k a g e isomers Csee Table I ) . The l a t t e r can u s u a l l y be accomplished by ion-exchange chromatography s i n c e the charge of the sundry isomers v a r i e s d i f f e r e n t l y with the pH of the eluant b u f f e r (12-14,16). A p a r t i c u l a r l y d r a s t i c change i n the a c i d i t y of n u c l e o s i d e s i s seen i n the cases of Ru(III) c o o r d i n a t i o n to c y t i d i n e and adenosine i n which the proton i o n i z a t i o n constant i n c r e a s e s by a f a c t o r of at l e a s t 109 over that of the f r e e ligands and the p r e f e r r e d p r o t o n a t i o n s i t e i s a l t e r e d (19), The a f f i n i t y of tr^CS03)(H G)(NH )4Ru(II) f o r guanosine i s approximately 200 times greater than i t s a f f i n i t y f o r adenosine (24), The lower b i n d i n g constant f o r adenosine corresponds w e l l w i t h the r e l a t i v e i n s t a b i l i t y of (Ado) (NH3) Ru(II) at low pH and i t i s l i k e l y that the l i g a n d i n both complexes i s coordinated at the N ( l ) p o s i t i o n (12,19,24), The s e l e c t i v i t y f o r guanosine may be e x p l o i t e d f o r the s p e c i f i c l a b e l l i n g of such s i t e s on n u c l e i c a c i d s , so long as the metal i s r e s t r i c t e d to the lower o x i d a t i o n s t a t e when b i n d i n g to the macromolecule, In g e n e r a l , the Ru(II) c o o r d i n a t i o n s i t e i s i d e n t i c a l to that of Ru(III) s i n c e both are normally s u b s t i t u t i o n - i n e r t and have f a i r l y high a f f i n i t i e s f o r most types of n i t r o g e n l i g a n d s (12-14,16,25), However, t h i s i s not always the case and the r e d u c t i o n of 4-(Ado)(HH3)5Ru(III) r e s u l t s i n a r a p i d l i n k a g e i s o m e r l z a t i o n r e a c t i o n (k«l,6 s e c " ) w i t h the Ru(II) i o n presumably c o o r d i n a t i n g at the N ( l ) p o s i t i o n (19,26). S i m i l a r l y , r e d u c t i o n of 7-(l,3-Me Xan)(NH )5Ru(III) i n a c i d y i e l d s 8-(l,3-Me2Xan)(H 0)(NH ) Ru(II) (14,15). 2

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Spectra of various (NH ) Ru(III) complexes showing variations in absorption patterns with ligand and binding site 3 5


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Figure 7. ApK values of isomers of 7-[(Me Xan)(NH ) Ru(II and III)] (U). Values are reported in ApK units relative to the free ligand. Numbers in parentheses are for the Ru(II) complexes. a

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Table I.

Changes in Acidity of Hypoxanthine Complexes on Coordination of (NH ) Ru(II and III) (16) 3 3

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1.18

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1.51

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The carbenoid l i g a n d i n the l a t t e r case serves as a potent t r a n s - l a b i l i z e r so that the ammine group opposite i t q u i c k l y exchanges f o r water. Nor i s a change i n o x i d a t i o n s t a t e necessary to i n i t i a t e a change i n b i n d i n g s i t e s i n c e protonation of 3-(7-MeHyp~) ( N H ) R u ( I I I ) i n 1 M EC1 r e s u l t s i n a movement of the metal to the more e l e c t r o n r i c h N(9) p o s i t i o n with an observed h a l f - l i f e c f 1,45 h r s . at 37° (16,21). These v a r i o u s linkage i s o m e r i z a t i o n r e a c t i o n s suggest that even " s u b s t i t u t i o n - i n e r t " metal ions are not always h e l d to a s i n g l e p o s i t i o n once bound to a n u c l e o t i d e or n u c l e i c a c i d . In f a c t , i t i s p o s s i b l e to e n v i s i o n s e q u e n t i a l i s o m e r i z a t i o n s r e s u l t i n g i n metal migration over the perimeter of a s i n g l e base r e s i d u e or along the chain of a n u c l e i c a c i d . While c e r t a i n l y s p e c u l a t i v e , these ideas imply that the primary l e s i o n i n f l i c t e d on a n u c l e i c a c i d by metal c o o r d i n a t i o n need not n e c e s s a r i l y be the most damaging, and that subsequent metal movement to other c o o r d i n a t i o n p o s i t i o n s , p a r t i c u l a r l y those normally on the i n t e r i o r of the n u c l e i c a c i d , may y i e l d the a c t u a l therapeutic or t o x i c effect. Indeed, such metal m i g r a t i o n might be e s p e c i a l l y e f f e c t i v e i n producing i n t e r s t r a n d c r o s s l i n k s i n DNA. 3


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Ruthenium I n t e r a c t i o n s with N u c l e i c A c i d s . The s p e c t r a of samples of [(NH3) Ru(III)] -DNA prepared from normal and heat-denatured DNA are shown i n F i g u r e 8. Comparison with F i g u r e 6 r e v e a l s a coincidence of bands i n the v i s i b l e r e g i o n suggesting that h e l i c a l DNA binds Ru(III) p r i m a r i l y at N(7) s i t e s on guanine r e s i d u e s , while the s i n g l e - s t r a n d e d DNA coordinates the metal a d d i t i o n a l l y at the e x o c y c l i c nitrogens of c y t o s i n e and adenine. Subsequent a c i d h y d r o l y s i s of these samples followed by ion-exchange chromatography allows the s e p a r a t i o n and spectrophotometric i d e n t i f i c a t i o n of the i n d i v i d u a l ( N H ) R u ( I I I ) - p u r i n e complexes (Figure 9), which s u b s t a n t i a l l y confirms t h i s i n t e r p r e t a t i o n (17). However, the c y t o s i n e complex cannot be i s o l a t e d by the techniques employed so that the evidence f o r R u ( I I I ) - c y t o s i n e complexation i s e n t i r e l y s p e c t r o s c o p i c . I n t e r e s t i n g l y , the s p e c t r a of the Ru-DNA prepared using h e l i c a l DNA at the higher ruthenium concentrations e x h i b i t s i m i l a r i t i e s to those obtained f o r the s i n g l e - s t r a n d e d samples. T h i s i m p l i e s some metal-induced u n c o i l i n g of the n u c l e i c a c i d allowing subsequent metal a t t a c k on the " i n t e r i o r " adenine and c y t o s i n e s i t e s . Since ammineruthenium ions can coordinate the exo-N s i t e s of c y t o s i n e and adenine as w e l l as r i n g n i t r o g e n s , a v a r i e t y of options f o r i n t e r - and i n t r a s t r a n d c r o s s l i n k i n g of DNA become a v a i l a b l e (11). However, the s t a b i l i t y of these v a r i o u s modes of binding depends both on pH and the o x i d a t i o n 5

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400

600 Mnm)

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Chromatography of acid hydrolyzed [(NH ) Ru(III)] -DNA prepared from: a, helical and b, single-stranded DNA (11) 3 5

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MEDICINE

s t a t e of the metal. Long-lived c o o r d i n a t i o n of Ru(III) occurs with the exo-N s i t e s of adenine and c y t o s i n e and the N(7) of guanine, so that c r o s s l i n k i n g i n v o l v i n g these modes would appear most l i k e l y . In the case of Ru(II) only l i n k a g e s i n v o l v i n g the N(7) of guanine are expected to p e r i s t f o r s i g n i f i c a n t periods (13). However, t r a n s i e n t l i n k i n g i n v o l v i n g Ru(II) and the exo-N s i t e s of adenine and c y t o s i n e and the N ( l ) p o s i t i o n on adenine are p o s s i b l e (19).


A c t i v a t i o n by Reduction

of Ru(III) Prodrugs.

S i g n i f i c a n t d i f f e r e n c e s e x i s t between the chemistry of ammine Ru(II) and Ru(III) ions (11,25) which can be taken to advantage i n the design of a n t i c a n c e r pharmaceuticals. While both (NH )5Ru(II and I I I ) have comparable a f f i n i t i e s f o r imidazole (K* 2 X 1 0 ) , Ru(III) has a f i v e - f o l d higher a f f i n i t y f o r ammonia and the s t a b i l i t y constant of Ru(II) f o r p y r i d i n e i s A X 1 0 g r e a t e r than that of Ru(III) (28). In g e n e r a l , Ru(II) ions bind more f i r m l y to those l i g a n d s which can serve as good ir-acceptors of e l e c t r o n d e n s i t y from metal dir - o r b i t a l s , while Ru(III) ions e x h i b i t a r e l a t i v e preference f o r acido l i g a n d s such as c h l o r i d e and c a r b o x y l a t e s . A l s o the s u b s t i t u t i o n r a t e s of v a t e r or acido l i g a n d s from ammineruthenium(II) ions are u s u a l l y much more r a p i d than those i n v o l v i n g R u ( I I I ) . For example, the r a t e of aquation of C l ( N H ) R u C H ) i s approximately 5 s e c ^ , while that of the analogous Ru(III) complex can be estimated to be a f a c t o r of A X 1 0 slower at n e u t r a l pH (27-31). 3

6

3

1

3

5

6

The r e l a t i v e chemical p r o p e r t i e s of Ru(II) versus Ru(III) suggest that ammineruthenium(III) ions should be f a r l e s s a c t i v e toward binding biochemical l i g a n d s than analogous RuClI) complexes. In the case of most n i t r o g e n ligands a wealth of chemical evidence e x i s t s i n support of t h i s (11). Thus a r e l a t i v e l y i n a c t i v e and so, h o p e f u l l y , f a i r l y nont o x i c Ru(III) complex might be a c t i v a t e d toward b i n d i n g to n i t r o g e n h e t e r o c y c l e s by i n v i v o r e d u c t i o n . Innocuous a n i o n i c l i g a n d s such as c h l o r i d e or acetate could be employed as l e a v i n g groups to lower the charge of the complex and enhance l i p o p h i l i c i t y , so as to f a c i l i t a t e d e l i v e r y of the Ru(III) prodrug across membrane b a r r i e r s . A c t i v a t i o n of the drug should take p l a c e p r e f e r e n t i a l l y i n reducing environments. I n a c t i v a t i o n would be expected to r e s u l t should r e o x i d a t i o n of the Ru(II) species take p l a c e before binding to a n i t r o g e n l i g a n d occurred. This s i m p l i s t i c approach, t h e r e f o r e , p r e d i c t s g r e a t e r l e v e l s of drug b i n d i n g i n t i s s u e s high i n reducing power and low i n oxygen content. Recent s t u d i e s on tumor metabolism i n d i c a t e very low l e v e l s c f 02 to be a v a i l a b l e , even at very short d i s t a n c e s from blood c a p i l l a r i e s (31-33). T h i s appears to be due to a



10.

CLARKE

Ru Anticancer

Drugs

111

high r a t e of oxygen u t i l i z a t i o n by tumor c e l l s , so that O2 i s r a p i d l y depleted and l a r g e l y u n a v a i l a b l e to much o f the tumor t i s s u e . G y l c o l y t i c metabolism must then be r e l i e d upon t o generate the major p o r t i o n of the energy supply f o r much of the n e o p l a s t i c t i s s u e w i t h concomitant increase i n l a c t i c a c i d production and lowering o f pH (34>35)» Such anaerobic metabolism and g l y c o l y t i c production o f NADH should and does provide a more reducing environment than the normal surrounding t i s s u e (36). Therefore, production of the lower o x i d a t i o n s t a t e s of metal ions should be p a r t i c u l a r l y favored i n many types of n e o p l a s t i c t i s s u e s . Moreover, f o r those metal ions whose r e d u c t i o n p o t e n t i a l s are pH dependent, the more a c i d i c mileu provided by most tumors should a d d i t i o n a l l y favor the reduced species (11,37). Most organic reductants o c c u r r i n g i n v i v o , such as NADK or s u c c i n a t e , do not r a p i d l y reduce metal ions from the I I I to I I o x i d a t i o n s t a t e s s i n c e a two-electron t r a n s f e r i s r e q u i r e d f o r the organic molecule to reach a s t a b l e product, while the metal i o n r e q u i r e s but a s i n g l e e l e c t r o n . Owing to t h i s mismatch, some i n t e r f a c e , u s u a l l y s u p p l i e d by a f l a v o p r o t e i n , i s necessary between the organic and i n o r g a n i c r e a c t a n t s . This i s not meant t o imply that f l a v i n s are the p r e f e r r e d i n v i v o reductants o f Ru(III) i o n s , but only that such r e d u c t i o n would be expected to occur a t or subsequent t o the e l e c t r o n - p a i r s p l i t t i n g process in a b i o l o g i c a l e l e c t r o n t r a n s f e r system. S i m i l a r l y , d e a c t i v a t i o n o f Ru(II) species does not n e c e s s a r i l y have to i n v o l v e On; however, few r e l a t i v e l y strong biochemical oxidants are a v a i l a b l e i n t i s s u e i n i t s absence. The r e s u l t s of experiments employing s u b c e l l u l a r components t o c a t a l y z e the r e d u c t i o n of C K N H g ^ R u U l I ) and subsequent metal complexation by a n i t r o g e n h e t e r o c y c l e c a r r i e d out i n both the presence and absence of a i r a r e i l l u s t r a t e d i n Figures 10 and 11 (38). These studies show that the NADH r e d u c t i o n of Ru(III) proceeds smoothly under anaerobic c o n d i t i o n s when microsomal enzymes are present. The a c t u a l reductant i s not known but i s l i k e l y t o be NADH- o r NADPHdehydrogenase or cytochrome-b5, which accepts s i n g l e e l e c t r o n s from the former enzyme. The cytochrome P-450 enzymes, which apparently serve to reduce chromate (39), probably do not reduce the metal complex i n the present system, s i n c e a d d i t i o n of metyrapone, a s p e c i f i c i n h i b i t o r f o r these p r o t e i n s , d i d not a f f e c t the n e t r a t e o f Ru(II) complexation. In keeping with the a c t i v a t i o n by r e d u c t i o n hypothesis, the r a t e of formation of (isonicotinamide)(NH3)5Ru(II) s i g n i f i c a n t l y decreases when the r e a c t i o n i s run i n a i r . Moreover, when the r e a c t i o n i s run under N2, c r e a t i o n of the d i n i t r o g e n complex does not g r e a t l y i n t e r f e r e with b i n d i n g of the n i t r o g e n heterocycle. Thus, d i v e r s i o n o f p o t e n t i a l ruthenium-containing



MICROSOME


to


I 20

I

10

I 30

3 5

I 40

I 50

1 T(mln)

60

I 70

I 80

3 5

I 90

I

I 110

L_l 120 Journal of Inorganic Biochemistry

100

Figure 11. Mitochondria-catalyzed succinate reduction of Cl(NH ) Ru(IH) and subsequent formation of (Isonicotinamide)(NH ) Ru(II) (3S): A, reaction run in air; B, reaction run under Ar; C, reaction run under Ar in presence of rotenone; D, reaction run under Ar in presence of antimycin

I

MITOCHONDRIA



174


BIOLOGY AND

MEDICINE

drugs i n t h i s manner i s u n l i k e l y to present a problem (38)• Analogous experiments were c a r r i e d out using mitochondria as the e l e c t r o n - t r a n s f e r c a t a l y s t and succinate as the e l e c t r o n source. While l a r g e d i f f e r e n c e s between the aerobic and anaerobic r a t e s of metal complexation were a l s o observed i n t h i s system, only 1-3% of the metal was coordinated as Ru(II) even under r e l a t i v e l y f o r c i n g c o n d i t i o n s (38), A d d i t i o n of malonate, a s p e c i f i c i n h i b i t o r f o r succinate dehydrogenase, or antlmycin-A, which blocks the r e s p i r a t o r y e l e c t r o n t r a n s f e r chain between cyt-b and c y t - c ^ , r e s u l t e d i n severe i n h i b i t i o n of metal complexation (Figure 11), This i m p l i e s that the a c t u a l metal reductant occurs subsequent to cyt-b i n the e l e c t r o n - t r a n s f e r sequence and e i t h e r c y t - c ^ or c y t - c are l i k e l y candidates. Assuming that e i t h e r cytochrome serves to reduce R u ( l I I ) at l e a s t p a r t i a l l y e x p l a i n s the low y i e l d of the r e a c t i o n , s i n c e the r e d u c t i o n p o t e n t i a l s of these p r o t e i n s CO.225 and 0.254 V, r e s p e c t i v e l y ) are r a t h e r high r e l a t i v e to that of the metal complex (-0.042 V) and s t e r i c i n t e r a c t i o n s would probably prevent c l o s e contact w i t h the reducing heme moiety (40-41), B i o l o g i c a l Screening

of Ruthenium Compounds.

A p a r t i a l summary of the r e s u l t s of b i o l o g i c a l s t u d i e s performed i n c o l l a b o r a t i o n with other l a b o r a t o r i e s are summarized i n Tables I I and I I I . In v i t r o work on the mutagenic p r o p e r t i e s of a s e r i e s of ruthenium compounds has r e c e n t l y been c a r r i e d out by Yashin, Miehl and Matthews (42). Kelman, Edmonds and P e r e s i e have studied the i n h i b i t i o n of c e l l u l a r DNA and p r o t e i n s y n t h e s i s and were involved i n the submission of a number of ruthenium compounds to the NCI f o r screening i n animal tumor systems (43). The r e s u l t s of the Ames t e s t f o r mutagenesis i n d i c a t e that many ruthenium compounds introduce s e r i o u s l e s i o n s i n t o c e l l u l a r genetic m a t e r i a l so that an error-prone DNA r e p a i r mechanism i s induced. These r e s u l t s are s i m i l a r to those obtained f o r c i s p l a t i n (44) and suggest that these complexes probably bind d i r e c t l y to nuclear DNA. In concert with t h i s , many of the ruthenium complexes a l s o i n h i b i t c e l l u l a r DNA s y n t h e s i s (11,43), another property a l s o noted f o r the c i s - p l a t i n u m drugs. Unfortunately, however, there i s no c o r r e l a t i o n between e i t h e r of these s t u d i e s and the antitumor a c t i v i t y of ruthenium compounds t e s t e d i n animal systems. A high percentage of the compounds t e s t e d , which would be expected to f u n c t i o n as Ru(III)-prodrugs, have e x h i b i t e d antitumor a c t i v i t y i n r a t s . An exception to t h i s are those complexes c o n t a i n i n g b i p y r i d y l or o-phenanthroline l i g a n d s which s t r o n g l y s t a b i l i z e the lower v a l e n t s t a t e and which


Ru


CLARKE

Table II.

Anticancer

Drugs

Antitumor and Mutagenic Activity of Selected Ruthenium Complexes DOSE (MG/KG)

COMPOUND

ZT/C

% INHIB. DNA SYNTHESIS

AMES TEST

CL (NH )RU(III)

50

189

86

CIS- (CL2

bk-1980-0140.ch010

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