2
Chem. Res. Toricol. 1991, 4, 2-16
Persp e c t i u e Redox Chemistry of Anthracycline Antitumor Drugs and Use of Captodative Radicals as Tools for Its Elucidation and Control Giorgio Gaudiano and Tad H. Koch* Department of Chemistry and Biochemistry, The University of Colorado, Boulder, Colorado 80309-0215 Received September 12, 1990
Introduction The anthracycline antitumor drugs, most notably daunorubicin (daunomycin, la) and doxorubicin (adriamycin, lb), likely derive a portion of their biological activity through bioreductive activation (1,2). Several enzymes are known to reduce the quinone functionality to semiquinone (2) and hydroquinone (3) redox states (3,4). In aerobic medium both of these states react rapidly with molecular oxygen to produce superoxide with regeneration of the quinone state (Scheme I) (5-7). Subsequent reaction of superoxide in a Haber-Weiss cycle with Fe(III)/Fe(II) catalysis yields sequentially hydrogen peroxide and the cytotoxic hydroxyl radical. The iron catalysis is possibly facilitated through strong complexation of the anthracycline with iron (8). Hydroxyl radical may also result simply from reaction of semiquinone with hydrogen peroxide without iron catalysis (9). The overall process has come to be known as redox recycling, and the materials produced, as reactive oxygen species. The catalytic formation of reactive oxygen species likely contributes to the cytotoxicity of these anthracyclines. Two forms of toxicity are of primary concern to the oncologist, tumor cytotoxicity and cardiotoxicity. Chronic cardiotoxicity ultimately limits the chemotherapy and results in part from oxidation of cardiac cell membrane lipids, among other critical molecules, by the reactive oxygen species (IO). Heart cells are particularly vulnerable becuase they are poorly equipped to defend against reactive oxy radicals. Whether reactive oxygen species also contribute to tumor cytotoxicity is presently being debated (11, 12). Development of methodology for separation of cardiotoxicity from tumor toxicity remains an important goal. One approach in clinical development is the addition of a cell membrane transportable iron chelating agent, d - 1,2-bis(3,5-dioxopiperazin- 1-y1)propane (ICRF-1871, which slows the Haber-Weiss cycle, possibly by competing with the anthracycline for iron cation (13, 14). In an anaerobic medium a reduced state of the anthracyclines undergoes glycosidic cleavage. If the reduced state is the semiquinone, the aglycon transient formed is the 7-deoxy semiquinone methide (4), and if the reduced state is the hydroquinone, the aglycon transient is the 7-deoxy quinone methide (5). Semiquinone methide and quinone methide have both been discussed as reactive intermediates in coupling the aglycon to critical biological molecules (Scheme 11); interesting possibilities are DNA, DNA-topoisomerase complex, and proteins in cell membranes. Topoisomerase enzymes unwind DNA during replication, and inhibition, resulting in DNA strand breaks, is thought to be an important mechanism for anthracycline cytotox-
Scheme I. Catalytic Production of Superoxide
1
liavoprolein[ R
Meb
... R'=
-
OH, H, daunomycin, adriamycin, ll ab
1
b- bH
bR' 2 ba hone-electron reducing apent
1.1
Canaerobic medium prolic solvent
Me+
H
+
R'OH
daunosamine 7-deoxydaunomycinone 6 8 7-deoxyadriamycinone 6 b
Scheme 11. Proposed Radical and Nucleophilic Coupling to Macromolecules
g:
M e I0 F
::
semiquinone
H
1
semiquinone methide
~-~ macromolecule with a radical site
0 2 .< ,
~
macromolecule with a nucleophilic site
I
it
Me0 hydroquinone
AH qulnone melhide
icity (15). Inhibition of topoisomerase, however, may not necessarily involve redox chemistry and/or covalent bond formation. Cell membranes are implicated from the cytotoxicity of anthracyclines which are immobilized with respect to cell membrane transport (16). The redox state at which glycosidic cleavage actually occurs remains under continuous debate. The nature of the reactive state dictates subsequent chemical possibilities. Semiquinone
Qa93-228~/91/2704-oo02$02.50/00 1991 American Chemical Society
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 3
Perspective
methide, unless it is reduced to quinone methide, might logically couple with a radical site in a biological molecule. Quinone methide might couple with a nucleophilic or electrophilic site (vide infra). In competition with covalent bond formation is reaction with the ever-present electrophile, the proton. Protonation gives 7-deoxydaunomycinone (6a) which as an external drug is nontoxic and inactive (17). The possibility of covalent bond formation between the aglycon and, for example, DNA is an attractive proposal for tumor cell cytotoxicity. Because the aglycon is also capable of redox recycling, covalent bond formation would locate a catalyst for the production of hydroxyl radical adjacent to the DNA. Since hydroxyl radical does not diffuse far from its origin before it reacts, such a location is ideal for oxidative cleavage of DNA. Of course, covalently bound aglycon must be reducible by the available enzymes. Over the past decade, we have been exploring the redox chemistry of anthracyclines using primarily a chemical one-electron reducing agent developed in our laboratories. The reagent is the captodative free radical, 3,5,5-trimethyl-2-oxomorpholin-3-yl(TM-3), formed in solution through spontaneous bond homolysis of its dimer, bi(3,5,5-trimethyl-2-oxomorpholin-3-yl) (TM-3 dimer) (18-20). A more water-soluble version, 3,5-dimethyl-5(hydroxymethyl)-2-oxomorpholin-3-yl (DHM-3), from bond homolysis of bi [3,5-dimethyl-5-(hydroxymethyl)-2oxomorpholin-3-y1] (DHM-3 dimer), has also been developed (21, 22). These reducing agents are of particular interest because the redox chemistry with the anthracyclines is often quantitative, and the reagents can possibly be employed in vivo as well as in vitro. Both TM-3 dimer and DHM-3 dimer show low mouse toxicity. Goals of our investigations continue to be the elucidation of reaction pathways which might be possible in vivo, especially ones that might provide differential cytotoxicity to tumor cells, and the development of reagents and protocols that might be useful for modulating the redox chemistry of bioreductively activated antitumor drugs to therapeutic advantage. This paper does not attempt to review the subject of anthracycline chemistry, biochemistry, and/or medicinal chemistry but to describe a useful one-electron reducing agent as a tool for elucidating possible in vivo redox processes. Excellent comprehensive reviews on various aspects of the anthracyclines are available (23-28).
Captodative Radicals as One-Electron Reducing Agents Captodative radicals are radicals that have electronic stabilization through the synergistic interaction of an electron-donating and an electron-withdrawingsubstituent (29,30). Such radicals have also been called merostabilized (31)and push-pull stabilized (32). One of the best combinations of electron-donating and electron-withdrawing substituents is the amino and carboxyl functional groups. We have been particularly intrigued with the stability and properties of 2-oxomorpholin-3-ylradicals, which contain these two functional groups. Such radicals are best illustrated by 3,5,5-trimethyl-2-oxomorpholin-3-yl (TM-3), which also bears sterically stabilizing methyl substituents. Captodative resonance can be illustrated in valence bond terms by the zwitterionic resonance structure 7, among other structures. 2-Oxomorpholin-3-yl radical dimers are prepared in synthetically useful quantities by photoreduction of the corresponding 5,6-dihydro-1,4-oxazin-2-ones (33). For example, irradiation of the n-r* band of 5,6-dihydro-
3,5,5-trimethyl-1,4-oxazin-2-one (8a) in 2-propanol solvent yields the TM-3 radical, which dimerizes to yield a mixture of the meso and dl isomers of TM-3 dimer. When the irradiation is performed at reduced temperature, the dimers precipitate and are collected in 50-70% yield, almost analytically pure, by filtration. The meso and dl isomers are readily separated by low-temperature flash or suction chromatography (34). Pure stereoisomers are useful for experiments in which kinetics are measured (vide supra).
X = OH, O H M 4 Dimers
X
I
OH, DHM.3
TM-3 forms spontaneously in solution upon dissolving its dimers, meso- and dl-bi(3,5,5-trimethyI-2-0~0morpholin-3-y1),in solvents ranging from benzene to 10% methanol in water and is readily characterized from its relatively simple EPR spectrum. Three observations are consistent with the captodative nature of the electronic stabilization. The methyl EPR hyperfine coupling constant, which is a measure of the spin density localized at the 3-position, is solvent dependent, ranging from 13.7 G in ethanol to 18 G in benzene, and is linearly related to the free energy of bond homolysis (35). The equilibrium constant for bond homolysis is solvent dependent, ranging from 6 X M in benzene to 3 X lo-" M in ethanol at 25 "C, and the free energy is linearly related to the solvent 2 parameter. Inductively electron-withdrawing substituents located at the 3-position facilitate bond homolysis, and inductively electron-withdrawingsubstituents located at the 5-position suppress bond homolysis (36). The solvent and substituent effects are consistent with the dipolar nature of the radical predicted by the captodative interaction. Polar solvents, especially hydrogen-bonding solvents, stabilize the radical more than the dimer, resulting in a larger equilibrium constant for bond homolysis. The solvent stabilization results in a larger contribution from the dipolar resonance structure 7, delocalizing the odd electron and lowering the hyperfine coupling constant. The favorable radical solvation energy more than compensates for the resulting charge separation.
~J-JLG -
w G E-$>
EWG
EWG
-
electron withdrawing group
H WG
A continuing question is what is the resonance energy for the captodative effect. The estimation is clouded by uncertainty with regard to models, to additivity of substituent effects on radicals with no captodative stabilization, to the effect of substituents on radical precursors, and to medium effects. Recent experimental (37) and theoretical (38-40) evaluations suggest that captodative radical stabilization is not general; however, it does appear to be in the range of 4-5 kcal/mol with carbonyl and amino substituents and will be affected by medium, especially protic solvent. Protic solvent probably magnifies the effect by an additional 3-4 kcal/mol in TM-3 as indicated by the effect of solvent on the bond dissociation energy. The chemical property of the oxomorpholinyl radicals which is most interesting with respect to the anthracyclines is their ability to serve as selective one-electron reducing agents. Reductions which TM-3 will perform quantitatively include isatin (9) to isatide (lo), methyl viologen (11) to its radical cation (12), diphenylpicrylhydrazyl (13) to diphenylpicrylhydrazine (14), Ag+ to Ago, Fe3+ to Fe2+,
4 Chem. Res. Toxicol., Vol. 4, No.1, 1991
Gaudiano and Koch
Scheme 111. Reduction Reactions of TM-3 Dimer
Scheme IV. Mechanism of Reaction of TM-3 with Oxygen
HO
+M-3
TM-3
/
pe;oxy radical
amiAyl radical 2 1
'I peroxide
oxazinone 8 a
Scheme V. Rationale for Electron-Transfer Mechanism HQ
0
TM-3 Dimer,
,*Ph
+
ea
electron
ph%Ph 15
proton
+A-
+
AH
TM-3 ground state of 8 a hydrogen atom transfer
+
AH
n,x* state of 8a
29
Table I. Rate Constants for Bond Cleavage of meso- and CUI-TM-3Dimers rate stereoisotemp, constant, mer solvent "C s-l half-life ref dl CHROH 25 3.4 x 10-3 200 s 50 CH30H dl 20 1.9 x 10-3 360 s 50 CHSOH dl 15 9.0 X lo-' 770 s 50 dl CH30H 5 2.6 X lo-' 2700 s 50 dl EtOH 25 1.2 x 10-3 580 s 35 dl 25 7.8 X lo4 25 h 35 glyme CHC13 dl 30 3.7 X lo4 52 h 34 PhH dl 71 2.3 X 10"' 50 min 35 PhH dl 25 3.4 x 10-7 570 h 35 meso CHROH 25 2.2 x 10-3 315 s unpublished meso CHCls 30 1.0 X 10" 19 h 34
benzil(l5) to benzoin (16), di-tert-butyl nitroxide (17) to di-tert-butylhydroxylamine(18),molecular oxygen to hydrogen peroxide, 2-benzoyl-4,4-dimethyl-2-oxazoline (19) to 2-(hydroxyphenylmethyl)-4,4dimethyl-2-oxazoline (20), and anthracyclines to their hydroquinones (Schemes 111, IV, and VI) (20,34). Disproportionationof TM-3 to form (29), oxazinone 8a and 3,5,5-trimethyl-2-oxomorpholine which is also a redox process, occurs very slowly at ambient temperature. Electrochemical reduction of the oxazinone 8a is irreversible and does not yield a thermodynamic reduction potential. Bracketing experiments place the Eo' for the reduction of oxazinone 8a to TM-3 dimer in the range of -0.5 to 4 . 6 V vs the standard hydrogen electrode. Although oxomorpholinylradicals react as reducing agents, this is not a general property of captodative radicals. In simplistic terms, oxomorpholinyl radicals bear the odd electron in a ?r* orbital, and consequently, transfer of the electron to a low-energy vacant orbital of an acceptor molecule is exothermic. For many of the reactions, reduction is faster than recombination of TM-3 radicals, and consequently, the rate of reduction is the rate of bond homolysis. The rate of bond homolysis is a function of the stereoisomer, the solvent, and the pH. As reported in Table I, the rate constant for bond homolysis of the dl isomer varies from
in methanol at 25 3.4 X lO-'s-l in benzene to 3.4 X OC. Consequently, the half-life varies from 570 h in benzene to 200 s in methanol; in 10% methanol/90% water the half-life is approximately 50 s. Bond homolysis is inhibited at pH much less than 5 because at least one of the amine functions of the dimer is protonated and protonation eliminates the dative group. The mechanism by which the oxomorpholinyl radicals serve as reducing agents is not completely established. Isotope effects on the radical and substituent effects on the substrate point to single electron transfer (20). Certainly for reduction of metal ions, electron transfer appears reasonable. Mechanistic studies of reduction of molecular oxygen also suggest a covalent mechanism with two pathways as shown in Scheme IV (41). Both pathways start with covalent bond formation between molecular oxygen and the 3-position of TM-3. One pathway involves subsequent intramolecular transfer of a hydrogen from the nitrogen to the peroxy radical with formation of the transient aminyl radical 21, characterized by a strong three-line EPR signal. The other pathway involves combination of the peroxy radical with a second TM-3 radical. In some cases, although not with TM-3, the intermediate peroxides have been isolated and characterized spectroscopically. The overall process is quantitative. TM-3 does not react rapidly, if at all, with hydrogen peroxide. The exact pathway by which TM-3 transfers electrons to daunomycin is not known; however, characteristic semiquinone and hydroquinone EPR and visible spectra, respectively, have been observed for these reduced states during the reduction, suggesting single electron transfer (42). No evidence supports direct hydrogen atom transfer from TM-3 to a reducible substrate. A possible rationale is that direct hydrogen atom transfer correlates TM-3 with the n,r* excited state of oxazinone 8a. Correlation with an excited state is likely because the nitrogen of TM-3 is probably sp2 hybridized to accommodate the resonance interaction, and consequently, the N-H bond lies in the a-plane (Scheme V). Such an intended correlation would raise the activation energy for direct hydrogen atom transfer above that for electron transfer or covalent bond formation (43). For these latter mechanisms correlation is with a ground state.
Chem. Res. Toxicol., Vol. 4 , No. 1, 1991 5
Perspective
Because of our interest in conducting anthracycline redox chemistry in vivo with the oxomorpholinyl radicals (vide infra), a material more water soluble than TM-3 dimer was needed. After several possibilities were explored, the addition of a hydroxyl group to one of the methyls in the 5-position to achieve higher water solubility was pursued. The synthesis of bi[3,5-dimethyl-5-(hydroxymethyl)-2-oxomorpholin-3-yl] (DHM-3 dimer) was analogous to the synthesis of TM-3 dimer; however, the additional chiral centers resulted in the formation of six diastereoisomeric radical dimers, with one diastereoisomer predominating (21). Because separation of the stereoisomers is difficult and bond homolysis gives a single racemic radical from all six dimers, reductions are commonly performed with the mixture. The major diastereoisomer was isolated and the rate of bond homolysis measured. The rate constant for bond homolysis in methanol at 25 "C is 1.2 X s-l (half-life 580 s), and in water at 25 "C, 6.5 X s-l (half-life 110 s). The smaller rate constant relative to that for TM-3 dimer is consistent with the general effect of an inductively electron-withdrawing substituent at the 5-position (vide supra) (36). Although DHM-3 radical still reduces molecular oxygen to hydrogen peroxide, the reduction appears to be slower than the rate of bond homolysis for as yet unexplained reasons.
Reduction of Daunomycin: Formation of a Quinone Methide versus a Semiquinone Methide Reasonable circumstantial evidence exists for in vivo covalent bond formation between daunomycin and DNA (44,45). The results of in vitro experiments also point to covalent binding upon reductive activation (46,47). The mechanism for the bonding has been intensely investigated, and a t present no clear mechanism has been established. As indicated earlier, both semiquinone methide (4) and quinone methide (5) transients have been proposed as reactive intermediates, the semiquinone methide resulting from glycosidic cleavage of the semiquinone anion and the quinone methide resulting from glycosidic cleavage of the hydroquinone anion. Reduction of the anthracyclines especially in methanol solvent with TM-3 dimer has proven to be an excellent method for generating the redox transients and probing their chemical reactivity. Upon mixing 10 mol equiv of dl-TM-3 dimer with 1 X M daunomycin in freeze-pump-thaw degassed Trisbuffered methanol (apparent pH of 8) a t 25 "C and scanning the visible absorption spectrum, destruction of daunomycin ,,A,( 480 nm) with initial formation of the hydroquinone anion 3a (Amu 420 nm) is apparent (Figure 1). The semiquinone anion 2a, although formed first, does not exist in sufficient concentration for visible spectroscopic observation in protic solvent; absorption at 382 and 510 nm has been reported for the semiquinone anion of daunomycinone in dimethylformamide solvent (48) and a t 480 and 700 nm for the semiquinone of adriamycin in water (49). The semiquinone anion is readily observed by EPR spectroscopic monitoring of reaction of 1mol equiv of TM-3 dimer with daunomcyin in methanol at 0 "C and characterized from the hyperfine coupling (50). A similar EPR spectrum has been observed upon chemical or enzymatic reduction of daunomycin in DMSO-water solvent (51). Semiquinone is never present in high concentration either because it is rapidly reduced by TM-3 radical or because it rapidly disproportionates to hydroquinone and quinone (49, 5 2 ) . Soon after visible absorption by hydroquinone 3a appears (Figure la), it disappears with absorption by quinone methide 5a at 380 and 610 nm. The new absorption, which is substantially intense, was as-
Scheme VI. Reductive Activation of Daunomycin with TM-3Dimer 9'
semiquinone
daunomycin la 480 nm
hydroquinone 3a
8
PH
28
420 nm
380, 610 nm
7-deoxydaunomycinone 6a 480 nm
la \
/
Me0
0-
23
0
H
W H
'OH-, -CH2
AcNH-AH-COOH
Me0
O 24
H
t 38
-CHt
AcNH-&H-COOH
490 nm
signed to the quinone methide rather than the semiquinone methide because its intensity does not correlate with any new EPR signal, and the chemical reactivity is consistent with the structure. Although the structure for the quinone methide is commonly shown as a C-ring quinone methide (Scheme VI), it has never been fully characterized and a B-ring quinone methide is also possible (53). This experiment provided the first glimpse of the quinone methide predicted by the theory of bioreductive activation (1, 2). Further monitoring of the visible absorption (Figure l b ) showed the disappearance of the quinone methide bands with reappearance of a band a t A, 480 nm for formation of 7-deoxydaunomycinone (6a). The 7-deoxyaglycon results from solvent protonation a t the 7-position; the process has been characterized by deuterium incorporation from deuterated protic solvent (54). Consequently, the quinone methide is simply a higher energy tautomer of the 7-deoxyaglycon. With excess reducing agent 7-deoxydaunomycinone is reduced to its hydroquinone, which also absorbs at 420 nm as shown in Figure IC. Spectroscopic monitoring of a reaction mixture equimolar in daunomycin and dl-TM-3 dimer at 2 X lo4 M showed no buildup of a band for hydroquinone and clean isosbestic points for formation and destruction of quinone methide. With these conditions the kinetics of the reaction could be modeled with the consecutive firstorder rate law, with the two slow steps being bond homolysis of dl-TM-3 dimer and solvent protonation of quinone methide. The protonation occurred with a rate constant of 1.3 X s-l, giving a half-life for the quinone methide of 53 s at 25 O C . The rate constant as a function of temperature gave an enthalpy of activation for protonation of 18 kcal/mol and an entropy of activation of -6 cal/ (degmol) (50). Reduction in methanol-d solvent showed a substantial primary deuterium kinetic isotope effect of 9 with a half-life for the quinone methide of 490 s. Similar chemistry has been observed in aqueous medium; however, 7-deoxydaunomycinone is not water soluble, and precipitation inhibits the complete spectroscopic monitoring of the reaction (54). At 25 "C in aqueous medium the half-life of the quinone methide is 15 s. Reductions of adriamycin (1b) and 4-demethoxydaunomycin (idarubicin) occur similarly with small differences in the rate constants for protonation as shown in Table I1 (55).
Gaudiano and Koch
6 Chem. Res. Toxicol., Vol. 4, No. 1, 1991 +l. 500
la
a
WAVELENGTH
+l. 500 -,
b
+l.200-
+O. w Z
m 0
m
+O.
+O.
1
I
0. c1000
I
300
3
*
. , 400
,
,
,
,
,
,
,
500
,
,
, . 600
,
7bQ
WAVELENGTH +l. 500
IC
WAVELENGTH
Figure 1. UV-vis spectral changes during reaction of a methanolic solution, 1.0 X loa M in daunomycin and 1.0 X M in TM-3 dimer, buffered to an apparent pH of 8 and thermostated at 25 "C. Panel a shows scans every 10 s for the first 60 s; absorption at 480 nm is by daunomycin (la),at 420 nm by daunomycin hydroquinone (3a), and at 380 and 610 nm by 7-deoxydaunomycinone quinone methide (5a). Panel b shows scans from 70 to 240 s; absorption at 420 nm is by the hydroquinone of 7-deoxydaunomycinone,at 480 nm by 7-deoxydaunomycinone (6a), and at 380 and 610 nm by 5a. Panel c shows scans from 240 to 400 s with the bands assigned
as in panel b.
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 7
Perspective Table 11. Rate Constants for Isomerization of Quinone Met hides to 7-Deoxyaglycons rate constant," anthracycline solvent s-l half-life, s ref 32 84 5-iminodaunomycin (38) MeOH 0.022 15 42, 50 HZO 0.046 daunomycin (la) 53 50 MeOH 0.013 daunomycin (la) 490 50 MeOD 0.00142 daunomycin (la) 23 54 H20 0.030 adriamycin (lb) 63 55 MeOH 0.011 adraimycin (lb) 86 55 4-demethoxydaunomycin MeOH 0.008 6 780 66 MeOH 0.000 89 aclacinomycin A (25) 7300 55 11-deoxydaunomycin (11) MeOH 0.000095
Scheme VII. Reduction of Aclacinomycin A and Formation of Aglycon Dimer TM-3 \
0
OH
44
H H dR aclacinomycin A 2 5 430 nm
+$-
+ Ron
quinone methide 350, 550 nm
-f&+
7-deoxyaklavlnone 2 7 430 nm
a The pH in water and the apparent pH in MeOH was 8, and the reaction temperature was 25 O C . Structures not given elsewhere are as follows:
0
R"
0 "*OH
+ 0.'
OH 613'
4demethoxydaunomycin: R" = OH; R"' = H lldeoxydaunomydn (11): W'= H; W"= OMe Was defined in Scheme I for b d h anthracydines
Although the visible and EPR spectroscopic data are most consistent with glycosidic cleavage occurring at the hydroquinone state, with enzymatic, one-electron reduction in vivo at very low concentration, glycosidic cleavage could occur at the semiquinone state to give the semiquinone methide. Proving or disproving such a possibility remains a challenge. Results from recent experiments suggest an additional pathway to semiquinone methide (vide infra). The structure of the quinone methide suggests a balance between reactivity as a nucleophile and as an electrophile. On the bottom side of the structure, the functionality is an unsaturated ketone, a Michael acceptor, and on the top side it is a nucleophilic and basic enol. Facile protonation to form the 7-deoxyaglyconconfirms the base character; aldol reaction with the electrophiles benzaldehyde (56)and p-carboxybenzaldehyde (54) supports the nucleophilic character. The aldol product 22 from reaction with the re face predominates; the diastereomer of 22 has never been characterized. Reaction at the re face is possibly directed thermodynamically by the acetyl group at the 13-position through subsequent hemiacetal formation. To date, no biological electrophiles including the disulfide functional group have had sufficient reactivity to trap the quinone methide. The quinone methide also reacts with nucleophiles, most notably mercaptans such as N-acetylcysteine (57). The Michael addition at the 7-position yields the adduct in its hydroquinone state (23). Because the nucleophile is also a good leaving group, re-formation of the quinone methide also occurs. The adduct can be stabilized through subsequent oxidation to form the adduct in its quinone state (24). For in vitro experiments the oxidizing agent is commonly the starting quinone (54,57). Consequently, the overall process is a chain reaction. In vivo, molecular oxygen might serve as the oxidizing agent. The concentration of molecular oxygen as a function of time is crucial because molecular oxygen also inhibits formation of the quinone methide. No nucleotides or nucleic acid bases have been sufficiently reactive to trap the quinone methide in vitro.
Reduction of 11-Deoxyanthracyclines Three anthracyclines missing the hydroxyl group in the 11-position are of interest with regard to understanding formation and reactivity of the anthracycline quinone
bi(7-deoxyaklavlnon-7-yl) 430 nm
28
methide. These are 11-deoxydaunomycin (24), aclacinomycin A (25), and 7-con-0-methylnogarol (menogaril, 26) (58,59). Aclacinomycin A and menogaril (60)are in various stages of clinical investigation. An important initial observation was formation of the 7,7'-aglycon dimer, bi(7-deoxyaklavinon-7-yl) (28), as well as 7-deoxyaklavinone (27), upon anaerobic reduction of aclacinomycin A (Scheme VII) (61, 62). Aglycon dimer formation has been interpreted as evidence for the intermediacy of semiquinone methide from glycosidic cleavage at the semiquinone state (63,64). Subsequently, aglycon dimers were characterized from reduction of 11-deoxydaunomycin (55)and menogaril (65). EPR and visible spectroscopic monitoring of the reduction of aclacinomycin A by TM-3 dimer in methanol as described for the reduction of daunomycin again revealed the formation of semiquinone and quinone methide (A, 350, 550 nm) transients, respectively, but not semiquinone methide (66). The rate of disappearance of the quinone methide followed a combined first- and secondorder rate law consistent with pseudo-first-order protonation to form 27 and second-order dimerization to form 28. Actually the aglycon dimer is formed in a half quinone-half hydroquinone state from one quinone methide serving as a nucleophile and the other as an electrophile. Upon admission of molecular oxygen the mixed-state dimer is rapidly oxidized to the bis-quinone 28. These mechanistic results now suggest that aglycon dimer formation does not establish the intermediacy of semiquinone methide. The behavior of menogaril (26) upon reduction differs in some respects. Initial inspection of the structure revealed the possibility that the quinone methide functionality could be formed on both sides of the molecule by elimination of the methoxy group at the 7-position and by breaking the C5'-0 bond and that menogaril might function as a bioreductively activated cross-linking agent. However, no evidence for reductive cleavage of the C5'-0 bond has been found. Menogaril semiquinone formed from reaction with TM-3 in methanol is never present in sufficient concentration for complete EPR characterization as has been accomplished for daunomycin and aclacino-
Gaudiano and Koch
8 Chem. Res. Toricol., Vol. 4, No. 1, 1991 Scheme VIII. Reductive Activation of Menogaril with TM-3 Dimer QH
- TM-3
OH
menogaril semiquinone
TM.3
475 nm
menogaril hydroquinone 420 nm
TM-~ d
hydroquinone catalyst
-----*
7-deoxynogaroi
+ hydroquinone (catalyst) 420 nm
quinone methide
7-deoxynogarol
375, 604 nm
475 nm
I
*'
\o
30
P
n
o
0
HO
0
PI HO
OH
OH
?H
.
O 0 475 nm -*, /bi(7-deoxynogarol-7-yi) 3 1
mycin semiquinones. A signal was observed, but only the benzylic hyperfine splittings were resolved. The primary difference in menogaril redox chemistry is that the hydroquinone (Amm 420 nm) and quinone methide (Ama 375, 604 nm) transients are much longer lived (67). In fact, the quinone methide formed in methanol solvent at an apparent pH of 8 is not protonated by the solvent to give 7-deoxynogarol (30) but only dimerizes to form bi(7deoxynogarol-7-yl) (31) in the mixed redox state (Scheme VIII). Surprisingly, 7-deoxynogarolis formed as the major reduction product in vitro when an excess of reducing agent is employed. Under this condition, some materials are always present in hydroquinone states, and these catalyze the formation of 7-deoxynogarolfrom the quinone methide. The kinetics of decay of the quinone methide in the presence of excess reducing agent can actually be fit with an autocatalytic rate law with the product, 7deoxynogarol (in its hydroquinone state), catalyzing formation of additional 7-deoxynogarol. The autocatalytic effect is probably only relevant to in vitro reductions. The second-order rate constants for dimerization of the quinone methides from reductive cleavage of aclacinomycin A, menogaril, and ll-deoxydaunomycin in methanol at 25 O C are 23, 11, and 1 M-ls-l, respectively. The presence of water greatly diminishes the observability of the quinone methide from reduction of menogaril. The predominant effect is upon the rate of formation from the hydroquinone. With water present or as the solvent, the rate of formation of the quinone methide from hydroquinone becomes much slower than the rates of dimerization and tautomerization. Aglycon dimer formation is still a major pathway in water with substoichiometric amounts of reducing agent, suggesting that the quinone methide, although not observable, is sufficiently long lived for bimolecular reactivity.
Aclacinomycin A and ll-deoxydaunomycin were the f i t anthracyclines with which nucleophilic addition of mercaptans to the quinone methides was observed (68),and with these quinone methides the yields of adducts were substantial. Trapping experiments with less nucleophilic materials such as nucleosides remain unsuccessful. The quinone methide from reduction of menogaril, because it is the longest lived, has recently been successfully trapped by the nitrogen nucleophiles imidazole (69) and 2'deoxyguanosine (70) by using the chain process described earlier. With a large excess of 2'-deoxyguanosine present in aqueous medium, reduction with 20 mol % DHM-3 dimer yielded 17% of the adduct (32) along with 30% 7-deoxynogarol(30) and 23% bi(7-deoxynogarol-7-yl) (31) (Scheme IX). This successful trapping provided the first model reaction for possible reductive linkage of an anthracycline aglycon to DNA.
Formation of Cyclomers from Reduction of Daunomycin Derivatives As established by the results described above, the quinone methides from reductive cleavage of daunomycin and adriamycin are too reactive with protons from solvent to bind competitively with nitrogen and oxygen nucleophilic sites in small molecule models for biological macromolecules. In vivo, covalent binding by nucleophilic addition might be competitive if the quinone methide were generated in association with the macromolecule. This possibility has been tested by locating a nucleophile with propitious geometry within the daunomycin molecule through derivatization of the carbonyl a t the 13-position (71). Oxygen and nitrogen nucleophiles were introduced through preparation of oxime (33a) and semicarbazone (33b) derivatives, respectively.
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 9
Perspective
Scheme IX. Reaction of 7-Deoxynogarol Quinone Methide with Guanosine QH
tl
.n
HzN\rc"Y
.."b OH
II HA
bH
!Me
quinone methide
Hb l'-deoxyguanosl ne
HO HO
Anaerobic reduction of the oxime derivative 33a in water at pH 9.0 with an excess of DHM-3 dimer yielded 10% 7-deoxydaunomycinone oxime (34a) and 90% 7,13-dideoxy-7,13-(epoxynitrilo)daunomycinone(cyclooxime, 35a) upon aerobic workup. The ratio of 35a to 34a was pH dependent, ranging from 9/1 in methanol at apparent pH of 9.3 to 1/10 at apparent pH of 7.0. The pH dependency is consistent with the effect of pH on the availability of protons for formation of 34a and the nucleophilicity of the oxime for formation of 35a, both from the quinone methide 36a (Scheme X). Similar reduction of the semicarbazone derivative 33b at pH 11 in aqueous medium containing 10% methanol with TM-3 dimer followed by aerobic workup yielded 20% 7-deoxydaunomycinone semicarbazone (34b), 50% 7,13dideoxy-7,13-(iminocarbonyliminonitrilo)daunomycinone (cyclosemicarbazone, 35b), and an unidentified product. Again the product ratio (35b to 34b) was pH dependent, ranging from 3/1 at pH 11 to 1/5 at pH 8.2. The semicarbazone derivative 33b bears two nucleophilic nitrogens and could have formed both three- and five-atom bridges to the 7-position of the quinone methide; the major pathway was to form the five-atom bridge. The possibility of equilibrium between the cyclomers in their hydroquinone states and the quinone methides 36a and 36b is of interest because of the biological importance of creating irreversible linkages. Reduction of cyclooxime 35a in methanol at apparent pH of 8.2 at ambient temperature with TM-3 dimer yielded 60% of 7-deoxydaunomycinone oxime (34a) after 12 min and 96% of 34a after 90 min. Similar reduction of cyclosemicarbazone (35b) yielded only recovered 35b. The irreversibility of cyclosemicarbazone formation may result from the higher stability of the 5-atom bridge and the poorer leaving ability of the semicarbazone substituent. Reductive cleavage has been discussed earlier in terms of stereoelectronic effects on the leaving group (72). The electronic nature of the amino group of the semicarbazone is similar to the electronic nature of the 2-amino group of guanosine. Consequently, the irreversible formation of the cyclosemicarbazonesuggests the possibility
Scheme X. Intramolecular Trapping of the Quinone Methide
.w4 fH
0
daunomycin
I N
PH
TM-3 ;r
H2N-ZH
MOO 33s 33b
Z Z
--
DHM-3F
in H 2 0 o r M e O H
R'
-0-F-CONHg
HZ
fH
/ \
daunosamlns
It
36s 36b
34s
Z
34b
Z
-
I
-0-N-CONHz
TM-3
z Z
I(
-0-
I
-NHCONH-
358 35b
-
Z = -0-cycio-oxime Z -NHCONHcycio-armicarbazone
of covalent bond formation between nucleic acids and daunomycin aglycon if the reactants are propitiously located a t the time of formation of the quinone methide. The proper location might logically be daunomycin intercalated in DNA. Both daunomycin and adriamycin show high binding constants for intercalation (73). However, enzymatic reduction is inhibited by intercalation (74). Furthermore, even reduction of daunomycin intercalated in DNA with the small molecule reducing agent, carbon dioxide radical anion, does not lead to covalent bond formation but only to production of intercalated 7-deoxydaunomycinone (75). Reduction with carbon dioxide radical anion was performed anaerobically, and the lack of covalent binding may have resulted from the lack of an oxidizing agent such as molecular oxygen to prevent cleavage of the covalent bond at the hydroquinone state. Alternatively, the geometry of intercalated anthracycline
10 Chem. Res. Toxicol., Vol. 4 , No. 1, 1991
Gaudiano and Koch
Scheme XI. Formation and Reaction of Leucodaunomycin
,
O H Na2s20$
0
,,
I
Me0
H daunomycin 480 nm
OR
-
Me0
1a
3a
H OR 420 nm
I
OH
, Me0 2 4 490 nm
-
pH3 H e PH 7
,
0
-0H
Me0 H 0 OR leucodaunomycin 3 7 420, 440 nm
OH
-0H
0
OH
0
S-CHp
I
AcNH-CH-COOH
quinone methide 5 a 620 nm + ROH daunosamine
may not be favorable for covalent bond formation.
Formation of Leucodaunomycin, a Semistable Form of Daunomycin Hydroquinone A problem associated with the proposal of the quinone methide as a reactive intermediate in covalent binding to DNA is that the reductive activation may occur in a region of the cell remote from the DNA. The quinone methide may then be protonated by the medium to form the 7deoxyaglycon before it can diffuse to the DNA. This would be particularly problematical with daunomycin and adriamycin, which form short-lived quinone methides. The chemistry that occurs upon reduction of daunomycin in aprotic medium and a t lower pH suggests a solution. Reduction of daunomycin with sodium dithionite in water a t pH 3.5 gives, in addition to 7-deoxydaunomycinone, a mixture of four diastereoisomers which are tautomers of daunomycin hydroquinone (Scheme XI) (76). A similar result occurs when daunomycin is reduced with TM-3 dimer in acetonitrile solvent. These stereoisomers now bear the common name leucodaunomycin (37)because they are less colored than daunomycin. The process of tautomerization of the hydroquinone state was first observed in the reduction of 7-deoxydaunomycin to its hydroquinone (77). Leucodaunomycin is characterized by twin visible absorption bands a t 420 and 440 nm. The leucodaunomycin isomers can be isolated and separated chromatographically. When they are redissolved in anaerobic pH 7 water, they form quinone methide as shown by the isolation of its tautomer 6a and by trapping with N-acetylcysteine to form 7-(N-acetyl-~-cystein-Syl)-7-deoxydaunomycinonehydroquinone. Oxidation of the hydroquinone by daunomycin or by molecular oxygen yields 7-(N-acetyl-~-cystein-S-y1)-7-deoxydaunomycinone (24). The rates at which two of the leuco isomers yield 7-deoxydaunomycinone have been measured at 25 " C at pH 7.4. One of the isomers has a half-life of 530 s, which is 24 times the half-life of the quinone methide 5a. Possibly, one of the other isomers has a half-life even longer. Tautomerization of daunomycin hydroquinone to leucodaunomycin in competition with glycosidic cleavage at low pH or in an aprotic environment is consistent with the glycosidic cleavage occurring a t a zwitterionic state and tautomerization occurring at a cationic state. The cationic center is the protonated amino group of the daunosamine. A scenario that might explain covalent binding of daunomycin or adriamycin to DNA is as follows. The anthracycline is reduced enzymatically in one part of the cell where the appropriate enzymes reside. The environment, either acidic or hydrophobic, facilitates tautomerization
7-deoxydaunomycinone 480 nm
to a relatively stable isomer of leucodaunomycin. Some preliminary results suggest that the pH necessary to achieve a leuco isomer may be as high as 6. The leucodaunomycin then diffuses to the DNA or other critical biological molecule and subsequently tautomerizes, with formation of the reactive quinone methide 5a. Covalent binding then proceeds. Interestingly, the environment of the tumor cell is in general more acidic than that of the normal cell (78). Possibly, the difference in pH of the two types of cells provides differential cytotoxicity.
Reduction of 5-Iminodaunomycin Because chronic cardiotoxicity is the predominant total dose limiting side effect of chemotherapy with adriamycin, significant effort has been devoted to elucidating the origin of the chronic cardiotoxicity. The prevailing theory implicates redox cycling and the production of reactive oxygen species. An important observation consistent with this theory is that 5-iminodaunomycin (38) is much less cardiotoxic than daunomycin and is not an efficient catalyst for the in vivo or in vitro production of reactive oxygen species (79-81). Inefficient catalysis of oxygen reduction is proposed to result from the more negative reduction potential and/or some difficulty in reoxidizing reduced 38. 5-Iminodaunomycin is a little more difficult to reduce than daunomycin;the half-wave potentials versus SCE are -0.67 and -0.64 V, respectively (82). However, 38 is reduced in vitro by NADPH cytochrome P-450 prepared from rat liver microsomes (83). Reduction with sodium dithionite in methanol solvent yields a quinone methide transient which has a half-life with respect to protonation to form 5-imino-7-deoxydaunomycinone (39) of 32 s, similar to the half-life of 5a (84). The reduction is suppressed by molecular oxygen analogously to the suppression of the reduction of daunomycin. The primary difference in the chemical reduction of 38 resides in the next phase, the reduction of 5-imino-7-deoxydaunomycinone. Reduction of 39 with dithionite leads to rapid deamination to form 2-acetyl-2,1l-dihydroxy-7-methoxy1,2,3,4-tetrahydro-5,12-naphthacenedione (40) (Scheme XII) (84, 85). Reductive deamination occurs with a half-life of only 17 s at 25 "C in methanol. Although naphthacenedione 40 is a quinone, a B-ring quinone, it is a poor catalyst for the production of reactive oxygen species. Reduction of 40 rapidly yields 8-acetyl-1-methoxy-7,9,10,12-tetrahydro-6,8,1 l-trihydroxy-5(8H)-naphthacenone (41), which reoxidizes with a half-life of 35 min. In contrast, the hydroquinone of 7-deoxydaunomycinone is relatively stable and is rapidly oxidized by molecular oxygen. Consequently, a rationale for the poor catalytic ability of 5-iminodaunomycin to produce reactive oxygen
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 11
Perspectioe
Scheme XIII. Reaction of Quinone Methide with Molecular Oxygen
Scheme XII. Reductive Activation and Deactivation of 5-Iminodaunomy cin
H
+ N a Z s 2+ 04 ROH
+
@&$
daunoramlne
38 5-iminodaunomycin
+ ROH
Meb NH quinone methide
H OR daunomycin 1 a
Me
daunosamlne
550, 552 nm
Me quinone methide S a
02
Me
Me0 NH H 5.imino-7.deoxydaunomycinone 30
Me0
NHz
H
7-deoxydaunomycinone
daunomycinone
68
42
w+ @&++
540, 580 nm
Me
H
7-epidaunomycinone
Me
H
H
7-deoxy-7-ketodaunomycinone
43
44
naphihacenedione 4 0 480 nm
OH
Me0 35 min
naphthacenone 4 1 397 nm
species is the facile reductive deamination in its 7-deoxy hydroquinone state. Although production of reactive oxygen species has been implicated in the cardiotoxicity of adriamycin, it has also been implicated in at least part of the cytutoxicity to tumor cells. Correspondingly, 5-iminodaunomycin is less cardiotoxic and less toxic to some tumor cells. These observations together with the studies of chemical reduction suggest that part of the tumor cell toxicity of adriamycin and daunomycin resides in the ability of their 7deoxyaglycons to serve as catalysts for the intracellular production of reactive oxygen species. As extracellular materials, the 7-deoxyaglycons are inactive, possibly because they are not easily transported. Another possibility that remains to be investigated is that 5-iminodaunomycin is unable to form a leuco isomer upon reduction a t lower pH because of competitive deamination.
Formation of a Semiquinone Methide from Daunomycin As mentioned above, the earliest proposed mechanism for covalently linking the aglycon of daunomycin and adriamycin to DNA invoked the respective semiquinone methides 4a and 4b. The in vitro studies reviewed here and those of others provide little or no evidence for formation of the semiquinone methide from reduction of the anthracyclines in one- or two-electron steps. Even the formation of aglycon dimers from reduction of the 11deoxyanthracyclines, often thought to implicate semiquinone methide, occurs from reaction of the quinone methide. Semiquinone methide is theoretically an attractive transient for the coupling to macromolecules. It should be a stabilized radical which would efficiently combine irreversibly with radical sites in macromolecules rather than abstract hydrogen atoms. The radical sites might logically be created by H-atom abstraction by reactive oxygen species formed through anthracycline catalysis. Investigation of the reaction of the quinone methide 5a with molecular oxygen has revealed a logical pathway to semiquinone methide (86). Significant concentrations of
7-deoxy-7,13-ep1d10xydaunomyc1noi
45
bi(7-deoxydaunomycinon-7-yl) 4 6
5a are produced upon anaerobic reduction of daunomycin in methanol-d solvent with sodium dithionite. Admission of molecular oxygen results in almost instantaneous consumption of the quinone methide, as indicated by the color change from dark gray to orange. The products formed are 7-deoxydaunomycinone (6a),daunomycinone (42)) 7-epidaunomycinone (43),7-deoxy-7-ketodaunomycinone (44), 7-deoxy-7,13-epidioxydaunomycinol(45), and two diastereomers of bi(7-deoxydaunomycinon-7-yl)(46) (Scheme XIII). The major products are 6a,45,and 46. The ratio of 46 to 42 43 44 45 is dependent upon the partial pressure of oxygen, increasing with decreasing partial pressure. The yield of daunomycinone plus epidaunomycinone significantly exceeds the yield of 7-deoxy-7-ketodaunomycinone, and the same products are formed when the quinone methide is generated in water. When quinone methide is generated by raising the pH of an aqueous solution of leucodaunomycin in the presence of air, the same products are formed except 46 (87). 7-Deoxydaunomycinonemost likely results from solvent protonation of the quinone methide. Pathways that rationalize formation of the other products are shown in Scheme XIV. Quinone methide reacts by combination with molecular oxygen to form the semiquinone peroxy radical 47 and/or by electron transfer to molecular oxygen to form superoxide and the semiquinone methide 4a. Reaction of 47 with another molecule of quinone methide gives a bis-semiquinone peroxide, 48,which is at the proper redox state to fragment to daunomycinone and epidaunomycinone. Stepwise combination of two semiquinone methides with two molecules of oxygen yields the bisquinone tetraoxide 49, which would likely fragment to ketodaunomycinone and a mixture of daunomycinone and epidaunomycinone plus singlet or triplet molecular oxygen. Tetraoxides have been implicated as intermediates in hydrocarbon oxidations (88). Combination of semiquinone methide with superoxide produces diastereomeric quinone hydroperoxides (50),one with the proper stereochemistry to cyclize to epidioxydaunomycinol and the other which must fragment to ketodaunomycinone. Combination of two semiquinone methides is now the logical pathway to bi(7-deoxydaunomycinon-7-yl)(46) because 46 is not formed from quinone methide in the absence of oxygen.
+ + +
12 Chem. Res. Toxicol., Vol. 4 , No.
I, 1991
Gaudiano and Koch
Scheme XIV. Proposed Mechanism for Reaction of Quione Methide with Molecular Oxygen 0 2
02
quinone methide (sa)
semiquinone peroxy radical (47) quinone methide (4a)
bi(7.deoxydaunomycinon-7-yl) (46) \
Me
0
.
0
OH
bis-semiquinone peroxide (48)
c
daunomyclnone (42)
c
daunomycinone (42)
+
7-epidaunom~cinone (43)
' 0 H
+ O2
7-epidaunomycinone (43)
7.deoxy-7-keiodaunomycinone
Variation in the relative yield of aglycon dimer as a function of oxygen partial pressure and the quinone methide concentration results from the competition between semiquinone methide combining with another semiquinone methide or with molecular oxygen. Aglycon dimer is not observed when quinone methide is produced from leucodaunomycin because the concentration of the quinone methide and, correspondingly, semiquinone methide is never high. With these observations, semiquinone methide reemerges as a contender for the transient involved in covalent binding to biological macromolecules. The scenario begins with radical sites in the macromolecules being created by reactive oxygen species formed through anthracycline catalysis. Upon reduction of the oxygen concentration, competitive reductive glycosidic cleavage occurs to form quinone methide. Quinone methide reacts with the available oxygen to form semiquinone methide, which then combines with radical sites in the macromolecule. The bond formed is likely a carbon-carbon bond which will not cleave upon subsequent reduction of the attached aglycon. Further catalytic production of reactive oxygen species by the attached aglycon will cleave the macromolecule. Unless the redox enzymes are in the vicinity of the target macromolecule, the probability of the above scenario leading to efficient covalent bond formation is low. The problem with radicals, probably including semiquinone methide, is that they do not diffuse very far. TM-3 and DHM-3 are exceptions. Of course, leucodaunomycin could serve as a protected source of semiquinone methide and be formed in a region of the cell remote from the macromolecular target. Also radical sites in the target could be formed by production of superoxide in one region of the cell, diffusion of superoxide to the target, and Haber-Weiss chemistry in the vicinity of the target. A cartoon illustrating this possibility is shown in Scheme XV. Another possibility which remains to be explored is that the actual cytotoxic species is the quinone hydroperoxide 50, epidioxydaunomycinol45, or some other anthracycline peroxide. These species bear functionality which can both create a radical site and bind to it. For example, Fe(I1)-catalyzed decomposition of 50 might yield a hydroxyl radical for H-atom abstraction and 7-deoxydaunomycinon-7-yloxy radical for combination with the radical site in the macromolecule. As mentioned earlier, cytotoxicity may require more than covalent binding of the aglycon to a macromolecule.
+
+ (44)
quinone hydroperoxide (50)
c
7-deoxy-7-keiodaunomycinone (44) + 7-deoxy-7,13-epidioxydaunomycinol (45)
A possibility is subsequent cutting of the macromolecule. If reducing enzymes are not available at the site of the macromolecule, aglycon-catalyzedredox recycling will not occur. However, the aglycon is known to be a strong chelating agent for iron, competitive with iron storage proteins (89). Consequently, the purpose of covalent binding may only be to locate an iron cation adjacent to the macromolecule. The hydrogen peroxide which diffuses to the macromolecule will then be efficiently converted to hydroxyl radical for subsequent cutting of the macromolecule (90).The hydrogen peroxide would be produced by other anthracyclines in the reducing region of the cell. This scenario also relates to an aspect of the chemistry of 5-iminodaunomycin and its lesser cytotoxicity. Although 5-iminodaunomycin forms an Fe(1II) complex, the complex is less efficient at producing hydroxyl radical from hydrogen peroxide (91). Utilization of Captodative Radicals in Vivo As already mentioned several times, a problem with bioreductive activation as a mechanism for cytotoxicity is that reducing enzymes are bulky and are probably not located sufficiently near target molecules for efficient reaction of the transients with the targets. This raises the possibility of in vivo activation with a chemical reducing agent such as DHM-3 dimer. DHM-3 dimer is relatively nontoxic in mice when administered both intraperitoneally and intravenously as single bolus injections (92). The first application explored was the use of TM-3 and DHM-3 dimers as antidotes for the anthracyclines because they reduce the anthracyclines to the inactive and nontoxic 7-deoxyaglycons (17). The goal was to effect extracellular reductive cleavage because intracellularly, the aglycons may still be cytotoxic. The initial application was with regard to the medical problem of extravasation necrosis (93). If intravenous administration results in some leakage around the catheter, significant necrosis occurs. The model employed was the swine model because of the similarity with human skin. When DHM-3 dimer was introduced 15 min after a subcutaneous injection of adriamycin, the total toxicity was reduced by 95%. Subsequent to administration of DHM-3 dimer, precipitation of 7-deoxyadriamycinone was apparent. DHM-3 dimer was also effective a t reducing the skin toxicity of daunomycin, 4demethoxydaunomycin, 4'-epidoxorubicin, B-iminodaunomycin, aclacinomycin A, menogaril, and mitomycin C (94). Mitomycin C is another quinone type antitumor drug
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 13
Perspective
Scheme XV. Logistics of Covalent Bond Formation to Macromolecule and Subsequent Cutting
reducing e n z y m t low pH
Me0
H OR daunomycin
rem lq uinonr cation
lo2 diffusion
leucodaunomycin
,,I ,,,,,,,,,,
, , , , , I
hydroquinone cation
I
1 1 1 1 1 * 1 ~ " ~ , 1 , , 1 1 1 ~ ' ~
diffusion
, , 1 ( , 1 1 1 - ~ ~ ~
I
-"""""membrane
, I , , I I I a * '
Haber-Welsr cycle
I
*
. . I I ~ * ~
I"""'
Me
macromolecule
oxidatively cleaved macromolecule leucodaunomycin iron complex
higher pH
f Fe(l1l) 'D H
quinone methlde
semiquinone methide
which is bioreductively activated; with this drug, covalent binding to DNA has been unambiguously established (95). If the time before administration of the antidote exceeded an hour, the antidote was ineffective. No other antidote to quinone antitumor drug extravasation necrosis has proven to be as effective as DHM-3 dimer in animal studies. Next, the concept of adriamycin chemotherapy modulated with DHM-3 dimer was explored (96). Mice were given L1210 tumor intraperitoneally on day 0, 25 mg/kg of body weight adriamycin on day 1,and 50 mg/kg DHM-3 dimer 15 min later, both intraperitoneally. The dose of adriamycin was a lethal intraperitoneal dose in the absence of DHM-3 dimer. This protocol resulted in a 5 times improvement in therapeutic index with greater than 70% long-term survivors versus the optimum sublethal dose of adriamycin without modulation with DHM-3 dimer. In general, sublethal doses of adriamycin with DHM-3 modulation were ineffective. Injection of the DHM-3 dimer more than 30 min after the adriamycin was also ineffective. A control experiment showed that the DHM-3 dimer itself is not an active antitumor drug. Heart, lung, liver, and kidney tissues of animals receiving adriamycin plus DHM-3 dimer showed lower levels of adriamycin and higher levels of adriamycin aglycons relative to animals receiving only adriamycin. Clearly, the result of this experiment poses more questions than it answers. Of particular relevance to this discussion, does the improved therapeutic index involve reductive activation of adriamycin by DHM-3? Does DHM-3 dimer even pass through cell membranes? An explanation of the improved therapeutic index may simply be that the modulated therapy achieved higher intratumor levels of the adriamycin be-
Me
cause the tumor was localized in the peritoneal cavity and that the excess toxic adriamycin never left the peritoneal cavity because it was reductively cleaved to its 7deoxyaglycon by DHM-3. Even if this is the explanation, techniques for achieving higher intratumor levels of anthracyclines without increasing toxic side effects may be of significant value in chemotherapy. Further progress with in vivo experiments depends upon developing drugs and methodology for intravenous administration of modulating and/or antidote materials. A major problem will likely be the deactivation of drugs such as DHM-3 by molecular oxygen and/or hemoglobin-bound molecular oxygen (vide supra). Thermodynamically, reduction of molecular oxygen is easier than reduction of the anthracyclines. Consequently, the problem must be solved kinetically.
Summary The oxomorpholinyl radicals are unique materials in organic and medicinal chemistry. Their closest parallel lies in inorganic chemistry with dithionite, which exists in equilibrium with sulfur dioxide radical anion, also a oneelectron reducing agent (97). However, dithionite is a more powerful reducing agent and is probably more toxic. The rate of release of the oxomorpholinyl radical from its dimer is medium and is structure dependent, which provides for some level of control. The oxomorpholinyl radicals TM-3 and DHM-3 are selective one-electron reducing agents for the anthracyclines, generating sequentially semiquinone and hydroquinone redox states. Formation of the reduced states of the anthracyclines is probably relevant to their cytotoxic activity. Semiquinones and hydroquinones react
14 Chem. Res. Toxicol., Vol. 4, No. 1, 1991
rapidly with molecular oxygen to yield superoxide. Hydroquinone redox states with anaerobic conditions in protic media at pH 7-8 undergo glycosidic cleavage to form quinone methides; in aprotic media or a t pH less than 4, they tautomerize to leuco forms. Quinone methides react with protons from solvent to form 7-deoxyaglycons, with some nucleophiles to form adducts, and with molecular oxygen to form semiquinone methide. The reactivity of the quinone methide is a function of substitution; nucleophilic addition is facilitated by the absence of a hydroxyl group at the 11-position and by proper location of the nucleophile. Quinone methides and semiquinone methides are both viable transients for covalently linking anthracycline aglycons to biological macromolecules. DHM-3 dimer is of possible pharmaceutical value for the detoxification of quinone antitumor drugs and for the improvement of chemotherapy through modulating the redox chemistry of the quinone antitumor drugs:
Acknowledgment. We gratefully acknowledge financial support from the USPHS in the form of Grant CA 24665, from the NSF in the form of Grant CHE-8903637, and from the University of Colorado Council on Research and Creative Work in the form of a Faculty Fellowship. We thank Barbara Schweitzer for preparing Figure 1.
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