DNA strand cleavage by the phenazine di- N -oxide natural product myxin under both aerobic and anaerobic conditions

Article abstract of DOI:10.1021/tx2004213

Heterocyclic N-oxides are an interesting class of antitumor agents that selectively kill the hypoxic cells found in solid tumors. The hypoxia-selective activity of the lead compound in this class, tirapazamine, stems from its ability to undergo intracellu

Full text of DOI:10.1021/tx2004213

Article
pubs.acs.org/crt
DNA Strand Cleavage by the Phenazine Di-N-oxide Natural Product
Myxin under Both Aerobic and Anaerobic Conditions
Goutam Chowdhury,† Ujjal Sarkar,† Susan Pullen,‡ William R. Wilson,‡ Anuruddha Rajapakse,†
Tarra Fuchs-Knotts,† and Kent S. Gates*,†
†Departments of Chemistry and Biochemistry, University of MissouriColumbia, 125 Chemistry Building, Columbia, Missouri
65211, United States
‡Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand
ABSTRACT: Heterocyclic N-oxides are an interesting class of
antitumor agents that selectively kill the hypoxic cells found in
solid tumors. The hypoxia-selective activity of the lead
compound in this class, tirapazamine, stems from its ability
to undergo intracellular one-electron reduction to an oxygen-
sensitive drug radical intermediate. In the presence of
molecular oxygen, the radical intermediate is back-oxidized
to the parent molecule. Under hypoxic conditions, the extended lifetime of the drug radical intermediate enables its conversion to
a highly cytotoxic DNA-damaging intermediate via a “deoxygenative” mechanism involving the loss of oxygen from one of its N-
oxide groups. The natural product myxin is a phenazine di-N-oxide that displays potent antibiotic activity against a variety of
organisms under aerobic conditions. In light of the current view of heterocyclic N-oxides as agents that selectively operate under
hypoxic conditions, it is striking that myxin was identified from Sorangium extracts based upon its antibiotic properties under
aerobic conditions. Therefore, we set out to examine the molecular mechanisms underlying the biological activity of myxin. We
find that myxin causes bioreductively activated, radical-mediated DNA strand cleavage under both aerobic and anaerobic
conditions. Our evidence indicates that strand cleavage occurs via a deoxygenative metabolism. We show that myxin displays
potent cytotoxicity against the human colorectal cancer cell line HCT-116 under both aerobic and anaerobic conditions that is
comparable to the cell-killing properties of tirapazamine under anaerobic conditions. This work sheds light on the processes by
which the naturally occurring aromatic N-oxide myxin gains its potent antibiotic properties under aerobic conditions.
Furthermore, these studies highlight the general potential for aromatic N-oxides to undergo highly cytotoxic deoxygenative
metabolism following enzymatic one-electron reduction under aerobic conditions.
conversion to highly cytotoxic DNA-damaging intermediates
via a “deoxygenative” mechanism involving loss of oxygen from
the N-oxide group in the 4-position.22,23 DNA damage is
central to the bioactivity of tirapazamine,24−27 but the exact
nature of the reactive intermediate(s) generated following
bioreductive activation of tirapazamine remains under inves-
tigation. Mechanisms involving homolytic fragmentation to
generate the known cell-killing agent hydroxyl radical28−31 or
dehydration to yield a highly oxidizing benzotriazinyl
radical32−36 have been proposed. The mono-N-oxide metabo-
lites such as 3 are diagnostic byproducts of the cell-killing,
deoxygenative metabolism that can follow one-electron
enzymatic activation of tirapazamine.37
There are only a handful of natural products that contain
aromatic N-oxide functional groups.38−43 The compound, 1-
hydroxy-6-methoxyphenazine-N5,N10-dioxide (7, Scheme 2),
commonly known as myxin, is an interesting example. Myxin
was first isolated from Sorangium sp. by Peterson et al.44 This
natural product displays potent antimicrobial activity against a
wide variety of organisms including Streptococcus agalactiae,
INTRODUCTION
■
Heterocyclic aromatic N-oxides are an interesting class of
antitumor agents exhibiting bioreductively activated, hypoxia-
selective DNA-damaging properties.1−7 Interest in these
compounds has been driven by the fact that they display
selective toxicity toward the hypoxic cells found in solid
tumors.8 Tirapazamine (1, TPZ, Scheme 1) is the lead
compound in this class of drugs and has been examined in a
variety of phase I, II, and III clinical trials for the treatment of
various cancers.9−11 Bioreductively activated aromatic N-oxides
also have the potential to kill Mycobacterium tuberculosis12 and
hypoxic bacteria in the gastrointestinal and urinary tracts.13−16
The hypoxia-selective cytotoxicity of tirapazamine and other
structurally related aromatic N-oxides stems from their ability
to undergo intracellular one-electron reduction to an oxygen-
sensitive drug radical intermediate (2, Scheme 1).17−20 In the
presence of molecular oxygen, the radical intermediate is
rapidly oxidized to regenerate the parent molecule and an
equivalent of superoxide radical.20,21 In the case of medicinally
interesting N-oxides such as 1, this futile cycling process is
evidently substantially less toxic than processes that occur
under hypoxic conditions. Under hypoxic conditions, the
extended lifetime of the drug radical intermediate enables its
Received: October 1, 2011
Published: November 15, 2011
© 2011 American Chemical Society
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Scheme 1
(Brookfield, CN); and bromophenol blue, United States Biochemicals
(Cleveland, OH).
Scheme 2
Synthesis of 1,6-Dimethoxyphenazine (5). This compound
was prepared previously by Pachter et al., and we used a slight
modification of the published approach.53 To a solution of benzene
(180 mL) was added o-anisidine (28.4 mL, 0.25 mol), o-nitroanisole
(55.8 mL, 0.45 mol), and powdered potassium hydroxide (70 g; 1.25
mol). The mixture was heated at reflux for 6.5 h with vigorous stirring
(not under inert gas). Upon cooling, the dark-brown benzene solution
was decanted off, and water (500 mL) was added to the remaining
solid in the reaction flask. After breaking up the chunky residue with a
spatula or glass rod, the solid was collected and washed twice with
ethanol (250 mL) to give a yellow crystalline solid. The original
benzene solution from above was diluted with additional benzene and
extracted with 15% HCl. The aqueous layer was then made basic, and
the resulting yellow solid was collected and washed with ethanol. The
yellow crystalline solids from above were combined and recrystallized
from hot ethanol to give 5 (8.7 g, 14%): Rf = 0.51 (100% ethyl
acetate). 1H NMR (CDCl3) δ 7.98 (d, 2H), 7.74 (t, 2H), 7.08 (d, 2H),
4.18 (s, 6H); 13C NMR (CDCl3) δ 155.6, 143.7, 137.6, 130.8, 111.7,
107.6, 57.2.
Synthesis of 1,6-Dimethoxyphenazine-N5,N10-dioxide (6).
The preparation of this compound was achieved using a modification
of the route described by Weigele et al.54 To a solution of methylene
chloride (25 mL) was added 5 (120 mg, 0.5 mmol) and m-CPBA (0.5
g, Aldrich, 77% max). The mixture was stirred for 2 h. Removal of
solvent by rotary evaporation, followed by column chromatography on
silica gel eluted with 100% ethyl acetate, afforded 6 (18 mg; 13%) as a
red solid: Rf = 0.06 (100% ethyl acetate). 1H NMR (CDCl3) δ 8.32 (d,
2H), 7.63 (t, 2H), 7.08 (d, 2H), 4.09 (s, 6H); 13C NMR (CDCl3) δ
153.6, 139.5, 130.5, 112.1, 110.2, 57.2. Spectral data matched that
reported by Weigele et al.54
Synthesis of 1-Hydroxy-6-methoxyphenazine (8). Aluminum
chloride (410 mg) was added to a solution of 5 (151 mg, 0.62 mmol)
in methylene chloride (50 mL) at room temperature. The reaction
mixture was stirred at room temperature for 20 min and quenched by
the addition of 2 N HCl (100 mL). The resulting mixture was
extracted with methylene chloride (5 × 100 mL), the combined
organic extracts dried over sodium sulfate, and then evaporated under
reduced pressure. Column chromatography on silica gel eluted with
1:1 ethyl acetate/hexane gave 8 (25 mg, 17%) as a yellow solid: Rf =
0.67 (100% ethyl acetate). 1H NMR (CDCl3) δ 8.17 (s, 1H), 7.92 (d,
1H), 7.74 (m, 3H), 7.24 (d, 1H), 7.06 (d, 1H), 4.17 (s, 3H); 13C
NMR (CDCl3) δ 155.1, 151.3, 142.4, 141.8, 137.4, 134.5, 131.2, 130.3,
120.8, 120.4, 109.1, 106.7, 56.4. HRMS (ESI) calcd for C14H13N2O4
[M + H+]+ 273.0875 found 273.0876.
Synthesis of 1-Hydroxy-6-methoxyphenazine-N5,N10-diox-
ide (7). To a solution of 6 (23 mg, 0.08 mmol) in methylene chloride
(10 mL) was added aluminum chloride (50 mL). The mixture was
stirred for 1.5 h. After the reaction was complete, 2 N HCl (40 mL)
was added to the mixture, and a color change from blue to red-orange
occurred. The aqueous layer was then extracted with methylene
chloride (8 × 30 mL) and the combined organic extracts dried by
rotary evaporation. Column chromatography over silica gel eluted with
100% ethyl acetate, followed by 4:1 ethyl acetate/hexane, afforded 7 (5
mg, 24%): Rf = 0.58 (100% ethyl acetate). 1H NMR (CDCl3) δ 14.59
Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, and
Candida albicans, with minimum inhibitory concentrations in
the range of 1−20 μM.45,46 Several structurally related synthetic
phenazine di-N-oxides that display bioreductively activated
cytotoxicity under aerobic conditions have been identified.3,47
Several lines of evidence from early studies indicated that
interactions with DNA may be important in the biological
activity of myxin. Myxin inhibits both DNA and RNA synthesis
and causes extensive degradation of cellular DNA in E. coli.48,49
In Salmonella, myxin causes frameshift mutations.50 Myxin also
was found to noncovalently associate with DNA via
intercalation.51 It was also noted that myxin undergoes
reduction in E. coli to biologically inactive products, though
structural characterization of the metabolites was not presented
in this early work.46,52
In light of the current view of heterocyclic N-oxides as agents
that selectively operate under hypoxic conditions, it is striking
that myxin was identified from Sorangium extracts based upon
its antibiotic properties under aerobic conditions. Therefore, we
set out to examine potential molecular mechanisms underlying
the biological activity of myxin. Our evidence suggests that
myxin causes bioreductively activated, radical-mediated DNA
strand cleavage under aerobic conditions via a deoxygenative
mechanism analogous to that characterized for tirapazamine
under anaerobic conditions.
MATERIALS AND METHODS
■
Materials. Materials were obtained from the following suppliers
and were of the highest purity available. Unless otherwise mentioned,
all chemicals were from Aldrich Chemical Co. (Milwaukee, WI);
sodium acetate, NADPH, desferal, and NADPH/cytochromeP450
reductase, Sigma Chemical Co. (St. Louis, MO); HPLC grade solvents
acetonitrile, methanol, ethanol, t-butanol, ethyl acetate, hexane, and
acetic acid, Fisher (Pittsburgh, PA); xanthine oxidase, catalase, SOD,
and ethidium bromide, Roche Molecular Biochemicals (Indianapolis,
IN); Seakem ME agarose, FMC; ethanol, McCormick Distillation Co.
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(s, 1H), 8.25 (d, 1H), 8.02 (d, 1H), 7.68 (t, 1H), 7.63 (t, 1H), 7.12 (d,
1H), 7.09 (d, 1H), 4.09 (s, 3H); 13C NMR (CDCl3) δ 154.5, 154.4,
139.4, 136.6, 133.1, 132.4, 130.6, 126.6, 115.6, 111.3, 110.4, 109.6,
58.0. The material was pure as judged by NMR, TLC, and HPLC.
Spectral data matched that in the published literature.54,55
EDTA (TAE) buffer and then stained in a solution of aqueous
ethidium bromide (0.3 μg/mL) for 1−2 h. DNA was visualized by UV-
transillumination and quantitated by digital imaging. The values
reported are not corrected for differential staining of form I and form
II DNA by ethidium bromide.56 DNA cleavage assays containing
radical scavengers were performed as described above with the
exception that radical scavengers like methanol, ethanol, t-butanol,
DMSO, or mannitol (500 mM) were added to the reaction mixture
before the addition of xanthine oxidase. Aerobic reactions were
performed as described above, with the exception that the solutions
were not degassed, and the incubations were performed under air.
Characterization of Products Arising from in Vitro Metab-
olism of Myxin. In a typical assay, a solution of myxin (7, 250 μM),
desferal (1 mM), and xanthine (250 μM) in sodium phosphate buffer
was mixed with xanthine oxidase (0.4 U/mL). The assays were
incubated for 3 h at room temperature. After incubation, the reaction
mixture was extracted with methylene chloride (2 × 100 μL) and then
analyzed by normal phase HPLC employing a Rainin Microsorb-MV
amino column (100 Å sphere size, 5 mm pore size, 25 cm length, 4.6
mm i.d.) eluted with an isocratic mobile phase of 70% ethyl acetate
and 30% heptane (each solvent containing 0.5% acetic acid), at a flow
rate of 1 mL/min. The elution of products from the column was
monitored by UV absorbance at 284 nm. The identity of the major
metabolites was determined by comparison of the retention time to
that of authentic standards and by coinjection with the authentic
synthetic samples.
Cytotoxicity Assay. The colon carcinoma line HCT116, sourced
from ATCC, was grown in AlphaMEM plus 5% fetal calf serum (FCS)
from frozen stocks confirmed to be mycoplasma-free by PCR-ELISA
(Roche Diagnostics). Late log-phase cultures were harvested by
trypsinization and centrifuged to prepare cell pellets for parallel
exposure to compounds under aerobic and anaerobic conditions. For
the latter, the cell pellet was transferred to a Pd catalyst anaerobic
chamber (Bactron II, Shellab) with an atmosphere of 5% H2/5%CO2/
90% N2. The pellet was resuspended in Alpha MEM with 10% FCS
plus 10 mM added glucose and 0.2 mM 2-deoxycytidine. The medium
and plasticware were previously equilibrated in the chamber for ≥3
days to remove residual oxygen. Cells were plated in 96-well plates
(1000 cells/well) and allowed to attach at 37 °C for 2 h before the
addition of tirapazamine (1) or myxin (7) from DMSO stocks (final
DMSO concentration <0.5%) by making 3-fold serial dilutions across
the plate in duplicate wells. Plates were incubated for a further 4 h,
removed from the chamber, and washed 3× with fresh medium (in
parallel with plates exposed under oxic conditions). After growth in a
CO2 incubator for a further 5 days, total cellular protein was
determined by sulforhodamine B staining57 using a ELx808 plate
reader (BioTek Instruments). IC50 values (concentration for 50%
inhibition relative to 8 controls on the same plate) were interpolated
by four-parameter logistic regression (KC4 v3.4 software).
Synthesis of 1-Hydroxy-6-methoxyphenazine-N10-oxide (9)
and 1-Hydroxy-6-methoxyphenazine-N5,N10-dioxide (7) by
Oxidation of 1-Hydroxy-6-methoxyphenazine (8). To a solution
of 8 (45 mg, 0.2 mmol) in methylene chloride (10 mL), was added m-
CPBA (250 mg) and the resulting mixture stirred at room temperature
for 12 h. The solvent was then removed under reduced pressure, and
column chromatography on silica gel eluted with 4:1 ethyl acetate/
hexane gave 9 (5 mg, 10%) as the first eluting compound: Rf = 0.65
1
(100% ethyl acetate). H NMR (CDCl3) δ 13.68 (s, 1H), 8.17 (d,
1H), 7.85 (d, 1H), 7.71 (q, 2H), 7.11 (d, 2H), 4.19 (s, 3H); 13C NMR
(CDCl3) δ 156.4, 152.7, 145.9, 139.6, 134.2, 133.1, 132, 125.7, 120.8,
113.6, 110.4, 108.8, 57.8. The second eluting compound was myxin, 7
(4 mg, 8%).
Synthesis of t-Butyldiphenylsilyl-Protected 1-Hydroxy-6-
methoxyphenazine (10). To a solution of 8 (23 mg, 0.1 mmol)
in methylene chloride (5 mL) was added imidazole (34 mg, 0.5 mmol)
and the mixture allowed to stir at room temperature for 10 min. To
the solution t-butyldiphenylsilyl chloride (TBDPS-Cl, 41 μL, 0.15
mmol) was added and the reaction stirred for another 4 h. The
reaction mixture was extracted with a saturated aqueous solution of
sodium chloride (10 mL). The aqueous layer was back-extracted with
methylene chloride (3 × 10 mL), the organic fractions pooled, dried
over sodium sulfate, and evaporated under reduced pressure. Finally,
column chromatography on silica gel eluted with 2:1 hexane/ethyl
1
acetate gave 10 (22 mg, 47%): Rf = 0.82 (100% ethyl acetate). H
NMR (CDCl3) δ 7.95 (d, 1H), 7.84 (d, 4H), 7.64 (t, 1H), 7.54 (m,
2H), 7.63−7.38 (m, 6H), 7.09 (d, 1H), 7.03 (d, 1H), 4.16 (s, 3H),
1.27 (s, 9H); 13C NMR (CDCl3) δ 154.7, 151.6, 142.9, 142.8, 138.1,
136.4, 135.3, 133.3, 130.1, 129.5, 129.4, 127.4, 122.6, 121.8, 116.3,
106.4, 56.2, 26.7, 20.0.
Synthesis of 1-Hydroxy-6-methoxyphenazine-N5-oxide
(12). To a solution of 10 (22 mg, 0.047 mmol) in methylene
chloride (5 mL) was added m-CPBA (17 mg) and the mixture stirred
for 5 h. Additional m-CPBA (17 mg) was added to the reaction
mixture and stirred for another 10 h. Tetrabutylammonium fluoride
(TBAF, 1 M solution in THF, 50 μL, 0.05 mmol) was added to the
reaction mixture and allowed to stir for 3 h. The reaction was
quenched by the addition of saturated aqueous solution of sodium
chloride (20 mL) and extracted with methylene chloride (3 × 50 mL).
The organic extracts were dried over sodium sulfate and evaporated
under reduced pressure. Column chromatography on silica gel eluted
with a 1:1 ethyl acetate/hexane gave 12 (6 mg, 55%) as a pale violet
1
solid: Rf = 0.51 (100% ethyl acetate). H NMR (CDCl3) δ 8.09 (bs,
1H), 8.07 (d, 1H), 7.74 (d, 1H), 7.65 (t, 1H), 7.59 (t, 1H), 7.20 (d,
1H), 7.00 (d, 1H), 4.08 (s, 3H); 13C NMR (CDCl3) δ 154.0, 153.2,
146.3, 137.8, 137.2, 131.45, 131.43, 130.2, 122.8, 111.3, 110.4, 108.7,
57.7. HRMS (ESI) calcd for C13H11N2O3 [M + H+]+ 243.0770; found,
243.0771.
RESULTS
■
Synthesis of Myxin. Preparation of the myxin precursor
1,6-dimethoxyphenazine (5) was carried out using the method
of Patcher and Kloetzel, involving a modified version of the
Wohl-Aue reaction.53,58 Briefly, 2-nitroanisole and 2-anisidine
were dissolved in benzene and heated at reflux in the presence
of KOH (Scheme 2). A black tar was obtained which, upon
repeated washing with ethanol, gave yellow crystals of 1,6-
dimethoxyphenazine. The resulting 1,6-dimethoxyphenazine
was then oxidized by meta-chloroperbenzoic acid (m-CPBA) to
give the 1,6-dimethoxyphenazine-N5,N10-dioxide (6). Myxin
(7) was prepared by the method of Weigele and Liemgruber
involving partial demethylation of 6 by AlCl3.54 The yield of
this reaction was low due to the formation of the 1,6-dihydroxy
compound as a side product (the dihydroxy compound is a
different natural product, known as iodinin.59).
Cleavage of Supercoiled Plasmid DNA. Typical DNA cleavage
reactions contained supercoiled plasmid DNA (750 ng, pGL2-basic), 1
(25−225 μM) or 7 (50−250 μM), xanthine (500 μM), xanthine
oxidase (0.4 U/mL), catalase (100 μg/mL), superoxide dismutase (10
μg/mL), sodium phosphate buffer (50 mM, pH 7.0), and desferal (1
mM). In the case of the anaerobic reactions, individual components,
with the exception of DNA and enzyme, were degassed prior to use by
three freeze−pump−thaw cycles in Pyrex tubes and then sealed under
vacuum. Sealed tubes were scored, transferred to a glovebag purged
with argon, and opened. The degassed solutions were used to prepare
individual reactions mixtures (final volume 30 μL). Reactions were
initiated by the addition of xanthine oxidase, wrapped with aluminum
foil to prevent exposure to light, and incubated for 16 h in the
glovebag at room temperature (24 °C). Following incubation, the
reactions were stopped by the addition of 3 μL of 50% glycerol loading
buffer, and the resulting mixture loaded onto 0.9% agarose gel. The gel
was electrophoresed for approximately 3 h at 80 V in 1 × Tris-acetate-
Myxin and its metabolites also were synthesized by an
alternative route (Scheme 3). This method involved partial
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DNA strand cleavage causes conversion of the supercoiled
plasmid to the open circular form.60−63 These two forms of
plasmid DNA can be separated using agarose gel electro-
phoresis, followed by staining with the DNA-binding dye
ethidium bromide. In these studies, we used xanthine/xanthine
oxidase for reductive activation of 7 and other compounds. This
enzyme system has been used for the activation of tirapazamine
in a number of in vitro studies.13,20,28−30,64−66 For reactions
carried out under hypoxic conditions, molecular oxygen was
removed from the solutions by freeze−pump−thaw degassing
and the assay mixtures prepared and incubated in an inert
atmosphere glovebag. Unless otherwise noted, catalase, super-
oxide dismutase, and desferal were added to prevent strand
cleavage stemming from the conversion of molecular oxygen to
the superoxide radical as described previously.29,65 To prevent
the potential photocleavage of DNA mediated by these
compounds,67,68 the reactions were shielded from light using
foil.
Scheme 3
demethylation of 1,6-dimethoxyphenazine by AlCl3 to give 1-
hydroxy-6-methoxyphenazine (8) in the first step. Compound
8 was then oxidized by m-CPBA to give the mono-N-oxide 9
and myxin (7). Although two mono-N-oxide isomers are
possible from the partial oxidation of 8, the reaction gave 9 as
the sole mono-N-oxide product. The position of N-oxidation in
9 was indicated by a diagnostic chemical shift for the phenolic
proton that appears as a sharp singlet at approximately 13 ppm
in CDCl3, presumably due to hydrogen bonding with the
oxygen of the N-oxide group.54 The fact that the “down” 5-
oxide 12 was not observed in the oxidation of 8 suggested that
the 1-hydroxy group may direct N-oxidation to the N10
position. We wished to prepare the 5-N-oxide analogue for use
as a standard for the in vitro metabolism experiments described
below. To enable selective oxidation at N5, the hydroxyl group
in 8 was protected with a t-butyldiphenylsilyl (TBDPS) group
(Scheme 4). This was intended to mask the directing effect of
Myxin was found to induce DNA cleavage in the presence of
the xanthine/xanthine oxidase enzyme system under anaerobic
conditions (Figure 1). The efficiency of DNA cleavage of myxin
Scheme 4
Figure 1. Efficiency of DNA cleavage of tirapazamine (1, TPZ) and
myxin (7) under anaerobic or aerobic conditions in the presence of the
xanthine/xanthine oxidase system. Supercoiled plasmid DNA (750 ng)
was incubated with 1 or 7, xanthine (500 μM), xanthine oxidase (0.4
U/mL), catalase (100 μg/mL), superoxide dismutase (10 μg/mL),
sodium phosphate buffer (50 mM, pH 7.0), and desferal (1 mM)
under anaerobic or aerobic conditions at room temperature for 16 h,
followed by agarose gel electrophoretic analysis. Strand breaks per
plasmid DNA molecule (S) was calculated using the equation S = −ln
f1, where f1 is the fraction of plasmid present as form I.
the hydroxyl group and also was expected to sterically hinder
oxidation at the N10 position. Accordingly, oxidation of the
TBDPS-protected compound 10 by m-CPBA gave the 5-N-
oxide 11 as the major product. Finally, deprotection of the
hydroxyl group by tetrabutylammonium fluoride (TBAF) gave
compound 12 in 26% overall yield. In contrast to the NMR of
9, the phenolic proton in 12 appears as a broad singlet at about
8 ppm versus tetramethylsilane (TMS).
DNA Cleavage by Myxin under Aerobic and
Anaerobic Conditions. We used a plasmid-based assay to
assess the ability of the myxin to cause bioreductively activated
DNA strand cleavage. This assay exploits the fact that oxidative
is comparable to that generated by the medicinally relevant N-
oxide, tirapazamine, under anaerobic conditions (Figure 1A).
Surprisingly, when DNA cleavage was performed under aerobic
conditions, myxin retained substantial DNA-cleaving power
(Figure 1B). In contrast, tirapazamine generates little or no
strand cleavage under aerobic conditions (Figure 1B).29
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Control reactions showed that xanthine + myxin (no enzyme),
myxin alone, or xanthine oxidase + xanthine (no myxin) did not
induce significant strand cleavage under these conditions
(Figure 2).
To better understand the mechanism of DNA strand
cleavage by myxin under both aerobic and anaerobic
conditions, the plasmid-based DNA-cleavage assay was
performed in the presence of various radical scavengers (Figure
2). DNA strand cleavage by tirapazamine and structurally
related analogues has been shown to proceed via a radical
mechanism, and addition of radical scavengers such as
methanol, ethanol, t-butanol, mannitol, and DMSO can quench
bioreductively activated DNA strand cleavage by these
drugs.13,29,30,65,66 Here, we find that addition of the radical
scavenging agents methanol, ethanol, t-butanol, mannitol, or
DMSO significantly decreased bioreductively activated strand
cleavage caused by myxin under both aerobic and anaerobic
conditions (Figure 2).
Interestingly, myxin causes DNA strand scission in the
presence of xanthine oxidase, even in the absence of the
enzyme’s usual electron source, xanthine (Figure 2A, lane 4).
Under anaerobic conditions, the xanthine-free strand cleavage
yields are clearly above background, although they are markedly
less than those resulting from the process that is driven by the
complete xanthine/xanthine oxidase enzyme system. A similar
phenomenon was observed previously for quinoxaline 1,4-di-N-
oxide.13 In that case, oxidation of the quinoxaline heterocycle
by xanthine oxidase generated reduced enzyme that, in turn,
carried out reductive activation of the 1,4-di-N-oxide. Thus,
some heterocyclic N-oxides are able to serve as substrates for
xanthine oxidase, in effect fueling their own reductive
activation. Xanthine oxidase is able to oxidize a variety of
heterocyclic nitrogen substrates,69−71 and it appears that myxin,
or adventitious traces of the mono-N-oxides derivatives, can
serve this role. The xanthine oxidase-catalyzed “self-fueled
activation” of myxin is more effective under anaerobic
conditions than it is under aerobic conditions presumably
because, under aerobic conditions, molecular oxygen competes
with the N-oxide to accept electrons from the reduced
enzyme.72 This enzyme-mediated, self-fueled activation process
was well characterized in the case of quinoxaline di-N-oxide13
but has not been studied in detail here.
Figure 2. DNA cleavage by myxin (7, 250 μM) under anaerobic (A)
and aerobic conditions (B) in the presence of various radical
scavengers. Xanthine/xanthine oxidase was used as a one-electron
reducing agent to activate the compounds. Supercoiled plasmid DNA
(750 ng) was incubated with 7, xanthine (500 μM), xanthine oxidase
(0.4 U/mL), catalase (100 μg/mL), superoxide dismutase (10 μg/
mL), sodium phosphate buffer (50 mM, pH 7.0), and desferal (1 mM)
under anaerobic (panel A) or aerobic conditions (panel B) in the
presence of various radical scavengers (500 mM) at room temperature
for 16 h, followed by analysis using agarose gel electrophoresis. Lane 1,
DNA alone; lane 2, DNA + xanthine/xanthine oxidase; lane 3, DNA +
7 + xanthine; lane 4, DNA + 7 + XO; lane 5, DNA + 7 + X/XO; lanes
6−10, DNA + 7 + X/XO + methanol, ethanol, t-butanol, DMSO (500
mM), and mannitol; and lane 11, DNA + 7. Strand breaks per plasmid
DNA molecule (S) was calculated using the equation S = −ln f1, where
f1 is the fraction of plasmid present as form I.
Figure 3. Aerobic DNA cleavage caused by redox cycling of menadione in the presence of xanthine/xanthine oxidase as an activating system can be
quenched by the presence of superoxide dismutase, catalase, and desferal. (A) Treatment of supercoiled plasmid DNA with menadione and
xanthine/xanthine oxidase. Numbers in parentheses following each lane description are the number of strand breaks, S, measured under each
condition, where S = −ln f1, where f1 is the fraction of plasmid present as form I.84 For lane 4, containing form III DNA, the number of strand breaks
was calculated using the equation form I + form II = [1 − S(2 h + 1)/(2 L)]S/2, where h is the maximum distance between breaks on opposing
strands that can yield linearized plasmid via spontaneous melting of the resulting sticky ends (16 bp), and L is the total number of base pairs in the
plasmid (5598, pGL2-basic).84 Lane 1, supercoiled plasmid DNA (750 ng) (0.1 0.1); lane 2, DNA + xanthine (500 μM) and xanthine oxidase (0.4
U/mL) (1.0 0.1); lane 3, DNA + menadione (250 μM) (0.02 0.01); lane 4, DNA + menadione (250 μM) + xanthine (500 μM) and xanthine
oxidase (0.4 U/mL) (7.6 5.3); lane 5, DNA + menadione (250 μM) + xanthine (500 μM) and xanthine oxidase (0.4 U/mL) + desferal (1 mM),
SOD (10 μg/mL), and catalase (100 μg/mL) (0.3 0.1). Assays were conducted in sodium phosphate buffer (pH 7.0, 50 mM) and were incubated
at room temperature for 16 h under aerobic conditions. (B) No significant strand cleavage above background was generated by the incubation of
DNA + menadione (0−500 μM) + xanthine (500 μM) and xanthine oxidase (0.4 U/mL) in the presence of desferal (1 mM), SOD (10 μg/mL), and
catalase (100 μg/mL) in sodium phosphate buffer (pH 7.0, 50 mM) incubated at room temperature for 16 h under aerobic conditions.
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In principle, the DNA strand cleavage observed for myxin
under aerobic conditions could result either from deoxygena-
tive metabolism processes or from redox cycling as shown on
the left side of Scheme 1 in the context of tirapazamine.20,21,66
Redox cycling has the potential to generate the superoxide
radical, hydrogen peroxide, and, ultimately, the DNA-cleaving
hydroxyl radical.73 However, it is important to reiterate that our
assays contained superoxide dismutase, catalase, and desferal
that are intended to squelch strand cleavage arising from redox-
cycling processes. Our early results suggested that these
additives did indeed perform the desired function. Specifically,
despite the fact that tirapazamine is known to undergo enzyme-
driven redox cycling under aerobic conditions,20,21,66 little
strand cleavage above background was observed in the aerobic
tirapazamine reactions (Figure 1). Nonetheless, the possibility
remained that myxin might be a more efficient redox cycling
agent that overwhelmed the ability of superoxide dismutase,
catalase, and desferal to protect against strand cleavage arising
via superoxide, hydrogen peroxide, and Fenton-type generation
of the hydroxyl radical. To investigate this issue, we examined
whether the additives employed in our assays were capable of
quenching superoxide-derived strand cleavage by the archetypal
redox-cycling agent menadione.74,75 First, we demonstrated
that menadione (250 μM), in the absence of the additives
superoxide dismutase, catalase, and desferal, but under
conditions otherwise identical to those used in the myxin
assays, caused complete nicking of the plasmid substrate along
with the generation of some extensively cleaved and linearized
form III plasmid (Figure 3A, lane 4). The xanthine/xanthine
oxidase enzyme system is known to generate strand cleavage via
one-electron reduction of O2 to the superoxide radical;72 but
strand cleavage by the menadione−xanthine/xanthine oxidase
combination was more than seven times greater than that
generated by the xanthine/xanthine oxidase enzyme system
alone (Figure 3A, lanes 2 and 4). We showed that the additives,
superoxide dismutase, catalase, and desferal effectively suppress
strand cleavage by the menadione−xanthine/xanthine oxidase
system (Figure 3A, lane 5 and Figure 3B). These control
experiments illustrate that superoxide dismutase, catalase, and
desferal effectively quench strand cleavage processes arising via
enzyme-driven redox-cycling processes. Taken together, the
results indicate that the bioreductively activated strand cleavage
observed for myxin under aerobic conditions likely does not
proceed via redox cycling processes that generate superoxide
radical.
Accordingly, we performed HPLC analysis of the metabolites
formed during reductive bioactivation of myxin by xanthine/
xanthine oxidase under aerobic conditions (Figure 4). We
Figure 4. HPLC trace of the products generated by in vitro
metabolism of myxin (7) in the presence of xanthine/xanthine
oxidase under aerobic conditions. In a typical assay, a solution of 7
(250 μM) in sodium phosphate buffer (pH 7.0, 50 mM) was
incubated with xanthine (250 μM) and xanthine oxidase (0.4 U/mL)
at room temperature for 3 h.
found that reductive metabolism of myxin by xanthine/xanthine
oxidase under aerobic conditions gave 9 as the major
metabolite (Scheme 5). The identity of the metabolite was
confirmed by coinjection with authentic synthetic standards.
Significant amounts of the other possible mono-N-oxide, 12
(Scheme 4), and the nor-oxide metabolite 8 were not detected
in these experiments. Interestingly, previous work showed that
electrochemical reduction of myxin similarly afforded 9 as the
sole mono-N-oxide product.76
DNA Cleavage by the Deoxygenated Myxin Ana-
logues 8, 9, and 12. The DNA-cleaving properties of the
deoxygenated analoguess of myxin were examined using the
plasmid-based DNA cleavage assay under aerobic conditions
using xanthine/xanthine oxidase as the one-electron activating
system (Figure 5). Under these conditions, the mono-N-oxide
12 and the nor-oxide metabolite, 1-hydroxy-6-methoxyphena-
Enzymatic Metabolism of Myxin. We anticipated that
examination of the products arising from in vitro aerobic
bioreductive metabolism of myxin would shed light on the
chemical mechanisms underlying DNA strand cleavage by the
natural product. Specifically, strand cleavage mechanisms
proceeding via deoxygenative mechanisms will generate
characteristic mono-N-oxide metabolites (Scheme 5). However,
redox cycling processes will not generate the deoxygenated,
reduced metabolites.
Figure 5. Aerobic DNA cleavage by myxin (7) and its deoxygenated
analogues 8, 9, and 12 in the presence of xanthine/xanthine oxidase as
a one-electron activating system. In a typical assay, a solution of the
compound (250 μM), DNA (750 ng), desferal (1 mM), SOD (10 μg/
mL), catalase (100 μg/mL), and sodium phosphate buffer (pH 7.0, 50
mM) was incubated with xanthine (500 μM) and xanthine oxidase
(0.4 U/mL) at room temperature for 16 h under aerobic conditions.
Strand breaks per plasmid DNA molecule (S) was calculated using the
equation S = −ln f1, where f1 is the fraction of plasmid present as form
I.
Scheme 5
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Article
zine (8), were found to cleave DNA, albeit less effectively than
myxin. No detectable cleavage was observed for the major
metabolite generated in the bioreductive activation of myxin,
the mono-N-oxide 9.
hypoxic cytotoxicity of tirapazamine. In vitro metabolism
studies showed that, under aerobic conditions, myxin under-
goes deoxygenative transformation to the mono-N-oxide
analogue following one-electron reduction in a manner
analogous to the anaerobic metabolism of tirapazamine and
other hypoxia-selective N-oxides.13,22,23,30,65,66
We have previously suggested a general mechanism involving
the release of hydroxyl radical to account for the bioreductively
activated DNA damage caused by hypoxia-selective heterocyclic
N-oxides (Scheme 5), but the exact nature of the DNA-
damaging species remains a subject of ongoing study.28−36 In
the context of tirapazamine, at least, the deoxygenative
metabolism stemming from one-electron reduction of aromatic
N-oxides is substantially more cytotoxic than the redox cycling
that occurs under aerobic conditions (hence the hypoxia-
selective cytotoxicity of this agent).66 It is interesting that the
deoxygenative metabolism of myxin produces only one of the
possible mono-N-oxide metabolites (9, Schemes 5 and 6).
Along these lines, it may be important to note that the
Cell Killing Assay under Aerobic and Anaerobic
Conditions. Unlike tirapazamine, myxin is able to carry out
bioreductively activated strand cleavage under aerobic con-
ditions. The strand cleaving ability of myxin under aerobic
conditions is comparable to that of tirapazamine under hypoxic
conditions. Together, these findings suggested that myxin
should possess cytotoxic activity comparable to that of
tirapazamine with the important distinction that myxin should
be active under both aerobic and anaerobic conditions. The
antibiotic properties of myxin have been characterized under
aerobic conditions,45,46 but the effects of oxygen on the
cytotoxicity of myxin have not previously been examined. In
addition, to the best of our knowledge, the activity of myxin
against human cancer cell lines has not been reported. With
these things in mind, we examined the activity of myxin against
the HCT-116 human colorectal cancer cell line under both
aerobic and hypoxic conditions. The activity of myxin was
compared to that of the hypoxia-selective agent tirapazamine.
We found that the activities of myxin (7) and tirapazamine (1)
against HCT-116 cells were comparable under hypoxic
conditions, with IC50 values of 4.2 and 1.8 μM, respectively.
The activity of myxin was similar under aerobic and anaerobic
conditions, with IC50 values of 1.8 and 4.2 μM, respectively.
The slightly superior activity of myxin under aerobic conditions
may reflect the ability of O2 to capture (or “fix”) DNA radical
intermediates more effectively than the N-oxide itself.77,78 In
contrast, tirapazamine was approximately 30-fold less cytotoxic
to the HCT-116 cells under aerobic conditions (Table 1). The
metabolite 9 produced by in vitro metabolism of myxin showed
Scheme 6
Table 1. Aerobic and Anaerobic Cytotoxicity of
Tirapazamine (1), Myxin (7), and the Myxin Metabolite 9
against Human Colorectal Cancer Cell Line HCT-116
deoxygenation reaction proceeds via the protonated N-oxide.
Importantly, protonation of the radical anion 13 seems more
likely to occur at the lower N-oxide (as drawn in Scheme 6)
rather than the upper oxide because hydrogen bonding with the
neighboring phenol likely renders the upper N-oxide of the
radical anion much less basic than the lower oxide. By way of
supporting analogy, the first pKa of 1,8-naphthalene diol is 6.6,
compared to 9.4 for 1-naphthol.79 It is also important to
consider structural reasons why the deoxygenative metabolism
of myxin can proceed in the presence of molecular oxygen.
Why is the radical anion 13 stable to molecular oxygen while
the radical anions derived from tirapazamine and at least some
other phenazine di-N-oxides are oxygen-sensitive?3,17−20,47,80 It
is possible that the radical anion 13 possesses some phenoxyl
radical character (structure C, Scheme 6) which lends
exceptional stability.81 Phenoxyl radicals do not react rapidly
with O2.82 Finally, it is interesting to point out that a recent
study identified other substituted phenazines with activity
against cancer cell lines under aerobic conditions and provided
evidence for the release of HO•; however, the mechanisms
underlying oxygen sensitivity in this series remain uncertain.80
This work may have some broad implications related to the
medicinal chemistry and toxicology of drug candidates that
contain nitrogen heterocycles. Nitrogen heterocycles are
common pharmacophores, and metabolism of these structures
in humans can generate aromatic N-oxide products.83 The
results presented here highlight the potential for aromatic N-
aerobic IC50
anaerobic IC50
hypoxic cytotoxicity
ratio
compd
(μM)
(μM)
1
7
9
58.6 5.5
1.75 0.17
60.4 4.6
1.77 0.15
4.2 0.6
225 25
33
2
0.44 0.07
0.29 0.05
relatively low aerobic cytotoxicity comparable to that of
tirapazamine (60.4 4.6 and 58.6 5.5 μM IC50 values,
respectively, Table 1). The cytotoxicity of 9 under anaerobic
conditions was even weaker, showing an IC50 value of 225 25
μM. This data suggests that 9 may engage in redox cycling
similar to that of tirapazamine under aerobic conditions but
that the radical anion is incapable of decomposing to release
cytotoxic species (e.g., hydroxyl radical) under either aerobic or
hypoxic conditions.
DISCUSSION
■
The results show that one-electron enzymatic reduction of
myxin leads to radical-mediated DNA strand cleavage under
both aerobic and anaerobic conditions. Strand cleavage by
myxin under both aerobic and hypoxic conditions is
comparable to that by the medicinally relevant compound
tirapazamine under hypoxic conditions. Likewise, myxin
displays activity against a human cancer cell line under both
aerobic and hypoxic conditions that is comparable to the
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Chemical Research in Toxicology
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(12) Vicente, E., Villar, R., Burguete, A., Solano, B., Per
́
ez-Silanes, S.,
oxides to undergo highly cytotoxic deoxygenative metabolism
under aerobic conditions. Thus, cycles of N-oxidation and
bioreductively activated one-electron deoxygenation could lead
to hepatic or systemic toxicity for some nitrogen heterocycles.
Further studies are necessary to elucidate the structural features
that enable both one-electron bioreductive activation of
aromatic N-oxides and their subsequent conversion to cytotoxic
reactive intermediates in normal aerobic tissue.
Aldana, I., Maddry, J. A., Lenaerts, A. J., Franzblau, S. G., Cho, S.-h.,
Monge, A., and Goldman, R. C. (2008) Efficacy of quinoxaline-2-
carboxylate 1,4-di-N-oxide derivatives in experimental tuberculosis.
Antimicrob. Agents Chemother. 52, 3321−3326.
(13) Ganley, B., Chowdhury, G., Bhansali, J., Daniels, J. S., and Gates,
K. S. (2001) Redox-activated, hypoxia-selective DNA cleavage by
quinoxaline 1,4-di-N-oxide. Bioorg. Med. Chem. 9, 2395−2401.
(14) McIlwain, H. (1943) Bacterial inhibition by metabolite
analogues. Part V. Reactions and antibacterial properties of p-diazine
di-N-oxides. J. Chem. Soc. No. part I, 322−327.
(15) Hennessey, T. D., and Edwards, J. R. (1972) Antibacterial
properties of quindoxin: a new growth promoting agent. Vet. Rec. 90,
187−190.
(16) Suter, W., Rosselet, A., and Knusel, F. (1978) Mode of action of
quindoxin and substituted quinoxaline-di-N-oxides on E. coli.
Antimicrob. Agents Chemother. 13, 770−783.
(17) Priyadarsini, K. I., Tracy, M., and Wardman, P. (1996) The one-
electron reduction potential of 3-amino-1,2,4-benzotriazine 1,4-dioxide
(tirapazamine): A hypoxia-selective bioreductive drug. Free Radical Res.
25, 393−399.
(18) Lloyd, R. V., Duling, D. R., Rumyantseva, G. V., Mason, R. P.,
and Bridson, P. K. (1991) Microsomal reduction of 3-amino-1,2,4,-
benzotriazine 1,4-dioxide to a free radical. Mol. Pharmacol. 40, 440−
445.
AUTHOR INFORMATION
Corresponding Author
*Phone: (573) 882-6763. Fax: (573) 882-2754. E-mail:
gatesk@missouri.edu.
■
ACKNOWLEDGMENTS
We thank Chad Inman and Zachary Parsons for experimental
assistance.
■
REFERENCES
■
(1) Xia, Q., Zhang, L., Zhang, J., Sheng, R., Yang, B., He, Q., and Hu,
Y. (2011) Synthesis, hypoxia-selective cytotoxicity of new 3-amino-
1,2,4-benzotriazine 1,4-dioxide derivatives. Eur. J. Med. Chem. 46, 919−
926.
(19) Wardman, P., Priyadarsini, K. I., Dennis, M. F., Everett, S. A.,
Naylor, M. A., Patel, K. B., Stratford, I. J., Stratford, M. R. L., and
Tracy, M. (1996) Chemical properties which control selectivity and
efficacy of aromatic N-oxide bioreductive drugs. Br. J. Cancer 74, S70−
S74.
(20) Laderoute, K. L., Wardman, P., and Rauth, M. (1988) Molecular
mechanisms for the hypoxia-dependent activation of 3-amino-1,2,4-
benzotriazine 1,4-dioxide (SR4233). Biochem. Pharmacol. 37, 1487−
1495.
(21) Silva, J. M., and O’Brien, P. J. (1993) Molecular mechanisms of
SR 4233-induced hepatocyte toxicity under aerobic versus hypoxic
conditions. Br. J. Cancer 68, 484−491.
(22) Laderoute, K., and Rauth, A. M. (1986) Identification of two
major reduction products of the hypoxic cell toxin 3-amino-1,2,4-
benzotriazine-1,4-dioxide. Biochem. Pharmacol. 35, 3417−3420.
(23) Walton, M. I., and Workman, P. (1993) Pharmacokinetics and
bioreductive metabolism of the novel benzotriazine di-N-oxide hypoxic
cell cytotoxin tirapazamine (WIN 59075, SR 4233, NSC 130181) in
mice. J. Pharmacol. Exp. Ther. 265, 938−947.
(24) Zeman, E. M., Brown, J. M., Lemmon, M. J., Hirst, V. K., and
Lee, W. W. (1986) SR 4233: a new bioreductive agent with high
selective toxicity for hypoxic mammalian cells. Int. J. Radiat. Oncol. Biol.
Phys. 12, 1239−1242.
(25) Zeman, E. M., Baker, M. A., Lemmon, M. J., Pearson, B. A.,
Adams, J. A., Brown, J. M., Lee, W. W., and Tracy, M. (1989)
Structure-activity relationships for benzotriazine di-N-oxides. Int. J.
Radiat. Oncol. Biol. Phys 16, 977−981.
(26) Brown, J. M. (1993) SR4233 (Tirapazamine): a new anticancer
drug exploiting hypoxia in solid tumours. Br. J. Cancer 67, 1163−1170.
(27) Evans, J. W., Chernikova, S. B., Kachnic, L. A., Banath, J. P.,
Sordet, O., Delahoussaye, Y. M., Treszezamsky, A., Chon, B. H., Feng,
Z., Gu, Y., Wilson, W. R., Pommier, Y., Olive, P. L., Powell, S. N., and
Brown, J. M. (2008) Homologous recombination is the principal
pathway for the repair of DNA damage induced by tirapazamine in
mammalian cells. Cancer Res. 68, 257−265.
(28) Birincioglu, M., Jaruga, P., Chowdhury, G., Rodriguez, H.,
Dizdaroglu, M., and Gates, K. S. (2003) DNA base damage by the
antitumor agent 3-amino-1,2,4-benzotriazine 1,4-dioxide
(tirapazamine). J. Am. Chem. Soc. 125, 11607−11615.
(2) Ismail, M. M. F., Amin, K. M., Naoman, E., Soliman, D. H., and
Ammar, Y. A. (2010) New quinoxaline 1,4-dioxides: anticancer and
hypoxia-selective agents. Eur. J. Med. Chem. 45, 2733−2738.
́
(3) Lavaggi, M. L., Cabrera, M., Gonzalez, M., and Cerecetto, H.
(2008) Differential enzymatic reductions governing the differential
hypoxia-selective cytotoxicities of phenazine 5,10-dioxides. Chem. Res.
Toxicol. 21, 1900−1906.
(4) Hicks, K. O., Siim, B. G., Jaiswal, J. K., Pruijin, F. B., Fraser, A. M.,
Patel, R., Hogg, A., Liyanage, H. D. S., Dorie, M. J., Brown, J. M.,
Denny, W. A., Hay, M. P., and Wilson, W. R. (2010) Pharmacokinetic/
pharmacodynamic modeling identifies SN30000 and SN29751 as
tirapazamine analogues with improved tissue penetration and hypoxic
cell killing in tumors. Clin. Cancer Res. 16, 4946−4957.
(5) Hay, M. P., Gamage, S. A., Kovacs, M. S., Pruijn, F. B., Anderson,
R. F., Patterson, A. V., Wilson, W. R., Brown, J. M., and Denny, W. A.
(2003) Structure-activity relationships of 1,2,4-benzotriazine 1,4-
dioxides as hypoxia-selective analogues of tirapazamine. J. Med.
Chem. 46, 169−182.
(6) Hay, M. P., Hicks, K. O., Pchalek, K., Lee, H. H., Blaser, A.,
Pruijn, F. B., Anderson, R. F., Shinde, S. S., Wilson, W. R., and Denny,
W. A. (2008) Tricyclic [1,2,4]triazine 1,4-dioxides as hypoxia selective
cytotoxins. J. Med. Chem. 51, 6853−6865.
(7) Solano, B., Junnotula, V., Marin, A., Villar, R., Burguete, A.,
Vicente, E., Perez-Silanes, S., Monge, A., Dutta, S., Sarkar, U., and
Gates, K. S. (2007) Synthesis and biological evaluation of new 2-
arylcarbonyl-3-trifluoromethylquinoxaline 1,4-dioxide derivatives and
their reduced analogues. J. Med. Chem. 50, 5485−5492.
(8) Wilson, W. R., and Hay, M. P. (2011) Targeting hypoxia in
cancer therapy. Nat. Rev. Cancer 11, 393−409.
(9) Marcu, L., and Olver, I. (2006) Tirapazamine: from the bench to
clinical trials. Curr. Clin. Oncol. 1, 71−79.
(10) Rischin, D., Hicks, R. J., Fisher, R., Binns, D., Corry, J.,
Porceddu, S., and Peters, L. J. (2006) Prognostic significance of [18F]-
misonidazole positron emission tomography−detected tumor hypoxia
in patients with advanced head and neck cancer randomly assigned to
chemoradiation with or without tirapazamine: a substudy of Trans-
Tasman Radiation Oncology Group Study 98.02. J. Clin. Oncol. 24,
2098−2104.
(11) Rischin, D., Peters, L. J., O’Sullivan, B., Giralt, J., Fisher, R.,
Yuen, K., Trotti, A., Bernier, J., Bourhis, J., Ringash, J., Henke, M., and
Kenny, L. (2010) Tirapazamine, cisplatin, and radiation versus
cisplatin and radiation for advanced squamous cell carcinoma of the
head and neck (TROG 02.02, HeadSTART): a phase III trial of the
trans-tasman radiation oncology group. J. Clin. Oncol. 28, 2989−2995.
(29) Daniels, J. S., and Gates, K. S. (1996) DNA cleavage by the
antitumor agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (SR4233):
evidence for involvement of hydroxyl radical. J. Am. Chem. Soc. 118,
3380−3385.
204
dx.doi.org/10.1021/tx2004213 | Chem. Res. Toxicol. 2012, 25, 197−206

Chemical Research in Toxicology
Article
(30) Junnotula, V., Sarkar, U., Sinha, S., and Gates, K. S. (2009)
Initiation of DNA strand cleavage by 1,2,4-benzotriazine 1,4-dioxides:
mechanistic insight from studies of 3-methyl-1,2,4-benzotriazine 1,4-
dioxide. J. Am. Chem. Soc. 131, 1015−1024.
(31) Chowdhury, G., Junnutula, V., Daniels, J. S., Greenberg, M. M.,
and Gates, K. S. (2007) DNA strand damage analysis provides
evidence that the tumor cell-specific cytotoxin tirapazamine produces
hydroxyl radical and acts as a surrogate for O2. J. Am. Chem. Soc. 129,
12870−12877.
(32) Shinde, S. S., Anderson, R. F., Hay, M. P., Gamage, S. A., and
Denny, W. A. (2004) Oxidation of 2-deoxyribose by benzotriazinyl
radicals of antitumor 3-amino-1,2,4-benzotriazine 1,4-dioxides. J. Am.
Chem. Soc. 126, 7865−7874.
(48) Behki, R. M., and Lesley, S. M. (1972) Deoxyribnucleic acid
degradation and the lethal effect by myxin in E. coli. J. Bacteriol. 109,
250−261.
(49) Hollstein, U., and Butler, P. L. (1972) Inhibition of ribonucleic
acid synthesis by myxin. Biochemistry 11, 1345−1350.
(50) Hartman, P. E., Berger, H., and Hartman, Z. (1973)
Comparison of hycanthone(Etrenol), some hycanthone analogs,
myxin, and 4-nitroquinoline 1-oxide as frameshift mutagens. J.
Pharmacol. Exp. Ther. 186, 390−398.
(51) Hollstein, U., and Van Gemert, R. J. J. (1971) Interaction of
phenazines with polydeoxyribonucleotides. Biochemistry 10, 497−504.
(52) Lesley, S. M., and Behki, R. M. (1967) Mode of action of myxin
on Escherichia coli. J. Bacteriol. 94, 1837−1845.
(33) Shinde, S. S., Hay, M. P., Patterson, A. V., Denny, W. A., and
Anderson, R. F. (2009) Spin trapping of radicals other than the
hydroxyl radical upon reduction of the anticancer agent tirapazamine
by cytochrome P450 reductase. J. Am. Chem. Soc. 131, 14220−14221.
(34) Shinde, S. S., Maroz, A., Hay, M. P., Patterson, A. V., Denny, W.
A., and Anderson, R. F. (2010) Characterization of radicals formed
following enzymatic reduction of 3-substituted analogues of the
hypoxia-selective cytotoxin 3-amino-1,2,4-benzotriazine 1,4-dioxide
(tirapazamine). J. Am. Chem. Soc. 132, 2591−2599.
(35) Anderson, R. F., Shinde, S. S., Hay, M. P., Gamage, S. A., and
Denny, W. A. (2003) Activation of 3-amino-1,2,4-benzotriazine 1,4-
dioxide antitumor agents to oxidizing species following their one-
electron Reduction. J. Am. Chem. Soc. 125, 748−756.
(36) Anderson, R. F., Shinde, S. S., Hay, M. P., Gamage, S. A., and
Denny, W. A. (2005) Radical properties governing the hypoxia-
selective cytotoxicity of antitumor 3-amino-1,2,4-benzotriazine 1,4-
dioxides. Org. Biomol. Chem. 3, 2167−2174.
(37) Fuchs, T., Chowdhary, G., Barnes, C. L., and Gates, K. S. (2001)
3-Amino-1,2,4-benzotriazine 4-oxide: characterization of a new
metabolite arising from bioreductive processing of the antitumor
agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine). J. Org.
Chem. 66, 107−114.
(38) Terao, M. (1963) On a new antibiotic, emimycin. II. On the
structure of emimycin. J. Antibiot. (Tokyo), Sec. A 16, 182−186.
(39) Ohmoto, T., Tanaka, R., and Nikaido, T. (1976) Studies on the
constituents of Ailanthus altissima Swingle. On the alkaloidal
constituents. Chem. Pharm. Bull. 24, 1532−1536.
(40) Antkowiak, W. Z., and Gessner, W. P. (1979) The structure of
orellanine and orelline. Tetrahedron Lett. 20, 1931−1934.
(41) O’Donnell, G., Poeschl, R., Zimhony, O., Gunaratnam, M.,
Moreira, J. B. C., Neidle, S., Evangelopoulos, D., Bhakta, S., Malkinson,
J. P., Boshoff, H. I., Lenaerts, A., and Gibbons, S. (2009) Bioactive
pyridine-N-oxide disulfides from Allium stipitatum. J. Nat. Prod. 72,
360−365.
(53) Pachter, I. J., and Kloetzel, M. C. (1951) The Wohl-Aue
reaction. I. Structure of benzo [a] phenazine oxides and syntheses of
1,6-dimethoxyphenazine and 1,6-dichlorophenazine. J. Am. Chem. Soc.
73, 4958−4961.
(54) Weigele, M., and Leimgruber, W. (1967) The structure of
myxin. Tetrahedron Lett. 8, 715−718.
(55) Edwards, O. E., and Gillespie, D. C. (1966) N-Dioxides, a new
class of amine oxides. Tetrahedron Lett. 40, 4867−4870.
(56) Bauer, W., and Vinograd, J. (1968) The interaction of closed
circular DNA with intercalative dyes. I. The superhelix density of SV40
DNA in the presence and absence of dye. J. Mol. Biol. 33, 141−171.
(57) Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J.,
Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., and Boyd, M. R.
(1990) New colorimetric cytotoxicity assay for anticancer-drug
screening. J. Natl. Cancer Inst. 82, 1107−1112.
̈
(58) Wohl, A., and Aue, W. (1901) Uber die Einwirkung von
Nitrobenzol auf Anilin bei Gegenwart von Alkali. Chem. Ber. 34,
2442−2450.
(59) Clemo, G. R., and Daglish, A. F. (1950) The constitution of the
pigment of Chromobacterium iodinum. J. Chem. Soc., 1481−1485.
(60) Jonson, P. H., and Grossman, L. I. (1977) Electrophoresis of
DNA in agarose gels. Optimizing separations of conformational
isomers of double and single-stranded DNAs. Biochemistry 16, 4217−
4224.
(61) Mirabelli, C. K., Huang, C. H., Fenwick, R. G., and Crooke, S. T.
(1985) Quantitative measurement of single- and double-strand
breakage of DNA in Escherichia coli by the antitumor antibiotics
bleomycin and talisomycin. Antimicrob. Agents Chemother. 27, 460−
467.
(62) Oppenheim, A. (1981) Separation of closed circular DNA from
linear DNA by electrophoresis in two dimensions in agarose gels.
Nucleic Acids Res. 9, 6805−6812.
(63) Hintermann, G., Fischer, H. M., Crameri, R., and Hutter, R.
(1981) Simple procedure for distinguishing CCC, OC, and L forms of
plasmid DNA by agarose gel electrophoresis. Plasmid 5, 371−373.
(64) Jones, G. D. D., and Weinfeld, M. (1996) Dual action of
tirapazamine in the induction of DNA strand breaks. Cancer Res. 56,
1584−1590.
(65) Chowdhury, G., Kotandeniya, D., Barnes, C. L., and Gates, K. S.
(2004) Enzyme-activated, hypoxia-selective DNA damage by 3-amino-
2-quinoxalinecarbonitrile 1,4-di-N-oxide. Chem. Res. Toxicol. 17,
1399−1405.
(66) Junnotula, V., Rajapakse, A., Abrillaga, L., Lopez de Cerain, A.,
Solano, B., Villar, R., Monge, A., and Gates, K. S. (2010) DNA strand
cleaving properties and hypoxia-selective cytotoxicity of 7-chloro-2-
thienylcarbonyl-3-trifluoromethylquinoxaline 1,4-dioxide. Bioorg. Med.
Chem. 18, 3125−3132.
(67) Fuchs, T., Gates, K. S., Hwang, J.-T., and Greenberg, M. M.
(1999) Photosensitization of guanine-specific DNA damage by a
cyano-substituted quinoxaline di-N-oxide. Chem. Res. Toxicol. 12,
1190−1194.
(68) Daniels, J. S., Chatterji, T., MacGillivray, L. R., and Gates, K. S.
(1998) Photochemical DNA cleavage by the antitumor agent 3-amino-
1,2,4-benzotriazine 1,4-dioxide (Tirapazamine, WIN 59075, SR4233).
J. Org. Chem. 63, 10027−10030.
(42) Nicholas, G. M., Blunt, J. W., and Munro, M. H. G. (2001)
Cortamidine oxide, a novel disulfide metabolite from the New Zealand
Basidiomycete (mushroom) Cortinarius species. J. Nat. Prod. 64, 341−
344.
(43) Nishioka, H., Imoto, M., Imaoka, T., Sawa, T., Takeuchi, T., and
Umezawa, K. (1994) Antitumor effect of piericidin B1 N-oxide. J.
Antibiot. 47, 447−452.
(44) Peterson, E. A., Gillespie, D. C., and Cook, F. D. (1966) A wide
spectrum antibiotic produced by a species of Sorangium. Can. J.
Microbiol. 12, 221−230.
(45) Weigele, M., Maestrone, G., Mitrovic, M., and Leimgruber, W.
(1970) Antimicrobial agents structurally related to myxin. Antimicrob.
Agents Chemother. 10, 46−49.
(46) Lesley, S. M., and Behki, R. M. (1973) Sensitivity to myxin of
Escherichia coli treated with ethylenediaminetetraacetic acid. Can. J.
Microbiol. 19, 531−533.
́
(47) Lavaggi, M. L., Cabrera, M., de los Angeles Aravena, M., Olea-
Azar, C., Lopez de Cerain, A., Monge, A., Pachon, G., Cascante, M.,
Bruno, A. M., Pietrasanta, L. I., Gonzalez, M., and Cerecetto, H.
(2010) Study of benzo[a]phenazine 7,12-dioxide as selective hypoxic
cytotoxin-scaffold. Identification of aerobic-antitumoral activity
through DNA fragmentation. Bioorg. Med. Chem. 18, 4433−4440.
205
dx.doi.org/10.1021/tx2004213 | Chem. Res. Toxicol. 2012, 25, 197−206

Chemical Research in Toxicology
Article
(69) Rastelli, G., Costantino, L., and Albasini, A. (1997) A model of
the interaction of substrates and inhibitors with xanthine oxidase. J.
Am. Chem. Soc. 119, 3007−3016.
(70) McCormack, J. J., Allen, B. A., and Hodnett, C. N. (1978)
Oxidation of quinazoline and quinoxaline by xanthine oxidase and
aldehyde oxidase. J. Heterocyclic Chem. 15, 1249−1254.
(71) Krenitsky, T. A., Neil, S. M., Elion, G. B., and Hitchings, G. H.
(1972) A comparison of the specificities of xanthine oxidase and
aldehyde oxidase. Arch. Biochem. Biophys. 150, 585−599.
(72) Brawn, K., and Fridovich, I. (1981) DNA strand scission by
enzymatically generated oxygen radicals. Arch. Biochem. Biophys. 206,
414−419.
(73) Halliwell, B., and Gutteridge, J. M. C. (1990) Role of free
radicals and catalytic metal ions in human disease: an overview.
Methods Enzymol. 186, 1−85.
(74) Nakamura, M. (1991) Allopurinol-insensitive oxygen radical
formation by milk xanthine oxidase systems. J. Biochem. 110, 450−456.
(75) Yee, S. B., and Pritsos, C. A. (1997) Comparison of oxygen
radical generation from the reductive activation of doxorubicin,
streptonigrin, and menadione by xanthine oxidase and xanthine
dehydrogenase. Arch. Biochem. Biophys. 347, 235−241.
(76) Donahue, J. J., and Oliveri-Vigh, S. (1973) Electrochemical
characterization of 6-methoxy-1-phenazinol-5,10-dioxide. Anal. Chim.
Acta 63, 415−425.
(77) Daniels, J. S., Gates, K. S., Tronche, C., and Greenberg, M. M.
(1998) Direct evidence for bimodal DNA damage induced by
tirapazamine. Chem. Res. Toxicol. 11, 1254−1257.
(78) Hwang, J.-T., Greenberg, M. M., Fuchs, T., and Gates, K. S.
(1999) Reaction of the hypoxia-selective antitumor agent tirapazamine
with a C1′-radical in single-stranded and double-stranded DNA: the
drug and its metabolites can serve as surrogates for molecular oxygen
in radical-mediated DNA damage reactions. Biochemistry 38, 14248−
14255.
(79) Forconi, M., and Williams, N. H. (2002) Mimicking
metallophosphatases: revealing a role for an OH group with no
libido. Angew. Chem., Int. Ed. Engl. 41, 849−852.
́
(80) Lavaggi, M. L., Cabrera, M., Pintos, C., Arredondo, C., Pachon,
G., Robríquez, J., Raymondo, S., Pacheco, J. P., Cascante, M., Olea-
Azar, C., Lopez de Cerain, A., Monge, A., Cerecetto, H., and Gonzalez,
́
́
M. (2011) Novel phenazine 5,10-dioxides release •OH in simulated
hypoxia and induce reduction of tumour volume in vivo. ISRN
Pharmacol., DOI: 10.5402/2011/314209.
(81) Minisci, F., Punta, C., Recupero, F., Fontana, F., and Pedulli, G.
F. (2002) Aerobic oxidation of N-alkylamides catalyzed by N-
hydroxyphthalimide under mild conditions. Polar and enthalic
effects. J. Org. Chem. 67, 2671−2676.
(82) Hunter, E. P. L, Derosiers, M. F., and Simic, M. G. (1989) The
effect of oxygen, antioxidants, and superoxide radical on tyrosine
phenoxyl radical dimerization. Free Radical Biol. Med. 6, 581−585.
(83) Guengerich, F. P. (2001) Common and uncommon cytochrome
P450 reactions related to metabolism and chemical toxicity. Chem. Res.
Toxicol. 14, 611−650.
(84) Hertzberg, R. P., and Dervan, P. B. (1984) Cleavage of DNA
with methidiumpropyl-EDTA-Iron(II): Reaction conditions and
product analyses. Biochemistry 23, 3934−3945.
206
dx.doi.org/10.1021/tx2004213 | Chem. Res. Toxicol. 2012, 25, 197−206