Isotopic labeling experiments that elucidate the mechanism of DNA strand cleavage by the hypoxia-selective antitumor agent 1,2,4-benzotriazine 1,4-Di- N -oxide

Article abstract of DOI:10.1021/tx400356y

The 1,2,4-benzotriazine 1,4-dioxides are an important class of potential anticancer drugs that selectively kill the low-oxygen (hypoxic) cells found in solid tumors. These compounds undergo intracellular one-electron enzymatic reduction to yield an oxygen-sensitive drug radical intermediate that partitions forward, under hypoxic conditions, to generate a highly reactive secondary radical that causes cell killing DNA damage. Here, we characterized bioreductively activated, hypoxia-selective DNA-strand cleavage by 1,2,4-benzotriazine 1,4-dioxide. We found that one-electron enzymatic activation of 1,2,4-benzotriazine 1,4-dioxide under hypoxic conditions in the presence of the deuterium atom donor methanol-d₄ produced nondeuterated mono-N-oxide metabolites. This and the results of other isotopic labeling studies provided evidence against the generation of atom-abstracting drug radical intermediates and are consistent with a DNA-damage mechanism involving the release of hydroxyl radical from enzymatically activated 1,2,4-benzotriazine 1,4-dioxides.

Full text of DOI:10.1021/tx400356y

Article
pubs.acs.org/crt
Isotopic Labeling Experiments That Elucidate the Mechanism of DNA
Strand Cleavage by the Hypoxia-Selective Antitumor Agent 1,2,4-
Benzotriazine 1,4-Di‑N‑oxide
Xiulong Shen,† Anuruddha Rajapakse,† Fabio Gallazzi,§ Venkatraman Junnotula,† Tarra Fuchs-Knotts,†
Rainer Glaser,† and Kent S. Gates*,†,‡
†Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, United States
‡Department of Biochemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, United States
§Structural Biology Core, University of Missouri, Columbia, Missouri 65211, United States
S
* Supporting Information
ABSTRACT: The 1,2,4-benzotriazine 1,4-dioxides are an important class of
potential anticancer drugs that selectively kill the low-oxygen (hypoxic) cells
found in solid tumors. These compounds undergo intracellular one-electron
enzymatic reduction to yield an oxygen-sensitive drug radical intermediate that
partitions forward, under hypoxic conditions, to generate a highly reactive secondary
radical that causes cell killing DNA damage. Here, we characterized bioreductively
activated, hypoxia-selective DNA-strand cleavage by 1,2,4-benzotriazine 1,4-dioxide. We found that one-electron enzymatic
activation of 1,2,4-benzotriazine 1,4-dioxide under hypoxic conditions in the presence of the deuterium atom donor methanol-d4
produced nondeuterated mono-N-oxide metabolites. This and the results of other isotopic labeling studies provided evidence
against the generation of atom-abstracting drug radical intermediates and are consistent with a DNA-damage mechanism
involving the release of hydroxyl radical from enzymatically activated 1,2,4-benzotriazine 1,4-dioxides.
Scheme 1
INTRODUCTION
■
In cancer chemotherapy, there is a continuing need for the
development of drugs that selectively target malignant tissue.1
Toward this end, the poorly developed vascular system in
tumors creates a low oxygen environment that may be exploited
by some small molecules for the selective killing of cancer
cells.2 The 1,2,4-benzotriazine 1,4-di-N-oxides are an important
class of hypoxia-selective cytotoxins.2−7 Tirapazamine (1a) is
the lead compound in this class of drugs, but many analogues in
this structural family display similar bioactivities,3−6 and the
relatively new agent SN30000/CEN-209 (4) is advancing
toward clinical development.8,9
accounts for the DNA-damaging properties and the hypoxia-
selective cytotoxicity of many different heterocyclic N-oxides
including 1,2,4-benzotriazine 1,4-dioxides,21,24−26,29 quinoxaline
1,4-dioxides,30−33 1,2-dihydro-4-phenylimidazo[1,2-a]pyrido-
The medicinal action of tirapazamine and other 1,2,4-
benzotriazine 1,4-dioxides involves intracellular one-electron
enzymatic reduction to yield an oxygen-sensitive drug radical
intermediate (2).10−13 In normally oxygenated tissue, 2 is
simply oxidized by O2 back to the parent compound 1.13,14
However, under hypoxic conditions, the radical intermediate 2
partitions forward to generate a highly reactive secondary
radical that kills cells via abstraction of hydrogen atoms from
the 2′-deoxyribose phosphate backbone of DNA.15−22 The
mono-N-oxide 3 is the major drug metabolite generated in this
process.15,16,23 The exact nature of the ultimate DNA-damaging
radical generated following bioreductive activation of the 1,2,4-
benzotriazine 1,4-dioxides remains a topic of ongoing study,
with two different possibilities considered in the recent
literature. First, evidence has been presented that is consistent
with the release of hydroxyl radical (HO·) from the protonated
drug radical 2 (Scheme 1).21,24−28 This mechanism adequately
[3,2-e]pyrazine 5-oxides,34 and phenazine 5,10-dioxides.35
A
second mechanistic proposal suggests that the tirapazamine
radical 2 can undergo dehydration to generate a highly reactive
benzotriazinyl radical 5 that abstracts hydrogen atoms from
DNA.36−40 To account for the bioactivities of other 1,2,4-
benzotriazine 1,4-dioxide analogues such as 1b, the dehydration
mechanism was expanded to include a family of hypothetical
processes that generate aryl radical intermediates such as 6.38
Isotopic labeling experiments have the potential to
distinguish which of these two mechanistic pathways is
Received: September 27, 2013
Published: December 11, 2013
© 2013 American Chemical Society
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3.3% v/v), and desferal (deferoxamine mesylate salt, 10 mM) under
anaerobic conditions at 25 °C for 4 h. All components of the reactions
except enzymes, NADPH, and DNA were degassed by three freeze−
pump−thaw cycles. Enzymes, NADPH, and DNA were diluted with
degassed water in an argon-filled glovebag to prepare stock solutions.
Reactions were initiated by the addition of cytochrome P450
reductase, wrapped in aluminum foil to prevent exposure to light,
and incubated in an argon-filled glovebag. Following incubation, the
reactions were quenched by the addition of 4 μL of loading buffer
containing bromophenol blue dye and were loaded onto a 0.9%
agarose gel. The gel was electrophoresed for approximately 2 h at 83 V
in 1 × TAE buffer. The gel was then stained in a solution of aqueous
ethidium bromide (0.5 μg/mL) for 15 min, destained in fresh distilled
water for 15 min, and the DNA in the gel visualized by UV-
transillumination. The amount of DNA in each band was quantified
using an Alpha Innotech IS-1000 digital imaging system. Buffers for
the reactions performed in D2O were prepared using the same mixture
of phosphate buffer salts used in the H2O experiments, as described by
others.45,46
operative. For example, the proposed generation of the aryl
radical 6 from 2b in the presence of a deuterium atom donor
would yield the corresponding deuterated drug metabolite
3c,41,42 whereas release of the hydroxyl radical from 2b will
produce the nondeuterated metabolite 3b. Here, we report the
results of such isotopic labeling studies carried out in the
context of compound 1b. We reasoned that 1b was a good
subject for mechanistic studies because this agent displays
hypoxia-selective cytotoxicity comparable to that of tirapaz-
amine (1a)3 and has been proposed to generate the DNA-
damaging aryl radical 6.38 However, the ability of 1b to carry
out in vitro reductively activated, hypoxia-selective DNA strand
cleavage in a manner similar to that of tirapazamine had not
been previously demonstrated. Therefore, before undertaking
isotopic labeling studies, we characterized the DNA-damaging
properties of 1b.13,21,25,43 We found that 1b displays
bioreductively activated, hypoxia-selective DNA-cleaving prop-
erties analogous to those of the lead compound tirapazamine
(1a). The results of several different isotopic labeling studies
provided evidence against the generation of atom-abstracting
drug radicals such as 6 following one-electron enzymatic
activation of 1b under hypoxic conditions.
Synthesis of 4-Deutero-1,2,4-benzotriazine 1,4-Dioxide (1c).
In a 25 mL round-bottomed flask, tert-butyl nitrite (0.25 mL, 2.10
mmol) was added to DMF-d7 (2 mL) and heated to 65 °C. To this
mixture, a solution of 1a (100 mg) in DMF-d7 (2 mL) was added
dropwise over the course of 15 min. The reaction was monitored by
thin layer chromatography every 10 min until all starting material was
consumed. The reaction mixture was then cooled to room
temperature, and the DMF-d7 was removed by high vacuum. Column
chromatography on silica gel eluted with 1:1 ethyl acetate−hexane
gave 1c as a yellow solid (23 mg, 25%). Rf 0.60 (100% ethyl acetate).
1H NMR (DMSO, 500 MHz,): δ 8.39 (d, J = 8.5 Hz, 1H), 8.36 (d, J =
8.5 Hz, 1H), 8.14 (ddd, J = 8.5, 7.5, 1.0 Hz, 1H), 8.10 (ddd, J = 8.5,
7.5, 1.0 Hz, 1H). 13C NMR (DMSO, 125.8 MHz): δ 142.2 (t, J = 33.3
Hz), 141.0, 136.0, 135.6, 133.3, 121.5, 119.4. HRMS (ES+, [M + H])
m/z calcd C7H5DN3O2 calcd mass, 165.0523; actual mass, 165.0515.
Synthesis of 1,2,4-Benzotriazine (7). Benzotriazine 7 was
prepared via a modification of the procedures described by Fuchs et
al. and Mason and Tennant.23,47 To a solution of 3b (300 mg, 2.04
mmol) in 70% ethanol−water (40 mL) was added sodium dithionite
(710 mg, 4.08 mmol). The resulting suspension was refluxed for 2 h,
an additional aliquot of sodium dithionite (305 mg, 2.04 mmol) added,
and the suspension refluxed for another 30 min, at which time all
starting material was consumed (as judged by TLC). The solvent was
removed by rotary evaporation and the resulting pale yellow residue
purified by column chromatography on silica gel eluted with 30% ethyl
acetate−hexane to yield 7 as a pale yellow solid (154 mg, 58%). Rf =
MATERIALS AND METHODS
■
Materials. Materials were of the highest purity available and were
obtained from the following sources: Sephadex G-25, cytochrome
P450 reductase, NADPH, sodium phosphate, mannitol, DMSO,
desferal, catalase, superoxide dismutase (SOD), tert-butyl nitrite, silica
gel (0.04−0.063 mm pore size) for column chromatography, and silica
gel plates for thin layer chromatography from Sigma Chemical Co. (St.
Louis, MO); agarose from Seakem; HPLC grade solvents (acetonitrile,
methanol, ethanol, tert-butyl alcohol, ethyl acetate, hexane, and acetic
acid) from Fischer (Pittsburgh, PA); ethidium bromide from Roche
Molecular Biochemicals (Indianapolis, IN); and deuterated NMR
solvents (deuterium oxide, D 99.96%, methanol-d4, D 99.8%, N,N-
dimethylformamide-d7, D 99.5%; and acetone-d6, D 99.8%) were from
Cambridge Isotope Laboratories (Andover, MA). Compounds 1a and
1b were prepared by routes described previously in the literature.3,23,44
Compound 3b was prepared via the same general procedure used for
1b,3 and the spectral data of this product matched that published
previously.38
1
0.57 (30% ethyl acetate/hexane). H NMR (DMSO, 500 MHz,): δ
10.13 (s, 1H), 8.61 (d, J = 8.5 Hz, 1H), 8.18−8.24 (m, 2H), 8.10 (ddd,
J = 8.5, 6.5, 1.5 Hz, 1H). 13C NMR (DMSO, 125.8 MHz): δ 154.1,
148.1, 140.1, 136.8, 132.2, 129.5, 129.0. Spectral data matched that
reported by Boyd et al.48
LC-MS/MS Analysis of Mixtures Generated by in Vitro
Hypoxic Metabolism of 1b and 1c. In the LC-MS isotope
incorporation assay, all stock solutions were made in D2O except for
1b. Stock solutions used in the assays were prepared as follows: a
solution of 1b (15 mM) was prepared by dissolving 10 mg of 1b in 4
mL of acetonitrile, a solution of sodium phosphate (500 mM) was
prepared from 409 mg of Na2HPO4 and 254 mg of NaH2PO4
dissolved in 10 mL of D2O, a solution of desferal (10 mM) was
prepared by dissolving 3 mg of desferal in 455 μL of D2O), a solution
of NADPH (30 mM) was prepared by dissolving 1 mg of NADPH
sodium salt in 40 μL of D2O, the stock of cytochrome P450 reductase
(120 U/mL) was prepared by dissolving 3 μL of a 260.4 U/mL stock
solution of enzyme dissolved in 3.5 μL of D2O, and a solution of
CD3OD (15 M) was prepared from 610 μL of CD3OD in 390 μL
D2O. All stock solutions except cytochrome P450 reductase were
degassed by three cycles of freeze−pump−thaw on a vacuum line. For
preparation of the assays, the dioxide 1b (10 μL) was mixed with
desferal (30 μL), sodium phosphate buffer (30 μL), NADPH (10 μL),
CD3OD (10 μL of the 15 M CD3OD stock in D2O), and 209 μL of
D2O. Finally, the reactions were inititated by the addition of
DNA Cleavage Assays. DNA strand cleavage reactions were
carried out as described previously.32,35 Briefly, in a hypoxic DNA-
cleavage assay, agents such as 1a, 1b, or 1d (25−150 μM) were
incubated with supercoiled plasmid DNA (33 μg/mL, pGL-2 Basic),
NADPH (500 μM), cytochrome P450 reductase (33 mU/mL), where
one unit is defined as the amount of enzyme required to cause the
reduction of 1.0 μmole of cytochrome c by NADPH per minute at pH
7.4 at 37 °C), catalase (100 μg/mL), superoxide dismutase (10 μg/
mL), sodium phosphate buffer (50 mM, pH 7.0), acetonitrile (0.5−
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cytochrome P450 reductase stock solution (1 μL). The final volume of
the assay mixture was 300 μL. These assays contained small amounts
of residual exchangeable protium. The final mixtures contained
approximately 99.5% D content in the water, and thus, small amounts
(∼0.5%) of CD3OH will be present alongside much larger amounts of
CD3OD. Nonetheless, these traces of protium in the deuterium atom
donor are not expected to represent a confounding factor in the
interpretation isotope incorporation assays. Bond enthalpies signifi-
cantly favor atom abstraction from the methyl group of methanol
rather than the hydroxyl group (see Supporting Information for
further discussion of this topic). Experimental evidence presented
below confirmed that atom abstraction in this system occurs at the
methyl group of methanol. Specifically, the formation of significant
amounts of metabolites 8 clearly indicated that ·CD2OH/D and ·
CH2OH were generated. In addition, assays in which additional steps
were taken to remove the residual exchangeable protium sources gave
identical results (Figure S8, Supporting Information).
dismutase, and desferal were included as described previ-
ously21,32 to prevent potential background strand cleavage
stemming from the conversion of adventitious molecular
oxygen to reactive oxygen species.
We found that 1b caused DNA strand cleavage when
incubated with the plasmid substrate and the NADPH/
NADPH:cytochrome P450 reductase enzyme system under
hypoxic conditions (Figure 1). Across a range of concentrations
After 4 h of incubation under argon at 25 °C, the proteins in the
assay mixtures were removed by centrifugation through Amicon
Microcon (YM3) filters. The filtrate was analyzed by Beckman Coulter
HPLC employing a C18 reverse phase Betabasic column (5 μm
particle size, 150 Å pore size, 25 cm length, and 4.6 mm i.d.) eluted
with gradient starting from 5% B (0.1% trifluoacetic acid in
acetonitrile) and 95% A (0.1% trifluoacetic acid in water) for 5 min
followed by linear increase to 11% B in another 24 min, then the
gradient was increased linearly to 80% of B within in 6 min, and finally
80% of B was used to wash the column for 10 min. A flow rate of 1.0
mL/min was used, and the products were monitored by their UV-
absorbance at 240 nm. LC/ESI-MS experiments were carried out using
an ion trap mass analyzer, on a LCQ FLEET instrument (Thermo
Fisher Scientific). Positive ion electrospray was used as the means of
ionization. The heated inlet capillary temperature was 375 °C, and
electrospray needle voltage was 5 kV. Nitrogen sheath gas was
supplied at 45 psi, and the LC/ESI-MS analysis was done in the
positive ion mode. Relative collision energies of 35% were used when
the ion trap mass spectrometry was operated in the MS/MS mode.
Parent ions were selected manually (164, 165, 148, 149, 132, 133, 178,
and 180), and subsequent tandem mass spectrometry was performed
automatically by XCalibur software (Thermo Fisher Scientific).
Figure 1. Bioreductively activated DNA strand cleavage by 1b under
hypoxic conditions. (Panel A) Concentration-dependent DNA strand
cleavage by 1b. Supercoiled plasmid DNA (pGL2-basic, 33 μg/mL)
was incubated with 1b (25−150 μM), NADPH:cytochrome P450
reductase (33 mU/mL), NADPH (500 μM), catalase (100 μg/mL),
superoxide dismutase (10 μg/mL), sodium phosphate buffer (50 mM,
pH 7.0), and desferal (1 mM) under anaerobic conditions at room
temperature for 4 h, followed by agarose gel electrophoretic analysis.
Strand breaks per plasmid DNA molecule (S) were calculated using
the equation S = −ln f I, where f I is the fraction of plasmid present as
form I. Lane 1, DNA alone (S = 0.35 0.14); lane 2, enzyme system
alone (S = 0.32 0.11); lane 3, 1b alone (150 μM, S = 0.38 0.17);
lanes 4−9, enzyme system + 1b at 25 μM (S = 0.49 0.13), 50 μM (S
= 0.76 0.05), 75 μM (S = 1.07 0.08), 100 μM (S = 1.30 0.09),
125 μM (S = 1.42 0.07), and 150 μM (S = 1.62 0.14). (Panel B)
Comparison of concentration-dependent DNA strand breakage by 1a,
1b, and 1d under hypoxic conditions. Strand cleavage by various
concentrations (25−150 μM) of the three different compounds was
carried out as described above. Background cleavage present in the
untreated plasmid was subtracted to allow direct comparison of DNA
cleavage yields between different experiments.
RESULTS AND DISCUSSION
■
Bioreductively-Activated, Hypoxia-Selective DNA
Strand Cleavage by 1b. We synthesized 1b by a literature
route involving treatment of 1a with t-butyl nitrite in DMF.3,44
We prepared the expected metabolite 3b via the analogous
reaction on 3a.38 In addition, we prepared a second
expected15,16,23 metabolite 7 by treatment of 3b with sodium
dithionite in ethanol−water. We employed a plasmid-based
assay to measure the ability of 1b to cause bioreductively
activated DNA strand cleavage under hypoxic conditions. In
this assay, oxidative DNA strand cleavage causes conversion of
a supercoiled plasmid substrate (form I) to the open circular
form II.49−52 These two forms of plasmid DNA are then
separated using agarose gel electrophoresis and visualized by
staining with ethidium bromide.49−52 This assay is especially
useful for monitoring the direct DNA strand breaks generated
by radical intermediates.53,54 Direct strand breaks induced by
radicals typically arise via abstraction of hydrogen atoms from
the 2-deoxyribose phosphate backbone of DNA.53,54 We used
recombinant human NADPH:cytochrome P450 reductase to
carry out one-electron reductive activation of 1b because this or
a related enzyme55 is thought to be responsible for the
intracellular activation of 1,2,4-benzotriazine 1,4-dioxides.43,56,57
For reactions carried out under hypoxic conditions, molecular
oxygen was removed from stock solutions by freeze−pump−
thaw degassing and the assay mixtures assembled and incubated
in an inert atmosphere glovebag. Catalase, superoxide
(25−150 μM), the yields of DNA strand breaks generated by
1b were comparable to those produced by 1a and 1d, two
biologically active compounds whose DNA-strand cleaving
properties have been characterized previously (Figure
1).3,13,21,25,43 Control experiments showed that neither 1b
alone nor the NADPH/NADPH:cytochrome P450 reductase
enzyme system (without 1b) caused significant amounts of
DNA strand cleavage (Figure 1). Likewise, when 1b was
incubated with the enzyme alone (no NADPH), NADPH alone
(no enzyme), or with the complete enzymatic reducing system
under aerobic conditions, no significant amounts DNA strand
cleavage were observed (Figure S1, Supporting Information).
Further control experiments showed that compounds 3b and 7,
two major metabolites generated in the hypoxic metabolism of
1b (see below), did not cause DNA strand cleavage either alone
or in the presence of the NADPH:cytochrome P450 reductase
enzyme system under hypoxic conditions. We found that DNA
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strand cleavage by 1b was substantially inhibited (60−90%) by
the classical radical-scavenging agents58 ethanol, methanol, t-
butanol, DMSO, and mannitol (500 mM, Figure 2), analogous
In Vitro Bioreductive Metabolism of 1b. In the case of
other 1,2,4-benzotriazine 1,4-dioxides that have been charac-
terized, DNA strand cleavage is accompanied by the generation
of the corresponding 1,2,4-benzotriazine 1-oxide 3 as the major
metabolite (Scheme 1).15,23,25,27,28 Here, we used LC-MS/MS
analysis to characterize the products generated by in vitro, cell-
free, hypoxic metabolism of 1b by NADPH:cytochrome P450
reductase in the presence of methanol as a hydrogen atom
source. UV−vis detection of the resulting products (240 nm)
revealed a mixture consisting of the starting material 1b
alongside several new metabolites (Figure 3). MS/MS analysis
of the products, along with comparison to authentic synthetic
standards, showed that the 1,4-dioxide 1b eluted at 6.5 min, the
expected mono-N-oxide 3b eluted at 18.8 min, and that the
expected “no-oxide” metabolite 7 eluted at 17.7 min (Table 1).
Figure 2. Radical-scavenging agents inhibit bioreductively activated
DNA strand cleavage by 1b. Column 1 shows the strand cleavage yield
generated by 1b (50 μM) under the standard hypoxic reaction
conditions described in the Materials and Methods and the legend to
Figure 1. Columns 2−6 illustrate the cleavage yields under those same
standard conditions except in the presence of the indicated radical
scavenging agent (500 mM). Background cleavage present in the
untreated plasmid was subtracted to allow direct comparison of DNA
cleavage yields between different experiments. The number of strand
breaks per plasmid DNA molecule (S) was calculated using the
equation S = −ln f I, where f I is the fraction of plasmid present as form
I.
Table 1. LC-MS/MS Properties of the Mixture Generated by
in Vitro Metabolism of 1b under Hypoxic Conditions in the
Presence of CH3OH
retention
time
metabolites
[M + H]+
MS/MS fragments
1b
7
6.5
164
132
148
178
147, 137, 119, 93, 92, 65
105, 77
17.7
18.8
14.6
3b
8a
120, 93, 92, 65
160, 132, 120, 104, 93, 92, 77, 65
The minor metabolite eluting at 14.6 min was tentatively
assigned the structure 8a based upon its m/z of 178 in the LC-
MS. Such a product could arise by a number of different
mechanisms including addition of the methanol-derived radical,
·CH2OH, to 1b, followed by either dehydration or loss of
HO·.59 An analogous mono-N-oxide product was previously
observed to arise via the reaction of the methyl radical with
1a.59
to the results observed previously for 1a and 1d. Of special
importance to the studies described below, we found that
methanol-d4 (500 mM) efficiently quenched the DNA-cleaving
intermediate generated upon enzymatic activation of 1b
(Figure S2, Supporting Information). Overall, the reductively
activated, hypoxia-selective DNA-cleaving properties of 1b
closely resemble those of tirapazamine and other 1,2,4-
benzotriazine 1,4-dioxides.13,21,25,43
Figure 3. Reverse-phase HPLC chromatogram (UV 254 nm) of the products generated by in vitro hypoxic metabolism of 1b in the presence of
methanol.
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Figure 4. Hypoxic metabolism of 1b in phosphate-buffered D2O/CD3OD does not lead to incorporation of deuterium into metabolite 3. LC-MS of
the mono-N-oxide metabolite 3 generated by hypoxic metabolism of 1b. (Panel A) Isotope cluster for the [M + H]+ ion of 3 generated in phosphate-
buffered H2O/CH3OH (relative peak intensities for m/z 148:149:150 = 100:7.9:0.5). (Panel B) Isotope cluster for the [M + H]+ ion of 3 generated
in phosphate-buffered D2O/CD3OD (relative peak intensities for m/z 148:149:150 = 100:8.2:0.6).
In Vitro Bioreductive Metabolism of 1b in Phosphate-
Buffered D2O-methanol-d4. The results described above set
the stage for isotopic labeling experiments designed to probe
whether bioreductive activation of 1b generates an atom-
abstracting drug radical intermediate such as 6. As noted above,
the generation of an aryl radical such as 6 from 2b in the
presence of a deuterium atom donor will yield the
corresponding deuterated drug metabolite 3c,41,42 whereas
the release of the hydroxyl radical from 2b will generate the
nondeuterated metabolite 3b. It may be important to reiterate
that the experiments described above demonstrated that
methanol-d4 efficiently quenches bioreductively activated
DNA strand cleavage by 1b, thus providing a clear indication
that the DNA-cleaving intermediate generated following
bioreductive activation of 1b does indeed react with
methanol-d4.
Thus, 1b was incubated with the NADPH:cytochrome P450
reductase enzyme system under hypoxic conditions in
phosphate-buffered D2O containing methanol-d4 (500 mM),
and the metabolites generated in this reaction were analyzed by
LC-MS/MS. We observed that the spectrum of metabolites
generated in the presence of phosphate-buffered D2O/
methanol-d4 was identical to that observed in the H2O/
methanol experiment shown in Figure 3 (Figure S3, Supporting
Information). Importantly, LC-MS analysis revealed no
significant deuterium incorporation into the major metabolite
3 (Figure 4). The relative abundances of ions in the isotope
cluster for the [M + H]+ ion of 3 observed in these experiments
matched that expected to arise from the natural abundances of
2H and 13C in a molecule of this molecular formula. Similarly,
the no-oxide metabolite showed no deuterium incorporation.
Our LC-MS analysis revealed that the mass-to-charge ratio of
the metabolite eluting at 14.7 min generated in the D2O/
methanol-d4 experiment was two Daltons greater than the
analogous product generated in the presence of nondeuterated
methanol (Figure S4, Supporting Information). This observa-
tion is consistent with the notion that the 14.7 min metabolite
contains a CH2OH or CD2OH fragment derived from
methanol or methanol-d4 (the deuterium on the hydroxyl
group of the CD2OD fragment presumably was exchanged with
protons from trifluoroacetic acid in the HPLC eluent during the
LC-MS analysis). As noted above, 8a and 8b could reasonably
arise via multiple reaction pathways; therefore, we cannot
consider the formation of these products diagnostic for any
particular mechanism.
Bioreductive Metabolism of the Deuterium-Contain-
ing Analogue 1c. Finally, we synthesized the deuterated
analogue 1c via treatment of 1a with t-butyl nitrite in DMF-
d7.44,60 Availability of this labeled compound enabled us to
undertake isotope-tracking experiments complementary to the
solvent-labeling experiments described above. Specifically, the
dehydration mechanism proposed38 to generate radical 6 would
result in the loss of deuterium content during the conversion of
1c to 3 when the reaction is carried out in the presence of
protic solvents and hydrogen atom sources. In contrast, the
hydroxyl radical mechanism will proceed with complete
retention of the label in the conversion of 1c to 3. LC-MS
analysis of the mixture generated by NADPH:cytochrome P450
reductase-catalyzed metabolism of the deuterated analogue 1c
revealed no significant loss of deuterium content in the
enzymatic transformation of 1c to the corresponding mono-N-
oxide metabolite 3 (Figure S5, Supporting Information). The
observation that the deuterium of the starting material 1c was
completely retained in 3 had the side benefit of demonstrating
that a deuterium label does not “wash out” of metabolite 3
under our reaction conditions. This serves as a control for the
methanol-d4 experiments described in the previous section,
ruling out the possibility, albeit remote, that in those
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experiments, deuterium was incorporated into metabolite 3 but
subsequently was completely washed out prior to the mass
spectrometric analysis.
Officer of the Cancer Etiology study section of CSR, NIH, for
his wisdom, compassion, integrity, his love of sciences and the
arts, and, above all, his contributions to the career development
of so many investigators during his own distinguished career.
CONCLUSIONS
■
The results presented here show that 1,2,4-benzotriazine 1,4-
dioxide (1b) is able to carry out enzyme-activated, hypoxia-
selective DNA strand cleavage analogous to the other
benzotriazine di-N-oxides that have been characterized.21,25,43
Two complementary isotopic labeling experiments provided
evidence against the generation of atom-abstracting drug
radicals such as 6 following one-electron reductive activation
of 1,2,4-benzotriazine 1,4-dioxide under hypoxic conditions.
Accordingly, the data is consistent with a mechanism involving
the release of the well-known DNA-damaging agent, the
hydroxyl radical from the reductively activated drug inter-
mediate 2b.
REFERENCES
■
(1) Morgan, G., Wardy, R., and Barton, M. (2004) The contribution
of cytotoxic chemotheraphy to 5-year survival in adult malignancies.
Clin. Oncol. 16, 549−560.
(2) Wilson, W. R., and Hay, M. P. (2011) Targeting hypoxia in
cancer therapy. Nat. Rev. Cancer 11, 393−409.
(3) Kelson, A. B., McNamara, J. P., Pandey, A., Ryan, K. J., Dorie, M.
J., McAfee, P. A., Menke, D. R., Brown, J. M., and Tracy, M. (1998)
1,2,4-Benzotriazine 1,4-dioxides. An important class of hypoxic
cytotoxins with antitumor activity. Anti-Cancer Drug Des. 13, 575−592.
(4) 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.
(5) 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.
(6) Hay, M. P., Hicks, K. O., Pruijn, F. B., Pchalek, K., Siim, B. G.,
Wilson, W. R., and Denny, W. A. (2007) Pharmaokinetic/
pharmacodynamic model-guided identification of hypoxia-selective
1,2,4-benzotriazine 1,4-dioxides with antitumor activity: the role of
extravascular transport. J. Med. Chem. 50, 6392−6404.
(7) 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.
(8) 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/
phamacodynamic modeling identifies SN30000 and SN29751 as
tirapazamine analogues with improved tissue penetration and hypoxic
cell killing in tumors. Clin. Cancer Res. 16, 4946−4957.
(9) Chitneni, S. K., Bida, G. T., Yuan, H., Palmer, G. M., Hay, M. P.,
Melcher, T., Wilson, W. R., Zalutsky, M. R., and Dewhirst, M. W.
(2013) 18F-EF5 PET imaging as an early response biomarker for the
hypoxia-activated prodrug SN30000 combined with radiation treat-
ment in a non-small cell lung cancer xenograft model. J. Nucl. Med. 54,
1−8.
It may be useful to consider how the results described here
mesh with the earlier suggestion that spin-trapped adducts of 2
and 6 were detected following microsomal reduction of 1b in
the presence of the nitrone spin-trap PBN (N-tert-butylphenyl-
α-nitrone).38 Importantly, recent computational analyses
suggested that spin-trapping agents may not serve as “neutral
observers” that report on the normal reactions of the
bioreductively activated radical intermediate 2.28 Rather, spin-
trapping agents have the potential to react directly with 2, in
effect, short-circuiting its normal reactivity. Thus, various
reactions of spin-trapping agents with 2b, rather than with 6,
may yield the observed ESR spectra.28,37,38 It is difficult to
rigorously define the structure of a spin-trapped radical using
ESR, and further work is required to determine the exact nature
of the radicals captured by spin-trapping reagents during the
bioreduction of 1b and other analogues.
Overall, the results are consistent with the hypothesis that
bioreductive activation of heterocyclic N-oxides under hypoxic
conditions leads to the release of the highly cytotoxic reactive
species, hydroxyl radical. This molecular mechanism may be
relevant to the biological properties of many, structurally varied
heterocyclic N-oxides under investigation as potential ther-
apeutic agents for the treatment of cancer,7,61,62 tuber-
culosis,63,64 malaria,65,66 leishmaniasis,66 Chagas disease,67 and
bacterial infections.68
(10) 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.
(11) 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.
(12) 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.
(13) 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.
ASSOCIATED CONTENT
* Supporting Information
Results of control experiments related to DNA strand cleavage,
LC-MS/MS data, and NMR data. This material is available free
of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: 573-882-6763. Fax: 573-882-2754. E-mail: gatesk@
missouri.edu.
Funding
We thank the National Institutes of Health for partial support
of this work (CA 100757).
Notes
■
(14) 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.
(15) 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.
The authors declare no competing financial interest.
DEDICATION
■
This article is dedicated to the memory of Victor Fung, Ph.D., a
former Program Officer at NCI and a former Scientific Review
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Article
(16) 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.
(17) 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.
(18) 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.
(19) Brown, J. M. (1993) SR4233 (Tirapazamine): a new anticancer
drug exploiting hypoxia in solid tumours. Br. J. Cancer 67, 1163−1170.
(20) 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.
(21) 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.
(22) Kotandeniya, D., Ganley, B., and Gates, K. S. (2002) Oxidative
base damage by the antitumor agent 3-amino-1,2,4-benzotriazine 1,4-
dioxide (tirapazamine). Bioorg. Med. Chem. Lett. 12, 2325−2329.
(23) 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.
(24) 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 (tirapaz-
amine). J. Am. Chem. Soc. 125, 11607−11615.
(25) 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.
(26) 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.
(27) Yin, J., Glaser, R., and Gates, K. S. (2012) On the Reaction
mechanism of tirapazamine reduction chemistry: unimolecular N-OH
homolysis, stepwise dehydration, or triazene ring-opening. Chem. Res.
Toxicol. 25, 634−645.
(28) Yin, J., Glaser, R., and Gates, K. S. (2012) Electron and spin-
density analysis of tirapazamine reduction chemistry. Chem. Res.
Toxicol. 25, 620−633.
(29) 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.
(30) 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.
(31) 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.
cyano-substituted quinoxaline di-N-oxide. Chem. Res. Toxicol. 12,
1190−1194.
(34) Priyadarsini, K. I., Dennis, M. F., Naylor, M. A., Stratford, M. R.
L., and Wardman, P. (1996) Free radical intermediates in the
reduction of quinoxaline N-oxide antitumor drugs: redox and
prototropic reactions. J. Am. Chem. Soc. 118, 5648−5654.
(35) Chowdhury, G., Sarkar, U., Pullen, S., Wilson, W. R., Rajapakse,
A., Fuchs-Knotts, T., and Gates, K. S. (2012) DNA strand cleavage by
the phenazine di-N-oxide natural product myxin under both aerobic
and anaerobic conditions. Chem. Res. Toxicol. 25, 197−206.
(36) 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.
(37) 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.
(38) 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.
(39) 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.
(40) 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.
(41) Chin, D.-H., and Goldberg, I. H. (1993) Sources of hydrogen
abstraction by activated neocarzinostatin chromophore. Biochemistry
32, 3611−3616.
̌
(42) Stefane, B., and Polanc, S. (2008) CAN-Mediated oxidation of
electron-deficient aryl and heteroaryl hydrazines and hydrazides.
Synlett, 1279−1282.
(43) Fitzsimmons, S. A., Lewis, A. D., Riley, R. J., and Workman, P.
(1994) Reduction of 3-amino-1,2,4-benzotriazine-1,4,-di-N-oxide to a
DNA-damaging species: a direct role for NADPH:cytochrome P450
oxidoreductase. Carcinogenesis 15, 1503−1510.
(44) Doyle, M. P., Dellaria, J. F. J., Siegfried, B., and Bishop, W. W.
(1977) Reductive amination of arylamine by alkyl nitries in N,N-
dimethylformamide. A direct conversion of arylamines to aromatic
hydrocarbons. J. Org. Chem. 42, 4394−3498.
́
(45) Bender, M. L., Clement, G. E., Kezdy, F. J., and Heck, H. D. A.
(1964) The correlation of the pH (pD) dependence and the stepwise
mechanism of α-chymotrypsin-catalyzed reactions. J. Am. Chem. Soc.
86, 3680−3690.
(46) Walde, P., Peng, Q., Fadnavis, N. W., Battistel, E., and Luisi, P.
L. (1988) Structure and activity of trypsin in reverse micelles. Eur. J.
Biochem. 173, 401−409.
(47) Mason, J. C., and Tennant, G. (1970) Heterocyclic N-oxides.
Part VI. Synthesis and nuclear magnetic resonance spectra of 3-
aminobenzo-1,2,4-triazines and their mono and di-N-oxides. J. Chem.
Soc. B, 911−916.
(48) Boyd, M., Hay, M. P., and Boyd, P. D. W. (2006) Complete 1H,
13C, and 15N NMR assignment of tirapazamine and related 1,2,4-
benzotriazine N-oxides. Magn. Reson. Chem. 44, 948−954.
(49) 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.
(50) 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.
(32) 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.
(33) Fuchs, T., Gates, K. S., Hwang, J.-T., and Greenberg, M. M.
(1999) Photosensitization of guanine-specific DNA damage by a
117
dx.doi.org/10.1021/tx400356y | Chem. Res. Toxicol. 2014, 27, 111−118

Chemical Research in Toxicology
Article
(51) 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.
(68) Gund, P. H., and Berkelhammer, G. (1971) New antibacterial
quinoxaline N,N-dioxide derivatives. J. Med. Chem. 14, 992.
(52) 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.
(53) Greenberg, M. M. (2007) Elucidating DNA damage and repair
processes by independently generating reactive and metastable
intermediates. Org. Biomol. Chem. 5, 18−30.
(54) Gates, K. S. (2009) An overview of chemical processes that
damage cellular DNA: spontaneous hydrolysis, alkylation, and
reactions with radicals. Chem. Res. Toxicol. 22, 1747−1760.
(55) Delahoussaye, Y. M., Evans, J. M., and Brown, J. M. (2001)
Metabolism of tirapazamine by multiple reductases in the nucleus.
Biochem. Pharmacol. 62, 1201−1209.
(56) Patterson, A. V., Saunders, M. P., Chinje, E. C., Patterson, L. H.,
and Stratford, I. J. (1998) Enzymology of tirapazamine metabolism: a
review. Anti-Cancer Drug Des. 13, 541−573.
(57) Walton, M. I., Wolf, C. R., and Workman, P. (1992) The role of
cytochrome P450 and cytochrome P450 reductase in the reductive
activation of the novel benzotriazine di-N-oxide hypoxic cytotoxin 3-
amino-1,2,4-benzotriazine 1,4-dioxide (SR 4233, WIN 59075) by
mouse liver. Biochem. Pharmacol. 44, 251−259.
(58) 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.
(59) Fuchs, T., Barnes, C. L., and Gates, K. S. (2001) Crystal
structure of 3-amino-5-methyl-1,2,4-benzotriazine 1-oxide: evidence
for formation of a covalent attachment between a carbon-centered
radical and the antitumor agent tirapazamine. J. Chem. Crystallog. 31,
387−391.
(60) Markgraf, J. H., Chang, R., Cort, J. R., Durant, J. L. J.,
Finkelstein, M., Gross, A. W., Lavyne, M. H., Peterson, R. C., and Ross,
S. D. (1997) Protodediazoniation of aryldiazonium fluoroborates by
dimethylformamide. Tetrahedron 53, 10009−10018.
(61) 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.
(62) Monge, A., Martinez-Crespo, F. J., Lopez de Cerain, A., Palop, J.
A., Narro, S., Senador, V., Marin, A., Sainz, Y., Gonzalez, M., Hamilton,
E., Barker, A. J., Clarke, E. D., and Greenhow, D. T. (1995) Hypoxia-
selective agents derived from 2-quinoxalinecarbonitrile 1,4-di-N-
oxides. 2. J. Med. Chem. 38, 4488−4494.
(63) Chopra, S., Koolpe, G. A., Tambo-ong, A. A., Matsuyama, K. N.,
Ryan, K. J., Tran, T. B., Doppalapudi, R. S., Riccio, E. S., Iyer, L. V.,
Green, C. E., Wan, B., Franzblau, S. G., and Madrid, P. B. (2012)
Discovery and optimization of benzotriazine di-N-oxides targeting
replicating and nonreplicating Mycobacterium tuberculosis. J. Med.
Chem. 55, 6047−6060.
(64) Vicente, E., Perez-Silanes, S., Lima, L. M., Ancizu, S., Burguete,
A., Solano, B., Villar, R., Aldana, I., and Monge, A. (2009) Selective
activity against Mycobacterium tuberculosis of new quinoxaline 1,4-di-
N-oxides. Bioorg. Med. Chem. 17, 385−389.
(65) Barea, C., Pabon
́
, A., Castillo, D., Zimic, M., Quillano, M.,
Galiano, S., Perez-Silanes, S., Monge, A., Deharo, E., and Aldana, I.
́
(2011) New salicylamide and sulfonamide derivatives of quinoxaline
1,4-di-N-oxide with antileishmanial and antimalarial activities. Bioorg.
Med. Chem. Lett. 21, 4498−4502.
́ ́
(66) Barea, C., Pabon, A., Perez-Silanes, S., Galiano, S., Gonzalez, G.,
Monge, A., Deharo, E., and Aldana, I. (2013) New amide derivatives of
quinoxaline 1,4-di-N-oxide with leishmanicidal and antiplasmodial
activities. Molecules 18, 4718−4727.
(67) Vicente, E., Cuchowicz, P. R., Benítez, D., Castro, E. A.,
Cerecetto, H., Gonzalez, M., and Monge, A. (2010) Anti-T. cruzi
́
activities and QSAR studies of 3-arylquinoxaline-2-carbonitrile di-N-
oxides. Bioorg. Med. Chem. Lett. 20, 4831−4835.
118
dx.doi.org/10.1021/tx400356y | Chem. Res. Toxicol. 2014, 27, 111−118