Transferring oxygen isotopes to 1,2,4-benzotriazine 1-oxides forming the corresponding 1,4-dioxides by using the HOF·CH3CN complex

Article abstract of DOI:10.1016/j.tet.2012.08.017

Heterocyclic benzotriazine N-oxides are an interesting class of experimental anticancer and antibacterial agents. Analogs with ¹⁸O incorporated into the N-oxide group may offer useful mechanistic tools. We describe the use of H₂¹⁸OF·CH₃CN in a fast, readily executed and high-yielding preparation of 1,2,4-benzotriazine 1,4-dioxides containing an ¹⁸O-label at the 4-oxide position.

Full text of DOI:10.1016/j.tet.2012.08.017

Tetrahedron 68 (2012) 8942e8944
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Transferring oxygen isotopes to 1,2,4-benzotriazine 1-oxides forming the
corresponding 1,4-dioxides by using the HOF$CH3CN complex
,
,
*
Julia Gatenyo a y, Kevin Johnson b, Anuruddha Rajapakse b, Kent S. Gates b, Shlomo Rozen a
a School of Chemistry, Tel-Aviv University, Tel-Aviv 69978, Israel
b Departments of Chemistry and Biochemistry, University of Missouri, Columbia, MO 65211, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 May 2012
Received in revised form 20 July 2012
Accepted 7 August 2012
Available online 14 August 2012
Heterocyclic benzotriazine N-oxides are an interesting class of experimental anticancer and antibacterial
agents. Analogs with 18O incorporated into the N-oxide group may offer useful mechanistic tools. We
describe the use of H218OF$CH3CN in a fast, readily executed and high-yielding preparation of 1,2,4-
benzotriazine 1,4-dioxides containing an 18O-label at the 4-oxide position.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Oxygen transfer
18O isotope
Tirapazamine
HOF$CH3CN
F2/N2
N-oxide
H218O
1. Introduction
released in the form of water or hydroxyl radical.6 The 4-N-oxide in
1,2,4-benzotriazine 1,4-dioxides compounds are generally installed
Heterocyclic benzotriazine N-oxides are an interesting class of
experimental anticancer1 and antibacterial therapeutic agents.2 One
of their important features is their ability to capitalize on the low
oxygen (hypoxic) environment found in many solid tumors. The lead
compound in this class of potential drugs is tirapazamine: 3-amino-
1,2,4-benzotriazine 1,4-dioxide 2a (x¼16).3 Tirapazamine and re-
lated N-oxides undergo intracellular one-electron reduction cata-
lyzed by enzymes, such as NADPH:cytochrome P450 reductase.4 In
normally-oxygenated tissue, the resulting drug radical primarily
undergoes relatively harmless back-oxidation to give the starting
compound. In contrast, under hypoxic conditions the lifetime of the
drug radical is extended, enabling a decomposition reaction that
yields a highly reactive DNA-damaging radical.5 The nature of the
key DNA-damaging radical generated by the bioreductively-
activated N-oxides remains a subject of ongoing investigation.6
Analogs bearing isotopic-labeled oxygens in the N-oxide func-
tional groups may provide powerful tools for elucidating the
mechanism(s), by which these potential drugs generate DNA-
damaging radicals. For example, careful metabolic studies of 2
(x¼18) could shed light on whether the 4-oxide of tirapazamine is
via reaction of the parent heterocyclic with H2O2eacetic acid
mixtures or peracids, such as m-chloroperbenzoic acid. These re-
actions can be sluggish and low yielding.1eeg,4c,7 Furthermore, in-
troduction of 18O via those routes generally necessitates
preparation of H128O2 from 18O2, a tedious, expensive, and inefficient
procedure.8 Here we describe the use of H218OF$CH3CN in an easy
and high-yielding preparation of 1,2,4-benzotriazine 1,4-dioxides
containing an 18O-label oxide at the 4-position (Scheme 1).
The acetonitrile complex of the hypofluorous acid HOF$CH3CN,
is readily prepared by passing dilute fluorine through aqueous
acetonitrile,9 and is considered today as one of the best oxygen-
transfer agents available to organic chemists.10 The oxygen atom
is a potent electrophile because it is weakly bonded to the most
electronegative elementdfluorine. The complex is able to transfer
an oxygen atom to very deactivated double bonds11 and to very
weak nucleophilic centers under mild conditions.12 Following our
work on the oxidation of the quinoxaline system with
HOF$CH3CN,13 we felt that this reagent could be useful for N-oxi-
dation of the benzotriazine ring system as well.
2. Results and discussion
* Corresponding author. Tel.: þ972 3 640 8378; fax: þ972 3 640 9293; e-mail
Indeed we found that HOF$CH3CN generated the desired 1,4-
dioxides (2) from the 1-oxide precursors 1 rapidly and in good
address: rozens@post.tau.ac.il (S. Rozen).
y
Tel.: þ972 3 640 8378; fax: þ972 3 640 9293.
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.08.017

J. Gatenyo et al. / Tetrahedron 68 (2012) 8942e8944
8943
large excess of the reagent probably encourages epoxidation re-
actions that eventually yield the triazolone derivative 4 (Scheme 2).
It should be noted that this mechanism is only a plausible specu-
lation as we have not isolated the oxaziridine byproduct (Scheme
2), which is anticipated to be very unstable.
O
N
O
N
x
F2 + H2 O + CH3CN
N
N
HxOF CH3CN
N
R
N
R
XO
O
N
O
N
1
2: x = 16
3: x = 18
HxOF CH3CN
N
N
a: R = NH2
b: R = H
c: R = NHAc
d: R = Me
2a (73%)
2b (70%)
3a (70%)
3b (70%)
Large excess,
fast adding
N
R
N
R
+ OH
F-
x = 16,18
O
1
2d (70%)
2e (75%)
2f (90%, 30% conversion)
e: R = OMe
f: R = 4-NO2C6H4
Scheme 1. Synthesis of 1,2,4-benzotriazine 4-dioxides with 16O and 18O isotopes.
N
N
H+/H2O
? -HF, -ROH?
R = H, NH2, Cl, Me
p-C6H4NO2
N
H
xO
4
Scheme 2. Amidation of 1,2,4-benzotriazine 1-oxide.
yield. Thus 3-amino-1,2,4-benzotriazine 1-oxide (1a) was con-
verted to tirapazamine (TPZ) (2a)7 in 60% yield and 1,2,4-
benzotriazine 1-oxide (1b) produced 1,2,4-benzotriazine 1,4-
dioxide (2b)6e in 70% yield. Somewhat improved yield of 2a (73%)
could be achieved if the reaction was performed on the acetylated
amine 1c followed by spontaneous hydrolysis (Scheme 1). It should
be noted that with reaction times of about 2 min, the N-oxidations
with HOF$CH3CN are much faster than any other method that has
been reported.7
The electrophilic oxygen in the HOF$CH3CN complex originates
from water. This should enable preparation of H18OF$CH3CN from
the most readily available and easy to handle precursor of 18O
isotopeeH128O.14 Thus, passing dilute fluorine through a solution of
acetonitrile and H128O, resulted in the labeled H18OF$CH3CN, which
was then reacted with 1a or 1c. The product, identified by HRMS
(APPI) m/z¼181.0609 (Mþ1) (calcd for C7H6N146O18O, 181.0611)
clearly showed that 3a, labeled with 18O isotope, was obtained for
the first time in comparable yield as with the common 16O isotope.
The heavy oxygen in the Ne18O moiety is not readily exchanged
with the common 16O isotope when treated with regular water or
exposed to air, enabling it to serve as a probe for metabolic studies.
Reaction of 1,2,4-benzotriazine 1-oxide (1b) with H18OF$CH3CN
produced 3b having a molecular ion in HRMS (APPI) m/z¼166.0500
(Mþ1) (calcd for C7H5N136O18O, 166.0502).
The described reaction is of general nature. Treating 3-methyl-
1,2,4-benzotriazine 1-oxide (1d) with the oxidizing solution pro-
duced 3-methyl-1,2,4-benzotriazine 1,4-dioxide (2d)15 in 70% yield.
The strong electron donating methoxy substituent in 3-methoxy-
1,2,4-benzotriazine 1-oxide (1e) provided 75% yield of 3-methoxy-
1,2,4-benzotriazine 1,4-dioxide (2e).15 Electron-withdrawing moi-
eties, such as the one found in 3-(4-nitrophenyl)-1,2,4-
benzotriazine 1-oxide (1f) deactivate the molecule as far as elec-
trophilic oxidation is concerned and while the yield of 3-(4-
nitrophenyl)-1,2,4-benzotriazine 1,4-dioxide (2f) was higher than
90% the conversion was only 30%. The remaining starting material
could be easily separated by flash-column chromatography and
recycled (Scheme 1).
3. Conclusion
There are many cases where it is highly desirable to have 18O
isotope attached to an organic molecule. It has been shown here
that the best, easiest, and most efficient way to achieve such goal is
through H18OF$ complex made readily from commercial diluted
fluorine, the best and cheapest source of 18O:H128O and acetonitrile.
The above reactions were conducted at 0 ꢀC using solutions
containing 2 mol-equivalent of the oxidizing complex slowly added
to the various substrates in small portions. However, when a large
excess of HOF$CH3CN solution was added in one portion to 1,2,4-
benzotriazine 1-oxide (1b) at room temperature, only 3-oxo-3,4-
dihydrobenzo-1,2,4-triazine 1-oxide (4) was obtained in almost
quantitative yield. Among the common analytical methods, which
supported this structure, one could observe a strong IR absorption
at 1663 cmꢁ1 characteristic of an amide. The same outcome was
observed when 1b was reacted with H18OF$CH3CN leading to the 3-
oxo derivative (4) having the 18O isotope. When other substrates
were treated with an excess of the reagent, the presence of the
triazolone derivative 4 was also observed. Thus with 3-chloro-
1,2,4-benzotriazine 1-oxide (1 R¼Cl) the transformation to 4 took
place in quantitative yield. In case of 3-amino, 3-methyl and 3-(4-
nitrophenyl)-1,2,4-benzotriazine 1-oxide (1R¼NH2, Me, p-
C6H4NO2) the 3-oxo derivative was obtained as a byproduct with
increasing yield proportionally to the excess of the reagent added.
These results suggest that while low temperature and addition of
small portions of oxidizing solution promoted the N-oxidation,
4. Experimental
4.1. General
1H NMR spectra were recorded using a 200 MHz spectrometer
with CDCl3 as a solvent and Me4Si as an internal standard. The
proton broad-band decoupled 13C NMR spectra were recorded at
100.5 MHz. Here too, CD3OD or CDCl3 served as a solvent and Me4Si
as an internal standard. MS was measured under EI, or APPI
conditions.
Preparation of 3-substituted 1,2,4-benzotriazine 1-oxides. Com-
pounds 1a,7 1b, 1d, 1e,15 and 1f16 were synthesized as previously
described.
4.2. General procedure for working with fluorine
Fluorine is a strong oxidant and a corrosive material. It should be
used with an appropriate vacuum line. For the occasional user,
however, various premixed mixtures of F2 in inert gases are

8944
J. Gatenyo et al. / Tetrahedron 68 (2012) 8942e8944
commercially available, thereby simplifying the process. Unreacted
fluorine should be captured by a simple trap containing a solid base,
such as soda-lime located at the outlet of the glass reactor. A de-
tailed setup for working with F2 could be found in the literature.17 If
elementary precautions are taken, work with fluorine is relatively
simple and we have never experienced any difficulties or un-
pleasant situations.
purified by chromatography, eluting with a gradient (0e15%) of
MeOH/DCM to give the 3-oxo-3,4-dihydrobenzo-1,2,4-triazine 1-
oxide (4) as a pale yellow solid: mp 228e229 ꢀC; Rf (E.A) 0.64; IR
1663 cmꢁ1; H NMR (CD3OD) 8.23 (1H, dd, J¼8.2, 1.2 Hz), 7.83 (1H,
ddd, J¼8.7, 7.8, 1.3 Hz), 7.36e7.42 (2H, m); 13C NMR (DMSO) 153.3,
136.9, 136.8, 129.6, 124.2, 121.2, 116.6; HRMS (APPI) m/z calcd for
C7H5N3O2 164.0460 (MþH)þ, found 164.0463. Anal. Calcd for
C7H5N3O2: C, 51.54; H, 3.09; N, 25.76. Found: C, 51.29; H, 2.80; N,
26.00.
4.3. General procedure for producing HOF$CH3CN
A mixture of 10e20% F2 in nitrogen was used throughout this
work. The gas mixture was prepared in a secondary container prior
to the reaction and passed at a rate of about 400 mL per minute
through a cold (ꢁ15 ꢀC) mixture of 100 mL of CH3CN and 10 mL of
H2O (or 10 mL CH3CN and 1mL H218O) in a regular glass reactor. The
development of the oxidizing power was monitored by reacting
aliquots with an acidic aqueous solution of KI. The liberated iodine
was then titrated with thiosulfate. Typical concentrations of the
oxidizing reagent were around 0.4e0.6 M.
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4.4. General procedure for working with HOF$CH3CN
A mono oxide derivative 1 was dissolved in DCM, and the
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chromatography, eluting with a gradient (0e15%) of MeOH/DCM.
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4.4.1. Synthesis of 3-substituted 1,2,4-benzotriazine 1,4-dioxides.
Compounds 2a,7 2b,6e 2d, and 2e15 were prepared from the corre-
sponding 1,2,4-benzotriazine 1-oxides as described above, using
2 equiv of the oxidizing agent. The physical and spectral properties
of all products completely matched those appearing in the
literature.
4.4.2. Synthesis of 3-(4-nitrophenyl)-1,2,4-benzotriazine 1,4-dioxide
(2f). Compound 2f was prepared from 3-(4-nitrophenyl)-1,2,4-
benzotriazine 1-oxide (1f) (0.15 g, 0.56 mmol) as described above,
using 2 equiv of the oxidizing agent. A pale beige solid (0.05 g, 95%
yield, 30% conversion) was obtained: mp>300 ꢀC; Rf (DCM) 0.38;
1H NMR (CDCl3) 8.84 (2H, d, J¼8.8 Hz), 8.67 (1H, d, J¼8.6 Hz), 8.57
(1H, d, J¼8.4 Hz), 8.39 (2H, d, J¼8.8 Hz), 8.09 (1H, t, J¼8.6 Hz), 7.95
(1H, t, J¼8.4 Hz); 13C NMR 120.5, 124.1, 129.6, 129.8, 131.3, 134.0,
136.3, 140.0, 147.6, 150.1 158.8; HRMS (APPI) m/z calcd for
C13H8N4O4 285.0624 (MþH)þ, found 285.0629.
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A large excess of HOF$CH3CN solution was added in one portion
to the mono oxide derivative 1 (0.09 g, 0.6 mmol) in DCM at room
temperature. The reaction was stopped after a few minutes by
evaporation of the solvent almost to dryness. The residue was