Electrochemical reduction of 3-phenyl-1,2-benzisoxazole 2-oxide on boron-doped diamond

Article abstract of DOI:10.1002/poc.3288

The bioreduction of N-oxide compounds is the basis for the mode of action of a number of biologically active molecules. These compounds are thought to act by forming a reactive oxygen species through an intracellular reduction and subsequent redox cycling process within the organism. With these results in mind, the preliminary investigation into the electrochemical reduction of the benzisoxazole 2-oxide ring system was undertaken, with the thought that this class of compounds would reduce in a similar fashion to other N-oxide heterocycles. The electrochemical reduction of 3-phenyl-1,2-benzisoxazole 2-oxide on boron-doped diamond was studied using cyclic and square wave voltammetry as well as controlled potential electrolysis and HPLC for qualitative identification of the reaction products. It was found that the reduction proceeded with an initial quasi-reversible one-electron reduction followed by the very fast cleavage of either the endocyclic or exocyclic N-O bond. Subsequent electron transfer and protonation resulted in an overall two-electron reduction and formation of the 2-hydroxyaryl oxime and benzisoxazole. These results are analogous to those observed in the electrochemical reduction of other heterocyclic N-oxides albeit the reduction of the benzisoxazole N-oxides takes place at a more negative potential. However, these encouraging results warrant further investigation into the reduction potential of substituted benzisoxazole N-oxides as well as to elucidate and characterize the nature of the intermediate species involved.

Full text of DOI:10.1002/poc.3288

Short Communication
Received: 10 August 2013,
Revised: 12 December 2013,
Accepted: 05 January 2014,
Published online in Wiley Online Library: 31 January 2014
(wileyonlinelibrary.com) DOI: 10.1002/poc.3288
Electrochemical reduction of 3-phenyl-1,
2-benzisoxazole 2-oxide on boron-doped
diamond†
Martin Kocioleka*, Jason Bennetta and Jerry Casbohma
The bioreduction of N-oxide compounds is the basis for the mode of action of a number of biologically active molecules.
These compounds are thought to act by forming a reactive oxygen species through an intracellular reduction and subsequent
redox cycling process within the organism. With these results in mind, the preliminary investigation into the electrochemical
reduction of the benzisoxazole 2-oxide ring system was undertaken, with the thought that this class of compounds would
reduce in a similar fashion to other N-oxide heterocycles. The electrochemical reduction of 3-phenyl-1,2-benzisoxazole
2-oxide on boron-doped diamond was studied using cyclic and square wave voltammetry as well as controlled potential
electrolysis and HPLC for qualitative identification of the reaction products. It was found that the reduction proceeded with
an initial quasi-reversible one-electron reduction followed by the very fast cleavage of either the endocyclic or exocyclic N–O
bond. Subsequent electron transfer and protonation resulted in an overall two-electron reduction and formation of the
2-hydroxyaryl oxime and benzisoxazole. These results are analogous to those observed in the electrochemical reduction of
other heterocyclic N-oxides albeit the reduction of the benzisoxazole N-oxides takes place at a more negative potential.
However, these encouraging results warrant further investigation into the reduction potential of substituted benzisoxazole
N-oxides as well as to elucidate and characterize the nature of the intermediate species involved. Copyright © 2014 John
Wiley & Sons, Ltd.
Keywords: N-oxides; electrochemical reduction; boron-doped diamond; cyclic voltammetry; square wave voltammetry
Boron-doped microcrystalline diamond (BDD) electrodes were
chosen due to their known anti-fouling properties and wide
INTRODUCTION
potential window.
The bioreduction of N-oxide compounds is the basis for the
mode of action of a number of biologically active molecules.
These compounds are thought to act by forming a reactive
oxygen species through an intracellular reduction and subsequent
redox cycling process within the organism. This is the basis for the
anti-infective activity exhibited by a number of different classes of
N-oxide compounds including quinoxaline 1,4-di-N-oxides[1]
(anti-infectious), benzofuroxans[2,3] (antiprotozoal) and indolone-
N-oxides[4] (antimicrobial). While the mode of action of these
compounds has been proposed, the exact mechanism by which
these drugs act in biological systems has not been fully elucidated.
In order to shed light on how these systems behave, a more
detailed investigation of the possible reaction mechanism is
warranted. For this reason, the electrochemical reduction of a
number of these compounds has been investigated. The reduction
of benzofuroxans has been proposed to proceed via a two-step
reduction mechanism, first forming a radical species and subse-
quently reducing to result in either ring opening of the furoxan
or deoxygenation of the ring.[2] Isoxazoles and related compounds
have also been shown to undergo electrochemical ring opening.[5]
In a similar manner, oximes have been shown to reduce by way of
a facile N–O bond cleavage.[6] With these results in mind, the
preliminary investigation into the electrochemical reduction of
the benzisoxazole 2-oxide ring system by cyclic voltammetry,
square wave voltammetry (SWV) and constant potential electroly-
sis was undertaken, with the thought that this class of compounds
would reduce in a similar fashion to other N-oxide heterocycles.
RESULTS
Initial investigation involved cyclic voltammetry of a 3 mM
solution of 3-phenyl-1,2-benzisoxazole 2-oxide (1a) dissolved in
0.1M tetrabutylammonium tetrafluoroborate and acetonitrile
(TBATFB/ACN) at a BDD electrode. Figure 1 shows a representative
cyclic voltammogram (CV). The potential was scanned positive
from ꢀ1.0 V to 1.0 V, then negative to ꢀ3.0V at a rate of 0.5 V/s.
The voltammogram exhibits no distinguishable features during
the initial forward scan. Scanning in the negative direction shows
a reduction at approximately ꢀ2.6 V (I), which can be attributed
to the reduction of the N-oxide. Upon scanning forward, an oxida-
tion is observed at ꢀ0.15 V (II). Due to the large peak separation, it
seems unlikely that II is due to the reoxidation of the N-oxide
radical anion. To examine this relationship closer, SWV was
performed, the results of which are shown in the Fig. 1 inset. The
* Correspondence to: Martin Kociolek, Penn State Erie, The Behrend College,
School of Science, Erie, USA.
E-mail: mgk5@psu.edu
a M. Kociolek, J. Bennett, J. Casbohm
Penn State Erie, The Behrend College, School of Science, Erie, USA
J. Phys. Org. Chem. 2014, 27 540–544
Copyright © 2014 John Wiley & Sons, Ltd.

ELECTROCHEMICAL REDUCTION OF 3-PHENYL-1,2-BENZISOXAZOLE 2-OXIDE
Figure 1. Cyclic voltammogram for 3 mM 1a in 0.1 M TBATFB/ACN at a scan
rate of 0.5 V/s. Inset is a square wave voltammogram for the same solution
Figure 3. Plot showing the linearity of the peak I current from
voltammograms in Fig. 2 with the square root of the scan rate
potential was stepped from ꢀ3.0V (to ensure reduction of the N-
oxide) to 1.0V at 5 mV increments, 25 mV amplitude and 15 Hz fre-
quency. The SWV exhibits two oxidation peaks, one similar to the
CV at approximately ꢀ0.10 V (II) and an additional peak at ꢀ2.5 V
(III). Peak III likely corresponds to the reoxidation of the radical an-
ion of 1a, which supports that II originates from the oxidation of a
different radical anion species. These results are consistent with
the electrochemical reduction (E step) of 1a being followed by a
fast chemical (C) step (and hence not being available for
reoxidation back to 1a), referred to as an EC mechanism.
To further investigate the electrochemical behavior of the
compounds, CVs were obtained at various scan rates as shown
in Fig. 2. Note that the magnitude of the N-oxide reduction (I)
increases linearly with the square root of scan rate, illustrated
in Fig. 3, which denotes a diffusion-limited process. According
to Savéant[7] the peak current (ip in A) for an EC reduction mech-
anism would be expected to increase linearly with the square
root of the scan rate (ν1/2) as shown in Eqn (1):
from Fig. 3 (adjusted to A/(V1/2 ꢀ1/2)), the number of electrons
s
transferred can be calculated through a simple rearrangement as
shown in Eqn (2).
jslopejꢁR1=2T1=2
n ¼
(2)
1=2
0:496F3=2AC D
∘
Using the slope from Fig. 3, a T of 295 K, an electrode area of
0.2190 cm2, a bulk concentration of 3 × 10ꢀ6 mol/cm3 and an
estimated diffusion coefficient of 2 × 10ꢀ5 cm2/s[8] resulted in a
calculated one-electron transfer (n = 1.01). This was further
supported via potential step chronoamperometry by stepping
the potential from 0 V to ꢀ3 V and using the well-known Cottrell
equation (n = 0.77). In addition, preliminary kinetic parameters
were calculated and are detailed in the supporting information.
It should be noted that the iIp : iIpI ratio in Fig. 2 varies with scan
rate as indicated in the inset table. The observation that the
current of II decreases (to a greater extent relative to I) with
decreasing scan rate further supports an EC mechanism. This will
be discussed further in the discussion section. The degree of
reversibility (RDPS) for the initial electrochemical reduction of 1a can
be approximated using double potential step chronoamperometry
by following the initial reduction step (0 V to ꢀ3V for a given time
tR) with an anodic step (ꢀ3V to 0V for the same amount of time).
The RDPS can then be calculated using Eqn ((3)) where ic(tR) is
the current measured at the end of the initial reduction step
and ia(2tR) is the current measured in the anodic step after the
same amount of time.[7]
1=2
0:496nF3=2AC D ν1=2
∘
ꢀ ꢀ
ꢀ ꢀ
ip
¼
(1)
R1=2T1=2
where n is the number of electrons transferred, F is Faraday’s
constant, A is the electrode area (cm2), Co is the bulk concentration
of 1a (mol/cm3), D is the diffusion coefficient of 1a (cm2/s), R is the
gas constant and T is the temperature (K). Since ip=ν = j is the slope
ꢀ
1
ꢀ
2
ꢀiað2tRÞ=icðtRÞ
pffiffiffi
RDPS
¼
(3)
1 ꢀ 1=
2
A system is considered more reversible as the RDPS value
approaches 1. The double potential step chronoamperometric ex-
periment (not shown) yielded a –ia(2tR)/ic(tR) ratio of 0.07366, which
resulted in a calculated RDPS value of 0.25. This quasi-reversibility
explains why the re-oxidation of 1a is only visible on the SWV (III
in Fig. 1 inset) and not on any of the CVs.
The effect of increasing water concentration was investigated by
examining both CV and SWV in various ACN/H2O ratios. These are
shown in Fig. 4 and its inset, respectively. There are two significant
results from this study. First, the oxidation II potential (CV) shifts
anodically with increasing water concentration, confirming the
Figure 2. Cyclic voltammogram for 3 mM 1a in 0.1 M TBATFB/ACN at
various scan rates
J. Phys. Org. Chem. 2014, 27 540–544
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/poc

M. KOCIOLEK, J. BENNETT AND J. CASBOHM
DISCUSSION
Given these results and what has been previously reported for the
electrochemical reduction of similar heterocyclic N-oxides, the
reduction mechanism shown in Scheme 1 can be proposed. The
initial reduction I can be attributed to the one-electron reduction
of N-oxide 1a to give radical anion 2a. Although the reverse oxida-
tion was not observed in the CV, SWV yielded an oxidation peak
(III) that can be attributed to the oxidation of 2a to 1a.
Further analysis of this reaction in aqueous acetonitrile
solutions was undertaken, so the effect of either protonation
or hydrogen bonding on different species could be observed
by changes in the potential for the corresponding waves.
Reduction I in the CV showed only small changes in potential
as the percentage of water was increased. This is consistent
with the fact that the equilibrium between neutral 1a and its
protonated form 1b would likely not change much with the
addition of water. The small changes observed would likely
be from a small degree of H-bonding to 1a. In contrast, the
SWV showed significant changes with an additional oxidation
peak appearing at approximately ꢀ2.9 V. These observations
can be rationalized by a shifting of equilibrium between 2a
and 2b, due to protonation of the radical anion. Also likely is
that the electron-rich species 2a possesses a greater degree
of H-bonding. Both these factors could explain the presence
of two oxidation peaks (III and IV) in the SWV at higher
water content.
Oxidation II, observed in the voltammograms, only appears on
the return scan after going to ꢀ3.0 V. The large difference in
potential along with the SWV results suggests that this oxidation
is not the reverse of reduction I but is consistent with the initial
electron transfer being followed by a very fast chemical step
resulting in a new species which is then oxidized. In addition,
an analysis of the CV at different scan rates was performed. While
the magnitudes of peaks I and II both decrease with decreasing
scan rate as expected, oxidation II decreased to a greater extent
(as given by the ratio of iIp : ipII). This would be consistent with an
Figure 4. Cyclic voltammogram for 3 mM 1a in 0.1 M TBATFB/ACN as a
function of water content. Inset is the square wave voltammograms for
the same solutions
participation of H+ in the reaction. Comparatively, only a minimal
change was observed in the reduction I potential in the CV as well
as in its corresponding reoxidation III in the SWV. The second
important result from this study is the emergence of a second
oxidation peak (IV) at a slightly more negative potential (ꢀ2.9 V)
than peak III at 5 and 10% water concentration.
In order to identify the species produced by the reduction, a
qualitative analysis using controlled potential electrolysis was
carried out in a custom thin-layer cell using a BDD working
electrode. The cell was filled with a 1.0 mM solution of 1a in
0.1 M TBATBF/ACN and the potential held at ꢀ3 V for 1500 s,
after which the cell was flushed and the mixture analyzed by
HPLC with a PDA detector. In addition to unreacted N-oxide,
the chromatogram revealed two peaks whose retention times
and UV spectra were identical to authentic samples of oxime 3
and benzisoxazole 4. It should be noted that increasing electrol-
ysis times resulted in a darkening of the reaction mixture, some
deposition and peaks early in the chromatogram that were
attributed to butylamine from the breakdown of the TBATFB. It
is important to note that a two-electron reduction is required
to convert the original compound to either of these products;
however, the exact nature of the second reduction cannot be
elucidated from these results.
EC mechanism. At slower scan rates, the product of the chemical
reaction is likely diffusing away from the surface and undergoing
further reaction before it could be oxidized; however, higher
scan rates allow for reoxidation before further reaction. One
hypothesis is that the odd electron species 2a is undergoing a
very fast endocyclic N–O bond cleavage to give
the ring-opened species 2c. A similar process has
been reported for oximes, which upon electro-
chemical reduction, form the radical anion that
quickly cleaves to form hydroxide ion and an
imine radical.[6] The rate of the N–O bond cleav-
age in oximes is fast enough that the oxidation
wave for the initial radical anion is not observed.
In this case, it is also reasonable to believe that
the chemical step would be faster than subse-
quent electron transfer on BDD, which is known
to possess semi-metal characteristics.[9] Subse-
quent reduction of 2c (or a similar ring-opened
species) could result in formation of oxime 3,
which was observed as a product of the con-
trolled potential electrolysis. The exact nature
of the species being oxidized (Peak II) and its
resulting product are still under investigation;
however, it has been previously reported that
2-hydroxyaryl oximes analogous to 3 have been
Scheme 1. Proposed mechanism for reduction of N-oxide 1a
wileyonlinelibrary.com/journal/poc
Copyright © 2014 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2014, 27 540–544

ELECTROCHEMICAL REDUCTION OF 3-PHENYL-1,2-BENZISOXAZOLE 2-OXIDE
of the electrochemical cell was reported previously.[13] BDD electrodes
electrochemically oxidized to give benzisoxazole N-oxides
1a,[10] making it conceivable that the species being oxidized
could be 2c, 2d or a related species.
were obtained from Greg Swain at Michigan State University and were
grown using a CH4/H2 ratio of 0.5%. Details of the electrode growing
conditions have been reported previously.[14,15] The diamond working
electrode was pressed against the bottom of the glass cell and a
Previously reported in the literature, the ortho quinone methide
2e intermediate has been proposed in the chemical oxidation of
oxime 3 to benzisoxazole N-oxide 1a.[10] A mechanism involving
the oxidation of 2c to 2e and subsequent cyclization to regenerate
1a would be consistent with our observed results. Modeling of the
proposed square scheme involving 1a, 2a, 2c and 2e yielded a CV
consistent with the experimental results, supporting a one-electron
reduction (see supporting information).
Analysis of the products of controlled potential electrolysis also
showed the presence of benzisoxazole 4. Formation of this prod-
uct can be rationalized, by protonation of initial radical anion 2a
to 2b, which can undergo a fast exocyclic N–O bond cleavage,
analogous to oximes, giving benzisoxazole 4 and hydroxyl radical.
An alternate pathway to 4 could involve recyclization and loss of
hydroxyl radical from 2d.
Chemraz® O-ring (Ace Glass, Inc.) was used to contain the solution and
define the exposed electrode area, 0.22 cm2. A non-aqueous Ag/AgCl
reference electrode (0.01 M AgNO3 + 0.1 M tetrabutylammonium perchlo-
rate in acetonitrile; CH Instruments) was positioned in close proximity to
the working electrode using a cracked-glass capillary filled with electro-
lyte solution. All potentials reported herein are referenced to this elec-
trode. Either a large-area carbon rod or a Pt wire served as the auxiliary
electrode. Electrical connection was made to the working electrode by
rubbing a graphite rod on the back side of the cleaned Si substrate prior
to contacting with a clean Cu plate. Compound 1a was dissolved in 0.1M
tetrabutylammonium tetrafluoroborate (TBATFB; Alfa Aesar) in acetonitrile
(ACN), to yield a final analyte concentration of 3.0mM. The TBATFB and
ACN were reagent grade or better and used without additional
purification. The proton study was performed using TBATFB that was dried
at room temperature under vacuum and a fresh bottle of extra dry acetoni-
The formation of 3 and 4 from N-oxide 1a must require an
overall two-electron reduction. Because the CV revealed only a
single one-electron reduction, it cannot be unequivocally
concluded that these products are being formed on the fast time
scale of the CV experiment. However, the observation of a one-
electron transfer would not be contradictory to a possible
secondary solution-electron transfer mechanism,[11] in which
case the second electron could be coming from the oxidation
of another single electron species in solution. In the case of the
controlled potential electrolysis, the much longer reaction times
and smaller solution volume (limiting diffusion) make it more
likely that the secondary reduction is happening at the electrode
surface, resulting in formation of 3 and 4.
trile 99.9% stored over molecular sieves and acrosealed® (Acros Organics).
Thin-layer electrolysis measurements
The products of the electrochemical reduction were isolated using
controlled potential electrolysis at a BDD electrode in a custom thin-
layer flow cell. The cell consisted of an Al bottom plate (in contact with
the back of the BDD) and a Teflon top piece, sandwiched together. A
gasket was cut out of an ethylene propylene diene monomer (EPDM)
sheet (McMaster-Carr, Cleveland, OH) to isolate the Al plate from
contacting the electrolyte solution. A channel for the solution was cut
into the Teflon piece and an inlet and outlet line was fed into/out of
the cell using steel tubing, with the latter serving as the auxiliary elec-
trode. A Luggin capillary for the non-aqueous Ag/AgCl electrode was
constructed out of a glass pipette and a Pt wire was extended through
the Teflon block to make electrical contact to the solution adjacent to
the BDD electrode. The electrolysis solution was fed into the cell using
a syringe pump so the reaction could be investigated on both flowing
and stationary aliquots.
CONCLUSION
The results of the electrochemical analysis of 3-phenyl-1,2-
benzisoxazole 2-oxide (1a) using a combination of cyclic and
SWV showed a quasi-reversible one-electron reduction followed
by a very fast chemical step involving cleavage of either the
exocyclic or endocyclic N–O bonds. Subsequent electron transfer
and protonation resulted in an overall two-electron reduction and
formation of oxime 3 and benzisoxazole 4. These results are analo-
gous to those observed in the electrochemical reduction of other
heterocyclic N-oxides albeit the reduction of the benzisoxazole
N-oxides takes place at a more negative potential. This could
in part be a function of the BDD electrode and not just that of
the substrate. However, these encouraging results warrant
further investigation into the reduction potential of substituted
benzisoxazole N-oxides as well as to elucidate and characterize
the nature of the intermediate species involved.
HPLC/PDA analysis
Reaction mixtures were analyzed on a Shimadzu 20LC with CBM 20A PDA
detector. Separation (40 μL injection) was accomplished on a 25 cm × 4.6
mm, 5 μm C18 column using 60:40 water:methanol mobile phase at a
flow rate of 2.5 mL/min with detection at 254 nm. The chromatograms
showed three identifiable peaks attributed to 3 (rt 7.72 min; λmax 206,
258, 305 nm), 1a (rt 9.07 min; λmax 200, 241, 288 nm) and
(rt 15.21 min; λmax 200, 232, 289 nm).
4
REFERENCES
[1] P. W. Crawford, R. G. Scamehorn, U. Hollstein, M. D. Ryan, P. Kovacic,
Chem. Biol. Interact. 1986, 60, 67.
[2] C. Olea-Azar, C. Rigol, F. Mendizabal, H. Cerecetto, M. R. Di,
M. Gonzalez, W. Porcal, A. Morello, Y. Repetto, J. D. Maya, Lett. Drug
Des. Discov. 2005, 2, 294.
[3] H. Cerecetto, M. R. Di, M. Gonzalez, M. Risso, P. Saenz, G. Seoane,
A.Denicola,G.Peluffo,C.Quijano,C.Olea-Azar,J. Med. Chem. 1999,42, 1941.
[4] K. Reybier, T. H. Y. Nguyen, H. Ibrahim, P. Perio, A. Montrose,
P.-L. Fabre, F. Nepveu, Bioelectrochemistry 2012, 88, 57.
[5] I. Surov, H. Lund, Acta Chem. Scand. 1986, 40, 831.
EXPERIMENTAL
Preparation of 3-phenyl 1,2-benzisoxaozle 2-oxide (1a)
Compound 1a was prepared as previously reported in the literature,
starting with 2-hydroxy benzophenone.[12]
[6] B. Soucaze-Guillous, H. Lund, Acta Chem. Scand. 1998, 52, 417.
[7] J.-M. Savéant, Elements of Molecular and Biomolecular Electrochemistry:
Electrochemical measurements
An Electrochemical Approach to Electron Transfer Chemistry, Wiley-
Interscience, Hoboken, NJ, 2006.
[8] D. P. Valencia, F. J. Gonzalez, Electrochem. Commun. 2011, 13, 129.
All electrochemical measurements were performed at room temperature
using a single-compartment glass cell and a CH Instruments model 842C
Series Electrochemical Analyzer (CH Instruments, Austin, TX). The design
J. Phys. Org. Chem. 2014, 27 540–544
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/poc

M. KOCIOLEK, J. BENNETT AND J. CASBOHM
[9] J. Wang, G. M. Swain, M. Mermoux, G. Lucazeau, J. Zak, J. W. Strojek,
New Diamond Front. Carbon Technol. 1999, 9, 317.
[10] A. J. Boulton, P. G. Tsoungas, J. Chem. Soc. Perkin Trans. 1986,
1, 1665.
[14] D. Y. Kim, J. C. Yang, H. W. Kim, G. M. Swain, Electrochim. Acta 2013,
94, 49.
[15] J. A. Bennett, G. M. Swain, J. Electrochem. Soc. 2010, 157, F89.
[11] D. H. Evans, Chem. Rev. 1990, 90, 739.
SUPPORTING INFORMATION
[12] M. G. Kociolek, O. Hoermann, Synthetic Comm. 2012, 42, 2632.
[13] M. C. Granger, M. Witek, J. Xu, J. Wang, M. Hupert, A. Hanks,
M. D. Koppang, J. E. Butler, G. Lucazeau, M. Mermoux, J. W. Strojek,
G. M. Swain, Anal. Chem. 2000, 72, 3793.
Additional supporting information may be found in the online
version of this article at the publisher’s website.
wileyonlinelibrary.com/journal/poc
Copyright © 2014 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2014, 27 540–544