Electrochemical and mARC-catalyzed enzymatic reduction of para-substituted benzamidoximes: Consequences for the prodrug concept "amidoximes instead of amidines"

Article abstract of DOI:10.1002/cmdc.201402437

The mitochondrial amidoxime reducing component (mARC) activates amidoxime prodrugs by reduction to the corresponding amidine drugs. This study analyzes relationships between the chemical structure of the prodrug and its metabolic activation and compares its enzyme-mediated vs. electrochemical reduction. The enzyme kinetic parameters K_M and V_max for the N-reduction of ten para-substituted derivatives of the model compound benzamidoxime were determined by incubation with recombinant proteins and subcellular fractions from pig liver followed by quantification of the metabolites by HPLC. A clear influence of the substituents at position 4 on the chemical properties of the amidoxime function was confirmed by correlation analyses of ¹H NMR chemical shifts and the redox potentials of the 4-substituted benzamidoximes with Hammett's σ. However, no clear relationship between the kinetic parameters for the enzymatic reduction and Hammett's σ or the lipophilicity could be found. It is thus concluded that these properties as well as the redox potential of the amidoxime can be largely ignored during the development of new amidoxime prodrugs, at least regarding prodrug activation.

Full text of DOI:10.1002/cmdc.201402437

DOI: 10.1002/cmdc.201402437
Full Papers
Electrochemical and mARC-Catalyzed Enzymatic Reduction
of para-Substituted Benzamidoximes: Consequences for
the Prodrug Concept “Amidoximes instead of Amidines”
Eva Bauch,[a] Debora Reichmann,[b] Ralf-Rainer Mendel,[b] Florian Bittner,[b] Anne-
Marie Manke,[c] Philipp Kurz,*[c] Ulrich Girreser,[a] Antje Havemeyer,[a] and Bernd Clement*[a]
The mitochondrial amidoxime reducing component (mARC) ac-
tivates amidoxime prodrugs by reduction to the corresponding
amidine drugs. This study analyzes relationships between the
chemical structure of the prodrug and its metabolic activation
and compares its enzyme-mediated vs. electrochemical reduc-
tion. The enzyme kinetic parameters Km and Vmax for the N-re-
duction of ten para-substituted derivatives of the model com-
pound benzamidoxime were determined by incubation with
recombinant proteins and subcellular fractions from pig liver
followed by quantification of the metabolites by HPLC. A clear
influence of the substituents at position 4 on the chemical
properties of the amidoxime function was confirmed by corre-
1
lation analyses of H NMR chemical shifts and the redox poten-
tials of the 4-substituted benzamidoximes with Hammett’s s.
However, no clear relationship between the kinetic parameters
for the enzymatic reduction and Hammett’s s or the lipophilici-
ty could be found. It is thus concluded that these properties as
well as the redox potential of the amidoxime can be largely ig-
nored during the development of new amidoxime prodrugs, at
least regarding prodrug activation.
Introduction
The mitochondrial amidoxime reducing component (mARC)
presents the fourth molybdenum-containing enzyme known in
humans, besides sulfite oxidase, aldehyde oxidase, and xan-
thine oxidoreductase.[1] It was discovered in 2006 in our re-
search group, as reported by Havemeyer et al.[2] All hitherto-an-
alyzed mammals show the two isoforms mARC1 and mARC2.
For both isoforms, protein variants encoded by non-synony-
mous single-nucleotide polymorphisms (SNPs) in the mARC
genes of healthy Caucasians are known, and in functional char-
acterizations by Ott et al., no relevant genetic polymorphisms
could be detected.[3] mARC1 and mARC2 are present in the
outer mitochondrial membrane of rat and porcine liver.[2,4] The
expression of human mARC2 is down-regulated in colon
tumors of human tissue samples and is up-regulated in diabet-
ic rat kidneys as well as by glucose in human and rat renal
cells.[5,6] Together with the mitochondrial isoform cytochro-
me b5 and cytochrome b5 reductase, mARC forms an N-reduc-
tive enzyme system with the nitric oxide (NO) precursor N4-hy-
droxy-l-arginine as a putative physiological substrate.[7,8] Under
anaerobic conditions, the mARC enzyme system catalyzes the
reduction of nitrite to NO.[9] Within this system, the two iso-
forms mARC1 and mARC2 show overlapping substrate specific-
ity, with the exception of N-oxides such as amitriptyline-N-
oxide or nicotinamide-N-oxide.[10] The participation of the
enzyme system in lipid synthesis in adipocyte mitochondria as
well as regulation by fasting and high-fat diet in mice was re-
cently discussed.[11,12] In addition, the mARC enzyme system
detoxifies N-hydroxylated base analogues, participates in the
metabolism of the antibiotic sulfamethoxazole, and is essential
for the activation of such amidoxime prodrugs as the serine
protease inhibitor Mesupron.[13–15] A number of drugs contain
amidine functions with high basicity; this results in insufficient
oral absorption because of protonation of the double-bonded
nitrogen atom at physiological pH. To improve oral absorption
and thus bioavailability, it has been shown that amidoximes
can be used as prodrugs instead of amidines.[16] Because of the
more electronegative oxygen atom as part of their structures,
the basicity is lowered, and non-protonated N-hydroxyami-
dines (amidoximes) are bioavailable at physiological pH.
[a] E. Bauch, Dr. U. Girreser, Dr. A. Havemeyer, Prof. Dr. B. Clement
Department of Pharmaceutical and Medicinal Chemistry
Christian Albrechts University Kiel
Gutenbergstraße 76, 24118 Kiel (Germany)
E-mail: bclement@pharmazie.uni-kiel.de
The strict dependence of enzymatic activity on a molybde-
num cofactor in the active site is well established.[17] The phys-
iological function of mARC as well as details of the protein’s
structure and mARC inhibitors are now studied intensively. Fur-
thermore, no structure–activity relationships for the reduction
of N-hydroxylated compounds by the mARC-containing
enzyme system have been described so far, making a more ra-
tional development of new amidoxime prodrugs difficult.
[b] Dr. D. Reichmann, Prof. Dr. R.-R. Mendel, Dr. F. Bittner
Department of Plant Biology, Technical University Braunschweig
Humboldtstraße 1, 38106 Braunschweig (Germany)
[c] A.-M. Manke, Prof. Dr. P. Kurz
Institute for Inorganic and Analytical Chemistry
Albert Ludwigs University Freiburg
Albertstraße 21, 79104 Freiburg (Germany)
E-mail: philipp.kurz@ac.uni-freiburg.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402437.
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The fact that the mARC enzyme system catalyzes a redox re-
action led us to the assumption that a link between the redox
potentials of structurally similar substrates and the enzymatic
reaction may exist. Thus, one might expect a relationship be-
tween the reduction potentials of the substrates and the rate
at which they are converted by mARC, to be similar to a linear
free-energy relationship.[18] If appropriate, higher values for
Vmax should be observed for compounds with higher Ered
values. To analyze this, we present herein a comparison of the
electrochemical and enzymatic reductions of ten 4-substituted
benzamidoximes with large differences in their electron densi-
ties. In each case, the Michaelis–Menten parameters Km and
Vmax for the N-reduction by outer mitochondrial membrane
vesicles (OMM) or by two recombinant enzymes were deter-
mined and compared. This was achieved by incubation of the
benzamidoximes followed by quantification of the correspond-
ing benzamidine metabolites using HPLC analysis. All investi-
gated compounds were enzymatically reduced by OMM as
well as by the recombinant proteins, with small differences in
N-reductive activity for OMM, and larger differences for the re-
combinant proteins. On the other side, correlations between
15N NMR chemical shifts of the 4-substituted benzamidoximes
with Hammett’s s showed that physicochemical properties of
the amidoxime function are affected by ring substituents.[19] To
support this finding, correlation analyses between 1H NMR
chemical shifts and the redox potentials of 4-substituted ben-
zamidoximes with Hammett’s s were performed in this study.
As presented below, we observed profound influences of the
Table 1. 1H NMR chemical shifts of 4-substituted benzamidoximes 1.
Substituent
s[a]
1
d [ppm]
NOH
NH2
NH2
OH
OCH3
CH3
H
Cl
Br
CF3
CN
NO2
À0.57
À0.38
À0.28
À0.14
0.00
0.24
0.26
0.53
0.70
1a
1b
1c
1d
1e
1 f
1g
1h
1i
9.16
9.33
9.43
9.50
9.60
9.71
9.72
9.90
10.01
10.12
5.49
5.61
5.70
5.72
5.78
5.85
5.85
5.96
5.99
6.05
0.81
1j
[a] Values taken from ref. [20].
Table 2. Correlations of 1H NMR chemical shifts (ppm) with Hammett
s values.
Functional group
Equation
r2[a]
Amino group of 1a–j
Oxime group of 1a–j
d(NH2)=(0.37Æ0.02)s+(5.76Æ0.01)
d(NOH)=(0.65Æ0.01)s+(9.57Æ0.01)
0.976
0.993
Confidence intervals, p=0.95, [a] n=10.
electronic interactions for the oxime protons than the amine
protons. Both regressions showed a linear dependence of the
chemical shifts from the electronic properties of the substitu-
ent, indicated by values for r2 of 0.976 for the amine protons
and 0.993 for the oxime protons (Table 2). This means that the
electronic situation at the amidoxime function is indeed
strongly affected by the para substituent.
1
para-substituents on both redox potentials and H NMR chemi-
cal shifts. However, comparison of electrochemical vs. enzymat-
ic reductions revealed fundamental differences between both,
because no relation between Vmax and Ered was observed. Fur-
thermore, clear correlations could also not be established be-
tween the electronic properties or the lipophilicity of the sub-
strate and the enzyme kinetic parameters. These results are im-
portant for the use of the prodrug principle “amidoximes in-
stead of amidines”, as we thus show that this concept can ap-
parently be used irrespective of the electronic properties of
the substituents at the benzamidoxime ring. They also support
the current mechanistic proposal for mARC of a molybdenum-
mediated reduction process taking place in a substrate-specific
binding pocket.[1]
Electrochemical behavior of benzamidoxime and determina-
tion of redox potentials of 4-substituted benzamidoximes
Prior to determination of the redox potentials of 4-substituted
benzamidoximes, the electrochemical behavior of the parent
compound (R=H) was analyzed in detail. Cyclic voltammetry
(CV) measurements for the reduction of benzamidoxime were
carried out in aqueous phosphate buffer (pH 6.0). To be able
to reach the necessary highly negative potentials, a hanging
mercury drop electrode (HMDE) was used to benefit from the
large over-potentials for proton reduction known for Hg. CV re-
vealed two irreversible reductions at À1.2 and À1.4 V (Fig-
ure 1A; NB: all potentials herein are given vs. normal hydrogen
electrode, NHE). Differential pulse voltammetry (DPV) of benza-
midoxime confirmed the presence of these two reduction
peaks detected by CV. Increasing concentrations of benzami-
doxime led to increased cathodic currents in the DPV, indicat-
ing that the detected cathodic peaks are indeed caused by the
reduction of benzamidoxime (Figure 1B). For both CV and DPV,
control experiments for the neat buffer electrolyte resulted in
hardly any reduction currents (Figure 1).
Results
1H NMR spectroscopy of 4-substituted benzamidoximes
1H NMR chemical shifts of the oxime and amine protons of 4-
substituted benzamidoximes were measured in [D6]DMSO
under identical experimental conditions to obtain comparable
data for a correlation analysis between chemical shifts and
Hammett constants s (Table 1). Electron-withdrawing substitu-
ents with a positive s led to downfield-shifted signals for
oxime and amine protons (Table 2). In the respective Hammett
correlation plots (Supporting Information, Figure S1), the slope
of 0.65 in the case of the oxime protons in contrast to only
0.37 for the amine protons indicated a greater susceptibility to
To identify the electrochemical reduction product, a prepara-
tive electrochemical reduction of benzamidoxime in phosphate
buffer (pH 6.0) was carried out at À1.2 V using a mercury pool
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Figure 1. Electrochemical reduction of benzamidoxime. A) Cyclic voltammogram for the reduction of benzamidoxime (5.5 mm) in 0.5m phosphate buffer
(pH 6.0); scan rate: 100 mVsÀ1. B) Differential pulse voltammograms for the reduction of benzamidoxime at various concentrations.
trolysis of benzamidoxime at a retention time of 3.9 min (Sup-
porting Information, Figure S2A,B). This signal was detected
for the electrolysis of a pure buffer solution too, indicating that
the peak seems not to be caused by benzamidoxime, but by
some unknown side reaction in the electrochemical cell.
Nothing is known so far about the second reduction event
at À1.4 V. Maybe the presence of the second reduction peak
could be explained with the reduction from the amidine to the
amine as described for ethanediamidoxime,[21] but further ex-
periments are necessary to clearly identify the product of the
second reduction step.
Having established the fundamental reduction chemistry of
benzamidoxime, we next determined the redox potentials of
the ten different 4-substituted benzamidoximes used in this
study by DPV. In each case, two reduction peaks were detected
Figure 2. HPLC analysis of the reaction product of the electrochemical re-
duction of benzamidoxime (0.5 mm) in 0.5m potassium phosphate buffer
similar to the case of R=H described above. Significant differ-
ences were found: the reduction of 4-cyanobenzamidoxime re-
(pH 6.0) at À1.2 V at various stages of the electrolysis. Retention times:
10.9 min: benzamidine; 11.7 min: benzamidoxime.
quired with À0.81 and À0.95 V the least, and 4-hydroxybenza-
midoxime with À1.17 and À1.49 V the most negative reduc-
tion potentials (Table 3).
electrode. Samples taken from the electrolysis cell at various
time intervals were analyzed by HPLC. The first sample, taken
and analyzed before starting the electrolysis, showed only ben-
zamidoxime with a retention time of 11.7 min in the HPLC
trace (Figure 2). During the course of the electrolysis, this HPLC
signal decreased while a new, well-separated peak at 10.9 min
appeared in the chromatogram. The injection of a benzamidine
reference sample confirmed this as the retention time of ben-
zamidine, and we thus conclude that the electrochemical
event at À1.2 V can be assigned to the two-electron reduction
of benzamidoxime to benzamidine (Scheme 1). However,
a second HPLC signal occurred and increased during the elec-
Analysis of the relationship between the electrochemical
properties of the substituent characterized by Hammett’s
Table 3. Reduction potentials of 4-substituted benzamidoximes.
Substituent
s[a]
1
Ered1 [V][b]
Ered2 [V][b]
NH2
OH
OCH3
CH3
H
Cl
Br
CF3
CN
NO2
À0.57
À0.38
À0.28
À0.14
0.00
0.24
0.26
0.53
0.70
1a
1b
1c
1d
1e
1 f
1g
1h
1i
À1.10
À1.17
À1.09
À1.08
À1.09
À1.01
À0.97
À0.99
À0.81
À1.10[c]
À1.36
À1.49
À1.42
À1.44
À1.36
À1.26
À1.24
À1.17
À0.95
À1.35[c]
0.81
1j
[a] Values taken from ref. [20]. [b] Potentials determined by differential
pulse voltammetry of degassed 1 mm solutions or 1:10 dilutions of satu-
rated solutions in 0.5m potassium phosphate buffer (pH 6.0) at HMDE.
[c] Identical potentials to 1a most likely due to the reduction of the nitro
group at À0.07 V.
Scheme 1. Reduction of benzamidoxime to benzamidine.
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s and the reduction potentials by linear regression analysis led
to the expected general trend that electron-withdrawing sub-
stituents with positive s values facilitate reduction of the re-
spective benzamidoximes. Linear fits for plots of Ered vs. s re-
sulted in positive slopes, supporting this hypothesis, although
the values for r2 with 0.845 and 0.929 for the first and second
reduction steps are relatively low (Table 4 and Supporting In-
formation, Figure S3).
formation, Figure S6). The enzyme kinetic parameters Km and
Vmax determined for OMM differed only slightly for the various
substrates. The highest conversion rate was detected for 4-
methylbenzamidoxime (1d) and the lowest for 4-nitrobenzami-
doxime (1j), showing an approximate twofold difference
(Table 5). The same trend was observed for the recombinant
proteins, but here much greater differences between the high-
est (1d) and lowest (1j) Vmax were observed, with values differ-
ing approximately 26-fold for mARC1 and 12-fold for mARC2
(Table 6).
Table 4. Correlations of reduction potentials (V) with Hammett s values.
Equation
r2[a]
Table 5. Enzyme kinetic parameters of the reduction of 4-substituted
E
E
red1 =(0.26Æ0.04)sÀ(1.06Æ0.02)
red2 =(0.44Æ0.04)sÀ(1.34Æ0.02)
0.845
0.929
benzamidoximes with OMM.
[c,d]
Compd
s[a]
p[b]
Vmax
Km [mm][d]
n[e]
Confidence intervals, p=0.95, [a] n=8 (1b–i).
1a
1b
1c
1d
1e
1 f
1g
1h
1i
À0.57
À0.38
À0.28
À0.14
0.00
0.24
0.26
0.53
0.70
À1.23
À0.67
À0.02
0.56
0.00
0.71
0.86
0.88
À0.57
À0.28
252Æ31
232Æ28
237Æ10
353Æ31
208Æ24
220Æ20
246Æ17
256Æ18
240Æ12
166Æ3
0.073Æ0.004
0.098Æ0.007
0.083Æ0.017
0.100Æ0.017
0.066Æ0.016
0.087Æ0.017
0.066Æ0.009
0.157Æ0.040
0.240Æ0.051
0.258Æ0.036
4
5
5
4
5
6
5
5
5
4
The electrochemical behavior of 4-nitrobenzamidoxime (1j)
proved to be different from the other compounds, as a third
reduction event at À0.07 V was observed in this case (Support-
ing Information, Figure S4). Because the other two reduction
potentials are identical to Ered1 and Ered2 determined for 4-ami-
nobenzamidoxime (1a), we assume that the nitro group of 1j
is first reduced to an amine to yield 1a, which is then convert-
ed into the corresponding benzamidine (Table 3). Therefore, 4-
nitrobenzamidoxime was not considered in the regression
analysis of Table 4/Figure S3, Supporting Information. Similarly,
the deviant redox behavior of the amino compound 1a might
be explained by formation of the phenylogues guanidine 4-
aminobenzamidine as reduction product. The resulting differ-
ent electrochemical properties of 1a were therefore also not
considered for the correlations of Ered vs. s.
1j
0.81
[a] Values taken from ref. [20]. [b] Values taken from ref. [22]. [c] Vmax
:
nmolminÀ1 (mg total protein)À1. [d] Values are the meanÆSD of n inde-
pendent experiments performed in single determination. [e] Number of
experiments; outliers detected by Nalimov test were excluded (Support-
ing Information, Table S1).[23]
Table 6. Enzyme kinetic parameters of the reduction of 4-substituted
benzamidoximes with the recombinant mARC enzyme system.
[a,b]
[a,b]
Compd
Vmax
Km [mm][b]
n[c]
Vmax
Km [mm][b]
n[c]
1a
1b
1c
1d
1e
1 f
1g
1h
1i
154Æ21
52Æ3
0.64Æ0.24
0.10Æ0.02
0.57Æ0.17
0.79Æ0.33
0.52Æ0.17
1.69Æ0.36
0.61Æ0.21
2.28Æ0.82
1.00Æ0.27
0.11Æ0.02
4
5
4
5
5
4
5
5
5
5
497Æ37
142Æ7
487Æ59
825Æ93
275Æ8
468Æ56
546Æ19
666Æ65
437Æ41
68Æ1
0.62Æ0.12
0.17Æ0.02
0.34Æ0.06
0.51Æ0.24
0.80Æ0.14
0.51Æ0.16
0.63Æ0.10
1.32Æ0.22
0.53Æ0.13
0.08Æ0.02
4
5
5
5
5
5
5
5
5
4
In vitro reduction assays
Incubation of all investigated 4-substituted benzamidoximes
with OMM or the recombinant proteins mARC1 or mARC2 led
to the formation of their corresponding benzamidine metabo-
lites (Scheme 2), which could be quantified by HPLC. The kinet-
283Æ37
523Æ74
161Æ30
351Æ36
313Æ91
495Æ87
129Æ31
20Æ2
1j
[a] Vmax : nmolminÀ1 (mg total protein)À1. [b] Values are the meanÆSD of n
independent experiments performed in single determination. [c] Number
of experiments; outliers detected by Nalimov test were excluded (Sup-
porting Information, Tables S2 and S3).[23]
Scheme 2. Enzymatic reduction of 4-substituted benzamidoximes by the
mARC-containing N-reductive system.
For OMM, correlations between the terms log(Vmax/Km) (a
measure of biological activity) and the substituent’s Hammett
constant s and Hansch’s lipophilicitiy constant p (representa-
tive for the physicochemical properties of the substrate) were
analyzed (Table 7). The respective r2 values of 0.611 and 0.878
as well as the slope of the equations of ~0.5 are all quite low,
indicating a questionable relationship between these parame-
ters. Regression analyses with the data obtained with mARC1
ic parameters Km and Vmax obtained for the unsubstituted com-
pound benzamidoxime (1e) were similar to those of previous
studies, indicating that the protein batches used were suitable
for performing in vitro assays.[3,7,17] The rate of product forma-
tion was linear with time (up to 15 min in the case of OMM
and 8 min in the case of recombinant enzymes; Supporting In-
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duction potentials below À0.8 V are required for the reduc-
tions of all studied benzamidoximes in aqueous solution at
near neutral pH. Reductive electrolysis of benzamidoxime cou-
pled to HPLC analyses verified that the corresponding benza-
midines (and thus the enzymatic product) are products of the
electrochemical reductions, yet they occur at much lower re-
duction potentials than within the enzyme.
Table 7. Enzyme kinetic parameter correlations for 1b–j.
Enzyme Equation
r2[a]
mARC1 log(Vmax/Km)=À(0.49Æ0.10)s+(2.59Æ0.04)
0.701
0.611
0.878
OMM
OMM
log(Vmax/Km)=À(0.48Æ0.11)s+(3.41Æ0.05)
log(Vmax
Km)=À(0.50Æ0.07)s+(0.22Æ0.05)p+(3.38Æ0.03)
/
Confidence intervals, p=0.95, [a] n=9.
Possible reasons for the differences between the electro-
chemical and enzymatic reduction on the one hand and the
expected and measured potentials on the other hand might
be the microenvironment of the substrate at the electrode sur-
face, which is, of course, not comparable to the reaction envi-
ronment within the enzyme.
led to similar results with a slope of 0.49 and r2 of 0.701,
whereas no dependence could be found for mARC2. As well as
in the electrochemical reduction, 4-aminobenzamidoxime
showed a deviant reduction behavior and was therefore ex-
cluded from the correlation analysis.
The postulated reaction mechanism for benzamidoxime re-
duction by mARC is based on coordination of the deprotonat-
ed substrate to the MoIV form of the molybdenum cofactor at
the catalytic site of mARC (Scheme 3).[1] This substrate bonding
Although differences were observed in the extent of N-re-
duction for the investigated derivatives as well as some corre-
lations with low r2 values between N-reductivity and Ham-
mett’s s, it seems to be more likely that no clear relationship
between these parameters exists. Furthermore, no correlation
between Vmax and Ered was detected (Supporting Information,
Figure S7). Thus, there seem to be considerable differences be-
tween the electrochemical and the enzymatic reduction path-
ways.
Scheme 3. Probable key intermediate for the molybdenum-mediated enzy-
matic reduction of benzamidoximes.[1]
Discussion
Several benzamidoxime derivatives were used as model sub-
strates to investigate effects of para substituents in amidoxime
prodrug activation. In this study we demonstrated for the first
time that differences in the enzymatic rates of N-reduction by
mARC are not caused by electronic properties or lipophilicity
of para substituents. The highest conversion rates were detect-
ed for 4-methylbenzamidoxime, and the lowest for 4-nitroben-
zamidoxime for incubation with OMM, mARC1, and mARC2.
could greatly facilitate the reduction process and thus explain
how benzamidoximes can be reduced with NADH as the elec-
tron source, despite the very low reduction potentials deter-
mined here for the free substrate in solution. The influence of
the substituent on this binding step might be much more im-
portant than the substituent-induced differences in redox po-
tentials. This could explain why electron-withdrawing substitu-
ents significantly facilitate the electrochemical reduction,
whereas the enzymatic reduction of, for example, 4-nitrobenza-
midoxime by OMM, mARC1, and mARC2 is clearly slowed rela-
tive to benzamidoxime itself. X-ray absorption structure analy-
sis indicates the presence of a dioxido MoVI site.[25] This sup-
ports the proposed catalytic cycle, with the Mo ion cycling be-
tween mono-oxido MoIV and di-oxido MoVI oxidation states.
Two sequential eÀ/H+ transfer steps catalyzed by cytochro-
me b5 and its reductase regenerate the catalytically active MoIV
state.
For OMM and mARC1, the weak correlation between log(Vmax
/
Km) and Hammett’s s suggests, counterintuitively, that elec-
tron-withdrawing substituents decrease, and electron-donating
groups increase, the extent of enzymatic N-reduction. Howev-
er, the values for r2 and the slope of the regression curves
were relatively low. As a consequence, we rather conclude that
there is no clear relationship between the electronic properties
of the substituent at the 4-position and the rate of enzymatic
N-reduction.
Even so, our experiments concerning the correlation of elec-
trochemical properties and enzymatic reaction rates revealed
two unexpected trends: First, one would expect that electron-
withdrawing groups facilitate benzamidoxime reduction. We
could indeed show such a relationship for the electrochemical
reduction, for which a good correlation between the deter-
mined redox potentials and Hammett’s s was observed. How-
ever, this trend seems to be unimportant for the enzymatic re-
duction reaction. Second, one would expect the reduction po-
tentials of the substrates for the mARC enzyme system to be
higher than the potential of the biochemical electron donor
for the reaction (NADH), which is À310 mV against NHE.[24]
However, our electrochemical analysis clearly reveals that re-
However, further experiments are needed to explain the ob-
served kinetic differences in greater detail. Furthermore, details
of the mechanism of the enzymatic reduction by mARC are
still unknown. Therefore, it might also be interesting to investi-
gate the reactions of MoIV model compounds with benzami-
doxime.[26] If suitable MoIV species are found to catalyze the re-
duction to benzamidines at mildly reducing potentials, this
would confirm the hypothesis that the formation of a coordina-
tive bond between MoIV and the benzamidoxime is crucial for
turnover in the enzyme. Moreover, it should be kept in mind
that our hypothesis mentioned above—that the electronic
properties or lipophilicity of the substituent are without signifi-
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1H NMR spectroscopy of 4-substituted benzamidoximes: All
spectra were recorded with a Bruker Avance III 300 instrument,
using the manufacturer’s pulse programs. The substituted benza-
midoximes (0.5–1.0 mg) were dissolved in [D6]DMSO (0.6 mL) and
measured at 300 MHz with [D5]DMSO (d=2.50 ppm) as internal
standard at 300 K.
cant influence on the enzymatic reduction—is based on in
vitro studies with a relatively small number of samples. Only
substituents at the para position were considered, and nothing
is known, for example, about the influence of steric effects. Ini-
tial experiments led to the assumption that the molybdenum
cofactor of mARC is localized at the surface of the enzyme, but
as soon as more details about the structure of mARC are
known, 3D-QSAR should be carried out to verify the hypothesis
and to simplify the rational development of new amidoxime
prodrugs.
Electrochemistry
Cyclic voltammetry and differential pulse voltammetry: Electro-
chemical measurements were performed on a Metrohm 797 VA
Computrace potentiostat equipped with a Metrohm Multi Mode
Electrode unit for measurements using mercury working electro-
des. The three-electrode setup consisted of a hanging mercury
drop electrode (HMDE) as working electrode, a Ag/AgCl (3m KCl,
+210 mV vs. NHE) reference electrode, and a platinum wire auxili-
ary electrode, all supplied by Metrohm. Cyclic voltammetry (CV)
and differential pulse voltammetry (DPV) experiments were carried
out at room temperature under an argon atmosphere. For the de-
termination of redox potentials by DPV, degassed 1 mm solutions
or 1:10 dilutions of saturated solutions of the substituted benzami-
doximes in 0.5m potassium phosphate buffer (pH 6.0) were used.
Conclusions
We demonstrated that the electronic properties of the sub-
stituents at the para position of benzamidoximes influence the
physicochemical properties of the amidoxime function, repre-
1
sented in this study by H NMR chemical shifts and redox po-
tentials, but differences in the enzymatic N-reduction activity
could not be explained by these electronic substituent effects.
The results furthermore indicate significant mechanistic differ-
ences between the electrochemical reduction of the free sub-
strate and the enzymatic reduction process at the active site of
mARC.
Electrolysis coupled with HPLC: A mercury pool, Ag/AgCl (3m
KCl), and a platinum wire were used as working, reference, and
auxiliary electrodes, respectively, in a coulometry cell provided by
Princeton Applied Research (Supporting Information, Figure S5).
Benzamidoxime (20 mL, 0.5 mm) in 0.5m potassium phosphate
buffer (pH 6.0) was degassed with nitrogen and electrolyzed at
À1.2 V for 45 min. Samples (250 mL) were taken after 0, 10, 30, and
45 min of electrolysis and analyzed by HPLC (20 mL sample loop). A
Kinetex C18 5 mm, 4.6 mmꢁ250 mm column (Phenomenex Inc.,
Aschaffenburg, Germany) as stationary phase and 20 mm K2HPO4
with 0.1% trifluoroacetic acid (TFA, pH 4.0) and 5% (v/v) CH3CN as
mobile phase were used. Retention times were: 10.9 min (benzami-
dine) and 11.7 min (benzamidoxime); eluent flow rate:
0.6 mLminÀ1; UV detection wavelength: 229 nm.
Besides, the applicability of the prodrug principle “amidox-
imes instead of amidines” is not influenced by the redox po-
tential of the amidoximes. This is consistent with previous
studies concerning the metabolic activation of a very broad
spectrum of N-hydroxylated prodrugs by the mARC enzyme
system.[1,14,16,27] Prodrugs containing N-hydroxy functional
groups are rapidly reduced by mARC to the active drugs with-
out P450-dependent interactions, making this prodrug princi-
ple valuable for further development of new prodrugs. The in-
fluence of substituents can be neglected. Thus, amidoximes
can be used as prodrugs for a large number of amidines. In ad-
dition, the independence of the N-reduction from the redox
potential of the substrate is highly relevant to the involvement
of mARC in detoxification pathways like the reduction of N-hy-
droxylated base analogues.
In vitro reduction assays and determination of the enzyme
kinetic parameters
Preparation of OMM: Pig liver mitochondria were obtained by
gradient centrifugation with Percoll as described by Hovius et al.[29]
and Krompholz et al.[13] The outer mitochondrial membrane was
purified by using the swell disruption method, and a sucrose den-
sity gradient centrifugation according to Havemeyer et al.[2]
Experimental Section
General
Expression and purification of recombinant human proteins:
mARC1 (reference sequence NP_073583) and mARC2 (reference se-
quence NP_060368) were expressed in Escherichia coli TP1000 and
mitochondrial cytochrome b5 (reference sequence NP_085056) as
well as NADH cytochrome b5 reductase isoform 2 (reference se-
quence NP_015565) in E. coli DL41 as previously described by Wahl
et al.[17]
Chemicals and reagents: 4-Trifluoromethylbenzamidoxime, 4-
methylbenzamidoxime and metabolites as well as 4-methoxyben-
zamidoxime were kindly supplied by Dr. Lehmann (Bayer Health-
Care Pharmaceuticals, Berlin, Germany). 4-Bromobenzamidoxime,
4-chlorobenzamidoxime, 4-cyanobenzamidoxime and 4-nitrobenza-
midoxime were obtained by reaction of hydroxylamine with the
benzonitriles according to Girreser et al.[19] The corresponding ben-
zamidines were prepared by base-catalyzed formation of the inter-
mediate imido ester starting from the benzonitriles and reaction
with ammonium chloride, as described by Moss et al.[28] All other
chemicals were purchased from AppliChem GmbH (Darmstadt,
Germany), J. T. Baker (Deventer, Holland), Merck KGaA (Darmstadt,
Germany), Carl Roth GmbH (Karlsruhe, Germany) and Sigma–Al-
drich Chemie GmbH (Taufkirchen, Germany), unless otherwise
stated.
Determination of protein concentrations: Protein concentrations
were determined with the BCA protein assay kit (Pierce, Rockford,
IL, USA) according to the manufacturer’s instructions, and bovine
serum albumin as standard.
Determination of prosthetic groups: The FAD and heme content
of cytochrome b5 reductase and cytochrome b5 was determined by
UV spectroscopy as described by Krompholz et al.[13]
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In vitro reduction assays: Incubations were carried out at 378C in
a shaking water bath under aerobic conditions. Experiments with
OMM were performed in 100 mm potassium phosphate buffer
(pH 6.0), with 2.5 mg protein and an incubation time of 15 min. For
incubations with recombinant proteins, 3.75 mg protein of mARC1
or mARC2, 37.5 pmol heme (cytochrome b5) and 3.75 pmol FAD
(cytochrome b5 reductase) in 20 mm MES (pH 6.0) and an incuba-
tion time of 8 min were used. With OMM and recombinant pro-
teins the total volume of the incubation mixture was 75 mL con-
taining 5% DMSO. After 3 min of pre-incubation in the water bath,
the reaction was started by addition of NADH (1 mm final concen-
tration). For termination, 75 mL ice-cold MeOH was added. Precipi-
tated proteins were sedimented by centrifugation (10000 rpm,
5 min, room temperature), and the supernatant was analyzed by
HPLC. All 4-substituted benzamidoximes were tested in parallel by
use of a self-constructed rack (Supporting Information, Figure S8).
For each compound, seven different substrate concentrations and
one control without protein were analyzed in one incubation. Each
experiment was repeated six times. Km and Vmax were calculated by
nonlinear regression analysis (Sigma Plot 11.0, SPSS Science, Chica-
go, IL, USA). The normal distribution of all raw data was tested by
the Shapiro–Wilk test. Outliers were detected with the Nalimov
test (Supporting Information, Tables S1–S3).[23]
Separation of 4-hydroxybenzamidoxime and 4-hydroxybenzami-
dine: Separations were carried out with 10 mm 1-octylsulfonate
sodium salt, 20 mm KH2PO4 (pH 3.0) and 15.0% (v/v) CH3CN as
mobile phase and UV detection at 258 nm. Retention times were
19.6Æ0.3 (4-hydroxybenzamidoxime) and 24.0Æ0.4 min (4-hydrox-
ybenzamidine).
Separation of 4-aminobenzamidoxime and 4-aminobenzami-
dine: Separations were carried out with 10 mm 1-octylsulfonate
sodium salt, 20 mm KH2PO4 (pH 3.0) and 15.0% (v/v) CH3CN as
mobile phase and UV detection at 299 nm. Retention times were
19.5Æ0.2 (4-aminobenzamidoxime) and 24.4Æ0.3 min (4-amino-
benzamidine).
Separation of 4-trifluoromethylbenzamidoxime and 4-trifluoro-
methylbenzamidine: Separations were carried out with 10 mm 1-
octylsulfonate sodium salt, 20 mm KH2PO4 (pH 3.0) and 27.5% (v/v)
CH3CN as mobile phase and UV detection at 224 nm. Retention
times were 14.6Æ0.1 (4-trifluoromethylbenzamidine) and 17.1Æ
0.1 min (4-trifluoromethylbenzamidoxime).
Separation of 4-chlorobenzamidoxime and 4-chlorobenzami-
dine: Separations were carried out with 20 mm KH2PO4 containing
0.1% TFA (pH 4.0) and 20.0% (v/v) CH3CN as mobile phase and UV
detection at 243 nm. Retention times were 3.9Æ0.1 (4-chloroben-
zamidine) and 6.3Æ0.1 min (4-chlorobenzamidoxime).
Separation of 4-bromobenzamidoxime and 4-bromobenzami-
dine: Separations were carried out with 20 mm KH2PO4 containing
0.1% TFA (pH 6.0) and 30.0% (v/v) CH3CN as mobile phase and UV
detection at 245 nm. Retention times were 3.2Æ0.1 (4-bromoben-
zamidine) and 6.2Æ0.1 min (4-bromobenzamidoxime).
Separation of 4-cyanobenzamidoxime and 4-cyanobenzamidine:
Separations were carried out with 20 mm KH2PO4 containing 0.1%
TFA (pH 4.0) and 5.0% (v/v) CH3CN as mobile phase and UV detec-
tion at 244 nm. Retention times were 8.0Æ0.1 (4-cyanobenzami-
dine) and 15.7Æ0.1 min (4-cyanobenzamidoxime).
Separation of 4-nitrobenzamidoxime and 4-nitrobenzamidine:
Separations were carried out with 20 mm KH2PO4 containing 0.1%
TFA (pH 4.0) and 15.0% (v/v) CH3CN as mobile phase and UV de-
tection at 256 nm. Retention times were 3.7Æ0.1 (4-nitrobenzami-
dine) and 8.2Æ0.1 min (4-nitrobenzamidoxime).
HPLC analysis
Separation and quantification of the substituted benzamidoximes
and benzamidines was performed with a Waters HPLC system (Mil-
ford, MA, USA), containing a controller (Waters 600), autosampler
(Waters 717 plus) and UV detector (Waters 2487 Dual l Absorbance
Detector). Peaks were integrated and analyzed with EZ Chrom
Chromatography Data System (EZChrom Elite ver. 2.8.3, Scientific
Software Inc., San Ramon, CA, USA). Separations were carried out
isocratically at a flow rate of 1.0 mLminÀ1 (0.8 mLminÀ1 for 4-cya-
nobenzamidoxime) and injected sample volumes of 10 mL. The
aqueous part of the mobile phase was filtered through a Sartorius
polyamide membrane filter (0.45 mm, Sartorius AG, Gçttingen, Ger-
many) before adding the organic portion (HPLC grade) and degass-
ing by sonication. For separation and quantification of benzami-
doxime and benzamidine, the HPLC method of Clement et al. was
used.[30] Separation and quantification of 4-methyl-, 4-methoxy-, 4-
hydroxy-, 4-amino-, and 4-trifluoromethylbenzamidoxime and -ben-
zamidine were carried out using a Symmetry max RP 4 mm,
4.6 mmꢁ250 mm column with a security guard cartridge system
C18, 3 mmꢁ4 mm (Phenomenex, Torrance, CA, USA). For 4-chloro-,
4-bromo-, 4-cyano-, and 4-nitrobenzamidoxime and -benzamidine
a Nova-Pak C18 4 mm, 3.9ꢁ300 mm column (Waters, Milford, MA,
USA) with a Phenomenex security guard cartridge system was
used.
Acknowledgements
We thank Petra Kçster, Sven Wichmann (both CAU Kiel) and
Sabine Zuelsdorf (ALU Freiburg) for their technical assistance.
Keywords: amidoximes
·
biotransformation
·
enzyme
Separation of 4-metylbenzamidoxime and 4-methylbenzami-
dine: Separations were carried out with 10 mm 1-octylsulfonate
sodium salt, 20 mm KH2PO4 (pH 4.0) and 21.5% (v/v) CH3CN as
mobile phase and UV detection at 242 nm. Retention times were
17.0Æ0.1 (4-methylbenzamidoxime) and 22.4Æ0.1 min (4-methyl-
benzamidine).
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Received: October 10, 2014
Published online on && &&, 0000
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Fundamental differences between the
electrochemical and enzymatic reduc-
tion of para-substituted benzamidox-
imes were detected: The electrochemi-
cal properties of the substituent clearly
influence the redox potential, but not
the enzymatic bioactivation of amidox-
ime prodrugs.
E. Bauch, D. Reichmann, R.-R. Mendel,
F. Bittner, A.-M. Manke, P. Kurz,*
U. Girreser, A. Havemeyer, B. Clement*
&& – &&
Electrochemical and mARC-Catalyzed
Enzymatic Reduction of para-
Substituted Benzamidoximes:
Consequences for the Prodrug
Concept “Amidoximes instead of
Amidines”
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