Reactive metabolites of desipramine and clomipramine: The kinetics of formation and reactivity with DNA

Article abstract of DOI:10.1016/j.bmc.2011.10.075

Tricyclic antidepressants (TCAs), along with phenothyazines and some industrial chemicals, are shown to react with enzymes that exhibit peroxidase activity. These reactions result in the formation of reactive intermediates having unpaired electrons. The peroxidase oxidation and reactivity of two TCAs, desipramine and clomipramine, were investigated. As a model of peroxidase, horseradish peroxidase (HRP) was employed. The products of the peroxidase catalyzed oxidation of desipramine and clomipramine were identified as N-dealkylated compounds iminodibenzyl and 3-chloroiminodibenzyl using the GC/MS technique. Both drugs formed broad UV/vis absorption spectra in the presence of HRP and H₂O₂, indicating the formation of a radical cations - reactive intermediate of the oxidation reaction. The dynamics of the formation of the desipramine intermediate was studied using UV/vis spectroscopy. The extinction coefficient was measured for the reactive intermediate, 7.80 × 10³ M^-1 cm^-1, as well as the apparent Michaelis-Menten and catalytic constants, 4.4 mM and 2.3 s^-1, respectively. Both desipramine and clomipramine degraded DNA in the presence of HRP/H₂O₂, as was revealed by agarose gel electrophoresis and PCI extraction. Manipulating the kinetic parameters of drug's radical formation and determining the extent of degradation to biomolecules could be potentially used for designing effective agents exhibiting specific reactivity.

Full text of DOI:10.1016/j.bmc.2011.10.075

Bioorganic & Medicinal Chemistry 20 (2012) 340–345
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Reactive metabolites of desipramine and clomipramine: The kinetics
of formation and reactivity with DNA
⇑
Ekaterina A. Korobkova , John Nemeth, Mikeisha Cadougan, Abhishek Venkatratnam, Mohanram Bassit,
Nikolay Azar
Science Department, John Jay College of Criminal Justice, CUNY, 445 W 59th St., New York, NY 10019, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Tricyclic antidepressants (TCAs), along with phenothyazines and some industrial chemicals, are shown to
react with enzymes that exhibit peroxidase activity. These reactions result in the formation of reactive
intermediates having unpaired electrons. The peroxidase oxidation and reactivity of two TCAs, desipramine
and clomipramine, were investigated. As a model of peroxidase, horseradish peroxidase (HRP) was
employed. The products of the peroxidase catalyzed oxidation of desipramine and clomipramine were
identified as N-dealkylated compounds iminodibenzyl and 3-chloroiminodibenzyl using the GC/MS tech-
nique. Both drugs formed broad UV/vis absorption spectra in the presence of HRP and H2O2, indicating the
formation of a radical cations—reactive intermediate of the oxidation reaction. The dynamics of the forma-
tion of the desipramine intermediate was studied using UV/vis spectroscopy. The extinction coefficient was
measured for the reactive intermediate, 7.80 ꢀ 103 Mꢁ1 cmꢁ1, as well as the apparent Michaelis–Menten
and catalytic constants, 4.4 mM and 2.3 sꢁ1, respectively. Both desipramine and clomipramine degraded
DNA in the presence of HRP/H2O2, as was revealed by agarose gel electrophoresis and PCI extraction. Manip-
ulating the kinetic parameters of drug’s radical formation and determining the extent of degradation to bio-
molecules could be potentially used for designing effective agents exhibiting specific reactivity.
Published by Elsevier Ltd.
Received 4 August 2011
Revised 21 October 2011
Accepted 27 October 2011
Available online 3 November 2011
Keywords:
Tricyclic antidepressants
One-electron oxidation
Michaelis–Menten kinetics
Rate constants
DNA damage
1. Introduction
against the formation of lipid peroxide and to enhance antioxidant
defensive mechanisms in cells.12,13 Even though there is sufficient
The discovery of tricyclic antidepressant’s (TCAs) radicals traces
back to the 1960s. During this time, an EPR spectrum of an imipra-
mine radical was detected in the presence of one-electron oxidation
systems such as ceric ion and horseradish peroxidase (HRP)/H2O2
and in 1983, pentavalent vanadium ion.1,2 Consequently, a number
of studies have demonstrated the ability of TCAs to promote the for-
mation of free radicals and oxidative stress in vivo. Thus, clomipra-
mine, imipramine and nortriptyline increase the level of hydroxyl
radicals in rats.3–5 Also, imipramine and amitriptyline lead to oxida-
tive stress in astrocyte cell lines.6 It is even proposed that the ability
of amitriptyline to increase the level of reactive oxygen species
(ROS) in cancerous cells could be utilized in antitumor chemother-
apy.7 Clomipramine and desipramine are shown to inhibit the activ-
ity of mitochondrial complexes of cancerous cells.8–11 On the other
hand, TCAs are proven to be capable of performing protective tasks
evidence of the radical nature of TCA’s reactivity with biomolecules,
the mechanisms and dynamics of the formation of these reactive
species have not been studied. The dynamics of the radical forma-
tion, however, has been investigated for molecules having structural
motifs similar to those of TCAs such as phenothiazines14 and
aminopyrine.15 The focus of the present work was desipramine
and clomipramine (Fig. 1). Both drugs are used in the treatment of
Abbrevations: HRP, horseradish peroxidase; TCAs, tricyclic antidepressants; EPR,
electron paramagnetic resonance; ROS, reactive oxygen species; ALS-GC/MS,
automatic liquid sampler-gas chromatography/mass spectrometry; PCI, Phenol–
Chloroform–Isoamyl alcohol.
⇑
Corresponding author. Tel.: +1 212 237 8064; fax: +1 212 237 8318.
E-mail address: ekorobkova@jjay.cuny.edu (E.A. Korobkova).
Figure 1. The molecular structures of the experimental TCAs.
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2011.10.075

E. A. Korobkova et al. / Bioorg. Med. Chem. 20 (2012) 340–345
341
obsessive-compulsive disorder.16 Specifically, desipramine is found
2.4. Kinetics of the reaction of catalytic oxidation
to be effective in impeding cocaine dependence17,18 and relieving
postherpetic neuralgia.19 Clomipramine appears to be more suc-
cessful than other TCAs in the treatment of panic disorder20 and
trichotillomania.21 We performed structural and kinetic studies on
the formation of reactive intermediates of the drugs under the per-
oxidase oxidation. Reactivity with DNA was examined for the drugs
under the conditions of one-electron oxidation.
Kinetic studies were performed with a Shimazu UV–visible Spec-
trophotometer, UV-1700. 1000 l quartz cuvettes were used. The
temperature in the cuvette chamber was thermostated at 25 °C.
The reactions were carried out in a 500 l solution of 133 mM Soren-
son buffer (pH 7.0). An aliquot of desipramine was added to a solu-
tion of HRP and H2O2. The solution was thoroughly mixed via
pipetting immediately after the addition of the drug. The absorption
intensity was recorded over the time period of 1–20 min at
k = 523 nm.
l
l
2. Material and methods
2.1. Materials
2.5. Determination of the extinction coefficient of the product
of one-electron oxidation of desipramine
Desipramine hydrochloride, purity P98% (Saint Louis, MO);
clomipramine hydrochloride, purity P99% (Saint Louis, MO); per-
oxidase from horseradish type VI, 250–330 units/mg (Allentown,
PA); and deoxyribonucleic acid type I from calf thymus (Allentown,
PA) were purchased from Sigma Aldrich. Hydrogen peroxide, 35%
(New Jersey, USA) was purchased from Acros Organics.
The absorbance was monitored over two minutes at 251 and
523 nm for the desipramine reaction after the drug was mixed
with HRP/H2O2. At 251 nm, the absorbance decreased with time
indicating the disappearance of the drug in the oxidation reaction.
At 523 nm, the absorbance increased indicating the formation of
the one-electron oxidation product of the drug. The reactions were
performed at various concentrations of HRP and a fixed concentra-
tion of desipramine. The concentration of the drug was chosen
such that the initial absorbance did not exceed 1. The initial slope
of the drug’s decay kinetics was plotted against the initial slopes of
the kinetics of radical appearance. The linear fit of the data pro-
vided the resulted slope, which was equal to the ratio of the extinc-
tion coefficient of the drug and the extinction coefficient of the
radical.
2.2. Als-gc/ms
An Agilent Automatic Liquid Sample injector (ALS-G4513A) was
employed with a syringe size of 10
lL. Two microliters of sample
were injected at a speed of 6000 l/min and set at a fast plunger
l
speed. The injector was washed with acetone and methanol seven
times before and after every injection, utilizing three sample
washes, five sample pumps and a standard L1 air gap injection type
for all sample injections.
An Agilent 7890A GC was utilized. The inlet parameters were as
follows: heater, 250 °C isocratic; pressure, 10.523 psi; total He flow,
27 ml/min; septum purge flow, 1 ml/min; septum purge flow mode,
standard; injection mode, split; split ratio, 25:1; split flow, 25 ml/
2.6. Analysis of the kinetic data, the determination of rate
constants
The absorbance versus time curves were obtained at different
concentrations of desipramine and analyzed in the following
way. The linear fits were performed at the onsets of the kinetics
curves. The values of the rate of absorbance growth were divided
by the extinction coefficient of the desipramine radical and nor-
malized by the concentration of HRP. The values of the normalized
rates of product formation were plotted against the drugs concen-
tration. The error bars were determined using the formula of the
standard deviation of a ratio of two independent variables: absor-
bance growth rate and the extinction coefficient of the radical. The
apparent catalytic and Michaelis–Menten constants were deter-
mined by fitting the data with the equation derived by Wang
et al.23
min. The column parameters were 30 m ꢀ 250
lm ꢀ 0.25
lm, col-
umn out—to vacuum. The carrier gas (He, 5.0 UHP) flow rate was
1 ml/min with post run flow of 1 ml/min. The initial oven tempera-
ture was 100 °C, ramped at 5 °C/min for 40 min, to the final tempera-
ture of 300 °C that was held for 2 min, with a post run temperature of
100 °C. The transfer line was kept at 300 °C for the entire 42 min run.
An Agilent 5975C Mass Selective Detector (MSD) was used with
a GC sample inlet, a solvent delay of 6 min and an EMV mode gain
factor of 1.00 = 1129 V. The MSD was run in ‘scan’ acquisition
mode at normal scan speed with a scan mass range of 50.00–
800.00 amu. The MSD’s threshold was set at 150 amu with
1.99 scans/s and a sampling rate (2n) of 2. The MS source temper-
ature was set at 230 °C and MS quadrupole temperature was set at
150 °C.
2.7. Agarose gel electrophoresis
An Agilent MSD ChemStation, Version E.02.00.493, was used for
data acquisition and analysis with the NIST 98 library.
1% agarose gels were prepared in TAE buffer. Electrophoresis
was performed at 80 V for 120 min. The loading samples contained
2.3. UV/vis spectroscopy
20
l
l of the reaction mixture and 2.2
l
l of the 10ꢀ loading buffer.
The 10ꢀ loading buffer consisted of 50% glycerol, 0.25% bromophe-
UV/vis spectra were recorded with a Shimazu UV–visible Spec-
nol blue and 0.25% xylene cyanole FF in TAE buffer. The gel was
trophotometer UV-1700 in 1000
solutions were prepared in 500
l
l quartz cuvettes at 25 °C. The
stained in ethidium bromide (0.5 lg/ml) for 20 min and then dis-
tained in distilled water for 20 min. The gel images were captured
with the PerkinElmer Geliance 200 Imaging system.
l
l of 133 mM Sorenson buffer (pH
7.0) containing 133 mM dibasic sodium phosphate and 133 mM
monobasic potassium phosphate. The spectra were recorded at the
wavelength interval of 200–800 nm right after mixing desipramine
or clomipramine with HRP and H2O2. The concentration of HRP was
calculated using the extinction coefficients at 404 nm of 102,000 M
3. Results and discussion
ꢁ1 cmꢁ1 22
The extinction coefficient of the drugs were determined
.
3.1. UV/vis spectra of desipramine oxidation intermediate
to be 7.61 ꢀ 103 Mꢁ1 cmꢁ1 at k = 251 nm for desipramine and
7.32 ꢀ 103 Mꢁ1 cmꢁ1 at k = 252 nm for clomipramine. The extinc-
tion coefficientswere determinedby mass measurements combined
with UV/vis spectroscopy.
The incubation of desipramine (Fig. 1) in the presence of HRP and
H2O2 led to the initial formation of a pink color followed by an
increasing brown colorover time, which produced the corresponding

342
E. A. Korobkova et al. / Bioorg. Med. Chem. 20 (2012) 340–345
broad UV/vis spectra:Figure 2. Figure 2A shows the spectra recorded
as the reaction of desipramine with HRP and H2O2 proceeded for
24 min. The negative control reactions containing HRP/H2O2, HRP/
desipramine or desipramine/H2O2 did not result in the appearance
of new spectra. In the mixture containing HRP, H2O2 and clomipra-
mine, no color was observed in the buffer due to the drug’s lack of sol-
ubility with the buffer used. A spectrum with the absorption
maximum at 520 nm (Fig. 2B) was observed in water with a clomip-
ramine concentration 10 times greater than that of desipramine
(Fig. 2A). Broad absorption spectra were observed earlier under the
one-electron oxidation of aminopyrine,15 phenothyazines,14 imipra-
mine24 and some industrial arylamines.25,26 The spectra were associ-
ated with the formation of the products of one-electron oxidation,
radicalcations. The assignmentof thespectra toradical cations is also
supported by the stoichiometry studies of the redox transitions
between the oxidized forms of HRP. In this experiment, the ‘one-
one-electron oxidation of imipramine,24 crystal violet30 and amino-
pyrine.15 In these instances, radical species were detected as reac-
tion intermediates.
Based on the experimental observations presented, we pro-
posed the kinetic schemes shown in Figure 3.
3.3. Determination of the extinction coefficient of desipramine
radical cation
The extinction coefficient of desipramine radical cation was
determined by measuring the rates of the absorbance decrease
for the drug (251 nm for desipramine and 252 nm for clomipra-
mine) and applying the equation:
d½Drug radicalꢂ
d½Drugꢂ
¼ ꢁ
ð1Þ
dt
dt
electron-gain’ conversions—HRP-I to HRP-II and HRP-II to HRP27
—
The equation holds true for the initial times after the reagents
are mixed (when the contribution of the N-dealkylation process
is negligible). Using Beer’s law:
were observed upon the addition of one HRP equivalent of
desipramine or clomipramine (data not shown). A radical formed is
potentially very stable. This stability originates from the delocaliza-
tion of the unpaired electron over the benzene rings of desipramine
and clomipramine. This conjugation phenomenon is known to stabi-
lize nitrogen-centered radicals.28,29
dAradical
dt
dAdrug
dt
ꢀ
edrug ¼ ꢁ
ꢀ
eradical
ð2Þ
3.2. GC/MS studies of the oxidation products of desipramine and
clomipramine
where Adrug is the absorbance at 251 nm, while Aradical is the absor-
bance value at 523 nm, and radical and drug are the extinction coef-
e
e
ficients for drug radical and drug, respectively. The initial slopes of
The products of the oxidation of the drugs were studied with
GC/MS. In the case of desipramine, a precipitant formed after the
incubation of the drug with HRP and H2O2 in the Sorenson buffer.
The precipitant was dissolved in acetonitrile and injected into a GC
column. A single peak was observed at 20.5 min. The mass spec-
trum of the peak matched that of iminoibenzyl, an N-dealkylated
product from desipramine (m/z: 195.1).
In the case of clomipramine, the enzymatic oxidation reaction
could only be performed in water due to the insolubility of clomip-
ramine in the buffer. No precipitant was observed after the drug was
incubated with HRP and H2O2. A reaction mixture was loaded on the
GC column, which led to the detection oftwo major peaks at24.9 and
29.4 min. The mass spectrum of the first peak matched that of
3-chloroiminodibenzyl (systematic name—5H-dibenz[b,f]aze-
pine,3-chloro-10,11-dihydro-) as an N-dealkylated product from
clomipramine (m/z: 229.1), and the second peak was identified as
clomipramine (m/z: 314.1). The two molecules, iminodibenzyl and
3-chloroiminodibenzyl, were purchased and were analyzed with
GC/MS. The retention times and the mass spectra of the commercial
compounds matched those of the reaction products. The N-dealky-
lating products were previously observed in the reactions of
Figure 3. Reaction scheme of the drug’s oxidation in the presence of HRP and H2O2
and the structures of the products. Des—desipramine, Im—iminodibenzyl, Clom—
clomipramine and Cl-Im—3-chloriminodibenzyl.
Figure 2. UV/vis absorption spectra of the drug’s reactive intermediate formed during the oxidation by the HRP/H2O2 system. (A) Reaction with desipramine performed in
133 mM Sorenson buffer pH 7.0. [HRP] = 0.6 M; [H2O2] = 10 mM; [Desipramine] = 92 M. (B) Reaction with clomipramine performed in water. [HRP] = 1 M;
[H2O2] = 10 mM; [Clomipramine] = 1 mM. The indicated times were when data were taken after the reagents were mixed.
l
l
l

E. A. Korobkova et al. / Bioorg. Med. Chem. 20 (2012) 340–345
343
Figure 4. Determination of the extinction coefficients of desipramine radical cation. (A) Kinetic curves recorded at 251 nm and 523 nm; [HRP] = 2.48
lM, [H2O2] = 10 mM,
[desipramine] = 37
lM. (B) Initial slopes of the kinetic curves recorded at 251 nm versus initial slopes of the kinetic curves recorded at 523 nm. The concentration of
desipramine was 37
l
M, and the concentration of H2O2 was 10 mM. The concentration of HRP varied from 0.44 to 3.5 M.
l
value is consistent with the extinction coefficients found for pheno-
thiazines that range between 6507 Mꢁ1 cmꢁ1 for promazine (kmax
=
518 nm) and 12,500 Mꢁ1 cmꢁ1 for chlorpromazine (kmax = 530 nm).14
3.4. Kinetic studies of desipramine peroxidase oxidation
Figure 5 shows the mechanisms underlying reactions (1) and
(3) from Figure 3. These mechanisms involve the formation of
the oxidation forms of HRP, HRP-I and HRP-II. In a reduced form
of peroxidase, an iron atom sitting on the porphyrine ring is in
its ferric state. In HRP-I, the iron atom is complexed with oxygen
and is in its ferryl state. The heme group of HRP-I has an unpaired
electron and a unit positive charge. In an HRP-II state, the ferryl
iron atom forms a covalent complex with oxygen.27,31 Transitions
HRP-I?HRP-II and HRP-II?HRP are associated with the transmis-
sion of one electron from the substrate (drug) and the peroxidase
porphyrine ring.27 The formation of complexes between the perox-
idase active site and the substrate is necessary for the electron
transfer (HRP-I-Drug and HRP-II-Drug, Fig. 5). Ling et al. (2008) ap-
plied spectroscopic measurements and computational docking
simulations to describe the structure of the complex intermediates
formed between HRP oxidized forms and N-(indole-3-ethyl) cyclic
amines. They found that the substrates bind to the active site of the
enzyme via their benzene rings.32 The benzene rings of both desi-
pramine and clomipramine represent the most electronegative
part of the molecule. Also, due to the electron overlap between
Figure 5. Kinetic scheme of the reactions of a drug’s oxidation by H2O2 catalyzed by
HRP.
the ‘absorbance versus time’ curves were measured at different con-
centrations of enzyme.
Figure 4A shows kinetic curves recorded at 251 and 523 nm,
respectively. The values of (dAdrug/dt) were plotted against (dAradi-
cal/dt). The slope was found to be ꢁ0.976 0.034, and eradical was
determined to be (7.80 0.29) ꢀ 103 Mꢁ1 cmꢁ1 (Fig. 4B). The eradical
the nitrogen atom electron pair and the
p orbitals of the benzene
rings, the tricyclic motif of the molecules possesses high reduction
Figure 6. The kinetics of the formation of the desipramine radical monitored by UV/vis spectroscopy at 523 nm. (A) The inicial slope (dAbs/dt) normalized by the extinction
coefficient of the radical and the concentration of HRP, 0.498 M, plotted against the concentration of desipramine. The error bars are standard deviations determined based
l
on the 6–8 measurements of the absorbance growth rate and combined with the standard deviation of the extinction coefficient of desipramine radical. The red line
represents the best fit curve obtained with Eq. (3). (B) The kinetics of the oxidation reactions of desipramine in the presence of HRP and H2O2 recorded for 20 min.
[Desipramine] = 47 lM, [H2O2] = 10 mM. The concentration of HRP for each curve is shown.

344
E. A. Korobkova et al. / Bioorg. Med. Chem. 20 (2012) 340–345
m0
½HRPꢂ0
jcat-app
jcat-app
ꢀ
þ
j1 ꢀ½H2O2ꢂ
j1 ꢀ½H2O2ꢂ
½Drugꢂ
¼ 2ꢀ
ꢀ
j1ꢀ½H2O2
ꢂ
jM-app
ꢀ
ꢂ þ½Drugꢂ
jcat-app
þj1ꢀ½H2O2
ð3Þ
where v0 is the initial rate of the formation of the product, and
kcat-app and KM-app are the apparent catalytic and Michaelis–Menten
constants, respectively. These constants represent functions of the
kinetics constants indicated in Figure 5.
The k1 constant (Fig. 5) was taken to be 1.8 ꢀ 107 Mꢁ1 sꢁ1 as
was determined by Wang and co-workers who studied HRP-
catalyzed oxidation of di-(N-acetyl-L-tyrpsin) and dipeptide
Gly–Tyr.23 The apparent Michaelis constant, KM-app, of desipramine
oxidation was found to be 4.4 0.3 mM (Fig. 6A). The apparent cat-
alytic constant for desipramine reaction was determined to be
2.3 0.1 sꢁ1 (Fig. 6A). Figure 6B shows the kinetic curves moni-
tored at different concentrations of HRP. The initial slope of the
curves proved to be a linear function of HRP concentration (data
not shown).
3.5. Studies of DNA damage cased by desipramine and
clomipramine in the presence of HRP/H2O2
Some TCAs are known to cause DNA degradation when present
with one-electron oxidation systems.24 Potential DNA damage
was examined for desipramine and clomipramine using calf thy-
mus DNA and agarose gel electrophoresis. Reaction mixtures con-
taining a drug, HRP and H2O2 were incubated at 37 °C for 1 h in a
water bath and gel-electrophoresed along with the control reac-
tions (Fig. 7). While DNA bands appeared in all control bands
(lanes 1, 2, 4 and 5), no DNA was visible in the gel when incu-
bated with one of the drugs in the presence of HRP/H2O2 (lanes
3 and 6), suggesting that reactive intermediates of both desipra-
mine and clomipramine degrade DNA. The DNA degradation
was confirmed by PCI extractions followed by UV/vis spectra
(Fig. 7B). The solid line represents DNA extracted from a control
sample with no other components added to the solution. The
other two spectra were measured from DNA extracted from the
reaction mixture containing HRP/H2O2 and desipramine (dashed
line) or clomipramine (dotted line). The absorbance at 260 nm
was essentially lower than that in a control sample (solid line).
PCI extractions were performed as described earlier.24 DNA dam-
age studies have been performed earlier with both drugs. Thus
DNA fragmentation has been observed with C6 glioma cells33
and human colon cancer cells.34 Desipramine was also shown to
induce chromosomal aberrations in mouse bone marrow cells.35
Clomipramine was shown to be genotoxic to wing cells of Dro-
sophila melanogaster.36 DNA fragmentation has been observed un-
der clomipramine treatment of human peripheral lymphocytes37
and human leukemic cells.38
Figure 7. Agarose gel electrophoresis of reactions with calf thymus DNA and PCI
extraction. For gel electrophoresis studies, all reactions mixtures were incubated for 1
hour at 37 °C in water. The concentrations of desipramine and clomipramine were
0.5 mM, concentrations of HRP and H2O2 were 2 and 10 mM, respectively. The
concentration of calf thymus DNA was 6 lM per bp in all reaction mixtures. (A)
Reactions with desipramine. Lane 1, DNA with clomipramine; lane 2, DNA with
clomipramine and H2O2; lane 3, DNA with clomipramine and HRP/H2O2; lane 4, DNA
with desipramine; lane 5, DNA with desipramine and H2O2; lane 6, DNA with
desipramine and HRP/H2O2. (B) PCI extraction of DNA reacted with drugs in the
presence of HRP/H2O2. Solid spectrum: 1 mM of DNA only; dashed spectrum:
[DNA] = 1 mM, [H2O2] = 10 mM, [Desipramine] = 2 mM, and [HRP] = 1
spectrum: [DNA] = 1 mM, [H2O2] = 10 mM, [Clomipramine] = 2 mM, and [HRP] =
M. The reaction mixtures were incubated at room temperature for 2 h after which
lM; dotted
4. Conclusion
1
l
the extraction was performed. The spectra were recorded with the NanoDrop 2000C
Spectrophotometer.
In this study, we have demonstrated that both desipramine and
clomipramine undergo one-electron oxidation in the presence of
HRP and H2O2, followed by the formation of reactive species. The
kinetics of the oxidation of desipramine was studied in detail,
and the apparent Michaelis and catalytic constants were found to
be 4.4 mM and 2.3 sꢁ1, respectively. We hypothesize that the reac-
tive intermediates of the oxidation of the drugs may play impor-
tant roles in living species. With identifying the toxicity and
dynamics behind the drugs oxidation in addition to previous re-
search that emphasizes the importance of these drugs in affecting
mitochondria of cancer cells, this mechanistic study can assist in
the design of molecules that would enable control over specific
activities in living systems.
(electron donating) potential, which makes it highly likely that the
drug binds to the enzyme heme pocket with its benzene ring.
We studied the kinetics of the oxidation of desipramine in the
presence of HRP and H2O2 by monitoring the absorbance at
523 nm. The absorption at kmax was recorded for a period of
1–20 min at various concentrations of desipramine and fixed con-
centrations of HRP and H2O2. The initial slopes were normalized by
the extinction coefficient of the radical and the concentration of
HRP. The normalized product formation rates plotted against the
concentration of the drug (Fig. 6), and the data were fitted with
the following equation:23

E. A. Korobkova et al. / Bioorg. Med. Chem. 20 (2012) 340–345
345
15. Perez-Gilabert, M.; Sanchez-Ferrer, A.; Garcia-Carmona, F. Free Radical Biol.
Med. 1997, 23, 548.
Acknowledgments
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This work was sponsored by the PRISM program at John Jay
College and the John Jay College Research Assistance Grant. The
authors cordially thank Professor Nathan Lents for technical help
and Zully Santiago for help with the editing of the manuscript.
19. Kishore-Kumar, R.; Max, M. B.; Schafer, S. C.; Gaughan, A. M.; Smoller, B.;
Gracely, R. H.; Dubner, R. Clin. Pharmacol. Ther. 1990, 47, 305.
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