Antioxidative properties of probucol estimated by the reactivity with superoxide and by electrochemical oxidation

Article abstract of DOI:10.1248/cpb.49.943

The reaction of probucol with superoxide (O₂^.- was investigated in acetonitrile using both electron spin resonance (ESR) and electrochemical techniques. The formation of phenoxyl radical was observed during the reaction of probucol with O₂^.- by ESR spectroscopy. The reaction of probucol with O₂^.- in acetonitrile was followed by cyclic voltammetry. With the addition of probucol, the oxidation peak current of O₂^.- decreased concentration dependently. This suggests that probucol reacts with O₂^.-, that is, probucol scavenges O₂^.- in acetonitrile. 2,6-Di-tertbutyl-p-benzoquinone was identified as the major product of the reaction of probucol with O₂^.- in acetonitrile. Electrochemical oxidation of probucol was also performed. Probucol gives an irreversible oxidation peak at ca.+1.4V vs. the saturated calomel electrode in the cyclic voltammogram. Controlled-potential electrolysis was carried out at +1.2V in a divided cell. 2,6-Di-tert-butyl-p-benzoquinone, 4,4′-dithiobis(2,6-di-tert-butylphenol), and 4,4′-trithiobis(2,6-di-tert-butylphenol) were identified as the products of anodic oxidation. These redox properties of probucol may correlate with the physiological activities.

Full text of DOI:10.1248/cpb.49.943

August 2001
Chem. Pharm. Bull. 49(8) 943—947 (2001)
943
Antioxidative Properties of Probucol Estimated by the Reactivity with
Superoxide and by Electrochemical Oxidation
Tetsuya ARAKI* and Hiroaki KITAOKA
Drug Metabolism and Physicochemical Property Research Laboratory, Daiichi Pharmaceutical Co., Ltd., 16–13, Kita-
Kasai 1-chome, Edogawa-ku, Tokyo 134–8630, Japan. Received January 12, 2001; accepted April 19, 2001
–
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The reaction of probucol with superoxide (O2 ) was investigated in acetonitrile using both electron spin reso-
nance (ESR) and electrochemical techniques. The formation of phenoxyl radical was observed during the reac-
–
tion of probucol with O2 by ESR spectroscopy. The reaction of probucol with O2– in acetonitrile was followed by
·
·
cyclic voltammetry. With the addition of probucol, the oxidation peak current of O2– decreased concentration de-
·
pendently. This suggests that probucol reacts with O2 , that is, probucol scavenges O2– in acetonitrile. 2,6-Di-tert-
–
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·
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butyl-p-benzoquinone was identified as the major product of the reaction of probucol with O2 in acetonitrile.
Electrochemical oxidation of probucol was also performed. Probucol gives an irreversible oxidation peak at ca.
؉1.4 V vs. the saturated calomel electrode in the cyclic voltammogram. Controlled-potential electrolysis was car-
ried out at ؉1.2 V in a divided cell. 2,6-Di-tert-butyl-p-benzoquinone, 4,4؅-dithiobis(2,6-di-tert-butylphenol), and
4,4؅-trithiobis(2,6-di-tert-butylphenol) were identified as the products of anodic oxidation. These redox properties
of probucol may correlate with the physiological activities.
Key words probucol; antioxidant; cyclic voltammetry; superoxide; ESR; anodic oxidation
Probucol (4,4Ј-isopropylidenedithio-bis(2,6-di-tert- are useful for investigating electron-transfer reactions. The
butylphenol),1) (Chart 1) is a hypolipidemic agent that has electrochemical behavior appears to be a good model for bio-
been widely used in the treatment of heart and blood vessel logical oxidation of pharmacologically active substances. In
disease.1) Oxidation of low-density lipoprotein (LDL) in this study, anodic oxidation of probucol was undertaken to
blood is implicated in the development of human atheroscle- determine the possible relationships between the electro-
rosis.2) Probucol is an effective antioxidant transported in chemical and physiological properties.
lipoproteins that blocks the oxidative modification of LDL in
vivo.3) A commonly discussed mechanism of the pharmaco-
Experimental
1
General H- and 13C-NMR spectra were recorded on a JEOL JNM-GSX-
logical effects of hypolipidemic agents, especially phenolic
antioxidants, is their antioxidative activities such as suppress-
500 spectrometer, ESR spectra on a JEOL JES-FE-2XG spectrometer, and
MS spectra on a JEOL JMS-HX-110 spectrometer. Cyclic voltammetry and
ing the formation of active species like reactive oxygens and
anodic oxidation were carried out by using a dual potentio-galvanostat
(DPGS-1, Nikko Keisoku, Kanagawa, Japan), a potential sweeper (NPS-2,
Nikko Keisoku), and a digital coulomb meter (NDCM-3, Nikko Keisoku).
The preparative high-performance liquid chromatography (HPLC) consisted
of a Hitachi L-7100 pump and a Hitachi L-4000 UV detector (Hitachi,
Tokyo, Japan). The single-crystal X-ray structure determinations were car-
ried out using a Rigaku AFC-7R diffractometer (Rigaku, Tokyo, Japan) with
monochromated CuKa radiation.
Materials Probucol was synthesized at Aventis Pharma AG (Frankfurt,
Germany). Potassium superoxide (KO2) and 18-crown-6 were purchased
from Sigma Co., Ltd., (St. Louis, MO, U.S.A.). Tetraethylammonium per-
chlorate (TEAP) and DL-alpha-tocopherol were purchased from Aldrich
Chemical Co., Ltd., (Milwaukee, WI, U.S.A.). 2,6-Di-tert-butylhydroxy-
toluene (BHT) was purchased from Kanto Kagaku Co., Ltd. (Tokyo, Japan).
These materials were used without further purification.
ESR Measurement Thirty milliliters of a dry acetonitrile solution con-
taining probucol 113.56 mg (0.22 mmol) and 18-crown-6 56.5 mg (0.21
mmol) was prepared, and then potassium superoxide powder approximately
100 mg was gradually added to the solution. The resultant solution was
transferred to capillary tubes [Drummond Microcaps, 50 ml, (Drummond
Scientific Co. PA, U.S.A.)], sealed with Terumoseal (Terumo, Tokyo, Japan),
and measured by ESR. ESR spectral conditions were as follows: magnetic
field, 327Ϯ5 mT; response, 0.3 s; modulation, 100 kHz; modulation ampli-
tude, 0.063 mT; temperature, ambient; microwave power, 8.0 mW; sweep
time, 2 min.
free radicals.4) Reactive oxygen species, such as superoxide
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·
anion radical (O2 ), hydroxyl radical, and nitric oxide, and
other reactive oxygen species, such as hydrogen peroxide, are
formed in vivo. An imbalance between the production of
these reactive oxygen species and antioxidant defense can re-
sult in oxidative stress. Among the reactive oxygen species,
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O2 , which is easily produced by the one-electron reduction
of molecular oxygen, is implicated in several harmful biolog-
ical processes such as lipid peroxidation and protein denatu-
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5)
ration. The reactivity of O2 is different in an aqueous media
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than in an aprotic solvent. O2 spontaneously and dispropor-
tionately forms hydrogen peroxide and molecular oxygen in
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aqueous systems. On the other hand, O2 shows many reac-
tivities, e.g., as an electrogenerated base, nucleophile, reduc-
tant, and oxidant in aprotic media.6)
The direct scavenging action of probucol on hydroxyl radi-
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·
cals, O2 , and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals
was examined by electron spin resonance (ESR) spectrome-
try.7) Probucol scavenged DPPH radicals dose dependently
but showed no effect on hydroxyl radicals or on superoxide
generated by Fenton reaction and by the hypoxanthine–xan-
thine oxidase system. From a pharmacological aspect, probu-
col is a lipophilic antioxidant; thus one of the antioxidative
properties of probucol plays a role in an aprotic system simi-
Cyclic Voltammetry The measurements were performed on a dual po-
lar to the lipophilic domain of the liposomal membrane. To
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clarify the reactivity of probucol with O2 in the lipophilic
phase, we studied the mechanism of redox reactions of
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probucol with O2 in acetonitrile. Electrochemical techniques
Chart 1
To whom correspondence should be addressed. e-mail: araki45s@daiichipharm.co.jp
© 2001 Pharmaceutical Society of Japan

944
Vol. 49, No. 8
tentio-galvanostat and potential sweeper in dry acetonitrile containing TEAP
0.1 M (supporting electrolyte). A glassy carbon electrode was used for the
working electrode, a platinum electrode for the counter electrode, and a satu-
rated calomel electrode (SCE) for the reference electrode. Probucol (50 mM)
was dissolved in dry acetonitrile containing 0.1 M TEAP. The scan speed was
5 s/V. The potential scan range was from Ϫ1.6 V to 2.2 V vs. SCE.
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O2 scavenging activity was estimated by cyclic voltammetry. The condi-
tions were as follows: The scan speed was 5 s/V. The potential scan range
was from Ϫ1.0 V to 0.0 V vs. SCE. The solution containing various concen-
trations of probucol (28.3, 56.4, 113, 226, 451, 902, 1804 mM) was previ-
ously saturated by air for 15 min to maintain a constant concentration of
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oxygen. O2 scavenging activity was determined by relative anodic current,
which was defined as the cathodic peak current of oxygen normalized
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against the anodic peak current of O2 .
Isolation of the Reaction Product of Probucol with Superoxide
Thirty milliliters of a dry acetonitrile solution containing probucol 113.56
mg (0.22 mmol) and 18-crown-6 56.5 mg (0.21 mmol) was prepared, and
then potassium superoxide powder approximately 100 mg was gradually
added to the solution. The reaction solution was allowed to stand at room
temperature for 1 d in the dark. Then the volume of the reaction solution was
concentrated to approximately 10 ml under reduced pressure, and the residue
was subjected to preparative HPLC. The preparative HPLC was conducted
on a reversed-phase column with TSKgel ODS-80TM (21.5 mm i.d.ϫ300
mm). Ninety-three percent (v/v) acetonitrile was used as the eluent at a flow
rate of 8.0 ml/min. Product detection was monitored at 242 nm. The product
yield was determined by HPLC.
Fig. 1. ESR Spectrum of the Reaction Mixture of Probucol with KO2 in
the Presence of 18-Crown-6 in Acetonitrile at Room Temperature
Magnetic field, 327Ϯ5 mT; response, 0.3 s; modulation, 100 kHz; modulation ampli-
tude, 0.063 mT; temperature, ambient; microwace power, 8.0 mW; sweep time, 2 min.
Controlled Potential Electrolysis of Probucol A platinum mesh was
used for the working electrode, a platinum wire for the counter electrode,
and SCE for the reference electrode. Probucol 106.9 mg (2.1 mmol) was dis-
solved into acetonitrile 100 ml containing TEAP 0.1 M in an H-shaped glass
cell divided by a methyl cellulose plug and a sintered glass disk. The solu-
tion was subjected to electrolysis at ϩ1.2 V vs. SCE until 350 °C, which cor-
responded to 1.7 F per mol of probucol, had been consumed. The electroly-
sis was carried out under nitrogen at atmospheric pressure.
Isolation of Anodic Oxidation Products The electrolyzed solution was
concentrated to approximately 10 ml under reduced pressure, and the residue
was subjected to preparative HPLC under the same HPLC conditions as de-
scribed above. The collected fractions were evaporated to dryness under re-
duced pressure at 40 °C. Three major products were isolated: product 2 10.5
mg (12% yield), product 3 29.0 mg (30%), and product 4 24.4 mg (23%).
X-Ray Structure Determination of Anodic Oxidation Product 4
A
yellowish, columnar crystal of C28H42O2S3 with the approximate dimensions
of 0.40ϫ0.15ϫ0.10 mm was grown from acetonitrile. The compound crys-
tallized in monoclinic space group P21/n with cell parameters aϭ10.152 Å,
bϭ27.771 Å, and cϭ12.462 Å. The structure was solved by the direct
method with the program TEXSAN. The final R value was 3.8%.
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Fig. 2. Cyclic Voltammograms of O2/O2 in Oxygen-Saturated Acetoni-
trile Containing TEAP 0.1 M in the Presence of Several Concentrations of
Probucol (28.3, 56.4, 113, 226, 451, 902, 1804 mM) at a Glassy Carbon at
Room Temperature
Voltage sweep rate, 5 s/V.
Results and Discussion
tonitrile. The reduction potential of the O2/O2– is Ϫ0.80 V vs.
·
ESR Spectrum of the Reaction of Probucol with Super-
oxide Figure 1 shows the ESR spectrum of the reaction
mixture of probucol with potassium superoxide in acetoni-
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SCE, and the oxidation potential of O2 /O2 is Ϫ0.65 V vs.
SCE. The reproducibility on cyclic voltammograms for
O2/O2 is quite high, because O2 is stable in an aprotic sol-
vent.5) Figure 2 shows cyclic voltammograms of various con-
centrations of probucol in acetonitrile containing TEAP 0.1
M. With the addition of probucol, the oxidation peak current
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trile at room temperature. A broad singlet was observed in
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the ESR spectrum. O2 is not detectable by ESR spectrome-
try at room temperature. The result shows that probucol re-
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acts with O2 in acetonitrile and forms stable free radicals
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during the reaction. Several reports have discussed the radi-
cals derived from probucol. A one-line spectrum formed dur-
ing the Ag2O-catalyzed oxidation of probucol and was as-
signed to the probucol phenoxyl radical.8) Probucol also re-
acted with DPPH radicals and gave a new signal in the ESR
spectra.7) Therefore the obtained signal was thought to be a
of O2 decreased, while the reduction current (cathodic cur-
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·
rent [O2 formation]) slightly increased. This suggests that
O2– is scavenged within the time scale of cyclic voltammetry
·
in acetonitrile in a concentration-dependent manner.
The scavenging activity of probucol was compared with
those of the other antioxidants BHT and DL-a-tocopherol
(Fig. 3). The scavenging concentration of the antioxidant
from the relative anodic current (SC50) was estimated. The
phenoxyl radical. The primary oxidation step of probucol by
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O2 is identified as the formation of free radicals.
Superoxide Scavenging Activity of Probucol Estimated
SC50 for these compounds was in the following order: probu-
ϳ
Using Cyclic Voltammetry We have already reported that
col (593 mM)ϾBHT (1052 mM)
ϭDL-a-tocopherol (1089 mM).
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the O2 scavenging activity of antioxidants in acetonitrile
Probucol scavenges O2 the most efficiently, because probu-
col has two phenolic groups. These results suggest that the
antioxidant property of probucol in acetonitrile is partly due
to its free radical scavenging effect.
could be simply estimated by cyclic voltammetry.9) A re-
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versible one-electron wave corresponding to the O2/O2
redox couple is seen in cyclic voltammograms in dried ace-

August 2001
945
Reaction of Probucol with Superoxide First, we at-
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tempted to generate O2 by electrolytic reduction of O2 in
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acetonitrile, but the reaction of probucol with O2 did not
proceed by this method because the concentration of O2– was
·
too low. Therefore we elected to use the commercially avail-
able potassium superoxide (KO2) in the presence of 18-
crown-6 in acetonitrile. The high-performance liquid chro-
matogram obtained from the reaction of probucol with O2– is
·
shown in Fig. 4. The reaction product was observed at tRϭca.
3.8 min. This product was isolated and identified as 2,6-di-
tert-butyl-p-benzoquinone (Chart 2) by NMR, MS: 1H-NMR
(CDCl3) d: 1.26 (18H, s), 6.51 (2H, s). 13C-NMR (CDCl3) d:
29.4, 35.6, 130.2, 157.9. MS (EI) m/z: 220 (Mϩ), (CI) 221
(MϩHϩ). The structure was confirmed by X-ray crystal
analysis. After the reaction mixture was stored for 1 d at
room temperature, 53% of the probucol was recovered and
34% of 2,6-di-tert-butyl-p-benzoquinone was produced.
Phenoxyl radical was generated in the reaction of probucol
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with O2 , and the stable reaction product of probucol with
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O2 was elucidated to be a 2,6-di-tert-butyl-p-benzoquinone.
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Fig. 3. O2 Scavenging Abilities of Antioxidants Determined by the
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The oxidation of hydroxynaphthalenes with O2 leads to the
Cyclic Voltammetry
formation of naphthoquinones.10) Therefore the mechanism
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᭹, probucol; ᭝, DL-a-tocopherol; ᮀ, BHT. O2 scavenging activity was determined
by relative anodic current, which was defined as the cathodic peak current of oxygen
normalized against the anodic peak current of O2
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of probucol with O2 was considered. The reaction is initi-
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·
.
ated by abstracting a hydrogen atom from phenol to give a
phenoxyl radical. The phenoxyl radical may be resonated
with the carbon-centered radical at the 4-position. The car-
bon-centered radical reacts with O2, and subsequently the
carbon–sulfur bond is cleaved. Finally 2,6-di-tert-butyl-p-
benzoquinone is formed. Chart 3 shows the proposed mecha-
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nism for the reaction of probucol with O2 . One of the an-
tioxidative properties of probucol is explained by the stable
phenoxyl radical that was formed by the attack of free radi-
cals in the aprotic system. The ability of probucol to partici-
pate in redox reactions probably plays a role in its pharmaco-
Fig. 4. High-Performance Liquid Chromatogram of the Reaction Solution
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of Probucol with O2
Chart 2
Chart 3

946
Vol. 49, No. 8
Chart 4
Fig. 7. Consumed Electricity vs. Peak Area of Probucol and Reaction
Products
Fig. 5. Cyclic Voltammograms of Probucol 50 mM in Oxygen-Saturated
Acetonitrile Containing TEAP 0.1 M at a Glassy Carbon Electrode at Room
Temperature
᭹, peak area of probucol; ᭺, product 2; ᭝, product 3; ᮀ, product 4.
(a) Voltage sweep started from anodic direction. (b) Voltage sweep started from ca-
thodic direcion. Volatge sweep rate, 5 s/V. The voltammetric peak A in (b) is due to the
oxidation as shown in Chart 4. Controlled potential electrolysis was done at the voltam-
metric peak B.
Fig. 8. X-Ray Crystal Structure of Anodic Oxidation Product 4
Fig. 6. High-Performance Liquid Chromatogram of an Electrolyzed Solu-
tion
matogram of probucol in during the electrolysis (0.74 F
molϪ1). Probucol was oxidized by electrolysis, yielding oxi-
dation products in addition to the three major products 2, 3,
and 4. The plot of the consumed electricity vs. peak area of
probucol and these oxidation products is shown in Fig. 7.
Product 2 is obtained in the highest yield by anodic oxida-
tion, followed by product 3.
logical activity.
Electrochemical Behavior of Probucol Figure 5 shows
the cyclic voltammograms of probucol in acetonitrile from
both anodic and cathodic scans. A voltammetric peak is seen
at ca. Ϫ0.2 V (Fig. 5b, A). The anodic peak voltage agrees
well with that reported by Ohmori et al.,11) and is ascribed to
the oxidation of the phenolate ion to the phenoxyl radical.
Product 2 has almost the same retention time as the reac-
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1
tion product of probucol with O2 . Further, the H-, 13C-
·
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The phenolate ion is formed by O2 as an electrogenerated
base,4) that is, a proton is abstracted from phenol by O2 .
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NMR, and EI-MS spectra of product 2 were similar to those
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Chart 4 shows this reaction.
of the reaction product of probucol with O2 . Thus product 2
A large irreversible anodic peak is seen at ca. ϩ1.4 V (Fig.
5, B). Controlled potential electrolysis of probucol was car-
ried out in acetonitrile containing TEAP 0.1 M at ϩ1.20 V vs.
SCE. Figure 6 shows the high-performance liquid chro-
was identified as 2,6-di-tert-butyl-p-benzoquinone.
A single crystal of product 4 was obtained and X-ray
analysis was performed. A perspective view of the molecule
is shown in Fig. 8. The anodic oxidation product 4 was iden-

August 2001
947
Chart 5
Chart 6
tified as 4,4Ј-trithiobis(2,6-di-tert-butylphenol). Products 3 othio radical or organothio cation radical and may form prod-
1
and 4 had very similar H-NMR spectral characteristics; uct 4 via a cyclic intermediate.
1
product 3: H-NMR (DMSO-d6) d: 1.33 (36H, s), 7.02 (2H,
We conclude that probucol has favorable antioxidant activ-
1
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·
s), 7.22 (4H, s); product 4: H-NMR (DMSO-d6) d: 1.31 ity such as O2 scavenging activity and redox properties in
(36H, s), 7.02 (2H, s), 7.27 (4H, s). A molecular ion of prod- aprotic media. The ability of probucol to participate in redox
uct 4 was observed at m/zϭ506 by EI-, FD- and FAB-MS, reactions probably plays a role in its pharmacological activ-
and a fragment ion was observed at m/zϭ476 by EI-MS. A ity.
peak at m/zϭ476 of product 3 in EI-, FD- and FAB-MS was
considered to be a molecular ion of product 3, with a mass
32 lower than that of product 4. Therefore product 3 was
Acknowledgments The authors are deeply grateful to M. Suzuki and C.
Nagata for performing the X-ray crystal structure analysis.
identified as 4,4Ј-dithiobis(2,6-di-tert-butylphenol). These
chemical structures of the anodic oxidation products are
summarized in Chart 5.
References
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2,6-Di-tert-butylphenols have often been employed as
model compounds for studies on the electrochemical oxida-
tion of phenols. For example, anodic oxidation of 2,6-di-tert-
butyl-4-methylphenol in acetonitrile predominantly gave the
cyclohexadienone, as shown in Chart 6.12) Product 2 was
formed where the phenoxonium ion was suggested to be in
the product yield. The formation of product 3 suggests a
mechanism involving the organothio radical. The initial one-
electron transfer produces the cation radical intermediate and
the carbon–sulfur bond is cleaved to give the corresponding
carbocation and the organothio radical. The organothio radi-
cal dimerizes to give product 3. Product 3 may undergo fur-
ther anodic oxidation to give product 4, but the mechanism 11) Ohmori H., Ueda C., Nakagawa T., Nishiguchi S., Jeong J., Masui M.,
Chem. Pharm. Bull., 34, 508—515 (1986).
12) Ohmori H., Ueda C., Tokuno Y., Maeda H., Masui M., Chem. Pharm.
remains unclear. There appears to be no report on the forma-
tion of the trisulfide form by electrochemical oxidation. One
possible mechanism is that probucol reacts with an organ-
Bull., 33, 4007—4011 (1985).