Chlorine atom substitution influences radical scavenging activity of 6-chromanol

Article abstract of DOI:10.1016/j.bmc.2012.05.008

Synthetic 6-chromanol derivatives were prepared with several chlorine substitutions, which conferred both electron-withdrawing inductive effects and electron-donating resonance effects. A trichlorinated compound (2), a dichlorinated compound (3), and three monochlorinated compounds (4, 5, and 6) were synthesized; compounds 2, 3, and 6 were novel. The antioxidant activities of the compounds, evaluated in terms of their capacities to scavenge galvinoxyl radical, were associated with the number and positioning of chlorine atoms in the aromatic ring of 6-chromanol. The activity of compound 1 (2,2-dimethyl-6-chromanol) was slightly higher than the activities of compounds 2 (2,2-dimethyl-5,7-dichloro-6-chromanol) or 3 (2,2-dimethyl-5,7,8-trichloro-6- chromanol), in which the chlorine atoms were ortho to the phenolic hydroxyl group of 6-chromanol. The scavenging activity of compound 3 was slightly higher than that of 2, which contained an additional chlorine substituted in the 8 position. The activities of polychlorinated compounds 2 and 3 were higher than the activities of any of the monochlorinated compounds (4-6). Compound 6, in which a chlorine was substituted in the 8 position, exhibited the lowest activity. Substitution of a chlorine atom meta to the hydroxyl group of 6-chromanol (compounds 2 and 6) decreased galvinoxyl radical scavenging activity, owing to the electron-withdrawing inductive effect of chlorine. Positioning the chloro group ortho to the hydroxyl group (compounds 4 and 5) retained antioxidant activity because the intermediate radical was stabilized by the electron-donating resonance effect of chlorine in spite of the electron-withdrawing inductive effect of chlorine. Antioxidant activities of the synthesized compounds were evaluated for correlations with the O-H bond dissociation energies (BDEs) and the ionization potentials. The BDEs correlated with the second-order rate constants (k) in the reaction between galvinoxyl radical and the chlorinated 6-chromanol derivatives in acetonitrile. This indicated that the antioxidant mechanism of the synthesized compounds consisted of a one-step hydrogen atom transfer from the phenolic OH group rather than an electron transfer followed by a proton transfer. The synthesized compounds also exhibited hydroxyl radical scavenging capacities in aqueous solution.

Full text of DOI:10.1016/j.bmc.2012.05.008

Bioorganic & Medicinal Chemistry 20 (2012) 4049–4055
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chlorine atom substitution influences radical scavenging activity
of 6-chromanol
Keiko Inami a,b, , Yuko Iizuka a, Miyuki Furukawa a, Ikuo Nakanishi c, Kei Ohkubo d, Kiyoshi Fukuhara e,
⇑
Shunichi Fukuzumi d,f, Masataka Mochizuki a,b
a Kyoritsu University of Pharmacy, Shibako-en 1-5-30, Minato-ku, Tokyo 105-8512, Japan
b Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan
c Radio-Redox-Response Research Team, Advanced Particle Radiation Biology Research Program, Research Center for Charged Particle Therapy, National Institute of
Radiological Sciences (NIRS), Inage-ku, Chiba 263-8555, Japan
d Department of Material and Life Science, Graduate School of Engineering, Osaka University, ALCA, Japan Science and Technology Agency, Suita, Osaka 565-0871, Japan
e Division of Organic Chemistry, National Institutes of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan
f Department of Bioinspired Science, Ewha Womans University, Seoul 120-750, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthetic 6-chromanol derivatives were prepared with several chlorine substitutions, which conferred
both electron-withdrawing inductive effects and electron-donating resonance effects. A trichlorinated
compound (2), a dichlorinated compound (3), and three monochlorinated compounds (4, 5, and 6) were
synthesized; compounds 2, 3, and 6 were novel. The antioxidant activities of the compounds, evaluated in
terms of their capacities to scavenge galvinoxyl radical, were associated with the number and positioning
of chlorine atoms in the aromatic ring of 6-chromanol. The activity of compound 1 (2,2-dimethyl-6-chro-
manol) was slightly higher than the activities of compounds 2 (2,2-dimethyl-5,7-dichloro-6-chromanol)
or 3 (2,2-dimethyl-5,7,8-trichloro-6-chromanol), in which the chlorine atoms were ortho to the phenolic
hydroxyl group of 6-chromanol. The scavenging activity of compound 3 was slightly higher than that of 2,
which contained an additional chlorine substituted in the 8 position. The activities of polychlorinated
compounds 2 and 3 were higher than the activities of any of the monochlorinated compounds (4–6).
Compound 6, in which a chlorine was substituted in the 8 position, exhibited the lowest activity. Substi-
tution of a chlorine atom meta to the hydroxyl group of 6-chromanol (compounds 2 and 6) decreased gal-
vinoxyl radical scavenging activity, owing to the electron-withdrawing inductive effect of chlorine.
Positioning the chloro group ortho to the hydroxyl group (compounds 4 and 5) retained antioxidant activ-
ity because the intermediate radical was stabilized by the electron-donating resonance effect of chlorine
in spite of the electron-withdrawing inductive effect of chlorine. Antioxidant activities of the synthesized
compounds were evaluated for correlations with the O–H bond dissociation energies (BDEs) and the ion-
ization potentials. The BDEs correlated with the second-order rate constants (k) in the reaction between
galvinoxyl radical and the chlorinated 6-chromanol derivatives in acetonitrile. This indicated that the
antioxidant mechanism of the synthesized compounds consisted of a one-step hydrogen atom transfer
from the phenolic OH group rather than an electron transfer followed by a proton transfer. The synthe-
sized compounds also exhibited hydroxyl radical scavenging capacities in aqueous solution.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 7 April 2012
Revised 4 May 2012
Accepted 5 May 2012
Available online 12 May 2012
Keywords:
Chlorine
Antioxidant
Chromanol
Electron-donating resonance effect
Electron-withdrawing inductive effect
1. Introduction
antioxidants is essential in the prevention of oxidative stress-re-
lated diseases.8–13
Antioxidants are of great scientific interest because of their
involvement in important biological processes such as the etiolo-
gies of cancers, cardiovascular diseases, and inflammation.1 Phe-
2,2-Dimethyl-6-chromanol (1) is the essential structure of vita-
min E. This compound and its derivatives inhibit lipid peroxidation
and lipoxygenase activity.14,15 Most lipoxygenase inhibitors used
as anti-inflammatory agents function as antioxidants or free radi-
cal scavengers, and their inhibitory activities are related to their
lipophilicities.16,17 The radical scavenging activity of vitamin E
increases with the substitution of electron-donating moieties into
its chromanol phenyl ring. In addition, the introduction of a halo-
gen atom into any molecule increases its lipophilicity.
nols and vitamin
E derivatives are regarded as particularly
significant antioxidants.2–7 The radical scavenging activity of the
⇑
Corresponding author. Tel./fax: +81 4 7121 3641.
E-mail address: inami@rs.noda.tus.ac.jp (K. Inami).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.008

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K. Inami et al. / Bioorg. Med. Chem. 20 (2012) 4049–4055
Figure 1. Structures of 6-chromanol (2,2-dimethyl-6-chromanol) and synthesized
chlorinated 6-chromanols.
Previous studies have described the synthesis of 2,2-dimethyl-
6-chromanols substituted with halogen atoms at the 5 or 7 posi-
tions;18–21 however, those reports did not focus on the radical
scavenging activities of the prepared compounds. We reasoned
that the introduction of one or more chlorine atoms into the phe-
nyl ring of 2,2-dimethyl-6-chromanol (compound 1) would retard
hydrogen atom abstraction due to the electron-withdrawing
inductive effect of chlorine. Whereas chlorine substitution would
predict to stabilize the intermediate radical due to its electron-
donating resonance effect, thereby enhancing antioxidative activ-
ity. We synthesized five derivatives of compound 1 (compounds
2–6) with chlorine atoms inserted at several positions in its chro-
man ring, and we evaluated the radical scavenging activities of
the compounds in an aprotic solvent and in aqueous solution
(Fig. 1).
Scheme 2. Synthesis of chlorinated chromanol 6.
The substitution of chlorine atom(s) into 6-chromanol increased
the acidity of the synthesized compounds. The pKa of each com-
pound was measured as the change in UV–visible absorption spec-
tra in aqueous buffer solution. Results were as follows: Compound
1, pKa 10.3; compound 2, 7.1; compound 3, 7.7; compound 4, 8.6;
compound 5, 9.1; compound 6, 9.8. The measured acidities were
consistent with the number and positioning of chlorine atom(s)
substituted into 6-chromanol.
The lipophilicities of the compounds were calculated as the
n-octanol-water partition coefficients (logP) calculated using
ChemDraw. LogP values increased significantly with the number
of chlorine group(s) carried by the synthesized compounds
(Table 1). Increased lipophilicities have been reported to influence
aqueous-phase radical scavenging properties and chain-breaking
antioxidant activities in biological membranes.17,23 The presence
of such compounds precludes the initial reaction between aqueous
radicals and biological membranes.24 In addition, compound lipo-
philicity is necessary for anti-inflammatory activity, as it affects
distribution, bioavailability, metabolism, and excretion of the
antioxidants.17,23
2. Results & discussion
2.1. Chemistry
Compound 1 has the basic structure of 2,2-dimethyl-6-chro-
manol. Compound 2 carries three chlorine atoms at positions 5,
7, and 8 on the aromatic ring; compound 3 carries two chlorine
atoms at positions 5 and 7. Compounds 4, 5, and 6 are substituted
with a chlorine atom at positions 5, 7, and 8, respectively, on the
chroman ring of compound 1.
2.2. Galvinoxyl radical scavenging activity of chlorinated 6-
chromanols
Chlorinated 6-chromanols 2, 3, 4, and 5 were synthesized
according to the methods of Nilsson et al.22 (Scheme 1). Briefly,
compounds 2, 3, 4, and 5 were prepared by condensation of the
corresponding chlorohydroquinones with 2-methyl-3-buten-2-ol.
Reaction mixtures were refluxed in formic acid under a nitrogen
atmosphere and were extracted with CH2Cl2. Extracts were washed
with water, dried over anhydrous Na2SO4, filtered, and evaporated
to yield oils, which then were dissolved in methanol and concen-
trated hydrochloric acid. Mixtures were refluxed for 1 h to induce
compound cyclization and were cooled to room temperature. The
reaction mixture was evaporated, extracted with CH2Cl2, and then
crude product was acetylated with acetic anhydride in pyridine
and was purified. Following acetylation of compound 1, compound
6 was synthesized by chlorination with sulfuryl chloride (Scheme
2). All acetylated 6-chromanols carrying chlorine atom(s) were
hydrolyzed with 1 M aqueous sodium hydroxide prior to measur-
ing radical scavenging activity.
The abilities of the halogenated 6-chromanols to scavenge gal-
vinoxyl radical were evaluated in acetonitrile. Galvinoxyl radical
is a reactive oxygen species with a strong absorption band at
Table 1
Second-order rate constant (k), BDE, IP, and logP of 6-chromanol derivatives
Compound
ka (Mꢀ1 sꢀ1
)
BDE (kcal/mol)
IP (eV)
LogPb
1
2
3
4
5
6
98 0.002
54 0.002
67 0.001
37 0.001
42 0.001
21 0.001
395.2
396.2
395.7
397.0
397.0
396.3
6.83
7.30
7.22
7.04
7.04
7.01
2.45
4.12
3.57
3.01
3.01
3.01
a
Standard error of regression line for each concentration.
LogP calculated using ChemDraw ver.8.0 (CambridgeSoft, Cambridge, USA)
b
Scheme 1. Synthesis of chlorinated chromanols 2–5.

K. Inami et al. / Bioorg. Med. Chem. 20 (2012) 4049–4055
4051
with a decreased second-order rate constant in the reaction with
galvinoxyl radical.
Halogen substitution confers aromatic rings with an electron-
withdrawing inductive effect and an electron-donating resonance
effect. The k values of all synthesized chlorinated chromanols were
lower than that of compound 1. The data indicated that, in the con-
text of 6-chromanol, chlorine atom(s) withdraw electrons via the
inductive effect more strongly than they donate electrons by reso-
nance. The inductive effect depends on the distance of substituent
from the halogen atom, whereas the resonance effect influences
atoms positioned ortho or para to the hydroxyl group. Compound
6 carried a chlorine in the meta position; therefore, the halogen ex-
erted only an electron-withdrawing effect, and this compound dis-
played the weakest relative scavenging activity. Compounds 4 and
5 had higher activities than 6 despite of having chlorines posi-
tioned where they would exert greater electron-withdrawing
effects.
Scavenging activity increased with the number of chlorine
atoms substituted into the 6-chromanol (compounds 2 and 3),
indicating that chlorine’s electron-donating resonance effect con-
ferred stability to the intermediate radical. In contrast, the chloro
group meta to the hydroxyl group (compound 6) reduced scaveng-
ing activity by destabilizing the intermediate radical via chlorine’s
electron-withdrawing inductive effect. Therefore, stability of the
intermediate radical was a key factor in radical scavenging activity.
To elucidate the antioxidant mechanisms of the chlorinated 6-
chromanols, the rate constants k for compounds 1–6 were deter-
mined and compared with the O–H bond dissociation enthalpies
(BDEs) and the ionization potentials (IPs). BDEs and IPs were
calculated according to density functional theory (see Section 4).
Phenolic compounds scavenge oxyl radicals via two primary mech-
anisms: one-step hydrogen atom transfer (HAT; Fig. 4A) or electron
transfer followed by proton transfer (ET–PT; Fig. 4B).29 One-step
HAT involves hydrogen atom abstraction from a phenolic hydroxyl
group to form a phenoxyl radical. By this mechanism, O–H bond
dissociation is homolytic, and the reaction is driven by the BDE.
Lower BDEs are associated with higher O–H bond dissociation
reactivities. In contrast to HAT, ET–PT involves electron loss fol-
lowed by exothermic proton loss and radical cation formation.
ET–PT is driven by the IP.
Figure 2. Spectral changes in the reaction of compound 1 (4.2 ꢂ 10ꢀ4 M) with
galvinoxyl radical (10 ꢂ 10ꢀ6 M) in deaerated acetonitrile at 298 K. (Inset) First-
order function describing the change in absorbance at 428 nm.
428 nm.25–28 Upon addition of compound 1 to a deaerated solution
of galvinoxyl radical in acetonitrile, the absorption band at 428 nm
decreased precipitously (Fig. 2). The decay in absorbance at
428 nm obeyed pseudo-first-order kinetics when the concentra-
tion of compound 1 was held at a >10-fold excess of galvinoxyl rad-
ical concentration (Fig. 2 inset). The observed pseudo-first-order
rate constant (kobs) was dependent on the concentration of com-
pound 1, as demonstrated by the linear correlation between kobs
and the concentration of compound 1. The second-order rate
constants (k) for the reaction between the halogenated 6-chroma-
nols and galvinoxyl radical were obtained from the slopes of the
linear functions of kobs versus the compound concentrations
(Fig. 3, Table 1).
The chlorinated 6-chromanols were ordered by radical scaveng-
ing activity as follows: 1 ꢁ 3 ꢁ 2 > 5 = 4 >> 6 (Fig. 3, Table 1). Com-
pound 3 exhibited slightly weaker activity than non-halogenated
compound 1, and halogenated compound 2 exhibited less activity
than 3. Monochlorinated compounds 4, 5, and 6 displayed lower
activities than compounds 1, 2, or 3. The monochlorinated 6-
chromanols with chloro groups positioned ortho to the hydroxyl
group (4 and 5), showed higher activities than compound 6, which
carried a chloro group in meta. In both polychlorinated and mono-
chlorinated 6-chromanols, the introduction of a chlorine atom
meta to the hydroxyl group in the chromanol ring was associated
Antioxidant capacity was inferred from galvinoxyl radical scav-
enging activity because galvinoxyl radical is stable at room tem-
perature and is commercially available. Table 1 lists the values of
k, BDEs, and IPs for the synthesized 6-chromanols. The logk values
of the halogenated 6-chromanols correlated linearly with the BDEs
(R = 0.98) except in the case of compound 6 (Fig. 5). In contrast, the
k values did not correlate with the IPs for any of the compounds.
These results suggest that the synthesized compounds scavenge
galvinoxyl radical via one-step HAT in acetonitrile.30–32
Several reports have calculated the BDEs of chlorinated phe-
nols;33–35 however, the BDEs of chlorinated 6-chromanols are not
well characterized. Other studies have reported correlations be-
tween BDEs and radical scavenging activities of paracetamols or
hydroxycinnamic acids carrying halogen atom(s) positioned ortho
to hydroxyl groups.36,37 Our findings support these results.
No correlation was observed between the logk value and the
BDE for compound 6, suggesting that the electron-withdrawing ef-
fect of chlorine positioned meta to the hydroxyl group was larger
than the calculated BDE of this compound and induced disadvanta-
geous hydrogen abstraction from the hydroxyl group.
2.3. Hydroxyl radical scavenging activity
Antioxidants must display radical scavenging activity in aque-
ous solution, as the hydroxyl radical is an extremely reactive and
short-lived species.38 We used electron spin resonance (ESR)39 to
Figure 3. Plot of the pseudo-first-order rate constant (kobs) versus the concentra-
tion of compound 1 (s), 2 (d), 3 (N), 4 (j), 5 (ꢀ), 6 (.).

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K. Inami et al. / Bioorg. Med. Chem. 20 (2012) 4049–4055
Figure 4. Mechanisms of HAT (A) and ET–PT (B).
Figure 6. Hydroxyl radical scavenging activities of compound 1 (s), 2 (d), 3 (N), 4
(j), 5 (ꢀ), 6 (.) determined by ESR spectroscopy.
Figure 5. Log k versus BDE for compound 1 (s), 2 (d), 3 (N), 4 (j), 5 (ꢀ). BDE
(kcal/mol) = BDE - 395.2 (BDE of compound 1).
measure the capacity of the antioxidants to compete with the
radical trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO),
in scavenging hydroxyl radical generated by the Fenton reaction.
ortho to the phenolic hydroxyl group (compound 3) retained radi-
cal scavenging activity through weak electron-donating resonance
effects in spite of electron-withdrawing inductive effect. However,
a chlorine atom positioned meta to the hydroxyl group (compound
6) decreased radical scavenging activity via the electron-with-
drawing inductive effect. The lipophilicity (logP) of the chlorinated
6-chromanols increased. It is important for antioxidants and anti-
inflammatory agents to increase the lipophilicity with having the
radical scavenging activity. Our results support the continued
investigation of substituted 6-chromanols as candidates for anti-
inflammatory agents based on their antioxidant activity.
Hydroxyl radical induces
a
a characteristic four-line (1:2:2:1,
N = 14.8 G) DMPO–OH signal. For concentration of each com-
pound, the relative hydroxyl radical scavenging activity was ex-
pressed as the ratio of the peak height of the DMPO–OH adduct
to the peak height of the Mn2+ internal standard. The DMPO–OH
adduct decreased in the presence of each synthesized compound
(Fig. 6).
Each of the chlorinated 6-chromanols inhibited the formation of
the DMPO–OH adduct in a dose-dependent manner and exhibited
scavenging activity in aqueous solution similar to compound 1. The
low solubility of the chlorinated 6-chromanols in aqueous solution
made the measurement at higher concentrations impossible. The
synthesized 6-chromanols all functioned as hydroxyl radical
scavengers.
4. Experimental methods
4.1. Reagents
2,6-Dichlorophenol, 2-methyl-3-buten-2-ol, and 2,3,6-trichlo-
rophenol were obtained from Tokyo Kasei Kogyo Co., Ltd (Tokyo,
Japan). Chlorohydroquinone was purchased from Kanto Chemical
Co., Inc. (Tokyo, Japan). DMPO was acquired from Labotec Co., Ltd
(Hiroshima, Japan). Galvinoxyl free radical was obtained commer-
cially from Sigma–Aldrich (St. Louis, USA). Acetonitrile, used for
spectral measurements, was obtained from Dojindo Laboratories
3. Conclusion
The radical scavenging activities of the synthesized 6-chro-
manol compounds were influenced by the number and positioning
of substituted chlorine(s). The presence of two chlorine atoms

K. Inami et al. / Bioorg. Med. Chem. 20 (2012) 4049–4055
4053
(Kumamoto, Japan). Other reagents were purchased from Wako
4.5. Acetylated compound 4
Pure Chemical Industries (Osaka, Japan). All reagents and solvents
used were the best quality commercially available and were not
further purified unless otherwise noted. Reaction progressions
were monitored using thin-layer chromatography (TLC) on silica
gel 60 F254 (0.25 mm, Merck). Column chromatography was per-
formed on silica gel 60 (0.063–0.200 mm, Merck). Melting points
were determined using a Yanagimoto micro melting-point appara-
tus without correction. Infrared spectra were recorded on a Nihon
Bunkou V-560 spectrophotometer (Tokyo, Japan). 1H NMR spectra
were recorded with a JEOL JNM A500 spectrometer (Tokyo, Japan).
Chemical shifts were expressed in terms of ppm downfield shifted
from tetramethylsilane (s = singlet, d = doublet, t = triplet, m =
multiplet, br = broad peak). Mass spectra were determined using
a Shimadzu GCMSQP5050A spectrometer (Kyoto, Japan). High res-
olution mass spectra were collected using a JEOL JMS700 mass
spectrometer. ESR spectra were obtained using a JEOL X-band
spectrometer (JES-RE1X) under nonsaturating microwave power
conditions. Compound 1 was synthesized as described previously
(mp: 74.2–75.2 °C).22
Brown oil; yield 3.5%; 1H NMR (500 MHz, CDCl3): d 1.32 (6H, s,
gem-CH3), 1.82 (2H, t, J = 6.7 Hz, H-3), 2.33 (3H, s, –COCH3), 2.78
(2H, t, J = 6.7 Hz, Ar-CH2–), 6.71 (1H, d, J = 9.2 Hz, Ar-H), 6.87 (1H,
d, J = 9.3 Hz, Ar-H); HR-MS 254.0709 (calcd for
C13H15ClO3:
254.0710).
4.6. Acetylated compound 5
Pale yellow oil; yield 7.1%; 1H NMR (500 MHz, CDCl3): d 1.31
(6H, s, gem-CH3), 1.77 (2H, t, J = 6.7 Hz, H-3), 2.31 (3H, s), 2.72
(2H, t, J = 6.7 Hz, Ar-CH2–), 6.81 (1H, s, Ar-H), 6.85 (1H, s, Ar-H);
HR-MS 254.0715 (calcd for C13H15ClO3: 254.0710).
4.7. Synthesis of 6-acetoxy-8-chloro-2,2-dimethylchroman
(acetylated compound 6)
Acetylated compound 1 was prepared with acetic anhydride
and pyridine. To a solution of compound 1 (1.0 g, 5.6 mmol) in pyr-
idine (5 mL), acetic anhydride (5 mL) was slowly added with stir-
ring under an argon atmosphere at room temperature. After 4 h,
the reaction mixture was poured into crushed ice, filtered, and a
white solid was isolated (yield 95.6%, mp 71.5–74.0 °C). To a solu-
tion of acetylated 1 (500 mg, 2.3 mmol) in CH2Cl2 (20 mL), SO2Cl2
(0.92 mL, 11.4 mmol) was added dropwise, and the solution was
stirred for 5 min at room temperature under a nitrogen atmo-
sphere. Water was added to the reaction mixture, and the mixture
was extracted three times with CH2Cl2. The combined organic
phase was serially washed with water, saturated NaHCO3 solution,
and water, and then was dried over Na2SO4, filtered, and evapo-
rated, yielding 652 mg of yellow oil. The crude product was puri-
fied by silica gel column chromatography to yield 277 mg of pale
yellow needles. Yield 48%; mp 47–49 °C; 1H NMR (500 MHz,
CDCl3): d 1.37 (6H, s, gem-CH3), 1.81 (2H, t, J = 6.7 Hz, H-3), 2.26
(3H, s, Ar-OCOCH3), 2.78 (2H, t, J = 7.0 Hz, Ar-CH2–), 6.72 (1H, d,
J = 2.4 Hz, Ar-H), 6.94 (1H, d, J = 3.1 Hz, Ar-H); HR-MS 254.0677
(calcd for C13H15ClO3: 254.0710).
4.2. Synthesis of 6-acetoxy-2,2-dimethylchroman substituted
with chlorine atom
2,3,6-Trichlorohydroquinone or 2,6-dichlorohydroquinone
were synthesized from the corresponding polychlorophenols using
PbO2 with HClO4 in acetic acid,40 followed by reduction of the poly-
chloro-p-benzoquinone ether solution with aqueous Na2S2O4 in a
separatory funnel.
A solution of the corresponding chlorohydroquinone in distilled
formic acid was refluxed for 1 h under a nitrogen atmosphere. 2-
Methyl-3-buten-2-ol (4.8 equiv for compound 2; 1.3 equiv for 3,
4 and 5) in distilled tetrahydrofuran was added dropwise over 2–
4 h, and the solution was refluxed overnight under a nitrogen
atmosphere. The reaction mixture was poured into crushed ice
and extracted twice with CH2Cl2. The combined organic phase
was serially washed with water, saturated NaHCO3 aqueous solu-
tion, and water. The organic phase was dried over Na2SO4, filtered,
and evaporated to yield a brown oil, which was dissolved in meth-
anol, combined with concentrated HCl, and refluxed for 1 h. The
methanol was evaporated to yield crude product, extracted with
CH2Cl2, washed with water, dried over Na2SO4, filtered, and evap-
orated. The crude product was fractionated by silica gel column
chromatography. A fraction was dissolved in excesses of pyridine
and acetic anhydride and stirred at room temperature for 5 h.
Water was added to the reaction mixture, and samples were ex-
tracted three times with diethyl ether following neutralization
with saturated Na2CO3 aqueous solution. The combined organic
phase was serially washed with water, 5% HCl, water, saturated
NaHCO3 aqueous solution, and water. It subsequently was dried
over Na2SO4, filtered, and evaporated to give an oil. The residue
was purified by silica gel column chromatography.
4.8. Hydrolysis of the acetylated 6-chromanol derivatives
The acetylated compounds were hydrolyzed immediately be-
fore measuring galvinoxyl radical scavenging activities. To a solu-
tion of the acetylated compound in acetonitrile, 1 M NaOH was
added, and the solution was stirred at room temperature under
an argon atmosphere until the starting material was no longer vis-
ible by TLC. Acetylated compounds 2 and 3 were stirred for 4 h.
Acetylated 4, 5, and 6 were stirred for 2 h. The reaction mixture
was neutralized with 5% aqueous HCl and extracted three times
with diethyl ether. The combined organic phase was washed with
water and dried over Na2SO4. Samples were filtered and evapo-
rated to yield crude products, which were then purified by column
chromatography. The yields exceeded 80% for all compounds.
Compound 2: White solid; mp 83–86 °C; 1H NMR (500 MHz,
CDCl3): d 1.36 (6H, s, gem-CH3), 1.85 (2H, t, J = 7.0 Hz, H-3), 2.77
(2H, t, J = 7.0 Hz, Ar-CH2–), 5.51 (1H, s, Ar-OH); HR-MS 279.9825
(calcd for C11H11Cl3O2: 279.9825).
4.3. Acetylated compound 2
White solid; yield 6.0%; mp 103–105 °C; 1H NMR (500 MHz,
CDCl3): d 1.38 (6H, s, gem-CH3), 1.86 (2H, t, J = 6.7 Hz, H-3), 2.39
(3H, s, Ar-OCOCH3), 2.79 (2H, t, J = 6.7 Hz, Ar-CH2–); HR-MS
321.9915 (calcd for C13H13Cl3O3: 321.9932).
Compound 3: Colorless oil; 1H NMR (500 MHz, CDCl3): d 1.30
(6H, s, gem-CH3), 1.81 (2H, t, J = 7.0 Hz, H-3), 2.72 (2H, t,
J = 6.7 Hz, Ar-CH2–), 6.77 (1H, s, Ar-H); HR-MS 246.0216 (calcd
for C11H12Cl2O2: 246.0214).
4.4. Acetylated compound 3
Compound 4: White solid; mp 68–71 °C; 1H NMR (500 MHz,
CDCl3): d 1.31 (6H, s, gem-CH3), 1.81 (2H, t, J = 6.7 Hz, H-3), 2.75
(2H, t, J = 7.0 Hz, Ar-CH2–), 5.10 (1H, s, Ar-OH), 6.65 (1H, d,
J = 8.6 Hz, Ar-H), 6.81 (1H, d, J = 8.5 Hz, Ar-H); HR-MS 212.0583
(calcd for C11H13ClO2: 212.0605).
White solid; yield 7.2%; mp 99–102 °C; 1H NMR (500 MHz,
CDCl3): d 1.32 (6H, s, gem-CH3), 1.81 (2H, t, J = 6.7 Hz, H-3), 2.37
(3H, s, Ar-OCOCH3), 2.74 (2H, t, J = 6.7 Hz, Ar-CH2–), 6.83 (1H, s,
Ar-H); HR-MS: 288.0281 (calcd for C13H14Cl2O3: 288.0321).

4054
K. Inami et al. / Bioorg. Med. Chem. 20 (2012) 4049–4055
Compound 5: Pale yellow oil; 1H NMR (500 MHz, CDCl3): d 1.30
4.12. Theoretical calculations
(6H, s, gem-CH3), 1.77 (2H, t, J = 6.7 Hz, H-3), 2.71 (2H, t, J = 6.7 Hz,
Ar-CH2–), 5.04 (1H, s, Ar-OH), 6.72 (1H, s, Ar-H), 6.76 (1H, s, Ar-H);
EI-MS: 212 (M+), 214 ([M+2]+).
Density functional theory calculations were performed using an
8 CPU workstation (PQS, Quantum Cube QS8-2400C-064). Geome-
try optimizations were carried out using the RB3LYP/6-31G(d)
basis set for the neutral forms of chromanols and the UB3LYP/6-
31G(d) basis set for the radical anion and phenoxyl radicals forms,
as implemented in the Gaussian 03 program, revision C.02 (Wal-
lingford, USA). The energies of the radical anion and phenoxyl rad-
ical were obtained from the single point energy calculations in the
ROB3LYP/6-31G(d) basis set using the geometry-optimized struc-
tures. IP values were calculated from the energy differences be-
tween the neutral and radical anion forms. Relative BDE values
were determined from the energy difference between the neutral
and phenoxyl radical forms, where the values are normalized by
the BDE of compound 1.
Compound 6: White solid; mp 34–37 °C; 1H NMR (500 MHz,
CDCl3): d 1.35 (6H, s, gem-CH3), 1.79 (2H, t, J = 6.7 Hz, H-3), 2.74
(2H, t, J = 6.7 Hz, Ar-CH2–), 4.43 (1H, s, Ar-OH), 6.48 (1H, d,
J = 3.6 Hz, Ar-H), 6.73 (1H, d, J = 3.1 Hz, Ar-H); HR-MS 212.0604
(calcd for C11H13ClO2: 212.0605).
4.9. Kinetics
Kinetics were determined by measuring the disappearance of
absorbance at 428 nm under pseudo-first-order conditions at
25 °C. An aliquot of chlorinated chromanols (final concentrations:
0.17, 0.25, 0.33, 0.42, 0.50
0.20, 0.27, 0.33, 0.40 M for compounds 2, 3) in deaerated acetoni-
trile was added to quartz cuvette (10 mm id) containing
galvinoxyl radical (final concentration 10.0 M) in deaerated ace-
lM for compounds 1, 4–6 and 0.13,
l
a
Acknowledgements
l
tonitrile (3 mL). UV–vis spectral changes following this reaction
were monitored using a Hewlett-Packard 8453 photo diode array
spectrophotometer (Hanover, USA). Radical scavenging rates were
determined by monitoring the absorbance change at 428 nm due
to the decrease in galvinoxyl radical. Pseudo-first-order rate con-
stants were determined by the method of least-squares. Pseudo-
This work was partially supported by Grant-in-Aid (Nos.
23590064, 23750014 and 20108010) from the Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan, the Global
COE (center of excellence) program ‘‘Global Education and Re-
search Center for Bio-Environmental Chemistry’’ of Osaka Univer-
sity from MEXT, Japan, and NRF/MEST of Korea through WCU
(R31-2008-000-10010-0) and GRL (2010-00353) programs.
first-order rate plots of ln(A–A ) versus time, where A and A refer
1
1
to the absorbance at the reaction time and the final absorbance,
respectively, were linear until three or more half-lives. First-order
rate constants were obtained from the slopes of plots of kobs versus
various concentrations of the compounds. All experiments were
conducted in triplicate.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.008.
4.10. Hydroxyl radical scavenging activity
References and notes
Hydroxyl radical scavenging capacity of the chlorinated chrom-
anols was determined by ESR, which was based on the competition
between the trapping agent (DMPO) and the chlorinated chroma-
nols. Hydroxyl radical was generated by the Fenton reaction. Ace-
tonitrile was used to dissolve each chlorinated chromanol.
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Reaction mixtures contained 10
FeSO4, 10 L of 200 mM DMPO, 10
0.1 M sodium phosphate buffer (pH 7.4), and 10
l
L of 10 mM freshly prepared
L of 10 mM H2O2, 180 L of
L of chlorinated
l
l
l
l
chromanols in acetonitrile solution (5, 10, 20, 50, 100, 250, and
500 nmol). Pure acetonitrile was used for the blank. The mixture
was transferred into a quartz cell and was incubated at room tem-
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settings were: magnetic field 335.5 5.0 mT, microwave power
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0.079 mT, sweep time 2.0 min, response time 0.1 s, receiver gain
50–100.
4.11. pKa measurement
An aliquot of chlorinated chromanols (1.3–2.5 ꢂ 10ꢀ4 M) in
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changes at the following wavelengths for each compound: 1,
312 nm; 2, 313 nm; 3, 321 nm; 4, 313 nm; 5, 319 nm; 6, 315 nm
(y axis) as a function of pH (x axis) (data not shown). The pKa
was obtained from the 50% value in the sigmoid curve, subtracting
the minimum absorbance from the maximum absorbance for each
compound.

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