Effects of electron-withdrawing substituents on DPPH radical scavenging reactions of protocatechuic acid and its analogues in alcoholic solvents

Article abstract of DOI:10.1016/j.tet.2005.06.040

The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of protocatechuic acid (3,4-dihydroxybenzoic acid) and its related catechols was examined. Compounds possessing strong electron-withdrawing substituents showed high activity. NMR analysis of the reaction mixtures of catechols and DPPH radical in methanol showed the formation of methanol adducts. The results suggest that high radical scavenging activity of catechols in alcohol is due to a nucleophilic addition of an alcohol molecule on o-quinones, which leads to a regeneration of a catechol structure. Furthermore, the radical scavenging activity in alcohols would largely depend on the electron-withdrawing/donating substituents, since they affect the susceptibility toward nucleophilic attacks on o-quinone.

Full text of DOI:10.1016/j.tet.2005.06.040

Tetrahedron 61 (2005) 8101–8108
Effects of electron-withdrawing substituents on DPPH radical
scavenging reactions of protocatechuic acid and its analogues
in alcoholic solvents
*
Shizuka Saito and Jun Kawabata
Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku,
Sapporo 060-8589, Japan
Received 2 May 2005; revised 16 June 2005; accepted 16 June 2005
Available online 5 July 2005
Abstract—The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of protocatechuic acid (3,4-dihydroxybenzoic acid) and
its related catechols was examined. Compounds possessing strong electron-withdrawing substituents showed high activity. NMR analysis of
the reaction mixtures of catechols and DPPH radical in methanol showed the formation of methanol adducts. The results suggest that high
radical scavenging activity of catechols in alcohol is due to a nucleophilic addition of an alcohol molecule on o-quinones, which leads to a
regeneration of a catechol structure. Furthermore, the radical scavenging activity in alcohols would largely depend on the electron-
withdrawing/donating substituents, since they affect the susceptibility toward nucleophilic attacks on o-quinone.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
found that regeneration of catechol structures by a
nucleophilic addition of a solvent alcohol molecule on
o-quinones is a key reaction for the higher radical
scavenging activity of protocatechuic esters in alcoholic
solvents than in non-alcoholic solvents.11,13 Interestingly, 1
showed significantly low activity in alcohol compared to its
methyl ester (2), and no alcohol adduct was formed. In
addition, the radical scavenging activity of 30,40-dihydroxy-
acetophenone (3), 3,4-dihydroxybenzaldehyde (4) and
3,4-dihydroxybenzonitrile (5), which bear electron-with-
drawing groups (–COMe, –CHO, –CN) at C-1 of the
catechol ring, was comparable to that of 2.14 It seems that
the radical scavenging activity is greatly affected by the C-1
substituents on the catechol ring. Therefore, in the present
study, we extended our investigation on radical scavenging
reactions to other C-1 substituted catechols. The objective
of this study is to determine whether the radical scavenging
mechanisms of protocatechuic ester analogues, possessing
electron-withdrawing/donating groups at C-1, are similar or
different to that of 2, and to examine the effects of the
electronic properties of the substituents on the radical
scavenging reactions beyond the formation of o-quinones.
In this study, the DPPH (2,2-diphenyl-1-picrylhydrazyl)
radical scavenging activity of protocatechuic acid and its
related compounds, which bear electron-withdrawing/
donating substituents on the catechol ring (Fig. 1), was
compared in acetonitrile and methanol. In addition, the
reaction mixtures of catechols and DPPH radical were
Polyphenols such as hydroxybenzoic and hydroxycinnamic
acids derivatives are known to exhibit potent antioxidant
activities. In particular, catechol-type o-diphenols such as
protocatechuic acid (3,4-dihydroxybenzoic acid, 1) and
caffeic acid show high antiradical activity,1–4 since they
would be readily converted to the corresponding o-quinones
and further complex products.1,5 In recent years, kinetic
studies on reactions of phenolic antioxidants with radicals,
including substituents effects,6–8 have been extensively
investigated.6–10 However, to understand the whole anti-
oxidant mechanisms of catechols and explain why they
show high radical scavenging activity, it is necessary to
study the reaction events after the formation of o-quinones.
Previously, we reported the solvent dependency of radical
scavenging activity of protocatechuic acid and its esters.11
In non-alcoholic acetone or acetonitrile, protocatechuic acid
and its esters consumed two radicals and were converted to
their quinones. In contrast, protocatechuic esters rapidly
scavenged more than four radicals with a concomitant
conversion to the corresponding quinones, 3-hemiacetals,12
and their alcohol adducts at C-2 in methanol or ethanol. We
Keywords: Protocatechuic acid; Radical scavenging mechanism; Anti-
oxidant; DPPH radical; Electron-withdrawing groups.
Corresponding author. Tel./fax: C81 11 706 2496;
e-mail: junk@chem.agr.hokudai.ac.jp
*
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.06.040

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S. Saito, J. Kawabata / Tetrahedron 61 (2005) 8101–8108
Figure 1. Structures of protocatechuic acid and its related compounds, and their oxidation products.
analyzed by NMR and HPLC, to determine what went on
after the quinone formation with the aid of molecular orbital
2. Results and discussion
The DPPH radical scavenging activity of catechols (1–13),
which possess electron-withdrawing/donating substituents
at C-1 of the aromatic ring except 11, was evaluated in
methanol and acetonitrile. The relative radical scavenging
equivalences of compounds 1–13, when that of DL-a-
tocopherol as standard was designated as 2.0, are listed in
Table 1. In inert acetonitrile, all test compounds scavenged
approximately two radicals in 30 min, and there was no
significant difference in activity among these compounds.
On the other hand, in nucleophilic methanol, all compounds
showed relatively high activity compared to that in
acetonitrile, and consumed more than two radical in
30 min. As shown in Figure 2, the DPPH radical scavenging
equivalences of compounds possessing electron-withdraw-
ing substituents (1–10) and 11 in methanol correlated well
with their Hammett substituents sp values.15 It clearly
shows that compounds bearing strong electron-withdrawing
substituents at C-1 exhibit high radical scavenging activity.
Considering that the high radical scavenging activity of
protocatechuic esters is due to their regeneration of the
catechol structures via a nucleophilic attack of an alcohol
molecule on o-quinones, it is indicated that the strong
electron-withdrawing substituents enhance the electrophilic
calculations, and the radical scavenging mechanism of proto-
catechuic ester analogues in alcoholic solvents is proposed.
Table 1. DPPH radical scavenging equivalence in methanol and
acetonitrile after 30 min, and the Hammett sp values
Radical scavenging equivalencea
b
sp
Compound
MeOH
MeCN
0.45 (COOH)
0.00 (COOK
0.45
0.50
)
1
2.5
2.2
2
3
4
5
6
7
8
9
10
11
12
13
5.0
4.8
5.7
5.5
4.9
4.9
5.2
3.0
2.5
2.7
2.8
2.1
2.2
2.2
2.2
2.1
0.42
0.66
0.36
—
—
0.23
0.06
0.00
c
—
2.1
2.3
2.0
1.8
1.9
1.9
1.8
K0.17
K0.27
a The equivalence is expressed as the values relative to that of DL-a-
tocopherol as 2.0.
b sp Values according to Ref. 15.
c Not tested due to low solubility.

S. Saito, J. Kawabata / Tetrahedron 61 (2005) 8101–8108
8103
Figure 3. 1H NMR spectra of 2 (a), 3 (b), 3c (c) and 12 (d) reacted with
DPPH radical in methanol-d4/acetone-d6 (3:1) 10 min after being
mixed.The intense signals in the range of 7.1–7.5 ppm are due to 2,2-
diphenyl-1-picrylhydrazine.
Figure 2. Correlation between DPPH radical scavenging equivalence in
methanol after 30 min and the Hammett sp values.
nature of the quinone rings, and thus facilitate the
nucleophilic addition of an alcohol molecule on o-quinones,
which leads to the high radical scavenging activity. Since
the Hammett sp value of COOK is much smaller than those
of COOH and COOMe, the low activity of 1 compared to its
methyl ester (2) in methanol can be due to the dissociation
of the free carboxylic group to the carboxylate ion, which is
accelerated by the strong electron-withdrawing property of
the quinone carbonyls. Moreover, the radical scavenging
equivalence of 3,4-dihydroxybenzoyloxyacetic acid (7),
which also has a free carboxylic acid (–CO2CH2CO2H) at
C-1, was comparable to that of 2 and 8, indicating that the
presence of a nonconjugated carboxylic group does not
affect the reactivity toward the nucleophilic attack on the
quinone ring. It is known that electron-donating substituents
reduce the O–H bond dissociation enthalpies of phenols,
and hence, facilitate hydrogen atom abstractions, whereas
electron-withdrawing groups have the opposite effect.6,7,16,17
In the present study, however, the radical scavenging
activity of compounds 12 and 13, which bear electron-
donating substituents at C-1, exhibited relatively low
activity compared to compounds, possessing strong elec-
tron-withdrawing groups (2–8). The result indicates that
although electron-withdrawing substituents have negative
effects toward initial hydrogen atom abstraction, they
contribute to the total radical scavenging ability of catechols
by enhancing the electrophilicity of the o-quinones.
5.85 (JZ10.3 Hz) was observed. The last doublet signal was
assumed to be a methanol adduct, since it corresponded to the
signal of H-5 of 2e (Fig. 3a). To confirm the formation of the
methanol adduct, the mixture of authentic 3c and DPPH
radical was analyzed(Fig. 3c). The1H NMR spectrumshowed
twosetsofdoubletsignalsatd6.20and7.49(JZ10.3 Hz),and
d 5.85 and 7.67 (JZ10.3 Hz), which correspond to the signals
of 2d and 2e, respectively.11 These doublet signals had typical
large coupling constants compared to the catechol (3c, JZ
8.6 Hz), indicating the formation of o-quinones and its
3-hemiacetals.14 Furthermore, the HMBC spectrum showed
a correlation between H-5 and C-3 acetal carbon at d 92.5.
Hence,thedoubletsignalatd5.85inFigure 3bwasassignedas
H-5 of 2-methoxy adduct (3e). Similarly, the NMR analysis of
the reaction mixture of 5 and DPPH radical revealed the
formation of a methanol adduct at C-2 (5e), together with 5a
and 5b. The results indicate that the radical scavenging
reactions of protocatechuic esters and analogues, which
possess strong electron-withdrawing substituents at C-1, are
very similar, and that methanol additions occur at C-2 of the
o-quinones.
1
The H NMR spectrum of the mixture of 12 and DPPH
radical in methanol-d4/acetone-d6 (3:1) showed peaks at
d 6.23 (1H, d, JZ2.0 Hz), d 6.32 (1H, d, JZ10.1 Hz), and d
7.05 (1H, dd, JZ10.1, 2.0 Hz) (Fig. 3d). The signals at d
3
6.32 and d 7.05 showed JCH HMBC correlation with two
To elucidate the radical scavenging mechanism, the reaction
mixtures of catechols and DPPH radical were directly
analyzed by NMR. Since the time course of the DPPH
radical scavenging activity of compounds 2–5 reached
steady state within 10 min,14 the reaction mixtures were
analyzed by NMR 10 min after mixing. The 1H NMR
spectrum of the mixture of 3 and DPPH radical in acetone-
d6 showed only signals of the corresponding o-quinone (3a),
distinct carbonyls of C-3 (d 180.4) and C-4 (d 181.3),
respectively, indicating the formation of the o-quinone
(12a). The result was in agreement with the report for the
formation of 12a from 12 and DPPH radical in aceto-
nitrile.18 Interestingly, unlike 1–5, no signal due to an acetal
(12b) was observed. Similarly, the spectrum of the mixture
of 11 (or 13) and DPPH radical in methanol-d4/acetone-d6
(3:1) after 10 min showed only the signals of its o-quinone
(11a, 13a). It can be speculated that the absence of an
electron-withdrawing substituent at C-1 increases the
stability of the o-quinone, and hence, a nucleophilic attack
of a methanol molecule at C-3 carbonyl of 12a to form the
corresponding 3-hemiacetal (12b) is unlikely to occur. The
1
as was seen for 1 and 2.14 As shown in Figure 3b, the H
NMR spectrum of the reaction mixture of 3 and DPPH
radical in methanol-d4/acetone-d6 (3:1) was also similar to
that of the mixture of 2 and DPPH radical (Fig. 3a), which
showed signals of 2a, 2b and its methanol adducts at C-2
(2d and 2e). Together with the characteristic signals of H-5 of
the o-quinone (3a) and its acetal (3b) at d 6.45 (d, JZ10.3 Hz)
and d 6.12 (d, JZ10.3 Hz), respectively, a doublet signal at d
1
stability of 12a was supported by the result of H NMR
analysis in which the signals of 12a remained unchanged for

8104
S. Saito, J. Kawabata / Tetrahedron 61 (2005) 8101–8108
Scheme 1. Plausible radical scavenging mechanism of methyl protocatechuate and its analogues in non-alcoholic solvents (a) and alcoholic solvents (b).
3 h, whereas those of 2a–5a disappeared within 1 h in
methanol-d4/acetone-d6 (3:1). Moreover, in the H NMR
produced from 2–5 by reacting with two radicals, 2c–5c
could largely contribute to the total radical scavenging
activity of 2–5. Previously, we reported that thiol adducts at
C-2 of 1–5, formed by oxidation of 1–5 in the presence of a
thiol nucleophile, undergo a second nucleophilic attack at
C-5.14 Therefore, a methanol addition on the o-quinones of
2c–5c would also occur. However, since 2c–5c consumed
less than four radicals, the second addition of a methanol
molecule might be limited by steric hindrance.
1
spectrum of the mixture of 12 and DPPH radical in
methanol-d4/acetone-d6 (3:1) after 5 h, new singlet signals
appeared at d 5.82 and d 6.29, which showed JCH HMBC
3
correlation with two distinct carbonyls of C-3 (d 181.1) and
C-4 (d 180.4), respectively, indicating the formation of a
methanol adduct at C-6 (12g). Suzuki et al. also reported the
formation of an ethanol adduct at C-6 of 12 by reacting 12
and DPPH radical in ethanol.18 This indicates that the
position of nucleophilic attack differs by the electron-
withdrawing/donating substituents on the quinone ring.
The plausible radical scavenging reaction of a protocate-
chuic ester and its analogues, bearing strong electron-
withdrawing groups at C-1, is shown in Scheme 1a and b. In
inert solvents, such as acetone and acetonitrile, catechols
only scavenge two radicals to yield the corresponding
o-quinones (Scheme 1a). In contrast, in alcoholic solvents,
catechols react with two radicals and are converted to their
quinones and 3-hemiacetals (Scheme 1b). Then, subsequent
nucleophilic addition of an alcohol molecule at C-2 of the
quinone leads to a regeneration of the catechol structure,
which can scavenge two additional radicals. In the present
study, o-quinones, which bear strong electron-withdrawing
groups at C-1, preferentially underwent nucleophilic attacks
at C-2. In addition, we previously reported that oxidations of
1–5 in the presence of thiols form adducts at C-2.14 To
substantiate the position of the nucleophilic attacks, electron
density of LUMO of o-quinones (1a–6a, 9a–13a) was
calculated by semiempirical method (Table 2). All of the
compounds 2a–6a, which possess strong electron-with-
drawing groups at C-1, had largest LUMO electron density
at C-2. The result indicates that C-2 of the o-quinones (2a–
6a) is the most susceptible position for the nucleophilic
attack. On the other hand, the LUMO electron density of C-
2 of 9a–13a was lower than that of 2a–6a and nearly equal
to the C-5 and C-6, hence the regioselectivity of 9a–13a
toward nucleophilic attacks seems to be lower than 2a–6a.
Formation of methanol adducts by oxidation of 3 and 5 in
methanol were further confirmed by HPLC analysis of the
reaction mixtures. After reacting catechols and DPPH
radical in methanol for 5 min, sodium dithionite was
added to the reaction mixture to reduce o-quinones and
3-hemiacetals to their catechol-forms. The HPLC analysis
of the mixture of 5 and DPPH radical showed a peak at
10.8 min, together with the peak of 5 at 9.2 min. The peak at
10.8 min was identical with that of authentic 5c. Similarly,
in the HPLC analysis of the reaction mixture of 3 and DPPH
radical, peaks at 6.5 and 7.2 min, which correspond to those
of authentic 3 and 3c, were observed. The NMR and HPLC
studies of the reaction mixtures clearly show that the radical
scavenging reactions of 3 and 5 proceed in the similar
manner as that of 2.
The DPPH radical scavenging activity of 2c–5c, which are
the oxidation products of 2–5, was evaluated in methanol.
The radical scavenging equivalence after 30 min was 2c,
3.1; 3c, 2.8; 4c, 3.7 and 5c, 3.5, which was approximately
2 equiv lower than that of 2–5 (Table 1). Taking into
account that 2c–5c are derived from an addition of a
methanol molecule to o-quinones (2a–5a), which were

S. Saito, J. Kawabata / Tetrahedron 61 (2005) 8101–8108
Table 2. LUMO energy and electron density at each carbon of o-quinones (1a–6a, 9a–13a)
8105
Compound
LUMO
1a
2a
3a
4a
5a
6a
9a
10a
11a
12a
13a
COOH
K2.339
COOK
1.656
K2.086
K2.041
K2.090
K2.184
K2.000
K1.904
K1.900
K1.640
K1.577
K1.612
C-1
C-2
C-3
C-4
C-5
C-6
0.34
0.41
0.19
0.14
0.22
0.16
0.21
0.17
0.26
0.29
0.18
0.31
0.30
0.41
0.20
0.14
0.21
0.16
0.31
0.40
0.20
0.14
0.20
0.16
0.31
0.41
0.20
0.14
0.20
0.15
0.29
0.36
0.20
0.16
0.23
0.19
0.32
0.36
0.20
0.16
0.23
0.19
0.28
0.29
0.20
0.19
0.27
0.24
0.26
0.25
0.18
0.20
0.30
0.27
0.26
0.27
0.21
0.21
0.27
0.26
0.26
0.26
0.21
0.21
0.26
0.26
0.25
0.19
0.18
0.23
0.30
0.30
This was supported by the report that catechin, an analogous
compound of 12 forms three isomers of mono-glutathione
adducts at C-20, 50, and 60.19 Moreover, 2a–6a had relatively
low LUMO energy compared to 11a–13a. The result
indicates that strong electron-withdrawing substituents at
C-1 of o-quinones facilitate the reactivity toward nucleo-
philic attacks. Furthermore, the low reactivity toward a
nucleophilic attack of 1 could be explained by the increase
of LUMO energy of 1a by dissociation of the free
carboxylic group to the carboxylate ion.
filtrate was concentrated in vacuo, and the residue was
subjected to silica gel column chromatography (hexane/ethyl
acetateZ3:1) to afford 30,40-dibenzyloxy-20-hydroxyaceto-
phenone (3.0 g, 86%). The dibenzyl ether (1.5 g, 4.3 mmol) in
acetonitrile/methanol (4:1, 15 mL) was methylated with 2 M
trimethylsilyldiazomethane solution in hexane (3.0 mL,
6 mmol, 1.4 equiv) in the presence of N,N-diisopropylethyl-
amine (1.0 mL, 6 mmol, 1.4 equiv) for 12 h at room
temperature. The reaction mixture was concentrated under
reduced pressure. After acidification with 2 M HCl, water was
added and the mixture was extracted with ethyl acetate. The
organic layer was evaporated under reduced pressure, and the
residue was subjected to silica gel column choromatagraphy
(hexane/ethyl acetateZ2:1) to afford 30,40-dibenzyloxy-20-
methoxyacetophenone (1.1 g, 70%). The resultant dibenzyl
methyl ether (1.1 g, 3.0 mmol) was deprotected by hydro-
genation at an atmospheric pressure with a catalytic amount of
10% palladium on carbon. The crude product was subjected to
silica gel column chromatography (hexane/ethyl acetateZ
2:1) to afford 3c (454 mg, 83%) as a yellow crystal; EI-HR-
MS, m/z [M]C182.0542, calcd C9H10O4, 182.0579; mp: 87–
88 8C; 1H NMR (methanol-d4): d 2.55 (3H, s, COCH3), 3.85
(3H, s, 20-OCH3), 6.60 (1H, d, JZ8.6 Hz, H-50), 7.18 (1H, d,
JZ8.6 Hz, H-60); 13C NMR (methanol-d4): d 30.4 (COCH3),
61.6 (20-OCH3), 111.7 (C-50), 122.5 (C-60), 125.1 (C-10),
139.6 (C-30), 150.6 (C-20), 152.8 (C-40), 200.6 (C]O).
3. Conclusion
In conclusion, the radical scavenging mechanisms of
catechols, bearing strong electron-withdrawing substituents
such as –COMe, –CN at C-1, were similar to that of methyl
protocatechuate. Moreover, the results suggested that the
radical scavenging activity largely depends on the electron-
withdrawing/donating properties of the substituents, since
they affect the susceptibility toward nucleophilic attack on
the o-quinones. In this study, nitrogen-centered DPPH
radical was used as a model radical. Therefore, it is of
interest to examine whether the reactions with oxygen-
centered peroxyl radicals in biological aqueous system are
similar to the DPPH radical scavenging reaction shown in
this paper.
4.1.2. 3,4-Dihydroxy-2-methoxybenzaldehyde (4c). To a
suspended solution of 2,3,4-trihydroxybenzaldehyde (2.3 g,
15 mmol) in dichloromethane (20 mL) was added N,N-
diisopropylethylamine (5.6 mL, 32 mmol, 2.1 equiv), and
stirred for 15 min at 0 8C. Methoxymethylchloride (2.4 mL,
32 mmol, 2.1 equiv) was then added dropwise. The mixture
was stirred at 0 8C for 15 min, and subsequently at room
temperature for 45 min. The reaction mixture was poured
into water and extracted with chloroform. The organic layer
was concentrated under reduced pressure, and the residue
was subjected to silica gel column chromatography (hexane/
ethyl acetateZ2:1) to afford 2-hydroxy-3,4-bis(methoxy-
methoxy)benzaldehyde (2.7 g, 74%). The bismethoxy-
methyl ether (2.1 g, 8.7 mmol) in acetonitrile/methanol
(4:1, 20 mL) was methylated with 2 M trimethylsilyldiazo-
methane solution in hexane (6.0 mL, 12 mmol, 1.4 equiv) in
the presence of N,N-diisopropylethylamine (2.1 mL,
12 mmol, 1.4 equiv) for overnight at room temperature.
The reaction mixture was concentrated under reduced
pressure. After acidification with 2 M HCl, water was
added and the mixture was extracted with ethyl acetate.
The organic layer was evaporated under reduced
pressure, and the residue was subjected to silica gel
column chromatography (hexane/ethyl acetateZ3:1) to
4. Experimental
4.1. Chemicals
3,4-Dihydroxybenzaldehyde, 30,40-dihydroxyacetophenone,
4-fluoro-1,2-dimethoxybenzene, and 4-chlorocatechol were
purchased from Tokyo Kasei Kogyo Co. 3,4-Dihydroxy-
benzonitrile and 4-methylcatechol were obtained from
Aldrich Chemical Co. and protocatechuic acid from
Sigma Chemical Co. Methyl protocatechuate (2) and methyl
3,4-dihydroxy-2-methoxybenzoate (2c) were prepared by
the method described previously.11 2,2-Diphenyl-1-picryl-
hydrazyl (DPPH) radical and other reagents were purchased
from Wako Pure Chemical Industries. All solvents used
were of reagent grade.
4.1.1. 30,40-Dihydroxy-20methoxyacetophenone (3c). A
mixture of 20,30,40-trihydroxyacetophenone (1.68 g,
10 mmol), benzyl bromide (2.1 mL, 18 mmol, 1.8 equiv),
and potassium carbonate (2.48 g, 18 mmol, 1.8 equiv) in
acetone (30 mL) was refluxed for 6 h. After cooling to
room temperature, the reaction mixture was filtered. The

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S. Saito, J. Kawabata / Tetrahedron 61 (2005) 8101–8108
afford 2-methoxy-3,4-bis(methoxymethyloxy)benzalde-
hyde (1.6 g, 72%). The resultant 2-methoxy-3,4-bis-
(methoxymethyloxy)benzaldehyde (1.6 g, 6.3 mmol) was
suspended in methanol (4 mL) and 1 M HCl (6 mL), and
refluxed for 1 h. The reaction mixture was concentrated
under reduced pressure, and the crude product was subjected
to silica gel column chromatography (hexane/ethyl acetateZ
2:1) to afford 4c (0.93 g, 89%) as a pale yellow powder; EI-
HR-MS, m/z [M]C168.0469, calcd C8H8O4, 168.0423; mp:
110–111 8C; 1H NMR (methanol-d4): d 3.93 (3H, s, 2-OCH3),
6.66 (1H, d, JZ8.6 Hz, H-5), 7.23 (1H, d, JZ8.6 Hz, H-6),
10.1(1H, s, CHO);13C NMR (methanol-d4):d 61.4(2-OCH3),
112.4 (C-5), 121.7 (C-6), 123.7 (C-1), 139.3 (C-3), 153.2
(C-2), 155.0 (C-4), 190.8 (CHO).
4.1.6. (3,4-Dihydroxybenzoyloxy)acetic acid methyl ester
(8). Compound 8 was prepared by the method of Wijesekera
and Ratnayake.21 To a solution of protocatechuic acid
(1.54 g, 10 mmol) in water (10 mL) was added 10 M
aqueous sodium hydroxide (1 mL) and chloroacetic acid
methyl ester (1.75 mL, 20 mmol, 2 equiv), and refluxed for
24 h. After cooling, the reaction mixture was extracted with
ethyl acetate, and then washed with water and saturated
aqueous sodium hydrogen carbonate. The organic layer was
concentrated under reduced pressure, and the residue was
subjected to silica gel column chromatography (hexane/
ethyl acetateZ2:1) to afford 8 (0.70 g, 31%) as a pale
yellow powder; EI-HR-MS, m/z [M]C226.0486, calcd
C10H10O6, 226.0477; mp: 171–173 8C; 1H NMR
(methanol-d4): d 3.76 (3H, s, CH3), 4.80 (2H, s, CH2),
6.81 (1H, d, JZ8.4 Hz, H-5), 7.46 (2H, m, H-2 and 6).
4.1.3. 3,4-Dihydroxy-2-methoxybenzonitrile (5c). Com-
pound 5c was prepared by the method of Shirai et al.20 To a
solution of hydroxylamine-O-sulfonic acid (204 mg,
1.8 mmol, 1.2 equiv) in water (2 mL) was added 4c
(252 mg, 1.5 mmol) at 0 8C, and stirred for 30 min. Then,
the reaction mixture was stirred for another 2 h at 60 8C.
After cooling, the reaction mixture was extracted with ethyl
acetate. The crude product was subjected to silica gel
column chromatography (hexane/ethyl acetateZ1:1) to
afford 5c (220 mg, 89%) as a pale yellow powder; EI-HR-
MS, m/z [M]C165.0443, calcd C8H7NO3, 165.0426; mp:
136–138 8C; 1H NMR (methanol-d4): d 3.95 (3H, s,
2-OCH3), 6.62 (1H, d, JZ8.4 Hz, H-5), 6.96 (1H, d, JZ
8.4 Hz, H-6); 13C NMR (methanol-d4): d 61.9 (2-OCH3),
97.7 (C-1), 112.7 (C-5), 118.3 (CN), 125.3 (C-6), 139.8
(C-3), 152.0 (C-2), 153.3 (C-4).
4.1.7. 4-Fluorocatechol (10). To a solution of 4-fluoro-1,2-
dimethoxybenzene (1.0 g, 6.4 mmol) in dichloromethane
(20 mL) at K80 8C, 1 M boron tribromide dichloromethane
solution (38.4 mL, 6 equiv) was added, and stirred for 1 h.
The reaction mixture was kept for another 12 h at room
temperature. The mixture was poured into ice-water, and
extracted with ethyl acetate. The organic layer was washed
with water, and then evaporated under reduced pressure to
give 10 (0.71 g, 86%) as a white powder; EI-HR-MS, m/z
[M]C128.0278, calcd C6H5FO2, 128.0274; mp: 90–91 8C;
1H NMR (acetone-d6): d 6.42 (1H, ddd, JZ8.6, 3.0 Hz,
JHFZ8.6 Hz, H-5), 6.59 (1H, dd, JZ3.0 Hz, JHFZ9.8 Hz,
H-3), 6.77 (1H, dd, JZ8.6 Hz, JHFZ5.7 Hz, H-6), 7.83 (1H,
s, OH), 8.17 (1H, s, OH).
4.1.4. 3,4-Dihydroxybenzamide (6). To 12 M HCl (15 mL)
was added 3,4-dihydroxybenzonitrile 675 mg (5.0 mmol),
and stirred for 3 h at 40 8C. The reaction mixture was poured
into ice-water and washed with ethyl acetate. The water
layer was concentrated in vacuo to afford 6 as a white
powder (616 mg, 81%); EI-HR-MS, m/z [M]C153.0446,
calcd C7H7NO3, 153.0426; mp: 216–218 8C; 1H NMR
(methanol-d4): d 6.81 (1H, d, JZ8.1 Hz, H-5), 7.27 (1H, dd,
JZ8.1, 2.2 Hz, H-6), 7.32 (1H, d, JZ2.2 Hz, H-2).
4.1.8. 4-Methoxycatechol(13). A mixture of 3,4-dihydroxy-
benzaldehyde (5.5 g, 40 mmol), potassium carbonate
(11.0 g, 80 mmol, 2 equiv), and benzyl bromide (9.5 mL,
80 mmol, 2 equiv) in acetone (100 mL) was refluxed for 4 h.
After cooling, the reaction mixture was filtered. The filtrate
was concentrated under reduced pressure, and the residue
was subjected to silica gel column chromatography (hexane/
ethyl acetateZ2:1) to afford 3,4-dibenzyloxybenzaldehyde
(10.7 g, 84%). The resultant dibenzyl ether was converted to
the corresponding phenol by the method of Roy et al.22 To a
mixture of boric acid (3.1 g, 50 mmol, 5 equiv) and 30%
hydrogen peroxide (2.5 g, 22 mmol, 2.2 equiv) in THF
(30 mL) was added concentrated H2SO4 (1 mL), and stirred
at room temperature for 0.5 h. A solution of 3,4-dibenzyl-
oxybenzaldehyde (3.18 g, 10 mmol) in THF (10 mL) was
added, and the reaction mixture was further stirred at room
temperature for 12 h. The mixture was filtered, and the
filtrate was neutralized with aqueous saturated sodium
hydrogen carbonate solution and extracted with ethyl
acetate. The organic layer was washed with water, and
evaporated under reduced pressure. The residue was
subjected to silica gel column chromatography (hexane/
ethyl acetateZ2:1) to afford 3,4-dibenzyloxyphenol (2.3 g,
75%). To a solution of 3,4-dibenzyloxyphenol (1.0 g,
3.3 mmol) in acetone (15 mL) was added potassium
carbonate (0.46 g, 3.3 mmol, 1 equiv) and iodomethane
(0.2 mL, 3.3 mmol, 1 equiv), and refluxed for 6 h. After
cooling, the reaction mixture was filtered. The filtrate was
concentrated under reduced pressure, and the residue was
subjected to silica gel column chromatography (hexane/ethyl
acetateZ2:1) to afford 1,2-dibenzyloxy-4-methoxybenzene
4.1.5. (3,4-Dihydroxybenzoyloxy)acetic acid (7). Com-
pound 7 was prepared by the method of Wijesekera and
Ratnayake.21 To a solution of protocatechuic acid (1.54 g,
10 mmol) in water (10 mL) was added 10 M aqueous
sodium hydroxide (1 mL) and benzyl chloroacetate
(3.1 mL, 20 mmol, 2 equiv), and refluxed for 24 h. After
cooling, the reaction mixture was extracted with ethyl
acetate, and then washed with water and saturated aqueous
sodium hydrogen carbonate. The organic layer was
concentrated under reduced pressure, and the residue was
subjected to silica gel column chromatography (hexane/
ethyl acetateZ2:1) to afford (3,4-dihydroxybenzoyloxy)-
acetic acid benzyl ester. The resultant benzyl ester was
deprotected by hydrogenation at an atmospheric pressure
with a catalytic amount of 10% palladium on carbon. The
crude product was subjected to silica gel column chroma-
tography (hexane/ethyl acetateZ1:2) to afford 7 (0.84 g,
40%) as a white powder; EI-HR-MS, m/z [M]C212.0280,
calcd C9H8O6, 212.0321; mp: 194–196 8C; 1H NMR
(methanol-d4): d 4.75 (2H, s, CH2), 6.84 (1H, d, JZ
8.4 Hz, H-5), 7.51 (2H, m, H-2, and 6).

S. Saito, J. Kawabata / Tetrahedron 61 (2005) 8101–8108
8107
1
(0.70 g, 67%). The resultant 1,2-dibenzyloxy-4-methoxy-
benzene (0.70 g, 2.2 mmol) was deprotected by hydrogen-
ation at an atmospheric pressure with a catalytic amount of
10% palladium on carbon. The crude product was subjected
to column chromatography (hexane/ethyl acetateZ2:1) to
afford 13 (0.24 g, 77%) as a yellow oil; EI-HR-MS, m/z
[M]C, 140.0499, calcd C7H8O3, 140.0474; 1H NMR
(acetone-d6): d 3.66 (3H, s, OMe), 6.24 (1H, dd, JZ8.6,
3.0 Hz, H-5), 6.42 (1H, d, JZ3.0 Hz, H-3), 6.70 (1H, d, JZ
8.6 Hz, H-6).
H-5), 2e: H NMR (methanol-d4/acetone-d6 (3:1)): d 5.83
(1H, d, JZ10.3 Hz, H-5), 7.53 (1H, d, JZ10.3 Hz, H-6).
4.4.1.2. Reaction of 3 and DPPH radical. 3a: 1H NMR
(methanol-d4/acetone-d6 (3:1)): d 6.45 (1H, d, JZ10.3 Hz,
H-5), 6.94 (1H, d, JZ2.2 Hz, H-2), 3b: 1H NMR (methanol-
d4/acetone-d6 (3:1)): d 6.12 (1H, d, JZ10.3 Hz, H-5), 3e: 1H
NMR (methanol-d4/acetone-d6 (3:1)): 5.85 (1H, d, JZ
10.3 Hz, H-5), 7.67 (1H, d, JZ10.3 Hz, H-6).
4.4.1.3. Reaction of 5 and DPPH radical. 5a: 1H NMR
(methanol-d4/acetone-d6 (3:1)): d 6.49 (1H, d, JZ10.3 Hz,
H-5), 5b: H NMR (methanol-d4/acetone-d6 (3:1)): d 6.19
4.2. Apparatus
1
(1H, d, JZ10.1 Hz, H-5), 5e: 1H NMR (methanol-d4/
acetone-d6 (3:1)): d 5.90 (1H, d, JZ10.1 Hz, H-5).
NMR spectra were recorded on a Bruker AMX500
spectrometer (1H, 500 MHz; 13C, 125 MHz); chemical
shifts are expressed relative to the residual signals of
methanol-d4 (dH 3.30, dC 49.0) and acetone-d6 (dH 2.04, dC
29.8). Electron ionization mass spectra (EI-MS) were
obtained with a JEOL JMS-AX500 instrument. Optical
absorbance was acquired using a HITACHI U-3210
spectrophotometer. Melting point data were measured
with a hot-stage apparatus and are uncorrected. Analytical
thin-layer chromatography was performed on silica gel
plates Merck 60 F254 (0.25 mm thickness). Ordinary phase
column chromatography was performed with silica gel,
Wakogel C-300 (Wako Pure Chemical Industries).
1
4.4.1.4. Reaction of 11 and DPPH radical. 11a: H
NMR (methanol-d4/acetone-d6 (3:1)): d 6.35 (2H, m, H-2
and 5), 7.14 (2H, m, H-1 and 6); 13C NMR (methanol-d4/
acetone-d6 (3:1)): d 131.7 (C-2 and 5), 140.9 (C-1 and 6),
181.3 (C-3 and 4); HMBC correlation peaks: H-1 (6)/C-3
(4), C-5 (2), H-2 (5)/C-4 (3), C-6 (1).
1
4.4.1.5. Reaction of 12 and DPPH radical. 12a: H
NMR (methanol-d4/acetone-d6 (3:1)): d 2.17 (3H, s, CH3),
6.23 (1H, d, JZ2.0 Hz, H-2), 6.32 (1H, d, JZ10.1 Hz, H-5),
7.05 (1H, dd, JZ10.1, 2.0 Hz, H-6); 13C NMR (methanol-
d4/acetone-d6 (3:1)): d 21.3 (CH3), 128.4 (C-2), 129.4 (C-5),
144.3 (C-6), 153.6 (C-1), 180.4 (C-3), 181.3 (C-4); HMBC
correlation peaks: H-2/C-4, C-6, H-5/C-1, C-3, H-6/C-2,
C-4, CH3/C-1, C-2, C-6, 12g: 1H NMR (methanol-d4/
acetone-d6 (3:1)): d 2.14 (3H, s, CH3), 5.82 (1H, s, H-5),
6.29 (1H, s, H-2); 13C NMR (methanol-d4/acetone-d6 (3:1)):
d 17.3 (CH3), 102.5 (C-5), 129.3 (C-2), 151.5 (C-1), 171.3
(C-6), 180.4 (C-4), 181.1 (C-3); HMBC correlation peaks:
H-2/C-1, C-4, H-5/C-1, C-3, C-6, CH3/C-1, C-2, C-6.
4.3. Colorimetric radical scavenging tests
DPPH radical scavenging activity was measured as
described previously.11,14 To a solution of a test compound
(12.5 mM, 4 mL) was added 1 mL of DPPH radical
(500 mM) in a test tube. The solution was immediately
mixed vigorously for 10 s by a Vortex mixer and transferred
to a cuvette. The absorbance reading at 517 nm was taken at
30 min after initial mixing. Acetonitrile and methanol were
chosen as inert non-alcoholic and nucleophilic alcoholic
solvents, respectively. A solution of DL-a-tocopherol in the
same concentration was measured as a positive control.
A reduction of the absorbance, 0.228, by the positive control
was regarded as corresponding to the consumption of two
molecules of DPPH radical. All experiments were
performed in triplicate.
1
4.4.1.6. Reaction of 13 with DPPH radical. 13a: H
NMR (methanol-d4/acetone-d6 (3:1)): d 3.91 (3H, s, CH3),
5.86 (1H, d, JZ2.7 Hz, H-2), 6.43 (1H, d, JZ10.3 Hz, H-5),
7.00 (1H, dd, JZ10.3, 2.7 Hz, H-6); 13C NMR (methanol-
d4/acetone-d6 (3:1)): d 57.5 (CH3), 103.5 (C-2), 131.8 (C-5),
140.7 (C-6), 171.1 (C-1), 179.2 (C-3), 182.0 (C-4); HMBC
correlation peaks: H-2/C-4, C-6, H-5/C-1, C-3, H-6/C-2,
C-4, CH3/C-1, C-2, C-6.
4.4. NMR analyses
4.4.2. NMR measurements of the reaction mixtures of
catechols (2c, 3c, and 5c) and DPPH radical. To a
catechol (2.5 mmol) was added DPPH radical (3.0 mg,
7.6 mmol, 3 equiv) in methanol-d4/acetone-d6 (3:1, 0.4 mL).
The mixture was immediately transferred to a NMR tube
4.4.1. NMR measurements of the reaction mixtures of
catechols and DPPH radical. To a catechol (2.5 mmol)
was added DPPH radical (5.0 mg, 13 mmol, 5 equiv) in
methanol-d4/acetone-d6 (3:1) or acetone-d6 (0.4 mL). In the
case of the methanol-d4 solution, acetone-d6 was added as a
cosolvent for enhancing a solubility of DPPH radical. The
mixture was immediately transferred to a NMR tube and
mixed vigorously. 1H NMR spectra were recorded at 10 min
after mixing.
1
and mixed vigorously. H NMR spectra were recorded at
10 min after mixing.
4.4.2.1. Reaction of 2c and DPPH radical. 2d: 1H NMR
(methanol-d4/acetone-d6 (3:1)): d 6.19 (1H, d, JZ10.3 Hz,
H-5), 7.38 (1H, d, JZ10.3 Hz, H-6), 2e: 1H NMR
(methanol-d4/acetone-d6 (3:1)): d 5.83 (1H, d, JZ10.3 Hz,
H-5), 7.53 (1H, d, JZ10.3 Hz, H-6).
4.4.1.1. Reaction of 2 and DPPH radical. 2a: 1H NMR
(methanol-d4/acetone-d6 (3:1)): d 6.44 (1H, d, JZ10.3 Hz,
H-5), 6.94 (1H, s, H-2), 7.51 (1H, d, JZ10.3 Hz, H-6), 2b:
1H NMR (methanol-d4/acetone-d6 (3:1)): d 6.11 (1H, d, JZ
10.3 Hz, H-5), 7.43 (1H, d, JZ10.3 Hz, H-6), 2d: 1H NMR
(methanol-d4/acetone-d6 (3:1)): d 6.19 (1H, d, JZ10.3 Hz,
4.4.2.2. Reaction of 3c and DPPH radical. 3d: 1H NMR
(methanol-d4/acetone-d6 (3:1)): d 2.52 (3H, s, COCH3), 4.16
(3H, s, 2-OCH3), 6.20 (1H, d, JZ10.3 Hz, H-5), 7.49 (1H, d,

8108
S. Saito, J. Kawabata / Tetrahedron 61 (2005) 8101–8108
JZ10.3 Hz, H-6), 3e: 1H NMR (methanol-d4/acetone-d6
(3:1)): d 2.44 (3H, s, COCH3), 4.33 (3H, s, 2-OCH3), 5.85
(1H, d, JZ10.3 Hz, H-5), 7.67 (1H, d, JZ10.3 Hz, H-6).
3. Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Free Radic. Biol.
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´
S.; Boersma, M. G.; Lemanska, K.; Vervoort, J.; Rietjens, I.
4.4.2.3. Reaction of 5c and DPPH radical. 5e: 1H NMR
(methanol-d4/acetone-d6 (3:1)): d 5.90 (1H, d, JZ10.1 Hz,
H-5).
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4.5. HPLC analyses
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4.5.1. HPLC analyses of the reaction mixtures of
catechols and DPPH radical. To a catechol (1.7 mmol) was
added DPPH radical (2.0 mg, 5.1 mmol, 3 equiv) in methanol
(0.2 mL). After 5 min, an aqueous solution (10 mL) of sodium
dithionite (0.9 mg, 5.2 mmol, 3 equiv) was added to the
reaction mixture. The resulting solution was passed through a
filter (0.45 mm), and analyzed by HPLC (Inertsil ODS-2 (GL
Sciences Inc.), 4.6 mm i.d.!250 mm, 20% MeCN/H2O
containing 0.1% HCOOH, detected by UV at 254 nm).
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4.6. Molecular orbital calculations
Electron density and energy of LUMO were calculated by
AM1 method using MOPAC 2000 program combined in
Chem3D package, CambridgeSoft Co.
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Acknowledgements
16. Nenadis, N.; Zhang, H. Y.; Tsimidou, M. Z. J. Agric. Food
Chem. 2003, 51, 1874–1879.
We are grateful to Mr. Kenji Watanabe and Dr. Eri Fukushi,
of the GC–MS and NMR Laboratory of our school, for
measuring mass spectra. This work was supported by
research fellowship for young scientists from the Japan
Society for the Promotion of Science (to S.S.).
17. Nenadis, N.; Wang, L. F.; Tsimidou, M. Z.; Zhang, H. Y. J.
Agric. Food Chem. 2005, 53, 295–299.
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