DPPH (=2,2-diphenyl-1-picrylhydrazyl) radical-scavenging reaction of protocatechuic acid (=3,4-dihydroxybenzoic acid): Difference in reactivity between acids and their esters

Article abstract of DOI:10.1002/hlca.200690139

Prolocatechuic acid (=3,4-dihydroxybenzoic acid; 1) exhibits a significantly slow DPPH (=2,2-diphenyl-1-picrylhydrazyl) radical-scavenging reaction compared to its esters in alcoholic solvents. The present study is aimed at the elucidation of the difference between the radical-scavenging mechanisms of protocatechuic acid and its esters in alcohol. Both protocatechuic acid (1) and its methyl ester 2 rapidly scavenged 2 equiv. of radical and were converted to the corresponding o-quinone structures 1a and 2a, respectively (Scheme). Then, a regeneration of catechol (=benzene-1,2-diol) structures occurred via a nucleophilic addition of a MeOH molecule to the o-quinones to yield alcohol adducts 1f and 2c, respectively, which can scavenge additional 2 equiv. of radical. However, the reaction of protocatechuic acid (1) beyond the formation of the o-quinone was much slower than that of its methyl ester 2. The results suggest that the slower radical-scavenging reaction of 1 compared to its esters is due to a dissociation of the electron-withdrawing carboxylic acid function to the electron-donating carboxylate ion, which decreases the electrophilicity of the o-quinone, leading to a lower susceptibility towards a nucleophilic attack by an alcohol molecule.

Full text of DOI:10.1002/hlca.200690139

Helvetica Chimica Acta – Vol. 89 (2006)
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DPPH (=2,2-Diphenyl-1-picrylhydrazyl) Radical-Scavenging Reaction of
Protocatechuic Acid (=3,4-Dihydroxybenzoic Acid): Difference in Reactivity
between Acids and Their Esters
by 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
(phone: +81-11-706-2496; fax: +81-11-706-2496; e-mail: junk@chem.agr.hokudai.ac.jp)
Protocatechuic acid (=3,4-dihydroxybenzoic acid; 1) exhibits a significantly slow DPPH (=2,2-
diphenyl-1-picrylhydrazyl) radical-scavenging reaction compared to its esters in alcoholic solvents. The
present study is aimed at the elucidation of the difference between the radical-scavenging mechanisms
of protocatechuic acid and its esters in alcohol. Both protocatechuic acid (1) and its methyl ester 2 rapidly
scavenged 2 equiv. of radical and were converted to the corresponding o-quinone structures 1a and 2a,
respectively (Scheme). Then, a regeneration of catechol (=benzene-1,2-diol) structures occurred via a
nucleophilic addition of a MeOH molecule to the o-quinones to yield alcohol adducts 1f and 2c, respec-
tively, which can scavenge additional 2 equiv. of radical. However, the reaction of protocatechuic acid (1)
beyond the formation of the o-quinone was much slower than that of its methyl ester 2. The results sug-
gest that the slower radical-scavenging reaction of 1 compared to its esters is due to a dissociation of the
electron-withdrawing carboxylic acid function to the electron-donating carboxylate ion, which decreases
the electrophilicity of the o-quinone, leading to a lower susceptibility towards a nucleophilic attack by an
alcohol molecule.
Introduction. – Polyphenols are known to exhibit potent antioxidant activities.
Recently, numerous studies have been reported on radical-scavenging activities of phe-
nolic compounds. Particularly, structureꢀactivityꢀrelationship studies of polyphenols
and kinetic studies on reactions of phenolic compounds with radicals, including sub-
stituent effects, have been extensively investigated [1–3]. It is well-known that the rad-
ical-scavenging activity of phenol-derived acids largely depends on the number and
arrangement of phenolic OH groups in the molecule [4–6]. Among them, o- or p-ben-
zenediol derivatives exhibit high radical-scavenging activity compared to m-benzene-
diol derivatives, since they could be converted to the corresponding stable o- or p-qui-
none derivatives [7].
Protocatechuic acid (=3,4-dihydroxybenzoic acid; 1) and its esters are o-benzene-
diols, ubiquitously found in edible plants, vegetables, and fruits, and are known to
exhibit potent antioxidant activities [4][8]. Additionally, it has been demonstrated
that protocatechuic acid has preventive effects on carcinogenesis and cardiovascular
diseases that are associated with free radicals [9][10].
Previously, we have found that the radical-scavenging reactions of protocatechuic
acid (1) and its esters depend on the applied solvents [11]. In non-alcoholic solvents,
1 and its esters scavenge 2 equiv. of radical to yield the corresponding o-quinones. In
alcoholic solvents, protocatechuic acid esters rapidly react with more than 4 equiv. of
© 2006 Verlag Helvetica Chimica Acta AG, Zürich

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radical with a concomitant conversion to the corresponding o-quinones, 3-hemiacetals
[12], and their alcohol adducts at C(2) [11]. Interestingly, unlike protocatechuic esters,
1 scavenged only 2 equiv. of radical within 30 min at room temperature. The previous
study has demonstrated that the higher radical-scavenging activity of protocatechuic
acid esters in alcoholic solvents than in non-alcoholic solvents is due to a regeneration
of catechol (=benzene-1,2-diol) structures via a nucleophilic addition of a solvent mol-
ecule to the o-quinones [11][13]. However, it is still unclear why sterically unhindered
protocatechuquinone (1a) scarcely undergoes a nucleophilic attack by an alcohol mol-
ecule, in contrast to its methyl ester 2a, which leads to low activity of 1. Kimura et al.
[14] have also shown the low activity of 1 compared to methyl protocatechuate (2) in
EtOH. Furthermore, caffeic acid (=3,4-dihydroxycinnamic acid) is reported to exhibit
lower radical-scavenging activity than its esters [15][16].
Therefore, the aim of this study was to elucidate why 1 exhibits low activity com-
pared to its esters in alcoholic solvents. To confirm whether acids generally show
lower activity than their esters or not, 3,4-dihydroxybenzenesulfonic acid (4), (3,4-dihy-
droxyphenyl)phosphonic acid (6), and their esters 5, 7, and 8 were synthesized (Fig. 1),
and the DPPH and ABTS (diammonium salt of 2,2’-azinobis[(3-ethyl-2,3-dihydroben-
zothiazole-6-sulfonic acid]) radical-scavenging activities of acids 1, 4, and 6 were com-
pared with those of their corresponding esters 2, 5, 7, and 8. DPPH and ABTS radicals
are strongly electrophilic radicals which act as a one-electron oxidant and hence, they
can readily oxidize catechols into the corresponding quinones via semiquinone radicals.
In addition, the radical-scavenging reactions of 1 and its sodium salt 3 were directly
analyzed by NMR spectroscopy. Finally, isolation of an oxidation product of 1 from
the reaction mixture was attempted, with the aim to explain the difference between
the radical-scavenging mechanisms of protocatechuic acid and its esters in alcoholic
solvents.
Results and Discussion. – Radical-Scavenging Activity. The DPPH-radical-scaveng-
ing activity of protocatechuic acid (1), 3,4-dihydroxybenzenesulfonic acid (4), and (3,4-
dihydroxyphenyl)phosphonic acid (6) was compared with that of the corresponding
esters 2, 5, 7, and 8 in MeOH and MeCN. The relative radical-scavenging equivalences
of compounds 1, 2, and 4–8, when that of (2RS)-a-tocopherol as standard was desig-
nated as 2.0, are listed in Table 1. In inert MeCN, all test compounds scavenged approx-
imately 2 equiv. of radical within 30 min at room temperature, and no significant differ-
ence in activity between acids and their esters was observed. In nucleophilic MeOH,
the acids 1, 4, and 6 also consumed ca. 2 equiv. of radical, whereas their esters 2, 5,
and 8 scavenged 5 equiv. of radical. The radical-scavenging activity of salt 3 was com-
parable to that of 1. In addition, the activity of phosphonic acid 6 and its monoethyl
ester 7 and diethyl ester 8 increased as the number of the Et groups increased. The
results clearly show that not only protocatechuic [11][14] and caffeic [15][16] acids
but also 3,4-dihydroxybenzenesulfonic acid (4) and (3,4-dihydroxyphenyl)phosphonic
acid (6) have significantly lower DPPH-radical-scavenging activity in MeOH as com-
pared to their esters.
Fig. 2 shows time courses of the DPPH-radical-scavenging activity of 1–3 in MeOH
and MeCN over 6 h. In MeCN, acid 1 and ester 2 readily reacted with 2 equiv. of radical
and reached steady states in 30 min. It is indicated that although dimerization of the

Helvetica Chimica Acta – Vol. 89 (2006)
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Fig. 1. Structures of protocatechuic acid (1) and related compounds 2–8, as well as their oxidation
products
formed o-quinones 1a and 2a might occur similarly to the reaction in acetone [17], the
resultant dimers are unlikely to scavenge radicals. In MeOH, ester 2 rapidly scavenged
5 equiv. of radical and reached a plateau within 30 min. In contrast, acid 1 and salt 3
scavenged only 2 equiv. of radical in 30 min, but they gradually consumed another 3
equiv. of radical and reached steady states within 6 h. The DPPH-radical-scavenging
equivalences of 1 and 3 in MeOH after 6 h were comparable to that of 2. These results
suggest that the radical-scavenging reactions beyond the formation of o-quinones 1a
and 3a might proceed in a similar manner as that of 2a, although the reaction rates
are significantly slower.
To establish whether the difference in reactivity between acid and its esters also
occurs in aqueous solution, the ABTS-radical-scavenging activity of 1–8 was evaluated
in aqueous solution. The radical-scavenging equivalence is expressed as the values rel-

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Table 1. DPPH and ABTS Radical-Scavenging Equivalences of 1–8 after 30 min at Room Temperature
DPPH radicala)
MeOHc)
ABTS radicalb)
MeCNc)
Oc)
1% EtOH/H2ACHTREUNG
1
2
3
4
5
6
7
8
2.5
5.0
2.7
2.3
5.2
1.6
1.9
4.8
2.2
2.2
–d)
2.0
2.8
–d)
1.7
2.1
2.4
5.9
2.2
2.8
5.6
1.8
3.0
5.9
a) The equivalence is expressed as the values relative to that of (2RS)-a-tocopherol taken as 2.0. b) The
equivalence is expressed as the values relative to that of Trolox taken as 2.0. c) Solvent. d) Not tested due
to low solubility.
^
*
Fig. 2. Time courses of DPPH-radical-scavenging activity at room temperature of 1 ( ), 2 ( ), and 3
&
^
*
( ) in MeOH, and of 1 ( ) and 2 ( ) in MeCN. The equivalence is expressed as the values relative
to that of (2RS)-a-tocopherol taken as 2.0.
ative to the standard value 2.0 of Trolox, and the results are shown in Table 1. Com-
pounds 1, 4, and 6 consumed approximately 2 equiv. of radical, whereas their esters
2, 5, and 8 scavenged more than 5 equiv. of radical. In addition, 3 had an activity similar
to that of 1. Thus, the tendency of the ABTS-radical-scavenging activity in H2O was
similar to that of the DPPH-radical-scavenging activity in MeOH, suggesting that the
radical-scavenging reaction in an aqueous solution is similar to that in an alcohol.
It is well-known that the pH of the solution affects the radical-scavenging activity of
phenols [5]. Therefore, the pH of the reaction mixtures was measured. Since the pH of
the mixtures was in the range of 6.63–6.77, the difference in radical-scavenging activity
between acids and esters is not caused by the pH of the reaction mixtures.
Furthermore, the dissociation of the free carboxylic group of quinone 1a to the car-
boxylate ion is expected to be facilitated by the strong electron-withdrawing property
of the quinone carbonyl groups. This was supported by comparing the dissociation
enthalpy of the carboxylic acid function of 1 and of protocatechuquinone (1a), which

Helvetica Chimica Acta – Vol. 89 (2006)
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was estimated from MOPAC calculations to be ꢀ27.9 and ꢀ39.7 kcal/mol, respectively.
We have previously demonstrated that catechols bearing a strong electron-withdrawing
substituent at C(1) exhibit high DPPH-radical-scavenging activity in alcoholic solvents,
since electron-withdrawing substituents enhance the electrophilicity of the o-quinone
intermediate [13]. Therefore, it is suggested that the low reactivity of 1 compared to
its ester 2 in MeOH is due to the dissociation of the electron-withdrawing carboxylic
acid function to the electron-donating carboxylate ion.
NMR Analyses of the Reaction Mixtures. To elucidate the radical-scavenging mech-
anism of 1 and 3, the reaction mixture of 1 (or 3) and DPPH was directly analyzed by
NMR. The 1H-NMR spectrum of the reaction mixture 1/DPPH in CD
3ACHTREOUNG D/(D6)acetone
3 :1 after 10 min showed signals of the corresponding o-quinone 1a and of its hemiace-
tal 1b [12], similar to those observed for 2/DPPH [11]. However, no signal due to a
MeOH adduct at the benzene ring was observed. Even after 2 h, the signals of 1a
and 1b remained unchanged. Apparently, 1a was stable compared to 2a, which disap-
peared within 1 h in CD3ACHTREOUNG D/(D6)acetone 3 :1 [13]. Since an electron-donating sub-
stituent at C(1) enhances the stability of such an o-quinone [13], the reason that 1a
is stable compared to 2a can be explained by the deprotonation of the carboxylic
acid function to the carboxylate ion, which enhances the electron-donating property
of the substituent.
1
The H-NMR spectrum of the reaction mixture 3/DPPH in CD
3A
3 :1 after 10 min showed peaks of the o-quinone 3a (Fig. 3,a), and the HMBC data con-
firmed the formation of the o-quinone 3a (see Exper. Part). In contrast to 1 and 2, no
signal due to a hemiacetal 3b was detected. After 2 h, the reaction mixture 3/DPPH
exhibited no d characteristic of a C(2) adduct. Instead, two new s appeared, which
were assumed to be HꢀC(5) and HꢀC(2) of the C(6) MeOH adduct 3g (Fig. 3,b).
The formation of 3g was confirmed by the 1H-NMR spectrum of the mixture of authen-
tic 3f and DPPH radical (Fig. 3,c), and the H,C-HMBC data of this mixture (see Exper.
Part) allowed to assign the two s to HꢀC(5) and HꢀC(2) of the C(6) adduct 3g. These
results revealed that unlike methyl protocatechuate (2), forming the C(2) adduct 2d,
protocatechuic acid sodium salt (3) preferentially formed a C(6) adduct.
Isolation of an Oxidation Product of 1. Although no signal of an adduct at the ben-
1
zene ring was observed in the H-NMR spectrum of the mixture 1/DPPH radical in
CD3ACHTREOUNG D/(D6)acetone 3 :1, it can be speculated that 1a undergoes a nucleophilic attack
similar to that of 3a, since the radical-scavenging activity of 1 was comparable to that of
3 (Fig. 2). To substantiate the formation of a MeOH adduct, an attempt was made to
isolate an oxidation product from the reaction mixture 1/DPPH in MeOH. For that pur-
pose sodium dithionite was added to the reaction mixture after 12 h to reduce o-qui-
nones and 3-hemiacetals to their catechol forms. Purification of the reaction mixture
afforded indeed the C(6) adduct 1f, and neither the C(2) adduct 1c nor the C(5) adduct
1i was detected by HPLC analysis. The result suggests that 1a preferentially undergoes
a nucleophilic attack at C(6) to form 1f, that the reaction mechanism of 1 is similar to
that of 3; and that 1a is present in a dissociated form in the reaction solution, i.e., as an
anion identical to that from 3a.
Molecular-Orbital Calculations. To substantiate the preference for the position of
the nucleophilic attacks, the electron densities of the LUMO of o-quinones 1a, 2a,
4a, and 5a were calculated by a semiempirical method (Table 2). Like the esters 2a

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Helvetica Chimica Acta – Vol. 89 (2006)
Fig. 3. 1H-NMR spectra (CD
3ACHTREOUNG D/(D6)acetone 3 :1) of the reaction mixture 3/DPPH after a) 10 min
and b) 120 min, and c) of a mixture 3f/DPPH after 10 min. The intense signals in the range d 7.1–7.4
are due to DPPH.
Table 2. LUMO Energy and Electron Density at the Atoms C(1)–C(6) of the o-Quinone Derivatives 1a,
2a, 4a, and 5a
LUMO energy [eV]
Electron density
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
1a
1a’ (CO2H)
1a’’ (COꢀ2 )
ꢀ2.339
1.656
ꢀ2.086
ꢀ2.494
1.423
0.34
0.21
0.30
0.34
0.22
0.33
0.41
0.17
0.41
0.55
0.23
0.55
0.19
0.26
0.20
0.20
0.27
0.20
0.14
0.29
0.14
0.11
0.26
0.11
0.22
0.18
0.21
0.18
0.17
0.18
0.16
0.31
0.16
0.12
0.27
0.12
2a
4
a
’ (S4O3H) a
4a’’ (SOꢀ3 )
5a
ꢀ2.423
and 5a, the undissociated acids 1a’ (ꢀCOOH) and 4a’ (ꢀSO3H) had the largest LUMO
electron density at C(2). On the other hand, the dissociated acids 1a’’ (ꢀCOOꢀ) and 4a’’
(ꢀSOꢀ3 ) had the largest LUMO electron density at C(6). The values predict that 2a, 5a,
1a’ (ꢀCOOH), and 4a’ (ꢀSO3H) undergo a nucleophilic attack at C(2), and 1a’’
(ꢀCOOꢀ) and 4a’’ (ꢀSOꢀ3 ) at C(6), in agreement with the results from the NMR
experiments (Fig. 3). However, we have previously reported that both 1a and 2a under-
went a nucleophilic attack by a thiol to yield C(2) adducts in acetone [18]. We suggest
that the difference in the position of the nucleophilic attacks of 1a by thiols and alcohols
are due to the solvent used. That is, in the less polar acetone, deprotonation of the car-
boxylic acid group would be suppressed compared to that in alcoholic solvents, and
hence 1a’ undergoes a nucleophilic attack by a thiol at C(2), which is the position of
the highest LUMO electron density. In addition, the LUMO energies of 1a’’
(ꢀCOOꢀ) and 4a’’ (ꢀSOꢀ3 ) were higher than those of 2a, 5a, 1a’ (ꢀCOOH), and 4a’
(ꢀSO3H), suggesting that the lower reactivity of the acids 1, 4, and 6 as compared to
their esters toward a nucleophilic attack is due to the increase of LUMO energy of
the o-quinones by deprotonation of the free acid functions.

Helvetica Chimica Acta – Vol. 89 (2006)
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Radical-Scavenging Activity of the Oxidation Products. The DPPH radical-scaveng-
ing activity of 1f, 2c, and 3f, which are the oxidation products of 1, 2, and 3, was eval-
uated in MeOH (Fig. 4). Compound 2c readily scavenged more than 3 equiv. of DPPH.
In contrast, 1f and 3f rapidly reacted with 2 equiv. of DPPH, and then slowly consumed
more of it. The radical-scavenging equivalences of 1f, 2c, and 3f after 6 h were approx-
imately 2 equiv. lower than those of 1, 2, and 3. Considering that 1f, 2c, and 3f are
derived from an addition of a MeOH molecule to the o-quinones 1a, 2a, and 3a,
which were produced from 1, 2, and 3 by reaction with 2 equiv. of DPPH, 1f, 2c, and
3f could largely contribute to the total radical-scavenging activity of 1, 2, and 3.
^
*
Fig. 4. Time courses of DPPH-radical-scavenging activity at room temperature of 1f ( ), 2c ( ), and
&
3f ( ) in MeOH. The equivalence is expressed as the values relative to that of (2RS)-a-tocopherol
taken as 2.0.
Plausible Radical-Scavenging Mechanism. The difference in radical-scavenging
mechanisms of protocatechuic acid (1) and its ester 2 in MeOH are proposed in the
Scheme. Both acid 1 and its ester 2 react with 2 equiv. of radical and are converted
to their o-quinones and 3-hemiacetals. Then, in the case of ester 2, a MeOH molecule
readily attacks C(2) of the o-quinone 2a, which leads to a regeneration of a catechol
structure, i.e., 2c (Scheme,b). The MeOH adduct 2c scavenges additional 2 equiv. of rad-
ical to yield o-quinone 2d and its 3-hemiacetal 2e. In contrast, o-quinones 1a and 3a
slowly undergo a nucleophilic attack by a MeOH molecule at C(6), leading to a regen-
eration of the catechol structures 1f and 3f, respectively, which could scavenge addi-
tional 2 equiv. of radical (Scheme,a).
Conclusion. – In conclusion, the overall results suggest that the slow radical-scav-
enging reaction of protocatechuic acid (1) compared to its esters is due to the dissoci-
ation of the carboxylic acid function, since it decreases the electron-withdrawing prop-
erty of the substituent, and thus decreases the susceptibility of the first formed o-qui-
none towards nucleophilic attack by a solvent molecule. The difference in reactivity
between protocatechuic acid (1) and its ester 2 described in this paper might also
play a role in an aqueous biological system.

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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 fellowships of the Japan
Society for the Promotion of Science for young scientists (to S. S.).
Experimental Part
1. General. Protocatechuic acid (1) and diammonium 2,2’-azinobis[3-ethyl-2,3-dihydrobenzothia-
zole-6-sulfonate] (ABTS) were obtained from Sigma Chemical Co. The 2,4,5-trimethoxybenzoic acid
and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Aldrich
Chemical Co. Methyl protocatechuate (2), methyl 3,4-dihydroxy-2-methoxybenzoate (2c), and methyl
3,4-bis(benzyloxy)-2-methoxybenzoate were prepared by the methods described previously [11]. The
2,2-diphenyl-1-picrylhydrazyl (=2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl; DPPH) and other
reagents were purchased from Wako Pure Chemical Industries. All solvents used were of reagent
grade. Anal. TLC, silica gel plates Merck 60 F254 (0.25 mm thickness). Column chromatography (CC):
silica gel, Wakogel C-300 (Wako Pure Chemical Industries); reversed phase CC with Cosmosil 75C18-
OPN (Nacalai Tesque Inc.). Anal. HPLC: LC-10AD-vp pump and SPD-10A-vp detector (Shimadzu).
M.p.: hot-stage apparatus; uncorrected. UV/VIS Spectra: Hitachi U-3210 spectrophotometer. NMR
Spectra: Bruker-AMX-500 spectrometer (1H, 500 MHz; 13C, 125 MHz); chemical shifts d in ppm, rel.
to the residual signals of CD3ACHTREUOGN D (d(H) 3.30, d(C) 49.0) and (D6)acetone (d(H) 2.04, d(C) 29.8). MS:
Jeol-JMS-AX-500 (for EI) and Jeol-JMS-SX-102A (for FD) instruments.
2. Syntheses. Sodium 3,4-Dihydroxybenzoate (3). To a suspension of 1 (0.50 g, 3.2 mmol) in H2O (10
ml) was added dropwise Na2CO3 (0.17 g, 1.6 mmol, 0.50 equiv.) in H2O (5 ml). The resultant colorless
soln. was concentrated i.v. to afford 3 (0.56 g, 98%). White powder. 1H-NMR (CD
J=8.1, HꢀC(5)); 7.34 (dd, J=8.1, 2.0, HꢀC(6)); 7.41 (d, J=2.0, HꢀC(2)).
3ACHTREOUGN D): 6.71 (d,
3,4-Dihydroxy-2-methoxybenzoic Acid (1c). To a suspension of methyl 3,4-bis(benzyloxy)-2-
methoxyACTHREbUNG enzoate [11] (0.76 g, 2 mmol) in EtOH (40 ml) was added 20% aq. KOH soln. (8 ml), and stir-
red for 6 h at 508. After cooling, the mixture was concentrated i.v. The residue was diluted with H2O and
washed with AcOEt. The aq. phase was acidified with 1M HCl to pH 2, and the mixture was then
extracted with AcOEt and the org. phase concentrated i.v. to afford 3,4-bis(benzyloxy)-2-methoxyben-
zoic acid (0.63 g, 86%). This acid (0.63 g, 1.7 mmol) was deprotected by hydrogenation under 1 atm H2
in the presence of a catalytic amount of 10% Pd/C in MeOH (15 ml). The mixture was filtered through
Celite, the filtrate concentrated i.v., and the crude product subjected to CC (silica gel, CHCl3/MeOH
10 :1): 1c (0.26 g, 83%). Yellow powder. M.p. 183–1858. 1H-NMR (CD
3A
J=8.6, HꢀC(5)); 7.24 (d, J=8.6, HꢀC(6)). HR-EI-MS: 184.0396 (M+, C
CHRTEUNHG CHTREUNG
4,5-Dihydroxy-2-methoxybenzoic Acid (1f). To a soln. of 2,4,5-trimethoxybenzoic acid (4.2 g, 20
mmol) in CH2Cl2 (30 ml) at ꢀ808 was added 1M BBr3 in CH2Cl2 (100 ml, 5.0 equiv.), and stirred for 1
h. The mixture was kept for another 12 h at r.t. The mixture was poured into ice-water and extracted
with AcOEt. The org. layer was washed with H2O and then concentrated i.v. to give 2,4,5-trihydroxyben-
zoic acid (3.2 g, 19 mmol, 95%). Then 1f was prepared by the same method as described for 2c [11]: A
mixture of 2,4,5-trihydroxybenzoic acid (1.70 g, 10 mmol), benzyl bromide (3.2 ml, 27 mmol, 2.7
equiv.), and potassium carbonate (3.7 g, 27 mmol, 2.7 equiv.) in acetone (30 ml) was refluxed for 6 h.
After cooling to r.t., the mixture was filtered and the filtrate concentrated i.v. The residue was subjected
to CC (silica gel, hexane/AcOEt 3 :1): 4,5-bis(benzyloxy)-2-hydroxybenzoic acid benzyl ester (3.1 g,
70%). To a suspension of this ester (3.1 g, 7.0 mmol) in acetone (50 ml) were added MeI (0.48 ml, 7.7
mmol, 1.1 equiv.) and potassium carbonate (1.1 g, 7.7 mmol, 1.1 equiv.), and the mixture was refluxed
for 6 h. The mixture was filtered, the filtrate concentrated i.v., and the residue subjected to CC (silica
gel, hexane/AcOEt 3 :1): 4,5-bis(benzyloxy)-2-methoxybenzoic acid benzyl ester (2.5 g, 79%). The
methoxy ester (1.0 g, 2.2 mmol) was deprotected by hydrogenation under 1 atm H2 in the presence of
a cat. amount of 10% Pd/C in MeOH (15 ml). The mixture was filtered through Celite, the filtrate con-
centrated i.v., and the crude product subjected to CC (silica gel, hexane/AcOEt 1:2): 1f (0.36 g, 91%).
1
Pale brown powder. M.p. 193–1948. H-NMR (CD
3A

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Helvetica Chimica Acta – Vol. 89 (2006)
C(6)). 13C-NMR (CD
3ACHTREUONG D): 57.2 (MeO); 101.4 (C(3)); 109.7 (C(1)); 119.4 (C(6)); 140.3 (C(5)); 153.2
(C(4)); 155.9 (C(2)); 169.0 (C=O). HMBC: HꢀC(3) $ C(1), C(5); HꢀC(6) $ C(2), C(4), C=O;
MeO $ C(2). HR-EI-MS: 184.0391 (M+, C
Oþ5 ; calc. 184.0372).
8ACHTREUHGN 8ACHTREUNG
Sodium 4,5-Dihydroxy-2-methoxybenzoate (3f). As described for 3, from 1f: 3f (97%). Brown pow-
der. 1H-NMR (CD
3A
3,4-Dihydroxy-5-methoxybenzoic Acid (1i). Methyl 3,4-dihydroxy-5-methoxybenzoate (2i) was pre-
pared by the method of Chang et al. [19]. To a suspension of 2i (0.99 g, 5.0 mmol) in EtOH (50 ml) was
added 20% aq. KOH soln. (8 ml), and the mixture was stirred for 3 h at 508. After cooling, the mixture
was concentrated i.v. The residue was suspended in H2O, the suspension washed with AcOEt, the aq.
phase acidified with 1M HCl to pH 2, and the mixture extracted with AcOEt. The org. phase was concen-
trated i.v., and the crude product recrystallized from H2O: 1i (0.79 g, 86%). Pale brown powder. M.p.
220–2218. 1H-NMR (CD
184.0372 (M+, C Oþ5 ; calc. 184.0372).
8A 8A
3A
CHRTEUNGH CHTREUNG
3,4-Dihydroxybenzenesulfonic Acid (4). Compound 4 was prepared by the method of Ban et al. [20]:
To a soln. of catechol (=benzene-1,2-diol; 1.1 g, 10 mmol) in dimethyl carbonate (5 ml) was added drop-
wise chlorosulfonic acid (=chlorosulfic acid; 0.66 ml, 10 mmol, 1.0 equiv.) in dimethyl carbonate (2.8 ml),
and the mixture was stirred for 2 h at 08. Then, the mixture was allowed to warm to r.t., stirred for 12 h,
and concentrated i.v. The crude product was subjected to reversed phase CC (H2O): 4 (1.87 g, 98%). Red
1
oil. H-NMR (CD
3A
C(2)). HR-EI-MS: 189.9933 (M+, C
CHTREUNGH CHTREUNGO CHTREUNG
A
A
S+; calc. 189.9936).
6
6
5
3,4-Dihydroxybenzenesulfonic Acid Methyl Ester (5). To a soln. of catechol (1.1 g, 10 mmol) in
dimethyl carbonate (5 ml) was added fluorosulfonic acid methyl ester (=fluorosulfuric acid methyl
ester; 0.80 ml, 10 mmol, 1.0 equiv.), and the mixture was refluxed for 12 h. After cooling, the mixture
was poured into ice-water and extracted with Et
product subjected to CC (silica gel, hexane/AcOEt 1:1): 5 (1.01 g, 50%). Red oil. H-NMR (CD
3.67 (s, MeO); 6.91 (d, J=8.3, HꢀC(5)); 7.23 (d, J=2.2, HꢀC(2)); 7.25 (dd, J=8.3, 2.2, HꢀC(6)).
HR-EI-MS: 204.0092 (M+, C S+; calc. 204.0092).
7A 8A 5A
2ACHTREOUNG . The org. phase was concentrated i.v. and the crude
1
3ACHTREOUNG D):
CTHREUNGH CHTREUNGO CHTREUNG
[3,4-Bis(benzyloxy)phenyl]phosphonic Acid Diethyl Ester. To a soln. of 3,4-bis(benzyloxy)phenol
(3.83 g, 12.5 mmol) in pyridine (10 ml) at 08 was added trifluoromethanesulfonic anhydride (2.5 ml, 15
mmol, 1.2 equiv.), and the mixture was stirred at 08 for 1 h, allowed to warm to r.t., and stirred for further
3 h. The resulting mixture was adjusted to pH 2 with 2M HCl and extracted with AcOEt. The org. phase
was concentrated i.v. to afford trifluoromethanesulfonic acid 3,4-bis(benzyloxy)phenyl ester (4.8 g, 88%)
which was used without further purification.
[3,4-Bis(benzyloxy)phenyl]phosphonic acid was prepared by the method of Thurieau et al. [21]: To a
soln. of trifluoromethanesulfonic acid 3,4-bis(benzyloxy)phenyl ester (2.5 g, 5.7 mmol) in MeCN (20 ml)
were added N-methylmorpholine (0.95 ml, 8.6 mmol, 1.5 equiv.), tetrakis(triphenylphosphine)palladium
(330 mg, 0.29 mmol, 0.05 equiv.) and phosphonic acid diethyl ester (1.1 ml, 8.6 mmol, 1.5 equiv.). The
mixture was stirred at 708 for 24 h and then the reaction stopped by an addition of AcOEt. The mixture
was washed successively with 5% citric acid and 5% aq. NaHCO3 solns., the org. phase concentrated i.v.,
and the residue subjected to CC (silica gel, hexane/AcOEt 1:1): 3,4-bis(benzyloxy)phenylphosphonic
acid diethyl ester (1.9 g, 78%). Yellow oil. 1H-NMR ((D6)acetone): 1.22 (t, J(HH)=6.9, 2 MeCH2);
3.97 (m, 2 MeCH2); 5.21 (s, PhCH2); 5.25 (s, PhCH2); 7.20 (dd, J(HH)=8.6, J(HP)=4.4, HꢀC(5));
7.29–7.53 (m, 2 PhCH2, HꢀC(2), HꢀC(6)). HR-FD-MS: 426.1589 (M+, C
P+; calc. 426.1596).
24ACHTREUNGH27AHCERTUNGO5ACHTREUNG
(3,4-Dihydroxyphenyl)phosphonic Acid (6). A soln. of [3,4-bis(benzyloxy)phenyl]phosphonic acid
diethyl ester (200 mg, 0.47 mmol) in conc. HCl soln. (10 ml) was refluxed for 3 h. After cooling, the mix-
ture was poured into H2O, and washed with AcOEt, the aq. phase concentrated i.v., and the crude product
subjected to reversed-phase CC (H2O): 6 (73 mg, 82%). Colorless oil. 1H-NMR (CD
3ACHTREOUGN D): 6.82 (dd,
J(HH)=8.1, J(HP)=4.4, HꢀC(5)); 7.14 (ddd, J(HH)=8.1, 1.5, J(HP)=13.3, HꢀC(6)); 7.20 (dd,
J(HH)=1.5, J(HP)=14.0, HꢀC(2)). HR-FD-MS: 191.0113 (M+, C
P+; calc. 191.0109).
6ACHTREUNGH8ACHERTUNGO5ACHTRENUG
(3,4-Dihydroxyphenyl)phosphonic Acid Monoethyl Ester (7). To a suspension of [3,4-bis(benzyloxy)-
ACHTREUNG

Helvetica Chimica Acta – Vol. 89 (2006)
1405
residue subjected to CC (silica gel, hexane/AcOEt 1:5): [3,4-bis(benzyloxy)phenyl]phosphonic acid
monoethyl ester (0.30 g, 75%). The resultant monoethyl ester (0.30 g, 0.75 mmol) was deprotected by
hydrogenation under 1 atm H2 in the presence of a cat. amount of 10% Pd/C in MeOH (15 ml). The mix-
ture was filtered through Celite, the filtrate concentrated i.v., and the crude product subjected to reversed-
phase CC (H2O): 7 (0.14 g, 86%). White powder. M.p. 179–1818. 1H-NMR ((D6)acetone): 1.23 (t,
J(HH)= 7.1, MeCH2); 3.90 (qd, J(HH)=7.1, J(HP)=7.1, MeCH2); 6.83 (dd, J(HH)=8.1, J(HP)=4.4,
HꢀC(5)); 7.12 (ddd, J(HH)=8.1, 1.5, J(HP)=13.0, HꢀC(6)); 7.17 (dd, J(HH)=1.5, J(HP)=13.8, Hꢀ
C(2)). HR-EI-MS: 218.0319 (M+, C P+; calc. 218.0344).
8ACHTREUNGH11ACHERTUNGO5ACHTRENUG
(3,4-Dihydroxyphenyl)phosphonic Acid Diethyl Ester (8). To a soln. of [3,4-bis(benzyloxy)phenyl]-
phosphonic acid diethyl ester (0.21 g, 0.5 mmol) in MeOH (10 ml) was added a cat. amount of 10%
Pd/C, and the mixture was stirred under 1 atm of H2 for 24 h. The mixture was filtered through Celite,
the filtrate concentrated i.v., and the residue subjected to CC (silica gel, hexane/AcOEt 1:2): 8 (0.10 g,
81%). Yellow oil. 1H-NMR ((D6)acetone): 1.26 (t, J(HH)=7.1, 2 MeCH2); 4.04 (m, 2 MeCH2); 6.94
(dd, J(HH)=8.1, J(HP)=4.7, HꢀC(5)); 7.14 (ddd, J(HH)=8.1, 1.7, J(HP)=13.0, HꢀC(6)); 7.38 (dd,
J(HH)=1.7, J(HP)=13.8, HꢀC(2)). HR-EI-MS: 246.0684 (M+, C P+; calc. 246.0657).
10ACHTREUNGH15AHCERTUNGO5ACHTREUNG
3. Colorimetric Radical-Scavenging Tests. 3.1. DPPH-Scavenging Test. To a soln. of a test compound
(12.5 mM, 4 ml) in a test tube, 1 ml of DPPH (500 mM), was added. The soln. was immediately mixed vig-
orously for 10 s by a Vortex mixer and transferred to a cuvette. The absorbance reading at 517 nm was
taken 1, 2, 5, 10, and 30 min, then 1, 2, 4, and 6 h after initial mixing. MeCN and MeOH were chosen
as inert non-alcoholic and nucleophilic alcoholic solvents, resp. A soln. of (2RS)-a-tocopherol of the
same concentration was measured as a positive control. A reduction of the absorbance, 0.228, by the pos-
itive control was regarded as corresponding to the consumption of 2 equiv. of DPPH. All experiments
were performed in triplicate at 23–258.
3.2. ABTS-Radical-Scavenging Test. ABTS-Radical-scavenging activity was measured by the method
of Re et al. [22] with a modification. The ABTS radical cation (ABTSC+) was produced by treating a 7mM
aq. ABTS soln. with 2.45mM potassium persulfate (final concentration). The mixture was allowed to
stand in the dark at r.t. for 12–16 h prior to use. The ABTSC+ soln. was diluted with dist. H2O to an
absorbance of 0.70 (ꢁ0.02) at 734 nm. To a diluted ABTSC+ soln. (2.97 ml) was added an EtOH soln.
of a test compound (0.5mM, 0.03 ml). The soln. was immediately mixed vigorously for 10 s by a Vortex
mixer and transferred to a cuvette. The absorbance reading was taken 30 min after initial mixing.
EtOH was used in place of an antioxidant soln. as a control. An EtOH soln. of Trolox was measured
as a positive control. A reduction of the absorbance by the positive control was regarded as correspond-
ing to the consumption of 2 equiv. of ABTSC+. The pH of the reaction solns. was measured 10 min after
mixing. All experiments were performed in triplicate at 23–258.
4. NMR Analyses. 4.1. NMR Measurements of the Reaction Mixtures 1/DPPH and 3/DPPH. To a cat-
echol derivative 1 or 3 (2.5 mmol) was added DPPH (5.0 mg, 13 mmol, 5.0 equiv.) in CD3ACHTREOUNG D/(D6)acetone
3 :1, (0.4 ml). (D6)acetone was added as a cosolvent to enhance the solubility of the DPPH radical. The
mixture was immediately transferred to an NMR tube and mixed vigorously. 1H-NMR spectra were
recorded 10, 60, and 120 min after mixing.
4.1.1. Reaction Mixture 1/DPPH: 3,4-Dioxocyclohexa-1,5-diene-1-carboxylic Acid (1a) and 3-Hy-
droxy-3-methoxy-4-oxocyclohexa-1,5-dienecarboxylic Acid (1b). Quinone 1a: 1H-NMR (CD
3ACHTREOUNG D/
(D6)acetone 3 :1): 6.43 (d, J=10.3, HꢀC(5)); 6.91 (s, HꢀC(2)); 7.53 (d, J=10.3, HꢀC(6)). 13C-NMR
(CD3ACHTREOUNG D/(D6)acetone 3 :1): 131.5 (C(5)); 133.3 (C(2)); 138.8 (C(6)); 141.7 (C(1)); 167.2 (COOH);
181.2 (C(4)); 182.3 (C(3)). HMBC: HꢀC(2) $ C(4), C(6), COOH; HꢀC(5) $ C(1), C(3); HꢀC(6) $
C(2), C(4).
1
Quinone Hemiacetal 1b: H-NMR (CD
OD/(D6)acetone 3 :1): 6.10 (d, J=10.3, HꢀC(5)); 7.21 (s,
HꢀC(2)); 7.43 (d, J=10.3, HꢀC(6)). 13C-NMR (CD
3ACHTREOUGN D/(D6)acetone 3 :1): 89.5 (C(3)); 126.1 (C(5));
128.6 (C(1)); 139.5 (C(6)); 145.1 (C(2)); 166.5 (COOH); 198.0 (C(4)). HMBC: HꢀC(2) $ C(4), C(6),
COOH; HꢀC(5) $ C(1), C(3); HꢀC(6) $ C(2), C(4).
4.1.2. Reaction Mixture 3/DPPH: Sodium 3,4-Dioxocyclohexa-1,5-diene-1-carboxylate (3a) and
Sodium 6-Methoxy-3,4-dioxocyclohexa-1,5-diene-1-carboxylate (3g). Quinone 3a: 1H-NMR (CD
CHTREOUNG D/
3A
(D6)acetone 3 :1): 6.33 (d, J=10.1, HꢀC(5)); 6.68 (d, J=2.0, HꢀC(2)); 7.61 (dd, J=10.1, 2.0, Hꢀ

1406
Helvetica Chimica Acta – Vol. 89 (2006)
C(6)). 13C-NMR (CD
3ACHTREOUNG D/(D6)acetone 3 :1): 129.3 (C(2)); 130.0 (C(5)); 142.4 (C(6)); 148.7 (C(1)); 170.6
(COONa); 181.0 (C(4)); 183.1 (C(3)). HMBC: HꢀC(2) $ C(4), C(6), COONa; HꢀC(5) $ C(1), C(3);
HꢀC(6) $ C(2), C(4).
6-Methoxyquinone 3g: 1H-NMR (CD
NMR (CD3ACHTREOUNG D/(D6)acetone 3 :1): 103.8 (C(5)); 123.5 (C(2)); 151.6 (C(1)); 170.1 (C(6)); 170.1
3A
(COONa); 179.6 (C(4)); 182.9 (C(3)). HMBC: HꢀC(2) $ C(4), C(6), COONa; HꢀC(5) $ C(1),
C(3); MeO $ C(6).
4.2. NMR Measurements of the Reaction Mixtures 1f/DPPH and 3f/DPPH. To a catechol derivative
1f or 3f (2.5 mmol) was added DPPH (3.0 mg, 7.6 mmol, 3.0 equiv.) in CD3ACHTREOUNG D/(D6)acetone 3 :1 (0.4 ml).
The mixture was immediately transferred to an NMR tube and mixed vigorously. 1H-NMR spectra were
recorded 10 min after mixing.
4.2.1. Reaction Mixture 1f/DPPH: 6-Methoxy-3,4-dioxocyclohexa-1,5-diene-1-carboxylic Acid (1g)
and 3-Hydroxy-3,6-dimethoxy-4-oxocyclohexa-1,5-diene-1-carboxylic Acid (1h). 6-Methoxyquinone 1g:
1H-NMR (CD
(CD3A
C
168.1 (C(6)); 179.1 (C(4)); 180.8 (C(3)). HMBC: HꢀC(2) $ C(4), COOH; HꢀC(5) $ C(1), C(3);
MeO $ C(6).
6-Methoxyquinone Hemiacetal 1h: 1H-NMR (CD
N
C(5)); 6.83 (s, HꢀC(2)). 13C-NMR (CD
3ACHTREOUNG D/(D6)acetone 3 :1): 57.6 (MeO); 89.7 (C(3)); 99.4 (C(5));
129.6 (C(1)); 142.9 (C(2)); 166.7 (COOH); 168.4 (C(6)); 195.6 (C(4)). HMBC: HꢀC(2) $ C(4),
COOH; HꢀC(5) $ C(1), C(3); MeO $ C(6).
4.2.2. Reaction Mixture 3f/DPPH. 6-Methoxyquinone 3g: 1H-NMR (CD
3ACHTREOUNG D/(D6)acetone 3 :1): 3.90
(s, MeO); 5.85 (s, HꢀC(5)); 6.14 (s, HꢀC(2)). 13C-NMR (CD
(C(5)); 123.5 (C(2)); 151.6 (C(1)); 170.1 (C(6)); 170.1 (COONa); 179.6 (C(4)); 182.9 (C(3)). HMBC: Hꢀ
C(2) $ C(4), C(6), COONa; HꢀC(5) $ C(1), C(3); MeO $ C(6).
5. Isolation of an Oxidation Product of 1 after Reaction with DPPH in MeOH. To a soln. of 1 (77 mg,
0.50 mmol) in MeOH/acetone 3 :1 (50 ml) was added DPPH (788 mg, 2.0 mmol, 4.0 equiv.), and the mix-
ture was stirred for 12 h at r.t. Then sodium dithionite (348 mg, 2.0 mmol, 4.0 equiv.) in H2O (15 ml) was
added and the mixture stirred for 30 min. The mixture was concentrated i.v., and the residue subjected to
CC (silica gel, CHCl3/MeOH 100 :7). The crude product was further purified by prep. TLC (CHCl3/
MeOH/formic acid 100 :7:0.1; Rf 0.27): 1f (1.9 mg, 2.1%).
6. Molecular-Orbital Calculations. The electron densities and energies of LUMOs were calculated by
the AM1 method with the MOPAC 2000 program included in the Chem3D package (CambridgeSoft Co).
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Received April 18, 2006