Preparation and antioxidant activity of α-pyridoin and its derivatives

Article abstract of DOI:10.1016/j.bmc.2005.07.065

Focusing on α-pyridoin (1, 1,2-di(2-pyridyl)-1,2-ethenediol) as the lead compound of the novel antioxidative enediol, we synthesized 5,5′- or 6,6′-bis-substituted derivatives of 1 from disubstituted pyridines. The antioxidant activity of 1 and its synthetic derivatives 2-7 was evaluated by DPPH (1,1-diphenyl-2-picrylhydrazyl radical) scavenging assay and inhibition of lipid peroxidation. In the DPPH assay, 1 exhibited an activity stronger than that of ascorbic acid, and 5,5′-dimethyl-(5) or 5,5′-dimethoxy- substituted derivatives (6) exhibited more potent activity than 1. The DPPH scavenging activities of α-pyridoins were correlated with their oxidation potential and thus the electron density of enediol. 5 and 6 effectively inhibited lipid peroxidation in the rat liver microsome/tert-butyl hydroperoxide system. Therefore, 5 and 6 serve as good candidates for a pharmacologically useful enediol antioxidant.

Full text of DOI:10.1016/j.bmc.2005.07.065

Bioorganic & Medicinal Chemistry 13 (2005) 6763–6770
Preparation and antioxidant activity of a-pyridoin and
its derivatives
Masashi Hatanaka, Kyoko Takahashi, Shigeo Nakamura and Tadahiko Mashino*
Kyoritsu University of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo 105-8512, Japan
Received 16 May 2005; revised 20 July 2005; accepted 21 July 2005
Available online 24 August 2005
Abstract—Focusing on a-pyridoin (1, 1,2-di(2-pyridyl)-1,2-ethenediol) as the lead compound of the novel antioxidative enediol, we
synthesized 5,50- or 6,60-bis-substituted derivatives of 1 from disubstituted pyridines. The antioxidant activity of 1 and its synthetic
derivatives 2-7 was evaluated by DPPH (1,1-diphenyl-2-picrylhydrazyl radical) scavenging assay and inhibition of lipid peroxida-
tion. In the DPPH assay, 1 exhibited an activity stronger than that of ascorbic acid, and 5,50-dimethyl-(5) or 5,50-dimethoxy-substi-
tuted derivatives (6) exhibited more potent activity than 1. The DPPH scavenging activities of a-pyridoins were correlated with
their oxidation potential and thus the electron density of enediol. 5 and 6 effectively inhibited lipid peroxidation in the rat liver
microsome/tert-butyl hydroperoxide system. Therefore, 5 and 6 serve as good candidates for a pharmacologically useful enediol
antioxidant.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
HO
OH
OH
N
Reactive oxygen species (ROS) and free radicals are
considered to be implicated in a variety of pathological
events, such as cancer and aging.1–3 ROS, including
superoxide anion, hydrogen peroxide, and hydroxyl rad-
ical, are thought to be generated subsequent to the
reduction of molecular oxygen in aerobic organisms.4,5
Under normal conditions, cells and tissues are protected
against ROS by an array of enzyme defense systems,
such as superoxide dismutase, catalase, and glutathione
peroxidase or free radical scavengers.6 Among these rad-
ical scavengers, ascorbic acid (AsA, Fig. 1) shows very
effective activity7 and has often served as a lead com-
pound for the design and synthesis of pharmacologically
effective antioxidants.8 The active site of AsA is the 2,3-
enediol moiety conjugated with the carbonyl group of a
five-membered lactone. The anionic form of AsA is very
stable and has a strong electron-donating ability to act
as an effective radical scavenger.9 Many O-acyl, O-alkyl,
and imine analogues of AsA have been synthesized and
their antioxidant activity been studied.8 Almost all these
AsA derivatives have a c-lactone formula, and a few
papers have reported the antioxidant activities of even
O
O
N
OH
OHOH
AsA
1
Figure 1. Structure of a-pyridoin (1) and ascorbic acid (AsA).
simpler enediols, such as reductic acid (2,3-dihydroxy-
2-cycropentenone)10 or a kind of sugar analogue.11
In the current study, we focused on a-pyridoin (1,
Fig. 1), having a coplanar E-enediol form, as a
candidate for enediol antioxidant. The enediol form is
stabilized by the intramolecular hydrogen bonding of
pyridine nitrogen and hydroxyl group, and no a-hydrox-
yketone form exists.12,13 The enediol form is conjugated
with the electron-withdrawing pyridine ring. Actually,
the pKa value of 1 is approximately 0.5. Therefore, it
is thought that the anion form (i, Fig. 2) is easily formed,
such as AsA, under physiological conditions. Further-
more, the electron density of the enediol can be easily
controlled by substitution on a pyridine ring. The phys-
ical and chemical properties of 1 have been well-studied
since the 1950s.14–16 The inhibition of mild steel corro-
sion in hydrochloric acid by 1 has been reported.17
However, the biological activities of 1 have been
Keywords: Antioxidant; a-Pyridoin; Enediol; Radical scavenger.
*
Corresponding author. Tel.: +81 3 5400 2694; fax: +81 3 5400
2691; e-mail: mashino-td@kyoritsu-ph.ac.jp
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.065

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M. Hatanaka et al. / Bioorg. Med. Chem. 13 (2005) 6763–6770
OH
N
O
N
O
N
-H+
+H+
N
OH
N
OH
(i)
N
OH
e
1
R
R
Figure 2. Proposed mechanism of free radical scavenging by a-pyridoin (1).
reported only with regard to the inhibition activity of
aldehyde dehydrogenase as an analogue of metyra-
pone.18 In this paper, we report the preparation of
a-pyridoin derivatives and the antioxidant activity of
these a-pyridoins.
6'
5'
R
OH
N
2'
N
CHO
2
6
5
NaCN
2
R
R
N
OH
5
6
8: R=6-CH3
2: R=6-, 6'-CH3
9: R=6-OCH3
10: R=6-COCH3
11: R=5-CH3
12: R=5-OCH3
13: R=5-COCH3
3: R=6-, 6'-OCH3
4: R=6-, 6'-COCH3
5: R=5-, 5'-CH3
6: R=5-, 5'-OCH3
7: R=5-, 5'-COCH3
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of a-pyridoin derivatives. The structures
of synthetic a-pyridoin derivatives 2–7 are illustrated
in Figure 3. a-Pyridoin derivatives were prepared in a
modification of a-pyridoin (1) synthesis using the benzo-
in condensation (Scheme 1).19 6-Methoxy-2-pyridinecar-
boxaldehyde (9) was prepared by monomethoxylation
of 2,6-dibromopyridine (14), followed by formylation
of the remaining bromine (Scheme 2).20 6-Acetyl-2-pyri-
dinecarboxaldehyde (10) was prepared in four steps
(Scheme 3). 2,6-Dibromopyridine (14) was treated with
n-butyllitium and N,N-dimethylacetamide to give 6-ace-
tyl-2-bromopyridine (16).21 The acetyl carbonyl of 16
was protected to form acetal 17.22 Formylation of 17
and subsequent deprotection of acetal gave 10. 5-Ace-
tyl-2-pyridinecarboxaldehyde (13) was prepared by the
same procedure as that followed for 10 using 2,5-dib-
romopyridine (18) as a starting material (Scheme 3).
Monoacetylation of 18 predominantly gave 5-acetyl-
2-bromopyridine (19).23 5-Methyl-2-pyridinecarbox-
aldehyde (11) was prepared by the formylation
of 6-bromo-3-picoline (23, Scheme 4). 5-Methoxy-
2-pyridinecarboxaldehyde (12) was prepared from 5-hy-
droxy-2-picoline (24) in five steps (Scheme 5). The
O-methylation and subsequent oxidation of 24 afforded
5-methoxy-2-picoline-N-oxide (26).24 N-Oxide 26 was
converted to pyridylmethanol 28 via trifluoroacetate
Scheme 1. Benzoin condensation of substituted 2-pyridinecarbox-
aldehydes 8–13.
Br
N
Br
CH3O
CHO
N
Br
a
14
15
CH3O
N
b
9
Scheme 2. Synthesis of aldehyde 9. Reagents and conditions: (a)
CH3ONa/DMF, 80 ꢁC, 2 h, 50%; (b) n-BuLi, DMF/THF, ꢀ78 ꢁC to
rt, 53%.
intermediate 27, and then 28 was oxidized to aldehyde
12 by manganase dioxide.25 Finally, benzoin condensa-
tion of these substituted pyridinecarboxaldehydes 8–13
was carried out to give pyridoins 2–7. 1H and 13C
NMR spectra in DMSO-d6 revealed that all a-pyridoin
derivatives, except for 4, existed in an E-enediol form
and 4 was a mixture of E-enediol and Z-enediol forms
(7:3).
2.1.2. Lipophilicity of a-pyridoins. The biomembrane
consists largely of lipids, one of the main components
of which are unsaturated fatty acids, and it is easily per-
oxidized with ROS. Thus, it is thought that the lipophil-
ic antioxidant acts effectively. To elucidate the ability to
penetrate a biomembrane, the lipophilic property of a-
pyridoins 1–7 was measured. The logkw value is nearly
in agreement with the partition coefficient logP and
can be calculated by measuring the retention time on
the reverse-phase HPLC using methanol/3-(N-morpholi-
no) propanesulfonate (MOPS) buffer (pH 7.4).26–28 The
log kw value, which is considered to be the lipophilic
property of 1–7, is shown in Table 1. a-Pyridoin (1)
6'
5'
OH
N
2'
R
2
R
N
OH
5
6
2: R=6-, 6'-CH3
3: R=6-, 6'-OCH3
4: R=6-, 6'-COCH3
5: R=5-, 5'-CH3
6: R=5-, 5'-OCH3
7: R=5-, 5'-COCH3
Figure 3. Structure of a-pyridoin derivatives 2–7.

M. Hatanaka et al. / Bioorg. Med. Chem. 13 (2005) 6763–6770
Table 1. Lipophilic property of a-pyridoins
6765
R1
R2
N
Br
Compound
log kw
1
2
3
4
5
6
7
3.15
3.94
5.10
3.51
4.13
4.68
3.33
14: R1=Br, R2=H
16: R1=Ac, R2=H
c
d
17: R1=2-methyl-1,3-dioxolan-2-yl, R2=H
18: R1=H, R2=Br
19: R1=H, R2=Ac
c
d
20: R1=H, R2=2-methyl-1,3-dioxolan-2-yl
R1
N
CHO
e
2.1.3. Oxidation potential. The reducing ability of enediol
is related to its electron density. Thus, as an index of the
electron density, oxidation potential of a-pyridoins was
measured by cyclic voltammetry (Table 2) in a mixture
of DMF and 2-(N-morpholino)ethanesulfonate (MES)
buffer (3:2 (v/v), pH 7.4). 1, 2, and 5–7 had greater nega-
tive potential than AsA. These data indicated that 1, 2,
and 5–7 could be oxidized easily, as their reducing abili-
ties are higher than that of AsA. It is thought that a
methyl or a methoxy group at the 5-position of the pyr-
idine ring increased the electron density of enediol due to
its electron-donating effects. On the other hand, 3 and 4
had more positive potential than 1. This result suggested
that the electron density on the enediol of 3 and 4 was
lower than that of 1 and the methoxy group of 3 worked
as an electron-withdrawing group. C-6 of a-pyridoin is in
R2
21: R1=2-methyl-1,3-dioxolan-2-yl, R2=H
10: R1=Ac, R2=H
f
22: R1=H, R2=2-methyl-1,3-dioxolan-2-yl
13: R1=H, R2=Ac
f
Scheme 3. Synthesis of aldehydes 10 and 13. Reagents and conditions:
(c) n-BuLi, AcN(CH3)2/Et2O, ꢀ78 ꢁC to rt, 17: 76%, 18: 50%; (d)
ethylene glycol, p-TsOH/toluene, reflux, 19: 4 h, 79%, 20: 6 h, 87%; (e):
n-BuLi, DMF/Et2O, ꢀ78 ꢁC to rt, 21: 54%, 22: 53%; (f) HCl, reflux,
10: 2 h, 82%, 13: 10 min, 48%.
N
Br
N
CHO
g
CH3
CH3
23
11
Table 2. DPPH radical scavenging activity and oxidation potential of
a-pyridoins
Scheme 4. Synthesis of aldehyde 11. Reagents and conditions: (g)
n-BuLi, DMF/Et2O, ꢀ78 ꢁC to rt, 31%.
Compound DPPH radical scavenging Oxidation potential
rate k (103 Mꢀ1 sꢀ1
)
E1/2 (mV vs Ag/AgCl)
1
45.1
94.2
9.37
6.99
130
ꢀ10
40
had high lipophilicity because it contains two internal
hydrogen bonds between pyridine nitrogen and hydrox-
yl group. A methyl and a methoxy substituent augment-
ed lipophilicity by 1.3- to 1.6-fold. In particular, 6,
60-dimethoxy derivative 3 showed an extremely high
lipophilicity. On the other hand, acetyl substitution
showed little effect on lipophilicity.
2
3
190
240
5
4
5
6
108
ꢀ20
125
180
7
AsA
43.7
15.1
O
N
CH3
N
CH3
N
CH3
h
i
CH3O
HO
CH3O
24
25
26
N
CH2OH
N
CH2OCOCF3
j
l
k
CH3O
CH3O
27
28
N
CHO
CH3O
12
Scheme 5. Synthesis of aldehyde 12. Reagents and conditions: (h) NaH, CH3I/DMF, 180 ꢁC, 2 h, 75%; (i) H2O2/AcOH, 100 ꢁC, 5 h, 35%;
(j) (CF3CO)2O/CH2Cl2, 0 ꢁC to rt, 24 h; (k) MeOH, reflux, 5 h; (l) MnO2/CH2Cl2, rt, 30 min.

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M. Hatanaka et al. / Bioorg. Med. Chem. 13 (2005) 6763–6770
a meta position to the enediol-binding site. Thus, it was
thought that the methoxy group showed its electron-
withdrawing inductive effect rather than its electron-
donating resonance effect, which can be accounted for
by HammettÕs substituent constant (rmꢀOCH = 0.115,
3
rpꢀOCH = ꢀ0.268).29
3
2.2. Antioxidant activity
2.2.1. DPPH radical scavenging activity and oxidation
potential. DPPH (1,1-diphenyl-2-picrylhydrazyl) assay is
the simplest method to measure the ability of antioxi-
dants to quench free radicals.30 The DPPH radical scav-
enging activities of a-pyridoins were evaluated by the
second-order rate constant with DPPH obtained by
pseudo first-order rate constant in the mixture of etha-
nol and MES buffer (3:2 (v/v), pH 7.4). As shown in
Table 2, all a-pyridoins 1–7 showed DPPH radical scav-
enging activity. In particular, the activity of 1 was three
times greater than that of AsA, and the activities of 5,
and 6 were 8.6- and 7.2-fold greater than that of AsA,
respectively. On the other hand, 3 and 4 showed activity
lower than that of AsA.
Figure 5. Inhibition activity on microsomal lipid peroxidation by t-
BuOOH. Values are means SEM.
to its high lipophilicity. On the other hand, 1, 2, 4, 7,
and AsA exerted little inhibition on lipid peroxidation.
These results indicated that some modification on the
pyridine ring is the important factor in antioxidant
activity under the heterogeneous system and they
demonstrated that 5 and 6 had excellent antioxidant
activity.
Figure 4 shows the relation between the oxidation poten-
tial and DPPH radical scavenging rate. Though the cor-
relation coefficient was not satisfactory, compounds
showing a negative oxidation potential tended to show
a higher radical scavenging activity. This result indicated
that one-electron transfer reaction step, but not the
deprotonation, was the rate-determining step of radical
scavenging by a-pyridoins. This kind of correlation is
also demonstrated by the reaction of a -tocopherol ana-
logues with peroxyradical species.31
3. Conclusion
To find novel antioxidative enediols, we have investigat-
ed the antioxidant activity of a-pyridoin (1) and its syn-
thetic derivatives 2–7. The DPPH radical scavenging
activities of 1 and its derivatives 2 and 5–7 were more
potent than the activity of AsA. In the tert-butyl hydro-
peroxide/rat liver microsome system, 5 and 6 effectively
inhibited lipid peroxidation. Therefore, we propose that
5 and 6 are good candidates for the novel antioxidative
enediols discussed here.
2.2.2. Inhibition of lipid peroxidation. To investigate the
antioxidant activity of biomembrane, 1–7 and AsA were
evaluated for the inhibition of lipid peroxidation in rat
liver microsomes, initiated by cytochrome P450 and
tert-butyl hydroperoxide.32 Lipid peroxidation was mea-
sured by the formation of thiobarbituric acid reactive
substances (TBARS, Fig. 5). The most potent DPPH
radical scavengers, 5, and 6, inhibited lipid peroxidation
effectively in a dose-dependent manner. Similarly, 3
inhibited lipid peroxidation effectively; nevertheless, 3
showed a DPPH radical scavenging activity lower than
that of AsA. The high activity of 3 may have been due
4. Experimental
4.1. Preparation of a-pyridoin and its derivatives
1
4.1.1. General. H NMR spectra (500 MHz) were mea-
sured on a JEOL JNM-A500 FT-NMR spectrometer
with tetramethylsilane as an internal standard
(d = 0.00) in CDCl3 or DMSO-d6. 13C NMR spectra
(125 MHz) were obtained on the same spectrometer
and the chemical shifts were referenced to the signals
of CDCl3 (d = 77.0) or DMSO-d6 (d = 39.5). Mass spec-
tra were recorded on a JEOL AUTOMASS SUN200
quadrupole mass spectrometer or a JEOL JMS-700
mass spectrometer. Melting points were determined with
a Yanagimoto MP-J3 micro-melting point apparatus
and are uncorrected. Column chromatography was per-
formed using Merck Silica gel 60. a-Pyridoin (1), 2,5-
dibromopyridine (18), and 6-bromo-3-picoline (23) were
purchased from Aldrich Chemical Co. 6-Methyl-2-pyri-
dinecarboxaldehyde (8) and 2,6-dibromopyridine (14)
were purchased from Tokyo Kasei Kogyo Co., Ltd. 5-
Hydroxy-2-picoline (24) was purchased from Acros
Organics.
Figure 4. Correlation between DPPH scavenging activity and oxida-
tion potential.

M. Hatanaka et al. / Bioorg. Med. Chem. 13 (2005) 6763–6770
6767
4.1.2. Preparation of 6,60-dimethyl-2,20-pyridoin (2).
Sodium cyanide (196 mg, 4 mmol in H2O, 2 mL) was
added to a stirred mixture of 6-methyl-2-pyridinecar-
boxaldehyde (8, 1.0 g, 8.0 mmol) and methanol (5 mL).
The mixture was refluxed at 80 ꢁC for 10 min. The
resulting orange solid was collected by filtration, washed
with methanol and H2O successively, and dried under
2H, –OH); 13C NMR (DMSO-d6): d 53.64, 108.93,
112.00, 134.53, 141.09, 152.45, 161.30; MS: m/z 274
(M+), 259, 231, 138, 109, 94; HRMS calcd for
C14H14N2O4, 274.0954; found: 274.0933 (M+); mp:
183–184 ꢁC (pyridine).
4.1.4. Preparation of 6,60-diacetyl-2,20-pyridoin (4)
1
reduced pressure to afford 2 (946 mg, 95%). H NMR
4.1.4.1. 2-Acetyl-6-bromopyridine (16). Under N2
atmosphere, n-BuLi (10.6 mL, 17 mmol, 1.6 M in n-hex-
ane) was added to a mixture of 2,6-dibromopyridine (14,
4.0 g, 17 mmol) and anhydrous Et2O (40 mL) at
–78 ꢁC. The reaction mixture was stirred for 30 min.
Then, N,N-dimethylacetamide (2.4 mL, 28 mmol) was
added, and the mixture was warmed to ambient temper-
ature within 1 h. The reaction mixture was quenched by
adding saturated NH4Cl, and then the whole mixture
was extracted with Et2O (3· 150 mL). The combined
organic layer was washed with brine, dried, and concen-
trated. The crude product was chromatographed on sil-
ica-gel column using n-hexane/AcOEt (25:1) to give 2.6 g
(DMSO-d6): d 2.55 (s, 6H, –CH3), 7.26 (d, 4H,
J = 7.9 Hz, 5 and 50-H), 7.61 (d, 4H, J = 7.9 Hz, 3 and
30-H), 7.92 (t, 4H, J = 7.9 Hz, 4 and 40-H), 13.3 (s, 2H,
–OH); 13C NMR (DMSO-d6): d 23.36, 116.11, 121.55,
135.01, 138.63, 154.87, 155.08; MS: m/z 242 (M+), 122,
93, 66; mp: 189–191 ꢁC (dec) (pyridine).
4.1.3. Preparation of 6,60-dimethoxy-2,20-pyridoin (3)
4.1.3.1. 2-Bromo-6-methoxypyridine (15). Under N2
atmosphere, sodium methoxide (6.5 mL, 8.4 mmol,
28% in methanol) was added to a mixture of 2,6-dib-
romopyridine (14, 2.0 g, 8.4 mmol) and DMF (33 mL).
The mixture was stirred at 80 ꢁC for 2 h. After cooling
to ambient temperature, the reaction mixture was
poured into H2O (100 mL) and then extracted with
Et2O (3· 100 mL). The combined organic layer was
washed with brine, dried, and concentrated. The crude
product was chromatographed on silica-gel column
using a mixture of n-hexane/AcOEt (30:1) to give
788 mg of 15 as a colorless oil (50%). 1H NMR (CDCl3):
d 3.94 (s, 3H, –OCH3), 6.68 (d, 1H, J = 7.9 Hz, 5-H),
7.05 (d, 1H, J = 7.9 Hz, 3-H), 7.41 (t, 1H, J = 7.9 Hz,
4-H); 13C NMR (CDCl3): d 54.07, 109.40, 120.19,
138.63, 140.32, 163.77.
1
of 16 as a colorless solid (76%). H NMR (CDCl3): d
2.70 (s, 3H, –COCH3), 7.64–7.72 (m, 2H, 4 and 5-H),
7.99 (d, 1H, J = 7.0 Hz, 3-H); 13C NMR (CDCl3): d
25.71, 120.45, 131.75, 139.11, 141.34, 154.30, 198.57;
MS: m/z 201 (M++2), 199 (M+), 173, 171, 158, 156,
131, 129, 106, 76.
4.1.4.2. 2-Bromo-6-(2-methyl-1,3-dioxlane-2-yl)pyri-
dine (17). A mixture of 16 (1.5 g, 7.5 mmol), ethylene
glycol (0.88 mL, 1.6 mmol), p-TsOH–H2O (428 mg,
2.3 mmol), and toluene (30 mL) was stirred for 4 h at
140 ꢁC with a Dean–Stark apparatus. The reaction mix-
ture was washed with saturated Na2CO3 and with brine
successively, dried, and concentrated. The crude product
was chromatographed on silica-gel column using n-hex-
ane/AcOEt (12:1) to give 1.45 g of 17 as a colorless oil
4.1.3.2. 6-Methoxy-2-pyridinecarboxaldehyde (9).
Under N2 atmosphere, n-BuLi (2.9 mmol, 1.9 mL,
1.6 M in n-hexane) was added to a mixture of 15
(536 mg, 2.9 mmol) and anhydrous THF (15 mL) at
ꢀ78 ꢁC. The reaction mixture was stirred for
30 min. Then DMF (0.25 mL) was added, and the
mixture was warmed to ambient temperature within
1 h. The reaction mixture was quenched by adding
5% NaHCO3, and then the whole mixture was
extracted with Et2O (3· 50 mL). The combined
organic layer was washed with brine, dried, and con-
centrated. The crude product was chromatographed
on silica-gel column using a mixture of n-hexane/
AcOEt (20:1) to give 206 mg of 9 as a yellow oil
(53%). 1H NMR (CDCl3): d 4.03 (s, 3H, –OCH3),
6.98 (d, 1H, J = 8.2 Hz, 5-H), 7.05 (d, 1H,
J = 7.3 Hz, 3-H), 7.41 (t, 1H, J = 7.6 Hz, 4-H), 9.96
(s, 1H, –CHO); 13C NMR (CDCl3): d 53.64, 115.51,
116.33, 139.06, 150.44, 164.42, 193.12.
1
(79%). H NMR (CDCl3): d 1.72 (s, 3H, CH3), 3.84–
3.94 (m, 2H, –OCH2–), 4.04–4.14 (m, 2H, –OCH2–),
7.41 (d, 1H, J = 7.3 Hz, 5-H), 7.48–7.56 (m, 2H, 3 and
4-H); 13C NMR (CDCl3): d 25.11, 65.08, 108.02,
118.21, 127.51, 138.75, 142.04, 162.51; MS: m/z 230
([M++2]–CH3), 228 ([M+]–CH3), 214, 212, 202, 200,
186, 184, 173, 171, 158, 156, 87, 76.
4.1.4.3. 6-(2-Methyl-1,3-dioxolan-2-yl)-2-pyridinea-
carboxaldehyde (21). Under argon atmosphere, n-BuLi
(3.7 mL, 6.0 mmol, 1.6 M in n-hexane) was added to a
stirred mixture of 17 (1.45 g, 5.9 mmol) and anhydrous
Et2O (45 mL) at ꢀ78 ꢁC and stirred for 30 min. Then,
DMF (0.5 mL, 7.1 mmol) was added and the whole mix-
ture was stirred at ꢀ78 ꢁC to ambient temperature with-
in 1 h. The whole mixture was poured into 5% NaHCO3
and extracted with Et2O (3· 50 mL). The combined
organic layer was washed with brine, dried, and concen-
trated. The crude product was chromatographed on sil-
ica-gel column using n-hexane/AcOEt (4:1) to give
625 mg of 21 as a colorless oil (54%). 1H NMR (CDCl3):
d 1.79 (s, 3H, CH3), 3.88–4.02 (m, 2H, –OCH2–), 4.10–
4.20 (m, 2H, –OCH2–), 7.80 (d, 1H, J = 7.3 Hz, 5-H),
7.86–7.95 (m, 2H, 3 and 4-H), 10.13 (s, 1H, –CHO);
13C NMR (CDCl3): d 25.18, 65.18, 108.39, 120.56,
123.59, 137.66, 152.65, 161.92, 193.79; MS: m/z 178
(M+–CH3), 150, 134, 107, 87, 78.
4.1.3.3. 6,60-Dimethoxy-2,20-pyridoin (3). Sodium cya-
nide (196 mg, 4 mmol in H2O, 2 mL) was added to a
stirred mixture of 9 (232 mg, 1.7 mmol) and methanol
(2 mL). The mixture was refluxed at 80 ꢁC for 10 min.
The resulting yellow solid was collected by filtration,
washed with methanol and H2O successively, and dried
1
under reduced pressure to afford 3 (65 mg, 28%). H
NMR (DMSO-d6): d 3.94 (s, 6H, –OCH3), 6.86 (d,
2H, J = 8.2 Hz, 5 and 50-H), 7.40 (d, 2H, J = 7.6 Hz, 3
and 30-H), 7.90 (t, 2H, J = 7.9 Hz, 4 and 40-H), 12.3 (s,

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M. Hatanaka et al. / Bioorg. Med. Chem. 13 (2005) 6763–6770
4.1.4.4. 6-Acetyl-2-pyridinecarboxaldehyde (10). Un-
The resulting orange solid was collected by filtration,
washed with methanol and H2O successively, and dried
under reduced pressure to afford 5 (238 mg, 73%). H
der N2 atmosphere, a mixture of 21 (625 mg, 3.2 mmol)
and HCl (3 M, 20 mL) was stirred at 100 ꢁC for 2 h.
After cooling to ambient temperature, the reaction mix-
ture was alkalified with saturated NaHCO3 and extract-
ed with Et2O (3· 50 mL). The combined organic layer
was washed with brine, dried, and concentrated to give
397 mg of 10 as a colorless solid (82%). 1H NMR
(CDCl3): d 2.81 (s, 3H, –COCH3), 8.03 (t, 1H,
J = 7.6 Hz, 4-H), 8.14 (d, 1H, J = 7.6 Hz, 3-H), 8.26
(d, 1H, J = 7.6 Hz, 5-H), 10.13 (s, 1H, –CHO); 13C
1
NMR (DMSO-d6): d 2.36 (s, 6H, –CH3), 7.69 (d, 2H,
J = 8.2 Hz, 3 and 30-H), 7.85 (d, 2H, J = 7.9 Hz, 4 and
40-H), 8.44 (s, 2H, 6 and 60-H), 12.9 (s, 2H, –OH); 13C
NMR (DMSO-d6): d 17.83, 118.45, 131.35, 134.43,
138.82, 146.13, 153.00; MS: m/z 242 (M+), 183, 138,
122, 92, 65; HRMS calcd for C14H14N2O2, 242.1055;
found: 242.1043 (M+); mp: 203–205 ꢁC (pyridine).
NMR (CDCl3):
d
25.63, 124.51, 125.49, 138.10,
4.1.6. Preparation of 5,50-dimethoxy-2,20-pyridoin (6)
4.1.6.1. 5-Methoxy-2-picoline (25). Under argon
atmosphere, 5-hydroxy-2-picoline (24, 5.0 g in 60 mL
DMF) was added to a stirred mixture of sodium hy-
dride (1.8 g, 60% dispersed in oil, 45 mmol) and
DMF (60 mL), and the mixture was heated to
180 ꢁC for 2 h. After cooling to ambient temperature,
methyl iodide (2.9 mL, 46 mmol) was added to the
mixture, which was stirred for 4 h. The reaction mix-
ture was diluted with isopropyl alcohol (20 mL) and
H2O (20 mL), and then was extracted with AcOEt
(3· 100 mL). The combined organic layer was washed
with saturated NaHCO3 and brine successively, dried,
and concentrated. The crude product was chromato-
graphed on silica-gel column using n-hexane/AcOEt
(3:2) to give 4.2 g of 25 as a yellow oil (75%). 1H
NMR (CDCl3): d 2.49 (s, 3H, –CH3), 3.83 (s, 3H, –
OCH3), 7.06 (d, 1H, J = 8.5 Hz, 3-H), 7.11 (dd, 1H,
J = 8.5 Hz, 2.4 Hz, 4-H), 8.19 (d, 1H, J = 2.4 Hz, 6-
H); 13C NMR (CDCl3): d 23.25, 55.54, 121.31,
123.20, 136.13, 150.30, 153.63.
152.13, 153.82, 192.86, 199.16; MS: m/z 149 (M+), 121,
107, 79.
4.1.4.5. 6,60-Diacetyl-2,20-pyridoin (4). Sodium cya-
nide (196 mg, 4 mmol in H2O, 2 mL) was added to a
stirred mixture of 10 (300 mg, 2 mmol) and methanol
(3 mL). The mixture was refluxed at 80 ꢁC for 20 min.
The resulting yellow solid was collected by filtration,
washed with methanol and H2O successively, and dried
under reduced pressure to afford 4 (300 mg, 100%). The
1H NMR spectra of product indicated that the afforded
4 existed as a mixture of E-enediol and Z-enediol (70%
and 30%, respectively). 1H NMR (DMSO-d6): E-enediol
d 2.71 (s, 6H, –COCH3), 8.00 (d, 2H, J = 7.6 Hz, 3 and
30-H), 8.12 (d, 2H, J = 7.6 Hz, 5 and 50-H), 8.26 (t, 2H,
J = 7.9 Hz, 4 and 40-H), 12.9 (s, 2H, –OH), Z-enediol d
2.14 (s, 6H, –COCH3), 8.19 (d, 2H, J = 7.6 Hz, 3 and 30-
H), 8.35 (t, 2H, J = 7.9 Hz, 4 and 40-H), 8.45 (t, 2H,
J = 7.9 Hz, 5 and 50-H), 10.2 (s, 2H, –OH); 13C NMR
(DMSO-d6): as a mixture of E-enediol and Z-enediol d
24.78, 26.08, 120.46, 122.83, 124.81, 125.62, 135.14,
139.93, 139.96, 149.42, 150.02, 150.19, 152.26, 154.63,
196.22, 197.74; MS: m/z 298 (M+), 269, 255, 225, 150,
122, 121, 106, 93, 78; HRMS calcd for C16H14N2O4,
298.0954; found: 298.0966 (M+); mp: 183–185 ꢁC (dec)
(pyridine).
4.1.6.2. 5-Methoxy-2-picoline-N-Oxide (26). Thirty
percent of aqueous H2O2 (1.8 mL, 16 mmol) was added
to a stirred mixture of 25 (2.0 g, 16 mmol) and AcOH
(12 mL). After being stirred for 5 h at 100 ꢁC, the reac-
tion mixture was alkalified with 10% NaOH and extract-
ed with CHCl3 (3· 100 mL). The combined organic
layer was dried and concentrated to give 787 mg of 26
as a yellow semi-solid (35%). No further purification
4.1.5. Preparation of 5,50-dimethyl-2,20-pyridoin (5)
4.1.5.1. 5-Methyl-2-pyridinecarboxaldehyde (11). Un-
der argon atmosphere, n-BuLi (5.4 mL, 8.7 mmol,
1.6 M in n-hexane) was added to a stirred mixture of
6-bromo-3-picoline (23, 1.5 g, 8.7 mmol) and anhydrous
Et2O (35 mL) at –78 ꢁC and stirred for 30 min. Then
DMF (0.8 mL, 11 mmol) was added to the mixture,
which was stirred at –78 ꢁC to ambient temperature
within 1 h. The whole mixture was poured into 5%
K2CO3 and extracted with Et2O (4· 50 mL). The com-
bined organic layer was washed with brine, dried, and
concentrated. The crude product was chromatographed
on silica-gel column using n-hexane/AcOEt (7:1 to 4:1)
1
was performed. H NMR (DMSO-d6): d 2.27 (s, 3H,
–CH3), 3.79 (s, 3H, –OCH3), 6.97 (dd, 1H, J = 8.5 Hz,
1.5 Hz, 4-H), 7.36 (d, 1H, 3-H), 8.08 (d, 1H,
J = 1.5 Hz, 6-H); 13C NMR (DMSO-d6): d 16.28,
56.14, 112.37, 125.94, 126.63, 140.49, 155.91.
4.1.6.3. 5-Methoxy-2-pyridylmetanol (28). Trifluoro-
acetic anhydride (4.5 mL, 32 mmol) was added to a stir-
red mixture of 26 (894 mg, 6.4 mmol) and CH2Cl2
(30 mL) in two portions at 0 ꢁC. After being stirred
for 30 min, the mixture was stirred at ambient tempera-
ture for 24 h. Then, the whole mixture was diluted with
methanol (12 mL), refluxed for 5 h, alkalified with satu-
rated NaHCO3, and extracted with CH2Cl2 (3· 50 mL).
The combined organic layer was washed with brine,
dried, and concentrated. The crude product was chro-
matographed on silica-gel column using AcOEt/metha-
nol (20:1) to give 573 mg of 28 as a yellow paste
(64%). 1H NMR (CDCl3): d 3.45 (br s, 1H, –CH2–
OH) 3.87 (s, 3H, –OCH3), 4.70 (s, 2H, –CH2–OH),
7.18 (d, 1H, J = 8.5 Hz, 3-H), 7.22 (dd, 1H, J = 8.5 Hz,
2.4 Hz, 4-H), 8.25 (d, 1H, J = 2.4 Hz, 6-H); 13C NMR
1
to give 324 mg of 11 as a yellow oil (31%). H NMR
(CDCl3): d 2.45 (s, 3H, –CH3), 7.68 (d, 1H, J = 7.6 Hz,
4-H), 7.88 (d, 2H, J = 7.9 Hz 3-H), 8.62 (s, 1H, 6-H),
10.05 (s, 1H, –CHO); 13C NMR (CDCl3): d 18.81,
121.43, 137.34, 138.61, 150.65, 150.74, 193.17; MS: m/z
121 (M+), 93, 65.
4.1.5.2. 5,50-Dimethyl-2,20-pyridoin (5). Sodium cya-
nide (196 mg, 4 mmol in H2O, 2 mL) was added to a
mixture of 11 (324 mg, 2.7 mmol) and methanol
(3 mL). The mixture was refluxed at 80 ꢁC for 20 min.

M. Hatanaka et al. / Bioorg. Med. Chem. 13 (2005) 6763–6770
6769
1
(CDCl3): d 55.16, 63.79, 121.11, 121.24, 135.40, 152.08,
154.35; MS: m/z 139 (M+), 138, 124, 110, 95, 78, 68.
(87%). H NMR (CDCl3): d 1.65 (s, 3H, –CH3), 3.74–
3.81 (m, 2H, –OCH2–), 4.03–4.10 (m, 2H, –OCH2–),
7.46 (d, 1H, J = 7.9 Hz, 3-H), 7.64 (dd, 2H, J = 8.2 Hz,
2.1 Hz, 4-H); 13C NMR (CDCl3): d 27.52, 64.65,
107.25, 127.57, 135.97, 138.25, 141.62, 147.74; MS: m/z
230 ([M++2]–CH3), 228 ([M+]–CH3), 186, 184, 158,
156, 149, 134, 104, 87, 77.
4.1.6.4. 5-Methoxy-2-pyridinecarboxaldehyde (12).
Under argon atmosphere, MnO2 (2.1 g, 24 mmol) was
added to a stirred mixture of 28 (258 mg 1.9 mmol)
and CH2Cl2 (20 mL). After being stirred for 30 min at
ambient temperature, the whole mixture was filtered
through a Celite pad, and the filtrate was concentrated.
The crude product was chromatographed on silica-gel
column using n-hexane/AcOEt (3:1) to give 153 mg of
12 as a colorless solid (60%). 1H NMR (CDCl3): d
3.96 (s, 3H, –OCH3), 7.31 (dd, 1H, J = 8.5 Hz, 2.4 Hz,
4-H), 7.97 (d, 1H, J = 8.5 Hz, 3-H), 8.44 (d, 1H,
J = 2.4 Hz, 6-H), 10.00 (s, 1H, –CHO); 13C NMR
(CDCl3): d 55.92, 119.97, 123.33, 138.53, 146.39,
158.98, 192.05; MS: m/z 137 (M+), 109, 93, 79, 66
4.1.7.3. 5-(2-Methyl-1,3-dioxolan-2-yl)-2-pyridinecar-
boxaldehyde (22). Under argon atmosphere, n-BuLi
(1.6 M in n-hexane, 3.5 mL, 5.7 mmol) was added to a
stirred mixture of 20 (1.4 g, 5.7 mmol) and anhydrous
Et2O (30 mL) at ꢀ78 ꢁC and stirred for 30 min. Then,
DMF (0.5 mL, 15 mmol) was added to the mixture
and stirred at –78 ꢁC to ambient temperature within
1 h. The reaction mixture was poured into 5% NaHCO3
and extracted with Et2O (3· 50 mL). The combined
organic layer was washed with brine, dried, and concen-
trated. The crude product was chromatographed on sil-
ica-gel column using n-hexane/AcOEt (6:1) to give
4.1.6.5. 5,50-Dimethoxy-2,20-pyridoin (6). Sodium cya-
nide (196 mg, 4 mmol in H2O, 2 mL) was added to a
stirred mixture of 12 (300 mg, 2.2 mmol) and methanol
(3 mL). The mixture was refluxed for 20 min at 80 ꢁC.
The resulting yellow solid was collected by filtration,
washed with methanol and H2O successively, and dried
1
568 mg of 22 as a yellow oil (53%). H NMR (CDCl3):
d 1.69 (s, 3H, –CH3), 3.77–3.83 (m, 2H, –OCH2–),
4.07–4.14 (m, 2H, –OCH2–), 7.93–7.98 (m, 2H, 3 and
4-H), 8.91 (s, 1H, 6-H), 10.09 (s, 1H, –CHO); 13C
NMR (CDCl3) d 27.55, 64.83, 107.42, 121.30, 134.23,
143.43, 147.85, 152.45, 193.06.
1
under reduced pressure to afford 6 (190 mg, 63%). H
NMR (DMSO-d6): d 3.90 (s, 6H, –OCH3), 7.64 (dd,
2H, J = 8.8 Hz, 2.5 Hz, 4 and 40-H), 7.72 (d, 2H,
J = 8.8 Hz, 3 and 30-H), 8.33 (d, 2H, J = 2.5 Hz, 6 and
60-H), 12.5 (s, 2H, –OH); 13C NMR (DMSO-d6): d
55.89, 119.63, 123.40, 133.13, 133.52, 148.46, 153.69;
MS: m/z 274 (M+), 259, 215, 138, 136, 109, 108, 93;
HRMS calcd for C14H14N2O4, 274.0954; found:
274.0918 (M+); mp: 158–159 ꢁC (pyridine).
4.1.7.4. 5-Acetyl-2-pyridinecarboxaldehyde (13). Un-
der N2 atmosphere, a mixture of 22 (314 mg, 1.6 mmol)
and HCl (3 M, 20 mL) was stirred at 100 ꢁC for 10 min.
After cooling to ambient temperature, the reaction mix-
ture was alkalified with saturated NaHCO3 and extract-
ed with Et2O (3· 50 mL). The combined organic layers
were washed with brine, dried, and concentrated to give
117 mg of 13 as a yellow solid (48%). 1H NMR (CDCl3):
d 2.70 (s, 3H, –COCH3), 8.07 (d, 1H, J = 8.2 Hz, 3-H),
8.39 (d, 1H, J = 8.2 Hz, 4-H), 9.31 (s, 1H, 6-H), 10.15
(s, 1H, –CHO); 13C NMR (CDCl3): d 27.07, 121.42,
134.76, 136.74, 150.13, 154.85, 192.51, 195.95; MS: m/z
149 (M+), 134, 121, 106, 78.
4.1.7. Preparation of 5,50-diacetyl-2,20-pyridoin (7)
4.1.7.1. 5-Acetyl-2-bromopyridine (19). Under N2
atmosphere, n-BuLi (7.9 mL, 13 mmol, 1.6 M in n-hex-
ane) was added to a mixture of 2,5-dibromopyridine
(18, 3.0 g, 13 mmol) and anhydrous Et2O (50 mL) at
ꢀ78 ꢁC. The reaction mixture was stirred for 30 min
and then N,N-dimethylacetamide (1.3 mL, 15 mmol)
was added and stirred at –78 ꢁC to ambient temperature
within 1 h. The whole mixture was poured into saturated
NH4Cl and then extracted with Et2O (3· 150 mL). The
combined organic layer was washed with brine, dried,
and concentrated. The crude product was chromato-
graphed on silica-gel column using n-hexane/AcOEt
4.1.7.5. 5,50-Diacetyl-2,20-pyridoin (7). Sodium cya-
nide (98 mg, 2 mmol in H2O, 1 mL) was added to a mix-
ture of 13 (117 mg, 0.78 mmol) and methanol (3 mL).
The mixture was refluxed for 20 min at 80 ꢁC, and the
resulting red-orange solid was collected by filtration,
washed with methanol and H2O successively, and dried
1
1
(7.5:1) to give 1.3 g of 19 as a colorless solid (50%). H
under reduced pressure to afford 7 (15 mg, 13%). H
NMR (CDCl3): d 2.63 (s, 3H, –CH3), 7.62 (d, 1H,
J = 8.5 Hz, 3-H), 8.08 (dd, 1H, J = 8,5 Hz, 2.4 Hz, 4-
H), 8.90 (d, 1H, J = 2.4 Hz, 6-H); 13C NMR (CDCl3):
d 26.72, 128.43, 131.45, 137.65, 146.97, 150.40, 195.57;
MS: m/z 201 (M++2), 199 (M+), 158, 156, 131, 129, 76.
NMR (DMSO-d6): d 2.67 (s, 6H, –COCH3), 7.97 (d,
2H, J = 8.4 Hz, 3 and 30-H), 8.51 (d, 2H, J = 8.4 Hz, 4
and 40-H), 9.20 (s, 2H, 6 and 60-H), 13.3 (s, 2H, –OH);
13C NMR (DMSO-d6): d 26.87, 119.26, 130.11, 136.78,
137.64, 147.21, 157.87, 195.98; MS: m/z 298 (M+), 296,
269, 255, 225, 212, 198, 150, 121, 106, 78; HRMS calcd
for C16H14N2O4, 298.0954; found: 298.0912 (M+); mp:
198–201 ꢁC (dec) (pyridine).
4.1.7.2. 2-Bromo-5-(2-methyl-1.3-dioxolan-2-yl)pyri-
dine (20). A mixture of 19 (1.3 g, 6.3 mmol), ethylene
glycol (1.1 mL, 20 mmol), and p-TsOH–H2O (364 mg,
1.9 mmol), and toluene (25 mL) was stirred for 6 h at
140 ꢁC with a Dean–Stark apparatus. The reaction mix-
ture was washed with saturated Na2CO3 and brine suc-
cessively and concentrated. The crude product was
chromatographed on silica-gel column using n-hexane/
AcOEt (7:1) to give 1.4 g of 20 as a colorless solid
4.2. Measurement of log kw
The measurement procedure is a modification of the
method of Tsantili-Kakoulidou et al.26 Sample solution
contained 1–7 (3 mM) and acetone (0.1 lM) as an unre-
tained substance in CH3CN. The mobile phase was

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M. Hatanaka et al. / Bioorg. Med. Chem. 13 (2005) 6763–6770
made up volumetrically using 41.8 mM MES buffer (pH
7.4), instead of a MOPS buffer, and different propor-
tions of methanol in the range of 85–65%. Two microli-
ters of the sample was injected on an octadecyl-silica-
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*
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Oxidation potential was measured by a Hokuto HA-301
electrochemical apparatus equipped with glassy carbon
as a working electrode, platinum as a counter electrode,
and Ag/AgCl as a reference electrode. The sample
mixture contained 1–7 or AsA (0.5 mM) and tetra-n-
butylammonium hexafluorophosphate (60 mM) as a
supporting electrolyte in the mixture of DMF and
MES buffer, pH 7.4 (3:2, v/v), and it was scanned from
ꢀ300 to 900 mV at 50 mV/s to gain a voltammogram.
4.4. DPPH radical scavenging activity
The measurement procedure is a modification of the
method of Yamaji et al.11 Sample 1–7 in DMSO solu-
tion (1 lM) was added to DPPH (16.7–50 lM) in a
mixture of MES buffer (pH 7.4) and ethanol (3:2,
v/v) at 25 ꢁC. The decrease in absorbance at 517 nm
was recorded on a JASCO V-560 UV–vis spectrometer
for 60 s to gain the pseudo-first order reaction rate
constant. The resulting pseudo first-order reaction
rate constant was converted to a second-order reaction
rate constant by plotting against the initial DPPH
concentration.
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incubation mixtures contained microsomes (0.56 mg
protein) and the indicated amount of sample in a mix-
ture of 680 lL of 0.1 M sodium phosphate buffer (pH
7.4) and 300 lL ethanol. Peroxidation was initiated by
the addition of tert-butyl hydroperoxide (1 mM) and
continued for 15 min at 37 ꢁC. The peroxidation was
terminated by an addition of 100 mL of 2,6-di-tert-
butyl-p-cresol in ethanol (2%). The solution was then
mixed with 2.0 mL of 15% trichloroacetic acid and
0.375% thiobarbituric acid in 0.25 M HCl. After
incubation at 80 ꢁC for 15 min, the precipitate was
removed by centrifugation (3000 rpm, 20 ꢁC, 20 min),
and the difference between the absorbance at 535 nm
and 600 nm was measured to gain a relative TBARS
formation.34
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