Synthesis of selenium-containing polyphenolic acid esters and evaluation of their effects on antioxidation and 5-lipoxygenase inhibition

Article abstract of DOI:10.1248/cpb.53.1402

Six novel selenium-containing polyphenolic acid esters were synthesized and evaluated as antioxidants and 5-lipoxygenase inhibitors. Synthesis of the title compounds involved the Mitsunobu reaction of polyphenolic acids (4-8, 14) with 2-phenylselenoethanol (3). Compounds 22, 23, and 25 were found to be very effective antioxidants and 5-lipoxygenase inhibitors with activity comparable to or better than caffeic acid (3,4-dihydroxycinnamic acid) phenethyl ester (CAPE).

Full text of DOI:10.1248/cpb.53.1402

1402
Chem. Pharm. Bull. 53(11) 1402—1407 (2005)
Vol. 53, No. 11
Synthesis of Selenium-Containing Polyphenolic Acid Esters and
Evaluation of Their Effects on Antioxidation and 5-Lipoxygenase
Inhibition
a
,b
c
c
,a
*
*
Chi-Fu LIN, Tsu-Chung CHANG Chih-Chia CHIANG, Hou-Jen TSAI, and Ling-Yih HSU
a School of Pharmacy, National Defense Medical Center; b Department of Biochemistry, National Defense Medical Center;
P. O. Box 90048–508, Neihu, Taipei: and c Department of Applied Chemistry, Chung Cheng Institute of Technology; Ta-shi,
Tao-yuan, Taiwan, Republic of China. Received May 10, 2005; accepted August 5, 2005
Six novel selenium-containing polyphenolic acid esters were synthesized and evaluated as antioxidants and
5-lipoxygenase inhibitors. Synthesis of the title compounds involved the Mitsunobu reaction of polyphenolic acids
(4—8, 14) with 2-phenylselenoethanol (3). Compounds 22, 23, and 25 were found to be very effective antioxidants
and 5-lipoxygenase inhibitors with activity comparable to or better than caffeic acid (3,4-dihydroxycinnamic
acid) phenethyl ester (CAPE).
Key words polyphenolic acid ester; organoselenium compound; antioxidant; free radical scavenging activity; 5-lipoxygenase
inhibition
redox state and induces apoptosis.14) It has been reported that
Free radicals are highly reactive molecules generated pre-
CAPE suppresses lipid peroxidation,15) displays antioxidant
dominantly during cellular respiration and normal metabo-
activity,16) and inhibits ornithine decarboxylase, protein tyro-
lism. Imbalance between the cellular production of free radi-
sine kinase (PTK), and lipoxygenase activities.17—20) CAPE
cals and the ability of cells to defend against them is referred
can also inhibit phorbol ester-induced H2O2 production and
tumor promotion.21,22)
to as oxidative stress (OS). Biological systems use enzymatic
(superoxide dismutase, glutathione peroxidase, etc.) and
nonenzymatic (uric acid, creatinine, polyamine, retinal, etc.)
antioxidant systems to prevent OS. However, once the sys-
tems are disturbed the uncontrolled oxidative stresses initiate
a series of harmful biochemical events or generate them as a
consequence of earlier tissue injury, thus aggravating the
final damages. Such damages include brain dysfunction, can-
cer, and cardiovascular disease and inflammation.1,2)
Selenium is an essential trace element and has been known
to be intimately involved in the activity of enzymes such as
glutathione peroxidase (GSH Px) and thioredoxin reductase,
which catalyze chemistry essential to the protection of bio-
molecules against OS and free radical damage.3) Although
possessing potent antioxidative activity, like other protein
drugs GSH Px is limited for clinical use due to the instability
and immunogenicity of the endogenous forms. To circum-
vent the intrinsic difficulties of using natural enzymes as
drugs, several attempts have been made to produce synthetic
compounds that mimic the properties of GSH Px. Ebselen,4)
2-phenylbenzisoselenazole-3(2H)-one, was the most success-
ful compound and showed anti-inflammatory, anti-athero-
sclerotic, and cytoprotective activities. These biological ac-
tivities are attributed partly to the strong tendency of sele-
nium to undergo redox reactions. Therefore, in the design of
novel antioxidants as therapeutic agents there are a number
of approaches utilizing the redox properties of selenium and
incorporating selenium into the molecule skeleton. Review
articles have summarized the synthesis and properties of a
considerable number of organoselenium compounds pre-
pared as potential pharmaceuticals.5—7)
In recent years, studies in our laboratory have been di-
rected at synthetic antioxidants with diverse anti-oxidative
functionalities.23—25) Based on the unique anti-oxidative
properties of selenium, we speculated that ester analogues of
polyphenolic acid and phenylselenoethanol, such as 2 (Fig.
1) might provide synergistic antioxidative activity. In this
study, a series of selenium-containing polyphenolic acid es-
ters were prepared and their efficacies as radical scavengers,
antioxidants, and inhibitors of 5-lipoxygenase were investi-
gated as well.
Chemistry
Selenium-containing aliphatic alcohol (2-phenylseleno-
ethanol; 3) was prepared from 2-chloroethanol with phenyl-
slenol produced in situ by a reduction of diphenyl diselenide
with sodium borohydride (Chart 1). Although Fischer esteri-
Fig. 1
Caffeic acid (3,4-dihydroxycinnamic acid) phenethyl ester
(CAPE, 1; Fig. 1), a kind of polyphenolic acid, is an active
component of propolis from honeybee hives. It has antiviral,
anti-inflammatory, and immunomodulatory properties8) and
has been shown to inhibit the growth of different types of
transformed cells.8—13) In transformed cells, CAPE alters the
Chart 1
∗ To whom correspondence should be addressed. e-mail: hly@ndmcsgh.edu.tw
© 2005 Pharmaceutical Society of Japan

November 2005
1403
Chart 2
fication9) of polyphenolic acid and aliphatic alcohol provides
the ester products in one step, alternative synthetic methods
are still needed because the harsh reaction conditions and the
low yield make the esterification of limited applicability. A
general synthetic route for the preparation of target com-
pounds (20—25) is depicted in Chart 2. The phenolic hy-
Table 1. Scavenging Activity of Antioxidants for DPPH· Radicals
Compound
SC50 (mM)a)
Inhibition (%)b)
20
21
22
23
24
610
490
70.5
100
290
76.3
38
5.6
6.7
40.9
27.8
8.9
droxyl groups of polyphenolic acids were protected from com-
mercially available polyphenolic acids (4—8) by acetylation
with acetic anhydride in pyridine in the dark at room temper-
ature because acids activated ex situ (chlorides, anhydrides,
and mixed anhydrides) in the next step showed poor chemo-
selectivity between phenolic and selenium-containing ali-
phatic alcohol. The acids were activated to the corresponding
acid chlorides by reaction of 4—8 with freshly distilled
thionyl chloride in dichlomethane at reflux temperature. The
25
CAPE
36.9
75.3
Data are shown as SC50 (mM) and % inhibition at 50 mM of antioxidants from two in-
dependent experiments. a) SC50, The concentration of test compounds needed to re-
duce DPPH absorption by 50% at 517 nm. The values were calculated from the slope
equation of the dose response curves. Values are means of three independent determi-
nations. b) Inhibition (%), indicates the percent inhibition at 50 mM of antioxidant.
acid chloride intermediates were also obtained by using a Results and Discussion
reagent of oxalyl chloride instead of thionyl chloride. We
The reducing abilities of the examined compounds at dif-
though that oxalyl chloride should be superior to thionyl ferent concentrations were determined by their interaction
chloride because the boiling point of oxalyl chloride (63 °C) with 0.5 mM of the stable free radical 1,1-diphenyl-2-picryl-
is lower than that of thionyl chloride (79 °C), so that after re- hydrazyl (DPPH). Antioxidants can react with DPPH and
action the excess oxalyl chloride is easily removed. Without produce 1,1-diphenyl-2-picryl-hydrazine.27) Due to its odd
purification, the acid chlorides reacted with selenium-con- electron DPPH gives a strong absorption band at 517 nm. As
taining aliphatic alcohol to give the ester products. Removal this electron becomes paired in the presence of a free radical
of protective groups was then achieved in a process initiated scavenger, the absorption vanishes and the resulting decol-
by 3 N HCl hydrolysis of the esters to yield target compounds orization is stoichiometric with respect to the number of
(21—25). Although the synthesis via acid chloride was electrons taken up. The change in absorbance produced in
straightforward, a one-step esterification in which acids react this reaction is assessed to evaluate the antioxidant potential
directly with alcohol is practical because of the troublesome of test samples, and this assay is useful as a primary screen-
protection and deprotection steps. The recent study by Ap- ing system. The free radical scavenging profiles of the six
penedino et al.26) indicated that the Mitsunobu reaction is es- novel selenium-containing polyphenolic acid esters and ref-
pecially suitable for the esterification of phenolic acids and erence compound CAPE are shown in Table 1. The activities
alcohols. Because phenolic carbons are not substrates for the of these compounds, in the order of SC50 values from low to
SN2-type reaction, the Mitsunobu reaction shows good high was CAPEꢀ22ꢀ25ꢀ23ꢀ24ꢀ21ꢀ20. Chen et al.28) re-
chemoselectivity between hydroxyl groups bound to aliphatic ported that the structural requirement of polyphenolic acid
and aromatic carbons. Thus, polyphenolic acids (4—8, 14) for potent scavenging activity were compounds containing an
were reacted directly with 3 in the presence of triphenylphos- o-dihydroxy group; and when the o-dihydroxy group was
phine (TPP), diisopropyl azodicarboxylate (DIAD), and substituted with a methoxy group, the activity decreased dra-
tetrahydrofuran to give the target compounds (20—25).
matically. We also found this trend in activity in our study
(25ꢀ23ꢀ24ꢀ21ꢀ20). It was of interest to note that without
the o-dihydroxy group, 22 also showed an inhibition of free

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Vol. 53, No. 11
Table 2. Antioxidant Activity of the Compounds against 2,2ꢁ-Azobis(2-
amidinopropane) Dihydrochloride (AAPH)-Induced Lipid Peroxidation of a
Tween-Emulsified Linoleic Acid System
Rate of peroxide formation
Compound
Inhibition (%)a)
(DA500/min)
CAPE
22
23
7.6ꢃ10ꢄ4
6.7ꢃ10ꢄ4
1.7ꢃ10ꢄ3
9.3ꢃ10ꢄ4
76.9
87.7
43.3
80.2
25
a) Inhibition of peroxidation (%)ꢂ(1ꢄrate of test compound/rate of control)ꢃ100%.
Peroxidation was initiated by the addition of AAPH (0.1 M) solution to the Tween-emul-
sified linoleic acid mixture. Degree of peroxidation was estimated by measuring ab-
sorption at 500 nm for the formation of complex [Fe(SCN)]2ꢅ. All tests were performed
in triplicate. Data shown here represent the slope of the time course-absorption curves
of each compound analyzed. The control for the assay was carried out identically but in
the absence of the test compound and the slope set as 100%.
Fig. 2. Plot of D0/DA against [Antioxidant]0/[PR]0
Reactions were carried out at room temperature by adding peroxynitrite (12.5 mM) to
tubes containing phosphate buffer (50 mM, pH 7.0) and 50 mM of [PR]0. Results shown
are the mean of two independent experiments.
radicals with activity similar to that of 25.
To verify the antioxidative activity of these compounds,
compounds 22, 23, and 25 were further analyzed for their ca-
pacity to inhibit the lipid peroxidation induced by 2,2ꢁ-azo-
bis(2-amidinopropane) dihydrochloride (AAPH). The in vitro
model using AAPH-induced lipid peroxidation of Tween-
emulsified linoleic acid is a common method29) used to mea-
sure the antioxidative activity of synthetic antioxidants. In
this assay, the oxidation is carried out under conditions rela-
tively similar to in vivo biological systems. The inhibitory ef-
fects on lipid peroxidation or the antioxidative activity of
these compounds are listed in Table 2. The relative rate of
lipid peroxidation initiated by AAPH radical is defined the
antioxidative activity of test compounds. The smaller the rate
of lipid peroxidation, the stronger the antioxidative activity.
As shown in Table 2, the inhibitory activity of these com-
pounds in decreasing order was 22ꢀ25ꢀCAPEꢀ23, and
compounds 22 and 25 exhibited similar anti-oxidative activ-
ity comparable to that of CAPE. Generally, these results are
in accordance with the DPPH radical scavenging activity of
these synthetic analogues.
The increasing evidence for a role of peroxynitrite in bio-
logical processes prompted us to investigate the reaction of
compounds with peroxynitrite. Studies30) also demonstrated
that polyphenolic and flavonoid compounds exhibited effi-
cient peroxynitrite-scavenging activity and prevented oxida-
tion of macromolecules elicited by peroxynitrite. In this
study, the peroxynitrite-scavenging activities were deter-
mined according to the method reported by Balavoine and
Geletii31) and Radi et al.32) Briefly, peroxynitrite induced the
bleaching of Pyrogallol red (PR) dye, which was measured at
550 nm. Consumption of PR in the presence and absence of
the test compounds was measured over a range of peroxyni-
trite concentrations (0—62.5 mM). Figure 2 shows the D0/DA
data for different concentrations of antioxidant plotted
against [antioxidant]0/[PR]0. D0 and DA are the stoichiome-
tries for the reaction of peroxynitrite with PR in the absence
and presence of the tested antioxidant compounds, respec-
tively. kA and kPR are the rate constants for the reaction of
peroxynitrite with the tested antioxidants and PR, respec-
tively. The ratio kA/kPR, which represents the relative antioxi-
dant activities, was calculated from the slope of the straight
line plotting D0/DAꢂagainst [A]0/[PR]0. The greater the ratio
of kA/kPR, the more potent the peroxynitrite-scavenging activ-
ity of the test compounds. In this study, compound 25 exhib-
Table 3. Inhibition of Test Compounds Peroxynitrite-Mediated Oxidation
a)
Test compound
kA/kPR
Activity relative to CAPE
CAPE
22
23
0.25
0.15
0.1
1.00
0.60
0.40
1.36
25
0.34
a) The ratio kA/kPR represents the relative antioxidant activities and was calculated
from the slope of the straight line plotting D0/DA against [A]0/[PR]0 from Fig. 1.
ited greater or comparable kA/kPR values than the standard
compound CAPE (Fig. 2). The peroxynitrite-scavenging po-
tency was in the order of 25ꢀCAPEꢀ22ꢀ23, and is listed in
Table 3. The data shown here for these compounds and
CAPE are similar to the previously published data31) for caf-
feic acid, with kA/kPRꢂ0.29.
Caffeic acid and its synthetic derivatives, including CAPE,
had been shown19,33) to inhibit efficiently the 5-, 12-, and 15-
lipoxygenase activities that are suggested to be involved in
cellular inflammation. To understand the anti-inflammatory
effects of our Se-CAPE analogues, we analyzed the 5-lipoxy-
genase inhibitory activity of these compounds. We showed
that our compounds, similar to CAPE, displayed typical
uncompetitive inhibitory patterns for 5ꢁ-lipoxygenase. As
shown in Fig. 3 with test compounds as inhibitor and
linoleate as the varied-concentration substrate, a series of
parallel lines were obtained, conforming to an uncompetitive
inhibition pattern. A straight line can be obtained by replot-
ting the concentration of the inhibitory test compounds and
the vertical intercept (1/Vmax), and the Ki value was deter-
mined from the horizontal intercept of the replotted figure
and listed in Table 4. The representative inhibitory pattern
and kinetic replot were shown in Fig. 3 for CAPE and com-
pound 25. Compounds 22 and 23 displayed almost identical
results to that of compound 25 or CAPE. Our results demon-
strate that compounds 22, 23, and 25 exhibit an inhibitory
constant (Ki) against 5ꢁ-lipoxygenase that is similar to that of
CAPE and the IC50 are almost identical to those of caffeic
acid derivatives as described.33) The uncompetitive inhibitory
patterns of these series of compounds demonstrate that they
are nonspecific antioxidants. Caffeic acid derivatives were
shown to be effective inhibitors of 5-, 12-, and 15-lipoxyge-

November 2005
1405
better than those of CAPE. These three compounds also had
inhibitory activity and acted in an uncompetitive manner on
5-lipoxygenase. The role of selenium in the enhanced activi-
ties on inhibiting AAPH-induced lipid peroxidation and per-
oxynitrite-scavenging activities requires further investigation.
Experimental
Melting points (mp) were recorded on a BUCHI 530 apparatus and are
uncorrected. Merck Art No.105554 plates precoated with Silica gel 60 con-
taining a fluorescent indicator were used for thin-layer chromatography, and
Silica gel 60 (Merck Art No 109385, 230—400 mesh) was employed for
column chromatography. Evaporations were carried out at ꢆ50 °C using a
rotary evaporator at reduced pressure (water aspirator). 1H- and 13C-NMR
spectra were obtained with a Varian 300 NMR spectrometer at 300 and
75 MHz, respectively. Where necessary, deuterium exchange experiments
were used to obtained proton shift assignments. Mass spectra were recorded
on a JEOL J.M.S-300 spectrophotometer. Analytical samples were dried
under reduced pressure at 78 °C in the presence of P2O5 for at least 12 h un-
less otherwise specified. Elemental analyses were obtained using a Perkin-
Elmer 2400 Elemental Analyzer.
2-Phenylselenoethanol (3) Sodium borohydride (0.5 g, 15 mmol) was
added in portions to a stirred solution of diphenyl diselenide (1.5 g, 5 mmol)
in absolute ethanol at 0 °C. To the resulting colorless solution was added a
solution of the appropriate 2-chloroethanol (0.7 ml, 10 mmol) dissolved in
the minimum quantity of ethanol. The mixture was stirred under reflux for
3 h. Solvent was filtered and removed in a vacuum. The residue was purified
by flash chromatography on silica gel with n-hexane/ethyl acetate (4/1) to
give 2-phenylselenoethanol (0.96 g, 96%) as pale yellow oil: Rf: 0.2 (n-
hexane/ethyl acetateꢂ4/1). 1H-NMR (DMSO-d6) d: 7.487.21 (m, 5H, ArH),
4.95 (s, 1H, OH), 3.60 (t, 2H, Jꢂ6.4 Hz, CH2), 3.00 (t, 2H, Jꢂ14.2 Hz,
SeCH2). 13C-NMR (DMSO-d6) d: 131.2, 130.2, 129.2, 126.4, 60.7, 29.4.
FAB-MS m/z 202 (MꢅHꢅ). Anal. Calcd for C8H10OSe: C, 47.77; H, 5.01.
Found: C, 47.67; H, 5.03.
Fig. 3. Inhibition of 5-Lipoxygenase by CAPE and Compound 25
Enzymatic activity was assayed as described in Experimental except that linoleate
and test compounds were varied as indicated. From bottom to top, the test compound
concentrations were as indicated. The inset shows the vertical intercept replotted
against concentrations of test compounds. Compounds 22 and 23 exhibited identical in-
hibitory patterns and kinetic parameters against 5ꢁ-lipoxygenase as shown in this figure.
Acetylation of Phenols of Polyphenolic Acid, General Procedure to
Obtain Compounds 9—13 To a solution of polyphenolic acid (20 mmol)
was added acetic anhydride (6 eq) and pyridine (2 ml). The mixture was
stirred at room temperature in the dark for 4 h and then poured onto 1 M
H3PO4 (10 ml) cold solution. The mixture was extracted with ethyl acetate.
The layers were washed with brine and aqueous saturated sodium bicarbon-
ate. The combined organic phase was dried under magnesium sulfate, fil-
tered, and the solvent was removed under a vacuum. The residue was crys-
tallized from proper solvent to afford the corresponding acetoxy polypheno-
lic acids: 2,4-diacetoxy benzoic acid (9), 2,5-diacetoxy benzoic acid (10),
3,4-diacetoxy benzoic acid (11), 3,5-diacetoxy benzoic acid (12), and 3,4-di-
acetoxy cinnamic acid (13). These acetoxy polyphenolic acids were identi-
Table 4. IC50 and Ki Values of the Iinhibitory Effects against 5-Lipoxyge-
nase
Compound
IC50 (mM)a)
Ki (mM)b)
22
23
25
0.18
0.21
0.24
0.22
0.27
0.29
0.22
0.30
CAPE
1
fied by melting point and H-NMR. Data were in agreement with literature
a) The test compound concentration that gave 50% inhibition of enzyme activity.
b) The horizontal intercept was obtained from the plot of inhibitor concentrations
against 1/Vmax, as shown in the inset of Fig. 2.
values.34,35)
Esterification of Acetoxy Polyphenolic Acids with 2-Phenylseleno-
ethanol via Thionyl Chloride, General Procedure to Obtain Compounds
15—20 To a solution of acetoxy polyphenolic acid (4 mmol) in dry
dichlomethane (20 ml) was added thionyl chloride (5 ml) and the mixture
was refluxed for 3 h. The mixture was concentrated, and was added dry
dichlomethane (20 ml), dimethylamino pyridine (0.1 eq), pyridine (1 ml),
and 2-phenylselenoethanol (1.2 eq) were added at 0 °C. The reaction mixture
was stirred for 3 h and poured onto ice water. The mixture was extracted
three times with ethyl acetate. The organic layers were washed with brine
and water. The combined organic phase was dried under magnesium sulfate,
filtered, and the solvent was removed in a vacuum. Without purification, the
residue was used for the next reaction.
nases.19) Therefore, these compounds are lipoxygenase in-
hibitors with antioxidant activity and with action mecha-
nisms similar to that of CAPE. Based on this point, it is justi-
fied to observe that these compounds showed almost identi-
cal inhibitory constants and IC50 values.
Conclusion
Interest in the health-promoting effects of polyphenolic
acid esters, including CAPE, and the unique chemopreven-
tive properties of selenium prompted us to prepare ester ana-
logues of polyphenolic acid and phenylselenoethanol and de-
termine their antioxidant activities. We measured the antioxi-
dant capacity of the six test compounds in terms of their ef-
fects on DPPH free radical scavenging, AAPH-induced lipid
peroxidation as well as scavenging of peroxynitrite radical.
The inhibitory effects of these compounds against 5-lipoxy-
genase were also analyzed in addition to the antioxidative ac-
tivity. Among the compounds analyzed, 22, 23, and 25 are
Esterification of Acetoxy Polyphenolic Acids with 2-Phenylseleno-
ethanol via Oxalyl Chloride, General Procedure to Obtain Compounds
15—20 To a solution of acetoxy polyphenolic acid (5 mmol) and in dry
dichlomethane (10 ml), oxalyl chloride (0.7 ml) was added and the mixture
was stirred for 8 h at room temperature. The mixture was concentrated, and
dry dichlomethane (10 ml), triethylamine (5 ml), and 2-phenylselenoethanol
(1.2 eq) were added at 0 °C. The reaction mixture was stirred over night and
then poured onto ice water. The mixture was extracted three times with ethyl
acetate. The combined organic phase was dried under magnesium sulfate,
filtered, and the solvent was removed in a vacuum. Without purification, the
residue (15—19) was used for the next reaction.
3,4-Dimethoxy-cinnamic Acid-(2-phenylseleno-ethyl ester) (20) The
residue was purified by flash chromatography on silica gel with CH2Cl2/n-
the most potent, with antioxidant activities comparable to or hexane (9/1) to give the product as a pale yellow powder in a yield of 79%:

1406
Vol. 53, No. 11
Rf: 0.34 (CH2Cl2/n-hexaneꢂ9/1). mp 113—115 °C. 1H-NMR (DMSO-d6):
d: 7.54—7.19 (m, 8H, ArH), 6.97 (d, 1H, Jꢂ9.9 Hz, COCH), 6.48 (d, 1H,
Jꢂ15.9 Hz, CH), 4.33 (t, 2H, Jꢂ12 Hz, COOCH2), 3.78 (s, 6H, CH3), 3.22
(t, 2H, Jꢂ13.8 Hz, SeCH2). 13C-NMR (DMSO-d6): d: 166.3, 151.2, 149.1,
145.0, 131.7, 129.5, 126.9, 123.0, 115.3, 111.8, 110.7, 63.3, 55.8, 25.0. IR
(KBr) cmꢄ1: 2954, 1701, 1134. UV lmax (MeOH) nm (log e): 207 (4.26).
FAB-MS m/z: 392 (MꢅHꢅ). Anal. Calcd for C15H14O4Se: C, 58.32; H, 5.15.
Found: C, 58.15; H, 5.14.
Hydrolysis of the Acetate of the Polyphenolic Acid Esters, General
Procedure to Obtain Compounds 21—25 A solution of the polyphenolic
acid esters (5 mmol) was dissolved in a mixture of THF (30 ml) and aqueous
3 N HCl (30 ml) at room temperature. The reaction mixture was stirred for
2 d at room temperature. The mixture was extracted three times with ethyl
acetate. The organic layers were washed with brine and water. The combined
organic phase was dried under magnesium sulfate, filtered, and the solvent
was removed in a vacuum to give the residue.
Mitsunobu Esterification of Acetoxy Polyphenolic Acids with 2-
Phenylselenoethanol To a solution of polyphenolic acids (6 mmol) and 2-
phenylselenoethanol (6 mmol) in dry tetrahydrofuran (15 ml) were added
TPP (6 mmol) and DIAP (6 mmol) at 0 °C. After stirring at room tempera-
ture for 48 h, the reaction was worked up by removal of the solvent, and the
residue was partitioned between ethyl acetate and saturated NaHCO3. The
organic phase was washed with brine and then dried over Na2SO4, and the
solvent was evaporated. The residue was purified by flash chromatography
on a silica gel column.
2,4-Dihydroxy-benzoic Acid-(2-phenylseleno-ethyl ester) (21) The
residue was purified by flash chromatography on silica gel with dichlo-
methane/n-hexane (2/1) to give the product as pale yellow oil in a yield of
68%: Rf: 0.27 (dichlomethane/n-hexaneꢂ2/1). 1H-NMR (DMSO-d6) d:
10.51 (s, 1H, OH), 10.42 (s, 1H, OH), 7.54—6.26 (m, 8H, ArH), 4.45 (t, 2H,
Jꢂ13.2 Hz, COOCH2), 3.29 (t, 2H, Jꢂ10.2 Hz, SeCH2). 13C-NMR (DMSO-
d6) d: 168.8, 164.4, 162.8, 131.8, 129.4, 126.9, 108.4, 103.9, 102.5, 64.1,
24.9. IR (KBr) cmꢄ1: 3416, 3090, 1666, 1095. UV lmax (MeOH) nm (log e):
208 (4.41). FAB-MS m/z: 338 (MꢅHꢅ). Anal. Calcd for C15H14O4Se: C,
53.42; H, 4.18. Found: C, 53.75; H, 4.51.
2,5-Dihydroxy-benzoic Acid-(2-phenylseleno-ethyl ester) (22) The
residue was purified by flash chromatography on silica gel with n-
hexane/dichlomethane/ethyl acetate (4/1/1) to give the product as pale yel-
low oil in a yield of 56%: Rf: 0.55 (n-hexane/dichlomethane/ethyl acetateꢂ
4/1/1). 1H-NMR (CDCl3) d: 7.63—6.88 (m, 8H, ArH), 4.61 (t, 2H, Jꢂ
13.8 Hz, COOCH2), 3.26 (2 t, H, Jꢂ17.1 Hz, SeCH2). 13C-NMR (CDCl3) d:
169.2, 155.8, 147.7, 133.1, 129.3, 129.2, 127.4, 127.3, 125.1, 118.4, 114.6,
64.7, 25.2. IR (KBr) cmꢄ1: 3425, 3120, 1676, 1076. UV lmax (MeOH) nm
(log e): 213 (4.35). FAB-MS m/z: 338 (MꢅHꢅ). Anal. Calcd for
C15H14O4Se: C, 53.42; H, 4.18. Found: C, 53.08; H, 4.30.
3,4-Dihydroxy-benzoic Acid-(2-phenylseleno-ethyl ester) (23) The
residue was purified by flash chromatography on silica gel with n-
hexane/ethyl acetate/acetic acid (70/29/1) to give the product as pale yellow
powder in a yield of 50%: Rf: 0.33 (n-hexane/ethyl acetate/acetic acidꢂ
70/29/1). mp 90—92 °C. 1H-NMR (CDCl3) d: 7.61—6.89 (m, 8H, ArH),
4.52 (t, 2H, Jꢂ14.4 Hz, COOCH2), 3.23 (t, 2H, Jꢂ13.2 Hz, SeCH2). 13C-
NMR (CDCl3) d: 166.0, 156.2, 148.8, 132.9, 129.1, 127.1, 123.6, 116.5,
114.6, 64.0, 25.6. IR (KBr) cmꢄ1: 3253, 3023, 1738, 1119. UV lmax
(MeOH) nm (log e): 206 (2.84). FAB-MS m/z: 338 (MꢅHꢅ). Anal. Calcd
for C15H14O4Se: C, 53.42; H, 4.18. Found: C, 53.13; H, 4.02.
56.21; H, 4.44. Found: C, 56.01; H, 4.08.
Determination of the Scavenging Effect on 1,1-Diphenyl-2-pycryl-hy-
drazyl Radical (DPPH·) The ethanolic solution of DPPH·27) was added
to 2 ml of the test compounds at different concentrations in ethanol (12.5,
25, 37.5, 50 mM). Each mixture was then shaken vigorously and kept for
30 min at room temperature in the dark. The decrease in absorption of
DPPH· at 517 nm was measured. Ethanol was used as a blank solution and
DPPH· solution in ethanol served as the control. The percentage of remain-
ing DPPH· was then calculated, and the radical-scavenging effects of the
test compounds were compared in terms of IC50 (the concentration needed to
reduce 50% of the initial amount of DPPH· and expressed as the molar ratio
of each compound to the radical). All tests were performed in triplicate.
Determination of Antioxidative Activity The antioxidative activity
was evaluated using AAPH-induced lipid peroxidation of a Tween-emulsi-
fied linoleic acid system and measured by the ferric thiocyanate assay as de-
scribed.28) Briefly, 0.2 ml of distilled water, 0.5 ml of 0.2 M phosphate buffer
(pH 7.0), and 0.5 ml of 0.25% Tween-20 (in buffer solution) were mixed
with 0.5 ml of 2.5% (w/v) linoleic acid in ethanol. The mixture was then
stirred for 1 min. The peroxidation was initiated by the addition of 50 ml of
AAPH solution (0.1 M). The stock solution of antioxidant or test compounds
in DMSO (final concentrations for the test compounds and DMSO are
10ꢄ4 M and 0.1%, respectively) was then added, and the reaction was carried
out at 37 °C for 375 min in the dark. The degree of inhibition of oxidation
was measured by the ferric thiocyanate method for each interval of 75, 150,
225, 300, and 375 min. To 0.1 ml of peroxidation reaction mixture at each
interval, 0.1 ml of 30% ammonium thiocyanate and 0.1 ml of 2ꢃ10ꢄ2
M
freshly prepared FeCl2 (in 3.5% aqueous HCl) were added. Precisely 3 min
after addition, the absorbance of the red complex [Fe(SCN)]2ꢅ was mea-
sured at 500 nm. The control for the assay was prepared in the same manner
by mixing all of the chemicals and reagents except the test compound. All
tests were performed in triplicate.
Determination of the Scavenging Effect on Peroxynitrite Peroxyni-
trite synthesis was carried out as described by Radi et al.32) Briefly, acidified
hydrogen peroxide (1 M in 0.7 M HCl, 20 ml) and sodium nitrite (0.2 M,
20 ml) solution were drawn into two separate syringes. The contents of both
syringes were simultaneously injected into an ice-cooled beaker containing
1.5 M potassium hydroxide (40 ml). Manganese dioxide was added to the so-
lution to remove excess hydrogen peroxide. The solution was filtered and the
concentration of the resulting stock was determined spectrophotometrically
at 302 nm (eꢂ1670 Mꢄ1 cmꢄ1). The typical yield of freshly prepared peroxy-
nitrite was 30 mM. The peroxynitrite was diluted in 0.1 M NaOH. Experi-
ments were conducted at 25 °C in 50 mM phosphate-buffered saline contain-
ing 1 mM diethylenetriaminepentaacetic acid, 90 mM NaCl, and 5 mM KCl,
pH 7.4. Blanks using DMSO alone in the absence of test compounds and
peroxynitrite allowed to degrade for 5 min in phosphate-buffered saline, pH
7.4, were also examined. There was no interference by DMSO and degraded
peroxynitrite on the PR. Peroxynitrite induced the bleaching of PR dye,
which was measured at 542 nm (eꢂ24000 Mꢄ1 cmꢄ1). Consumption of PR
(50 mM) in the presence and absence of test compounds (1.25—125 mM) was
measured over a range of peroxynitrite concentrations (0—62.5 mM). Antiox-
idative activities were determined according to the methods reported by Bal-
avoine et al.31) The ratios of rate constants kA/kPR, which represent the rela-
tive antioxidant activities, were determined by plotting D0/DA against [an-
tioxidant]0/[PR]0. kA and kPR are the rate constants for reaction of peroxyni-
trite with the antioxidants and PR, respectively. D0 and DA are the stoi-
chiometries for the reaction of peroxynitrite with PR in the absence and
presence of the antioxidant compounds, respectively.
Assay of 5ꢀ-Lipoxygenase Activity The rate of the 5ꢁ-lipoxygenase-cat-
alyzed formation of linoleic acid hydroperoxides was monitored spectropho-
tometrically at 234 nm.19) The reaction was started by addition of the sub-
strate to the otherwise complete assay mixture. The standard reaction mix-
ture contained 0.27 mg/ml of 5-lipoxygenase, 100 mM of ammonium linolete,
and 50 mM sodium phosphate, pH 6.8. The inhibition study was performed
by adding CAPE and its structural analogues to the reaction mixture, and the
resulting enzyme activity was compared with that of the control without in-
hibitor added. Inhibition with the test compounds was analyzed by varying
the concentration of linoleate substrate from 0.05 to 0.25 mM, and test com-
pound from 0 to 1.25 mM. Concentrations of other components were held
constant.
3,5-Dihydroxy-benzoic Acid-(2-phenylseleno-ethyl ester) (24) The
residue was purified by flash chromatography on silica gel with n-hexane/
ethyl acetate (9/1→4/1→1/1) to give the product as pale yellow oil in a yield
of 52%: Rf: 0.45 (n-hexane/ethyl acetateꢂ1/1). 1H-NMR (CDCl3) d: 7.62—
6.99 (m, 8H, ArH), 4.55 (t, 2H, Jꢂ14 Hz, COOCH2), 3.24 (t, 2H, Jꢂ14 Hz,
SeCH2). 13C-NMR (CDCl3) d: 165.9, 157.0, 156.4, 153.0, 133.0, 132.0,
129.1, 127.1, 108.9, 107.5, 64.5, 25.4. IR (KBr) cmꢄ1: 3410, 3080, 1650,
1080. UV lmax (MeOH) nm (log e): 207 (4.10). FAB-MS m/z: 338 (MꢅHꢅ).
Anal. Calcd for C15H14O4Se: C, 53.42; H, 4.18. Found: C, 53.49; H, 4.11.
3,4-Dihydroxy-cinnamic Acid-(2-phenylseleno-ethyl ester) (25) The
residue was purified by flash chromatography on silica gel with n-hexane/
ethyl acetate (9/1→4/1→1/1) to give the product as pale yellow powder in a
yield of 57%: Rf: 0.45 (n-hexane/ethyl acetateꢂ1/1). mp 70—72 °C. 1H-
NMR (CDCl3) d: 7.61—7.09 (8m, H, ArH), 7.01 (d, 1H, Jꢂ10.2 Hz,
COCH), 6.90 (d, 1H, Jꢂ10.2 Hz, CH), 4.45 (t, 2H, Jꢂ14.7 Hz, COOCH2),
3.19 (2H, t, Jꢂ14.7 Hz, SeCH2). 13C-NMR (CDCl3) d: 166.9, 156.3, 146.4,
145.0, 144.0, 132.9, 129.1, 127.5, 127.2, 122.3, 115.4, 115.2, 114.2, 63.7,
Acknowledgments This study was supported by research grants from
the National Science Council (NSC) and the National Defense Medical Cen-
25.5. IR (KBr) cmꢄ1: 3251, 2979, 1737, 1109. UV lmax (MeOH) nm (log e): ter of Taiwan, Republic of China (DOD 93-1-11 and NSC 92-2320-B-016-
209 (3.15). FAB-MS m/z: 364 (MꢅHꢅ). Anal. Calcd for C15H14O4Se: C,
048 to L. Y. Hsu and NSC 93-NU-7-016-003 and DOD-93-17 and to T.-C.

November 2005
1407
Chang). We thank Ms. Shu-Yun Sun and Ching-Wei Lu, Instrumentation
Center, NSC, for their help in obtaining the mass spectra and elemental
analysis.
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