Significant enhancement in radical-scavenging activity of curcuminoids conferred by acetoxy substituent at the central methylene carbon

Article abstract of DOI:10.1016/j.bmc.2011.04.055

For a compound to be a radical-trapping antioxidant, the antioxidant-derived radical must be sufficiently inert to molecular oxygen as this would generate harmful chain-propagating peroxyl radicals. Curcumin has a unique structure with phenolic hydroxyl g

Full text of DOI:10.1016/j.bmc.2011.04.055

Bioorganic & Medicinal Chemistry 19 (2011) 3793–3800
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Significant enhancement in radical-scavenging activity of curcuminoids
conferred by acetoxy substituent at the central methylene carbon
⇑
Mi Kyoung Kim , Wooseong Jeong , Jihoon Kang, Youhoon Chong
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
For a compound to be a radical-trapping antioxidant, the antioxidant-derived radical must be sufficiently
inert to molecular oxygen as this would generate harmful chain-propagating peroxyl radicals. Curcumin
has a unique structure with phenolic hydroxyl group as well as b-diketone moiety in the same molecule,
both of which are able to donate electrons to free radicals. However, due to the reactivity toward molec-
ular oxygen, the carbon-centered radical derived from b-diketone moiety do not serve as radical-trapping
antioxidants. In this study, we reasoned that stabilization of the carbon-centered radical through substi-
tution with an electron-withdrawing group would enhance the radical-scavenging antioxidative activity
of the resulting curcuminoids. Thus, various substituents (methyl, allyl, methoxy, xanthate, and acetoxy)
covering broad spectrum of the polar substituent effect were introduced to the central methylene posi-
tion of both phenolic and non-phenolic curcuminoids. With the free phenolic hydroxyl groups present,
the methylene-substituent did not exert significant effect on the antioxidant activity of the curcuminoids
Received 14 March 2011
Revised 28 April 2011
Accepted 29 April 2011
Available online 6 May 2011
Keywords:
Radical-scavenging activity
Curcuminoids
Carbon-centered radical
Acetoxy
(EC50 = 23.2–30.3
lM) with the exception of the acetoxy-substituted derivative (EC50 = 8.7
lM) which
showed more potent activity than curcumin (EC50 = 22.6
lM). When substituted to the non-phenolic cur-
cumin scaffold, however, the methylene-substituent enhanced antioxidant activity of the otherwise inac-
tive curcuminoids in the increasing order of methyl < allyl < methoxy < xanthate ꢀ acetoxy, which is well
correlated with the polar inductive effects of the substituents.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
antioxidant activity of curcumin is mainly attributed to its free
phenoxyl group. Formation of the phenoxy radical is dependent
Free radical-induced chain reaction of oxidation has been impli-
cated to play a major role in progression of pathological distur-
bances, which eventually leads to cardiovascular disease, cancer,
Alzheimer’s disease, and Parkinson’s disease. Antioxidants break
the chain reaction of oxidation by giving up their own electrons
to free radicals. Curcumin (diferuloylmethane, 1; Fig. 1), a natural
yellow pigment derived from rhizomes of Curcuma longa, is en-
dowed with typical radical-scavenging as well as chain-breaking
antioxidant activity by its characteristic structure with both phenol
and b-diketone moieties in the same molecule. Phenols and b-dike-
tones are both able to donate electrons to free radicals but, unlike
phenoxyl radicals, most carbon-centered radicals including the one
derived from b-diketone moiety do not serve as radical-trapping
antioxidants due to their instability and reactivity to molecular
oxygen to create harmful chain-propagating peroxyl radicals.1
For this reason, the radical-scavenging as well as chain-breaking
upon the reaction conditions and proceed either through direct
hydrogen atom transfer (HAT) of the phenolic hydrogen to a reac-
tive radical source, or by way of more efficient sequential proton
loss electron transfer (SPLET) mechanism2 which includes sequen-
tial events of (i) proton loss from the central b-methylene to form
an enolate anion, (ii) electron transfer to a reactive radical source
to form a carbon-centered radical, (iii) second proton loss from
the phenolic hydroxyl group, and finally, (iv) electron transfer from
the resulting phenoxide ion to carbon-centered radical to form a
stable phenoxy radical.2 Thus, the free phenolic hydroxyl group is
considered to be a prerequisite for the radical-trapping antioxidant
activity of the curcuminoids, and this claim has been supported by
many studies in which non-phenolic compounds showed no anti-
oxidant activity.3–7
However, recently, Weber et al., made unprecedented report on
the antioxidant activity of the non-phenolic curcuminoids.8 The
curcumin analogues with no aromatic substituent but an alkyl
(methyl or ethyl) group attached to the central methylene moiety
(2 and 3, Fig. 1) showed significant antioxidant activity, and the
authors claimed that formation of a stable tertiary carbon-centered
radical was responsible for this unique property.8 Related to this
⇑
Corresponding author.
E-mail address: chongy@konkuk.ac.kr (Y. Chong).
These two authors contributed equally on this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.055

3794
M. K. Kim et al. / Bioorg. Med. Chem. 19 (2011) 3793–3800
OH
O
O
OH
MeO
HO
OMe
R
OH
Curcumin (1)
2
3
R = Me
R = Et
Figure 1. Structures of curcumin (1) and non-phenolic curcuminoids with antioxidant activity (2 and 3).
issue, it is worth to note that stable carbon-centered radicals,
especially the resonance-stabilized ones, exhibit attenuated reac-
tivity toward oxygen.9–11 Therefore, it can be anticipated that the
otherwise unstable reactive radicals derived from b-diketone moi-
ety might serve as a radical-trapping antioxidant by introduction
of a substituent stabilizing the carbon-centered radical. Unfortu-
nately, studies on the curcuminoids with a substituent at the cen-
tral methylene moiety have been focused as anticancer, antiviral,
or antibacterial agents but their antioxidant activity has rarely
been reported.12–15 Thus, the lack of systematic study of the curc-
uminoids with central methylene-substituents prompted us to
study the substituents’ effect on the antioxidant activity of the cor-
responding curcuminoids. In this study, various substituents
(methyl, allyl, methoxy, xanthate, and acetoxy) covering broad
spectrum of the polar substituent effect as well as radical stabiliz-
ing effect were introduced to the central methylene position of
both phenolic and non-phenolic curcuminoids, and their antioxi-
dant activity was evaluated by DPPH (1,1-diphenyl-2-pic-
rylhydrazyl) radical-scavenging activity.16
tuted acetylacetones (6d) were obtained by substitution of 3-chlo-
roacetylacetone (5) with sodium acetate and potassium xanthate,
respectively (Scheme 1).
A series of curcuminoids with no methylene-substituents (7)
were prepared by condensation of the commercially available ace-
tylacetone (4) with various aromatic aldehydes (Scheme 2). Like-
wise, condensation of the acetylacetone derivatives obtained
above (6, Scheme 1) with aromatic aldehydes provided the curc-
uminoids with various methylene-substituent (8, Scheme 2).
Briefly, the acetylacetones (4 and 6) were treated with boric
anhydride (B2O3) in the presence of tributylborate to provide the
corresponding acetylacetone–boron complexes which were al-
lowed to react with variously substituted aromatic aldehydes
(ArCHO) using 1,2,3,4-tetrahydroquinoline as a catalyst.7 The free
curcuminoids (7 and 8, Scheme 2) were then obtained after decom-
position of the resulting boron complex by aqueous AcOH.
2.2. DPPH radical-scavenging activity
The ability to scavenge stable 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical is a widely used and accurate method to evaluate
antioxidant activity.21,22 The radical-scavenging activity of the
curcuminoid prepared in this study was evaluated by monitoring
reduction of light absorption at 517 nm by DPPH radical upon reac-
tion with curcuminoids in methanol solution (Fig. 2), and, after a
plateau has been reached, the radical-scavenging activity of the
curcuminoids was characterized by their EC50 values (Table 1).
2. Results and discussion
2.1. Chemistry
As an aldol-type condensation of acetylacetone and vanillin is
known to be the most efficient method for preparation of curcu-
min,17 substituted acetylacetones (6, Scheme 1) are required for
preparation of curcuminoids with substituents at the central meth-
ylene unit. In this study, in order to study the electronic effect of
the methylene-substituents on the antioxidant activity of the curc-
uminoids, functionalities such as methyl, allyl, methoxy, xanthate,
and acetoxy were introduced to the acetylacetone scaffold
(Scheme 1). Thus, nucleophilic substitution of the acetylacetone
(4) provided alkyl- (6a and 6b)18 and methoxy- (6c)19 substituted
acetylacetones, whereas the acetoxy- (6e)20 and xanthate-substi-
As anticipated, curcumin (7a, EC50 = 22.6
l
M) and a curcumi-
2,3-dihydroxy-4-methoxybenzene moiety (7d,
M) showed potent radical-scavenging activity
noid with
EC50 = 10.4
a
l
(Fig. 2a, Table 1), whereas the non-phenolic curcuminoids (7b–
7c, 7f–7g) failed to reduce the amount of DPPH radical even at con-
centration of 100 lM (Fig. 2a, Table 1). Interestingly, the curcumi-
noid with a monohydroxyphenyl moiety (7e) was not an active
radical scavenger (Fig. 2a, Table 1), and this result indicates that,
in addition to the phenolic hydroxyl group, the adjacent methoxy
(7a) or hydroxyl (7d) group is also required for formation of the
stable phenoxy radical which is believed to play the critical role
in radical-scavenging activity of the curcuminoids.3–7 The struc-
ture–activity relationship presented above indicates that the phe-
nolic hydroxyl group is prerequisite, but alone insufficient, for
radical-scavenging effect of the curcuminoids.
The methylene-substituents such as alkyl (8a, 8b, 8e, and 8f),
methoxy (8c, 8g), and xanthate (8d, 8h) did not change the nature
of the radical-scavenging activity of the curcuminoids, which is
highly dependent on the phenolic hydroxyl group (Fig. 2b). Thus,
the radical-scavenging activity of the methylene-substituted curc-
uminoids with dimethoxyphenyl moiety (8e–8h) was significantly
lower than that of the corresponding analogues with free phenolic
hydroxyl groups (8a–8d) (Fig. 2b, Table 1). Nevertheless, slight to
moderate increase in radical-scavenging activity of the non-pheno-
lic methylene-substituted curcuminoids (8e–8h) compared to
their unsubstituted counterpart (7b) should be noted (Fig. 2b).
More interestingly, the radical-scavenging activity was well corre-
lated with the polar inductive effect of the substituent, in which 8g
with electron-withdrawing methoxy group (Hammett constant,
O
O
O
O
a
4
R1
R1 = Me
6b R1 = CH2CH=CH2
6a
6c
R1 = OMe
O
O
O
O
b
R1
Cl
5
6d
6e
R
R
1 = SC(S)OEt
1 = OAc
Scheme 1. Synthesis of substituted acetoacetates. Reagents and conditions: (a) for
6a, MeI, K2CO3, acetone, reflux, 6 h; for 6b, allyl bromide, K2CO3, acetone, reflux, 6 h;
for 6c, PhI(OAc)2, BF3ꢁOEt2, MeOH, rt, 5 h; (b) for 6d, KSC(S)OEt, acetone, rt, 4 h; for
6e, NaOAc, DMSO, rt, 3 h.

M. K. Kim et al. / Bioorg. Med. Chem. 19 (2011) 3793–3800
3795
O
O
R2
R3
R2
R3
R1
R4
R4
O
O
a
R1
O
OH
4
R1 = H
R1 = Me
R2
R3
R2
R3
6a
6b
6c
R1
R
R
1 = CH2CHCH2
1 = OMe
R4
R4
6d R1 = SC(S)OEt
6e
7a
R1 = H,
R2 = OMe, R3 = OH,
R2 = OMe, R3 = OMe, R4 = H
R2 = OMe, R3 = OMe, R4 = OMe
R4 = H
R
1 = OAc
7b R1 = H,
7c
7d
7e
7f
R
R
R
1 = H,
1 = H,
1 = H,
R
R
R
R
2 = OH,
2 = H,
2 = H,
2 = H,
R3 = OH,
R3 = OH,
R3 = OMe,
R3 = H,
R
R
R
R
4 = OMe
4 = H
4 = H
R1 = H,
1 = H,
7g
R
4 = H
8a R1 = Me,
R1 = CH2CH=CH2,
8c R1 = OMe,
8d R1 = SC(S)OEt,
8e R1 = Me,
R2 = OMe, R3 = OH,
R2 = OMe, R3 = OH,
R2 = OMe, R3 = OH,
R2 = OMe, R3 = OH,
R4 = H
R4 = H
R4 = H
R4 = H
8b
R2 = OMe, R3 = OMe, R4 = H
8f
8g
8h
8i
8j
8k
8l
R
R
R
R
R
R
1 = CH2CH=CH2, R2 = OMe, R3 = OMe,
R
R
R
R
R
R
R
4 = H
4 = H
4 = H
4 = H
4 = H
4 = OMe
4 = OMe
1 = OMe,
1 = SC(S)OEt,
1 = OAc,
R
R
R
R
R
R
R
R
R
2 = OMe, R3 = OMe,
2 = OMe, R3 = OMe,
2 = OMe, R3 = OH,
2 = OMe, R3 = OMe,
2 = OMe, R3 = OMe,
1 = OAc,
1 = OAc,
R1 = OAc,
2 = OH,
2 = H,
2 = H,
2 = H,
R3 = OH,
R3 = OH,
R3 = OMe,
R3 = H,
8m R1 = OAc,
8n
8o
R4 = H
4 = H
R4 = H
R
1 = OAc,
R
R1 = OAc,
Scheme 2. Synthesis of curcuminoids. Reagents and conditions: (a) (i) B2O3, (nBuO)3B, DMF, 60 °C, 20 min; (ii) ArCHO, 65 °C, 10 min; (iii) 1,2,3,4,-tetrahydroquinoline, AcOH,
95 °C, 4 h; (iv) aq. AcOH, 70 °C, 1 h.
r
m = +0.12) showed significantly higher activity compared with 8e
min ionization is rate-limiting and the reaction rate is dependent
upon the equilibrium concentration of the anion.2 In this context,
the remarkable radical-scavenging activity of the acetoxy-substi-
tuted curcuminoids (8h–8o) might be attributed to the increased
anion concentration due to the stabilizing effect of the electron-
withdrawing acetoxy group.
or 8f with electron-donating methyl (
ent (
enging effect of the acetoxy-substituted curcuminoids (8i–8o) is
r
m = ꢂ0.07) or allyl substitu-
r
m = ꢂ0.11).23 In line with this observation, the radical-scav-
of particular interest (Fig. 2c, Table 1). Acetoxy group has a higher
Hammett constant (r
m = +0.39)23 than methoxy group and it can
be easily introduced to the central methylene position of the curc-
uminoids by simple substitution of the 3-chloroacetylacetone (5)
with acetate anion (Scheme 1). To our surprise, even the non-phe-
nolic curcuminoids were endowed with potent radical-scavenging
Ionization is suppressed in acidic conditions, and at sufficiently
high concentration of the acid, the SPLET mechanism is blocked
and the residual slow reaction occurs by HAT from a phenolic hy-
droxyl group of the neutral curcumin to the radical.2 By the same
token, the favored ionization of the curcuminoids due to the stabil-
ization by the methylene-substituted acetoxy group would also be
suppressed in acidic conditions resulting in lower level of radical-
scavenging effect. For these reasons, antioxidant effect of the acet-
effect (Fig. 2c, Table 1; EC50 values of 47.8–22.1 lM) by the acetoxy
group substituted at the central methylene position (8j, 8k, 8n, and
8o). The inactive curcuminoid with a monohydroxyphenyl moiety
(7e) also gained radical-scavenging effect upon substitution with
the acetoxy group, and the resulting curcuminoid 8m was as po-
oxy-substituted curcuminoids (100 lM) was evaluated in metha-
tent (EC50 = 26.3
l
M) as curcumin (7a) (Fig. 2c, Table 1). The influ-
nol solution containing large excess of acetic acid (100 mM),2 and
the relative change in radical-scavenging activity (Fig. 4) was cal-
culated from the equation shown below where Eacid and Eneutral
indicate radical-scavenging effect in acidic and neutral media,
respectively.
ence of the acetoxy group on the radical-scavenging effect of
curcuminoids was synergistic with the phenolic hydroxyl group,
and the activity of 8i and 8l (EC50 = 8.7 and 2.9
significantly improved compared with their counterparts with no
methylene-substituents, 7a and 7d (EC50 = 22.6 and 11.3 M, Ta-
lM, Table 1) was
l
ble 1), respectively. The SPLET mechanism for antioxidant effect
of curcumin (Fig. 3)2 support these experimental observations
regarding the role of the acetoxy group. According to this mecha-
nism, DPPH radical reacts with the curcumin anion (II, Fig. 3)
formed by proton loss from the central b-methylene.2 Thus, curcu-
Relative change ¼ ðEacid ꢂ EneutralÞ=Eneutral
It is well known that the main factor controlling the antioxidant
activity of ascorbic acid and catechol derivatives such as 8l is
the stabilization by intramolecular hydrogen bonding of the

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M. K. Kim et al. / Bioorg. Med. Chem. 19 (2011) 3793–3800
curcumin (8i) were decreased about 10–15% in acidic media
(a) 100
(Fig. 4). In contrast, the non-phenolic acetoxy-substituted curcumi-
noids (8j, 8k, 8n, and 8o) showed significant decrease (25–35%) in
radical-scavenging activity, and this result clearly indicates that
the antioxidant mechanism is anion-dependent and, even in the
presence of the stabilizing acetoxy group, the HAT from the b-
methylene moiety is less efficient than that from the phenolic hy-
droxyl group. The monohydroxyl group in 8m seems to give slight
but significant contribution to the HAT mechanism when ioniza-
tion is suppressed in acidic media showing intermediate decrease
(13%) in antioxidant activity (Fig. 4).
7a ( ), 7b ( ), 7c ( ), 7d ( ), 7e ( ), 7f ( ), 7g ( ),
Ascorbic acid (
)
80
60
40
20
0
3. Conclusion
Autoxidation is a radical chain reaction which occurs in the
presence of molecular oxygen to form hydroperoxides, and rad-
ical-trapping antioxidants break the chain reaction of oxidation
by giving up their own electrons to free radicals. For a com-
pound to be a radical-trapping antioxidant, the antioxidant-de-
rived radical must be sufficiently inert to molecular oxygen as
this would generate harmful chain-propagating peroxyl radicals.
Phenoxyl radicals are stable enough not to react with molecular
oxygen whereas most carbon-centered radicals including the one
derived from b-diketone moiety readily react with oxygen to cre-
ate peroxyl radicals.1 For this reason, the generally accepted
antioxidant mechanism of curcumin includes HAT from phenol
rather than from the neutral central methylene unit, which enti-
tles the free phenolic moiety as the prerequisite for antioxidant
activity of curcumin (7a–7g, Table 1).2 However, it is also known
that stable carbon-centered radicals, especially the resonance-
stabilized ones, exhibit attenuated reactivity toward oxygen.9–11
Therefore, it can be anticipated that the otherwise unstable reac-
tive radicals derived from b-diketone moiety might serve as a
radical-trapping antioxidant by introduction of a substituent sta-
bilizing the carbon-centered radical. The methylene-substituents
used in this study (methyl, allyl, methoxy, xanthate, and acet-
0
20
40
60
80
100
Concentration (uM)
(b) 100
8a ( ), 8b ( ), 8c ( ), 8d ( ), 8e ( ), 8f ( ), 8g ( ),
8h ( ), 7b ( ),Ascorbicacid (
)
80
60
40
20
0
0
20
40
60
80
100
Concentration (uM)
oxy) cover broad spectrum of the polar substituent effect (rm
,
100
(c)
ꢂ0.11 to +0.39), and the observed antioxidant activity in the
increasing order of methyl- < allyl- < methoxy- < xanthate-
ꢀ acetoxy-substituted curcuminoids (Fig. 2b and c) is well
7a ( ), 7b ( ), 8i ( ), 8j ( ), 8k ( ), 8l ( ), 8m ( ),
8n ( ), 8o ( ),Ascorbic acid (
)
80
60
40
20
0
correlated with the polar inductive effects (rm) of the substitu-
ents. In particular, the acetoxy-substituted curcuminoids with
free phenolic hydroxyl groups (8i and 8l, Table 1) showed more
potent activity (EC50 = 8.7
cumin (7a, EC50 = 22.6 M) and ascorbic acid (EC50 = 15.7
More importantly, the acetoxy substituent provided potent anti-
oxidant activity (EC50 = 22.1–47.8 M) even to the non-phenolic
lM for 8i and 2.9
lM for 8l) than cur-
l
l
M).
l
curcuminoids (8j, 8k, and 8m–8o), which demonstrated that
the carbon-centered radicals are working as radical-trapping
antioxidants. In addition, the radical-scavenging effect of the
non-phenolic acetoxy-substituted curcuminoids was shown to
be substantially diminished in acidic environment, which indi-
cates that the antioxidant mechanism is anion-dependent
(Fig. 4). Taken together, the non-phenolic curcuminoids seem
to generate the carbon-centered radicals via electron transfer
from the enolate anion, which is reminiscent of the initial stage
of the SPLET mechanism. According to the SPLET mechanism, the
equilibrium formation of the enolate anion is the rate-limiting
step in the antioxidant mechanism of the curcuminoids. In
non-phenolic curcuminoids in which the b-diketone moiety is
the only plausible source of the radical, the anion concentration
would be more crucial, and thus, stabilization of the enolate an-
ion by the polar electron-withdrawing substituents such as the
acetoxy group is likely to be the key factor for the antioxidant
mechanism.
0
20
40
60
80
100
Concentration (uM)
Figure 2. Radical-scavenging activity of curcuminoids with (a) no methylene-
substituents (7a–7g), (b) methylene-substituents of methyl (8a, 8e), allyl (8b, 8f),
methoxy (8c, 8g), and xanthate (8d, 8h) group, and (c) methylene-substituents of
acetoxy group (8i–8o).
oxyradicals formed after direct HAT,24 which explains the acid-
independent radical-scavenging effect of ascorbic acid and 8l
(Fig. 4). As anticipated by the SPLET mechanism, the radical-scav-
enging effects of both curcumin (7a) and acetoxy-substituted

M. K. Kim et al. / Bioorg. Med. Chem. 19 (2011) 3793–3800
3797
Table 1
EC50 values (amount of curcuminoids necessary to decrease the initial DPPH concentration by 50%) of curcuminoids prepared in this study
O
O
O
OH
R2
R3
R2
R3
R2
R3
R2
R3
R1
R1
R4
R4
R4
R4
Curcuminoid
R1
R2
R3
R4
EC50 (lM)
7a
7b
7c
7d
H
H
H
H
OMe
OMe
OMe
OH
H
OH
H
H
OMe
OMe
H
22.6 2.3
>100
>100
11.3 1.2
>100
OMe
OMe
OH
7e
H
OH
7f
7g
H
H
H
H
OMe
H
H
H
>100
>100
8a
8b
8c
8d
Me
OMe
OH
H
23.2 1.7
23.2 2.3
30.3 1.5
29.7 2.0
>100
CH2CHCH2
OMe
SC(S)OEt
Me
8e
OMe
OMe
H
8f
8g
8h
8i
8j
8k
8l
8m
CH2CHCH2
OMe
SC(S)OEt
OAc
OAc
OAc
OAc
OAc
OAc
OAc
>100
75.5 1.7
>100
OMe
OMe
OMe
OH
H
OH
H
H
OMe
OMe
H
8.7 0.6
22.1 2.7
27.4 0.8
2.9 0.3
26.3 0.6
32.8 1.6
47.8 2.1
15.7 2.3
OMe
OMe
OH
OH
OMe
H
8n
8o
H
H
H
H
Ascorbic acid
O
OH
O
O
O
O
-H+
DPPH-
DPPH
+
R
R
R
+H+
R'
R'
R'
I
II
III
Figure 3. SPLET mechanism.
4. Experimental
4.1. Materials and general methods
Nuclear magnetic resonance spectra were recorded at 400 MHz
for 1H NMR and at 100 MHz for 13C NMR with tetramethylsilane as
the internal standard. Chemical shift are reported as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), or br s (broad
singlet). Coupling constants are reported in hertz (Hz). The chem-
ical shifts are reported as parts per million (d) relative to the sol-
vent peak. All tested compounds were P95% purity as
determined by reverse phase HPLC. HPLC was performed on equip-
ment with variable wavelength (VW) UV detector and C18—A
250 ꢃ 4.6 mm column. Analytical conditions were as follows: Gra-
dient used was 20% acetonitrile in water containing 0.1% formic
acid (0–8 min), 25–35% acetonitrile in water containing 0.1% for-
mic acid (8–18 min), 35% acetonitrile in water containing 0.1% for-
mic acid (18–25 min), 35–80% acetonitrile in water containing 0.1%
formic acid (25–40 min), 80–100% acetonitrile in water containing
0.1% formic acid (40–45 min), and 100% acetonitrile in water
Figure 4. Relative change in radical-scavenging effect of the curcuminoids
(100 M) in the presence of acetic acid (100 mM). Absorbance was measured
30 min after mixing the curcuminoids with DPPH.
l

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M. K. Kim et al. / Bioorg. Med. Chem. 19 (2011) 3793–3800
containing 0.1% formic acid (45–50 min). Flow was 1 mL/min. UV
was detected at two different wavelengths (340 and 254 nm).
4.3. General procedures for preparation of curcumin analogues
(7 and 8)
Tributyl borate (10 mmol) was added to a solution of boric
oxide (5.0 mmol) and acetylacetone (4 or 6, 5.0 mmol) in DMF
(1.0 mL), and the resulting solution was stirred at 60 °C for
20 min. The mixture was treated with aromatic aldehyde
4.2. Synthesis of substituted acetoacetates (6a–6e)
4.2.1. 4-Hydroxy-3-methylpent-3-en-2-one (6a)
A solution of acetylacetone (5, 0.45 mL, 4.4 mmol) in acetone
(10 mL) was treated with potassium carbonate (608 mg, 4.4 mmol)
and iodomethane (0.28 mL, 4.4 mmol) and stirred under reflux for
12 h. After cooling to room temperature, the reaction mixture was
filtered and concentrated under reduced pressure to give 4-hydro-
xy-3-methylpent-3-en-2-one (6a) as pale yellow syrup, which was
used for the next step without further purification.
(10 mmol) and stirred at 65 °C. After 10 min,
a solution of
1,2,3,4-tetrahydroquinoline (0.1 mL) and acetic acid (0.3 mL) in
DMF (1 mL) was added dropwise to the mixture, which was al-
lowed to stir at 95 °C for 4 h. After cooling, aqueous acetic acid
(20%, 50 mL) was added and the mixture was stirred at 70 °C for
1 h. The reaction mixture were extracted with ethyl acetate three
times (200 mL ꢃ 3), washed with water, dried over MgSO4, and
then concentrated under reduced pressure. The residue was puri-
fied by column chromatography on silica gel (hexanes/ethyl ace-
tate = 2:1) followed by recrystallization from CHCl3 to afford the
desired curcuminoids as a yellow solid.
4.2.2. 3-(1-Hydroxyethylidene)hex-5-en-2-one (6b)
A solution of acetylacetone (5, 0.37 mL, 3.6 mmol) in acetone
(10 mL) was treated with potassium carbonate (498 mg, 3.6 mmol)
and allyl bromide (0.28 mL, 3.6 mmol) and stirred under reflux for
12 h. After cooling to room temperature, the reaction mixture was
filtered and concentrated under reduced pressure to give 3-(1-
hydroxyethylidene)hex-5-en-2-one (6b) as pale yellow syrup,
which was used for the next step without further purification.
4.3.1. 1,7-Bis(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-
trien-3-one (7b)
(68% yield): 1H NMR (400 MHz, CD3COCD3) d (ppm) 7.62 (d,
J = 15.8 Hz, 2H), 7.32 (d, J = 1.9 Hz, 2H), 7.24 (dd, J = 1.9, 8.3 Hz,
2H), 7.01 (d, J = 8.3 Hz, 2H), 6.75 (d, J = 15.8 Hz, 2H), 6.00 (s, 1H),
3.88 (d, J = 6.8 Hz, 12H).
4.2.3. 4-Hydroxy-3-methoxypent-3-en-2-one (6c)
Boron trifluoride etherate (1.2 mL, 9.7 mmol) was added to a
suspension of (diacetoxyiodo)benzene (3.127 g, 9.7 mmol) in
methanol (20 mL). To the resulting mixture was added acetylace-
tone (5, 1 mL, 9.7 mmol). The contents were allowed to stir at room
temperature for 5 h. After addition of aqueous sodium hydrogen
carbonate solution (60 mL), the reaction mixture was extracted
with dichloromethane (100 mL ꢃ 3), dried over MgSO4, and con-
centrated under reduced pressure. The crude product was then
purified by column chromatography on silica gel (hexanes/ethyl
acetate = 9:1) to afford 4-hydroxy-3-methoxypent-3-en-2-one
(6c) as clear oil in 85% yield (100% enol-form): 1H NMR
(400 MHz, CDCl3) d (ppm) 3.57 (s, 3H), 2.17 (s, 6H).
4.3.2. 5-Hydroxy-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4,6-
trien-3-one (7c)
(72% yield): 1H NMR (400 MHz, CDCl3) d (ppm) 7.57 (d,
J = 15.5 Hz, 2H), 6.78 (br s, 4H), 6.53 (d, J = 15.7 Hz, 2H), 5.86 (d,
J = 1.1 Hz, 1H), 3.89–3.94 (m, 18H).
4.3.3. 1,7-Bis(3,4-dihydroxy-5-methoxyphenyl)-5-
hydroxyhepta-1,4,6-trien-3-one (7d)
(35% yield): 1H NMR (400 MHz, CDCl3) d (ppm) 7.52 (d,
J = 15.6 Hz, 2H), 6.88 (br s, 2H), 6.62 (br s, 2H), 6.48 (d,
J = 15.6 Hz, 2H), 5.80 (s, 1H), 3.91 (s, 6H).
4.3.4. 5-Hydroxy-1,7-bis(4-hydroxyphenyl)hepta-1,4,6-trien-3-
one (7e)
4.2.4. O-Ethyl-S-2-hydroxy-4-oxopent-2-en-3-yl
(40% yield): 1H NMR (400 MHz, CD3COCD3) d (ppm) 7.55–7.67
(m, 6H), 6.91 (d, J = 8.6 Hz, 4H), 6.65 (d, J = 15.8 Hz, 2H), 5.99 (s,
1H).
carbonodithioate (6d)
3-Chloroacetylacetone (4, 0.3 mL, 2.7 mmol) was treated with
potassium ethyl xanthate (426 mg, 2.7 mmol) in acetone (3 mL)
and the reaction mixture was stirred at room temperature. After
4 h, the resulting solution was concentrated under reduced pres-
sure, and the residue was purified by column chromatography on
silica gel (hexane/diethyl ether = 15:1) to afford O-ethyl-S-2-hy-
droxy-4-oxopent-2-en-3-yl carbonodithioate (6d) as clear oil in
75% yield (100% enol-form): 1H NMR (400 MHz, CDCl3) d (ppm)
4.69 (q, J = 7.1 Hz, 2H), 2.24 (s, 6H), 1.42 (t, J = 7.1 Hz, 3H).
4.3.5. 5-Hydroxy-1,7-bis(4-methoxyphenyl)hepta-1,4,6-trien-3-
one (7f)
(72% yield): 1H NMR (400 MHz, CDCl3) d (ppm) 7.93 (d,
J = 15.6 Hz, 2H), 7.57 (d, J = 8.8 Hz, 4H), 6.94 (d, J = 8.8 Hz, 4H),
6.58 (d, J = 15.5 Hz, 2H), 6.00 (s, 1H), 3.87 (s, 6H).
4.3.6. 5-Hydroxy-1,7-diphenylhepta-1,4,6-trien-3-one (7g)
(55% yield): 1H NMR (400 MHz, CD3COCD3) d (ppm) 7.68–7.72
(m, 6H), 7.40–7.47 (m, 6H), 6.89 (d, J = 16.0 Hz, 2H), 6.13 (s, 1H).
4.2.5. 3-Acetoxy acetylacetone (6e)
3-Chloroacetylacetone (4, 0.5 mL, 4.4 mmol) in DMSO (5 mL) was
treated with sodium acetate (726 mg, 8.8 mmol), and the resulting
mixture was stirred at room temperature for 3 h. After addition of
water(20 mL), thereactionmixturewasextractedwithdiethylether
(30 mL ꢃ 3), dried over MgSO4, and concentrated under reduced
pressure. The residue was then purified by column chromatography
on silica gel (hexanes/ethyl acetate = 8:1) to afford 3-acetoxy acetyl-
acetone (6e) as clear oil (60% yield): Inseparable mixture (2.7:1) of
the keto–enol tautomers: For keto-form; 1H NMR (400 MHz, CDCl3)
d (ppm) 5.44 (s, 1H), 2.29 (s, 6H), 2.24 (s, 3H); for enol-form; 1H NMR
(400 MHz, CDCl3) d (ppm) 2.26 (s, 3H), 2.02 (s, 6H).
4.3.7. (1E,4Z,6E)-5-Hydroxy-1,7-bis(4-hydroxy-3-
methoxyphenyl)-4-methylhepta-1,4,6-trien-3-one (8a)
(35% yield): 1H NMR (400 MHz, CDCl3) d (ppm) 7.68 (d,
J = 15.4 Hz, 2H), 7.18 (dd, J = 1.5, 8.3 Hz, 2H), 7.06 (d, J = 1.5 Hz,
2H), 6.93–6.99 (m, 4H), 3.96 (s, 6H), 2.21 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) d (ppm) 13.6, 57.1 (2C), 107.1, 112.7, 112.9,
117.0 (2C), 119.2, 123.6, 124.7, 125.1, 127.1, 128.1, 142.9, 145.3,
149.3 (2C), 150.7, 151.1, 183.7, 197.7; HRMS (ESI) m/z calcd for
C
22H23O6 [M+H]+: 383.14946, found: 383.15061.

M. K. Kim et al. / Bioorg. Med. Chem. 19 (2011) 3793–3800
3799
4.3.8. (1E,4Z,6E)-4-Allyl-5-hydroxy-1,7-bis(4-hydroxy-3-
methoxyphenyl)hepta-1,4,6-trien-3-one (8b)
HRMS (ESI) m/z calcd for C24H27O7 427.17568 [M+H]+, found
427.17605.
(30% yield): 1H NMR (400 MHz, CDCl3) d (ppm) 7.69 (d,
J = 15.4 Hz, 2H), 7.15 (dd, J = 1.5, 8.2 Hz, 2H), 7.02 (d, J = 1.5 Hz,
2H), 6.93 (d, J = 8.2 Hz, 2H), 6.85 (d, J = 15.4 Hz, 2H), 5.99–6.09
(m, 1H), 5.14–5.18 (m, 2H), 3.94 (s, 6H), 3.33 (d, J = 5.0 Hz,
2H); 13C NMR (100 MHz, CDCl3) d (ppm) 30.0, 56.0 (2C), 107.7,
107.7, 110.2 (2C), 114.9 (2C), 116.0, 118.4 (2C), 122.5 (2C),
128.1 (2C), 134.6, 141.6 (2C), 146.7 (2C), 147.8 (2C), 194.7;
HRMS (ESI) m/z calcd for C24H25O6 409.16511 [M+H]+, found
409.16566.
4.3.14. S-(1E,3E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-3-hydroxy-5-
oxohepta-1,3,6-trien-4-yl O-ethyl carbonodithioate (8h)
(65% yield): 1H NMR (400 MHz, CDCl3) d (ppm) 7.79 (d,
J = 15.5 Hz, 2H), 7.38 (d, J = 15.5 Hz, 2H), 7.32 (br s, 1H), 7.29 (br
s, 3H), 7.04 (d, J = 8.1 Hz, 2H), 4.67 (q, J = 7.1 Hz, 2H), 3.90 (s,
12H), 1.37 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CD3COCD3) d
(ppm) 14.0, 55.0, 56.2 (4C), 71.7 (2C), 112.2 (2C), 112.7 (2C),
118.8 (2C), 124.0 (2C), 128.7 (2C), 144.9 (2C), 150.7 (2C), 153.2,
187.8, 188.5, 215.1; HRMS (ESI) m/z calcd for
C26H29O7S2
517.13547 [M+H]+, found 517.13595.
4.3.9. (1E,4E,6E)-5-Hydroxy-1,7-bis(4-hydroxy-3-
methoxyphenyl)-4-methoxyhepta-1,4,6-trien-3-one (8c)
(55% yield): 1H NMR (400 MHz, CD3COCD3) d (ppm) 7.64 (d,
J = 15.8 Hz, 2H), 7.37 (d, J = 1.9 Hz, 2H), 7.26 (dd, J = 1.9, 8.2 Hz,
2H), 7.16 (d, J = 15.8 Hz, 2H), 6.91 (d, J = 8.2 Hz, 2H), 3.94 (s, 6H),
3.72 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d (ppm) 55.9 (2C),
64.21, 112.1 (2C), 115.1 (2C), 115.9 (2C), 123.2 (2C), 126.6 (2C),
139.5, 141.9 (2C), 148.1 (4C), 149.7, 177.7; HRMS (ESI) m/z calcd
for C22H23O7 399.14438 [M+H]+, found 399.14479.
4.3.15. 3-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-5-
oxohepta-1,3,6-trien-4-yl acetate (8i)
(65% yield): Inseparable mixture (2.2:1) of keto–enol tautom-
ers; for keto-form, 1H NMR (400 MHz, CDCl3) d (ppm) 7.73 (d,
J = 15.9 Hz, 2H), 7.16 (dd, J = 1.8, 8.2 Hz, 2H), 7.07 (d, J = 1.8 Hz,
2H), 6.92 (d, J = 8.3 Hz, 2H), 6.89 (d, J = 16.0 Hz, 2H), 5.99 (s, 1H),
3.94 (s, 6H), 2.02 (s, 3H); for enol-form, 1H NMR (400 MHz, CDCl3)
d (ppm) 15.48 (s, OH), 7.67 (d, J = 15.7 Hz, 2H), 7.16 (dd, J = 1.8,
8.2 Hz, 2H), 7.00 (d, J = 1.7 Hz, 2H), 6.94 (d, J = 8.2 Hz, 2H), 6.62
(d, J = 15.6 Hz, 2H), 3.94 (s, 6H), 2.30 (s, 3H); as a mixture of tau-
tomers, 13C NMR (100 MHz, CD3COCD3) d (ppm) 20.6 (2C), 56.4
(4C), 100.9 (2C), 102.7 (2C), 112.0 (2C), 112.4 (2C), 115.8 (2C),
116.3 (4C), 120.0 (2C), 124.1 (2C), 125.0 (2C), 127.3 (2C), 128.0
(2C), 143.3 (2C), 146.3 (2C), 148.7 (2C), 148.8, 150.5 (2C), 151.0,
168.5, 171.1 (2C), 190.5, 206.2 (2C); HRMS (ESI) m/z calcd for
4.3.10. O-Ethyl S-(1E,3E,6E)-3-hydroxy-1,7-bis(4-hydroxy-3-
methoxyphenyl)-5-oxohepta-1,3,6-trien-4-yl carbonodithioate
(8d)
(53% yield): 1H NMR (400 MHz, CD3COCD3) d (ppm) 7.78 (d,
J = 15.5 Hz, 2H), 7.35 (d, J = 15.5 Hz, 2H), 7.31 (br s, 2H), 7.25 (d,
J = 8.2 Hz, 2H), 6.91 (d, J = 8.2 Hz, 2H), 4.67 (q, J = 7.1 Hz, 2H),
3.91 (s, 6H), 1.37 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, DMSO-
d6) d (ppm) 12.1, 54.2 (4C), 69.8, 100.2 (2C), 110.9 (2C),114.4
(2C),114.9, 121.8 (2C),124.4 (2C), 142.9, 146.4 (4C),148.6, 185.5;
HRMS (ESI) m/z calcd for C24H26O7S2 489.10417 [M+H]+, found
489.10438.
C
23H23O8 427.13929 [M+H]+, found 427.13938.
4.3.16. 1,7-Bis(3,4-dimethoxyphenyl)-3-hydroxy-5-oxohepta-
1,3,6-trien-4-yl acetate (8j)
(52% yield): Inseparable mixture (1:4.8) of keto–enol tautom-
ers; for keto-form, 1H NMR (400 MHz, CDCl3) d (ppm) 7.75 (d,
J = 15.9 Hz, 2H), 7.20 (dd, J = 1.6, 8.3 Hz, 2H), 7.09 (d, J = 1.6 Hz,
2H), 6.91 (d, J = 15.9 Hz, 2H), 6.87 (d, J = 8.3 Hz, 2H), 6.00 (s, 1H),
3.92 (s, 12H), 2.37 (s, 3H); for enol-form, 1H NMR (400 MHz, CDCl3)
d (ppm) 15.48 (s, OH), 7.69 (d, J = 15.6 Hz, 2H), 7.17 (dd, J = 1.5,
8.3 Hz, 2H), 7.04 (d, J = 1.5 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 6.63
(d, J = 15.6 Hz, 2H), 3.92 (s, 12H), 2.40 (s, 3H); as a mixture of tau-
tomers, 13C NMR (100 MHz, DMSO-d6) d (ppm) 22.3 (6C), 57.2 (8C),
83.5, 112.2 (4C), 112.9, 113.2 (4C), 116.9, 121.9, 125.0, 125.8,
128.3, 128.9, 144.1 (2C), 146.7 (2C), 150.5 (4C), 150.6 (4C), 153.0,
153.4 (2C), 171.0, 172.5, 178.3, 191.5 (2C); HRMS (ESI) m/z calcd
for C25H27O8 455.17059 [M+H]+, found 455.17089.
4.3.11. (1E,4Z,6E)-1,7-Bis(3,4-dimethoxyphenyl)-5-hydroxy-4-
methylhepta-1,4,6-trien-3-one (8e)
(45% yield): 1H NMR (400 MHz, CDCl3) d (ppm) 7.70 (d,
J = 15.4 Hz, 2H), 7.19 (dd, J = 1.8, 8.3 Hz, 2H), 7.09 (d, J = 1.8 Hz,
2H), 7.35 (d, J = 15.4 Hz, 2H), 6.89 (d, J = 8.3 Hz, 2H), 3.97 (s, 6H),
3.93 (s, 6H), 2.19 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d (ppm)
11.9, 55.9 (2C), 56.0 (2C), 106.5 (2C), 110.8, 111.0, 111.9, 119.4,
123.5, 123.6, 123.9, 127.3, 128.3, 141.6, 143.9, 149.4 (2C), 151.3,
151.7, 182.8, 196.8; HRMS (ESI) m/z calcd for
C24H27O6
411.18076 [M+H]+, found: 411.18125.
4.3.12. (1E,4Z,6E)-4-Allyl-1,7-bis(3,4-dimethoxyphenyl)-5-
hydroxyhepta-1,4,6-trien-3-one (8f)
(42% yield): 1H NMR (400 MHz, CDCl3) d (ppm) 7.71 (d,
J = 15.4 Hz, 2H), 7.17 (dd, J = 1.9, 8.3 Hz, 2H), 7.06 (d, J = 1.9 Hz,
2H), 6.86–6.90 (m, 4H), 6.00–6.08 (m, 1H), 5.19 (d, J = 0.7 Hz,
1H), 5.14–5.19 (m, 1H), 3.94 (s, 6H), 3.93 (s, 6H), 3.34 (d,
J = 5.1 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) d (ppm) 29.8, 54.8
(4C), 62.7, 109.7, 110.0, 110.9, 122.3, 122.7, 122.9, 126.2, 127.1,
138.1, 140.7, 143.2, 148.2 (4C), 150.2, 162.1, 182.3, 193.8, 228.3;
HRMS (ESI) m/z calcd for C26H29O6 437.19641 [M+H]+, found
437.19690.
4.3.17. 3-Hydroxy-5-oxo-1,7-bis(3,4,5-trimethoxyphenyl)hepta-
1,3,6-trien-4-yl acetate (8k)
(60% yield, 100% enol-form): 1H NMR (400 MHz, CDCl3) d (ppm)
15.34 (s, OH), 7.67 (d, J = 15.6 Hz, 2H), 6.77 (s, 4H), 6.65 (d,
J = 15.6 Hz, 2H), 3.89 (s, 18H), 2.40 (s, 3H); as a mixture of tautom-
ers, 13C NMR (100 MHz, DMSO-d6) d (ppm) 21.1 (2C), 56.1 (8C),
60.5 (4C), 82.0 (2C), 107.0 (8C), 117.5, 122.5 (4C), 129.8 (4C),
130.3, 140.4, 140.6 (2C), 143.0, 145.4 (2C), 153.5 (8C), 169.8,
171.2, 177.0, 190.4, 207.1 (2C); HRMS (ESI) m/z calcd for
C
27H31O10 515.19172 [M+H]+, found 515.19135.
4.3.13. (1E,4E,6E)-1,7-Bis(3,4-dimethoxyphenyl)-5-hydroxy-4-
methoxyhepta-1,4,6-trien-3-one (8g)
(40% yield): 1H NMR (400 MHz, CD3COCD3) d (ppm) 7.65 (d,
J = 15.9 Hz, 2H), 7.37 (d, J = 1.9 Hz, 2H), 7.32 (dd, J = 1.9, 8.3 Hz,
2H), 7.19 (d, J = 15.8 Hz, 2H), 7.03 (d, J = 8.3 Hz, 2H), 3.90 (s, 6H),
3.88 (s, 6H), 3.73 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d (ppm)
56.9 (2C), 57.0 (2C), 65.4, 112.3 (2C), 113.0 (2C), 117.3 (2C),
124.3 (2C), 129.0 (2C), 137.8, 142.8 (2C), 150.4 (4C), 152.5, 179.0;
4.3.18. 1,7-Bis(3,4-dihydroxy-5-methoxyphenyl)-3-hydroxy-5-
oxohepta-1,3,6-trien-4-yl acetate (8l)
(35% yield): Inseparable mixture (1.25:1) of keto–enol tautom-
ers; for keto-form, 1H NMR (400 MHz, CDCl3) d (ppm) 7.64 (d,
J = 15.6 Hz, 2H), 6.90 (br s, 2H), 6.86 (d, J = 15.6 Hz, 2H), 6.70 (br
s, 2H), 5.73 (s, 1H), 3.96 (s, 6H), 2.12 (s, 3H); for enol-form, 1H

3800
M. K. Kim et al. / Bioorg. Med. Chem. 19 (2011) 3793–3800
NMR (400 MHz, CDCl3) d (ppm) 15.49 (s, OH), 7.51 (d, J = 15.6 Hz,
2H), 6.87 (br s, 2H), 6.60 (br s, 2H), 6.46 (d, J = 15.6 Hz, 2H), 3.91
(s, 6H), 2.12 (s, 3H); as a mixture of tautomers, 13C NMR
(100 MHz, CD3COCD3) 42.7 (2C), 57.0 (4C), 110.5 (4C), 111.5 (4C),
112.6, 114.9, 115.2 (2C), 120.4 (2C), 126.8 (2C), 127.6 (2C), 127.7
(2C), 138.2, 139.1 (2C), 141.7, 143.0 (2C), 146.7 (2C), 146.9 (2C),
147.0 (2C), 147.4 (2C), 190.9, 191.8, 191.9, 195.4, 200.7 (2C); HRMS
(ESI) m/z calcd for C23H23O10 459.12912 [M+H]+, found 459.12875.
96-well plate, a freshly prepared methanol solution of DPPH
(190 L, 0.1 mM) was mixed with 10 L of a methanol (1% DMSO)
l
l
solution of the curcuminoids at different concentrations (0.01,
0.05, 0.1, 0.2, 0.5, 1.0, and 2.0 mM). The absorption spectra at
517 nm were recorded every 2 min. For each curcuminoid concen-
tration tested, the percentage of DPPH remaining reached the stea-
dy state after 30 min. Therefore, the percentage of residual DPPH
after 30 min was plotted against concentration of the curcumi-
noids. The values EC50 were determined from this plot.
4.3.19. 3-Hydroxy-1,7-bis(4-hydroxyphenyl)-5-oxohepta-1,3,6-
trien-4-yl acetate (8m)
Acknowledgments
(52% yield): Inseparable mixture (1:2.3) of keto–enol tautom-
ers; for keto-form, 1H NMR (400 MHz, CD3COCD3) d (ppm) 7.70
(d, J = 15.9 Hz, 2H), 7.57–7.64 (m, 4H), 7.02 (d, J = 15.9 Hz, 2H),
6.89–6.93 (m, 4H), 6.02 (s, 1H), 2.44 (s, 3H); for enol-form, 1H
NMR (400 MHz, CD3COCD3) d (ppm) 7.68 (d, J = 15.7 Hz, 2H),
7.57–7.64 (m, 4H), 6.89–6.93 (m, 4H), 6.86 (d, J = 15.7 Hz, 2H),
2.44 (s, 3H); as a mixture of tautomers, 13C NMR (100 MHz,
CD3COCD3) 22.29 (2C), 33.7 (2C), 38.2 (2C), 117.1, 118.4 (4C),
118.5 (4C), 121.1, 128.4, 129.1 (2C), 129.8, 131.8, 133.2 (4C),
133.5 (4C), 144.7 (2C), 147.7, 162.5 (2C), 163.1, 165.2 (2C), 171.6,
172.8, 179.6, 192.3 (2C); HRMS (ESI) m/z calcd for C21H19O6
367.11816 [M+H]+, found 367.11784.
This work was supported by the Konkuk University, the Korea
Science and Engineering Foundation (KOSEF) grant funded by the
Korea government (MEST) (No. 2009-0071110), 2nd Biogreen 21
program (PJ007982, the Korean Rural Development Administra-
tion), and by Priority Research Centers Program through the Na-
tional Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology (2009-0093824).
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4.3.20. 3-Hydroxy-1,7-bis(4-methoxyphenyl)-5-oxohepta-1,3,6-
trien-4-yl acetate (8n)
(60% yield, 100% enol-form): 1H NMR (400 MHz, CDCl3) d (ppm)
15.49 (s, OH), 7.70 (d, J = 15.7 Hz, 2H), 7.50 (d, J = 8.6 Hz, 4H), 6.91
(d, J = 8.6 Hz, 4H), 6.65 (d, J = 15.7 Hz, 2H), 3.85 (s, 6H), 2.41 (s, 3H);
as a mixture of tautomers, 13C NMR (100 MHz, CD3COCD3) 20.4
(2C), 55.7 (4C), 113.5, 113.6, 115.1 (8C), 115.2 (2C), 116.0 (4C),
120.1, 127.7, 127.9, 128.4 (2C), 131.1 (8C), 131.5 (2C), 142.5 (2C),
145.4, 162.6 (2C), 163.1, 170.9, 206.0 (2C); HRMS (ESI) m/z calcd
for C23H23O6 395.14946 [M+H]+, found 395.14985.
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2002, 15, 1576.
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4.3.21. 3-Hydroxy-5-oxo-1,7-diphenylhepta-1,3,6-trien-4-yl
acetate (8o)
(40% yield): Inseparable mixture (1: 4.3) of keto–enol tautom-
ers; for keto-form, 1H NMR (400 MHz, CDCl3) d (ppm) 7.79 (d,
J = 16.0 Hz, 2H), 7.58–7.60 (m, 5H), 7.39–7.40 (m, 5H), 7.06 (d,
J = 16.0 Hz, 2H), 6.01 (s, 1H), 2.32 (s, 3H); for enol-form, 1H NMR
(400 MHz, CDCl3) d (ppm) 15.27 (s, OH), 7.75 (d, J = 15.6 Hz, 2H),
7.54–7.56 (m, 5H), 7.39–7.40 (m, 5H), 6.78 (d, J = 15.6 Hz, 2H),
2.42 (s, 3H); as a mixture of tautomers, 13C NMR (100 MHz, CDCl3)
20.6 (2C), 106.8, 114.2, 116.4 (2C), 118.2 (2C),121.2 (2C), 127.1
(8C), 128.2 (8C), 131.2, 132.4 (4C), 141.3 (2C), 142.5 (2C), 145.2,
170.3 (2C), 171.5, 178.5, 191.3 (2C); HRMS (ESI) m/z calcd for
C
21H19O4 335.12833 [M+H]+, found 335.12885.
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4.4. DPPH free radical-scavenging activity assay
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6226.
Free radical-scavenging activity of curcuminoid was measured
using DPPH radical.16 In a typical procedure, in each well of a