Coumarin moiety can enhance abilities of chalcones to inhibit DNA oxidation and to scavenge radicals

Article abstract of DOI:10.1016/j.tet.2014.08.063

Coumarin and chalcone are naturally occurring compounds, and coumarin as a functional group was combined with chalcone in this work, aiming to test the inhibitory effects of coumarin-substituted chalcones on the oxidation of DNA and on scavenging radicals. It was found that the antioxidant activity of hydroxyl group attaching to coumarin can be increased by hydroxyl groups attaching to chalcone. The double hydroxyl groups at adjacent position exhibited high abilities to inhibit Cu²⁺/glutathione-induced oxidation of DNA and to trap 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS⁺) as well as 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH). Especially, the double hydroxyl groups in chalcone were able to protect DNA against 2,2′-azobis(2-amidinopropanehydrochloride) (AAPH)-induced oxidation significantly. On the other hand, the hydroxyl group attaching to coumarin exhibited high ability to inhibit OH-induced oxidation of DNA. Therefore, coumarin-appended chalcones exhibited higher antioxidant effectiveness with only single or double phenolic hydroxyl groups contained.

Full text of DOI:10.1016/j.tet.2014.08.063

Tetrahedron 70 (2014) 8397e8404
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Coumarin moiety can enhance abilities of chalcones to inhibit DNA
oxidation and to scavenge radicals
*
Gao-Lei Xi, Zai-Qun Liu
Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130021, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2014
Received in revised form 16 August 2014
Accepted 26 August 2014
Available online 6 September 2014
Coumarin and chalcone are naturally occurring compounds, and coumarin as a functional group was
combined with chalcone in this work, aiming to test the inhibitory effects of coumarin-substituted
chalcones on the oxidation of DNA and on scavenging radicals. It was found that the antioxidant ac-
tivity of hydroxyl group attaching to coumarin can be increased by hydroxyl groups attaching to chal-
cone. The double hydroxyl groups at adjacent position exhibited high abilities to inhibit Cu2þ
/
glutathione-induced oxidation of DNA and to trap 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonate)
Keywords:
Coumarin
Chalcone
cationic radical (ABTSDꢀ) as well as 2,20-diphenyl-1-picrylhydrazyl radical (DPPH). Especially, the double
hydroxyl
groups
in
chalcone
were
able
to
protect
DNA
against
2,20-azobis(2-
amidinopropanehydrochloride) (AAPH)-induced oxidation significantly. On the other hand, the hy-
droxyl group attaching to coumarin exhibited high ability to inhibit ꢀOH-induced oxidation of DNA.
Therefore, coumarin-appended chalcones exhibited higher antioxidant effectiveness with only single or
double phenolic hydroxyl groups contained.
Antioxidant
Oxidation of DNA
Free radical
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
A large body of clinic evidences elucidates that the pathogenesis
for many fatal diseases correlates with the free radical-initiated
Coumarin, a natural compound,1 exhibits various biological
properties, such as anticancer,2,3 antimicrobial,4,5 antiviral,6 anti-
oxidant,7,8 anti-inflammatory,9 and enzymatic inhibitor,10 and are
thereby used as food additives and medicines. Especially, 4-
methylcoumarin is a popular scaffold for constructing novel anti-
oxidants.11e13 On the other hand, chalcones also possess various
biological properties14 including antioxidant15 and anticancer ac-
tivities,16 and are usually applied as a basic structure for preparing
novel antioxidants.17 In our previous work, we found that the in-
hibitory effect of 4-methylcoumarin on the radical-mediated oxi-
dation of DNA can be increased by the moiety of 4,5-
dihydropyrazole.18,19 The aforementioned backgrounds motivate
us to explore whether the combination of chalcone with coumarin
can be a novel antioxidant with high activity. Hence, as shown in
Scheme 1, 4-methylcoumarin was prepared by the reaction of
resorcinol with ethyl acetoacetate, followed by acetylizing to afford
chalcones via ClaiseneSchmidt condensation. The obtained
coumarin-substituted chalcones contain only one or two hydroxyl
groups in order to evaluate the antioxidant properties of coumarin-
modified chalcones in the case of few hydroxyl groups contained.
oxidations of membrane, lipid, protein, and DNA.20,21 Hence, the
inhibitory effect on the oxidation of DNA and on radicals are major
index for evaluating the antioxidant capacity.22 The oxidations of
DNA caused by 2,20-azobis(2-amidino propane hydrochloride)
(AAPH, ReN]NeR, R]eCMe2C(]NH)NH2),23 Cu2þ/glutathione
(GSH),24 and hydroxyl radical (ꢀOH)25 are usually used as the ex-
perimental systems for estimating the antioxidant ability. In addi-
tion, the radical-scavenging capacities of antioxidants can be
estimated by reacting with 2,20-azinobis(3-ethylbenzothiazoline-6-
sulfonate) cationic radical (ABTSD
)
and 2,20-diphenyl-1-
ꢀ
picrylhydrazyl radical (DPPH). Presented here is a study on the
inhibitory effects of seven coumarin-substituted chalcones on
Cu2þ/GSH-, ꢀOH-, and AAPH-induced oxidation of DNA and on
trapping ABTSDꢀ and DPPH.
2. Results and discussion
2.1. Effects of coumarin-substituted chalcones on Cu2D/GSH-
induced oxidation of DNA
The intracellular GSH and Cu(II) may destroy DNA because the
produced GSH radical (GSꢀ) can oxidize DNA to form carbonyl
species, which can be detected after reacting with TBA. The formed
* Corresponding author. Tel.: þ86 431 88499174; fax: þ86 431 88499159; e-mail
address: zaiqun-liu@jlu.edu.cn (Z.-Q. Liu).
http://dx.doi.org/10.1016/j.tet.2014.08.063
0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.

8398
G.-L. Xi, Z.-Q. Liu / Tetrahedron 70 (2014) 8397e8404
Scheme 1. Synthetic routine of coumarin-substituted chalcones.
TBA reactive substance (TBARS) can be measured at 535 nm.24 As
shown in Fig. 1, the increase of the absorbance line in the blank
experiment indicates that the TBARS is produced successively in
the mixture of DNA, Cu2þ, and GSH with the reaction period in-
creasing. Although the increasing tendency of the absorbance line
cannot be inhibited completely by adding 0.4 mM coumarin-
substituted chalcones, all the absorbance lines locate below that
of the blank experiment. So, all the coumarin-substituted chalcones
used herein can retard GSꢀ-induced destroy of DNA. The potential
antioxidant group in CC is the hydroxyl group at coumarin moiety,
thus, hydroxyl group at coumarin just exhibits a weak activity in
this case. The absorbance lines of PMCC and MNCC approach to that
of CC, indicating that para-methoxyl group in PMCC together with
meta-nitro group in MNCC are not active groups, while the hydroxyl
group attaching to coumarin moiety in PMCC and MNCC still plays
the inhibitory role in GSꢀ-induced destroy of DNA. Moreover, the
additions of VCC and OPHCC do not obviously vary the position of
the absorbance lines, indicating that para-hydroxyl group at chal-
cone moiety cannot ameliorate the inhibition effects on GSꢀ-in-
duced degradation of DNA. On the other hand, relative low position
of the absorbance line of MPHCC reveals that MPHCC possesses
relative high activity in this case, implying that meta-, para-dihy-
droxyl groups are beneficial for MPHCC to chelate Cu2þ, and
therefore, inhibit the reaction between Cu2þ and GSH to form GSꢀ.
In addition, according to the classic concept on the antioxidant
effectiveness, ortho-dihydroxyl benzene can be easily oxidized to
form ortho-benzoquinone, during which the antioxidative activity
is markedly enhanced.
ꢀ
2.2. Effects of coumarin-substituted chalcones on OH-in-
duced oxidation of DNA
ꢀOH is produced from H2O2 in the presence of tetra-
chlorohydroquinone (TCHQ).25 The ribosyl moiety in DNA is the
target for ꢀOH-induced oxidation, and the oxidative products can be
detected as TBARS.26 The absorbance of TBARS in the blank ex-
ꢀ
periment of OH-induced oxidation of DNA is assigned as 100%.
Fig. 2 outlines the percentages of TBARS in the presence of 0.40 mM
coumarin-substituted chalcones, which can exhibit inhibition ef-
ꢀ
fects on OH-induced oxidation of DNA because the TBARS per-
centages are lower than 100%. The activities of CC, MNCC, and OHCC
are higher than other compounds, especially, CC can decrease the
percentage of TBARS to 64.0%, the lowest TBARS percentage reveals
that hydroxyl group attaching to coumarin moiety is beneficial for
ꢀ
inhibiting OH-induced oxidation of DNA. The inhibitory effect on
ꢀ
ꢀOH is generally regarded as OH adding to benzene ring.27 CC
contains few functional groups and can provide much more posi-
ꢀ
tions for the addition of OH, as a result, CC exhibits relative high
ꢀ
activity in inhibiting OH-induced oxidation of DNA, while more
hydroxyl groups contained do not increase the activities of OPHCC
and MPHCC.
Fig. 1. The increase of the absorbance at 535 nm in the absence and presence of
0.40 mM coumarin-substituted chalcones in the mixture of 2.0 mg/mL DNA, 5.0 mM
Cu2þ, and 3.0 mM GSH.
Fig. 2. The percentages of TBARS in the mixture of 2.0 mg/mL DNA, 4.0 mM H2O2,
2.0 mM TCHQ, and 0.40 mM coumarin-substituted chalcones at 37 ꢁC for 30 min.

G.-L. Xi, Z.-Q. Liu / Tetrahedron 70 (2014) 8397e8404
8399
As shown in Scheme 2, we have synthesized some natural
[AAPH] sꢅ1 31
) because both sodium salt of DNA and AAPH are dis-
ꢀ
licochalcones and evaluated their abilities to inhibit OH-induced
solved in water, and radicals resulting from AAPH can attack DNA at
the same phase.32 The values of n of coumarin-substituted chalcones
are the product of the coefficients in the equation and
oxidation of DNA.28 It was found that HMP, DHM, and HMT cannot
ꢀ
prohibit OH-induced oxidation of DNA as efficiently as coumarin-
substituted chalcones because the TBARS percentages of licochal-
cones (all the percentages of TBARS are higher than 90.0%) are
higher than those of coumarin-substituted chalcones (all the per-
centages of TBARS are lower than 85%). So, 7-hydroxylcoumarin is
able to enhance the inhibitory effects on ꢀOH-induced oxidation of
DNA even in the case of few hydroxyl group contained (as CC,
whose percentage of TBARS is 64.0%).
Ri¼Rg¼1.4ꢄ10ꢅ6ꢄ40 mM sꢅ1¼3.36
m
M minꢅ1 (see Table 1). For ex-
ample, the coefficient in tinhw[OHCC] is 0.61, which multiplies
3.36 m
M minꢅ1 to give the n of OHCC, 2.07. So, OHCC can trap w2
radicals in protecting DNA against AAPH-induced oxidation. Ac-
cordingly, the n values of MPHCC, VCC, and OPHCC are 2.92, 2.96, and
3.86, implying that MPHCC, VCC, and OPHCC can trap w3, w3, and
w4 radicals, respectively. The antioxidant effectiveness follows the
ꢀ
Scheme 2. Some natural licochalcones applied to inhibit OH- and AAPH-induced oxidation of DNA as reported in our previous work.28
2.3. Effects of coumarin-substituted chalcones on AAPH-
induced oxidation of DNA
order of OPHCC>VCCwMPHCC>OHCC. The characteristic structure
of the aforementioned compounds is to contain hydroxyl group at
chalcone moiety. The antioxidant activity of ortho-hydroxyl group (in
OHCC) is not as high as para-hydroxyl group (in VCC and MPHCC).
But ortho-, para-dihydroxyl groups (in OPHCC) increase the antiox-
idant activity more markedly than meta-, ortho-dihydroxyl groups
(in MPHCC). This is not in agreement with previous report that
dihydroxyl groups at adjacent position are beneficial for increasing
the antioxidant activity.33 However, as reported in our previous
study on licochalcones, the n values of HMP, DHM and HMT are 1.68,
2.42, and 5.61, respectively (see Scheme 2),28 while the n value of
OPHCC is 3.86, indicating that double hydroxyl groups at meta-po-
sition may increase the inhibitory effect on AAPH-induced oxidation,
and ortho-, para-dihydroxyl groups may interact with the hydroxyl
group at coumarin moiety via intramolecular synergistic effect to
enhance the antioxidant activity.
The inhibitory effects of coumarin-substituted chalcones on
Cu2þ/GSH-, ꢀOH-, and AAPH-induced oxidation of DNA can be
summarized by Scheme 3. The peroxyl radical resulting from AAPH
is able to abstract hydrogen atom at C0-4 position of DNA, and the
produced single electron at DNA can be recovered by the hydroxyl
group of CC (taken as the example compound). The hydroxyl group
and/or carbonyl group are able to chelate copper ion in inhibiting
Cu2þ/GSH-mediated oxidation of DNA, while CC can quench ꢀOH by
donating its hydrogen atom or by accepting ꢀOH as an electrophile.
The guanine bases in DNA can be oxidized by peroxyl radicals
generated from AAPH,29 and the oxidative process can also be fol-
lowed by measuring TBARS. The absorbance line in the blank ex-
periment indicates that the amount of TBARS increases with the
reaction period (see Fig. 3).
The additions of CC and MNCC cannot inhibit the increase of
TBARS even the concentration increases to 400
droxyl group at coumarin moiety is not active in this case. But the
addition of 400 M PMCC retards the increase of the absorbance line
mM. Hence, the hy-
m
for a period, and then the absorbance line recovers as the blank
experiment. The inhibition period (tinh) can be measured by the
cross-point from the tangent lines for the inhibition and oxidation
period. Although the para-methoxyl group in PMCC cannot form
a conjugation system with the hydroxyl group at coumarin moiety, it
is still beneficial for enhancing the antioxidant ability of PMCC. This
intramolecular synergistic effect among hydroxyl groups via a long
distance was found in our previous work, in which ferrocene group
can enhance the antioxidant effect of hydroxyl group in ailanthoidol
on AAPH-induced oxidation of DNA.30 The additions of VCC, OHCC,
OPHCC, and MPHCC can generate tinh (see Fig. 3), which is measured
and plotted versus the concentration as shown in Fig. 4. The lines in
Fig. 4 are expressed by the quantitative equations as listed in Table 1.
The tinh is proved to be proportionally related to the concen-
tration of the antioxidant as shown as Eq. 1.31
2.4. Rate constant of coumarin-substituted chalcones to
ꢀ
scavenge ABTSD and DPPH
tinh ¼ ðn=RiÞ ½antioxidantꢂ
(1)
ꢀ
The stoichiometric factor (n) means the number of the radical-
propagation terminated by one molecule of the antioxidant. Ri, the
initiation rate of the radical-induced reaction, is assumed to be equal
to the generation rate (Rg) of radicals (Rg¼(1.4ꢃ0.2)ꢄ10ꢅ6
ABTSþ and DPPH are usually used to test the ability of an an-
tioxidant to trap radicals.34,35 The rate constants (k) in trapping
ABTSþꢀ and DPPH are important index for antioxidative ability. The
additions of VCC, OHCC, OPHCC, and MPHCC lead to the decay of

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G.-L. Xi, Z.-Q. Liu / Tetrahedron 70 (2014) 8397e8404
Fig. 3. The variation of the absorbance of TBARS in the mixture of 2.0 mg/mL DNA, 40 mM AAPH, and various concentrations of coumarin-substituted chalcones at 37 ꢁC.
the concentrations of ABTSþꢀ and DPPH (see Fig. 5), indicating that
these chalcones can quench radicals, while others cannot. There-
fore, the hydroxyl group at coumarin moiety does not exhibit
radical-scavenging property, and the hydroxyl groups at chalcone
moiety play the major role in this case.
After the data in Fig. 5 are input into statistical software, the
ꢀ
relationship between the concentrations of ABTSþ and DPPH
([radical]) and reaction period (t) fits for a double exponential
function (Eq. 2), and the results are listed in Tables 2 and 3.
½radicalꢂ ¼ A eꢅðt=aÞ þ B eꢅðt=bÞ þ C
(2)
Moreover, as shown as Eq. 3, the differential style of Eq. 2 reveals
the variation of the reaction rate (r¼ꢅd[radical]/dt) with the re-
action period (t). The results are contained in Tables 2 and 3 as well.
ꢅd½radicalꢂ=dt ¼ r ¼ ðA=aÞeꢅðt=aÞ þ ðB=bÞeꢅðt=bÞ
The reaction rate at t¼0 (r0) can be calculated by Eq. 3 when t is
assigned to be0 (seeTables 2 and 3). According tothe kinetic equation
(Eq. 4), the reaction rate at t¼0 (r0) is related to the concentrations of
radical and antioxidant at the beginning of the reaction.
(3)
Fig. 4. The linear relationship between the concentrations of coumarin-modified chal-
cones and inhibition period (tinh) in protecting DNA against AAPH-induced oxidation.
Table 1
The equations of
tinhw[coumarin-substituted chalcones] and n of coumarin-
substituted chalcones in protecting DNA against AAPH-induced oxidationa
r0 ¼ k½radicalꢂ0½antioxidantꢂ0
The concentrations of radical and antioxidant at t¼0 together
with r0 are known, and rate constant (k) can be calculated by Eq. 5.
(4)
Antioxidant
tinh (min)¼(n/Ri) [coumarin-substituted
n
chalcones (
m
M)]þconstantb
OHCC
MPHCC
VCC
tinh¼0.61 (ꢃ0.03) [OHCC]þ66.96 (ꢃ3.35)
tinh¼0.87 (ꢃ0.04) [MPHCC]þ127.01 (ꢃ6.35)
tinh¼0.88 (ꢃ0.04) [VCC]þ150.94 (ꢃ7.55)
tinh¼1.15 (ꢃ0.06) [OPHCC]þ24.85 (ꢃ1.24)
2.07(ꢃ0.10)
2.92(ꢃ0.15)
2.96(ꢃ0.15)
3.86(ꢃ0.19)
r0
k ¼
(5)
OPHCC
½radicalꢂ0½antioxidantꢂ0
a
Ri¼Rg¼1.4ꢄ10ꢅ6 [AAPH] sꢅ1¼3.36
m
M
minꢅ1 when 40 mM AAPH was
This method has been applied to calculate the rate constant (k)
employed, thus, n¼coefficientꢄ3.36
m .
M minꢅ1
ꢀ
of dihydropyrimidine in trapping ABTSþ and DPPH,36 and herein,
b
The constant was generated from the linear regression analysis.

G.-L. Xi, Z.-Q. Liu / Tetrahedron 70 (2014) 8397e8404
8401
Scheme 3. A plausible mechanism for the oxidative modes of DNA by different initiators.
the k values of VCC, OHCC, OPHCC, and MPHCC are also calculated
and listed in Tables 2 and 3. Only one hydroxyl group at chalcone
moiety (in VCC and OHCC) exhibits a weak radical-scavenging
ability since the k values of VCC and OHCC are lower than those
ꢀ
of OPHCC and MPHCC in trapping either ABTSþ or DPPH. ortho-,
para-Dihydroxyl groups increase the ability of OPHCC to trap these
two radicals. In particular, meta-, para-dihydroxyl groups increase
ꢀ
the k of MPHCC to the highest value, especially, in trapping ABTSþ
.
Hence, dihydroxyl groups at adjacent position in chalcone moiety
possess high abilities to donate its hydrogen atom to N-centered
ꢀ
radical (as DPPH) and to reduce radical directly (as ABTSþ ).
The present result can be employed to justify the function of
coumarin moiety. The compound CC with a hydroxyl group
ꢀ
attaching to coumarin cannot trap ABTSþ and DPPH, indicating
that the coumarin moiety is not active group for trapping radicals.
This phenomenon is also found in PMCC and MNCC, in which only
one hydroxyl group is contained at coumarin moiety. On the con-
trary, OHCC contains a hydroxyl group at ortho-position of benzene
ring and is able to trap ABTSþꢀ and DPPH with the rate constants (k)
being 2.63 and 4.23 mMꢅ1 ꢅ1, respectively. Thus, the hydroxyl
s
group at benzene ring plays the major role in trapping radicals.
ꢀ
Furthermore, MPHCC traps ABTSþ and DPPH with the rate con-
stants (k) being 148.0 and 10.70 mMꢅ1 ꢅ1, respectively, while, as
s
reported in our previous work, the k values of catechol are only 2.58
ꢀ
and 1.30 mMꢅ1 sꢅ1 to trap ABTSþ and DPPH, respectively.37 As the
major functional group for trapping radicals, the activity of the
catechol moiety in MPHCC is markedly enhanced by the coumarin
moiety, and an intermolecular synergistic interaction is proved to
be existed in coumarin-substituted chalcones.
3. Conclusion
Integrating coumarin with chalcone is a useful way to construct
novel antioxidant. The antioxidant activity of hydroxyl group at
chalcone can be enhanced by coumarin even in the absence of
a conjugation system. The hydroxyl groups at different positions
display different activities in inhibiting the oxidation of DNA.
ꢀ
Fig. 5. Decay of 60
cones, and decay of 263
chalcones.
m
M ABTSþ in the presence of 10
m
M coumarin-substituted chal-
m
M DPPH in the presence of 15
mM coumarin-substituted

8402
G.-L. Xi, Z.-Q. Liu / Tetrahedron 70 (2014) 8397e8404
Table 2
ꢀ
ꢀ
Equation of [ABTSþ ]wt and its differential style (ꢅd[ABTSþ ]/dtwt), reaction rate at t¼0 (r0), and rate constant (k)a
ꢀ
ꢀ
Compound
OHCC
Equation of [ABTSþ
(
t
m
M)]wt (s)
Equation of ꢅd[ABTSþ ]/dtwt
r0
(
m
M sꢅ1
)
k (mMꢅ1 sꢅ1
2.63
)
þ$
t
t
t
ꢂ
½ABTSþ$ꢂ ¼ 18:83eꢅ
þ 12:73e
þ 28:11
þ 35:53
þ 25:82
þ 26:96
ꢅd½ABTS ¼ 1:54eꢅ
þ 0:04eꢅ
1.58
3.54
ꢅ340:11
12:24
12:24
340:11
dt
þ$
t
t
t
t
ꢂ
VCC
½ABTSþ$ꢂ ¼ 15:03eꢅ þ 9:50eꢅ
ꢅd½ABTS ¼ 3:52eꢅ þ 0:02eꢅ
5.89
4:27
480:74
4:27
480:74
dt
þ$
t
t
t
t
ꢂ
OPHCC
½ABTSþ$ꢂ ¼ 25:56eꢅ þ 8:67eꢅ
ꢅd½ABTS ¼ 6:50eꢅ þ 0:02eꢅ
6.52
10.86
3:93
523:51
3:93
523:51
dt
þ$
t
t
t
t
ꢂ
MPHCC
½ABTSþ$ꢂ ¼ 21:21eꢅ þ 10:53eꢅ
ꢅd½ABTS ¼ 88:38eꢅ þ 0:52eꢅ
88.90
148.0
0:24
20:06
0:24
20:06
dt
ꢀ
a
The concentration of coumarin-substituted chalcone is 10
m
M, and the concentration of ABTSþ is 60.06
m
M.
Table 3
Equation of [DPPH]wt and its differential style (ꢅd[DPPH]/dtwt), reaction rate at t¼0 (r0), and rate constant (k)a
Compound
Equation of [DPPH (
m
M)]wt
Equation of ꢅd[DPPH]/dtwt
r0
(
m
M sꢅ1
)
k (mMꢅ1 sꢅ1
)
t
t
t
t
½DPPHꢂ ¼ 89:35eꢅ þ 39:78eꢅ
5:36
1235:41
5:36
1235:41
OHCC
VCC
þ 133:60
þ 133:61
þ 57:04
þ 63:16
ꢅd½DPPHꢂ ¼ 16:67eꢅ þ 0:03eꢅ
16.70
22.57
30.81
42.22
4.23
5.72
dt
t
t
t
t
½DPPHꢂ ¼ 107:89eꢅ þ 41:39eꢅ
ꢅd½DPPHꢂ ¼ 22:52eꢅ þ 0:05eꢅ
4:79
827:33
4:79
827:33
dt
t
t
t
t
OPHCC
MPHCC
a
½DPPHꢂ ¼ 129:47eꢅ þ 76:45eꢅ
ꢅd½DPPHꢂ ¼ 30:75eꢅ þ 0:06eꢅ
7.81
4:21
1293:15
4:21
1293:15
dt
t
t
t
t
½DPPHꢂ ¼ 169:14eꢅ þ 30:67eꢅ
ꢅd½DPPHꢂ ¼ 42:18eꢅ þ 0:04eꢅ
10.70
4:01
720:02
4:01
720:02
dt
The concentration of coumarin-substituted chalcone is 15
m
M, and concentration of DPPH is 263.08
m
M.
Dihydroxyl groups at adjacent position can inhibit Cu2þ/GSH-in-
duced oxidation of DNA and trap radicals efficiently, while dihy-
droxyl groups at ortho-, para-position are useful for protecting DNA
against AAPH-induced oxidation. The hydroxyl group at coumarin
moiety plays the major role in inhibiting ꢀOH-induced oxidation of
DNA. Therefore, coumarin-substituted chalcones can be widely
applied as antioxidants to inhibit the various styles of the oxidation.
reagents, the hydroxyl group was etherized by reacting with benzyl
chloride. In brief, the preparation of 2-phenylmethoxyl
benzaldehyde was taken as an example. 2-Hydroxybenzaldehyde
(1.22 g, 10 mmol) and K2CO3 (2.07 g, 15 mmol) were refluxed in
10 mL of 95% ethanol for 0.5 h, then benzyl chloride (1.55 g,
12 mmol) was added and refluxed for 6 h. The reaction mixture was
cooled to room temperature and filtered to remove inorganic salt.
The organic solvent was removed under vacuum to yield 2-phe-
nylmethoxylbenzaldehyde. The yields of this reaction were gener-
ally >90%.
4. Materials and methods
4.1. Materials and instrumentation
4.2.5. ClaiseneSchmidt condensation. Acetyl coumarin (0.436 g,
2.0 mmol), hydroxyl protected-benzaldehyde (2.5 mmol), and pi-
peridine (0.25 mL) were mixed in 15 mL of ethanol and refluxed for
6 h. The reaction mixture was then cooled to room temperature,
and the precipitates were filtered to obtained crude product, fol-
lowed by recrystallization with 95% ethanol to afford hydroxyl-
protected coumarin-modified chalcones.
Diammonium salt of 2,20-azinobis(3-ethylbenzothiazoline-6-
sulfonate) (ABTS salt) and DPPH were purchased from Fluka
Chemie GmbH, Buchs, Switzerland. AAPH, GSH, and the naked DNA
sodium salt were purchased from Acros Organics, Geel, Belgium.
Other agents were of analytical grade and used directly. The
structures of coumarin-modified chalcones were identified by 1H
and 13C NMR (Varian Mercury 300 NMR spectrometer).
4.2.6. Debenzylation from hydroxyl group. The protective group for
the hydroxyl group was removed by TiCl4. In brief, hydroxyl-
protected coumarin-modified chalcone (1.0 mmol, dissolved in
10 mL of anhydrous CH2Cl2) was added dropwisely to 10 mL of
2.0 M CH2Cl2 solution of TiCl4 within 30 min at 0 ꢁC and stirred
overnight at room temperature. The reaction mixture was poured
into ice-cold water and extracted with ethyl acetate. The organic
phase was washed with brine and dried over Na2SO4. The solvent
was evaporated under vacuum. The crude product was purified by
silica chromatography with ethyl acetate/chloroform (1:1, v:v) be-
ing eluent. The NMR data of coumarin-modified chalcones were
listed as following.
4.2. Synthesis and structural characterization of coumarin-
substituted chalcones
4.2.1. Preparation of coumarin. A mixture of resorcinol (3.30 g,
30 mmol) and ethyl acetoacetate (3.90 g, 30 mmol) was added to
98% H2SO4 under stirring at 0e10 ꢁC for 2 h and at 25 ꢁC for 2 days.
Then the reaction mixture was poured into ice-cold water to obtain
the solid, which was recrystallized by 95% ethanol to afford cou-
marin at 80% yield. Mp 181e183 ꢁC.
4.2.2. Esterification of coumarin. Coumarin (4.36 g, 20 mmol) was
refluxed in acetic anhydride (10 mL) for 2 h. Then the reaction
mixture was poured into ice-cold water to obtain the solid, which
was recrystallized by 95% ethanol to afford coumarin acetate at 95%
yield. Mp 133e134 ꢁC.
CC, yield 76%. Mp 150e151 ꢁC. 1H NMR (300 MHz, CDCl3)
d: 13.81
(s,1H), 8.24e8.29 (m,1H), 7.95e8.00 (m,1H), 7.67e7.75 (m, 3H), 7.44
(s, 3H), 6.96 (d, J¼9 Hz, 1H), 6.20 (s, 1H), 2.44 (s, 3H). 13C NMR
(75 MHz, CDCl3) d: 193.4, 167.3, 159.3, 154.8, 153.1, 145.9, 134.8, 131.1,
131.0, 130.2, 129.1, 128.2, 126.1, 125.3, 115.2, 112.0, 111.1, 109.6, 19.3.
PMCC, yield 84%. Mp 176e178 ꢁC. 1H NMR (300 MHz, CDCl3)
14.01 (s, 1H), 8.15e8.20 (m, 1H), 7.95e8.00 (m, 1H), 7.66e7.72 (m,
3H), 6.93e6.98 (m, 3H), 6.19 (s, 1H), 3.87 (s, 3H), 2.44 (s, 3H). 13
NMR (75 MHz, CDCl3) : 193.1, 167.3, 162.1, 159.3, 154.7, 153.1, 146.0,
d
:
4.2.3. Formation of acetyl coumarin. Coumarin acetate (2.18 g,
10 mmol) was mixed with AlCl3 (2 g, 15 mmol) and heated at 160 ꢁC
for 2 h. The reaction mixture was then cooled to room temperature
and diluted by adding HCl aqueous solution. The obtained solid was
recrystallized by 95% ethanol to afford acetyl coumarin at 72% yield.
Mp 161e163 ꢁC.
C
d
130.9, 130.7, 127.6, 123.6, 115.1, 114.5, 111.9, 110.9, 109.7, 55.4, 19.2.
MNCC, yield 56%. Mp 224e226 ꢁC. 1H NMR (300 MHz, DMSO-d6)
d
: 10.98 (s, 1H), 8.58 (s, 1H), 8.26 (d, J¼7.8 Hz, 2H), 7.69e7.76 (m,
2H), 7.55e7.60 (m, 1H), 7.34e7.40 (m, 1H), 6.97 (d, J¼8.7 Hz, 1H),
6.20 (s, 1H), 2.42 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
: 192.3,
4.2.4. Protection of hydroxyl group in benzaldehyde. Before the
hydroxyl-substituted benzaldehydes were employed as the
d

G.-L. Xi, Z.-Q. Liu / Tetrahedron 70 (2014) 8397e8404
8403
159.5,158.2,153.5,151.6,148.3,143.1,136.1,134.2,130.3,127.4,124.9,
123.7, 115.0, 112.8, 112.1, 110.6, 18.3.
respectively. The effects of coumarin-substituted chalcones on ꢀOH-
induced oxidation of DNA were expressed by Adetect/A0ꢄ100.
VCC, yield 81%. M.p 240e241 ꢁC. 1H NMR (300 MHz, DMSO-d6)
d
: 10.92 (s, 1H), 9.74 (s, 1H), 7.71 (d, J¼8.7 Hz, 1H), 7.32 (d, J¼1.8 Hz,
4.5. AAPH-induced oxidation of DNA test
1H), 7.20e7.25 (m, 1H), 7.13 (dd, J¼8.4, 2.1 Hz, 1H), 7.00e7.05 (m,
1H), 6.95 (d, J¼8.7 Hz, 1H), 6.78 (d, J¼8.1 Hz, 1H), 6.18 (d, J¼1.2 Hz,
The experiment of AAPH-induced oxidation of DNA was per-
formed as the description in a literature.23 Briefly, 2.0 mg/mL DNA,
40 mM AAPH, and a certain concentration of coumarin-substituted
chalcones (dissolved in DMSO as the stock solution) were mixed to
form a solution. The solution was poured into test tubes, and each
test tube contained 2.0 mL. The test tubes were incubated at 37 ꢁC
to initiate the oxidation of DNA, and three of them were taken out
at every 2 h and cooled immediately. The following operation was
1H), 3.80 (s, 3H), 2.41 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
d: 192.2,
159.7, 158.2, 153.6, 151.5, 149.9, 148.0, 146.9, 127.0, 125.6, 125.2,
123.7, 115.6, 115.5, 112.8, 112.0, 111.8, 110.5, 55.7, 18.3.
OHCC, yield 88%. Mp 204e206 ꢁC. 1H NMR (300 MHz, DMSO-d6)
d
: 10.96 (s, 1H), 10.21 (s, 1H), 7.71 (d, J¼8.7 Hz, 1H), 7.65 (dd, J¼7.8,
1.2 Hz, 1H), 7.56e7.61 (m, 1H), 7.27 (td, J¼7.5, 1.5 Hz, 1H), 7.12e7.17
(m, 1H), 6.96 (d, J¼8.7 Hz, 1H), 6.89 (d, J¼8.1 Hz, 1H), 6.85 (t,
J¼7.5 Hz, 1H), 6.19 (d, J¼1.2 Hz, 1H), 2.42 (s, 3H). 13C NMR (75 MHz,
ꢀ
the same as in OH-induced oxidation of DNA except the heating
DMSO-d6)
d
: 192.6, 159.6, 158.2, 157.0, 153.7, 151.5, 141.4, 132.4,
period was 15 min after TBA and trichloroacetic acid were added.
The absorbance of TBARS was plotted versus the incubation period.
128.8, 127.3, 127.2, 120.8, 119.6, 116.3, 115.4, 112.8, 112.0, 110.6, 18.3.
OPHCC, yield 88%. Mp 197e198 ꢁC. 1H NMR (300 MHz, DMSO-
ꢀ
4.6. Scavenging DPPH and ABTSD
d6)
d
: 10.91 (s, 1H), 10.11 (s, 1H), 10.00 (s, 1H), 7.69 (d, J¼8.7 Hz, 1H),
7.49 (d, J¼5.4 Hz, 1H), 7.45 (d, J¼1.8 Hz, 1H), 6.96 (d, J¼3.3 Hz, 1H),
6.92 (d, J¼3.9 Hz, 1H), 6.33 (d, J¼2.4 Hz, 1H), 6.29 (dd, J¼8.7, 2.4 Hz,
DPPH was dissolved in 50 mL of ethanol to make the absorbance
ꢀ
1H), 6.18 (s, 1H), 2.41 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
d
: 192.3,
around 1.00 at 517 nm (εDPPH¼4.09ꢄ103 Mꢅ1 cmꢅ1). ABTSþ was
161.8, 159.7, 158.9, 158.2, 153.7, 151.5, 142.3, 130.5, 127.0, 123.9, 115.7,
112.8, 112.7, 112.0, 110.5, 108.3, 102.5, 18.3.
produced from 2.0 mL of a mixture containing 4.0 mM ABTS
aqueous solution and 1.41 mM K2S2O8 after kept for 16 h and di-
luted by 100 mL of ethanol. The absorbance of ABTSþꢀ solution was
MPHCC, yield 81%. Mp >266 ꢁC (decomp.). 1H NMR (300 MHz,
ꢀ
4
around 1.00 at 734 nm (εABTS ¼1.6ꢄ10 Mꢅ1 cmꢅ1). The DMSO
þ
DMSO-d6)
1H), 7.14e7.19 (m, 1H), 7.06 (s, 1H), 6.92e6.99 (m, 2H), 6.74e6.85
(m, 2H), 6.17 (s, 1H), 2.40 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
d: 10.93 (s, 1H), 9.72 (s, 1H), 9.17 (s, 1H), 7.70 (d, J¼8.7 Hz,
solutions of coumarin-modified chalcones (0.1 mL) were added to
ꢀ
d:
1.9 mL of DPPH or ABTSþ solution. The final concentrations of
191.9,159.6,158.2,153.6,151.5,149.0,146.8,145.7,127.1,125.5,124.6,
122.4, 115.8, 115.4, 114.9, 112.8, 112.0, 110.5, 18.3.
coumarin-substituted chalcones were 10 and 15 mM in trapping
ABTSþꢀ and DPPH, respectively. The decreases of the absorbance of
these radicals were recorded at 25 ꢁC at a certain time interval.
4.3. Cu2D/GSH-induced oxidation of DNA test
4.7. Statistical analysis
Cu2þ/GSH-induced oxidation of DNA was carried out following
a previous report24 with a slight modification. Briefly, DNA, CuSO4,
and GSH were dissolved in phosphate buffered solution (PBS1:
6.1 mM Na2HPO4, 3.9 mM NaH2PO4), and coumarin-substituted
chalcones were dissolved in dimethyl sulfoxide (DMSO). Then,
2.0 mg/mL DNA, 5.0 mM Cu2þ, 3.0 mM GSH, and 0.4 mM coumarin-
substituted chalcones were mixed to form a solution. The solution
was poured into test tubes, and each test tube contained 2.0 mL.
The test tubes were incubated at 37 ꢁC to initiate the oxidation of
DNA, and three of them were taken out at every 30 min and cooled
immediately. PBS1 solution of EDTA (1.0 mL, 30.0 mM) was added to
chelate Cu2þ, followed by adding 1.0 mL of thiobarbituric acid (TBA)
solution (1.00 g of TBA and 0.40 g of NaOH dissolved in 100 mL of
PBS1) and 1.0 mL of 3.0% trichloroacetic acid aqueous solution. The
test tubes were heated in boiling water for 30 min and cooled to
room temperature, 1.5 mL of n-butanol was added and shaken
vigorously to extract thiobarbituric acid reactive substance (TBARS)
whose absorbance was measured at 535 nm.
All the data were the average value from at least three in-
dependent measurements with the experimental error within 10%.
The equations were analyzed by one-way ANOVA in Origin 8 pro-
fessional software, and p<0.001 indicated a significant difference.
Acknowledgements
Financial support from Jilin Provincial Foundation for Natural
Science, China, (20130206075GX) is acknowledged gratefully.
Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2014.08.063.
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