Antioxidant effectiveness generated by one or two phenolic hydroxyl groups in coumarin-substituted dihydropyrazoles

Article abstract of DOI:10.1016/j.ejmech.2013.06.059

A cascade operation was designed to synthesize nine coumarin-substituted dihydropyrazoles with only one or two phenolic hydroxyl groups contained. Antioxidant abilities of the obtained compounds were evaluated by inhibiting 2,2′-azobis(2-amidinopropanehydrochloride) (AAPH)-, Cu²⁺/ glutathione (GSH)-, and ^.OH-induced oxidation of DNA. It was found that less phenolic hydroxyl groups can enhance the abilities of coumarin-substituted dihydropyrazoles to protect DNA against the oxidation. Moreover, these coumarin-substituted dihydropyrazoles were employed to scavenge 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS^+.), 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH), and galvinoxyl radical, respectively. It was found that double phenolic hydroxyl groups were more beneficial for enhancing the abilities of coumarin-substituted dihydropyrazoles to quench the aforementioned radicals. Therefore, dihydropyrazole linked with coumarin exhibited powerful antioxidant effectiveness even in the case of less phenolic hydroxyl groups involved.

Full text of DOI:10.1016/j.ejmech.2013.06.059

European Journal of Medicinal Chemistry 68 (2013) 385e393
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antioxidant effectiveness generated by one or two phenolic hydroxyl
groups in coumarin-substituted dihydropyrazoles
*
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:
A cascade operation was designed to synthesize nine coumarin-substituted dihydropyrazoles with only one
or two phenolic hydroxyl groups contained. Antioxidant abilities of the obtained compounds were eval-
uated by inhibiting 2,20-azobis(2-amidinopropanehydrochloride) (AAPH)-, Cu2þ/glutathione (GSH)-, and
.OH-induced oxidation of DNA. It was found that less phenolic hydroxyl groups can enhance the abilities of
coumarin-substituted dihydropyrazoles to protect DNA against the oxidation. Moreover, these coumarin-
substituted dihydropyrazoles were employed to scavenge 2,20-azinobis(3-ethylbenzothiazoline-6-
sulfonate) cationic radical (ABTSþ.), 2,20-diphenyl-1-picrylhydrazyl radical (DPPH), and galvinoxyl radical,
respectively. It was found that double phenolic hydroxyl groups were more beneficial for enhancing the
abilities of coumarin-substituted dihydropyrazoles to quench the aforementioned radicals. Therefore,
dihydropyrazole linked with coumarin exhibited powerful antioxidant effectiveness even in the case of less
phenolic hydroxyl groups involved.
Received 20 March 2013
Received in revised form
20 June 2013
Accepted 26 June 2013
Available online 13 August 2013
Keywords:
Coumarin
Dihydropyrazole
Antioxidant
Oxidation of DNA
Free radical
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
decomposition of DNA [10]. Hence, it is worthy to explore whether
a compound containing less than two phenolic hydroxyl groups can
The in vivo oxidative stress of DNA is regarded as the patho-
genesis of many fatal diseases [1]. The free radicals from the
metabolism [2] or the environment [3] may abstract hydrogen
atom from membrane, lipid, protein, and DNA, and consequently,
lead to damages of many biological species [4]. The supplementa-
tion of antioxidants is beneficial for avoiding oxidative damages [5],
keeping redox balance in vivo [6], and designing novel medicines
[7]. The natural antioxidants attract much research attentions
because of their high capacities and low toxicities. Natural poly-
phenols are antioxidants widely used to scavenge radicals [8]. On
the other hand, many efforts are contributed to synthesize anti-
oxidants containing phenolic hydroxyl groups [9]. Increasing the
amount of phenolic hydroxyl groups may enhance the antioxidant
effectiveness according to traditional concept. However, it was
recently found that more phenolic hydroxyl groups contained may
cause prooxidative effect and improve the risk in the biological
systems. For example, hydroxycinnamic acids containing more than
two phenolic hydroxyl groups accelerated the oxidation of DNA in
the presence of Cu(II) ions. This was because phenolic hydroxyl
groups can reduce Cu(II) to form Cu(I), and Cu(I) can cause the
also function as an antioxidant.
2. Chemistry
The modification on the structure of natural antioxidants [11]
and the recombination of known antioxidative structures [12]
are the efficient ways to increase the antioxidant ability. For
example, polyphenols were used to link with 4-amino
methylbenzylamine or tris(2-aminoethyl)amine to form dendritic
structure. As a result, the abilities of the obtained antioxidants to
scavenge 2,20-diphenyl-1-picrylhydrazyl radical (DPPH) [13] and to
inhibit 2,20-azobis(2-amidinopropane hydrochloride) (AAPH, ReN]
NeR, R ¼ -CMe2C(¼NH)NH2)- and Cu2þ-induced oxidations of low-
density lipoprotein and DNA were improved markedly [14]. More-
over, we applied pyrazole [15]and 1,2,4-oxadiazole [16] to bridge with
phenols. It has been found that single phenolic hydroxyl group in
these compounds exhibited high abilities to inhibit AAPH-induced
oxidation of DNA and to scavenge radicals. This result reveals that a
suitable arrangement of every structural feature can lead to high
antioxidant effectiveness even in the case of less phenolic hydroxyl
group involved. On the other hand, coumarin is an important skeleton
widely used to construct anticancer [17], antimicrobial [18], antiviral
[19], and anti-inflammatory drugs [20]. The aim of this work is to
synthesize coumarin-related compounds with less than two phenolic
* Corresponding author. Tel.: þ86 431 88499174; fax: þ86 431 88499159.
E-mail address: zaiqun-liu@jlu.edu.cn (Z.-Q. Liu).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.059

386
G.-L. Xi, Z.-Q. Liu / European Journal of Medicinal Chemistry 68 (2013) 385e393
hydroxyl groups contained, and to screen the antioxidant effective-
ness. Thus, as shown in Scheme 1, we herein design a series of com-
pounds with dihydropyrazole linking with coumarin and two
benzene rings for comparing the antioxidant effectiveness.
The peaks in 1H NMR spectra of these compounds can be
generally divided into three parts. The first part located around 8e
10 ppm, which can be assigned to the hydrogen atom in eOH. The
second part ranged from 6 to 8 ppm, which was contributed from
the hydrogen atoms in the aromatic rings. The last part ranged from
3 to 6 ppm, which was donated to CH2 and CH in dihydropyrazole.
Accordingly, the peaks in 13C NMR spectra of these compounds can
be divided into two parts. The first part ranged from 100 to
160 ppm, which can be assigned to the carbon atoms at aromatic
rings. Other peaks were lower than 100 ppm, which was contrib-
uted from CH2 and CH in dihydropyrazole.
3. Pharmacology
The antioxidant effectiveness can be estimated by many
methods [21]. We herein evaluated the abilities of the obtained
compounds to inhibit the oxidation of DNA induced by AAPH [22],
Cu2þ/glutathione (GSH) [23], and hydroxyl radical (.OH) [24] and to
scavenge 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonate) cation
ic radical (ABTSþ.), DPPH, and galvinoxyl radical, respectively.
4.2. Effects of coumarin-substituted dihydropyrazoles on
Cu2þ/GSH-induced oxidation of DNA
4. Results and discussion
The Cu(II) may oxidize GSH to form GSH radical (GS.), while the
produced Cu(I) combines with DNA to form a complex, DNA-Cu(I)e
OOH. The complex can be degraded in the presence of GS., leading
to the formation of the oxidative products of DNA [23].
4.1. Synthesis and spectral interpretation
Acetyl coumarin was prepared by the reaction of 2-
hydroxybenzaldehyde (10.0 mmol) with the same amount of
ethyl acetoacetate (10.0 mmol) in the presence of pyridine and
The oxidative products of DNA can be detected after colorized by
thiobarbituric acid [25]. As shown as the control line in Fig. 1 (the
upper line), the continuous increase of the absorbance reveals
much more oxidative products were produced from the mixture of
DNA, Cu2þ, and GSH with the increase of the reaction period.
However, all the absorbance lines locate below that of the control
experiment, indicating that the addition of all these coumarin-
substituted dihydropyrazoles can decrease the amount of oxida-
tive products of DNA, and thus, these dihydropyrazoles act as an-
tioxidants to protect DNA against Cu2þ/GSH-induced oxidation. It is
worthy to note that 1, 2, and 3 also function as antioxidants even
without phenolic hydroxyl group attaching. This finding is in
agreement with our previous result that 1,3,5-triphenyl-1H-pyr-
azole, a pyrazole derivative without phenolic hydroxyl group
involved, can inhibit Cu2þ/GSH-induced oxidation of DNA [15].
Moreover, the absorbance lines in the presence of other coumarin-
substituted dihydropyrazoles locate at lower positions than those
of non-hydroxyl-involving dihydropyrazoles, demonstrating that
the phenolic hydroxyl group enhances the antioxidant effects of
coumarin-substituted dihydropyrazoles. Especially, the additions of
diethylamine as the catalysts. This step was carried out at 0e10 ꢀ
C
for 3 h, and the yield was >95%. The following reaction was
ClaiseneSchmidt condensation taking place between acetyl
coumarin (2.0 mmol) and benzaldehyde (2.2 mmol) in the presence
of piperidine (0.25 mL) as the catalyst and ethanol as the solvent.
The catalyst used in the condensation between acetyl coumarin and
benzaldehyde was the same as in the synthesis of acetyl coumarin.
The difference was only on the operation. The preparation of acetyl
coumarin was performed at 0e10 ꢀC, and the ClaiseneSchmidt
condensation was carried out under refluxing for 6 h. The yield was
also >95%. Finally, excess phenylhydrazine was added, followed by
the addition of the catalyst (acetic acid) and refluxing for 3 h. Thus,
the isolation after the final step was to remove phenylhydrazine.
The cascade operation was just to change the catalyst from bases
(pyridine, diethylamine, and piperidine) to acid (acetic acid). The
yields of every step were so high that all the reagents were almost
exhausted. Thus, the amount of excess reagents in the first and the
second step was too little to influence the following reaction.
O
O
R1
R2
R3
H
NHNH2
O
R1
or
H
Fe
A
R2
R3
N
H
A
B
O
O
reflux
O
CH3COOH
O
O
O
OH
O
H
O
O
O
or
(C2H5)2NH
N
Fe
0 ~ 5oC
acetyl coumarin
2-hydroxybenzaldehyde
O
O
coumarin-substituted chalcone
Abbreviation
R1
H
R2
H
R3
1
2
4
5
6
7
8
9
H
B
H
H
CH3O
H
B
N
N
OH
H
H
R1
Fe
N
R2
R3
or
N
OH
H
H
A
O
O
H
OH
OH
OH
OH
O
O
H
CH3O
OH
H
3
H
coumarin-substituted dihydropyrazole
OH
Scheme 1. Synthetic routine of coumarin-substituted dihydropyrazoles.

G.-L. Xi, Z.-Q. Liu / European Journal of Medicinal Chemistry 68 (2013) 385e393
387
relative high antioxidant ability to inhibit .OH-induced oxidation of
DNA. On the other hand, the relative high percentage of TBARS in
the presence of 3 may be due to the Fe(II) in ferrocene moiety can
induce the decomposition of H2O2 and generate additional .OH. But
the additional .OH does not make 3 exhibit prooxidant effect
because the antioxidant effect deriving from the structural feature
of dihydropyrazole is high enough and can depress the side effect of
.OH. The methoxyl group in 2 is an electron-donating substituent,
the increase of the TBARS percentage of 2 (75.3%) implies that
electron-donating group is not beneficial for enhancing the ability
Blank
4
5
6
7
9
1
2
3
8
0.6
0.4
0.2
0.0
.
of coumarin-substituted dihydropyrazoles to prohibit OH-caused
oxidation of DNA. This deduction is proved by the increase of the
TBARS percentage of 6 (81.1%) because hydroxyl group in 6 is also
an electron-donating substituent. Moreover, it can be found that
the electron-donating substituent at para-position (R3 position)
does not benefit for the antioxidant capacity, and contrarily, the
hydroxyl group at ortho-position (R1 position) as in 4 and 9 (44.2%
and 35.5%, respectively) can improve the antioxidant effectiveness
markedly.
0
60
120
180
Reaction period (min)
Fig. 1. The increase of the absorbance at 535 nm in the absence and presence of
0.20 mM coumarin-substituted dihydropyrazoles in the mixture of 2.0 mg/mL DNA,
5.0 mM Cu2þ, and 3.0 mM GSH.
4.4. Effects of coumarin-substituted dihydropyrazoles on AAPH-
induced oxidation of DNA
8 and 9 make the absorbance lines locate at the lowest position,
demonstrating that double phenolic hydroxyl groups at either
ortho-, para- or meta-, para-positions are able to increase the abil-
AAPH can provide initiating peroxyl radical [27] for abstracting
hydrogen atom from the guanine bases in DNA [28]. The oxidative
process can be followed by measuring TBARS. As shown in Fig. 3,
the absorbance line of the blank experiment indicates that the
amount of TBARS increases with the reaction period of AAPH-
induced oxidation of DNA.
ities of coumarin-substituted dihydropyrazoles to inhibit Cu2þ
GSH-induced oxidation of DNA.
/
.
It can be seen from Fig. 3 that the additions of 1 and 2 did not
affect the increase of the TBARS absorbance even with the con-
4.3. Effects of coumarin-substituted dihydropyrazoles on OH-
induced oxidation of DNA
centration increasing to 400
mM, revealing that the structural
feature of dihydropyrazole cannot inhibit AAPH-induced oxidation
of DNA. On the other hand, the addition of 3 retarded the increase
of the absorbance line for a period, and then the absorbance line
ꢁOH is produced by the reaction between H2O2 and tetra-
chlorohydroquinone (TCHQ) [24] and can abstract hydrogen atom
from the ribosyl moiety in DNA [26]. As can be seen from Fig. 2, the
absorbance of TBARS was assigned as 100% when 2.0 mg/mL DNA
was incubated with 4.0 mM TCHQ and 2.0 mM H2O2 at 37 ꢀC for
30 min. Other absorbances of TBARS in the presence of 0.20 mM
coumarin-substituted dihydropyrazoles were compared with that
of the control experiment and listed in Fig. 2 as well.
The attractive results were also from the additions of 1, 2, and 3,
which do not involve phenolic hydroxyl groups but can decrease
the TBARS percentage to 67.8%, 75.3%, and 91.8%, respectively. 1, 2,
and 3 still play the antioxidant role in this case. The TBARS per-
centage of 1 is as low as 67.8%, demonstrating that the structural
feature of dihydropyrazole with the aid of coumarin can generate
increased as in the control experiment. This inhibition period (tinh
)
can be measured by the cross-point from the tangent lines for the
inhibition and oxidation period. The antioxidant effectiveness of 3
is ascribed to the introduction of ferrocene moiety. The additions of
other coumarin-substituted dihydropyrazoles exhibit the antioxi-
dant effectiveness, and tinh can be measured even in the case of low
concentration employed. Fig. 4 outlines the linear relationship
between the concentration of coumarin-substituted dihydropyr-
azole and tinh, and Table 1 lists the quantitative equations of these
linear relationships.
As shown as equation (1), chemical kinetics demonstrates that
tinh correlates proportionally with the concentration of the anti-
oxidant [29].
120
100
tinh ¼ ðn=RiÞ½antioxidantꢂ
(1)
91.8
In equation (1) n refers to the stoichiometric factor, revealing the
number of the radical-propagation terminated by one molecule of
the antioxidant, and Ri stands for the initiation rate of the radical-
induced reaction. When equation (1) is employed to treat the re-
sults from AAPH-induced oxidation of DNA, Ri can be regarded as
the radical-generation rate (Rg), Rg ¼ (1.4 ꢃ 0.2) ꢄ 10ꢅ6 [AAPH] sꢅ1
[29], because both sodium salt of DNA and AAPH are dissolved in
water, and radicals generated from AAPH attack DNA at the same
phase [30]. Thus, n of coumarin-substituted dihydropyrazole is the
81.1
75.3
67.8
66.4
70
53.9
44.2
43.4
35.5
20
1
2
3
4
5
6
7
8
9
product
of
the
coefficient
in
m
the
equation
and
Ri ¼ Rg ¼ 1.4 ꢄ 10ꢅ6 ꢄ 40 mM sꢅ1 ¼3.36
M minꢅ1 (see Table 1). For
Blank
example, the coefficient in tinh w [3] is 0.87, the n of 3 is
Fig. 2. The percentages of TBARS in the mixture of 2.0 mg/mL DNA, 4.0 mM H2O2,
2.0 mM TCHQ, and 0.20 mM coumarin-substituted dihydropyrazoles at 37 ꢀC for
30 min.
0.87 ꢄ 3.36
m
M minꢅ1 ¼ 2.92, implying that 3 can terminate 2.92
radical-propagation in AAPH-induced oxidation of DNA.

388
G.-L. Xi, Z.-Q. Liu / European Journal of Medicinal Chemistry 68 (2013) 385e393
1
2
0
M
M
M
M
M
0
M
M
M
M
M
100
200
300
400
100
200
300
400
1.2
0.8
0.4
3
0
M
M
M
M
M
100
200
300
400
tinh
0
300
600
0
300
600
0
300
600
Reaction period (min)
5
6
4
0
M
M
M
M
M
0
M
M
M
M
M
0
M
M
M
M
M
100
200
300
400
100
200
300
400
100
200
300
400
1.2
0.8
0.4
0
300
600
0
300
600
0
300
600
Reaction period (min)
7
8
9
0
50
100
150
200
M
M
M
M
M
0
50
100
150
200
M
M
M
M
M
0
50
100
150
200
M
M
M
M
M
1.2
0.8
0.4
0
300
600
0
300
600
0
300
600
Reaction period (min)
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 dihydropyrazoles at 37 ꢀC.
Accordingly, the n of other coumarin-substituted dihydropyrazoles
are calculated and listed in Table 1. It can be found in Table 1 that
the antioxidant effectiveness of coumarin-substituted dihydropyr-
azoles can be divided into two parts, 7, 8, and 9 can terminate >6
radical-propagation because of double phenolic hydroxyl groups
contained, and 4, 5, and 6 can terminate >3 radical-propagation
because of only one phenolic hydroxyl group contained. This
result indicates that double phenolic hydroxyl groups, especially, at
ortho- and para-position as in 9, are beneficial for increasing the
antioxidant effectiveness, while single phenolic hydroxyl group at
ortho-position as in 4 can enhance the antioxidant effectiveness.
The antioxidant effectiveness is almost proportionally related to the
number of phenolic hydroxyl group. Moreover, the n of 3 (2.93) is
close to that of 5, revealing that the antioxidant effectiveness of a
ferrocene moiety is the same as a meta-phenolic hydroxyl group. As
shown in Scheme 2, we have measured the n of dihydropyrazoles
under the same experimental condition [31].
It can be seen from Scheme 2 that the n of 8 (6.60) is similar to
that of 4a (6.15). The n of 4a is contributed from three phenolic
hydroxyl groups, while double phenolic hydroxyl groups increases
the n value of 8 to the same level, implying that meta-, para-
dihydroxyl groups play the major role in the generation of n value.
On the other hand, when the hydrogen atom in NeH of 3b is
replaced by a benzene ring to form 7, the n value increases

G.-L. Xi, Z.-Q. Liu / European Journal of Medicinal Chemistry 68 (2013) 385e393
389
4.5. Rate constant of coumarin-substituted dihydropyrazoles to
scavenge ABTSþ. And DPPH
6
4
ABTSD., DPPH, and galvinoxyl radical are usually used to test the
ability of an antioxidant to trap radicals [32e34]. As shown in
Figs. 5e7, the additions of these coumarin-substituted dihy-
dropyrazoles except 1, 2, and 3 can lead to the decay of the con-
centrations of these radicals. This fact demonstrates that the
8
5
450
300
150
9
7
3
.
antioxidant abilities of 1, 2, and 3 to inhibit Cu2þ/GSH and OH-
induced oxidation of DNA are not derived from the radical-
scavenging property but from redox behavior. Other coumarin-
substituted dihydropyrazoles are able to trap radicals directly.
We have developed a method to calculate k of antioxidants in
trapping radicals [35]. The first step is to input the data in Figs. 5e7
into statistical software. Consequently, the concentrations of these
radicals ([radical]) and reaction period (t) is suitable for the double
exponential function (equation (2)), and the results are listed in
Tables 2e4.
0
150
300
450
Concentration (µM)
½radicalꢂ ¼ Aeꢅðt=aÞ þ Beꢅðt=bÞ þ C
(2)
Fig. 4. The linear relationship between the concentrations of coumarin-substituted
dihydropyrazoles and inhibition period (tinh in protecting DNA against AAPH-
)
induced oxidation. The p < 0.001 indicated a significance difference of these equations.
Then, the equation (2) is carried out by differential operation to
convert into equation (3), which expresses the relationship be-
tween the reaction rate (r ¼ -d[radical]/dt) and the reaction period
(t).
Table 1
The equations of tinh
w [coumarin-substituted dihydropyrazoles] and n of
coumarin-substituted dihydropyrazoles in protecting DNA against AAPH-induced
ꢅd½radicalꢂ=dt ¼ r ¼ ðA=aÞeꢅðt=aÞ þ ðB=bÞeꢅðt=bÞ
The equation (3) can be used to calculate the reaction rate when
the reaction time is assigned. Hence, when t is assigned to 0, the
reaction rate at the beginning of the reaction (r0) can be obtained
and listed in Tables 2e4.
The equation (4) reveals that the reaction rate (r) is related to
the concentrations of the radical and the antioxidant, in which the
coefficient is the rate constant (k).
(3)
oxidation.a
Antioxidant tinh (min) ¼ (n/Ri) [antioxidant (
m
M)] þ constantb
n
3
4
5
6
7
8
9
tinh ¼ 0.87 (ꢃ0.04) [3] þ 85.50 (ꢃ4.28)
2.93(ꢃ0.15)
3.87(ꢃ0.19)
3.23(ꢃ0.16)
3.50(ꢃ0.17)
6.31(ꢃ0.32)
6.60(ꢃ0.33)
6.92(ꢃ0.35)
tinh ¼ 1.15 (ꢃ0.06) [4] þ 2.05 (ꢃ0.10)
tinh ¼ 0.96 (ꢃ0.05) [5] þ 69.10 (ꢃ3.46)
tinh ¼ 1.04 (ꢃ0.05) [6] þ 63.85 (ꢃ3.19)
tinh ¼ 1.88 (ꢃ0.09) [7] þ 39.70 (ꢃ1.99)
tinh ¼ 1.96 (ꢃ0.10) [8] þ 23.45 (ꢃ1.17)
tinh ¼ 2.06 (ꢃ0.10) [9] þ 18.25 (ꢃ0.91)
a
Ri ¼ Rg ¼ 1.4 ꢄ 10ꢅ6 [AAPH] sꢅ1 ¼ 3.36
m
M minꢅ1 when 40 mM AAPH was
m .
M minꢅ1
r ¼ k½radicalꢂ½antioxidantꢂ
(4)
employed, thus, n ¼ coefficient ꢄ 3.36
b
The constant was generated from the linear regression analysis, and p < 0.001
indicated a significance difference of these equations.
If the concentrations of the radical and the antioxidant at a
certain time are known, and the reaction rate (r) is also measured,
the rate constant (k) can be calculated. At present, the reaction rate
at t ¼ 0 (r0) is obtained, and the concentrations of the radical and
the antioxidant at the beginning of the reaction are known, the rate
constant (k) can be calculated by equation (5), and the results are
listed in Tables 2e4.
remarkably, indicating that benzene ring is beneficial for
increasing the antioxidant effectiveness. Furthermore, when single
phenolic hydroxyl group attaches to the skeleton of 1,3,5-
triphenyl-1H-pyrazole, the n value cannot exceed 2 under the
same experimental condition [15]. The aforementioned compari-
son indicates that the molecular structure containing coumarin
and double benzene rings is suitable for the less phenolic hydroxyl
groups to generate high antioxidant effectiveness. The antioxidant
mechanism can be further elucidated by the measurement of the
rate constant (k) when these coumarin-substituted dihydropyr-
azoles are applied to trap radicals.
r0
k ¼
(5)
½radicalꢂ0½antioxidantꢂ0
The reaction between an antioxidant and ABTSD. reveals the
ability of the antioxidant to reduce the radical, and all sorts of
phenolic hydroxyl group can reduce ABTSD.. 1, 2, and 3 cannot react
with these radicals because of no hydroxyl groups attached. This
Replacing acetyl by benzene
Replacing H by benzene
O
H
N
B
B
N
OH
OH
N
OCH3
OH
N
N
N
A
OCH3
OH
OH
OH
N
N
A
A
A
O
O
O
OH
O
OH
O
O
O
O
Three hydroxyl
groups contribute
to the n value.
n = 6.15
Double hydroxyl
groups contribute
to the n value.
Single hydroxyl
group contributes
to the n value.
n = 4.64
Double hydroxyl
n = 6.31
n = 6.60
groups contribute
to the n value.
VCP
MPCP
4a
3b
Scheme 2. The comparison of n values among structural analogs.

390
G.-L. Xi, Z.-Q. Liu / European Journal of Medicinal Chemistry 68 (2013) 385e393
4
5
6
7
8
9
12
Galvinoxyl
ABTS+.
4
5
6
7
8
9
60
40
8
4
0
500
1000
0
500
1000
Reaction period (s)
Reaction period (s)
Fig. 5. Decay of 60.75
m
M ABTSþꢁ in the presence of 5
mM coumarin-substituted
Fig. 7. Decay of 10.88
m
M galvinoxyl in the presence of 15 mM coumarin-substituted
dihydropyrazoles.
dihydropyrazoles.
trapping DPPH. The hydrogen atom in phenolic hydroxyl groups of
dihydropyrazoles is transferred to O-centered radical, forming a
hydroxyl group in galvinoxyl radical. There is no variation in the
bond energy in this process. One OeH bond is broken and one NeH
bond is formed in the reaction between an antioxidant and DPPH,
the bond energy changes in this process. Therefore, the k value
from the reaction of an antioxidant and galvinoxyl radical reveals
the real rate of the transfer of hydrogen atom.
fact indicates that phenolic hydroxyl group plays the role in trap-
ping radicals, while dihydropyrazole is not active to quench radi-
cals. The rate constants (k) of 4, 5, and 7 are lower than that of other
dihydropyrazoles, revealing that the ability of single phenolic hy-
droxyl group to reduce radical is lower than that of double phenolic
hydroxyl groups. Especially, 9 has the highest k in trapping ABTSD.
,
indicating that ortho-, para-dihydroxyl groups exhibit the strongest
ability to reduce ABTSD.. The reactions between an antioxidant and
DPPH and galvinoxyl radical reveal the abilities of the antioxidant
to donate its hydrogen atom to N-centered and O-centered radicals,
respectively. It can be seen from Table 3 that the k values of 4 and 5
are lower than that of other dihydropyrazoles, indicating that single
phenolic hydroxyl group cannot donate its hydrogen atom to N-
centered radical as fast as the dihydropyrazoles containing double
phenolic hydroxyl groups. In particular, 8 possesses the highest k
value, revealing that dihydroxyl groups at meta-, para-positions are
beneficial for donating hydrogen atoms to N-centered radical. But,
it can be found in Table 4 that the difference of k value among these
dihydropyrazoles in trapping galvinoxyl radical is not as large as in
5. Conclusion
A cascade method is designed to prepare coumarin-substituted
dihydropyrazoles. The continuous operation without the isolation
of the intermediate products increases the efficiency of the syn-
thesis. The coumarin moiety is an important structural feature for
enhancing the antioxidant effectiveness of single or double
phenolic hydroxyl groups in dihydropyrazole. Hence, the structural
style of coumarin and two benzene rings attaching to dihydropyr-
azole exhibits powerful antioxidant potential to inhibit DNA
oxidation. This structural style also exhibits the ability to protect
.
DNA against Cu2þ/GSH and OH-induced oxidation even in the
absence of phenolic hydroxyl group. In particular, 3, a dihy-
dropyrazole derivative without any phenolic hydroxyl groups
involving, still exhibit the antioxidant effectiveness in inhibiting
Cu2þ/GSH-induced oxidation of DNA. This result shows the po-
tential application of ferrocene moiety for designing antioxidant
molecules. The skeleton of coumarin-substituted dihydropyrazole
can be widely applied in the design of novel antioxidants.
4
5
6
7
8
9
DPPH
250
6. Experimental section
200
150
6.1. Materials and instrumentation
Diammonium salt of 2,20-azinobis(3-ethylbenzothiazoline-6-
sulfonate) (ABTS salt), DPPH, and galvinoxyl radical were pur-
chased from Fluka Chemie GmbH, Buchs, Switzerland. AAPH, GSH,
and the naked DNA sodium salt were purchased from Acros Or-
ganics, Geel, Belgium. Other agents were of analytical grade and
used directly. The structures of coumarin-substituted dihydropyr-
azoles were identified by 1H and 13C NMR (Varian Mercury 300
NMR spectrometer) and high solution mass spectra equipped with
ESI as the ionization mode (Agilent1290-micrOTOF Q II).
0
500
1000
Reaction period (s)
Fig. 6. Decay of 260.88
dihydropyrazoles.
m
M DPPH in the presence of 10
m
M coumarin-substituted

G.-L. Xi, Z.-Q. Liu / European Journal of Medicinal Chemistry 68 (2013) 385e393
391
Table 2
Equation of [ABTSD.] w t and its differential style (ꢅd[ABTSD.]/dt w t), reaction rate at t ¼ 0 (r0), and rate constant (k).a
Antioxidant
Equation of [ABTSþ.
(
m
M)] w t (s)
Equation of -d[ABTSþ.]/dt w t
r0(
m
M.sꢅ1
)
k (mMꢅ1 sꢅ1
)
d½ABTSþ,
dt
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
t
t
4
t
t
2.26
1.31
3.39
1.82
3.92
7.47
7.44
4.31
ꢅ
½ABTSþ,ꢂ ¼ 8:66eꢅ
½ABTSþ,ꢂ ¼ 6:64eꢅ
þ 5:55e
þ 3:30e
þ 46:53
þ 50:80
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
¼ 2:23eꢅ
¼ 1:30eꢅ
¼ 3:36eꢅ
¼ 1:80eꢅ
¼ 3:89eꢅ
¼ 7:44eꢅ
þ 0:03e
þ 0:01e
þ 0:03e
þ 0:02e
þ 0:03e
þ 0:03e
3:88
203:16
3:88
203:16
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
d½ABTSþ,
dt
t
t
5
6
7
8
9
t
t
ꢅ
5:11
328:90
5:11
328:90
d½ABTSþ,
dt
t
t
t
t
11.16
5.99
ꢅ
½ABTSþ,ꢂ ¼ 11:67eꢅ
þ 4:23e
þ 44:85
þ 44:66
þ 39:63
þ 37:89
3:47
167:98
3:47
167:98
d½ABTSþ,
dt
t
t
t
t
ꢅ
½ABTSþ,ꢂ ¼ 10:22eꢅ
þ 5:85e
þ 7:92e
þ 7:39e
5:69
308:25
5:69
308:25
d½ABTSþ,
dt
t
t
t
t
12.91
24.59
ꢅ
½ABTSþ,ꢂ ¼ 13:20eꢅ
½ABTSþ,ꢂ ¼ 15:47eꢅ
3:39
286:21
3:39
286:21
d½ABTSþ,
dt
t
t
t
t
ꢅ
2:08
216:43
2:08
316:43
a
The concentrations of coumarin-substituted dihydropyrazoles is 5 m m
M, and the concentration of ABTSD. is 60.75 M 1, 2, and 3 cannot react with ABTSD. radical. The
p < 0.001 indicated a significance difference of these equations.
6.2. A cascade synthetic operation of coumarin-substituted
dihydropyrazoles
purified by silica chromatography with ethyl acetate/petroleum
ether (1:1, v:v) being eluent. The NMR data of coumarin-substituted
dihydropyrazoles were listed as following.
The hydroxyl group in aldehyde A was protected by etherizing
with benzyl chloride before used in the following synthesis. Briefly,
hydroxyl-substituted benzaldehyde (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 benzoxyl-substituted benzaldehyde (>90%).
The following synthesis was carried out as a cascade operation
without purification. 2-Hydroxylbenzaldehyde (1.22 g, 10.0 mmol)
was mixed with ethyl acetoacetate (1.30 g, 10.0 mmol) and stirred
at 0e10 ꢀC for 0.5 h. Then, pyridine (0.05 mL) and (C2H5)2NH
(0.05 mL) were added dropwisely and stirred at 0e10 ꢀC for 3 h. To
above mixture, 40 mL of ethanol, the corresponding aldehyde
(12.0 mmol), and piperidine (0.5 mL) were added and refluxed for
6 h. Finally, phenylhydrazine (50.0 mmol) and acetic acid (15 mL)
were added and refluxed for 3 h. The reaction mixture was poured
into ice-cold water and extracted with ethyl acetate at room tem-
perature. The organic phase was washed with brine and dried over
Na2SO4. The solvent was evaporated under vacuum pressure. The
crude product was purified by silica chromatography with ethyl
acetate/petroleum ether (1:4, v:v) being eluent to afford the
hydroxyl-protected coumarin-substituted dihydropyrazoles.
The benzyl group was removed by TiCl4. In brief, hydroxyl-
protected coumarin-substituted dihydropyrazoles (1.0 mmol, dis-
solved 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
6.2.1. 3-(1,5-Diphenyl-4,5-dihydro-1H-pyrazol-3-yl)chromen-2-
one (1)
Yield 78%. m. p.: 204e206 ꢀC 1H NMR (300 MHz, CDCl3)
d: 8.42
(s, 1H, Hvinyl), 7.57e7.59 (m, 1H, phenyl), 7.48e7.54 (m, 1H, phenyl),
7.27e7.33 (m, 6H, phenyl), 7.23 (t, 1H, phenyl), 7.17e7.20 (m, 2H,
phenyl), 7.07e7.10 (m, 2H, phenyl), 6.83 (t, 1H, phenyl), 5.34 (dd,
J ¼ 12.9 Hz, J ¼ 7.2 Hz, 1H, CH), 4.10 (dd, J ¼ 18.6 Hz, J ¼ 12.9 Hz, 1H,
CH2), 3.39 (dd, J ¼ 18.3 Hz, J ¼ 7.2 Hz, 1H, CH2). 13C NMR (75 MHz,
CDCl3) d: 159.7, 153.5, 144.0, 142.9, 142.0, 137.6, 131.5, 129.1, 129.0,
128.2, 127.6, 125.7, 124.7, 120.8, 119.8, 119.5, 116.4, 113.6, 64.8, 45.2.
MS: m/z 367.1476 [M þ Hþ].
6.2.2. 3-(5-(4-Methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-
3-yl)chromen-2-one (2)
Yield 82%. m. p.: 131e133 ꢀC 1H NMR (300 MHz, CDCl3)
d: 8.40 (s,
1H, Hvinyl), 7.48e7.60 (m, 2H, phenyl), 7.27e7.39 (m, 3H phenyl),
7.08e7.23 (m, 5H, phenyl), 6.83e6.87 (m, 2H, phenyl), 5.30 (dd,
J ¼ 12.6 Hz, J ¼ 7.2 Hz, 1H, CH), 4.07 (dd, J ¼ 18.3 Hz, J ¼ 12.6 Hz, 1H,
CH2), 3.78 (s, 3H, OCH3), 3.36 (dd, J ¼ 18.3 Hz, J ¼ 7.2 Hz, 1H, CH2).
13C NMR (75 MHz, CDCl3)
d: 159.6, 159.0, 153.5, 144.0, 142.9, 137.4,
134.1, 131.4, 130.0, 129.0, 128.9, 128.1, 126.9, 125.4, 124.6, 120.8,
119.7, 119.5, 116.3, 114.4, 113.9, 113.6, 64.4, 55.2, 45.2. MS: m/z
397.1576 [M þ Hþ].
6.2.3. 3-(5-Ferrocenyl-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)
chromen-2-one (3)
Yield 73%. m. p.: 193e195 ꢀC 1H NMR (300 MHz, CDCl3)
d: 8.42
(s, 1H, Hvinyl), 7.50e7.60 (m, 2H, phenyl), 7.30e7.37 (m, 3H, phenyl),
7.18e7.22 (m, 3H, phenyl), 6.85 (t, 1H, phenyl), 5.20 (dd, J ¼ 10.8 Hz,
J ¼ 6.0 Hz, 1H, CH), 4.24e4.28 (m, 1H, CH2), 4.19 (s, 5H, ferrocenoyl),
Table 3
Equation of [DPPH] w t and its differential style (ꢅd[DPPH]/dt w t), reaction rate at t ¼ 0 (r0), and rate constant (k).a
Antioxidant
Equation of [DPPH (
m
M)] w t (s)
Equation of ꢅd[DPPH]/dt w t
r0
(
m
M sꢅ1
)
k (mMꢅ1 sꢅ1
0.54
)
t
t
t
t
4
5
6
7
8
9
1.41
1.83
6.37
6.62
23.04
9.71
½DPPHꢂ ¼ 37:89eꢅ
½DPPHꢂ ¼ 33:01eꢅ
½DPPHꢂ ¼ 41:36eꢅ
½DPPHꢂ ¼ 57:00eꢅ
½DPPHꢂ ¼ 80:69eꢅ
½DPPHꢂ ¼ 49:50eꢅ
þ 12:79e
þ 17:11e
þ 207:69
þ 208:90
ꢅd½DPPHꢂ ¼ 1:35eꢅ
þ 0:06e
þ 0:06e
ꢅ
ꢅ
ꢅ
28:03
229:76
28:03
229:76
dt
t
t
t
t
0.70
ꢅd½DPPHꢂ ¼ 1:77eꢅ
ꢅ
18:64
281:95
18:64
281:95
dt
t
t
t
t
2.44
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
6:96
77:70
6:96
77:70
þ 33:19e
þ 186:10
þ 176:98
þ 146:80
ꢅd½DPPHꢂ ¼ 5:94eꢅ
þ 0:43e
þ 0:28e
dt
t
t
t
t
2.54
þ 26:45e
þ 53:36e
þ 60:50e
ꢅd½DPPHꢂ ¼ 6:34eꢅ
ꢅ
8:99
95:08
8:99
95:08
dt
t
t
t
t
8.83
ꢅd½DPPHꢂ ¼ 22:35eꢅ
ꢅd½DPPHꢂ ¼ 9:12eꢅ
þ 0:69e
ꢅ
3:61
77:63
3:61
77:63
dt
t
t
t
t
3.72
ꢅ
5:43
102:60
5:43
102:60
þ 150:75
þ 0:59e
dt
a
The concentrations of coumarin-substituted dihydropyrazoles is 10 mM, and the concentration of DPPH is 260.88 mM 1, 2, and 3 cannot react with DPPH radical. The
p < 0.001 indicated a significance difference of these equations.

392
G.-L. Xi, Z.-Q. Liu / European Journal of Medicinal Chemistry 68 (2013) 385e393
Table 4
Equation of [galvinoxyl] w t and its differential style (ꢅd[galvinoxyl]/dt w t), reaction rate at t ¼ 0 (r0), and rate constant (k).a
Antioxidant
Equation of [galvinoxyl (
m
M)] w t (s)
Equation of ꢅd[galvinoxyl]/dt w t
r0
(
m
M.sꢅ1
)
k (mMꢅ1 sꢅ1
4.35
)
t
t
t
t
4
5
6
7
8
9
0.71
1.18
0.95
1.10
1.13
1.29
½galvinoxylꢂ ¼ 3:94eꢅ
½galvinoxylꢂ ¼ 2:52eꢅ
½galvinoxylꢂ ¼ 4:53eꢅ
½galvinoxylꢂ ¼ 4:41eꢅ
½galvinoxylꢂ ¼ 5:58eꢅ
½galvinoxylꢂ ¼ 5:13eꢅ
þ 1:24e
þ 1:66e
þ 1:33e
þ 1:69e
þ 0:79e
þ 1:11e
þ 5:69
ꢅd½galvinoxylꢂ ¼ 0:70eꢅ
þ 0:01e
þ 0:02e
þ 0:01e
þ 0:02e
þ 0:01e
þ 0:02e
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
5:59
113:48
5:59
113:48
dt
t
t
t
t
7.23
ꢅ
2:17
74:39
2:17
74:39
þ 6:69
ꢅd½galvinoxylꢂ ¼ 1:16eꢅ
dt
t
t
t
t
5.82
ꢅ
ꢅ
ꢅ
ꢅ
4:82
103:69
4:82
103:69
þ 5:02
þ 4:78
þ 4:50
ꢅd½galvinoxylꢂ ¼ 0:94eꢅ
dt
t
t
t
t
6.74
ꢅd½galvinoxylꢂ ¼ 1:08eꢅ
4:10
112:21
4:10
112:21
dt
t
t
t
t
6.92
ꢅd½galvinoxylꢂ ¼ 1:12eꢅ
4:98
158:45
4:98
158:45
dt
t
t
t
t
7.90
ꢂ
4:04
70:16
4:04
70:16
þ 4:63
ꢅd½galvinoxyl ¼ 1:27eꢅ
dt
a
The concentrations of coumarin-substituted dihydropyrazoles is 15 mM, and the concentration of galvinoxyl is 10.88 mM 1, 2, and 3 cannot react with galvinoxyl radical.
The p < 0.001 indicated a significance difference of these equations.
4.12e4.15 (m, 4H, ferrocenoyl), 4.00e4.03(m, 1H, CH2). 13C NMR
(75 MHz, CDCl3) : 159.9, 153.5, 144.3, 143.5, 137.5, 131.4, 128.8,
CH2). 13C NMR (75 MHz, DMSO-d6)
d: 158.0,152.7,147.8,145.7,143.7,
d
143.1, 138.0, 133.0, 131.6, 128.7, 128.6, 124.6, 119.8, 119.1, 117.8, 115.7,
128.1, 124.6, 120.8, 119.8, 119.5, 116.3, 114.3, 89.8, 68.7, 68.6, 68.1,
113.3, 109.8, 63.1, 55.4, 44.5. MS (ESI): m/z 413.1536 [M þ Hþ].
67.6, 66.7, 59.8, 43.5. MS: m/z 475.1116 [M þ Hþ].
6.2.8. 3-(5-(3,4-Dihydroxyphenyl)-1-phenyl-4,5-dihydro-1H-
6.2.4. 3-(5-(2-Hydroxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-
pyrazol-3-yl)chromen-2-one (8)
3-yl)chromen-2-one (4)
Yield 87%. m. p.: 233e234 ꢀC 1H NMR (300 MHz, DMSO-d6)
d:
Yield 85%. m. p.: 216e218 ꢀC 1H NMR (300 MHz, DMSO-d6)
d
:
8.93 (s, 1H, OH), 8.86 (s, 1H, OH), 8.45 (s, 1H, Hvinyl), 7.83 (d,
J ¼ 7.8 Hz, 1H, phenyl), 7.61 (t, 1H, phenyl), 7.35e7.42 (m, 2H,
phenyl), 7.05e7.21 (m, 4H, phenyl), 6.58e6.78 (m, 4H, phenyl), 5.32
(dd, J ¼ 12.3 Hz, J ¼ 6.0 Hz,1H, CH), 3.91 (dd, J ¼ 18.0 Hz, J ¼ 12.3 Hz,
1H, CH2), 3.18 (dd, J ¼ 18.0 Hz, J ¼ 6.3 Hz, 1H, CH2). 13C NMR
9.89 (s,1H, OH), 8.47 (s,1H, Hvinyl), 7.83 (dd, J ¼ 7.8 Hz, J ¼ 1.2 Hz,1H,
phenyl), 7.57e7.63 (m,1H, phenyl), 7.34e7.42 (m, 2H, phenyl), 7.16e
7.21 (m, 2H, phenyl), 7.00e7.10 (m, 3H, phenyl), 6.84e6.91 (m, 2H,
phenyl), 6.66e6.78 (m, 2H, phenyl), 5.62 (dd, J ¼ 12.3 Hz, J ¼ 6.3 Hz,
1H, CH), 3.95 (dd, J ¼ 18.0 Hz, J ¼ 12.6 Hz, 1H, CH2), 3.16 (dd,
(75 MHz, DMSO-d6) d: 157.9, 152.7, 145.5, 144.6, 143.5, 142.9, 138.0,
J ¼ 18.0 Hz, J ¼ 6.0 Hz, 1H, CH2). 13C NMR (75 MHz, DMSO-d6)
d:
133.1, 131.6, 128.7, 128.5, 124.6, 119.8, 119.1, 119.0, 116.6, 115.8, 115.7,
163.6, 159.5, 158.3, 149.0, 148.9, 143.6, 137.2, 134.4, 134.2, 133.8,
132.9, 131.6, 130.1, 125.5, 124.7, 124.5, 121.3, 121.0, 118.4, 63.2, 48.7.
MS: m/z 383.1421 [M þ Hþ].
113.2, 112.6, 62.7, 44.5. MS: m/z 399.1387 [M þ Hþ].
6.2.9. 3-(5-(2,4-Dihydroxyphenyl)-1-phenyl-4,5-dihydro-1H-
pyrazol-3-yl)chromen-2-one (9)
6.2.5. 3-(5-(3-Hydroxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-
Yield 84%. m. p.: 149e150 ꢀC 1H NMR (300 MHz, DMSO-d6)
d:
3-yl)chromen-2-one (5)
9.67 (s, 1H, OH), 9.21 (s, 1H, OH), 8.45 (s, 1H, Hvinyl), 7.83 (d,
J ¼ 7.5 Hz, 1H, phenyl), 7.57e7.62 (m, 1H, phenyl), 7.34e7.42 (m, 2H,
phenyl), 7.15e7.21 (m, 2H, phenyl), 7.02e7.05 (m, 2H, phenyl),
6.64e6.77 (m, 2H, phenyl), 6.35 (d, J ¼ 1.8 Hz, 1H, phenyl), 6.10 (dd,
J ¼ 8.4 Hz, J ¼ 2.1 Hz, 1H, phenyl), 5.51 (dd, J ¼ 12.3 Hz, J ¼ 6.0 Hz,
1H, CH), 3.88 (dd, J ¼ 18.0 Hz, J ¼ 12.3 Hz, 1H, CH2), 3.13 (dd,
Yield 80%. m. p.: 197e198 ꢀC 1H NMR (300 MHz, DMSO-d6)
d:
9.43 (s, 1H, OH), 8.47 (s, 1H, Hvinyl), 7.82 (d, J ¼ 6.6 Hz, 1H, phenyl),
7.58e7.63 (m, 1H, phenyl), 7.35e7.42 (m, 2H, phenyl), 7.04e7.22 (m,
5H, phenyl), 6.63e6.79 (m, 4H, phenyl), 5.42 (dd, J ¼ 12.0 Hz,
J ¼ 6.0 Hz, 1H, CH), 3.96 (dd, J ¼ 18.0 Hz, J ¼ 12.6 Hz, 1H, CH2), 3.20
(dd, J ¼ 18.0 Hz, J ¼ 6.3 Hz, 1H, CH2). 13C NMR (75 MHz, DMSO-d6)
d:
J ¼ 18.0 Hz, J ¼ 6.3 Hz, 1H, CH2). 13C NMR (75 MHz, DMSO-d6)
d:
158.1, 157.9, 152.9, 152.8, 143.8, 143.5, 143.1, 138.3, 131.8, 130.1, 128.9,
128.7, 124.7, 119.8, 119.2, 116.3, 115.8, 114.5, 113.2, 112.1, 63.0, 44.5.
MS: m/z 383.1425 [M þ Hþ].
157.6, 155.0, 152.8,143.6,143.4,137.9, 131.7, 128.8, 128.7, 127.0, 124.7,
120.2, 119.3, 118.9, 118.2, 115.8, 113.0, 106.6, 102.7, 64.9, 43.4. MS: m/
z 399.1383 [M þ Hþ].
6.2.6. 3-(5-(4-Hydroxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-
6.3. Cu2þ/GSH-induced oxidation of DNA test
3-yl)chromen-2-one (6)
Yield 86%. m. p.: 230e232 ꢀC 1H NMR (300 MHz, DMSO-d6)
d:
Cu2þ/GSH-induced oxidation of DNA was carried out following
a previous report [23] 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 dihydropyrazoles were dissolved in dimethyl sulf-
oxide (DMSO). Then, 2.0 mg/mL DNA, 5.0 mM Cu2þ, 3.0 mM GSH,
and 0.2 mM coumarin-substituted dihydropyrazoles 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 tempera-
ture; 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.
9.40 (s,1H, OH), 8.47 (s,1H, Hvinyl), 7.83 (dd, J ¼ 7.8 Hz, J ¼ 1.5 Hz,1H,
phenyl), 7.58e7.63 (m, 1H, phenyl), 7.35e7.42 (m, 2H, phenyl),
7.15e7.20 (m, 2H, phenyl), 7.04e7.10 (m, 4H, phenyl), 6.70e6.78 (m,
3H, phenyl), 5.41 (dd, J ¼ 12.6 Hz, J ¼ 6.3 Hz, 1H, CH), 3.93 (dd,
J ¼ 18.0 Hz, J ¼ 12.3 Hz, 1H, CH2), 3.18 (dd, J ¼ 18.0 Hz, J ¼ 6.3 Hz, 1H,
CH2). 13C NMR (75 MHz, DMSO-d6)
d: 158.0, 156.6, 152.7, 143.5,
142.9, 138.1, 132.3, 131.6, 128.7, 128.6, 126.9, 124.6, 119.9, 119.1, 119.0,
115.7, 115.6, 113.2, 62.6, 44.5. MS: m/z 383.1419 [M þ Hþ].
6.2.7. 3-(5-(4-Hydroxy-3-methoxyphenyl)-1-phenyl-4,5-dihydro-
1H-pyrazol-3-yl)chromen-2-one (7)
Yield 76%. m. p.: 185e187 ꢀC 1H NMR (300 MHz, DMSO-d6)
d:
8.97 (s, 1H, OH), 8.48 (s, 1H, Hvinyl), 7.84 (d, J ¼ 6.6 Hz, 1H, phenyl),
7.58e7.64 (m, 1H, phenyl), 7.35e7.43 (m, 2H, phenyl), 7.16e7.21 (m,
2H, phenyl), 7.06e7.09 (m, 2H, phenyl), 6.89 (d, J ¼ 1.5 Hz, 1H,
phenyl), 6.70e6.79 (m, 2H, phenyl), 6.62e6.66 (m, 1H, phenyl), 5.38
(dd, J ¼ 12.3 Hz, J ¼ 6.3 Hz, 1H, CH), 4.03 (dd, J ¼ 14.4 Hz, J ¼ 7.2 Hz,
1H, CH2), 3.72 (s, 3H, OCH3), 3.22 (dd, J ¼ 18.0 Hz, J ¼ 6.6 Hz, 1H,

G.-L. Xi, Z.-Q. Liu / European Journal of Medicinal Chemistry 68 (2013) 385e393
393
6.4. .OH-induced oxidation of DNA test
Acknowledgment
ꢁOH was generated by mixing H2O2 with tetrachlorohydro
quinone (TCHQ, dissolved in DMSO as the stock solution) as the
description in a literature [24]. DNA and H2O2 were dissolved in
phosphate buffered solution (PBS2: 8.1 mM Na2HPO4, 1.9 mM
Financial support from Jilin Provincial Science and Technology
Department, China, is acknowledged gratefully (20130206075GX).
Appendix A. Supplementary data
NaH2PO4, 10.0 mM EDTA). DNA (2.0 mg/mL), 4.0 mM TCHQ, 2.0 mM
H2O2, and 0.2 mM coumarin-substituted dihydropyrazoles (dis-
solved in DMSO as the stock solution) were mixed to form a solution.
The solution was poured into test tubes, and each test tube con-
tained 2.0 mL. The test tubes were incubated at 37 ꢀC for 30 min and
cooled immediately. The following operation was the same as in
Cu2þ/GSH-induced oxidation of DNA except EDTA was not added.
The absorbances in the control experiment and in the presence of
coumarin-substituted dihydropyrazoles were assigned as A0 and
Adetect, respectively. The effects of coumarin-substituted dihy-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.06.059.
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