Synthesis of 4-methylcoumarin derivatives containing 4,5-dihydropyrazole moiety to scavenge radicals and to protect DNA

Article abstract of DOI:10.1016/j.ejmech.2012.03.052

A series of 4-methylcoumarin derivatives containing 4,5-dihydropyrazole moiety were synthesized and their antioxidant activities were evaluated in AAPH (2,2′-azobis(2-amidinopropane hydrochloride))-induced oxidation of DNA, and in trapping DPPH (2,2′-diph

Full text of DOI:10.1016/j.ejmech.2012.03.052

European Journal of Medicinal Chemistry 53 (2012) 159e167
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 4-methylcoumarin derivatives containing 4,5-dihydropyrazole
moiety to scavenge radicals and to protect DNA
,
b
Chuan Xiao a, Xu-Yang Luo a, De-Jun Li a, Hang Lu a, Zai-Qun Liu a, Zhi-Guang Song a , Ying-Hua Jin
*
a Department of Organic Chemistry, College of Chemistry, Jilin University, No. 2699 Qianjin Road, Changchun 130012, China
b Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, Jilin University, Changchun 130012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-methylcoumarin derivatives containing 4,5-dihydropyrazole moiety were synthesized and their
antioxidant activities were evaluated in AAPH (2,20-azobis(2-amidinopropane hydrochloride))-induced
oxidation of DNA, and in trapping DPPH (2,20-diphenyl-1-picrylhydrazyl) and ABTSþꢀ (2,20-azinobis(3-
ethylbenzothiazoline-6-sulfonate) cationic radical), respectively. Among coumarin derivatives, 3aed and
4aec exhibited the termination of radical propagation-chains in AAPH-induced oxidation of DNA. The ortho
dihydroxyphenyl substitution at 5 position and 1-unsubstitution of the 4,5-dihydroxylpyrazole was found
enhancing the antioxidant activities of these coumarin derivatives.
Received 29 January 2012
Received in revised form
26 March 2012
Accepted 27 March 2012
Available online 5 April 2012
Keywords:
Synthesis
Ó 2012 Elsevier Masson SAS. All rights reserved.
4-Methylcoumarins
4,5-Dihydroxypyrazole
Free radical
Antioxidant
Oxidation of DNA
1. Introduction
[26e28], some coumarin derivatives containing pyrazole moiety
have been synthesized. Although these compounds exhibits
The importance of free radicals, especially reactive oxygen
species (ROS) in the pathogenicity of various diseases, has of late
received greater attention. Because a large body of evidence has
revealed the correlation of the in vivo oxidative stress with aging [1]
and some fatal diseases [2,3], many works devoted to the synthesis
of novel antioxidants and the evaluation of their activities in
various experimental systems [4,5].
Coumarins, a wide family of compounds present in remarkable
amounts in the nature, have been found to exhibit different biolog-
ical and pharmacological significance, including anticancer [6,7],
antioxidant [8,9], anti-inflammatory [10], antimicrobial [11], anti-
viral [12], and enzymatic inhibitory activities [13]. Compared to the
coumarins, 4-methylcoumarins not only possess similar various
biological activities, but known to be less toxic. In particular, 4-
methylcoumarins have been studied as novel antioxidants recently
[14e18]. On the other hand, pyrazole, has been found in numerous
pharmaceutically active compounds [19e21], and a large number of
pyrazoles have shown free radical scavenging ability [22e25].
Since the combination of two pharmacophores on the same
scaffold is a well established approach to more potent drugs
various biological activities [29e32], only very a few report
focused on the synthetic methodology and systematic evaluation
of antioxidant activities. Our group [33] has recently reported
on the antioxidant activity of coumarin derivatives xanthotoxol
and methyl-substituted xanthotoxol (Fig. 1). As an following
study, 4,5-dihydropyrazole ring was considered introducing into
the parent 7-hydroxy-4-methyl coumarin skeleton to design
novel structures with enhanced antioxidant activities. Herein,
a
series of 4-methylcoumarin derivatives containing 4,5-
dihydropyrazole moiety were synthesized (Scheme 1). Then
these 4,5-dihydropyrazole coumarins were employed to protect
DNA against the oxidation damage induced by AAPH, and radical
scavenging properties were screened by trapping ABTSþꢀ and
DPPH.
2. Results and discussion
2.1. Chemistry
The title compound, 4-methylcoumarin derivatives containing
4,5-dihydropyrazole moiety, was derived from 8-acetyl-7-hydroxy-
4-methylcoumrain 1 employed as the reagent (Scheme 1). The
* Corresponding author. Tel./fax: þ86 431 88499176.
E-mail address: szg@jlu.edu.cn (Z.-G. Song).
a,b-unsaturated ketone 2 was accomplished by ClaiseneSchmidt
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.052

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C. Xiao et al. / European Journal of Medicinal Chemistry 53 (2012) 159e167
2.2. Antioxidant profiling
2.2.1. Antioxidant effects on AAPH-induced oxidation of DNA
The peroxyl radical (ROO$) generated from the decomposition of
AAPH is able to abstract H atom from the C-40 atom of DNA,
resulting in strand breaks and further generating more than 20
carbonyl species called thiobarbituric acid reactive substance
(TBARS, lmax ¼ 535 nm) [34]. The oxidation of aforementioned
biological samples can also be detected by measuring TBARS [35].
Hence, we herein also applied this method in the in vitro assays of
the inhibitory activities of synthetic compounds 3aed and 4aec on
DNA oxidation (Fig. 2). xanthotoxol, a good natural coumarin
antioxidant [36,37], was used as positive control.
As shown in Fig. 2(A), the negtive control shows a continual
increase of the absorbance of TBARS, indicating that much more
carbonyl species are produced in AAPH-induced oxidation of DNA
with the incubation period increasing. However, the additions of
various concentrations of coumarin derivatives (3aed, 4aec) and
xanthotoxol remarkably suppress the increase of the absorbance of
TBARS at the beginning of the incubation period, indicating that
these compounds were able to inhibit the oxidation of DNA for
Fig. 1. Xanthotoxol and methyl-substituted xanthotoxol.
condensation of 1 and substituted-benzaldehyde in the presence of
mild catalyst piperidine, and simply obtained as precipitate. Further
purification by recrystallization was performed when necessary. The
treatment of 2 with hydrazine hydrate in refluxing ethanol or
acetic acid afforded 8-(4,5-dihydro-5-aryl-1-H-pyrazol-3-yl)-7-
hydroxy-4-methyl-2H-1-benzopyran-2-ones 3a, 3b and 8-(1-
acetyl-4,5-dihydro-5-aryl-1-H-pyrazol-3-yl)-7-hydroxy-4-methyl-
2H-1-benzopyran-2-ones 3cef, respectively. Finally, compound
4aec were obtained from the deprotection of 3def. We also
a period. This period could be designated as inhibition period (tinh
)
being related proportionally to the concentration of 4,5-
dihydropyrazole coumarins (3aed and 4aec) and xanthotoxol as
shown in Fig. 2(B). Moreover, the quantitative equations of tinh
versus the concentration of 3aed, 4aec and xanthotoxol were
calculated and listed in Table 1. The application of the linear corre-
lation between tinh and the concentration of the antioxidant for the
evaluation of the antioxidant efficacy was shown as Eq. (1) [38].
examined the viability of
a protecting-group-free route for
compound 4aec (Scheme 2). Unfortunately, the ClaiseneSchmidt
condensation between 1 and benzaldehyde partners with free
hydroxyl at ortho or meta positions led to complex mixtures. The
desired products could not be isolated as pure form.
tinh ¼ ðn=RiÞ½antioxidantꢁ
(1)
Scheme 1. Synthesis of title compounds Reagent and conditions: (a) substituted-benzaldehyde, piperdine, ethanol, reflux; (b) 80% hydrazine monohydrate, ethanol, reflux, 12 h; (c)
80% hydrazine monohydrate, acetic acid, reflux, 2h; (d) AlCl3, toluene, pyridine, reflux, 6 h; or BBr3, dichloromethane, ꢂ20e0 ꢃC; or Pd(PPh3)4, K2CO3, methanol, reflux, 6 h.

C. Xiao et al. / European Journal of Medicinal Chemistry 53 (2012) 159e167
161
Scheme 2. Attempt to synthesize compound 4 reagent and conditions: (a) piperdine, ethanol, reflux.
The n refers to the stoichiometric factor, expressing the number
of the radical propagation terminated by one molecule of the anti-
oxidant, and Ri is the initiation rate of the radical-induced reaction.
This equation has been employed in the study of the protection of
DNA against AAPH-induced oxidation by homoisoflavonoids [39]. Ri
is equal to the generation rate (Rg) from the decomposition of AAPH
(Rg ¼ (1.4 ꢄ 0.2) ꢅ 10 ꢂ 6 [AAPH] sꢂ1 [38]). This is because both
sodium salt of DNA and AAPH are dissolved in the same water phase,
and radicals generated from AAPH attack DNA at the water phase.
Thus, the n values of 3aed, 4aec and xanthotoxol are the products of
coefficients in the corresponding equation multiplied by
variation of the absorbance of ABTSþꢀ and DPPH decreases in the
presence of 4,5-dihydropyrazole coumarins and xanthotoxol.
Recently, the interaction between an antioxidant with ABTSþꢀ was
investigated by chemical kinetics [43]. As shown in Eq. (2),
[ABTSþꢀ
]
and [ABTSþꢀ
]
stand for the concentration of ABTSþꢀ at
0
N
the beginning and the end of the reaction, respectively. Thus,
[ABTSþꢀ [ABTSþꢀ means the concentration of ABTSþꢀ
]
ꢂ
]
N
0
depleted by a certain concentration of the antioxidant. The
depleted concentration of ABTSþꢀ is divided by the concentration of
the antioxidant to indicate the number of ABTSþꢀ (n) trapped by the
antioxidant at the primary period:
Ri ¼ Rg ¼ 1.4 ꢅ 10ꢂ6 ꢅ 40 mM sꢂ1 ¼ 3.36
m
M minꢂ1 and listed in
ꢀ
ꢁ
ꢀ
ꢁ
ABTSþ 0 ꢂ ABTSþ
½antioxidantꢁ
$
$
Table 1 as well.
N
n ¼
(2)
Table 1 shows an order of the inhibitory effect of assayed
compounds on the oxidation of DNA as 4a ꢆ 4b ꢆ 3b ꢆ 3d
z 4c ꢆ 3a ꢆ 3c ꢆ xanthotoxol. This reveals all of our synthesized
4,5-dihydropyrazole coumarins have higher activity than xantho-
toxol against DNA damage induced by AAPH.
If the oxidized product of the antioxidant can trap ABTSþꢀ, this
period is called the secondary period for the antioxidant to trap
ABTSþꢀ. In the secondary period of the reaction between the anti-
oxidant and ABTSþꢀ, the variation of the concentration of ABTSþꢀ
([ABTSþꢀ]) follows:
Among the N-acetylation compounds 3c, 3d, and 4aec, the order
of the antioxidant capacities is 4a > 4b > 3d z 4c > 3c. Apparently,
the n value of compound 4a (6.15) and 4b (5.55), both bearing with
more two hydroxyl groups on the phenyl rings, are much higher
than compound 3c (2.15). This indicates that the antioxidant activity
is largely depended on the number of hydroxyl group on the phenyl
ring. The protective activity of 4a (n ¼ 6.15) is higher than 4b
(n ¼ 5.55), indicating the ortho dihydroxyl groups on the benzene
ring could enhance the antioxidant activities of these 4,5-
dihydropyrazole coumarin derivatives. This could be rationalized
by the increased stabilization of the semiquinone radical with
intramolecular hydrogen bond (Scheme 3). The intramolecular
hydrogen bond, in fact, is stronger than in the parent phenol [40,41].
As to the 1-N-H compound 3a, 3b and their N-acetylated
derivatives 3c, 3d, the order of the antioxidant capacities was 3a
(3.36) ꢆ 3c (2.15) and 3b (4.64) ꢆ 3d (3.99), indicating that the NeH
in position 1-N-unsubstituted 4,5-dihydropyrazole ring could also
enhance ability to protect DNA against AAPH-induced oxidation.
The reason for the antioxidant activity of the NeH in 4,5-
dihydropazole coumarins can be concluded that the hydrogen
atom can be abstracted by radicals and the resulting single electron
can be stabilized via resonance (Scheme 4). In Table 1, the n value of
3b and 3d is higher than 3a and 3c, respectively, due to the
hydroxyl group on benzene ring (5 position of the 4,5-
dihydropazole ring).
h
i
h
i
ab
b þ t
ABTSþ$
¼
ABTSþ$
þ
(3)
N
where a ¼ napp[antioxidant]0, b ¼ 1/(kapp[antioxidant]0), and t is the
reaction time. In Eq. (3), napp refers to the apparent number of
ABTSþꢀ trapped by the oxidized products of the antioxidant, and
kapp is the apparent rate constant in this period. Therefore, n þ napp
is the total number of ABTSþꢀ trapped by the antioxidant [44]. In
this work we apply this method to study the reaction between 4,5-
dihydropyrazole coumarins, xanthotoxol and DPPH as well, and the
n and napp in ABTSþꢀ and DPPH trapping reactions were listed in
Table 2.
Firstly, in the evaluations of the N-acetylated compund 3c, 3d
and 4aec, the order of the total numbers of electrons donated in
trapping ABTSþꢀ and DPPH was found in the order:
4b > 4a > 4c > 3d > 3c and 4a > 3d > 4b z 4c > 3c, respectively
(Table 2). With the phenol moieties, the capacities of 4,5-
dihydropyrazole coumarins 3d, and 4aec to trap ABTSþꢀ (n þ napp
ranges from 1.52 to 2.48) and DPPH (n þ napp ranges from 1.01 to
4.53) are remarkably higher than the p-nitrophenyl substituted
analog 3c (n þ napp in trapping ABTSþꢀ and DPPH were 0.18 and
0.79, respectively).
On the other hand, among the compounds with phenol
substituents, the total number of electrons donated to DPPH by 4a
(n
þ
napp
¼
4.53) was also found much higher than 4b
2.2.2. Radical scavenging ability
(n þ napp ¼ 1.04). This indicated that the ortho dihydroxyl on the
benzene ring are more efficient to donate hydrogen atom in the
The radical scavenging ability of an antioxidant is usually eval-
uated by trapping ABTSþꢀ and DPPH [42]. Fig. 3 indicates the

162
C. Xiao et al. / European Journal of Medicinal Chemistry 53 (2012) 159e167
Fig. 2. (A) The variation of the absorbance of TBARS in the mixture of DNA (2.0 mg/ml) and 40 mM AAPH with various concentrations of 4,5-dihydropyrazole coumarins (3aed and
4aec) and xanthotoxol added. (B) The relationships between tinh and the concentrations of 4,5-dihydropyrazole coumarins and xanthotoxol.
hydroxyl group to N-centered radicals compared with meta
dihydroxyl counterpart. On the contrary, 4b is more efficient to
reduce radicals than 4a since n þ napp of 4b (2.48) is higher than
Secondly, in the survey of the effect of the N-substituents on the
pyrazole core, we found that the total numbers of electrons
transferred in ABTSþꢀ and DPPH trapping by 1-N-H compunds
3a (2.54/3.35) and 3b (4.41/5.13) are much higher than those of
those of 4a (1.68) in trapping ABTSþꢀ
.

C. Xiao et al. / European Journal of Medicinal Chemistry 53 (2012) 159e167
163
Table 1
ring at the 5 position of 4,5-dihydropyrazole and leave the 1 posi-
tion of the 4,5-dihydropyrazole core unsubstituted improve the
DNA protection activity against AAPH-induced oxidation via
ABTSþꢀ and DPPH trapping. Especially, with two hydroxyl groups
ortho to each other in the benzene ring of 4,5-dihydropyrazole
coumarins possess stronger antioxidant capacity. Compound 3b
and 4a are the two novel and promising lead compounds suitable
for further development for designing antioxidants.
The equations of tinhw[antixoidant] and n of antixoidant in protecting DNA against
AAPH-induced oxidationa.
Compound
tinh (min) ¼ (n/Ri) [Antioxidant (
m
M)] þ constantb
n
3a
3b
3c
3d
4a
4b
4c
tinh ¼ 1.09 [3a] þ 50.3
tinh ¼ 1.38 [3b] þ 49.3
tinh ¼ 0.64 [3c] þ 40.6
tinh ¼ 1.19 [3d] þ 31.4
tinh ¼ 1.83 [4a] þ 8.4
tinh ¼ 1.64 [4b]ꢂ3.1
tinh ¼ 1.18 [4c] þ 50.3
tinh ¼ 0.59 [3c] þ 24.6
3.36
4.64
2.15
3.99
6.15
5.55
3.96
2.00
xanthotoxolc
4. Experiment
a
Ri ¼ Rg ¼ 1.4 ꢅ 10ꢂ6 [AAPH] sꢂ1 ¼ 3.36
m
M minꢂ1 when 40 mM AAPH was
employed, thus, n ¼ coefficient ꢅ 3.36
m .
M minꢂ1
4.1. Materials and instrumentation
b
The constant is generated from the linear regression analysis.
Xanthotoxol was used as standard.
c
Diammonium salt of 2, 20-azinobis(3-ethylbenzothiazoline-6-
sulfonate) (ABTS), DPPH and galvinoxyl radicals were purchased
from Fluka Chemie GmbH, Buchs, Switzerland. AAPH, the naked DNA
sodium salt, and GSH were purchased from ACROS ORGANICS, Geel,
Belgium. Other agents were of analytical grade and used directly.
Other reagents were of analytical grade, and purchased from Beijing
Chemical Reagent Co., China. Flash column chromatography was
performed on a silica gel (300e400 mesh) and thin layer chroma-
tography (TLC) inspections were carried out on a silica gel GF254
plates. The 1H and 13C NMR data were recorded with a Varian
Mercury 300 NMR spectrometer, using TMS as an internal standard.
their 1-N-acetylated derivatives 3c(0.18/0.79) and 3d (1.52/2.09).
This results suggest that the NeH in position 1-N-unsubstituted
4,5-dihydropyrazole ring could trap ABTSþꢀ and DPPH more effi-
ciently, given the more stable resonance structures of radicals of 3b
(Scheme 4). As shown in Table 2, the higher radical scavenging
activities of 3b and 3d than 3a and 3c, respectively, is also attrib-
uted to the presence of phenol substituents at the 5 position of
4,5-dihydropazole core.
3. Conclusion
Chemical shifts (d) were given in parts per million and coupling
constants were given as absolute values expressed in Hertz. IR
spectra were obtained with a 360 FT-IR spectrophotometer of Nico-
let. Mass spectra were obtained using LC/MS 1100 of Agilent Tech-
nology Corporation and Alltech ELSD 2000 instrument. Melting
points were determined by an X-4 digital microscope. The synthetic
A novel and concise synthetic entry to the 4-methylcoumarin
derivatives containing 4,5-dihydropyrazole moiety from 8-acetyl-
7-hydroxy-4-methylcoumrain and substituted-benzaldehyde has
been developed. Introducing hydroxyl groups onto the benzene
Scheme 3. Stability of phenoxy radical via hydrogen bonding.

164
C. Xiao et al. / European Journal of Medicinal Chemistry 53 (2012) 159e167
Scheme 4. The NeH as antioxidative group in 4,5-dihydropyrazole coumarins.
compounds were analyzed by high performance liquid chromatog-
raphy, and the purities of these compounds were larger than 98.0%.
4.2.1.1. 7-Hydroxy-8-((2E)-3-(3-nitrophenyl)-1-oxo-2-propen-1-yl)-
4-methyl-2H-1-benzopyran-2-one (2a). The reaction was con-
ducted according to the general procedure A and the product 2a
was obtained as an orange yellow solid (0.175 g, 50% yield). m. p.
220e221 ꢃC. IR (KBr): 3428, 1724, 1638, 1596, 1525, 1390 cmꢂ1 1H
4.2. Synthesis of title compound 3aed and 4aec
NMR (300 MHz, CDCl3)
8.26e8.34 (m, 2 H), 8.11e8.13 (m, 1 H), 7.90e7.95 (m, 1 H),
7.65e7.75 (m, 2 H), 6.97e7.01 (m, 1 H), 6.22 (d, 1 H), 2.46 (s, 3 H).
d (ppm): 13.52 (s, 1 H, eOH), 8.51 (d, 1 H),
4.2.1. General procedure A for the synthesis of 7-hydroxy-8-((2E)-3-
(3-aryl)-1-oxo-2-propen-1-yl)-4-methyl-2H-1-benzopyran-2-ones
(2aed)
To 8-acetyl-7-hydroxy-4-methylcoumrain 1 (0.218 g, 1.0 mmol)
and substituted-benzaldehyde (1.1 mmol) in ethanol (10 mL) was
added piperidine (0.1 mL). The reaction mixture was refluxed for
12 h or until completion of the reaction indicated by TLC. After the
reaction mixture was cooled to room temperature, the precipitates
were filtered to give the product. Further purification was per-
formed by recrystallization from 95% ethanol.
4.2.1.2. 7-Hydroxy-8-((2E)-3-(3-methoxy-4-hydroxy)-1-oxo-2-
propen-1-yl)-4-methyl-2H-1-benzopyran-2-one (2b). The reaction
was conducted according to the general procedure A to afford 2b as
an orange yellow solid (0.282 g, 80% yield). m. p. 240e241 ꢃC. IR
(KBr): 3419, 1723, 1629, 1596, 1509, 1385 cmꢂ1 1H NMR (300 MHz,
CDCl3)
d
(ppm): 14.18 (s, 1 H, eOH), 8.28 (d, J ¼ 15.5 Hz,1 H), 7.94 (d,
J ¼ 15.5 Hz, 1 H), 7.69 (d, J ¼ 8.7 Hz, 1 H), 7.43 (d, 2 H), 6.96 (d,
J ¼ 8.7 Hz, 1 H), 6.18 (s, 1 H), 5.97 (s, 1 H), 4.03 (s, 3 H), 2.45 (s, 3 H).
3a
3b
3c
0.75
4.2.1.3. 7-Hydroxy-8-((2E)-3-(2,4-dimethoxy)-1-oxo-2-propen-1-
yl)-4-methyl-2H-1-benzopyran-2-one (2c). The reaction was con-
ducted according to the general procedure A to afford 2c as an
orange solid (0.30 g, 82% yield). m. p. 212e214 ꢃC. IR (KBr): 3421,
1720, 1631, 1600, 1512, 1385 cmꢂ1 1H NMR (300 MHz, CDCl3)
3d
4a
4b
0.50
4c
xanthotoxol
d
(ppm): 14.29 (s, 1 H, eOH), 8.39 (d, J ¼ 15.6 Hz, 1 H), 8.25 (d,
0.25
J ¼ 15.6 Hz, 1 H), 7.65 (d, 2 H), 6.93 (d, 1 H), 6.48e6.58 (m, 2 H), 6.17
(s, 1 H), 3.98 (s, 3 H), 3.87 (s, 3 H), 2.43 (s, 3 H).
0.00
4.2.1.4. 7-Hydroxy-8-((2E)-3-(2-allyloxy)-1-oxo-2-propen-1-yl)-4-
methyl-2H-1-benzopyran-2-one (2d). The reaction was conducted
according to the general procedure A to afford 2d as an orange solid
(0.25 g, 70% yield). m. p. 122e124 ꢃC. IR (KBr): 3427, 1722, 1630,
0
250
500
750
Incubation Period (s)
1599, 1509, 1387 cmꢂ1 1H NMR (300 MHz, CDCl3)
d (ppm): 13.90 (s,
3a
3b
3c
3d
4a
4b
4c
1.2
0.9
0.6
0.3
1 H, eOH), 8.28e8.42 (m, 2 H), 7.79e8.82 (m, 1 H), 7.67 (d, 1 H),
7.34e7.40 (m, 1 H), 7.00e7.05 (m, 1 H), 6.92e6.96 (m, 2 H), 6.18 (s,
1 H), 6.07e6.16 (m, 1 H), 5.44 (dd, J ¼ 17.3, 1.2 Hz, 1 H), 5.31 (dd,
J ¼ 10.5, 1.2 Hz, 1 H), 4.72 (d, 1 H), 2.44 (s, 3 H).
xanthotoxol
4.2.2. General procedure B for the synthesis of 8-(4,5-dihydro-5-
aryl-1H-pyrazol-3-yl)-7-hydroxy-4-methyl-2H-1-benzopyran-2-
ones (3a, 3b)
To a suspension of
a,b-unsaturated ketone 2 (0.5 mmol) in
ethanol (8 mL) was added hydrazine monohydrate (1 mmol), and
the reaction mixture was refluxed for 2 h. After the reaction
mixture was cooled, the precipitates were collected by filtration. It
was then washed by ethanol and dried to give the product.
0
400
800
1200
Incubation Period (s)
Fig. 3. The decrease of the absorbance of ABTSþꢀ (734 nm) in the presence of 0.04
4,5-dihydropyrazole coumarins and xanthotoxol, and the decrease of the absorbance of
mM
4.2.2.1. 8-(4,5-Dihydro-5-(3-nitrophenyl)-1H-pyrazol-3-yl)-7-
hydroxy-4-methyl-2H-1-benzopyran-2-one (3a). The reaction was
conducted according to the general procedure B to compound 3a as
DPPH (517 nm) in the presence of 0.40
xanthotoxol.
mM 4,5-dihydropyrazole coumarins and

C. Xiao et al. / European Journal of Medicinal Chemistry 53 (2012) 159e167
165
Table 2
The numbers of ABTSþꢀ and DPPH trapped by 3aed, 4aec and xanthotoxol in the primary stage (n), the secondary stage (napp), and the total number of trapped radicals
(n þ napp).
Compound
ABTSþꢀ
DPPH
n
napp
n þ napp
n
napp
n þ napp
3a
3b
3c
3d
4a
4b
4c
2.24 ꢄ 0.11
3.84 ꢄ 0.19
0.14 ꢄ 0.01
1.44 ꢄ 0.07
1.64 ꢄ 0.08
2.00 ꢄ 0.10
1.58 ꢄ 0.08
0.80 ꢄ 0.04
0.30 ꢄ 0.02
0.30 ꢄ 0.02
0.04 ꢄ 0.01
0.08 ꢄ 0.01
0.04 ꢄ 0.01
0.48 ꢄ 0.02
0.02 ꢄ 0.01
0.16 ꢄ 0.01
2.54 ꢄ 0.13
4.14 ꢄ 0.21
0.18 ꢄ 0.01
1.52 ꢄ 0.08
1.68 ꢄ 0.08
2.48 ꢄ 0.12
1.60 ꢄ 0.08
0.96 ꢄ 0.05
3.05 ꢄ 0.15
5.01 ꢄ 0.25
0.73 ꢄ 0.04
2.07 ꢄ 0.10
4.43 ꢄ 0.22
1.02 ꢄ 0.05
0.95 ꢄ 0.05
0.73 ꢄ 0.04
0.30 ꢄ 0.02
0.12 ꢄ 0.01
0.06 ꢄ 0.01
0.02 ꢄ 0.01
0.10 ꢄ 0.01
0.02 ꢄ 0.01
0.06 ꢄ 0.01
0.02 ꢄ 0.01
3.35 ꢄ 0.17
5.13 ꢄ 0.26
0.79 ꢄ 0.04
2.09 ꢄ 0.10
4.53 ꢄ 0.23
1.04 ꢄ 0.05
1.01 ꢄ 0.05
0.75 ꢄ 0.04
xanthotoxola
a
Xanthotoxol was used as standard.
a yellowewhite solid (0.173 g, 95% yield). m. p. 230e232 ꢃC. IR
(0.346 g, 85% yield). m. p. 210e213 ꢃC. IR (KBr): 3406, 1728, 1670,
1633, 1599, 1516, 1384 cmꢂ1 1H NMR (300 MHz, DMSO-d6)
(ppm):
(KBr): 3430, 3339, 1729, 1632, 1599, 1532, 1383 cmꢂ1 1H NMR
d
(300 MHz, CDCl3)
d
(ppm): 12.48 (s, 1 H, eOH), 8.33 (s, 1 H), 8.19 (d,
9.70 (s, 1 H, eOH), 7.15 (d, 1 H), 6.68e6.89 (m, 4 H), 6.10 (s, 1 H),
5.38e5.43 (m, 1 H), 3.75e3.90 (m, 1 H), 3.71 (s, 3 H), 3.15e3.23
(m,1 H), 2.37 (s, 3 H), 2.21 (s, 3 H). 13C NMR (75 MHz, DMSO-d6)
J ¼ 8.4 Hz, 1 H), 7.77e7.79 (m, 1 H), 7.49e7.60 (m, 2 H), 6.99 (d,
J ¼ 8.7 Hz, 1 H), 6.10 (s, 1 H), 5.03e5.10 (m, 1 H), 4.22e4.31 (m, 1 H),
3.53e3.63 (m, 1 H), 2.41 (s, 3 H). MS (ESI): m/z 364.3 [M-Hþ].
d
(ppm): 168.08, 162. 24, 159.84, 153.84, 151.71, 147.68, 145.81,
133.55, 127.28, 118.49, 115.24, 113.59, 111.01, 109.94, 109.44, 106.47,
58.44, 55.56, 21.93, 20.48.
4.2.2.2. 8-(4,5-Dihydro-5-(3-methoxy-4-hydroxy)-1H-pyrazol-3-yl)-
7-hydroxy-4-methyl-2H-1-benzopyran-2-one (3b). The reaction
was conducted according to the general procedure B, the product
was further purified by flash column chromatography with chlo-
roform and methanol (15:1, v:v) as eluent. 4,5-Dihydropyrazol
derivative 3b was obtained as a yellowewhite solid (0.170 g, 93%
yield). m. p. 228e230 ꢃC. IR (KBr): 3433, 3418, 1723, 1625, 1598,
4.2.3.3. 8-(1-Acetyl-4,5-dihydro-5-(2,4-dimethoxy)-1H-pyrazol-3-
yl)-7-hydroxy-4-methyl-2H-1-benzopyran-2-one (3e). The reaction
was conducted according to the general procedure C to give 3e as
a yellowewhite solid (0.176 g, 87% yield). m. p. 213e214 ꢃC. IR
(KBr): 3426, 1735, 1672, 1633, 1600, 1508, 1386 cmꢂ1 1H NMR
1514, 1385 cmꢂ1 1H NMR (300 MHz, DMSO-d6)
d
(ppm): 12.75 (s,
(300 MHz, CDCl3) d (ppm): 11.97 (s, 1H, OH), 7.55 (d, 1H), 6.99e7.04
1 H, eOH), 8.94 (s, 1 H, eOH), 7.92 (s, 1H, eNH), 7.15 (d, 1 H),
6.77e7.01 (m, 4 H), 6.19 (s, 1 H), 4.77e4.84 (m, 1 H), 3.83e3.93 (m,
1 H), 3.77 (s, 3 H), 3.22e3.28 (m, 1 H), 2.39 (s, 3 H). 13C NMR
(m, 2H), 6.40e6.43 (m, 2H), 6.12 (d, 1H), 5.64e5.569 (m, 1H),
4.19e4.30 (m, 1H), 3.75 (d, 6H), 3.64e3.72 (m, 1H), 2.39 (d, 6H).
(75 MHz, DMSO-d6)
d
(ppm): 160.69, 159.33, 153.85, 151.54, 149.03,
4.2.3.4. 8-(1-Acetyl-4,5-dihydro-5-(2-allyloxy)-1H-pyrazol-3-yl)-7-
hydroxy-4-methyl-2H-1-benzopyran-2-one (3f). The reaction was
conducted according to the general procedure C to give 3f as
a yellowewhite solid (0.129 g, 62% yield). m. p. 209e210 ꢃC. IR
(KBr): 3437, 1739, 1671, 1632, 1599, 1384 cmꢂ1 1H NMR (300 MHz,
147.59, 132.82, 125.95, 119.17, 115.34, 113.14, 111.95, 110.83, 110.19,
105.33, 62.27, 55.56, 44.89, 18.45. MS (ESI): m/z 366.1 [M ꢂ Hþ].
4.2.3. General procedure C for the synthesis of 8-(1-acetyl-4,5-dihydro-
5-aryl-1H-pyrazol-3-yl)-7-hydroxy-4-methyl-2H-1-benzopyran-2-
ones (3cef)
To a suspension of ketone 2 (1.0 mmol) in acetic acid (2.0 mL)
was added hydrazine monohydrate (4.0 mmol) and the reaction
mixture was refluxed for 2 h. The mixture was then cooled to room
temperature, poured into crush ice, and allowed to stand at room
temperature over night. The crude product was collected by
filtration and purified by flash column chromatography with
chloroform and ethyl acetate (9:1, v:v) as eluent.
CDCl3)
d
(ppm): 12.01 (s, 1 H, eOH), 7.55 (d, J ¼ 8.7 Hz, 1 H),
7.17e7.25 (m, 2 H), 7.00 (d, J ¼ 9.0 Hz,1 H), 6.85e6.93 (m, 1 H), 6.11
(s, 1 H), 5.86e5.97 (m, 1 H), 5.69e5.74 (m, 1 H), 5.27 (dd, J ¼ 1.2,
17.1 Hz, 1 H), 5.11 (dd, J ¼ 1.2, 10.2 Hz, 1 H), 4.48e4.52 (m, 2 H),
4.24e4.34 (m, 1 H), 3.73e3.81 (m, 1 H), 2.40 (d, 6 H).
4.2.4. 8-(1-Acetyl-4,5-dihydro-5-(3,4-dihydroxy)-1H-pyrazol-3-yl)-
7-hydroxy-4-methyl-2H-1-benzopyran-2-one (4a)
A suspension of 3d (0.204 g, 0.5 mmol) in toluene (10 mL), at
0e5 ꢃC, was added aluminum chloride (0.131 g, 1.0 mmol) lot wise.
Then the temperature was raised to 50e60 ꢃC, and pyridine
(166 mL, 2.0 mmol) was added drop-wise. Then the reaction was
heated to reflux for 6 h. Upon completion of the reaction (progress
of the reaction was monitored by TLC), the reaction mixture was
cooled to room temperature. Then aq. HCl (5.0%) was added drop-
wise and the mixture was stirred for 30 min. The organic phase was
separated and the aqueous phase was extracted with ethyl acetate,
and the combined organic phase was washed with water, dried
over anhydrous sodium sulfate. The solvent was removed and the
crude product was purified by flash chromatography column with
chloroform and methanol (9:1, v:v) as eluent. Compound 4a was
obtained as a white solid (0.138 g, 70% yield). m. p. 229e231 ꢃC. IR
(KBr): 3443, 3428, 1724, 1673, 1623, 1599, 1517, 1385 cmꢂ1 1H NMR
4.2.3.1. 8-(1-Acetyl-4,5-dihydro-5-(3-nitrophenyl)-1H-pyrazol-3-yl)-
7-hydroxy-4-methyl-2H-1-benzopyran-2-one (3c). The reaction was
conducted according to the general procedure C to give title
compounds 3c as a white solid (0.362 g, 89% yield). m. p.
235e237 ꢃC. IR (KBr): 3432, 1736, 1673, 1633, 1597, 1500, 1384 cmꢂ1
1H NMR (300 MHz, CDCl3)
d (ppm): 11.68 (s, 1H, eOH), 8.08e8.17
(m, 2 H), 7.52e7.66 (m, 3 H), 7.05 (d, 1 H), 6.13 (s, 1 H), 5.60e5.65
(m, 1 H), 4.40e4.51 (m, 1 H), 3.80e3.88 (m, 1 H), 2.42 (s, 6 H). 13C
NMR (75 MHz, CDCl3)
152.20, 147.65, 142.11, 131.45, 129.02, 126.89, 122.02, 119.68, 113.28,
113.27, 111.68, 110.27, 56.95, 45.47, 21.01, 18.10.
d (ppm): 166.86, 160.69, 158.59, 153.30,
4.2.3.2. 8-(1-Acetyl-4,5-dihydro-5-(3-methoxy-4-hydroxy)-1H-pyr-
azol-3-yl)-7-hydroxy-4-methyl 2H-benzopyran-2-one (3d). The
reaction was conducted according to the general procedure C the
eluent for purification by flash column chromatography is chloro-
form and methanol (15:1, v:v). 3d was obtained as a white solid
(300 MHz, DMSO-d6) d (ppm): 8.85 (s, 1 H, eOH), 8.84 (s, 2 H, eOH),
7.72 (d, J ¼ 8.7 Hz, 1 H), 6.99 (d, J ¼ 8.7 Hz, 1 H), 6.59e6.67 (m, 3 H),
6.21 (s, 1 H), 5.30e5.38 (m, 1 H), 3.89e3.99 (m, 1 H), 3.23e3.32 (m,
1 H), 2.39 (s, 3 H), 2.23 (s, 3 H). 13C NMR (75 MHz, DMSO-d6)

166
C. Xiao et al. / European Journal of Medicinal Chemistry 53 (2012) 159e167
d
(ppm): 166.94, 160.20, 159.18, 157.52, 154.88, 153.71, 153.05,
UV-absorbance of n-butanol layer was measured at 535 nm, and
152.72, 127.73, 126.97, 118.41, 113.16, 112.24, 110.49, 106.06, 102.74,
54.14, 45.18, 21.82, 18.39. MS (ESI): m/z 393.2 [M ꢂ Hþ], m/z 787.3
[2M ꢂ Hþ].
plotted versus incubation time.
4.4. Antioxidant effectiveness in chemical experimental systems
The experiments of 3aed and 4aec to trap ABTSþꢀ and DPPH
were carried out following the description in literature [46,47]. The
radical scavenging activity of xanthotoxol was also assayed as
positive control. Briefly, ABTS (2.00 mL, 4.0 mM) was oxidized by
1.41 mM K2S2O8 for 16 h to generate ABTSþꢀ, to which 100 mL of
ethanol was added to make the absorbance (Absref) around 0.70 at
4.2.5. 8-(1-Acetyl-4,5-dihydro-5-(2,4-dihydroxy)-1H-pyrazol-3-yl)-
7-hydroxy-4-methyl-2H-1-benzopyran-2-one (4b)
Compound 3e (0.211 g, 0.5 mmol) was dissolved in anhydrous
CH2Cl2 (15 mL) under N2, and cooled to ꢂ20 ꢃC. Then 1 M BBr3 in
CH2Cl2 (1.5 mL) was added drop-wise via a syringe. The mixture
was stirred at room temperature for the indicated time. The
mixture was poured cautiously to icy water and extracted with
ethyl acetate. The combined organic phase was washed with brine,
dried over anhydrous sodium sulfate, and. concentrated under
vacuum. The residue was purified by column chromatography with
chloroform and methanol (9:1, v:v) as eluent. Compound 4b was
obtained as a white solid (0.16 g, 81% yield). m. p. 240e241 ꢃC. IR
(KBr): 3451, 3429, 1721, 1670, 1629, 1598, 1519, 1386 cmꢂ1 1H NMR
734 nm (
3
¼ 1.6 ꢅ 104 Mꢂ1 cmꢂ1) [48]. DPPH was dissolved in
ABTSþꢀ
ethanol to make the absorbance (Absref) around 1.00 at 517 nm
(
3
¼ 4.09 ꢅ 103 Mꢂ1 cmꢂ1), to which ethanol solution of 3aed
DPPH
or 4aec was added with 40 mM as the final concentration in the
radical solutions. The decay of the absorbance for DPPH solution
(Absdetect) was recorded. The same operation was performed to
ABTSþꢀ with 40
mM as the final concentration for 3aed or 4aec.
(300 MHz, DMSO-d6)
d (ppm): 11.50 (s, 1 H, eOH), 9.48 (s, 1 H,
eOH), 9.16 (s, 1 H, eOH), 7.71 (d, J ¼ 8.7 Hz, 1 H), 6.98 (d, J ¼ 8.7 Hz,
1 H), 6.77 (d, J ¼ 8.4 Hz, 1 H), 6.14e6.29 (m, 3 H), 5.49e5.55 (m, 1 H),
3.91e4.02 (m, 1 H), 3.16e3.24 (m, 1 H), 2.39 (s, 3 H), 2.27 (s, 3H). 13C
NMR (75 MHz, DMSO-d6): 166.89, 160.18, 159.15, 157.51, 154.85,
153.68, 153.00, 152.73, 127.71, 126.95, 118.39, 113.12, 112.22, 110.49,
106.04, 105.19, 102.72, 54.11, 45.14, 21.79, 18.36. MS (ESI): m/z 393.1
[M ꢂ Hþ].
4.5. Statistical analysis
All the data were the average values from at least triple inde-
pendent measurements with the experimental error within 10%.
The equations were analyzed by one-way ANOVA in Origin 7.5
Professional software, and p < 0.001 indicated a significant
difference.
4.2.6. 8-(1-Acetyl-4,5-dihydro-5-(2-dihydroxy)-1H-pyrazol-3-yl)-
7-hydroxy-4-methyl-2H-1-benzopyran-2-one (4c)
Acknowledgments
To a stirred suspension of compound 3f (0.209 g, 0.5 mmol) in
MeOH (8 mL) was added Pd(PPh3)4 (6.2 mg, 5.0
m
mol) under
This work was supported by grants from the National Nature
Science Foundation of China (Projects 90813003 and 31070670).
a nitrogen atmosphere. The slightly yellow mixture was stirred for
5 min, and K2CO3 (0.204 g, 1.5 mmol) was added. The reaction was
then refluxed for 6 h, before cooled to room temperature and
concentrated in vacuo. The residue was treated with 2 N HCl, and
extracted with ethyl acetate. The combined organic phase was
washed with brine, dried over anhydrous sodium sulfate, and
concentrated in vacuo. The crude product was purified by flash
column chromatography with chloroform and methanol (15:1, v:v)
as eluent. Compound 4c was obtained as an off-white solid (0.124 g,
68% yield). m. p. 231e234 ꢃC. IR (KBr): 3430, 1731, 1670, 1633, 1599,
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