Synthesis and biological evaluation of a novel series of 1,3-dicoumarinyl-5-aryl-2-pyrazolines

Article abstract of DOI:10.3987/COM-11-12196

In the present paper novel 1,3,5-trisubstituted 2-pyrazolines (4a-q) were synthesized via condensation of different substituted 3-cinnamoyl-2-oxo-2H- chromenes (2a-q) with 2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetohydrazide (3). Chalcones were prepared vi

Full text of DOI:10.3987/COM-11-12196

HETEROCYCLES, Vol. 83, No. 7, 2011
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HETEROCYCLES, Vol. 83, No. 7, 2011, pp. 1553 - 1566. © The Japan Institute of Heterocyclic Chemistry
Received, 28th February, 2011, Accepted, 11th April, 2011, Published online, 13th April, 2011
DOI: 10.3987/COM-11-12196
SYNTHESIS AND BIOLOGICAL EVALUATION OF A NOVEL SERIES
OF 1,3-DICOUMARINYL-5-ARYL-2-PYRAZOLINES
Milan Čačić,* Maja Molnar, and Ivica Strelec
Department of Applied Chemistry and Ecology, Faculty of Food Technology, J. J.
Strossmayer University, Franje Kuhača 20, 31 000 Osijek, Croatia; e-mail:
mcacic@ptfos.hr
Abstract – In the present paper novel 1,3,5-trisubstituted 2-pyrazolines (4a-q)
were
synthesized
via
condensation
of
different
substituted
3-cinnamoyl-2-oxo-2H-chromenes (2a-q) with 2-(4-methyl-2-oxo-2H-chromen-7-
yloxy)acetohydrazide (3). Chalcones were prepared via Claisen-Schmidt
condensation by refluxing 3-acetyl-2-oxo-2H-chromen (1) with corresponding
aldehydes in ethanol, in the presence of piperidine. All of these compounds were
1
characterized by means of their IR, H NMR and LC/MS/MS spectroscopic data
and elemental microanalysis. Chalcones and pyrazolines were screened for their
antioxidant and iron chelating activity.
INTRODUCTION
Coumarins comprise a very large and important class of organic compounds found in various plants1 and
have been recognized to possess variety of biological activities, anti-inflammatory,2 antioxidant,3
antithrombotic,4 antiallergic, hepatoprotective,5 antiviral2 and anticarcinogenic,5 respectively. Natural,
semi-synthetic and synthetic coumarins are useful substances in drug research. Numerous coumarin
derivatives can be utilized for the synthesis of different valuable heterocyclic ring systems. Chalcones
with coumarin moiety obtained by the reaction 3-acetylcoumarins with corresponding aromatic
aldehydes6-12 proved to be especially important compounds for this purpose. Chalcones are an important
class of naturally occurring flavonoid compounds, largely distributed in plants, fruits and vegetables that
exhibit a wide spectrum of biological activities. Chalcones in many cases, serve in plant defense
mechanisms against reactive oxygen species (ROS), thus preventing molecular damage and damage by
microorganisms, insects, and herbivores.13 Chalcones have been used in the treatment of atherosclerosis
and they also prevent low density lipoproteins (LDL) oxidation. Some natural chalcones, as well as

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HETEROCYCLES, Vol. 83, No. 7, 2011
synthetic ones, possessing hydroxyl and methoxy substituents, are proven to exhibit significant
antioxidant activity.14 Natural chalcones which have exhibited antioxidant activity include
isoliquiretigenin, isoprenyl chalcone, butein, homobutein, and naringenin chalcone.14 Chalcones are
useful synthons in the synthesis of a large number of bioactive molecules, such as 1,5-benzodiazepines15
and 1,5-benzothiaazepines,16,17 pyrazolines18,19 and isoxazoline.12
Pyrazolines are important nitrogen-containing five-membered heterocycles and some of them possess
significant biological activitiy e.g., activity on the central nervous system of a mammalian host,20
immunosuppressive,21 and antimicrobial22 activity. 2-Pyrazolines display a broad spectrum of potential
pharmacological activities and are present in a number of pharmacologically active molecules such as
phenazone/amidopyrene/methampyrone (analgesic and antipyretic), azolid/ tandearil (anti-inflammatory),
indoxacarb (insecticidal), anturane (uricosuric), etc. and some 1,3,5-substituted pyrazolines have also
proven to be an excellent antioxidants, compared to ascorbic acid.23
Therefore, both coumarins and pyrazolines possess significant bioactivities, which single them out as
useful substances in drug research. On this basis, Levai et al.19 have recently described the synthesis of
1-substituted-5-aryl-3-(3-coumarinyl)-2-pyrazolines by reaction of (3-coumarinyl)-chalcones and
hydrazines. In view of these observations and in continuation of our previous research on the synthesis of
various heterocyclic compounds,24-26 we herein report a synthesis of some new 2-pyrazoline derivatives
with two coumarins moieties, along with their antioxidant and iron chelating activity investigation.
RESULTS AND DISCUSSION
In our work we have synthesized 5-aryl-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-
7-yloxyacetyl)-2-pyrazolines (4a-q) by reaction of 3-aryl-1- (2-oxo-2H-chromen-3-yl)-2-propen-1-ones
(2a-q) and 2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetohydrazide (3) in ethanol, with piperidine as
catalyst (Scheme 2). Starting materials 1, 2a-q and 3 were synthesized according to the known procedures
(Scheme 1).8-11, 25, 27, 28
The structures of all the new compounds 4a-q were elucidated by elemental analysis and spectroscopic
technique data. In their IR spectra a characteristic lactone carbonyl band at around 1730 cm-1 proves the
presence of the coumarin moiety. A C=N band at around 1600 cm-1 is in accordance with the 2-pyrazoline
1
frame (skeleton). H NMR spectra of compounds 4a-q gave a characteristic ABX spin system for the
protons on the C-4 and C-5 carbon atoms of the five-membered ring, which are typical for 2-pyrazoline
structure.18 Their mass spectra exhibit molecular ion peaks and contain fragments that confirm the
pyrazoline ring structure.29 Molecular ion peaks of compounds 4a-q are of relatively low intensity.

HETEROCYCLES, Vol. 83, No. 7, 2011
1555
O
O
O
O
O
H
solvent free
+
O
OH
O
O
1
Ar
ArCHO
1
O
O
2a-q
H2NNH2
H
N
O
H2N
O
O
O
O
O
O
O
O
3
Scheme 1. Synthetic route for preparation of 3-aryl-1-(2-oxo-2H-chromen-3-yl)2-propen-1-ones and
2-(4-methyl-2-oxo-2H-chromen-7-yloxy)acetohydrazide
O
O
O
O
2a-q
+
3
N
N
Ar
4a-q
O
O
Scheme 2. Synthetic route for preparation of 5-aryl-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-
chromen-7-yloxyacetyl)-2-pyrazolines (4a-q)
2,4
a
b
c
Ar
2,4
j
Ar
phenyl
4-fluorophenyl
3-hydroxyphenyl
4-hydroxyphenyl
2-methoxyphenyl
3-methoxyphenyl
4-methoxyphenyl
2-chlorophenyl
3-bromophenyl
4-bromophenyl
k
l
3-phenoxyphenyl
4-N,N-dimethylaminophenyl
4-styryl
d
e
m
n
o
4-hydroxy-3-methoxyphenyl
2,4-dihydroxyphenyl
3,4-dihydroxyphenyl
3,4,5-trimethoxyphenyl
f
g
h
i
p
q

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HETEROCYCLES, Vol. 83, No. 7, 2011
Antioxidant activity
Antioxidant activity can be expressed in a several ways, by suppression of the formation of ROS by
inhibiting the certain enzymes involved in its generation, by chelating the metal ions responsible for the
production of free radicals, by scavenging the ROS and regulating and protecting antioxidant defence
mechanism.14 Antioxidant capacity of any compound is related to its hydrogen or electron donation
capacity, its ability to stabilize and delocalize the unpaired electron and its potential to chelate transition
metal ions.14 In the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and phosphomolybdenum assay
methods hydrogen and electron transfer from antioxidants to DPPH radical and Mo(VI) complex occurs.
These transfers depend on the structure of antioxidants. The reason we used ascorbic acid as standard in
both antioxidant investigations is that ascorbic acid has the ability to donate hydrogen and electrons and
can be detected by both assay models.
Scavenging of 1,1-diphenyl-2-picrylhydrazyl radical
The unpaired electron in the DPPH free radical gives a strong absorption maximum at 517 nm and is
purple in colour. The colour turns from purple to yellow when the unpaired electron of DPPH radical
becomes paired with hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-H.
The method used in this work applies to the antioxidant capacity of the sample after 30 minutes of
incubation. DPPH scavenging activity was determined against 0.2 mM ascorbic acid as a standard
compound.
Table 1. DPPH scavenging activity of novel chalcones (2a-q) and pyrazolines (4a-q)a
DPPH scavenging activity
(%)
compound
AAb
compound DPPH scavenging activity (%)
88.2
AA2
88.2
2a
2b
2c
2d
2e
2f
2g
2h
2i
8.1
4a
4b
4c
4d
4e
4f
4g
4h
4i
13.2
20.8
7.4
10.2
13.3
11.5
5.3
12.4
19.1
18.7
15.7
17.1
6.4
19.0
55.0
14.4
22.0
42.7
20.3
15.2
5.5
2j
2k
4j
4k
2l
7.4
4l
48.3

HETEROCYCLES, Vol. 83, No. 7, 2011
1557
2m
2n
2o
13.6
27.0
20.0
36.3
10.8
4m
4n
4o
6.0
33.9
20.5
14.0
19.8
2p
2q
4p
4q
adata are means of three replicates
b0.2 mM ascorbic acid was used as standard
According to the data in Table 1 the best DPPH scavengers are pyrazolines 4e, 4l and 4h showing 55%,
48% and 43% scavenging activity, two of them 4e and 4l possessing electron donating groups and 4h
possessing electron withdrawing bromine substituent on a phenyl ring. Compounds possessing hydroxyl
groups on phenyl ring showed less activity than expected, since the previous studies showed that the
hydroxybenzoyl group has a key role as free radical scavenger in other classes of compounds.30 This
proves that the introduction of pyrazoline ring enhances the DPPH scavenging activity of the
corresponding chalcones they were synthesized from.
In the series of chalcones the best scavenger of DPPH radicals was 2p, possessing better activity than
corresponding pyrazoline 4p (14%), thus, with 2j and 2k, making an exception from the fact that the
introduction of pyrazoline ring enhances scavenging activity of corresponding chalcones. Compounds 2n
and 2o are the best DPPH scavengers, in a series of chalcones, after 2p, both possessing two electron
donating groups on a phenyl ring, which is in accordance with previous investigations.27, 30
Evaluation of antioxidant activity by phosphomolybdenum method
This method is based on the reduction of Mo(VI) to Mo(V) by the tested compounds followed by
formation of green phosphate/Mo(V) complex at acid pH. The mechanism responsible for reduction of
Mo(VI) to Mo(V) is dependent on the transfer of the single electron from the compound to molybdenum.
From the data in the Figure 1 it is evident that pyrazolines possess better antioxidant activity than the
corresponding chalcones, as it is a case with the DPPH scavenging activity too. Pyrazoline 4m showed
even better antioxidant activity than ascorbic acid, which was used as standard. Other pyrazolines
showing significant antioxidant activity, comparable to this of ascorbic acid are 4f, 4c, 4g, 4o and 4n. All
of these pyrazolines possess an electron donating substituent in p-position, except for 4g, possessing an
electron withdrawing -Cl substitutent in o-position of phenyl ring.
Among the chalcone series the one showing the highest activity was 2l, but in comparison with
pyrazolines, its activity is not significant. The introduction of a pyrazoline ring obviously increases the
antioxidant activity in this assay as well.

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HETEROCYCLES, Vol. 83, No. 7, 2011
Figure 1. Antioxidant activities of chalcones and pyrazolines relative to ascorbic acid (Am – activity
relative to ascorbic acid (AA) on a molar basis). 2 mM ascorbic acid was used as standard.
Iron chelating activity
Various compounds can act as antioxidants by chelating the metal ion responsible for the production of
free radical, Fe2+ being one of them. This investigation is based upon complex formation between
ferrozine and Fe2+, which is red in colour. In the presence of chelating agents, the complex formation is
disrupted with the result that the red colour of the complex is decreased. Therefore, the chelating activity
of the coexisting chelator is estimated by measurement of colour reduction at 562 nm.
Table 2. Chelating activity of chalcones and pyrazolinesa,c,e
compound
chelating activity (%)
EDTAd
93.46
1.49
2c
2l
5.43
4l
2.00
2o
61.13
adata are means of three replicates
ccompounds not listed are inactive
d2 mM EDTA was used as standard
ecompounds 4a, 4c, 4d, 4f, 2h, 4j, 4k, 4m are not completely soluble in the solvent used, thus not possible to
determine chelating activity under these conditions

HETEROCYCLES, Vol. 83, No. 7, 2011
1559
Of all the compounds investigated on iron chelating activity, only four of them showed some kind of
activity, with 2o being the best iron chelator of all, with 61% chelating activity, respectively. Compound
2o is coumarinyl chalcone, bearing 2,4-dihydroxyphenyl moiety, and its ability to chelate metal ions is in
accordance with inevestigation of Khatib et al.31 who have proven an importance of resorcinol group (2,4
dihidroxy phenyl ring) for tyrosinase inhibition and copper chelating activity.31 In general chalcones have
proven to possess better chelating activity than corresponding pyrazolines.
EXPERIMENTAL
General information
The melting points were taken on Electrothermal Capillary melting point apparatus and are uncorrected.
Thin-layer chromatographies were performed using fluorescent silica gel plates HF254 Merck, which were
examined under UV 254 and 365 nm light, using benzene:acetone:acetic acid (8:1:1) as eluent. The
elemental analysis for C, H and N were done on a Perkin-Elmer Analyzer 2440. Infrared spectra (/cm-1)
1
were recorded on a Shimadzu FT-IR 8400 S, using KBr discs. H NMR spectra were recorded on JEOL
EX-270 MHz NMR Spectrometer at 293 K in DMSO-d6. Spectra were internally referenced to TMS.
Peaks are reported in ppm downfield of TMS. The mass spectra were recorded on the LC/MS/MS (API
2000) Applied Biosystems. The absorbance was measured on a UV/Vis spectrophotometer Helios ,
(Thermo Spectronic, Cambridge UK).
Synthesis of 3-acetyl-2-oxo-2H-chromen (1)
To a cold mixture of salicylaldehyde (0.2 mole) and ethyl acetoacetate (0.2 mole), 2 ml piperidine was
added by rapid stirring. After 20-30 min the yellowish precipitate was filtered off and subsequently
washed with EtOH. Recystallization from water: EtOH mp 118-120 ºC (mp 117-119 ºC),32 yield 81%.
Procedure for synthesis of 3-aryl-1-(2-oxo-2H-chromen-3-yl)- 2-propen-1-ones (2a-q)
Mixture of 0.031 mole of 3-acetylcoumarin (1) and 0.03 mole of appropriate aromatic aldehyde is
dissolved in 30 mL of CHCl3. Piperidine (0.02 mole) is added and the solution is refluxed for 1.5 h.
CHCl3 is distilled off and the precipitate is washed with MeOH.7-10
Synthesis of 2-[(4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetohydrazide (3)27, 28
Methyl 2-[(4-methyl-2-oxo-2H-chromen-7-yl)oxy]acetate (0.01 mole) in EtOH (20 mL) was stirred for
30 min at room temperature. Hydrazine hydrate (0.014 mole) was added to the solution. This mixture was
stirred under reflux for 1 h, and the solid was filtered. The residue was dried and then desiccated to afford
a crystalline powder. The powder was recrystallized from CHCl3/MeOH to afford colourless needles with,

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HETEROCYCLES, Vol. 83, No. 7, 2011
mp 198-200 ºC (lit., mp 202-204 ºC)28 and yield 80-90%.
Synthesis of 5-aryl-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-
pyrazoline (4a-q)
Chalcones (2a-q) (0.05 mole) and 0.2 mole of hydrazide (3) is dissolved in 30 mL of pyridine and
refluxed for 6 h. Solution is poured into crushed ice and neutralized with 2N HCl. The precipitate is
filtered off, dried and recrystallized from appropriate solvent.
5-Phenyl-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-pyrazoline
(4a)
This compound was prepared as yellow crystals in 75% yield, mp 235-240 ºC; FT-IR (KBr), νmax 3173,
3049, 1724, 1685, 1616, 1560, 1392, 1277, 1201, 1142, 1078, 842, 748 cm-1; 1H NMR (DMSO-d6) δ 2.42
(s, 3H, CH3), 3.41 (dd, 1Htrans, CH2, pyr.), 3.96 (dd, 1Hcis, CH2, pyr.), 4.83 (s, 2H, CH2), 5.56 (s, 1H, CH),
6.90-7.54 (m, 12H, coum. and arom.), 8.40 (s, 1H, H-4 coum); MS m/z: 507.0 [M+H+], (M=506.51). Anal.
Calcd for C30H22N2O6: C, 71.14; H, 4.38; N, 5.53. Found: C, 71.08; H, 4.40; N, 5.50.
5-(3-Hydroxyphenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-
pyrazoline (4b)
This substance was prepared as yellow crystals in 85% yield, mp 268-269 ºC, FT- IR (KBr), νmax 3283,
1
3084, 1720, 1612, 1538, 1391, 1276, 1253, 1151, 1078, 841, 748 cm-1; H NMR (DMSO-d6) δ 2.42 (s,
3H, CH3), 3.44 (dd, 1Htrans, CH2, pyr.), 3.95 (dd, 1Hcis, CH2, pyr.), 4.85 (s, 2H, CH2), 5.59 (s, 1H, CH,
H-5), 7.14-7.54 (m, 12H, coum. and arom.), 8.45 (s, 1H, H-4 coum), 10.12 (s, 1H, OH); MS m/z: 521.3
[M-H+], (M=522.50). Anal. Calcd for C30H22N2O7: C, 68.96; H, 4.24; N, 5.36. Found: C, 68.90; H, 4.26;
N, 5.32.
5-(4-Hydroxyphenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-
pyrazoline (4c)
This substance was prepared as pale yellow crystals in 85% yield, mp 289 ºC; FT- IR (KBr), νmax 3286,
3074, 1720, 1693, 1612, 1558, 1512, 1390, 1269, 1153, 1078, 841, 750 cm-1. 1H NMR(DMSO-d6) δ 2.43
(s, 3H, CH3), 3.45 (dd, 1Htrans, CH2, pyr.), 3.96 (dd, 1Hcis, CH2, pyr.), 4.82 (s, 2H, CH2), 5.59 (s, 1H, CH,
H-5), 7.18-7.57 (m, 12H, coum. and arom.), 8.45 (s, 1H, H-4 coum), 10.05 (s, 1H, OH); MS m/z: 521.0
[M-H+], (M=522.50). Anal. Calcd for C30H22N2O7: C, 68.96; H, 4.24; N, 5.36. Found: C, 68.92; H, 4.22;
N, 5.30.
5-(2-Methoxyphenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-

HETEROCYCLES, Vol. 83, No. 7, 2011
1561
pyrazoline (4d)
This compound was prepared as yellow crystals in 65% yield, mp 273 ºC, FT- IR (KBr), νmax 3285, 3068,
1724, 1701, 1614, 1539, 1489, 1391, 1276, 1151, 1078, 842, 750 cm-1. 1H NMR (DMSO-d6) δ 2.44 (s, 3H,
CH3), 3.45 (dd, 1Htrans, CH2, pyr.), 3.95 (dd, 1Hcis, CH2, pyr.), 4.83 (s, 2H, CH2), 5.59 (s, 1H, CH, H-5),
7.12-7.56 (m, 12H, coum. and arom.), 8.42 (s, 1H, H-4 coum); MS m/z: 537.3 [M+H+], (M=536.53). Anal.
Calcd for C30H22N2O7: C, 68.96; H, 4.24; N, 5.36. Found: C, 68.97; H, 4.20; N, 5.36.
5-(3-Methoxyphenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-
pyrazoline (4e)
This substance was obtained as orange crystals in 80% yield, mp 213 ºC; FT- IR (KBr), νmax 3311, 3097,
1720, 1664, 1606, 1541, 1483, 1450, 1291, 1184, 1049, 978, 760 cm-1; 1H NMR(DMSO-d6) δ 2.42 (s, 3H,
CH3), 3.46 (dd, 1Htrans, CH2, pyr.), 3.73 (s, 3H, OCH3), 3.94 (dd, 1Hcis, CH2, pyr.), 4.83 (s, 2H, CH2), 5.59
(s, 1H, CH, H-5), 7.18-7.60 (m, 12H, coum. and arom.), 8.44 (s, 1H, H-4 coum); MS m/z: 537.0 [M+H+],
(M=536.53). Anal. Calcd for C31H24N2O7: C, 69.40; H, 4.51; N, 5.22. Found: C, 69.38; H, 4.49; N, 5.24.
5-(4-Methoxyphenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-
pyrazoline (4f)
This substance was prepared as pale yellow in 78% yield, mp 267 ºC; FT- IR (KBr), νmax 3282, 3174,
1
1724, 1685, 1620, 1537, 1429, 1392, 1300, 1153, 1078, 841, 748 cm-1; H NMR(DMSO-d6) δ 2.44 (s,
3H, CH3), 3.42 (dd, 1Htrans, CH2, pyr.), 3.71 (s, 3H, OCH3), 3.96 (dd, 1Hcis, CH2, pyr.), 4.83 (s, 2H, CH2),
5.59 (s, 1H, CH, H-5), 7.16-7.64 (m, 12H, coum. and arom.), 8.44 (s, 1H, H-4 coum); MS m/z: 537.2
[M+H+], (M=536.53). Anal. Calcd for C31H24N2O7: C, 69.40; H, 4.51; N, 5.22. Found: C, 69.41; H, 4.48;
N, 5.20.
5-(2-Chlorophenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-
pyrazoline (4g)
This substance was prepared as orange crystals in 81% yield, mp 205 ºC; FT- IR (KBr), νmax 3284, 3157,
1724, 1691, 1618, 1560, 1508, 1489, 1390, 1277, 1153, 1078, 841, 748 cm-1; 1H NMR (DMSO-d6) δ 2.44
(s, 3H, CH3), 3.46 (dd, 1Htrans, CH2, pyr.), 3.96 (dd, 1Hcis, CH2, pyr.), 4.82 (s, 2H, CH2), 5.58 (s, 1H, CH,
H-5), 7.16-7.64 (m, 12H, coum. and arom.), 8.45 (s, 1H, H-4 coum); MS m/z: 542.2 [M+H+], (M=540.95).
Anal. Calcd for C30H21ClN2O6: C, 66. 61; H, 3.91; N, 5.18. Found: C, 66.59; H, 3.90; N, 5.21.
5-(3-Bromophenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-
pyrazoline (4h)
This substance was prepared as orange crystals in 70% yield, mp 199 ºC; FT-IR (KBr), νmax 3287, 3064,

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HETEROCYCLES, Vol. 83, No. 7, 2011
1
1724, 1701, 1616, 1558, 1390, 1276, 1203, 1153, 1078, 843, 750 cm-1; H NMR (DMSO-d6) δ 2.42 (s,
3H, CH3), 3.42 (dd, 1Htrans, CH2, pyr.), 3.97 (dd, 1Hcis, CH2, pyr.), 4.84 (s,2H, CH2), 5.56 (s, 1H, CH, H-5),
7.16-7.64 (m, 12H, coum. and arom.), 8.45 (s,1H, H-4 coum); MS m/z: 587.0 [M+H+], (M=585.40). Anal.
Calcd for C30H21BrN2O6: C, 61.55; H, 3.62; N, 4.79. Found: C, 61.50; H, 3.64; N, 4.82.
5-(4-Bromophenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-
pyrazoline (4i)
This substance was prepared as yellow-orange crystals in 68% yield, mp 289 ºC; FT- IR (KBr), νmax 3288,
3080, 1724, 1614, 1557, 1487, 1390, 1277, 1201, 1151, 1076, 849, 750 cm-1; 1H NMR (DMSO-d6) δ 2.44
(s, 3H, CH3), 3.46 (dd, 1Htrans, CH2, pyr.), 3.97 (dd, 1Hcis, CH2, pyr.), 4.82 (s,2H, CH2), 5.56 (s, 1H, CH,
H-5), 7.16-7.64 (m, 12H, coum. and arom.), 8.44 (s,1H, H-4 coum); MS m/z: 587.4 [M+H+], (M=585.40).
Anal. Calcd for C30H21BrN2O6: C, 61.55; H, 3.62; N, 4.79. Found: C, 61.57; H, 3.61; N, 4.80.
5-(4-Fluorophenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-
pyrazoline (4j)
This substance was prepared as pale yellow crystals in 64% yield, mp 260 ºC; FT-IR (KBr), νmax 3285,
1
3084, 1724, 1685, 1620, 1537, 1489, 1392, 1300, 1277, 1201, 1153, 1078, 841, 748 cm-1; H NMR
(DMSO-d6) δ 2.44 (s, 3H, CH3), 3.45 (dd, 1Htrans, CH2, pyr.), 3.96 (dd, 1Hcis, CH2, pyr.), 4.83 (s, 2H, CH2),
5.54 (s, 1H, CH, H-5), 7.16-7.68 (m, 12H, coum. and arom.), 8.45(s,1H, H-4 coum); MS m/z: 525.60
[M+H+], (M=524.50). Anal. Calcd for C30H21FN2O6: C, 68.70; H, 4.04; N, 5.34. Found: C, 68.71; H,
4.07; N, 5.32.
5-(3-Phenoxyphenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-
pyrazoline (4k)
This substance was prepared as brown crystals in 70% yield, mp 225 ºC; FT-IR (KBr), νmax 3282, 3063,
1724, 1689, 1612, 1560, 1532, 1489, 1300, 1300, 1274, 1202, 1153, 1078,748 cm-1; 1H NMR (DMSO-d6)
δ 2.42 (s, 3H, CH3), 3.46 (dd, 1Htrans, CH2, pyr.), 3.96 (dd, 1Hcis, CH2, pyr.), 4.82 (s, 2H, CH2), 5.55 (s, 1H,
CH, H-5), 7.06-7.68 (m, 18H, coum. and arom.), 8.45 (s, 1H, H-4 coum); MS m/z: 599.80 [M+H+],
(M=598.60). Anal. Calcd for C36H26N2O7: C, 72.23; H, 4.38; N, 4.68. Found: C, 72.20; H, 4.40; N, 4.66.
5-(4-Dimethylaminophenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxy-
acetyl)-2-pyrazoline (4l)
This substance was prepared as red crystals in 84% yield, mp 173 ºC; FT-IR (KBr), νmax 3433, 3288, 1732,
1608, 1570, 1532, 1371, 1276, 1176, 1078, 987, 812, 760 cm-1; 1H NMR (DMSO-d6) δ 2.44 (s, 3H, CH3),
2.85 (s, 6H, N(CH3)2), 3.46 (dd, 1Htrans, CH2, pyr.), 3.97 (dd, 1Hcis, CH2, pyr.), 4.83 (s, 2H, CH2), 5.55 (s,

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1563
1H, CH, H-5), 7.16-7.68 (m, 13H, coum. and arom.), 8.44 (s, 1H, H-4 coum); MS m/z: 550.10 [M+H+],
(M=549.57). Anal. Calcd for C32H27N3O6: C, 69.93; H, 4.95; N, 7.65. Found: C, 69.90; H, 4.97; N, 7.61.
5-Styryl-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-2-pyrazoline
(4m)
This substance was prepared as pale yellow crystals in 84% yield, mp 280-281 ºC; FT-IR (KBr), νmax
3284, 3057, 1720, 1612, 1558, 1448, 1389, 1265, 1149, 1076, 972, 848, 752 cm-1; 1H NMR (DMSO-d6) δ
2.43 (s, 3H, CH3), 3.46 (dd, 1Htrans, CH2, pyr.), 3.97 (dd, 1Hcis, CH2, pyr.), 4.82 (s, 2H, CH2), 5.54 (s, 1H,
CH, H-5), 7.06-7.66 (m, 16H, HC=CH, coum. and arom.), 8.44 (s, 1H, H-4 coum); MS m/z: 531.40
[M-H+], (M=532.54). Anal. Calcd for C32H24N2O6: C, 72.17; H, 4.54; N, 5.26. Found: C, 72.10; H, 4.51;
N, 5.23.
5-(4-Hydroxy-3-methoxyphenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-
yloxyacetyl)-2-pyrazoline (4n)
This substance was prepared as brown crystals in 84% yield, mp 167 ºC; FT-IR (KBr), νmax 3288, 3078,
1724, 1691, 1618, 1559, 1512, 1392, 1274, 1201, 1155, 1080, 841, 748 cm-1; 1H NMR (DMSO-d6) δ 2.43
(s, 3H, CH3), 3.44 (dd, 1Htrans, CH2, pyr.), 3.73 (s, 3H, OCH3), 3.96 (dd, 1Hcis, CH2, pyr.), 4.83 (s, 2H,
CH2), 5.56 (s, 1H, CH, H-5), 7.16-7.66 (m, 12H, coum. and arom.), 8.44 (s, 1H, H-4 coum), 9.95 (s, 1H,
OH); MS m/z: 551.10 [M-H+], (M=552.53). Anal. Calcd for C31H24N2O8: C, 67.39; H, 4.38; N, 5.07.
Found: C, 67.34; H, 4.40; N, 5.00.
5-(2,4-Dihydroxyphenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-
2-pyrazoline (4o)
This substance was prepared as brown crystals in 65% yield, mp 184-185 ºC; FT-IR (KBr), νmax 3269,
3068, 1685, 1612, 1538, 1508, 1391, 1265, 1203, 1151, 1078, 842, 750 cm-1; 1H NMR (DMSO-d6) δ 2.42
(s, 3H, CH3), 3.46 (dd, 1Htrans, CH2, pyr.), 3.97 (dd, 1Hcis, CH2, pyr.), 4.83 (s, 2H, CH2), 5.52 (s, 1H, CH,
H-5), 7.14-7.68 (m, 11H, coum. and arom.), 8.46 (s, 1H, H-4 coum), 9.95 (s, 1H, OH), 10.05 (s, 1H, OH);
MS m/z: 537.20 [M-H+], (M=538.50). Anal. Calcd for C30H22N2O8: C, 66.91; H, 4.12; N, 5.20. Found C,
66.88; H, 4.13; N, 5.10.
5-(3,4-Dihydroxyphenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxyacetyl)-
2-pyrazoline (4p)
This substance was prepared as oker crystals in 60% yield, mp 264 ºC; FT-IR (KBr), νmax 3286, 3159,
1724, 1691, 1618, 1539, 1489, 1390, 1276, 1203, 1155, 1074, 843, 748 cm-1; 1H NMR (DMSO-d6) δ 2.43
(s, 3H, CH3), 3.46 (dd, 1Htrans, CH2, pyr.), 3.96 (dd, 1Hcis, CH2, pyr.), 4.83 (s,2H, CH2), 5.54 (s, 1H, CH,

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H-5), 7.14-7.68 (m, 11H, coum. and arom.), 8.44 (s,1H, H-4 coum.), 9.85 (s, 1H, OH), 10.08 (s, 1H, OH);
MS m/z: 537.10 [M-H+], (M=538.50). Anal. Calcd for C30H22N2O8: C, 66.91; H, 4.12; N, 5.20. Found: C,
66.89; H, 4.15; N, 5.23.
5-(3,4,5-Trimethoxyphenyl)-3-(2-oxo-2H-chromen-3-yl)-1-(4-methyl-2-oxo-2H-chromen-7-yloxy-
acetyl)-2-pyrazoline (4q)
This substance was prepared as yellow crystals in 78% yield, mp 277 ºC; FT-IR (KBr) νmax 3287, 3067,
1
2941, 1720, 1693, 1612, 1560, 1508, 1421, 1390, 1274, 1202, 1155, 1074, 846, 748 cm-1; H NMR
(DMSO-d6) δ 2.44 (s, 3H, CH3), 3.46 (dd, 1Htrans, CH2, pyr.), 3.73 (s, 9H, OCH3), 3.96 (dd, 1Hcis, CH2,
pyr.), 4.83 (s,2H, CH2), 5.54 (s, 1H, CH, H-5), 7.14-7.68 (m, 10H, coum. and arom.), 8.44 (s,1H, H-4
coum.); MS m/z: 597.10 [M+H+], (M=596.58). Anal. Calcd for C33H28N2O9: C, 66.44; H, 4.73; N, 4.70.
Found: C, 66.44; H, 4.73; N, 4.70.
Scavenging of 1,1-diphenyl-2-picrylhydrazyl radical
Determination of antioxidant activity was performed according to the procedure described in the
literature33,34 with some modifications. DMSO was used as a solvent,35 due to the low solubility of
synthesized compounds in EtOH and MeOH.
0.75 mL of DMSO solution of the corresponding synthesized compound (0.2 mM) was added to a DMSO
solution of DPPH radical (0.2 mM), so that the final concentration of DPPH radical and the synthesized
compound in a solution was 0.1 mM. The mixture was shaken and allowed to stand at room temperature.
After 30 min the absorbance at 517 nm was determined and the scavenging activity was calculated
according to the formula below. Ascorbic acid (AA) was used as a reference compound.
scavenging actvitiy (%) = ((Ab + As – Am)/Ab)*100
Ab – absorbance of 0.1 mM DMSO solution of DPPH radical at 517 nm
As – absorbance of 0.1 mM DMSO solution of test compound at 517 nm
Am – absorbance of DMSO mixture of test compound and DPPH radical at 517 nm
Evaluation of antioxidant activity by phosphomolybdenum method
The antioxidant activity of tested coumarin derivatives was evaluated by the phosphomolybdenum
method according to the procedure of Prieto et al.36 An aliquot of 100 L of sample solution (2mM in
DMSO) is mixed with 1 mL of the reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and
4 mM ammonium molybdate). The samples were incubated in a water bath at 95 C for 90 min. Samples
are cooled to room temperature and the absorbance was measured at 695 nm. The antioxidant activity was
expressed relative to the antioxidant activity of the same concentration of ascorbic acid.

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Iron chelating activity
The chelating activity of dipicolinic acid derivatives for ferrous ions Fe2+ was measured according to the
literature method37 with some modifications. 0.025 mL of FeCl2 (2 mM) was added to 1 mL 2mM
MeOH/DMSO solution (4:1) of the compound investigated. After 30 s, 0.05 mL of ferrozine (5 mM) was
added. Samples were incubated at room temperature for 10 min and the absorbance of the complex
formed between Fe2+ and ferrozine was measured at 562 nm. Metal chelating efficiency of samples was
compared to the chelating activtiy of EDTA disodium salt. The chelating activity of the extract for Fe2+
was calculated as:
Chelating rate = [(A0 –A1)/ A0]100%
A0 - absorbance of the control (blank, without samples) at 562 nm
A1 - absorbance in the presence of the MeOH/DMSO sample solution at 562 nm
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