Antioxidant activity of 3-[N-(acylhydrazono)ethyl]-4-hydroxy-coumarins

Article abstract of DOI:10.3390/molecules21020138

A series of 3-acylhydrazono-4-hydroxycoumarins were synthesized via condensation of 3-acetyl-4-hydroxycoumarinwith appropriate hydrazides. The structures of the newly-synthesized compounds were characterizedby spectral andelememental analysis orHRMSmeasur

Full text of DOI:10.3390/molecules21020138

molecules
Article
Antioxidant Activity of
3-[N-(Acylhydrazono)ethyl]-4-hydroxy-coumarins
Antigoni Kotali 1,*, Despina A. Nasiopoulou 1, Constantinos A. Tsoleridis 2, Philip A. Harris 3,
Christos A. Kontogiorgis 4 and Dimitra J. Hadjipavlou-Litina 4
1
Laboratory of Organic Chemistry, Department of Chemical Engineering,
Aristotle University of Thessaloniki, Thessaloniki 54124, Greece; dnasiopoulou@yahoo.gr
Laboratory of Organic Chemistry, Department of Chemistry, Aristotle University of Thessaloniki,
2
Thessaloniki 54124, Greece; tsolerid@chem.auth.gr
GlaxoSmithKline, 1250 South Collegeville Road, P. O. Box 5089, Collegeville, PA 19426-0989, USA;
3
philip.a.harris@gsk.com
4
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki,
Thessaloniki 54124, Greece; ckontogi@med.duth.gr (C.A.K.); hadjipav@pharm.auth.gr (D.J.H.-L.)
*
Correspondence: kotali@eng.auth.gr; Tel./Fax: +30-2310-996-253
Academic Editor: David D. Kitts
Received: 27 October 2015; Accepted: 15 January 2016; Published: 23 January 2016
Abstract: A series of 3-acylhydrazono-4-hydroxycoumarins were synthesized via condensation of
3-acetyl-4-hydroxycoumarin with appropriate hydrazides. The structures of the newly-synthesized compounds
werecharacterizedbyspectralandelemementalanalysisorHRMSmeasurements. Theirantioxidant properties
were evaluated by using scavenging effects on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical as well
as inhibition of lipid peroxidation. Moreover, their ability to inhibit in vitro soybean lipoxygenase has
been investigated. They were found to be capable of rapid inactivation of alkylperoxy radicals.
Keywords: antioxidants; 3-acetyl-4-hydroxycoumarin; acyl hydrazones; DPPH; trolox; lipid peroxidation;
soybean lipoxygenase
1. Introduction
3-Substituted-4-hydroxycoumarins constitute an important class of heterocycles, which occur
widely among natural products and have interesting biological properties and importance in medicine.
They have been reported to exhibit a variety of pharmacological activity as antibacterial, [1,2] antitumor, [3]
activity against HIV virus, [4] antithrombotic, [5–8], as well as antioxidant activity [9,10]. Coumarins are
one of the most important secondary metabolites of plants and are known as naturally occurring
benzo-
of coumarins have been isolated from natural sources. Robustic acid, [12] ferulenol and its analogues, [13
as well as the two sesquiterpenecoumarins isolated from Ferula pallida [15], are characteristic examples
of 4-hydroxycoumarins, which have been isolated as natural products. Furthermore, the 4-hydroxycoumari
moiety has been the molecular template for the synthesis of a variety of analogues with important
biological activity. Warfarin is a synthetic coumarin, which is widely used as anticoangulant, [16
whereas aminocoumarin analogues, such as novobiocin, chlorobiocin, coumermycin, and simocyclinon
are potent antibiotics [17 19]. Furthermore, the importance of hydrazone derivatives is well known
because of their use as synthons in organic synthesis [20 21] as well as because of their biological
α-pyrone derivatives from metabolism of phenylalanine [11]. More than 1000 different types
,
14
]
n
]
e
–
,
properties. They have been reported to possess among others anticonvulsant, antidepressant, analgestic
anti-inflammatory, antimicrobial, antimalarial, antitumoral, antileukemic, antiviral, antitubercular, as
well as antioxidant activity [22–26].
,
Molecules 2016, 21, 138; doi:10.3390/molecules21020138
www.mdpi.com/journal/molecules

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It is well known that the design of biological substrates with antioxidant activity to be used for
disease treatment or as food additives, as well as oxidative stress, have attracted many researchers’
interest. The potential activity of both coumarin, as well as hydrazone derivatives, as antioxidant
agents prompted us to synthesize a series of new coumarin analogues bearing the 3-acylhydrazono
functionality and a 4-hydroxy group on the coumarin ring. The combination of the pharmacophores
of two different biologically-active compounds in the same molecule could lead to a new product
exhibiting combined activity.
The formation of Reactive Oxygen Species (ROS) is characteristic of aerobic organisms that can
normally defend themselves against these highly reactive species. However, in many pathophysiologica
l
conditions the excessive production of ROS overwhelms the natural antioxidant defense mechanisms.
This imbalance is termed oxidative stress, which has been associated with the inflammation process.
ROS, like superoxide radical anion, hydrogen peroxide and hydroxyl radical, are produced during the
inflammation process by phagocytic leukocytes. Moreover, these reactive species are involved in the
biosynthesis of prostaglandins and in the cycloxygenase- and lipoxygenase-mediated conversion of
arachidonic acid. The rates of ROS production are increased in most pathophysiological conditions [27];
therefore, it is evident that the treatment of various diseases could benefit from the use of drugs that
combine antioxidant and anti-inflammatory activity.
Thus, based on the above literature findings and on our interest in coumarin [28
–
32] and
hydrazone derivatives [20 21], as well as in the biological activity of small molecules [23
,
,29], we
present here the synthesis and structural characterization of a series of 3-acylhydrazono-substituted
4-hydroxycoumarins, as well as their in vitro antioxidant and soybean lipoxygenase inhibitory activity.
2. Results and Discussion
3-Acetyl-4-hydroxycoumarin N-acylhydrazones 2a–l were prepared according to the literature [28]
via treatment of 3-acetyl-4-hydroxycoumarins
it is depicted in Scheme 1. The molar ratio of the reactants was 1:1. The reaction was performed
under reflux for 24 h to yield hydrazones 2a in excellent yields. Products 2b 2f 2g, and 2h are
new compounds, whereas 2a 2c 2e, and 2i 2l have been recently synthesized and identified [28].
Compound 2a has also been mentioned in the literature earlier [33] but its spectral data have been
only recently reported [28]. New hydrazones 2b 2f 2g, and 2h were obtained in 70%–94%, whereas
1 with the appropriate hydrazide in n-propanol, as
–
l
,
,
,
–
–
,
,
they have been alternatively afforded via reflux of ketone 1 with the appropriate hydrazide for 2 h in
very good to excellent yields (69%–94%), comparatively lower to those under 24 h reflux (70%–98%).
Hydrazones 2a–l were purified via recrystallization from n-propanol. The mother ketone 1 has been prepared
according to the literature by direct acetylation of 4-hydroxy-coumarin with acetyl chloride [34].
Scheme 1. Synthesis of 3-acetyl-4-hydroxycoumarin N-acylhydrazones 2a–l.

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The structure of the new compounds 2b
,
2f
,
2g, and 2h was identified by their 1H- and 13C-NMR
data as well as by their mass spectra and either their elemental analysis or high-resolution exact
1
mass measurement. In H-NMR a characteristic singlet at about 2.65–2.77 ppm is assigned to the
methyl protons attached at the 3-C=N carbon, whereas the proton at C-5 of the coumarin appears
as a doublet of doublets at about 7.95 to 8.02 ppm in accordance with the literature data for other
4-hydroxycoumarin derivatives [29,35]. Furthermore, full assignments of the proton and carbon
chemical shifts were based on coupling constants and on analogous coumarin derivatives assigned by
detailed study of their 2D NMR data [35,36].
Furthermore, hydrazones
mass spectra. It should be noted that according to the literature data [35] compound
2
show prominent peaks corresponding to the ion [M + 1] in their
derivatives
2
possibly exist as enols stabilized by hydrogen bond (as shown in Figure 1). Recently, the structure
of 3-{N-[(21-thienylcarbonyl)hydrazono]ethyl}-4-hydroxycoumarin 2k has been confirmed by X-ray
analysis [37].
Figure 1. Hydrogen bond in 3-acetyl-4-hydroxycoumarin N-acylhydrazones.
2.1. Pharmacology
Antioxidant Activity
Taking the multifactorial character of oxidative stress into account, we decided to evaluate the in vitro
antioxidant activity of the synthesized molecules using two different antioxidant assays. Therefore, the
radical scavenging ability of the compounds was tested against the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) stable free radical and their ability to inhibit lipid peroxidation induced by the thermal free
1
radical producer 2,2 -azobis(2-amidinopropane) dihydrochloride (AAPH) was evaluated.
It is well known that the interaction of the synthesized 3-acetyl-4-hydroxycoumarin N-acylhydrazones 2
with the stable free radical DPPH indicates their radical scavenging ability in an iron-free system [29].
In the present case, the interaction of the tested 3-acylhydrazono substituted 4-hydroxycoumarins
with DPPH was found to be concentration-dependent whereas, the time did not influence the reducing
radical scavenging ability. Furthermore, all the tested compounds at 100 M have presented similar
radical reducing abilities ranging from 23%–27% to 24%–31% for 20 and 60 min respectively whereas,
the interaction was found to be rather limited for the concentration of 50 M (as shown in Figure 2
and in the collective Table 1). Considering the antioxidant activity of 3-acetyl-4-hydroxy coumarin (
it seems to be higher than the hydrazone derivatives 2a in both concentrations and in relation with
2
µ
µ
1
)
–l
the time and it is correlated with the presence of 3-acetyl and 4-hydroxy groups in the lactone ring and
the possibility of tautomers (A–D) formation [38–40] as shown in Scheme 2. It has been reported that
3-acetyl-4-hydroxycoumarin mainly exists in endocyclic enol form (B) in polar solvents (methanol,
ethanol) and it is well known that enols show antioxidant activity e.g., it has been reported that enolic
and phenolic hydroxyl groups is beneficial for curcumin to protect erythrocytes against hemin-induced
hemolysis and to protect DNA against AAPH-induced oxidation [41]. The lower results of hydrazones
2a–l, are correlated with their stereochemistry which influence their interaction with DPPH.

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Table 1. Inhibition % of DPPH at different concentrations and times, calculated lipophilicity Clog P [41] and % inhibition of LP and (LOX) (IC50) for compound 2.
LOX b IC50 (µM)
Compd.
RA%, 50 µM, 20 min
RA%, 50 µM, 60 min
RA%, 100 µM, 20 min RA%, 100 µM, 60 min
Clog P
LP a 60 s, 100 µM
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
5 ˘ 0.2
4 ˘ 0.2
5 ˘ 0.1
7 ˘ 0.3
7 ˘ 0.2
10 ˘ 0.5
9 ˘ 0.3
5 ˘ 0.1
8 ˘ 0.5
4 ˘ 0.2
7 ˘ 0.1
6 ˘ 0.3
29 ˘ 0.5
84 ˘ 2.0
7 ˘ 0.3
7 ˘ 0.1
7 ˘ 0.4
10 ˘ 0.2
10 ˘ 0.5
14 ˘ 1.2
16 ˘ 0.6
8 ˘ 0.2
10 ˘ 0.1
8 ˘ 0.3
9 ˘ 0.2
10 ˘ 0.2
31˘ 0.3
83 ˘ 3.3
23 ˘ 3.0
25 ˘ 2.0
25 ˘ 1.2
27 ˘ 2.2
27 ˘ 0.9
24 ˘ 2.2
27 ˘ 0.8
27 ˘ 1.5
27 ˘ 0.2
25 ˘ 1.8
25 ˘ 2.1
27 ˘ 2.2
36˘ 1.3
81 ˘ 5.2
24 ˘ 2.0
29 ˘ 1.1
30 ˘ 2.8
27 ˘ 1.4
29 ˘ 0.8
26 ˘ 1.2
31 ˘ 1.4
31 ˘ 1.6
29 ˘ 1.7
27 ˘ 0.8
30 ˘ 2.2
29 ˘ 1.0
36 ˘ 0.8
83 ˘ 4.7
1.85
3.62
3.29
3.79
4.20
2.96
2.38
2.53
2.96
2.46
3.13
3.46
1.91
100 ˘ 9.8
100 ˘ 5.5
98 ˘ 5.4
100 ˘ 3.2
94 ˘ 4.8
98 ˘ 2.9
95 ˘ 7.2
99 ˘ 3.7
100 ˘ 8.2
95 ˘ 4.1
98 ˘ 3.9
95 ˘ 6.2
8 ˘ 0.2
62.5 ˘ 2.3
40 ˘ 0.5
58 ˘ 2.7
No c
55 ˘ 2.1
70 ˘ 4.3
46.5 ˘ 2.3
No c
49.5 ˘ 1.2
90 ˘ 5.1
43.5 ˘ 3.2
35 ˘ 0.2
44 (˘ 0.3) d
5.5 ˘ 0.1
1
NDGA
TROLOX
a
63 ˘ 0.2
% inhibition of LP induced by AAPH; b in vitro inhibition of soybean lipoxygenase (LOX); c no action under the reported experimental conditions; d the presented biological response
is given as % inhibition. The IC50 value was not be able to be determined.

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90
80
70
60
50
40
30
20
10
0
50 Μμ 20 min
50 μM, 60 min
100 µM, 20 min
100 μM, 60 min
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
1
NDGA
Figure 2. Effect of compounds 2 towards 2,2-diphenyl-2-picrylhydrazyl (DPPH).
Scheme 2. Tautomers (A–D) of 3-acetyl-4-hydroxycoumarin 1.
AAPH induced linoleic acid oxidation is based on the inhibition of lipid oxidation and provides
a measure of how efficiently antioxidants protect against lipid oxidation in vitro. Oxidation of exogenous
linoleic acid by a thermal free radical producer (AAPH) is followed by UV spectrophotometry in
a highly-diluted sample [36,42].
In general, all the studied compounds effectively inhibit AAPH induced lipid peroxidation, showing
higher activity than the reference compound trolox (63%, Figure 3 and Table 1). 3-Acetyl-4-hydroxy
coumarin (1) presents non-significant anti-lipid peroxidation activity. However, all the derivatives
exhibit very potent inhibition of lipid peroxidation and almost the same as a result of their combined
structural characteristics. Lipophilicity does not seem to play any significant role. For example, methyl
derivative 2a, is a good inhibitor of lipid peroxidation (100%), while it presents the lowest Clog P value
among all the analogues (Table 1). The tested derivatives possess a favorable electronic distribution for
reacting quickly with intermediate lipid peroxy radicals and sufficient lipid solubility to partition
effectively in lipid bilayers. Our preliminary results suggest that they are indeed capable to inactivate
rapidly alkylperoxy radicals.
100
90
80
70
60
50
60 s, 100 μM
40
30
20
10
0
Figure 3. Effect of compound 2 towards AAPH lipid peroxidation.

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2.2. In Vitro Inhibition of Soybean Lipoxygenase (LOX)
Coumarins as well as flavonoids are among the most potent 5-lipoxygenase inhibitors. The synthesized
coumarins were evaluated for inhibition of soybean lipoxygenase by the UV-absorbance-based
enzyme assay [29] and the results are presented in Figure 4 as well as in Table 1. The majority of
LOX inhibitors are antioxidants or free radical scavengers [43], since lipoxygenation occurs via
a carbon-centered radical. Some studies suggest a relationship between LOX inhibition and the ability
of the inhibitors to reduce the Fe3+ at the active site to the catalytically inactive Fe2+. Several LOX
inhibitors are excellent ligands for Fe3+
action is presumably related to their coordination with a catalytically crucial Fe3+. 3-Acetyl-4-hydroxy
coumarin (1) showed low inhibitory activity at 100 M and, thus, we did not proceed to determine
its IC50 value. In Table 1, its response is given, as a % inhibition value at 100 M. Among the tested
[44,45]. It has been demonstrated that their mechanism of
µ
µ
1
compounds, the 2 -NO2-substituted phenyl (2l) was found to exhibit superior LOX inhibitory activity,
followed by the benzyl substituted hydrazine (2b), the 21-thienyl-substituted derivative (2k), the
4-NH2-phenyl substituted hydrazine (2g) and the 21-OH-substituted phenyl (2i) (Figure 4, Table 1).
No sign for the role of overall lipophilicity is obvious. However, the three most potent derivatives
2l,
2b, and 2k present a mean value of Clog P = 3.4. The 21-thienyl-substituted derivative (2k) is
more potent than the corresponding 2’-furyl derivative (2j), whereas the 41-pyridyl-analogue 2h
and the 41-CH3-substituted phenyl hydrazone (2d) do not seem to present any activity under the
reported experimental conditions. The position of substitution is significant since the 2-substituted
derivative, e.g., the 21-OH-substituted phenyl (2i) is more potent than the corresponding 2f which is
1
a 4 -OH-substituted phenyl hydrazone. Small differences are observed when R is a phenyl or a small
alkyl group. Each in vitro experiment was performed at least in triplicate and the standard deviation
of absorbance was less than 10% of the mean.
100
90
80
70
60
50
40
IC50 (μΜ)
IC (µM)
50
30
20
10
0
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
1
NDGA
Figure 4. Effect of compounds 2 towards soybean lipoxygenase (LOX).
3. Experimental Section
3.1. General
3-Acetyl-4-hydroxycoumarin
1
was synthesized according to the literature [28,34]. Melting points
are uncorrected and were determined on a Fisher-Johns melting point apparatus. 1D-NMR spectra were
recorded at room temperature on a Bruker Avance 400 spectrometer (Bruker, Billerica, MA, USA) at
400.15 MHz for 1H-NMR and 100.62 MHz for 13C-NMR in DMSO-d6. The chemical shifts are expressed
in
δ
values (ppm) relative to TMS as internal standard for 1H and relative to TMS (0.00‘ppm) or to
DMSO-d6 (39.50 ppm) for 13C-NMR spectra. Coupling constants nJ are reported in Hz. Second order
1H spectra, where it was possible, were analyzed by simulation [46]. Either elemental analysis or
HRMS has been provided for all new products of 2. All the chemicals used for biological assays

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were of analytical grade and commercially available by Merck, 2,2-diphenyl-1-picrylhydrazyl (DPPH),
nordihydroguairetic acid (NDGA), trolox, and AAPH were purchased from the Aldrich Chemical Co.
(Milwaukee, WI, USA). Soybean Lipoxygenase and linoleic acid sodium salt were obtained from Sigma
Chemical, Co. (St. Louis, MO, USA).
3.2. Chemistry
Synthesis of 3-[1-(Acyl-hydrazono)ethyl]-4-hydroxycoumarins (2a–l)
To a solution of 3-acetyl-4-hydroxy-coumarin 1 (1 mmol) in n-propanol (15–20 mL) was added the
appropriate hydrazide (1 mmol). The mixture was refluxed for 24 h and cooled at room temperature.
The precipitate was collected by filtration and dried to give the 3-[1-(acyl-hydrazono)ethyl]-4-hydroxycoumarin
(2a–l) as solid and was then recrystallized from n-propanol in very good yields. The following
compounds have been prepared according to this procedure:
3-[N-(Acetylhydrazono)ethyl]-4-hydroxycoumarin (2a). Yield: 87% under reflux for 24 h and 81% under
˝
˝
reflux for 2 h; light yellow solid; mp 248–249 C (mp 250–251 C (from MeOCH2CH2OH/H2O) [30]);
1H-NMR (DMSO-d6, 400 MHz)
δ
2.07 (s, 3H, 3-COCH3), 2.66 (s, 3H, 3-CCH3), 7.29 (dd, J = 8.3, 1.0 Hz,
1H, 8-H), 7.32 (ddd, J = 7.9, 7.3, 1.0 Hz, 1H, 6-H), 7.66 (ddd, J = 8.3, 7.3, 1.8 Hz, 1H, 7-H), 7.97 (dd,
J = 7.9 Hz, 1.8 Hz, 1H, 5-H), 11.42 (s, 1H, NNH), 15.90 (br s, 1H, 4-OH); 13C-NMR (DMSO-d6, 100 MHz)
δ
17.3 (3-CCH3), 20.4 (3-COCH3), 94.9 (C-3), 116.3 (C-8), 119.5 (C-4a), 123.8 (C-5), 125.5 (C-6), 134.2 (C-7),
153.0 (C-8a), 161.8 (C-2), 167.0 (3-C=N), 169.2 (NHCO), 178.7 (C-4); HRMS (ESI+) calcd for C13H12N2O4
m/z: 261.08698 (M + H+); found 261.08688 (M + H+).
3-[N-(Phenylacetylhydrazono)ethyl]-4-hydroxycoumarin (2b). Yield: 87% under reflux for 24 h and 83%
˝
1
under reflux for 2 h; yellow solid; mp 216–217 C; H-NMR (DMSO-d6, 400 MHz)
δ 2.65 (s, 3H,
3-CCH3), 3.70 (s, 2H, CH2Ph), 7.28 (dd, J = 8.3, 1.0 Hz, 1H, 8-H), 7.28–7.36 (m, 6H, 6-H, C6H5), 7.64
(ddd, J = 8.3, 7.3, 1.0 Hz, 1H, 7-H), 7.95 (dd, J = 7.8, 1.5 Hz, 1H, 5-H), 11.7 (s, 1H, NNH), 15.9 (br, 1H,
4-OH); 13C-NMR (DMSO-d6, 100 MHz)
δ 17.8 (3-CCH3), 40.4 (CH2, masked under the septet of the
solvent), 95.5 (C-3), 116.8 (C-8), 119.9 (C-4a), 124.3 (C-5), 126.0 (C-6), 127.3 (C-41), 128.9 (C-31,51), 129.7
(C-21,61), 134.7 (C-7), 135.2 (C-11), 153.5 (C-8a), 161.8 (C-2), 168.4 (3-C=N), 170.3 (NHCO), 179.2 (C-4);
HRMS (ESI+) calcd for C19H16N2O4 m/z: 359.10023 (M + Na+), 695.21124 (2M + Na+); found 359.10031
(M + Na+), 695.21160 (2M + Na+).
˝
3-[N-(Benzoylhydrazono)ethyl]-4-hydroxycoumarin (2c). Yield: 95%; white solid; mp 225–226 C [28]
1
˝
3-{N-[(4 -Methylbenzoyl)hydrazono]ethyl}-4-hydroxycoumarin(2d). Yield: 87%; white solid; mp 251–252 C [28].
1
˝
3-{N-[(4 -Chlorobenzoyl)hydrazono]ethyl}-4-hydroxycoumarin(2e). Yield: 98%; white solid; mp 248–248.5 C [28].
1
3-{N-[(4 -Hydroxybenzoyl)hydrazono]ethyl}-4-hydroxycoumarin (2f). Yield: 98% under reflux for 24 h and
94% under reflux for 2 h; white solid; mp 287–288 ˝C; 1H-NMR (DMSO-d6, 400 MHz)
δ 2.75 (s, 3H,
3-CCH3), 6.91 (d, J = 8.7 Hz, 2H, 31,51-H), 7.30 (dd, J = 8.3, 1.0 Hz, 1H, 8-H), 7.33 (ddd, J = 7.8, 7.2,
1.0 Hz, 1H, 6-H), 7.67 (ddd, J = 8.3, 7.0, 1.7 Hz, 1H, 7-H), 7.84 (d, J = 8.7 Hz , 2H, 21,61-H), 8.00 (dd,
J = 7.9, 1.7 Hz, 1H, 5-H), 10.34 (br s, 1H, 41-OH), 11.55 (s, 1H, NNH), 15.72 (br, 1H, 4-OH); 13C-NMR
(DMSO-d6, 100 MHz) δ
18.1 (3-CCH3), 95.7 (C-3), 115.8 (C-31,51), 116.8 (C-8), 120.2 (C-4a), 122.0 (C-11),
124.3 (C-5), 126.2 (C-6), 130.6 (C-21,61), 134.8 (C-7), 153.6 (C-8a), 161.98 (C-41), * 162.04 (C-2), * 165.0
(3-C=N), 172.2 (NHCO), 179.7 (C-4); MS (ESI): m/z 338 (M+). Anal. calcd for C18H14N2O5: C, 63.90; H,
4.17; N, 8.28; found C, 63.70; H, 3.98; N, 8.44. (*: The assignments may be interchanged).
3-{N-[(41-Aminobenzoyl)hydrazono]ethyl}-4-hydroxycoumarin (2g). Yield: 70% under reflux for 24 h and
69% under reflux for 2 h; light yellow solid; mp 256–257 ˝1 C; 1H-NMR (DMSO-d6, 400 MHz)
δ 2.74 (s,
1
3H, 3-CCH3), 6.01 (br, 2H, NH2), 6.63 (d, J = 8.6 Hz, 2H, 3 ,5 -H), 7.29 (d, J = 8.4 Hz, 1H, 8-H), 7.33 (dd,
J = 7.8, 7.2 Hz, 1H, 6-H), 7.66 (ddd, J = 8.4, 7.2, 1.5 Hz, 1H, 7-H), 7.69 (d, J = 8.6 Hz, 2H, 21,61-H), 8.00
(dd, J = 7.8, 1.5 Hz, 1H, 5-H), 11.30 (s, 1H, NNH), 15.74 (br, 1H, 4-OH); 13C-NMR (DMSO-d6, 100 MHz)

Molecules 2016, 21, 138
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1 1 1
18.2 (3-CCH3), 95.5 (C-3), 113.2 (C-3 ,5 ), 116.8 (C-8), 117.2 (C-1 ), 120.3 (C-4a), 124.3 (C-5), 126.1 (C-6),
130.3 (C-2 ,6 ), 134.6 (C-7), 153.6 (C-8a), * 153.8 (C-4 ), * 162.0 (C-2), 165.2 (3-C=N), 171.4 (NHCO), 179.6
(C-4); MS (ESI) m/z 337 (M+). Anal. Calcd for C18H15N3O4 C, 64.09; H, 4.48; N, 12.46. Found C, 63.73;
H, 4.25; N, 12.46. (*: The assignments may be interchanged).
δ
1
1
1
3-[N-(Isonicotinoylhydrazono)ethyl]-4-hydroxycoumarin (2h). Yield: 95% under reflux for 24 h and 91%
under reflux for 2 h; orange solid; mp 274 ˝C; 1H-NMR (DMSO-d6, 400 MHz)
δ 2.78 (s, 3H, 3-CCH3),
7.31 (dd, J = 8.3, 1.0 Hz, 1H, 8-H), 7.34 (ddd, J = 7.8, 7.4, 1.0 Hz, 1H, 6-H), 7.67 (ddd, J = 8.3, 7.4, 1.6 Hz,
1H, 7-H), 7.95 (br d, J = 4.8 Hz, 2H, 21,61-H), 8.01 (dd, J = 7.9, 1.6 Hz, 1H, 5-H), 8.84 (br s, 2H, 31,51-H),
11.9 (br, 1H, NNH), 15.75 (br s, 1H, 4-OH); 13C-NMR (DMSO-d6, 100 MHz)
δ 17.6 (3-CCH3), 95.3 (C-3),
116.3 (C-8), 119.9 (C-4a), 122.0 (C-21,61), 123.8 (C-5), 125.6 (C-6), 134.2 (C-7), 140.6 (br, C-11), 149.3 (br,
C-31,51), 153.1 (C-8a), 161.50 (C-2), 163.2 (3-C=N), 171.0 (br, NHCO), 178.9 (C-4); HRMS (ESI+) Anal.
Calcd for C17H13N3O4 m/z: 324.09788 (M + H+); Found 324.09784 (M+H+).
1
˝
3-{N-[(2 -Hydroxybenzoyl)hydrazono]ethyl}-4-hydroxycoumarin(2i). Yield: 97%; white solid; mp 271–272 C [28].
1
˝
3-{N-[(2 -Furoyl)hydrazono]ethyl}-4-hydroxycoumarin (2j). Yield: 89%; yellow solid; mp 254.5–255.0 C [28].
3-{N-[(21-Thienylcarbonyl)hydrazono]ethyl}-4-hydroxycoumarin (2k). Yield: 94%; light yellow solid; mp
˝
228–228.5 C [28].
1
˝
3-{N-[(2 -Nitrobenzoyl)hydrazono]ethyl}-4-hydroxycoumarin (2l). Yield: 91%; yellow solid; mp 219 C [28].
3.3. Pharmacology
3.3.1. Determination of the Reducing Activity of the DPPH (RA%)
To an ethanolic solution of DPPH (0.05 mM) in absolute ethanol the new coumarin derivatives
dissolved in DMSO were added (final concentration 50 and 100
µM). The mixture was shaken
vigorously and allowed to stand for 20 min or 60 min; absorbance at 517 nm was determined
spectrophotometrically against the blank and the percentage of reducing activity (RA) was calculated
by the formula: RA% = [(A0
absorbance of the reaction mixture. All tests were undertaken on three replicates and the results
presented in Table 1 were averaged.
´
A1)/A0]
ˆ
100 where A0 is the absorbance of blank and A1 is the
3.3.2. Inhibition of Linoleic Acid Lipid Peroxidation
Production of conjugated diene hydroperoxide by oxidation of linoleic acid in an aqueous dispersion
is monitored at 234 nm. 2,21-Azobis(2-amidinopropane) dihydrochloride (AAPH) is used as a free
radical initiator. Ten microliters of the 16 mM linoleic acid sodium salt solution was added˝to
the UV cuvette containing 930 µL of 0.05 mM phosphate buffer, pH 7.4 prethermostated at 37 C.
˝
The oxidation reaction was initiated at 37 C under air by the addition of 50
µL of 40 mM AAPH
solution. Oxidation was carried out in the presence of aliquots (10 L) of the tested coumarins. In the
µ
assay without antioxidant, lipid peroxidation was measured in the presence of the same level of
˝
DMSO. The rate of oxidation at 37 C was monitored by recording the increase in absorption at 234 nm
caused by conjugated diene hydroperoxides.
3.3.3. Soybean Lipoxygenase Inhibition Study In Vitro
The tested compounds dissolved in DMSO were incubated at room temperature with sodium
linoleate (100
µ
L) and 200
µ
L of enzyme solution (1/9
ˆ
10´4 w/v in saline) in Tris buffer pH 9.
The conversion of sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded and
compared with the appropriate standard inhibitor.

Molecules 2016, 21, 138
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3.3.4. Physicochemical Studies
Since lipophilicity is a significant physicochemical property determining distribution, bioavailability,
metabolic activity, and elimination, the theoretically calculated [47] Clog P values in n-octanol-buffer
are included in Table 1. For their determination the C-QSAR program of Biobyte Corp. was used.
4. Conclusions
In this study, a series of 3-acylhydrazono substituted 4-hydroxycoumarins have been synthesized
and characterized. The antioxidant activity of the synthesized compounds has been studied in vitro
using two different assays. Moreover, in an attempt to identify the potential of the compounds
as anti-inflammatory agents, their ability to inhibit in vitro soybean lipoxygenase was evaluated.
Although the free 4-hydroxy coumarin was not found to present any antioxidant activity [48] its
combination with a 3-imino group [49] recently led to antioxidant properties. These results go in
parallel to our findings, where the combination of 4-hydroxy coumarin with the 3-acyl-hydrazone
group leads to potent inhibitors of lipid peroxidation.
Our study indicates that high LOX inhibitory activity is not accompanied by high DPPH radical
scavenging activity. This is in accordance with the finding of Curini et al. [50], who have studied the
antioxidant and LOX inhibitory activity of five natural prenyloxy-carboxylic acids and showed that the
most efficient LOX inhibitor (boropinic acid) is not the most active DPPH radical scavenger. However,
a better correlation exists between LOX inhibitory activity and lipid peroxidation inhibition.
It is of interest that compound 2l, the 2-nitro-substituted-3-acylhydrazono-4-hydroxy-coumarin,
is the most potent as it possesses an array of potentially beneficial characteristics: it inhibits lipid
peroxidation with satisfactory potency and it inhibits LOX (IC50 = 35 µM). It would, thus, be of special
interest to characterize this molecule in terms of its anti-inflammatory profile.
Acknowledgments: The authors are grateful to Royal Society of Chemistry for financial support of the chemical
synthesis and to Biobyte Corp. and Hansch and Leo for their support and free access to the C-QSAR program.
This research has been done using the above program via Internet. Biobyte Corp., 01 West 4th Street, Suite 204,
Claremont, CA 91711, USA.
Author Contributions: A.K. conceived the idea of this piece of research; A.K. and D.J.H. designed the experiments;
D.A.N. performed the chemical experiments; C.A.K. performed the pharmacology experiments; P.A.H. performed
the spectra analyses and provided HRMS and elemental analysis; A.K., D.J.H., C.A.T., P.A.H., D.A.N. and C.A.K.
analyzed the data; A.K., D.J.H., C.A.T. and P.A.H. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 2d, 2i and 2l are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).