Acetophenone benzoylhydrazones as antioxidant agents: Synthesis, in vitro evaluation and structure-activity relationship studies

Article abstract of DOI:10.1016/j.foodchem.2018.06.083

Acetophenone and its analogues are naturally-occurring compounds found in many foods and plants. In this study, a series of acetophenone benzoylhydrazones 5a-o were designed and synthesized as new potential antioxidant agents. Designed molecules contain hydrazone and phenolic hydroxyl moieties which possibly contribute to antioxidant activity. The antioxidant properties of compounds 5a-o in terms of reducing ability and radical-scavenging activity were assessed by using FRAP and DPPH tests, respectively. While the unsubstituted compound 5a had the superior capacity in the FRAP assay, the 2,4-dihydroxyacetophenone analogue 5g was the most potent radical scavenger in the DPPH method. The antioxidant potential of representative compounds 5a and 5g was further confirmed by TEAC and ORAC assays. Cell viability assays revealed that while the promising compounds 5a and 5g had no significant toxicity against HepG2 and NIH3T3 cells, they potently protected HepG2 cells against H₂O₂-induced oxidative damage at low concentrations. Furthermore, spectroscopic studies with different biometals demonstrated that 5g was able to interact with Cu²⁺ to form a 1:1 complex.

Full text of DOI:10.1016/j.foodchem.2018.06.083

FoodChemistry268(2018)292–299
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Acetophenone benzoylhydrazones as antioxidant agents: Synthesis, in vitro
evaluation and structure-activity relationship studies
Saeed Emamia,⁎, Zahra Esmailia, Gholamreza Dehghanb, Maryam Bahmanib,
Seyedeh Mahdieh Hashemia, Hassan Mirzaeic, Mohammad Shokrzadehd, Seyed Ershad Moradie
a
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
Department of Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran
Pharmaceutical Sciences Research Center, Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
b
c
d
e
Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
Antioxidant activity
Hydrazones
Acetophenone derivatives
Oxidative stress
Acetophenone and its analogues are naturally-occurring compounds found in many foods and plants. In this
study, a series of acetophenone benzoylhydrazones 5a-o were designed and synthesized as new potential anti-
oxidant agents. Designed molecules contain hydrazone and phenolic hydroxyl moieties which possibly con-
tribute to antioxidant activity. The antioxidant properties of compounds 5a-o in terms of reducing ability and
radical-scavenging activity were assessed by using FRAP and DPPH tests, respectively. While the unsubstituted
compound 5a had the superior capacity in the FRAP assay, the 2,4-dihydroxyacetophenone analogue 5g was the
most potent radical scavenger in the DPPH method. The antioxidant potential of representative compounds 5a
and 5g was further confirmed by TEAC and ORAC assays. Cell viability assays revealed that while the promising
compounds 5a and 5g had no significant toxicity against HepG2 and NIH3T3 cells, they potently protected
HepG2 cells against H2O2-induced oxidative damage at low concentrations. Furthermore, spectroscopic studies
with different biometals demonstrated that 5g was able to interact with Cu2+ to form a 1:1 complex.
1. Introduction
functional foods to prevent chronic diseases, such as cancer, athero-
sclerosis and heart disease (Lee, Woo, Ahn, & Je, 2014; Varadharaj
Oxidative stress has been described as the unbalance of reactive
oxygen species’ (ROS) generation and the organism's capacity to
counteract their action (Persson, Popescu, & Cedazo-Minguez, 2014).
High concentrations of free radicals and ROS, including hydrogen
peroxide, superoxide, hydroxyl radical and peroxynitrite may cause
damage to lipids, proteins and DNA in biological systems (Sugamura &
Keaney, 2011). It has been estimated that oxidative stress contributes to
the pathogenesis and pathophysiology of over 100 diseases, such as
cardiovascular and inflammatory diseases, cataracts, carcinogenesis,
Parkinson’s, Alzheimer's disease, Huntington's disease and amyotrophic
et al., 2017). Also, antioxidants are beneficial as supplements in foods
for maintaining redox balance and avoiding oxidative damage to pro-
tect against lipid oxidation and off-flavour development (Lönn, Dennis,
& Stocker, 2012). In particular, natural antioxidants, such as vitamin C,
α-tocopherol, ubiquinol, and polyphenols, are widely used to scavenge
free radicals and to combat the harmful effects of ROS (Choi, Lee, Hong,
& Lee, 2012). Recently, many efforts have been focussed on designing
antioxidants containing phenolic hydroxyl groups (Bandgar et al.,
2013). The potential antioxidant effect of polyphenols and phenolic
compounds is related to their reducing activity, hydrogen-donating,
and singlet oxygen-quenching properties (Prior, Wu, & Schaich, 2005).
Acetophenone and its substituted analogues are naturally occurring
compounds found in many foods, such as apples, apricots, bananas,
beef, cheese, and cauliflowers (Müller-Schwarze, & Houlihan, 1991), as
well as in many plants, such as Camellia sinensis (Dong et al., 2012).
Also, acetophenone has been approved by FDA for use as a flavouring
agent in non-alcoholic beverages, ice creams, candies, baked goods,
lateral sclerosis (Sahebkar, Panahi, Yaribeygi,
& Javadi, 2018;
Varghese, Patel, & Yadav, 2018). Furthermore, oxidative stress plays
critical roles in the impairment of physiological functions and in the
biology of aging (Franco & Vargas, 2018; Maulik, McFadden, Otani,
Thirunavukkarasu, & Parinandi, 2013).
Antioxidants are currently considered to be a potential treatment for
oxidative stress-related diseases and are widely used as ingredients of
⁎
Corresponding author.
E-mail address: semami@mazums.ac.ir (S. Emami).
https://doi.org/10.1016/j.foodchem.2018.06.083
Received 6 February 2018; Received in revised form 2 June 2018; Accepted 18 June 2018
Availableonline19June2018
0308-8146/©2018ElsevierLtd.Allrightsreserved.

S. Emami et al.
FoodChemistry268(2018)292–299
gelatins, puddings, and chewing gums (Hazardous Substances Data
Bank, http://toxnet.nlm.nih.gov/).
spots on TLC were visualized and detected by UV lamp (254 nm).
Melting points were determined in glass capillary tubes on a Stuart
Scientific apparatus and are uncorrected. IR spectroscopy was carried
out on a FT-IR Perkin Elmer spectrometer (KBr disks). All NMR spectra
were recorded on a Bruker ultrashield avance III spectrometer, working
at 400 MHz and chemical shifts are expressed as ppm in respect to the
internal standard tetramethylsilane (TMS). Elemental analyses were
carried out on a CHN-O-rapid elemental analyzer (GmbH-Germany) for
C, H and N, and the results are within 0.4% of the theoretical values.
The quantum mechanical calculation studies of hydro-
xyacetophenone derivatives by Bentes, Borges, Monteiro, de Macedo,
and Alves (2011) postulated that the presence of a carbonyl group in
phenolic derivatives may stabilize the radical formed during oxidation,
extending the conjugation via resonance effects (Bentes et al., 2011).
Furthermore, Rezk, Haenen, van der Vijgh, and Bast (2002) have re-
ported the potential antioxidant activity of 2,6-dihydroxyacetophenone
that is possibly explained by stabilization of the radical that is formed
after hydrogen abstraction (Rezk et al., 2002).
2.2. General procedure for the synthesis of compounds 5a-o
On the other hand, several hydrazones were reported as potent
antioxidants due to their free radical-scavenging activity (Mohammed
Khan et al., 2012). Structurally, the hydrazones are characterized by an
azomethine group, which has a critical rule in antioxidant activity
(Belkheiri et al., 2010).
To a solution of benzohydrazides 3a or 3b (1 mmol) and acet-
ophenone derivative 4a-h (1 mmol) in methanol (5 ml), a few drops of
glacial acetic acid were added and the reaction mixture was refluxed at
70 °C for 6–8 h. After consumption of starting materials (monitoring
with TLC), the reaction mixture was cooled to room temperature and
left in the refrigerator overnight. The precipitated crystals were sepa-
rated by filtration and washed with cooled methanol to give pure
compounds 5a-o.
N′-(1-Phenylethylidene)benzohydrazide (5a). Yield: 42%; mp:
147–148 °C; IR (νmax, cm−1): 3467, 3054, 1611, 1541, 1488, 1316,
1284, 1133, 1026, 799, 758, 693, 564; 1H NMR (400 MHz, DMSO‑d6) δ:
2.38 (s, 3H, CH3), 7.38–7.48 (m, 3H, H-3′, H-4′ and H-5′), 7.50–7.62
(m, 3H, H-3, H-4 and H-5), 7.78–7.95 (m, 4H, H-2′, H-6′, H-2 and H-6),
10.79 (br s, 1H, NH). Anal. Calcd for C15H14N2O: C, 75.61; H, 5.92; N,
11.76. Found: C, 75.49; H, 5.89; N, 11.73.
Based on these findings and in continuation of our works on anti-
oxidant agents (Emami, Hosseinimehr, Shahrbandi, Enayati,
&
Esmaeeli, 2012; Foroumadi et al., 2007; Khoobi et al., 2011, 2011), we
report here the synthesis and antioxidant properties of acetophenone
benzoylhydrazones containing a phenolic group and their structure-
activity relationships (Fig. 1). As depicted in Fig. 1, designed molecules
contain a phenolic hydroxyl group and hydrazone moiety which pos-
sibly contribute to radical-scavenging activity and the antioxidant
property.
2. Materials and methods
4-Hydroxy-N′-(1-phenylethylidene)benzohydrazide (5b). Yield: 81%;
mp: 246–247 °C; IR (νmax, cm−1): 3500, 3150, 1670, 1583, 1436, 1323,
1222, 1174, 903, 846, 773, 699, 504; 1H NMR (400 MHz, DMSO‑d6) δ:
2.36 (s, 3H, CH3), 6.86 (d, 1H, J = 8.4 Hz, H-3 and H-5), 7.38–7.46 (m,
3H, H-3′, H-4′ and H-5′), 7.79 (d, 2H, J = 8.8 Hz, H-2 and H-6),
7.80–7.88 (m, 2H, H-2′ and H-6′), 10.09 (s, 1H, 4-OH), 10.52 (s, 1H,
NH). Anal. Calcd for C15H14N2O2: C, 70.85; H, 5.55; N, 11.02. Found: C,
70.97; H, 5.53; N, 10.98.
2.1. Chemical reagents and instruments
The required starting materials and reagents were obtained from
Sigma-Aldrich and Merck. The intermediate compounds 2a,b and 3a,b
were prepared according to the reported methods (Bhole, & Bhusari,
2011; Masunari, & Tavares, 2007; Rando, Avery, Tekwani, Khan, &
Ferreira, 2008). 4-Methoxy-2-hydroxyacetophenone (4e) was synthe-
sized from 2,4-dihydroxyacetophenone as described previously (Safavi
et al., 2010). All reactions were monitored by thin-layer chromato-
graphy (TLC) using pre-coated silica gel plates (Kieselgel 60 F254). The
N′-(1-(2-Hydroxyphenyl)ethylidene)benzohydrazide (5c). Yield: 44%;
mp: 183–184 °C; IR (νmax, cm−1): 3211, 1637, 1612, 1577, 1485, 1304,
1285, 1025, 931, 834, 744, 712, 690; 1H NMR (400 MHz, DMSO‑d6) δ:
(a)
R'
R'= H, OH
Conjugated hydrazone
increasing free radical
scavenging potency
O
N
NH
Secondary
antioxidant
probe
Phenolic
CH3
radical scavenger
HO
R
R= H, OH, MeO
R'
(b)
NH2
O
N
O
OH
H2SO4
MeOH
O
OMe
O
NH
NH
CH3COOH (cat.)
EtOH, reflux
N2H4
CH3
EtOH,
reflux
O
R'
R'
CH3
R'
3a,b
R
R
5a-o
1a,b
2a,b
4a-h
Fig. 1. (a) Design of acetophenone benzoylhydrazones as antioxidant agents; (b) Synthesis of compounds 5a-o.
293

S. Emami et al.
FoodChemistry268(2018)292–299
2.50 (s, 3H, CH3), 6.88–6.94 (m, 2H, H-3′ and H-5′), 7.32 (td, 1H,
J = 7.6 and 1.2 Hz, H-4′), 7.55 (dd, 2H, J = 8.0 and 7.2 Hz, H-3 and H-
5), 7.60–7.67 (m, 2H, H-4 and H-6′), 7.95 (d, 2H, J = 7.2 Hz, H-2 and
H-6), 11.36 (br s, 1H, NH), 13.38 (s, 1H, 2′–OH). Anal. Calcd for
N′-(1-(2,5-Dihydroxyphenyl)ethylidene)benzohydrazide (5k). Yield:
44%; mp: 210–211 °C; IR (νmax, cm−1): 3500, 1655, 1641, 1538, 1488,
1319, 1289, 1204, 1172, 931, 821, 786, 710, 614; 1H NMR (400 MHz,
DMSO‑d6) δ: 2.42 (s, 3H, CH3), 6.71–6.79 (m, 2H, H-3′ and H-4′), 6.98
(d, 1H, J = 2.0 Hz, H-6′), 7.55 (t, 2H, J = 7.2 Hz, H-3 and H-5), 7.63 (t,
1H, J = 7.2 Hz, H-4), 7.93 (d, 2H, J = 7.2 Hz, H-2 and H-6), 8.95 (s,
1H, 5′–OH), 11.30 (s, 1H, NH), 12.60 (s, 1H, 2′–OH). Anal. Calcd for
C
15H14N2O2: C, 70.85; H, 5.55; N, 11.02. Found: C, 70.79; H, 5.54; N,
11.20.
4-Hydroxy-N′-(1-(2-hydroxyphenyl)ethylidene)benzohydrazide (5d).
Yield: 57%; mp: 258–259 °C; IR (νmax, cm−1): 3189, 3054, 1611, 1541,
1488, 1305, 1133, 1104, 932, 892, 799, 758, 564; 1H NMR (400 MHz,
DMSO‑d6) δ: 2.47 (s, 3H, CH3), 6.85–6.93 (m, 4H, H-3, H-5, H-3′ and H-
5′), 7.30 (td, 1H, J = 8.4 and 1.6 Hz, H-4′), 7.63 (dd, 1H, J = 8.4 and
1.6 Hz, H-6′), 7.84 (d, 2H, J = 8.4 Hz, H-2 and H-6), 10.21 (br s, 1H, 4-
OH), 11.08 (br s, 1H, NH), 13.44 (br s, 1H, 2′–OH). Anal. Calcd for
C
15H14N2O3: C, 66.66; H, 5.22; N, 10.36. Found: C, 66.49; H, 5.30; N,
10.18.
N′-(1-(2-Hydroxy-5-methoxyphenyl)ethylidene)benzohydrazide (5l).
Yield: 35%; mp: 151–152 °C; IR (νmax, cm−1): 3466, 3233, 1601, 1577,
1510, 1488, 1340, 1219, 1147, 1040, 921, 814, 715, 690; 1H NMR
(400 MHz, DMSO‑d6) δ: 2.49 (s, 3H, CH3), 3.75 (s, 3H, OCH3), 6.86 (d,
1H, J = 8.8 Hz, H-3′), 6.95 (dd, 1H, J = 8.8 and 2.8 Hz, H-4′), 7.14 (d,
1H, J = 2.8 Hz, H-6′), 7.55 (t, 2H, J = 7.4 Hz, H-3 and H-5), 7.63 (t, 1H,
J = 7.4 Hz, H-4), 7.95 (d, 2H, J = 7.2 Hz, H-2 and H-6), 11.34 (s, 1H,
NH), 12.83 (s, 1H, 2′–OH). Anal. Calcd for C16H16N2O3: C, 67.59; H,
5.67; N, 9.85. Found: C, 67.80; H, 5.66; N, 9.91.
4-Hydroxy-N′-(1-(2-hydroxy-5-methoxyphenyl)ethylidene)benzohy-
drazide (5m). Yield: 75%; mp: 260–261 °C; IR (νmax, cm−1): 3367, 2985,
2831, 1670, 1609, 1507, 1439, 1374, 1254, 1042, 921, 847, 784, 619;
1H NMR (400 MHz, DMSO‑d6) δ: 2.47 (s, 3H, CH3), 3.75 (s, 3H, OCH3),
6.84 (d, 1H, J = 8.8 Hz, H-3′), 6.89 (d, 2H, J = 8.4 Hz, H-3 and H-5),
6.93 (dd, 1H, J = 8.8 and 2.8 Hz, H-4′), 7.12 (d, 1H, J = 2.8 Hz, H-6′),
7.84 (d, 2H, J = 8.4 Hz, H-2 and H-6), 10.21 (br s, 1H, 4-OH), 11.07 (br
s, 1H, NH), 12.89 (br s, 1H, 2′–OH). Anal. Calcd for C16H16N2O4: C,
63.99; H, 5.37; N, 9.33. Found: C, 64.21; H, 5.59; N, 9.35.
N′-(1-(4-Hydroxy-3-methoxyphenyl)ethylidene)benzohydrazide (5n).
Yield: 30%; mp: 150–151 °C; IR (νmax, cm−1): 3400, 1637, 1509, 1423,
1311, 1274, 1226, 1097, 1026, 890, 713, 635; 1H NMR (400 MHz,
DMSO‑d6) δ: 2.31 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 6.82 (d, 1H,
J = 8.0 Hz, H-5′), 7.29 (d, 1H, J = 7.2 Hz, H-6′), 7.43–7.61 (m, 4H, H-
3, H-4, H-5 and H-2′), 7.88 (d, 2H, J = 6.8 Hz, H-2 and H-6), 9.45 (br s,
1H, 4′–OH), 10.70 (br s, 1H, NH). Anal. Calcd for C16H16N2O3: C, 67.59;
H, 5.67; N, 9.85. Found: C, 67.51; H, 5.48; N, 10.16.
4-Hydroxy-N′-(1-(4-hydroxy-3-methoxyphenyl)ethylidene)benzohy-
drazide (5o). Yield: 38%; mp: 207–208 °C; IR (νmax, cm−1): 3400, 1639,
1609, 1501, 1453, 1313, 1279, 1176, 1029, 850, 618; 1H NMR
(400 MHz, DMSO‑d6) δ: 2.29 (s, 3H, CH3), 3.81 (s, 3H, OCH3), 6.81 (d,
1H, J = 8.4 Hz, H-5′), 6.85 (d, 1H, J = 8.4 Hz, H-3 and H-5), 7.18–7.32
(m, 1H, H-6′), 7.38–7.49 (m, 1H, H-2′), 7.78 (d, 2H, J = 8.8 Hz, H-2 and
H-6), 9.40 (s, 1H, 4′–OH), 10.06 (s, 1H, 4-OH), 10.43 (s, 1H, NH). Anal.
Calcd for C16H16N2O4: C, 63.99; H, 5.37; N, 9.33. Found: C, 64.01; H,
5.29; N, 9.36.
C
15H14N2O3: C, 66.66; H, 5.22; N, 10.36. Found: C, 66.80; H, 5.23; N,
10.34.
N′-(1-(4-Hydroxyphenyl)ethylidene)benzohydrazide (5e). Yield: 48%;
mp: 241–242 °C; IR (νmax, cm−1): 3300, 1626, 1569, 1486, 1372, 1268,
1152, 1027, 979, 838, 708, 694, 666; 1H NMR (400 MHz, DMSO‑d6) δ:
2.30 (s, 3H, CH3), 6.82 (d, 2H, J = 8.0 Hz, H-3′ and H-5′), 7.45–7.60
(m, 3H, H-3, H-4 and H-5), 7.72 (d, 2H, J = 7.6 Hz, H-2′ and H-6′), 7.88
(d, 2H, J = 6.4 Hz, H-2 and H-6), 9.83 (s, 1H, 4′–OH), 10.67 (s, 1H,
NH). Anal. Calcd for C15H14N2O2: C, 70.85; H, 5.55; N, 11.02. Found: C,
70.74; H, 5.70; N, 10.88.
4-Hydroxy-N′-(1-(4-hydroxyphenyl)ethylidene)benzohydrazide (5f).
Yield: 75%; mp: 290–291 °C; IR (νmax, cm−1): 3300, 1625, 1534, 1502,
1440, 1286, 1248, 1172, 845, 764, 621, 538; 1H NMR (400 MHz,
DMSO‑d6) δ: 2.28 (s, 3H, CH3), 6.80 (d, 2H, J = 8.4 Hz, H-3′ and H-5′),
6.84 (d, 2H, J = 8.4 Hz, H-3 and H-5), 7.63–7.73 (m, 2H, H-2′ and H-
6′), 7.77 (d, 2H, J = 8.4 Hz, H-2 and H-6), 9.78 (s, 1H, 4′–OH), 10.06 (s,
1H, 4-OH), 10.39 (s, 1H, NH). Anal. Calcd for C15H14N2O3: C, 66.66; H,
5.22; N, 10.36. Found: C, 66.83; H, 5.20; N, 10.51.
N′-(1-(2,4-Dihydroxyphenyl)ethylidene)benzohydrazide (5g). Yield:
90%; mp: 221–222 °C; IR (νmax, cm−1): 3491, 3216, 1606, 1509, 1485,
1304, 1254, 1100, 991, 848, 796, 713, 651; 1H NMR (400 MHz,
DMSO‑d6) δ: 2.42 (s, 3H, CH3), 6.29 (d, 1H, J = 2.4 Hz, H-3′), 6.35 (dd,
1H, J = 8.6 and 2.4 Hz, H-5′), 7.46 (d, 1H, J = 8.8 Hz, H-6′), 7.54 (t,
2H, J = 8.0 Hz, H-3 and H-5), 7.61 (t, 1H, J = 7.4 Hz, H-4), 7.92 (d, 2H,
J = 7.2 Hz, H-2 and H-6), 9.91 (s, 1H, 4′–OH), 11.18 (s, 1H, NH), 13.57
(br s, 1H, 2′–OH). Anal. Calcd for C15H14N2O3: C, 66.66; H, 5.22; N,
10.36. Found: C, 66.87; H, 5.21; N, 10.22.
4-Hydroxy-N′-(1-(2,4-dihydroxyphenyl)ethylidene)benzohydrazide
(5h). Yield: 48%; mp: 292–293 °C; IR (νmax, cm−1): 3447, 1608, 1502,
1456, 1266, 1146, 1109, 990, 844, 789, 605, 527; 1H NMR (400 MHz,
DMSO‑d6) δ: 2.39 (s, 3H, CH3), 6.27 (d, 1H, J = 2.0 Hz, H-3′), 6.33 (dd,
1H, J = 8.8 and 2.4 Hz, H-5′), 6.87 (d, 2H, J = 8.4 Hz, H-3 and H-5),
7.43 (d, 1H, J = 8.8 Hz, H-6′), 7.81 (d, 2H, J = 8.4 Hz, H-2 and H-6),
9.86 (br s, 1H, 4′–OH), 10.13 (br s, 1H, 4-OH), 10.90 (br s, 1H, NH),
13.61 (s, 1H, 2′–OH). Anal. Calcd for C15H14N2O4: C, 62.93; H, 4.93; N,
9.79. Found: C, 62.68; H, 5.11; N, 9.87.
N′-(1-(2-Hydroxy-4-methoxyphenyl)ethylidene)benzohydrazide (5i).
Yield: 25%; mp: 216–217 °C; IR (νmax, cm−1): 3400, 1650, 1617, 1602,
1508, 1265, 1205, 1133, 828, 715, 678; 1H NMR (400 MHz, DMSO‑d6)
δ: 2.45 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 6.45–6.52 (m, 2H, H-3′ and H-
5′), 7.50–7.58 (m, 3H, H-3, H-5 and H-6′), 7.62 (t, 1H, J = 7.2 Hz, H-4),
7.93 (d, 2H, J = 7.2 Hz, H-2 and H-6), 11.25 (s, 1H, NH), 13.68 (s, 1H,
2′–OH). Anal. Calcd for C16H16N2O3: C, 67.59; H, 5.67; N, 9.85. Found:
C, 67.83; H, 5.68; N, 9.71.
2.3. DPPH free radical-scavenging assay
The DPPH radical-scavenging activity of compounds 5a-o was de-
termined according to the reported method (Nur Alam, Bristi, &
Rafiquzzaman, 2013). Briefly, the test compounds at various con-
centrations (30, 60, 100, and 150 µl) were added to 3 ml of DPPH so-
lution (0.1 mM in ethanol) and the reaction mixture was shaken vig-
orously. After incubation at room temperature for 10 min, the
absorbance of this solution was determined at 517 nm after 10, 30, and
60 min, using a spectrophotometer. The percent of scavenging activity
was obtained from the following equation:
Radical scavenging (%) = [OD(DPPH)−OD(DPPH+sample)]/OD(DPPH) × 100
4-Hydroxy-N′-(1-(2-hydroxy-4-methoxyphenyl)ethylidene)benzohy-
drazide (5j). Yield: 89%; mp: 271–272 °C; IR (νmax, cm−1): 3323, 1623,
1605, 1499, 1290, 1164, 1110, 1027, 847, 786, 652, 635, 559; 1H NMR
(400 MHz, DMSO‑d6) δ: 2.42 (s, 3H, CH3), 3.77 (s, 3H, OCH3),
6.43–6.50 (m, 2H, H-3′ and H-5′), 6.88 (d, 2H, J = 8.8 Hz, H-3 and H-
5), 7.54 (d, 1H, J = 8.8 Hz, H-6′), 7.82 (d, 2H, J = 8.4 Hz, H-2 and H-
6), 10.18 (s, 1H, 4-OH), 10.98 (s, 1H, NH), 13.74 (s, 1H, 2′–OH). Anal.
Calcd for C16H16N2O4: C, 63.99; H, 5.37; N, 9.33. Found: C, 63.81; H,
5.21; N, 9.35.
The radical-scavenging activity was expressed as IC50 value ob-
tained from the regression analysis.
2.4. FRAP assay
The total antioxidant capacity of compounds 5a-o was measured by
using the FRAP assay according to the method of Benzie and Strain with
some modifications (Benzie & Strain, 1999). This method measures the
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FoodChemistry268(2018)292–299
ability of the test compound to reduce Fe+3-TPTZ (2,4,6-tripyridyl-s-
triazine) to a ferrous form (Fe+2) that absorbs light at 593 nm. The
FRAP solution, prepared freshly, contained 25 ml of acetate buffer
(300 mM, pH 3.6) plus 2.5 ml of TPTZ solution (10 mM) in 40 mM HCl
and 2.5 ml of ferric chloride (FeCl3·6H2O, 20 mM). This solution was
used as blank. The sample was incubated at 37 °C throughout the
monitoring period. Absorbancies of the blank and samples were mea-
sured after 10 min at 593 nm, and the results were compared with the
standard curve provided using different concentrations of FeSO4. The
final FRAP value of each sample was the mean value of three replica-
tions. The linearity of the relationship between Fe+2 concentration and
absorbance was very good within this concentration range (r2 = 0.997).
Quercetin was used as standard antioxidant in this method.
spectrophotometer (Tokyo, Japan). The final volume of reaction mix-
ture was 5 ml, and the final concentrations of tested compound and
metals were 20 μM. The stoichiometry of the 5g–Cu2+ complex was
determined using the molar ratio method (Meyer & Ayres, 1957), as
follows: the final concentration of 5g was 20 μM, and the final con-
centration of Cu2+ (ranging from 2 to 32 μM), and the absorption
spectra of the solutions were recorded after 30 min. The UV spectra
were recorded and treated by numerical subtraction of CuSO4 and 5g at
corresponding concentrations, plotted versus the mole fraction of tested
compound. The absorbance of the absorption peak at 375 nm was
plotted against the molar concentration of Cu2+. The breakpoint re-
vealed the stoichiometry of the 5g–Cu2+ complex.
2.8. Cell lines and cytotoxicity assay
2.5. Trolox equivalent antioxidant capacity (TEAC) assay
The normal fibroblast cell line NIH3T3 (mouse embryo fibroblast)
and Human Hepatoma HepG2 cell line were purchased from Pasture
institute, Tehran, Iran. The cells were seeded at 104 cells/well in RPMI
1640 medium, supplemented with 10% fetal bovine serum (FBS), 100
unit/ml of penicillin-G, 100 μg/ml of streptomycin in each well of a 96-
well plate. The cells were cultured in an incubator containing 5% CO2
for 24 h in 37 °C.
The selected compounds 5a and 5g were dissolved in dimethyl
sulfoxide (DMSO) as a 100 µM stock solution and then diluted to the
different concentrations in the RPMI 1640 medium. The final con-
centration of DMSO was 0.4% in medium culture. After 24 h, the cells
were treated with various concentrations of compounds (200, 100, 50,
10 and 1 μg/ml). After 24 h of incubation, 10 μl of MTT solution (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (5 mg/ml) was
added to each well, and incubated for 4 h. Then the culture medium
from each well was carefully removed, and DMSO (200 μl) was added
into each well, gently shaken for 15 min and the absorbance was de-
termined with an ELISA reader (Bioteck) at 570 nm (Eghtedari et al.,
2017). Two independent experiments in triplicate were done for de-
termination of cell viability inhibition for each compound. The IC50
values were calculated from concentration–response curves by using
The 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) dia-
mmonium salt (ABTS) radical-scavenging activity of selected com-
pounds (5a, 5g, 5k and 5o) was determined, based on trolox equivalent
antioxidant capacity (TEAC). This assessment was according to the
method of Re et al. (1999). Briefly, ABTS powder (54.2 mg) was dis-
solved in 10 ml of phosphate buffer (5 mM, pH 7.0) and mixed with 1 g
of MnO2 and incubated at room temperature within 15 h for generation
of ABTS·+ radicals. The prepared solution was filtered and the filtrate
was diluted with phosphate buffer until the absorbance of solution
equals 0.70
0.01 at 734 nm. Different concentrations of desired
compounds (5a, 5g, 5k or 5o) were mixed with 4 ml of ABTS solution
and incubated for 10 min at room temperature. The decrease of ab-
sorbance was monitored at 734 nm after 10 min. The percentage of
radical inhibition activity (%) was calculated according to the following
equation:
1 - Af
Ao
Scavenging of A734nm(%) =
× 100
where Ao is the absorbance of the untreated radical and Af is the ab-
sorbance measured 10 min after addition of the samples.
Different concentrations of trolox (0.05–1 mM) were used to obtain
a standard curve. The ABTS radical-scavenging activity of samples was
calculated and plotted against the concentration of trolox and results
were expressed as TEAC (trolox equivalent antioxidant capacity).
the Prism 6, GraphPad Software and expressed as means
SD.
2.9. Viability assay against H2O2-induced cell death in HepG2 cells
The HepG2 cells were seeded at 104 cells/well in RPMI 1640
medium, supplemented with 10% fetal bovine serum (FBS), 100 unit/
ml of penicillin-G and 100 μg/ml of streptomycin in each well of a 96-
well plate. The cells were cultured in an incubator containing 5% CO2
for 24 h at 37 °C. After 24 h, the cells were treated with various con-
centrations (10 and 1 μg/ml) of selected compounds 5a and 5g.
Curcumin was used as positive control, at the same concentrations.
Then, two hours later H2O2 (200 μM) was added to the culture medium
for induction of cell death in HepG2 cells. The cell viability was mea-
sured after 24 h of incubation by using the MTT assay as described
previously (Jiang et al., 2014).
2.6. Oxygen radical absorbance capacity (ORAC) assay
ORAC assay was performed following the method of Kim, Jang, &
Kim (2007) with some modifications. First, solutions of 0.1 μM fluor-
escein and 90 mM AAPH (2,2′-azobis(2-amidinopropane) dihy-
drochloride), both in 75 mM phosphate buffer (pH 7.4), were prepared.
Then, 1.5 ml of fluorescein solution and 250 µl of sample (5a, 5g, 5k or
5o) were mixed and incubated for 15 min at 37 °C in the dark. After the
addition of 250 µl of AAPH, the decrease in the fluorescence intensity
per min (λex = 485 nm and λem = 535 nm) was monitored for 60 min
in a spectrofluorometer (Jasco, FP-750 spectrofluorometer, Kyoto,
Japan). Several concentrations of trolox (2–16 μM) were used to find a
calibration curve. The area under the curve (AUC) was calculated for
each; the difference in the area under the curves between each sample
and the blank were calculated and results were expressed as TEAC
value. The results were expressed as micromolar equivalents of trolox
for each microgramme of desired compounds (µM TE/µg).
3. Results and discussion
3.1. Synthesis
The synthetic route to acetophenone benzoylhydrazones 5a-o is il-
lustrated in Fig. 1. The benzoic methyl esters 2a,b were prepared from
appropriate benzoic acid derivatives 1a,b in refluxing methanol and in
the presence of H2SO4 as catalyst. The reaction of esters 2a,b with
hydrazine hydrate in ethanol afforded hydrazides 3a,b (Bhole &
Bhusari, 2011; Masunari & Tavares, 2007; Rando et al., 2008). Finally,
condensation of hydrazides 3a,b with different acetophenones 4a-h in
methanol under reflux produced the desired acetophenone benzoylhy-
drazones 5a-o.
2.7. Metal binding studies
The UV absorption spectra of selected compound 5g and its related
acetophenone 2,4-DHA (2,4-dihydroxyacetophenone), in the absence
or presence of sulfate salts of Cu2+, Zn2+, Fe2+ and Al3+ cations were
recorded with wavelength ranging from 200 to 400 nm after incubating
in methanol at room temperature for 30 min, using a JASCO V-630
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FoodChemistry268(2018)292–299
Table 1
Antioxidant activity of compounds 5a-o, and cytotoxicity of selected compounds against normal fibroblast cell line (NIH3T3) and human hepatoma cell line (HepG2).
R'
O
NH
N
CH3
R
Compound
R
R'
FRAP value (mmol Fe2+/l)
DPPH (IC50, µg/ml)
NIH-3T3 Cytotoxicity (IC50, µg/ml)
HepG2 Cytotoxicity (IC50, µg/ml)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
H
H
2-OH
2-OH
4-OH
4-OH
2,4-(OH)2
2,4-(OH)2
2-OH-4-OMe
2-OH-4-OMe
2,5-(OH)2
2-OH-5-OMe
2-OH-5-OMe
3-OMe-4-OH
3-OMe-4-OH
H
OH
H
OH
H
OH
H
OH
H
OH
H
H
OH
H
1.26
0.54
0.51
0.38
0.62
0.67
0.01
0.06
0.19
0.24
0.75
0.33
0.18
0.68
0.66
1.04
–
0.11
0.01
0.02
0.06
0.5
0.01
0.00
0.05
0.10
0.11
0.09
0.04
0.06
0.11
0.07
0.01
160
> 200
> 200
8.9
283.8
–
–
–
–
–
189
–
–
–
–
–
–
–
–
–
–
2.1
136.0
–
–
–
–
–
121
–
–
–
–
–
–
–
–
–
–
2.8
55
1.1
3.8
1.0
0.03
11.2
133
131
19
136
194
> 200
84
145
97
158
87
2.7
1321
2.6
1.4
17.1
14.3
4.0
1.2
21.3
6.2
0.1
OH
Quercetin
2,4-DHAa
Etoposide
74.2
118
0.1
9.0
0.1
a
2,4-DHA: 2,4-Dihydroxyacetophenone.
3.2. Antioxidant activity
3.2.1. General
The antioxidant properties of compounds 5a-o were determined in
vitro by evaluating their radical-scavenging activity and reducing
ability. There are different methods for evaluation of the antioxidant
potential of compounds (Nur Alam et al., 2013). In this study, two
distinct methods which are commonly used for antioxidant evaluation
were utilized: the FRAP (ferric reducing antioxidant power) assay and
DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging test.
3.2.3. Radical-scavenging activity on DPPH
The DPPH radical-scavenging activities of compounds 5a-o, along
with 2,4-dihydroxyacetophenone, are presented in Table 1. Most of
acetophenone benzoylhydrazones, with the exception of 5b, 5c and 5j,
showed significant radical-scavenging activity. The 2,4-dihydrox-
yacetophenone derivative 5g with IC50 value of 19 µg/ml was the most
potent compound. Besides 5g, compound 5d (R = 2-OH and R' = OH)
showed respectable activity in the DPPH test (IC50 = 55 µg/ml).
The comparison of 4-hydroxybenzoyl and 2-hydroxyacetophenone
derivatives (5b and 5c, respectively) with the parent acetophenone
benzoylhydrazone (5a) revealed that the hydroxyl substituent de-
creased the radical scavenging activity. Surprisingly, the simultaneous
substitution of R = 2-OH and R' = OH resulted in compound 5d with
increased activity with respect to 5b and 5c. Also, sole substitution of 2-
or 4-OH on the acetophenone system had no positive effect on the
potency but concurrent substitution of 2,4-dihydroxy dramatically im-
proved the radical-scavenging activity. The 2,4-dihydroxy derivative 5g
was 4-fold more potent than its 2,5-dihydroxy analogue 5k; thus the
2,4-dihydroxy pattern was better than the 2,5-dihydroxy one. The effect
of a 4-hydroxy substituent on the benzoyl moiety was dependent on the
substitution pattern in the acetophenone system.
3.2.2. Ferric-reducing antioxidant power (FRAP)
The FRAP assay is based on the reduction of the ferric 2,4,6-tri-
pyridyl-s-triazine complex to the coloured ferrous form at low pH and
monitoring the change in absorption at 593 nm. Accordingly, The FRAP
value of test compound is obtained by comparing the absorbance
changes in test reaction mixtures with those containing known con-
centrations of Fe2+ ions (Benzie & Strain, 1999; Schlesier, Harwat,
Böhm, & Bitsch, 2002). The obtained data from FRAP assay of com-
pounds 5a-o are expressed as mmol Fe2+/l in Table 1.
Based on the FRAP values listed in Table 1, the unsubstituted
compound 5a showed the highest reducing power, being more effective
than quercetin as a polyphenolic antioxidant. Furthermore, the 2,5-di-
hydroxyacetophenone derivative 5k with FRAP value of 0.75 mmol
Fe2+/l exhibited remarkable capacity for reducing of Fe3+ ions. Also,
compounds 5e, 5f, 5n and 5o, bearing a 4-hydroxyacetophenone fra-
mework, displayed FRAP values of 0.62–0.68 mM, close to that of
compound 5k. The FRAP value of 4-hydroxybenzoyl analogue 5b was
significantly less than that of parent unsubstituted compound 5a. Thus,
the introduction of the 4-hydroxy group on the benzoylhydrazone di-
minishes the reducing power of the molecule. Surprisingly, the presence
of oxygenated functional groups (OH or MeO) on the acetophenone part
of the molecules could not improve the reducing capacity of the com-
pounds 5c-o compared to their congeners 5a and 5b.
The kinetics behaviour of selected compound 5k in the DPPH free
radical-scavenging process was determined in comparison to quercetin.
To follow the kinetics behaviours, the methanolic solution of the
compounds were added to the reaction medium and the absorbance was
measured until it became constant. The obtained results are given in
Fig. S1 (Supplementary material). Indeed, immediately after adding
compound 5k to the reaction medium, the absorbance at 517 nm
dropped, due to the decrease of DPPH radicals in the medium. Similar
to quercetin, compound 5k reacted within a very short time, and a
steady state was reached almost after 150 s.
3.2.4. Antioxidant capacity determined by TEAC and ORAC assays
As reviewed by Prior et al. (2005), among the available methods for
the measurement of antioxidant capacity, three assays have been
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FoodChemistry268(2018)292–299
Table 2
Antioxidant capacity of representative compounds evaluated by the TEAC and
ORAC methods.a
Compound
TEAC values
ORAC values
μM/μg
μM TE/μg
5a
5g
5k
5o
28
25
26
27
0.6
0.5
0.5
1.3
33
31
18
22
2.0
1.1
0.4
1.4
a
The average values ( SD) of TEAC are expressed as antioxidant capacity
equivalent to micromolar of trolox per microgramme of compounds (TEAC μM/
μg). The average values ( SD) of ORAC are expressed as micromolar trolox
equivalents per microgramme of test compounds (µM TE/µg).
mostly considered for standardization: oxygen radical absorbance ca-
pacity (ORAC), trolox equivalent antioxidant capacity (TEAC) and
Folin-Ciocalteu methods. While ORAC represents a hydrogen transfer
reaction mechanism, the TEAC and Folin-Ciocalteu methods are elec-
tron transfer-based assays. In this study, two out of these methods were
taken for the evaluation of antioxidant capacity. Therefore, the re-
presentative compounds 5a, 5g, 5k and 5o were examined for their
antioxidant capacity by means of ABTS•+ cation radical (TEAC) and
ORAC assays (Table 2). As can be seen, the antioxidant capacity ob-
tained for compounds 5a and 5g by the ORAC assay was higher than
the value obtained by TEAC, while compounds 5k and 5o showed
higher capacity in the TEAC assay. Compound 5a, with TEAC value of
28
0.6 µM/µg and ORAC value of 33
2.0 µM TE/µg, was the best
one among the tested compounds.
3.3. Metal chelating properties of selected compound 5g
The ability of promising compound 5g to chelate biometals such as
Zn(II), Fe(II), Cu(II) and Al(III) was studied by UV–visible spectrometry
and compared with its corresponding acetophenone 2,4-DHA. The re-
sults are shown in Fig. 2. New optical bands were observed in the
UV–vis spectra of compound 5g upon the addition of CuSO4, suggesting
that 5g was able to interact with Cu2+ to form a complex. It could be
seen that the spectrum of 5g was slightly changed upon addition of
FeSO4, ZnSO4 and Al2(SO4)3 (Fig. 2). Moreover, no significant shift or
enhancement in absorption band occurred after addition of Zn(II), Fe
(II), Cu(II) and Al(III) to 2,4-DHA. The chelating ability could be at-
tributed to the imine and 2-hydroxyl groups in compound 5g.
Spectrophotometric titration was used for the Cu2+ binding. A
series of UV−vis spectra of compound 5g titrated by Cu2+ was re-
corded. In the absence of Cu2+, the UV−vis spectrum of compound 5g
showed an absorption maximum at 327 nm. When Cu2+ was added, a
new band at 375 nm appeared which was associated with the copper
complex. The presence of an isosbestic point revealed the formation of a
unique 5g–Cu2+ complex. Therefore, the binding stoichiometry of
compound 5g with CuSO4 was determined by studying the absorption
changes at 375 nm. The molar ratio method was performed to de-
termine the stoichiometry of the 5g–Cu2+ complex, by preparing the
methanol solutions of 5g with ascending amounts of CuSO4. The UV
spectra were used to obtain the absorbance of the 5g–Cu2+ complex
and different concentrations of CuSO4 at 375 nm. The results showed
that absorbance increased linearly initially and then plateaued (Fig. 3).
The points for the straight lines to intersect were determined to be at a
mole proportion of 1.0, revealing a 1:1 stoichiometry for the 5g–Cu2+
complex.
Fig. 2. The UV spectra of (a) compound 5g and (b) 2,4-DHA (20 μM) alone and
in the presence of FeSO4, ZnSO4, Al2(SO4)3 or CuSO4 in methanol.
Fig. 3. Determination of the stoichiometry of 5g (20 μM)-Cu2+ complex by
molar ratio method.
MTT assay. The IC50 values of tested compounds are listed in Table 1.
Neither of the compounds showed any significant activity against
HepG2 or NIH3T3 cells (IC50 values > 120 µg/ml). These results re-
vealed that the prototype compounds can display significant anti-
oxidant activity at low concentrations without inducing cytotoxicity.
3.4. Cytotoxicity of promising compounds 5a and 5g
3.5. Protection against H2O2-induced cell death in HepG2 cells
In order to check the safety profile of promising antioxidants, the
effect of compounds 5a and 5g on the viability of normal cells
(NIH3T3) and Human Hepatoma cells (HepG2) was investigated by
HepG2 cells with a high degree of differentiation have been widely
used as
a cellular model to investigate protective activity of
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FoodChemistry268(2018)292–299
hydroxy and methoxy substituents on the acetophenone benzoylhy-
drazone framework can modulate the antioxidant properties of the
prototype compounds.
Acknowledgment
This work was supported by a grant from the Research Council of
Mazandaran University of Medical Sciences, Sari, Iran.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.foodchem.2018.06.083.
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curcumin, a naturally occurring cytoprotective agent.
The cells were treated with test compounds at the concentrations of
1 and 10 µg/ml and then exposed to H2O2. The cell viability was
measured in comparison to untreated cells by using the MTT assay and
the obtained results are depicted in Fig. 4. It should be noted that
compounds 5a and 5g had no cytotoxic effect on HepG2 cells at the 1
and 10 µg/ml concentrations. As seen in Fig. 4, H2O2 significantly re-
duced the HepG2 cell viability compared with the negative control
(without H2O2). The pretreatment of HepG2 cells with the test com-
pounds at the concentration of 10µg/ml protected hepatocytes against
cell death and significantly increased cell viability. Notably, the pro-
tecting effect of compound 5a at the concentration of 1 µg/ml was
much higher than that of curcumin. Furthermore, the protective effect
of compound 5g was comparable to curcumin at each concentration.
4. Conclusion
We have designed and synthesized a number of polyoxygenated
acetophenone benzoylhydrazones 5a-o as new potential antioxidant
agents. The in vitro antioxidant properties of compounds 5a-o in terms
of reducing ability and radical scavenging activity were assessed by
using FRAP and DPPH tests, respectively. While the unsubstituted
compound 5a had a superior capacity in the FRAP assay, the 2,4-di-
hydroxyacetophenone analogue 5g was the most potent radical sca-
venger in the DPPH method. Furthermore, cytotoxicity assay showed
that compounds 5a and 5g had no toxicity against HepG2 and NIH3T3
cell lines. The promising antioxidant compounds 5a and 5g sig-
nificantly protect HepG2 cells against H2O2-induced oxidative damage
at low concentrations. Furthermore, spectroscopic studies with dif-
ferent cations revealed that compound 5g was able to interact with
Cu2+ to form a complex. The obtained data from 2,4-dihydrox-
yacetophenone-derived benzoylhydrazone 5g and its parent actophe-
none compound indicated that the introduction of a hydrazone moiety
offers potent antioxidant potency and metal binding ability to the
acetophenone scaffold. The SAR study revealed that the pattern of
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