Correlation of antioxidant activities with theoretical studies for new hydrazone compounds bearing a 3,4,5-trimethoxy benzyl moiety

Article abstract of DOI:10.1016/j.ejmech.2015.09.016

A new series of antioxidants, namely imines bearing the well-known free radical scavenger group 3,4,5-trimethoxybenzyloxy, was designed and synthesized. Theoretical calculations based on density functional theory (DFT) were performed to understand the antioxidant activities. Experimental studies evaluating the antioxidant activities of the compounds using DPPH and FRAP assays verified the predictions obtained by DMOL3 based on DFT.1. The DPPH radical scavenging activities depended on the substitution pattern of the aromatic aldehyde, with both the substitution type and position showing significant effects. Compounds 7b, 7c and 7d, which contain a phenolic hydroxyl group at the para position to the imine as well as, additional electron donating groups at the ortho-position to this hydroxyl group, exhibited IC₅₀ values of 62, 75 and 106 μg/mL, respectively, and potent antioxidant activities against DPPH, which were better than that of the reference compound BHT. With the exception of compounds 7a and 7h with a phenolic hydroxyl group at the ortho position, all of the investigated compounds exhibited ferric reducing activities above 1000 μM. Correlation analysis between the two antioxidant assays revealed moderate positive correlation (r = 0.59), indicating differing antioxidant activities based on the reaction mechanism. Therefore, imines bearing a 3,4,5-trimethoxybenzyloxy group can be proposed as potential antioxidants for tackling oxidative stress.

Full text of DOI:10.1016/j.ejmech.2015.09.016

European Journal of Medicinal Chemistry 103 (2015) 497e505
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Correlation of antioxidant activities with theoretical studies for new
hydrazone compounds bearing a 3,4,5-trimethoxy benzyl moiety
Huda S. Kareem a b, Azhar Ariffin a , Nurdiana Nordin , Thorsten Heidelberg ,
,
,
*
a
a
c
c
d
Azlina Abdul-Aziz , Kin Weng Kong , Wageeh A. Yehye
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia
General Directorate of Curricula, Ministry of Education, Baghdad 3310, Iraq
Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia
Nanotechnology & Catalysis Research Centre (NANOCAT), University of Malaya, Block 3A, Institute of Postgraduate Studies Building, Kuala Lumpur 50603,
a
b
c
d
Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 April 2015
Received in revised form
A new series of antioxidants, namely imines bearing the well-known free radical scavenger group 3,4,5-
trimethoxybenzyloxy, was designed and synthesized. Theoretical calculations based on density func-
tional theory (DFT) were performed to understand the antioxidant activities. Experimental studies
evaluating the antioxidant activities of the compounds using DPPH and FRAP assays verified the pre-
dictions obtained by DMOL3 based on DFT.1. The DPPH radical scavenging activities depended on the
substitution pattern of the aromatic aldehyde, with both the substitution type and position showing
significant effects. Compounds 7b, 7c and 7d, which contain a phenolic hydroxyl group at the para
position to the imine as well as, additional electron donating groups at the ortho-position to this hy-
droxyl group, exhibited IC50 values of 62, 75 and 106 mg/mL, respectively, and potent antioxidant ac-
tivities against DPPH, which were better than that of the reference compound BHT. With the exception of
compounds 7a and 7h with a phenolic hydroxyl group at the ortho position, all of the investigated
compounds exhibited ferric reducing activities above 1000 mM. Correlation analysis between the two
6
September 2015
Accepted 10 September 2015
Available online 12 September 2015
Keywords:
Hydrazone
Antioxidant
Hydrogen atom transfer
Single electron transfer
Spin density (SD)
Bond dissociation energy (BDE)
antioxidant assays revealed moderate positive correlation (r ¼ 0.59), indicating differing antioxidant
activities based on the reaction mechanism. Therefore, imines bearing a 3,4,5-trimethoxybenzyloxy
group can be proposed as potential antioxidants for tackling oxidative stress.
©
2015 Elsevier Masson SAS. All rights reserved.
1
. Introduction
prevent or reduce the impact of oxidative stress on cells [2,3].
Various compounds have been designed and chemically synthe-
sized based on data for the structural requirements of potent an-
tioxidants. These compounds are only required in minute amounts.
Generally, the free radical scavenging capacity of phenols is
attributed to the hydrogen atom on the hydroxyl group [4], while
nitrogen and sulphur analogues may provide alternatives for labile
hydrogen atoms [5e7].
Hydrazones are well-known compounds with a wide range of
pharmacological activities, including antitumoral [8,9], antifungal
[10e12], and antibacterial behaviour [13,14]. They have also shown
potent antioxidant activities with regards to scavenging free radi-
cals [15,16]. Typically, the hydrazones are characterized by an imine
ꢀ
Reactive oxygen species (ROS) such as hydroxyls (HO ), super-
ꢀ
ꢁ
ꢀ
ꢀ
oxides (O2 ), peroxyls (ROO ), alkoxyls (RO ), and nitric oxides
ꢀ
(
NO ) can lead to cellular injury in the form of damaged DNA, lipids
and proteins. Therefore, the human body is able to counterbalance
ROS by a variety of antioxidant defence mechanisms and immedi-
ately eliminate an excess of ROS from the cell by cellular antioxi-
dant enzymes and other redox molecules [1]. Natural and synthetic
antioxidants may protect against cells damage by virtue of their
ability to scavenge free radicals.
Scientists from various disciplines are interested in new anti-
oxidants compounds, of both synthetic and natural origin, to
(
[
azomethine) group, which is critical for their antioxidant activities
4,17].
Methoxy groups on aromatic systems have been extensively
investigated for their well-known antioxidant effects [18]. These
*
Corresponding author.
E-mail address: azhar70@um.edu.my (A. Ariffin).
http://dx.doi.org/10.1016/j.ejmech.2015.09.016
223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
0

498
H.S. Kareem et al. / European Journal of Medicinal Chemistry 103 (2015) 497e505
groups are important in cytotoxic and microtubule-binding agents
used for cancer chemotherapy [19,20]. For instance, structure ac-
tivity relationship studies on combretastatin A-4, which is an anti-
tumour drug from the combretastatin group [21], have shown that
the 3,4,5-trimethoxy phenyl groups are important for its cytotoxic
activity [22,23]. Moreover, recently 2,4,5-trimethoxy chalcones and
Scheme 2, provided the hydrazone derivatives 7aei in good yields.
Their structures were confirmed by IR, NMR and mass spectral
analyses.
The 1H NMR spectrum of compound 5 exhibited signals origi-
nating from the ethyl ester and methoxy groups at 1.30 & 4.27 ppm
and 3.67 & 3.79 ppm, respectively. Conversion of ester 5 into the
key intermediate 6 led to the disappearance of signals belonging to
the ethoxy group. Instead, new signals reflecting the hydrazide
structure appeared at 4.43 ppm (2H) and 9.61 ppm (1H). All of the
hydrazones 7aei showed a singlet peak in the region between 11.40
and 12.40 ppm due to the amide NH as well as, another singlet peak
between 8.20 and 8.90 ppm reflecting an up field shift of the
aldehyde due to the formation of the hydrazone. At the same time,
the signal for the amine group of hydrazide 6 disappeared, thus
confirming the presence of the imine in the hydrazone compounds.
Protons of phenolic groups for compounds 7b, 7c, 7d, 7g, and 7i
appear as a singlet peak in the region between 7.39 and 9.90 ppm.
Whereas proton of phenolic groups for compound 7a and 7h were
shifted further to lower field at 12.80 and 12.02 respectively. The
shifted in chemical for 7a and 7h is believed due to hydrogen
bonding between the OH group and the N atom of the imine, as
shown in Fig. 2. Base on this observation we believed that all of the
hydrazones, 7ae7i, have trans (E) configurations at the imine
double bonds. This observations are in agreement with the data
reported by Levrand et al. [29].
0
0
their analogues 2,4,5-trimethoxy-2 ,5 -dihydroxychalcone, have
shown superior DPPH radical scavenging activities [24].
In the present study, SAR and rational design strategies were
used to create molecules with multiple functions, which include a
radical scavenging ability and other biological activities. This
strategy was performed with and tested based on computational
studies using DMOL3 based on DFT.1. The synthesized compounds
have been characterized by IR, NMR and mass spectral analyses.
The antioxidant activities of the synthesized compounds were
experimentally verified by DPPH and FRAP assays.
2
. Results and discussion
2.1. SAR and the rational design of antioxidant hydrazones
The generic structure of the new hydrazones, as illustrated in
Fig. 1, consists of a well-known free radical scavenger, the 3,4,5-
trimethoxybenzyl group (ring A). The active group eCOeNHN]C
e enables resonance to the two adjacent aromatic rings B and C,
leading to multiple resonance structures, which allows this func-
tional group to act as an electron donating group to enhance the
radical scavenging activity. The imine group of the hydrazone that
contains a lone pair of electrons might be used to form a covalent
bond with a biological target [25].
It has been reported that electron-donating substituents,
such as alkyl or alkoxyl groups at the 2,4,6-positions, increase
the primary antioxidant activities of phenols [26]. This is due to
the lowering of the bond dissociation enthalpy (BDE) of the
phenol OeH group [27] and the stabilization of the phenoxyl
radical by inductive and hyperconjugative effects. Thus, hydra-
zones with a phenolic hydroxyl group at the para position on
ring C with additional EDGs at the ortho-position can exhibit
potent antioxidant activities, due to resonance-based stabilizing
effects.
2.3. In vitro antioxidant activities
In the present study, the antioxidant activities of synthesized
compounds 5 and 6 and their hydrazone derivatives 7aei were
tested in vitro by using DPPH and FRAP, the two most common
antioxidant assays [30,31].
The DPPH radical scavenging assay is often used as a quick and
reliable parameter to investigate the antioxidant activities of
phenolic compounds [32e35]. DPPH is a stable free radical that can
accept a hydrogen radical or an electron to become a stable mole-
cule [36]. In the methanolic medium, it is odd electron configura-
tion shows a strong absorption band at 515 nm. The absorbance
decreases in the presence of free radical scavengers resulting in a
colour change from deep purple to yellow [37,38]. The FRAP assay,
on the other hand, is a simple, versatile and low-cost test that is
commonly used to assess the antioxidant activities of extracts and
pure natural products [39,40].
2.2. Synthesis
The starting material, 3,4,5-trimethoxy benzyl bromide (2), was
prepared according to a procedure found in the literature [28].
2.3.1. DPPH free radical scavenging activities
Refluxing 3,4,5-trimethoxybenzyl bromide 2 with ethyl 4-hydroxy-
benzoate (4) in acetone in the presence of anhydrous K CO
2 3
The DPPH radical scavenging ability strongly depends on the
geometric accessibility of the radical trapping site. The presence of
steric hindrance may prevent a test compound from reaching the
radical site of DPPH, resulting in a low activity [41]. The DPPH assay
is based on either a hydrogen atom transfer (HAT) or a single
electron transfer (SET) mechanism.
resulted in ethyl 4-(3,4,5-trimethoxybenzyloxy)benzoate (5). The
ester was converted to the corresponding acid hydrazide (6) by
reaction with hydrazine hydrate in absolute ethanol. The synthetic
process is summarized in Scheme 1. Treatment of the latter with
various aromatic aldehydes in anhydrous ethanol, as shown in
In general, the two proposed mechanisms, by which the
Alternative antioxidant
Secondary aromatic amine
EDG or EWG
R
H CO
3
C
electron donating
groups
H CO
A
HN
N
OH
3
O
B
Primary antioxidants
H CO
O
3
Imin group increase the free radical
stabilization by conjugation
Fig. 1. SAR analysis of synthesized hydrazones.

H.S. Kareem et al. / European Journal of Medicinal Chemistry 103 (2015) 497e505
499
HO
O
HO
MeO
1)
OMe
OH
(
3)
OMe
PBr3,
(
Et O,
str.6h
EtOH,
H SO ,
2
2
4
ref lux 24h
Br
MeO
MeO
MeO
O
O
O
MeO
2 )
OMe
+
HO
K CO , acetone
2 3
OEt
OEt
OMe
reflux 30h
(
( 5 )
(
4 )
NH NH H O
2
2. 2
Reflux.12h
MeO
MeO
MeO
O
O
HN NH2
(
6)
Scheme 1. Synthesis of acid hydrazide 6.
R5
R1
R4
MeO
MeO
MeO
MeO
O
O
R3
aromatic aldehyde
MeO
HN
O
N
HN NH2
EtOH/ ref lux 5-6h.
O
R2
(6)
MeO
7(a- i)
7
7
7
a: R
b: R
c: R
1
= OH, R
= R
= R
2
= Cl, R
4
= Br
7d: R
7e: R
7f: R
3
=OH, R
4
=OEt
= OMe
= OMe
7g: R
7h: R
7i: R
2
= OH
= OH
= OH
2
4
= t-Bu, R
= OMe, R
3
= OH
3
= R
= R
4
= R
= R
5
1
2
4
3
= OH
2
3
4
3
Scheme 2. Synthesis of hydrazone derivatives.
MeO
MeO
MeO
ArXH þ R /ArXH þ Rꢁ
$
þ$
(2)
(3)
(4)
HN N
O
O
H O
ArXH /ArX þ Hþ
þ$
$
ꢁ
þ
R þH /RH
The synthesized compounds were tested against DPPH at
different concentrations and compared with standard antioxidants
Fig. 2. Intramolecular H-bond formation of compound 7h.
(
ascorbic acid and BHT). The choice of the reference compounds is
antioxidants may scavenge free radicals, are shown below:
based on typical antioxidant for both hydrophilic (Ascorbic acid)
[
37,42] and lipophilic media (BHT) [31,43,44].
The maximum inhibition of the DPPH radical by the synthesized
1. Hydrogen atom transfer (HAT), in which a phenolic H-atom is
transferred in a single step.
compounds at a concentration of 125
mg/mL. Related IC50 and
maximum inhibition values are presented in Table 1.
IC50 values could only be determined in four of the compounds,
whereas the remaining compounds did not reach 50% inhibition of
the DPPH radicals within the concentration range investigated in
this study. For the DPPH assay, a dominant HAT mechanism is
assumed. Preferred hydrogen abstraction sites are phenolic hy-
droxyl groups, preferably with conjugation to the hydrazone, as the
latter could stabilize the resulting radical by additional resonance
structures.
$
$
ArX ꢁ H þ R /ArX þ R ꢁ H
(1)
2
. Single electron transfer (SET), which requires the following two
steps: the initial formation of a radical cation from ArXH by
electron transfer (Equation (2)) and the rapid deprotonation of
ArXH (Equation (3)), which compensates for the charge of the
initial anion R (Equation (4)) [4].
þ
ꢀ

500
H.S. Kareem et al. / European Journal of Medicinal Chemistry 103 (2015) 497e505
Table 1
IC50, DPPH radical scavenging maximum inhibition and FRAP values of the synthesized compounds.
Compounds
DPPH IC50
/m
g/mL
Radical scavenging max. Inhibition %
FRAP mM
5
6
>100
264 ± 2
>100
62 ± 0.5
75 ± 1
106 ± 0.4
>100
>100
>100
>100
>100
10.8 ± 0.3
37.2 ± 0.2
15.6 ± 0.2
75.3 ± 0.3
57.9 ± 0.1
53.3 ± 0.3
10.4 ± 0.4
10.4 ± 0.2
9.3 ± 0.1
27 ± 7
3515 ± 29
125 ± 15
1096 ± 30
2087 ± 8
1844 ± 20
1609 ± 12
1365 ± 7
1819 ± 17
26 ± 8
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
g
h
i
9.6 ± 0.1
18.7 ± 0.2
90.6 ± 0.2
54.8 ± 0.4
3056 ± 34
2562 ± 10
2837 ± 13
*
AA
BHT
40 ± 0.1
99.7 ± 0.8
Results are expressed as a mean ± standard deviation (n ¼ 3). DPPH radical scavenging activities are expressed as IC50.concentrations of the compounds (
mg/mL)
required to inhibit 50% of the radicals and the maximum inhibition values. >100 ¼ did not reach 50% inhibition of the DPPH radicals at the concentrations used in this
study). FRAP, ferric reducing antioxidant power, expressed at 500 mg/mL concentration. *Ascorbic acid.
Polar aprotic solvents reduce the rates of many ArXH/R re-
phenolic hydroxyl group, but they may abstract a hydrogen atom at
the amide to form resonance stabilized radicals [6].
actions, which has been explained by considering that most of the
molecules of ArXH are hydrogen bonded to the solvent, and these
ꢀ
species are unable to react by HAT with R . Only the free (non-
2.3.2. DFT study of the DPPH radical scavenging activities
hydrogen-bonded) fraction of ArXH is capable of transferring a H-
The antioxidant abilities of the hydrazone compounds can be
ꢀ
atom to R [45,46].
related to a calculated spin density (SD) of related radical species
51]. A more delocalized spin density in the radical stabilizes the
Based on the structures and antioxidant activities, the hydra-
zone antioxidants in this study can be classified into three classes.
The first class consists of 7b, 7c and 7d, which exhibited potent
antioxidant activities that were equal to or better than that of the
reference compound BHT. All of these compounds contain a
phenolic hydroxyl group at the para position to the hydrazone and
additional EDGs at the ortho position to this hydroxyl group. The
high activity can be explained by the resonance-based stabilizing
effects of the EDG as well as the hydrazone on the phenoxy-radical.
The order of activity, 7b ~ 7c > 7d with IC50 values of 62, 75 and
[
latter, thus favouring radical formation [52]. Therefore, lower SD
values indicate better antioxidants. For the HAT mechanism, the
easiness of the homolytic cleavage of the XeH bond, reflecting the
efficacy of an antioxidant, can be estimated by calculating the bond
dissociation enthalpy (BDE) of the respective OeH or NeH bond.
Lower BDEs indicate weaker OeH or NeH bonds, from which the
hydrogen is more easily extracted [53]. The presence of sub-
stituents, including both electron withdrawing groups (EWG) and
electron donating groups (EDG), in aromatics and their respective
position affects the SD and BDE values [49].
1
06 mg/mL, respectively, is in line with this explanation, as the latter
only contains one stabilizing EDG ortho to the radical, whereas the
former two compounds involve two such groups. In addition, the
higher activity of compound 7b is due to an enhanced steric hin-
drance effect provided by the ortho tertiary butyl group, which
stabilized the phenolic antioxidant radical [47,48]. A mechanistic
explanation for the scavenging of the DPPH free radical by the
compounds is proposed in Scheme 3.
The second class covers compounds 7g, 7h and 7i. Like in the
first class a phenolic hydroxyl group is present, but without addi-
tional EDGs or EWGs. The DPPH radical scavenging activities for
this class are substantially lower than those for the first class. None
of the compounds reached 50% inhibition of the DPPH radicals.
The sequence of the DPPH radical scavenging activities is
To evaluate the reactivity of the NH and OH groups for HAT, the
spin densities of the respective radical species of the hydrazone
compounds were determined separately. The results for these two
separate calculations are summarized in Fig. 3. The results, as
shown in Table 2, indicated higher stabilities for the oxygen radicals
of almost all of the compounds. Thus the experimental data are in
agreement with the calculated data. An exception, however, was
compound 7a, for which the spin density of the N-radical was
slightly below that of the O-radical. As explain earlier, this could be
due to an intra molecular hydrogen bond of the phenolic hydrogen
with the hydrazone nitrogen as shown in Fig. 2.
As shown in Fig. 3, the OH group of compound 7b exhibits the
lowest spin density. This is in agreement with its experimental
DPPH scavenging activity. However, the spin densities for com-
pounds 7c do not match the experimental results. We could not
figure out a logical explanation for the observation at the moment.
The SD for the oxygen radicals of most of these compounds were
similar and reflect the experimental DPPH scavenging activities.
Nonetheless, the relative activities of 7gei are in agreement with
the spin densities.
7
i > 7h > 7g. The sequence indicates a higher activity for the hy-
droxyl groups in the para and ortho positions to the hydrazone
compared to the meta analogue 7g. This finding is in line with
resonance stabilizing effects [49]. The para substituted 7i was more
active than 7h with ortho substitution. It is postulated that an intra-
molecular hydrogen bond of the phenolic hydrogen with the
hydrazone nitrogen in a 6-membered ring disfavours the DPPH
scavenging activity of the ortho substituted phenol due to the
enhanced binding of the phenolic hydrogen atom [50], as displayed
in Fig. 2.
The introduction of substituents with dominant polarization
based electron withdrawing effects over potential electron
donating effects due to resonance slightly increased the antioxidant
activity [4], as seen by the comparison of compounds 7a and 7b.
The third class consists of compounds 7e and 7f, which are
practically inactive. Neither of these compounds contains a
Another important parameter to be considered is the BDE. The
antioxidant activity, reflected in the calculated BDE value [54], is
often attributed to
lization of the radicals obtained after H-abstraction. It is assumed
that if electron delocalization exists in the parent molecule, it also
p electron delocalization, leading to the stabi-
p
exists in the corresponding radical [55]. Therefore, a close rela-
tionship between the BDE and spin density is to be expected. BDE
values were calculated for the homolysis of NeH and OeH bonds.
The data are shown in Table 2. As expected, they reflect the spin

H.S. Kareem et al. / European Journal of Medicinal Chemistry 103 (2015) 497e505
501
R1
R1
DPPH
O
O
H
HN
O
N
+
DPPH-H
HN
O
N
R
R1
R
R
1
7(a- i)
R1
O
R
1
H
N
O
N
O
.
R
R1
N
N
DPPH
R
R1
O
DPPH-H
MeO
MeO
O
R=
R = t-Bu, OCH , H.
1 3
MeO
Scheme 3. Proposed reactions for scavenging of DPPH∙ by hydrazone derivatives.
0.442
MeO
MeO
OH
HN
O
N
O
MeO
0
.244
0
.430
Br
Cl
7b
OMe
OH
MeO
MeO
MeO
0.424
MeO
MeO
HN
O
N
HN N
O
HO
O
OMe
MeO
O
0
.293
OMe
0.530
0.419
7f
MeO
MeO
MeO
7c
7
a
OMe
OMe
HN
N
O
O
MeO
0.421
0
.434
MeO
MeO
MeO
OH
MeO
MeO
HN
O
N
HN
N
O
O
OH
0.292
O
0.450
7i
7g
0.297
MeO
MeO
MeO
HN
O
N
O
HO
7
h
0.295
Fig. 3. Spin densities for selected compounds; the structures represent two independently calculated radicals obtained by abstraction of a hydrogen atom from the OH or the NH
group.
compared to the ortho analogue 7h, which is in agreement with the
higher experimental antioxidant activity for the para compound.
This effect is attributed to an intramolecular hydrogen bond for 7h.
The intramolecular hydrogen bond increases the value of the BDE
are in agreement with previous studies [48,56], because the for-
mation of the radical requires not only the splitting of the OH-bond
but also the cleavage of the intramolecular hydrogen bond.
The calculated atomic distance of approximately 1.461 Å be-
tween the hydrazone nitrogen and the hydroxyl hydrogen confirms
the hydrogen bonding, as displayed in Fig. 4.
Table 2
Calculated properties for the antioxidant compounds.
Compounds
BDE (kJ/mol)
Spin density
N-radical
NeH
OeH
O-radical
7
7
7
7
7
7
7
a
b
c
f
g
h
i
428
365
383
444
459
455
404
e
431
365
382
e
0.43
0.44
0.42
0.42
0.43
0.45
0.42
e
0.53
0.24
0.29
e
458
456
402
269
364
0.30
0.30
0.29
e
AA
BHT
2.3.3. Ferric reducing antioxidant power (FRAP) activity
3
þ
e
e
e
The FRAP assay measures the reducing capacity of Fe ions,
3
þ
based on the reduction of the ferric tripyridyltriazine [Fe(TPTZ) ]
2
2
þ
complex to ferrous tripyridyltriazine [Fe (TPTZ)
2
]
by the transfer
density trends.
of a single electron from an antioxidant compound according to
Equation (5).
Based on the BDE values a comparison was made between
compounds 7h and 7i to determine the influence of the position of
the hydroxyl group at ring C (aldehyde component). Compound 7i
with the hydroxyl group at the para position has a lower BDE
Â
Ã
Â
Ã2þ þ Ar$þ
3þ
þe
FeðTPTZÞ2
þ Ar! FeðTPTZÞ
(5)
2

502
H.S. Kareem et al. / European Journal of Medicinal Chemistry 103 (2015) 497e505
Fig. 4. Optimized three-dimensional structures of compounds 7h and 7i.
The FRAP assay utilizes an SET mechanism to determine the
capacity of an antioxidant in the reduction of an oxidant, as dis-
played in Equations (2)e(4). Upon reduction, the ferric colour
changes [57,58], and the degree of the colour change can be
correlated with the concentration of antioxidants in the sample.
However, the reducing capacity of a sample is not directly
related to its radical scavenging capability. In addition, to accurately
measure the total reducing power, certain condition must be met.
3
þ
(
1) All antioxidants can reduce [Fe (TPTZ)
2
]
under the reaction
conditions (thermodynamics). (2) The reaction rate must be suffi-
ciently fast that the reaction can be completed in a short assay time
(
e.g., 4 min in the actual FRAP assay) (kinetics) (3). The oxidized
antioxidant ArXH and its secondary reaction products should have
no absorption at 595 nm, the maximum absorption of [Fe
2
þ
(
TPTZ)
2
]
. These conditions are very difficult to meet. Therefore,
not all antioxidants reduce ferric ions at a fast rate [59].
Fig. 5. Pearson correlation analysis between the DPPH radical scavenging activities
and ferric reducing activities of the synthesized compounds. Correlation is significant
at the 0.05 level.
For the SET mechanism, a strong solvent dependence is ex-
pected. The expected solvent effect for SET is an increase with an
increase in the polarity and hydrogen bonding ability of the solvent
[
60]. The FRAP values of the tested compounds, as displayed in
differences in the reaction mechanisms of the two assays. More-
over, the DPPH radical scavenging activity may be affected by steric
issues, whereas for the FRAP assay, solvent issues may apply.
In HAT, the OH group in the C ring of the active hydrazones is
considered the most reactive site for radical scavenging activity.
However, a SET process does not require such a specific group, as an
aromatic system is already sufficient to enable the electron transfer.
Therefore, SET reactions based on the hydrazones 7aei may origi-
nate either from the AB-ring-system or ring C.
Table 1, showed a different trend than the DPPH radical scavenging
results. Some of the compounds are inactive in the DPPH assay but
exhibit high activities in the FRAP assay.
With the exception of compounds 5, 7a and 7h (FRAP values
below 150
reducing activities above 1000
mildly active in the DPPH assay, showed the highest ferric reducing
activity, while compound 7b, which was highly active in the DPPH
assay, showed an unusually low FRAP value.
mM), all of the investigated compounds exhibited ferric
mM. Compound 6 which was only
In fact, the contour plots of the HOMO for different hydrazine
compounds 7aei suggest different reactivity sites for SET. For
example, the HOMO of compound 7g, which exhibited a low ac-
tivity in the DPPH assay but a high activity in FRAP, suggests SET at
trimethoxylated ring A, whereas for compound 7b, the electron
density is concentrated on ring C, as shown in Fig. 6. For compound
2
.4. Correlation analysis
A Pearson correlation analysis was performed to investigate the
relationship between the DPPH radical scavenging and ferric
reducing activities of the compounds [61], as shown in Fig. 5. It was
expected that the antioxidant activities of the compounds in both
assays would show similar trends and that the values of both could,
therefore, be correlated.
7i, which showed the highest FRAP activity, both ring systems
appear to be potentially active for SET.
However, the data revealed only a moderate positive correlation
between the two antioxidant assays, indicating that compounds
with high DPPH radical scavenging activities did not necessarily
show high ferric reducing activities and vice versa. Y. Unver et al.
reported a similar observation when they evaluate the activity on
3. Conclusion
In conclusion, this study describes the antioxidant activities of a
series of newly synthesized hydrazones. The only moderate cor-
relation between the antioxidant activities for the DPPH and FRAP
assays relate to their different mechanisms, i.e., HAT dominance for
1,2,4-triazole derivatives [62]. The different results probably reflect

H.S. Kareem et al. / European Journal of Medicinal Chemistry 103 (2015) 497e505
503
Fig. 6. Density distributions of the HOMOs for selected hydrazones.
the former and SET for the latter.
under reduced pressure and the resulting brown oil was extracted
with three portions (30 mL) of ethyl acetate against 5% NaOH.
The combined organic layer were washed with water (30 mL)
and dried over anhydrous Na SO . After evaporation of the solvent
2 4
the product 5 was crystallized from methanol to give colourless
Quantum-mechanical calculations indicate a favoured extrac-
tion of the hydrogen atom at the phenolic hydrogen over the
alternative amide NH. Compounds with a hydroxyl group at the
para position and EDG substituents on the aldehyde component
showed high antioxidant activities, whereas EWG substituents
decreased the antioxidant activity. The lower antioxidant activities
of the ortho substituted phenolic hydrazones compared to the para
analogues was explained based on the intramolecular hydrogen
bonding of the former.
ꢂ
crystals (0.80 g, 61%). m.p. 77e80 C. IR: 3066 (CHAr), 2941 (CHa-
1
liph.), 1714 (C]O), 1462 (C]C), 1233 (CeO), 1020 (OeCH
(400 MHz, DMSO-d
, ppm: 1.30 (t, J ¼ 6.9 Hz, 3H, CH
[3.66 (s, 3H), 3.78 (s, 6H)], 4.27 (q, J ¼ 6.9 Hz, 2H, CH ), 5.09 (s, 2H,
OeCH
), Ar.H [6.79 (s, 2H), 7.14 (d, J ¼ 8 Hz, 2H), 7.93 (d, J ¼ 8 Hz,
3
). H NMR
6
)
d
3 3
), OeCH
2
2
13
2
H)]. C NMR (100 MHz, DMSO-d
6
)
d
3
, ppm: 14.7 (1C, CH ), (3C,
OeCH ) [56.4, 60.5], 60.8 (1C, CH ), 70.3 OeCH
3
2
2
, Ar.C [105.9, 115.2,
4
. Experimental
1
22.8, 131.6, 132.4, 137.7, 153.4, 162.7], 165.8 (ester C]O). HREIMS.
þ
C
19
H
22
O
6
, m/z ¼ 347.1455 (M þ H ). Calcd. 347.1488.
4.1. General
Chemicals and solvents were obtained from SigmaeAldrich and
Merck and were used without purification. H and C NMR spectra
4.3. 4-(3,4,5-trimethoxybenzyloxy)benzohydrazide (6)
1
13
were recorded on a 400 MHz FT-NMR Bruker spectrometer in
DMSO-D . Chemical shifts are reported in (ppm) and coupling
6
Hydrazine hydrate (2 mL, 85%) was added to a solution of
compound 5 (1.0 g, 2.88 mmol) in absolute ethanol (3 mL). The
solution was refluxed for 24 h. After that, ethanol was evaporated
under reduced pressure and a small amount of water (2e3 mL) was
added to precipitate the hydrazide 6, which was filtered and dried
constants (J) are reported in Hz. The abbreviations s ¼ singlet,
d ¼ doublet, dd ¼ double doublet, t ¼ triplet, q ¼ quadruplet,
m ¼ multiplet and bs ¼ broad signal were used throughout.
HRESI mass spectra were recorded at the Department of
Chemistry at the University of Singapore on a MAT 95 x1-T spec-
trometer at 70 eV. Melting points were determined using a Mel-
Temp II melting point apparatus and are uncorrected. Thin layer
chromatography was applied using Si-60 plates from Merck, which,
were developed with an UV light and in an iodine chamber. IR
spectra were recorded on a PerkineElmer RX1 FT-IR spectrometer.
The absorbance of the reaction mixtures in the DPPH and FRAP
assays were determined by UV spectroscopy using a Power Wave
X340, BIO-TEK INC instrument.
ꢂ
in vacuum to give a shiny white solid (0.83 g, 87%). m.p.106e108 C.
IR: 3272, 3250 (m, NH, NH
2
), 3003 (CHAr), 2941 (CHaliph.), 1716 (C]
1
O), 1236 (CeO), 1120 (CeN), 1020 (OeCH
DMSO-d , ppm: OeCH
NHeNH ), 5.07 (s, 2H, OeCH
2H), 7.81 (d, J ¼ 8 Hz, 2H)], 9.60 (s, 1H, NH). C NMR (100 MHz,
DMSO-d , ppm: (3C, OeCH ) [56.4, 60.5], 70.1 (OeCH ), Ar.C
3
). H NMR (400 MHz,
6
)
d
3
[3.67 (s, 3H), 3.78 (s, 6H)], 4.43 (s, 2H,
), Ar.H [6.79 (s, 2H), 7.07 (d, J ¼ 8 Hz,
2
2
13
6
)
d
3
2
[105.8, 114.8, 126.2, 129.2, 132.7, 137.6, 153.4, 161.0], 166.0 (hydrazide
þ
C]O). HREIMS. C17
333.1365.
H
20
N
2
O
5
, m/z ¼ 333.1459 (M þ H ). Calcd.
4.2. Ethyl 4-[(3,4,5-trimethoxybenzyl)oxy]benzoate (5)
4.4. General procedure for the synthesis of the hydrazones of 4-
(3,4,5-trimethoxy-benzyloxy)benzohydrazide 7aei
The starting material, 3,4,5-trimethoxybenzyl bromide (2) was
prepared according to a procedure in the literature [28]. A mixture
of compound 2 (1.0 g, 3.8 mmol), anhydrous K CO (0.53 g,
.8 mmol) in acetone (25 mL) and ethyl 4-hydroxybenzoate (4)
0.63 g, 3.8 mmol) (prepared by esterfication of 4- hydroxyl-
benzoic acid) was refluxed for 30 h. Acetone was evaporated
A solution of the required aldehyde (1.2 mmol) in absolute
2
3
ethanol (15 mL) was added to a solution of compound 6 (0.40 g,
1.2 mmol) in absolute ethanol (10 mL) and the mixture was
refluxed for 5e6 h. The solution was concentrated to half of its
volume under reduced pressure and subsequently poured on
3
(

504
H.S. Kareem et al. / European Journal of Medicinal Chemistry 103 (2015) 497e505
crushed ice. The product was precipitated quickly separated, dried
functional of Perdew and Ernzerhof [67,68].
and finally recrystallized from ethanol.
The hydrazone molecules and the free radicals generated after
H-abstraction from the hydroxyls were modelled in Materials
Visualizer and were initially optimized by the “smart minimiza-
tion” method in Discover. Then, calculations in the gas phase
employed the DFT method provided by DMOL3. The energy opti-
mization was performed and the structural properties were ob-
tained at the Generalized Gradient Approximation (GGA) level,
with the spin unrestricted approach and PBE/DNP method.
4.4.1. N-(5-Bromo-3-chloro-2-hydroxybenzylidene)-4-(3,4,5-
trimethoxybenzyloxy)benzo-hydrazide (7a)
ꢂ
White solid (0.53 g, 81%), m.p. 235e236 C. IR: 3315 (OHphenol),
3
211 (NH), 2936 (CHAr), 2834 (CHaliph), 1642 (C]O), 1495 (C]C),
1
1238 (CeO), 1120 (CeN), 1023 (OeCH
3
). H NMR (400 MHz, DMSO-
d
6 3 2
) d, ppm: OeCH [3.67 (s, 3H), 3.79 (s, 6H)], 5.12 (s, 2H, OeCH ),
Ar.H [6.81 (s, 2H), 7.20 (d, J ¼ 8 Hz, 2H), 7.69 (d, J ¼ 2.4 Hz, 1H), 7.72
(
d, J ¼ 2.4 Hz, 1H), 7.96 (d, J ¼ 8 Hz, 2H)], 8.52 (s, 1H, CH), 12.41 (s,
5.4. Statistical analysis
13
1
H, NH), 12.80 (bs, 1H, OH). C NMR (100 MHz, DMSO-d
) [56.4, 60.5], 70.3(OeCH ), Ar.C [105.9, 111.2, 115.2,
20.9, 123.7, 124.8, 129.7, 130.3, 132.5, 133.3, 137.7, 147.0 (C]N),
53.4, 153.7, 162.0], 162.9 (C]O). HREIMS, C24 22BrClN , m/
z ¼ 549.0721, 551.0250 (M þ H ). Calcd. 549.0419, 551.0398.
6
) d, ppm:
(
3C, OeCH
3
2
All analyses were performed in triplicates. Results were
expressed as a means ± standard deviation. A Pearson correlation
test was utilized to study the relationship between the antioxidant
activities of the synthesized compounds in the DPPH and FRAP
antioxidant assays. Correlation is significant at the 0.05 level. The
data were statistically analysed using the SPSS statistical software,
version 15 (SPSS Inc., Chicago, Illinois, USA).
1
1
H
2 6
O
þ
5
. Analysis of the antioxidant activities
5.1. DPPH radical scavenging activity
Acknowledgements
The DPPH free radical scavenging assay was performed ac-
cording to the method of Brand-Williams [63] with slight modifi-
cations. The reaction mixture was prepared by mixing 195 l of a
00 M methanolic DPPH solution with 50 l of the test compounds
at different concentrations (0e1000 g/mL). The test compounds
We gratefully acknowledge the financial support from the Uni-
versity of Malaya/Postgraduate Research Grants (PPP): PG021-
m
1
m
m
2013A and PG030-2013B as well as from the Ministry of Education
m
of Iraq for the financial assistance of Huda.
were initially dissolved in dimethyl sulfoxide (DMSO). BHT and
ascorbic acid were used as positive controls and were run in par-
allel. After 30 min of incubation in the dark at room temperature,
the absorbance of the reaction mixture was determined at 515 nm.
The colour of the reaction mixture changed from purple to yellow
as a result of the decreased absorbance. The radical scavenging
activity was calculated by the following equation:
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.09.016.
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