Evaluation of malonic acid diamide analogues as radical scavenging agents

Article abstract of DOI:10.1039/c4nj01968g

The malonic acid diamide analogues such as N,N′-dimethyl-N,N′-dioctyl-malonamide (DMDOMA), N,N′-dimethyl-N,N′-dioctyl-2,(2′-hexyloxyethyl) malonamide (DMDOHEMA), N,N′-dimethyl-N,N′-dicyclohexyl-malonamide (DMDCMA) and N,N,N′,N′-tetraisopropyl malonamide (TiPMA) were synthesized by an ester amine coupling method. These synthesized diamides were evaluated for the scavenging activity of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide anion scavenging and ferric reducing antioxidant power (FRAP), by biochemical methods. Antioxidant properties exhibited by these diamides were compared with standard antioxidants like gallic acid and ascorbic acid. The pulse radiolysis technique was employed to generate 2-2′-azinobis 3-ethylbenzothioline-6-sulfonic acid (ABTS⁺), hydroxyl radicals (OH), and carbonate (CO₃^-) radicals for evaluating the scavenging activity of the diamides. DMDCMA, TiPMA, and DMDOMA were found to show better antioxidant potential as compared to DMDOHEMA due to their more hydrophilic nature. The pulse radiolysis technique was found to be advantageous for investigating the interaction of the diamide class of compounds with radicals generated under high energy radiation.

Full text of DOI:10.1039/c4nj01968g

View Article Online
View Journal
NJC
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: A. B. Patil, S.
Ghosh, S. D. Phadatare, P. N. PATHAK, G. K. Sharma, B. A. Chopade and V. S. Shinde, New J. Chem.,
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/njc

Page 1 of 22
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ01968G
Evaluation of Malonic acid diamide analogues as radical scavenging agents
a,c
b
e
c
Ajay B. Patil , Sougata Ghosh , Suvarna D. Phadatare , Priyanath Pathak , Geeta K.
e
b,d
a*
Sharma , Balu. A. Chopade , and Vaishali S. Shinde
0
0
0
0
0
0
.025
.020
.015
.010
.005
.000
Malonamide
CTRL
.1 mM
.2 mM
.3 mM
0.4 mM
0
0
0
-
0.005
0
20
40
Time (
60
s)
80
100
µ
Radical scavenging ability of malonamides has been explored by use of pulse radiolysis
technique.

New Journal of Chemistry
Page 2 of 22
View Article Online
DOI: 10.1039/C4NJ01968G
Evaluation of Malonic acid diamide analogues as radical scavenging agents
a,c
b
e
c
Ajay B. Patil , Sougata Ghosh , Suvarna D. Phadatare , Priyanath Pathak , Geeta K.
e
b,d
a*
Sharma , Balu A. Chopade , and Vaishali S. Shinde
a
Garware Research Centre, Department of Chemistry, University of Pune, Pune – 411 007,
India.
Institute of Bioinformatics and Biotechnology, University of Pune, Pune – 411 007, India.
b
c
Radiochemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbaiꢀ400 085,
India
d
Department of Microbiology, University of Pune, Pune – 411 007, India.
e
National Centre for Free Radical Research, Department of Chemistry, University of Pune,
Pune, Pune – 411 007, India.
Abstract
The malonic acid diamide analogues such as N,N′ꢀdimethylꢀN,N′ꢀdioctylꢀmalonamide
(DMDOMA),
N,N′ꢀdimethylꢀN,N′ꢀdioctylꢀ2,(2′ꢀhexyloxyethyl)
malonamide
(DMDOHEMA), N,N′ꢀdimethylꢀN,N′ꢀdicyclohexylꢀmalonamide (DMDCMA) and N,N,N′,N′ꢀ
tetraisopropyl malonamide (TiPMA) were synthesized by ester amine coupling method.
These synthesized diamides were evaluated for scavenging activity of 2,2ꢀdiphenylꢀ1ꢀ
picrylhydrazyl (DPPH) radical, superoxide anion scavenging and ferric reducing antioxidant
power (FRAP), by biochemical methods. Antioxidant properties exhibited by these diamides
were compared with standard antioxidants like gallic acid and ascorbic acid. Pulse radiolysis
technique was employed to generate 2ꢀ2′ꢀazinobis 3ꢀethylbenzothiolineꢀ6ꢀsulfonic acid
•+
•
•ꢀ
(
3
ABTS ), hydroxyl radical ( OH), and carbonate (CO ) radicals for evaluating the
scavenging activity of the diamides. The DMDCMA, TiPMA, DMDOMA were found to
show better antioxidant potential as compared to DMDOHEMA due to more hydrophilic
nature. Pulse radiolysis technique was found to be advantageous to know the interaction of
the diamides class of compounds with radicals generated under high energy radiations.
Key words: diamides, pulse radiolysis, antioxidants, metal chelation, free radical scavenging
*
Author to whom correspondence should be addressed;
*
Eꢀmail: vsshinde@chem.unipune.ac.in Fax: 91ꢀ20ꢀ25601395

Page 3 of 22
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ01968G
1. Introduction
Accumulation of free radicals at high level in cells causes oxidative stress. Reactive oxygen
•ꢀ
•
•
2
species (ROS) such as superoxide (O ), hydroxyl ( OH), and peroxyl ( OOR) are
responsible for oxidative damage to lipid, protein and DNA molecules. This causes
development of diseases such as cancer, rheumatoid arthritis, ageing, heart diseases and
1
-3
Alzheimer’s disease. Antioxidants act as radical scavengers, which react with reactive
4
oxygen species (ROS) and lower the risk of cancer and degenerative diseases. Metalꢀ
chelating capabilities of earlier reported antioxidants have enabled them as effective radical
5
-9
scavengers and inhibitors of lipid peroxidation. Another important aspect regarding the
antioxidant activity, is the lipophilicity of the compounds. It is well established in literature
1
0,11
that the lipophilic compounds exhibit good antioxidant behavior in lipid systems.
Recently, our group has focused on the synthesis of malonic acid diamides such as
N,N′ꢀdimethylꢀN,N′ꢀdioctylꢀmalonamide (DMDOMA)(Figure 1(1a)), N,N′ꢀdimethylꢀN,N′ꢀ
dicyclohexylꢀmalonamide (DMDCMA) (Figure 1(1c)) and N,N′ꢀdimethylꢀN,N′ꢀdioctylꢀ2,(2′ꢀ
hexyloxyethyl) malonamide (DMDOHEMA) (Figure 1(4)); and their application as an
extractant for the separation of radioactive actinide metal ions by solvent extraction method,
and by using liquid membranes under the different experimental conditions in radioactive
12-15
waste management.
During such applications, these ligands undergo radiolytic
degradation due to ionizing radiation. Therefore, their interaction with free radicals was
considered to be matter of greater interest for us, to get insights of their radiological stability
1
4
in actinide partitioning.
These compounds along with N,N,N′,N′ꢀtetraisopropyl malonamide (TiPMA) (Figure
1(1b)) are important ligand molecules. They form very stable complexes with a wide variety
of metal ions. However, in the case of malonamides, the complexation with metal ions would
significantly affect the ketoꢀenol equilibrium. If the concentration of malonamides is much

New Journal of Chemistry
Page 4 of 22
View Article Online
DOI: 10.1039/C4NJ01968G
more than that of radioactive actinide metal ions, the excess metalꢀfree malonamides can
14
scavenge ROS in the solutions. B. S. Jursic has studied the use of malonamide derivatives
1
6
for control of epileptic seizures. It was discovered that certain malonamides exhibit
anticonvulsant activity which is a lay symptom in epilepsy, a disease caused by oxidative
17,18
stress.
Malonamide analogues were tested for different biological activities such as κ
opioid receptors, DNA transfection, cytotoxicity, oxidative stress, and prostaglandinꢀH
1
9-21
synthase inhibition in recent literature.
Further, the lower basicity of substituted diamides
is expected to make them good drugs candidates for treatment of bone diseases, which could
be due to better binding to bone, improved bioꢀavailability and superior therapeutic
22,23
behavior.
Thus, they can lead to good therapeutic applications such as the antioxidant
24
agents. Recent reports proposed the good stability of malonamide class of compounds
towards ionizing radiations, which provided the impetus to study their ability to scavenge the
2
5
free radicals. Structureꢀactivity relationship in curcumin type of antioxidants proved that the
diketone moiety in their structure was responsible for stabilization of radicals and reduces the
26,27
oxidative damage and diabetic symptoms.
Though, Metalꢀchelating capability is not
directly correlated with the radicalꢀscavenging activity. The metal chelation would lead to the
inhibition of the Fenton reaction, i.e., the generation of hydroxyl radical. In this context, the
radicalꢀscavenging activity itself is also important for efficient antioxidants. Therefore, we
have envisioned antioxidant activity by malonamides due to presence of 1,3ꢀdiketone
skeleton which can undergo tautomerization to enol form easily. To investigate radical
scavenging and antioxidant activities, superoxide anion is one of the important species. All
these radicals play a key role in many naturally occurring processes, since almost all free
2
8,29
radicals generated in the biological systems react rapidly with oxygen causing damage.
Pulse radiolysis method is ideal for the selective generation of radicals of interest
under appropriate experimental conditions as the rate constants of a variety of reactions are

Page 5 of 22
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ01968G
3
0-34
well documented in the literature.
Precise knowledge about the yield of radicals makes
the evaluation of kinetics easier and thereby rate constants as well as the extinction
coefficients of the transients can be determined with good accuracy. It is reported that pulse
radiolysis of malonamides has been carried out for the identification of the possible site of
•
OH attack on Nꢀmethyl amides which was helpful for the study of radical formation in
35
peptides and proteins.
O
O
O
O
C H
8
17
C H
8
17
N
N
N
N
1b
1a
O
4
O
O
O
C H
8
17
C H
8
17
N
N
N
N
1c
O
C H
6
13
Figure 1. Malonamides used in present work
1a: DMDOMA, 1b: TiPMA, 1c: DMDCMA, 4: DMDOHEMA)
(
The present report deals with the radical scavenging studies of DMDOHEMA,
12,13
DMDOMA, DMDCMA, and TiPMA derivatives (Figure 1).
The studies towards the
antioxidant and radical scavenging activities of synthesized molecules were performed by
using pulse radiolysis and biochemical assay techniques. The radical scavenging activities
were evaluated by pulse radiolysis for 2ꢀ2′ꢀazinobis 3ꢀethylbenzothiolineꢀ6ꢀsulfonic acid
•+
•
•-
(
ABTS ), OH, CO radicals generated by Pune University Linear Accelerator Facility
3
(PULAF). However, 2,2ꢀdiphenylꢀ1ꢀpicrylhydrazyl (DPPH), superoxide anion scavenging
and ferric reducing antioxidant power (FRAP) study were done by laboratory biochemical
methods. The scavenging ability of the synthetic diamides was compared with that of the

New Journal of Chemistry
Page 6 of 22
View Article Online
DOI: 10.1039/C4NJ01968G
universal antioxidants such as gallic acid and ascorbic acid. The present study was also
intended to know the mechanism of interaction of malonamides with the free radicals under
high radiation. The set of data thus obtained with different radical solution conditions is
correlated for plausible mechanism of radical scavenging by the synthesized diamides.
2.
Experimental
2.1. Synthesis of Diamides derivatives:
Nꢀmethyl octyl amine, Nꢀmethyl cyclohexyl amine, and N,Nꢀdiisopropyl amine (synthesis
grade) procured from Alfa Aesar, Fluka, and spectrochem chemicals respectively and was
freshly distilled before use. Diethyl malonate (Synthesis grade), ethylene glycol (AR grade),
1ꢀbromohexane (Puriss Grade), dichloromethane (DCM) (AR grade), dimethyl formamide
(DMF) (AR grade), sodium hydride (60% suspension in mineral oil) were supplied by
Spectrochem Pvt. Ltd., Mumbai, India. IR spectra were recorded using Shimadzu FTꢀIR
spectrometer. NMR spectra were recorded on Varian 300MHz spectrometer. MS analysis
was performed on Shimadzu GCꢀMS Spectrometer.
We have synthesized four maloamide derivatives, N,N′ꢀdimethylꢀN,N′ꢀdioctylꢀ
malonamide (DMDOMA), N,N′ꢀdimethylꢀN,N′ꢀdioctylꢀ2,(2′ꢀhexyloxyethyl) malonamide
(DMDOHEMA), N,N′ꢀdimethylꢀN,N′ꢀdicyclohexylꢀmalonamide (DMDCMA), N,N,N′,N′ꢀ
1
2,13
tetraisopropyl malonamide (TiPMA) by known literature method
which is mentioned
briefly as: Ester ꢀ amine coupling strategy was used for getting the diamide backbone (for
DMDOMA, DMDCMA, TiPMA) (Scheme 1).
O
O
R1
O
O
R1
+
CH3
R1
HN
N
N
R2
H C
3
O
O
R2
1
R2
Diethyl malonate
secondary amine
Diamide derivative
1
1
1
a: DMDOMA; R = methyl, R = octyl
1
2
b: TiPrMA; R = R = Isopropyl
1
2
c: DMDCMA; R = methyl, R = Cyclohexyl
1
2
Scheme: 1

Page 7 of 22
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ01968G
For synthesis of DMDOHEMA (4), the oxyalkyl skeleton (2) was developed by the
selective monoprotection of the ethylene glycol using hexyl bromide. It was tosylated (3) to
facilitate alkylation to diamide backbone by the NaH (Scheme 2).
OH
H C
O
5
3
HO
NaOCH /CH OH
3 3
H C
Br
3
OH
+
1
ꢀBromohexane
ethylene glycol
3
ꢀ oxanonanol
2
O
O
S
H C
O
5
Pyridine, DCM H C
3
3
O
5
OH H C
ꢀ oxanonanol
S Cl
O
pꢀtoluene sulphonyl
chloride
+
O
CH3
3
0
°C ꢀ RT, 55%
3
O
1
ꢀTosyloxyꢀ 3ꢀ oxanonane
2
3
O
O
O
O
H C
3
CH3
O
S
H C
CH3
NaH/DMF
3
N
N
H
3
C
O
5
N
N
+
O
CH3
o
C H
8
C H
8 17
0 C ꢀRTꢀ reflux
17
C H
8
C H
8 17
O
17
58%
O
C6H
13
3
4
1a
Scheme: 2
The synthesized compounds were characterized by analytical techniques such as IR,
NMR, MS, etc. The characterization details are as follows:
The yield after column purification was nearly 65ꢀ70% for all derivatives.
ꢀ1
ꢀ1
1
Characterization for DMDOMA (1a): IR νmax/cm :1641cm (CO); H NMR (300MHz,
1
3
CDCl
3
): δ 0.9(t, 6H), 1.24(br s, 24H), 2.9, 3.1 (t, 6H), 3.7 (m, 4H) 3.45 (d, 2H); C NMR
(
75MHz, CDCl ): δ 48, 51( ꢀ CH ), 167 (ꢀCOꢀ); MS m/z C H N O : cal 354.32, found
N
3
3
21 42
2
2
3
54.
ꢀ1
ꢀ1
1
Characterization for TiPMA (1b): IR νmax/cm :1645cm (CO); H NMR (300MHz,
1
3
CDCl
3
): δ 1.2(d, 24H, ꢀNꢀCHꢀCH
3
), 3.1(s, 2H, ꢀCOꢀCH
2
ꢀCOꢀ), 3.94 (m, 4H, ꢀNꢀCHꢀ);
C
NMR (75MHz, CDCl ): δ 45(
N
ꢀ CH), 165 (ꢀCOꢀ); MS m/z C H N O : cal 270.41, found
3
15 32
2
2
2
70.

New Journal of Chemistry
Page 8 of 22
View Article Online
DOI: 10.1039/C4NJ01968G
ꢀ1
ꢀ1
1
Characterization DMDCMA (1c): IR
ν
max/cm :1643cm (CO); H NMR (300MHz,
CDCl ): 1.32–1.84 (20H, multiplet, 2 ꢀCH
3
δ
2
cyclohexyl), 2.81–2.92 (6H, double doublet, 2
CH ꢀN), 3.47–3.51 (2H, triplet, COꢀCH ꢀCO), 4.40–4.42 (2H, multiplet, 2 ꢀCH cyclohexyl);
3
2
1
3
C NMR (75MHz, CDCl ): δ 52, 56(
N
ꢀ CH ), 169 (ꢀCOꢀ); MS m/z C H N O : cal 294.43,
3
3
17 30
2
2
found 294.
ꢀ1
ꢀ1
1
Characterization DMDOHEMA (1(4)): IR νmax/cm : 1651cm (CO). H NMR (300MHz,
3
CDCl ): δ 0.85(t, 9H), 1.2ꢀ1.3(br s, 26H), 1.56 (br s, 8H), 2.9 (t, 6H), 3.4 (m, 8H), 3.9 (m,
13
1
H). C-NMR (75MHz, CDCl ): δ 49, 50 (N ꢀ CH ), 69, 71(ꢀOꢀCH ꢀ),170 (ꢀCOꢀ). MS m/z
3 3 2
29 58 2 3
C H N O : cal 482.78, found 483.
2
.2. Pharmacology
Folin–Ciocalteu reagent and quercetin was obtained from Qualigens, Mumbai, India. Gallic
acid, Lꢀascorbic acid, potassium thiocyanate, 2ꢀ2’ꢀazinobisꢀ3ꢀethylbenzothiolineꢀ6ꢀsulfonic
acid (ABTS), 2,2ꢀdiphenylꢀ1ꢀpicrylhydrazyl (DPPH), 2,4,6ꢀ[Tri(2ꢀpyridyl)ꢀsꢀtriazine]
(
TPTZ), phenazine methosulfate (PMS), nitroblue tetrazolium (NBT), 2ꢀdeoxyribose,
thiobarbituric acid (TBA), sodium nitroprusside, sulphanilic acid, ꢀ(1ꢀNaphthyl)
ethylenediamine dihydrochloride, potassium hexacyanoferrate (K Fe(CN) ), trichloroacetic
acid (TCA), ferric chloride were procured from HiMedia Laboratories, Mumbai, India.
N
3
6
2
.2.1 Pulse radiolysis study
The linear accelerator (LINAC) is an evacuated cylindrical pipe that functions as a
waveguide for the accelerating field. The electrons are injected in pulses into this straight
segmented waveguide (microwave cavities) and accelerated by the oscillating polarity of the
30
electric field of an electromagnetic wave that travels down the waveguide. Highꢀenergy
electrons in the range 7MeV can be easily obtained using LINAC, and such high energy

Page 9 of 22
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ01968G
provides more penetrating power to the electrons and also a uniform distribution of the
31
ionization events throughout the sample. In the present work the pulse radiolysis was done
by using Linear accelerator (LINAC) electron pulse radiolysis system at the ‘National Centre
32
for Free Radical Research’, University of Pune, Pune.
•
+
•
2
.2.1.1 Radiolysis generated ABTS and carbonate (CO
3
⁻
) radical scavenging assay
Scavenging of ABTS radical by diamides were determined using pulse radiolysis. The
reaction mixture (4 mL) contained 0.05 M sodium azide (NaN ), 2 mM 2ꢀ2’ꢀazinobis 3ꢀ
•+
3
ethylbenzothiolineꢀ6ꢀsulfonic acid (ABTS) and mili Q water. After purging with N O for 5
2
min, suprasil quartz cuvettes containing the samples were exposed to an electron beam of 100
•+
ns pulse width and dose/pulse 17 Gy generated hydroxyl radicals. The ABTS radical was
2−
produced by the reaction of radiolytically generated azide radicals with ABTS
. Scavenging
3
4
of the radical was estimated by recording the traces at 600 nm.
•
The CO
NaHCO
by an exposure to an electron pulse of 100 ns. The absorbance was recorded at 600 nm.
3
⁻
radicals were generated using a reaction mixture containing 0.5 M
3
and 0.5 M Na
2
CO saturated with N O for 5 min in presence of diamides followed
3
2
2
8
2
.2.1.2 Hydroxyl radical assay
•
34
Hydroxyl radicals ( OH) were generated by radiolysis of water using LINAC. Water when
exposed to 7 MeV electron pulse generates hydroxyl radicals, hydrated electrons and
•
hydrogen atoms. In order to generate selectively the OH, all solutions were preꢀsaturated
with nitrous oxide (N O) for removal of dissolved oxygen. Generated hydroxyl radicals were
2
ꢀ1
subjected to react with malonamide derivatives. The first order rate constants (
1
k = s ) for
radical formation were measured and found to vary with the malonamide derivatives. The
ꢀ1
3 ꢀ1
2
second order rate constants (k = mol dm s ) were obtained from the slope of linear plots.

New Journal of Chemistry
Page 10 of 22
View Article Online
DOI: 10.1039/C4NJ01968G
The ability to scavenge hydroxyl radicals was measured by comparing it with standard
•
potassium thiocyanate (KSCN) using competition kinetics. In this method, OH reacts with 1
•
mM KSCN in absence and presence of malonamide derivatives. The OH reacts completely
−
•−
36,37
•−
with SCN to produce (SCN)
2
which absorbs at 480 nm.
The rate constant of (SCN)
2
formation reaction with test malonamide compounds were measured at 480 nm.
2
2
.2.2 Chemical assay for antioxidant activity
.2.2.1 DPPH radical scavenging assay
Test compounds (20 ꢁL) at concentration of 1 mM to 100 mM were mixed with ethanolic
solution (80 ꢁL) of 2,2ꢀdiphenylꢀ1ꢀpicrylhydrazyl (DPPH) in 96 well plate. Changes in
absorbance were measured at 517 nm (after incubation for 20 min) in a 96ꢀwell plate reader
(Spectramax M5, Molecular devices corporation, Sunnyvale, CA). Radical scavenging
activity was expressed in terms of IC50 in mM. Vitamin C (Lꢀascorbic acid) was served as a
38
reference antioxidant compound.
2
.2.2.2 Superoxide anion scavenging activity assay
Superoxide anions were generated in a nonꢀenzymatic phenazine methosulfateꢀnicotinamide
adenine dinucleotide (PMSꢀNADH) system through the reaction of PMS, NADH, and
oxygen. It was assayed by the reduction of nitroblue tetrazolium (NBT). The 0.3 ml of test
malonamide compound solution was added in 3 mL of TrisꢀHCl buffer (100 mM, pH 7.4)
containing 0.75 mL of NBT (300 ꢁM) solution and 0.75 mL of NADH (936 ꢁM) solution.
The reaction was initiated by adding 0.75 mL of PMS (120 ꢁM) to the mixture. After 5 min
of incubation at room temperature, the absorbance at 560 nm was measured in
39
spectrophotometer.
The superoxide anion scavenging activity was calculated according to the following
equation:

Page 11 of 22
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ01968G
%
Inhibition = [(A
0
ꢀA
1 0
) / A × 100],
where, A
0
was the absorbance of the control (blank, without compound) and A was the
1
39
absorbance in the presence of the compound.
2
.2.2.3 Ferric Reducing Antioxidant Property
Ferric reducing antioxidant property (FRAP) assay was estimated according to the protocol of
40
Pulido et. al. with some modifications. FRAP solution was prepared freshly by mixing 25
ml of acetate buffer, 2.5 ml of TPTZ solution and 2.5 ml of FeCl3..6H O solution, and then
warmed to 37°C before use. The 30 l of individual compound solutions at different
2
ꢀ
concentrations were allowed to react with 0.9 ml of FRAP solution for 15 min in darkness.
The absorbance recorded at 595 nm was used to quantify the activity by extrapolating from
the standard calibration curve. The % scavenging was expressed in terms of gallic acid
equivalent antioxidant capacity (GAEAC).
3
3
. Results and discussion:
.1 Synthesis of malonamides:
The synthesis of malonamide derivatives with varying structural modifications and
hydrophilic nature has been accomplished using ester amine coupling method. The yield and
12,41,42
purity was better than earlier reports (~25ꢀ30%).
The synthesis of pentaalkyl derivative
has been achieved by selective monoprotection, tosylation and Cꢀalkylation methods. The
developed synthetic route can be applied to different tailor made alkyl analogues of malonic
acid diamides as radical scavenging motifs.

New Journal of Chemistry
Page 12 of 22
View Article Online
DOI: 10.1039/C4NJ01968G
3
3
.2 Radical scavenging studies:
.2.1 Pulse radiolysis:
•+
•
•-
Using linear accelerator, the different radicals such as ABTS , OH, and CO
3
has been
generated. The rate constant for the reaction of the any reductant with the free radicals
indicates its reactivity towards the free radical. Diamide analogues were found to scavenge
the generated radicals at their characteristic wavelength. The transient kinetic analysis
showed the decrease in second order rate constant with diamide analogues, indicating the
inhibition of radical formation. The appearance of the solution was same indicating the
stability of the malonamides under high radiation fields. For all pulse experiments, the
optimum concentration of malonamides, suitable to give a proper scavenging was 0.4 mM.
3.2.1.1 Carbonate radical scavenging assay:
Carbonate radicals present in the biological systems significantly contributes to the
2
8
oxidative damage. Therefore, it is necessary to study such radical species for evaluation of
the antioxidant activity of malonamides.
As observed in Figure 2, the extent of scavenging of carbonate radicals was increased
with concentration of malonamide. The highest activity was obtained at 0.4 mM
concentration of the malonamide derivatives. Therefore, further assays have been carried out
with this optimized diamides concentration. Among all the malonamide derivatives,
DMDOHEMA showed the least scavenging which can be attributed to its least hydrophilic
nature in the assay solution.

Page 13 of 22
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ01968G
0
0
0
0
0
0
.025
.020
.015
.010
.005
.000
0.025
0.020
0.015
0.010
0.005
0.000
-0.005
DMDOMA
DMDOHEMA
CTRL
0.1 mM
CTRL
0.1 mM
0.2 mM
0.3 mM
0.4 mM
0
.2 mM
0.3 mM
.4 mM
0
-
0.005
0
20
40
Time (
60
s)
80
100
0
20
40
Time (
60
s)
80
100
ꢀ
ꢀ
0
0
0
0
0
0
.025
.020
.015
.010
.005
.000
0.025
0.020
0.015
0.010
0.005
0.000
-0.005
TiPMA
DMDCMA
CTRL
.1 mM
.2 mM
.3 mM
.4 mM
CTRL
.1 mM
.2 mM
.3 mM
.4 mM
0
0
0
0
0
0
0
0
-
0.005
0
20
40
Time (
60
s)
80
100
0
20
40
Time (
60
s)
80
100
ꢀ
ꢀ
•ꢀ
Figure 2: Traces obtained in CO radical assay by pulse radiolysis at 620 nm using
3
increasing concentration of diamides
3.2.1.2 Hydroxyl radical scavenging assay:
Hydroxyl radical scavenging studies were performed using competition kinetics at 480 nm,
using pulse radiolysis. The transient traces were used to determine the second order rate
constants as the measure of radical scavenging activity (Figure 3 and 4). Malonamides were
found to exhibit significantly higher second order rate constant of transient radical as
compared to control experiment. DMDOMA and TiPMA were found to be more active in
•
scavenging OH radical than that of compounds DMDOHEMA and DMDCMA due to more
hydrophilicity.

New Journal of Chemistry
Page 14 of 22
View Article Online
DOI: 10.1039/C4NJ01968G
0.080
0.075
0.070
0.065
0.060
0.055
0.050
0.045
0.040
CNTRL
DMDOHEMA
DMDOMA
TiPMA
DMDCMA
2
4
6
8
10
Time (ꢀs)
•
Figure 3: Traces obtained in OH radical assay by pulse radiolysis at 480 nm using 0.4 mM
concentration of diamides
6
3.5x10
6
3.0x10
6
2.5x10
1
-
s
.
6
3
2.0x10
m
d
.
1
-
l
o
6
1.5x10
m
k
2
6
1.0x10
5
5.0x10
DMDOMA
TiPMA
DMDCMA
DMDOHEMA
Compounds
Figure 4: Scavenging of hydroxyl radicals in terms of second order rate constant of different
compounds by pulse radiolysis.
3.2.1.3 ABTS radical scavenging assay:
•+
ABTS radical was generated using pulse radiolysis technique and easily identified by
absorbance at 600 nm. The second order rate constants of transient traces recorded using

Page 15 of 22
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ01968G
LINAC showed that the extent of radical formation decreased in presence of the malonamide
derivatives (Figure 5).
7
2.0x10
1
-
s
.
7
3
1.5x10
m
d
.
1
-
l
o
7
1.0x10
M
2
k
6
5.0x10
0.0
DMDOMA DMDOHEMA
TiPMA
DMDCMA
Compounds
Figure 5: Second order rate constants of traces obtained in ABTS assay by pulse radiolysis at
00 nm using 0.4 mM concentration of diamides
6
The maximum scavenging efficiency was observed for TiPMA malonamide
7
ꢀ1
3 ꢀ1
derivative (second order rate constant,
2
k = 2.29 × 10 mol dm s ) while least scavenging
6
ꢀ1
3 ꢀ1
was seen for DMDCMA (second order rate constant, k2= 9.62 × 10 mol dm s ).
3
.2.2 Biochemical Assay:
3.2.2.1 DPPH and Superoxide anion scavenging assay:
Malonamides were found to possess comparatively lower scavenging potential against DPPH
radical. The least hydrophilic derivative DMDOHEMA showed superior scavenging of
DPPH radicals as compared to other counterparts.
A similar trend was observed even in case of the superoxide anion scavenging assay; which is
one of the major aspects for the antioxidant evaluation. Therefore, there is a need to study the
28
superoxide anion scavenging activity of the diamides. Superoxide anion scavenging activity

New Journal of Chemistry
Page 16 of 22
View Article Online
DOI: 10.1039/C4NJ01968G
confirmed the antioxidant properties of the diamide derivatives. Among the malonamides,
high % scavenging was found with the DMDOMA and DMDOHEMA (Table 1).
Compounds under studies were compared with that of the standard antioxidants such
as gallic acid and ascorbic acid. It can be seen that the IC50 values for the different
malonamide was found to be in similar range with that of gallic acid and ascorbic acid for
superoxide anion scavenging activity.
Table 1. Antioxidant activities of malonamide derivatives and their comparison with
universal antioxidants
Radical Scavenging activity,
IC50(mM)
Compound
Superoxide
DPPH
anion
DMDOMA
DMDOHEMA
TiPMA
0.85
0.62
0.97
1.05
0.61
0.64
0.21
0.22
0.27
0.24
0.20
0.23
DMDCMA
Gallic Acid
Ascorbic Acid
3.2.2.2 Ferric ion reducing antioxidant power (FRAP) assay:
Reducing power assay of the antioxidants is based on the reduction of Fe(III) to Fe(II). It was
evident that malonamides showed good antioxidant power in the present test solution
conditions (Figure 6). The FRAP was expressed in terms of gallic acid equivalents (GAEAC)
in mM concentration. All four malonamides were compared and found to show good
reducing property. Compound TiPMA was showed highest reducing power, while compound
DMDOMA showed the least ferric ion reducing antioxidant power.

Page 17 of 22
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ01968G
50
40
30
20
10
0
DMDOMA DMDOHEMA TiPMA
Compounds
DMDCMA
Figure 6: Ferric ion reducing antioxidant power (FRAP) of different malonamide compounds
3
.2.3 Plausible radical scavenging mechanism in radical scavenging activity of diamides
analogues
From all radical scavenging assays, it is evident that structural relationship of the diamides
analogues results in variable scavenging abilities. The least hydrophilic DMDOHEMA
possessed least scavenging activity due to presence of the longer alkyl chain substitution on
the malonamide skeleton. However, such nonpolar substrates can be exploited as the
1
0
antioxidant candidates in lipid systems. Also, the diamides polar head serves as the electron
rich centre with electron donating nature; which might be the source of scavenging ability as
proposed in Figure 7.
Similar scavenging mechanism has been proposed earlier for the curcumin’s antioxidant
activity and can be correlated here as in both the cases, the 1,3ꢀdiketone moiety is responsible
43
for radical scavenging. It is already evident that malonamides are present in their tautomeric
1
2
form, which supports the plausible scavenging pathway showed in Figure 7.

New Journal of Chemistry
Page 18 of 22
View Article Online
DOI: 10.1039/C4NJ01968G
R
H
O
O
O
O
O
O
R1
R1
R1
R1
R1
R1
N
N
N
N
N
N
+ RH
R2
R2
R2
R2
R2
R2
Figure 7: Plausible radical scavenging by malonamide analogues
4
. Conclusions
Pulse radiolysis has been applied successfully to study radical scavenging ability of
malonamide class of compounds. In present solution conditions, malonamides were found to
scavenge carbonate, hydroxyl, ABTS, DPPH radicals and superoxide anion. Metal chelating
ability of malonamides has been correlated with radical scavenging ability of malonamide
through the plausible tautomerizable moiety. Increasing ability of radical scavenging with
less substitution (DMDOHEMA to TiPMA) showed that hydrophilicity is the affecting
parameter of scavenging activity of the studied malonamides in the solution conditions.
Comparison with the universal antioxidants such as ascorbic acid and gallic acid clearly
demonstrated that the malonamides can be good targets for radical scavenging and
antioxidant studies. Metal complexes of the said malonamides and lipophilic substituted
malonamides can be explored in future as antioxidants for lipid systems.

Page 19 of 22
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ01968G
5
. Acknowledgments
The authors thank Prof. P. K. Mohapatra for helpful discussions. Authors also thank to Prof.
A. S. Kumbhar for allowing the use of PULAF facility. ABP great fully acknowledges the
financial support for his fellowship from Bhabha Atomic Research Centre, Mumbai, under
BARCꢀUniversity of Pune, Pune Collaborative Research Programme. SG and SDP thanks
Council of Scientific and Industrial Research (CSIR, Government of India) for Senior
Research fellowship.

New Journal of Chemistry
Page 20 of 22
View Article Online
DOI: 10.1039/C4NJ01968G
6
. References
1
2
3
. Y. He, P. Yao, S. Chen, Z. Huang, S. Huang, J. Tan, D. Li, L. Gu and Z. Huang, Eur.
J. Med. Chem., 2013, 63, 299ꢀ312.
. J.K. Willcox, S.L. Ash and G.L. Catignani, Crit. Rev. Food Sci. Nutr., 2004, 44, 275ꢀ
2
95.
. A. Pe´rezꢀGonza´lez, A. Galano and J. AlvarezꢀIdaboy, New J. Chem., 2014, 38,
639ꢀ2652.
2
4
5
. L.A. PhamꢀHuy, H. He and C. PhamꢀHuy, Int. J. Biomed. Sci., 2008, 4, 89ꢀ96.
. J. Terao, and M.K. Piskula, in Flavonoids as inhibitors of lipid peroxidation in
membranes, in C.A. RiceꢀEvans and L. Packer (editor), Flavonoids in health and
disease, Marcel Dekker, New York, 1997, p.p. 277ꢀ295.
6
7
. N.R. Perron and J.L. Brumaghim, Cell. Biochem. Biophys., 2009, 53, 75–100.
. S.K. Suthar, V. Jaiswal, S. Lohan, S. Bansal, A. Chaudhary, A. Tiwari, A.T. Alex and
A. Joesph, Eur. J. Med. Chem. 2013, 63, 589ꢀ602.
8
9
. D. HadjipavlouꢀLitina, G.E. Magoulas, M. Krokidis and D. Papaioannou, Eur. J. Med.
Chem., 2010, 45, 298–310.
. A. Barik, B. Mishra, A. Kunwar, R.M. Kadam, L. Shen, S. Dutta, S. Padhye, A.K.
Satpati, H. Zhang and K.I. Priyadarsini, Eur. J. Med. Chem., 2007, 42, 431ꢀ439.
0. L. Liu, C. Jin and Ying Zhang, RSC Adv., 2014, 4, 2879ꢀ2891.
1
1
1. M. Barontini, R. Bernini, I. Carastro, P. Gentili and A. Romani, New J.Chem., 2014,
3
8, 809ꢀ816.
1
1
2. A. B. Patil, V. S. Shinde, P. N. Pathak, P. K. Mohapatra and V. K. Manchanda,
Radiochim. Acta, 2013, 101, 93ꢀ100.
3. A. B. Patil, V. S. Shinde, P. N. Pathak, and P. K. Mohapatra, Sep. sci. Technol., 2014,
(In press DOI: 10.1080/01496395.2014.943772).

Page 21 of 22
New Journal of Chemistry
View Article Online
DOI: 10.1039/C4NJ01968G
1
1
4. A. B. Patil, P. N. Pathak, V. S. Shinde, S. V. Godbole and P. K. Mohapatra, Dalton
Trans., 2013, 42, 1519ꢀ1529.
5. A. B. Patil, P. Kandwal, V. S. Shinde, P. N. Pathak and P. K. Mohapatra, J. Membra.
Sci., 2013, 442, 48ꢀ56.
1
1
1
1
6. B. S. Jursic, Tet. Lett., 2005, 41, 325ꢀ328.
7. C. M. Darling, US Patent No. 1985, 4 537 781.
8. C. M. Darling and P. Pryor, In J. Pharm. Sci., 1979, 68, 108ꢀ110.
9. C. Wolk, M. Heinze, P. Kreideweiß, M. Dittrich, G. Brezesinski, A. Langner and B.
Dobner, Int. J. Pharmaceut., 2011, 409, 46–56.
2
0. G. Chu, M. Gu, J.A. Cassel, S. Belanger, T. M. Graczyk, R.N. DeHaven, N. Conwayꢀ
James, M. Koblish, P. J. Little, D. L. DeHavenꢀHudkins and Roland E. Dolle, Bioorg.
Med. Chem. Lett., 2007, 17, 1951–1955.
2
2
2
1. J. L. Vennerstrom and T. J. Holmes, J. Med. Chem., 1987, 30, 434ꢀ437.
2. M. M. A. Boojar and A. Shockravi, Bioorg. Med. Chem., 2007, 15, 3437–3444.
3. P. Sozio, E. D’Aurizio, A. Iannitelli, A. Cataldi, S. Zara, F. Cantalamessa, C. Nasuti
and A. Di Stefano, Arch. Pharm. Chem. Life Sci., 2010, 343, 133 – 142.
4. D. A. Stepinski and A. W. Herlinger, Syn. Comm., 2002, 32, 2683–2690.
5. L. Berthon, J. M. Morel, N. Zorz, C. Nicol, H. Virelizier and C. Madic, Sep. Sci.
Technol., 2001, 36, 709ꢀ728.
2
2
2
2
2
6. U. Singh, A. Barik, B. G. Singh and K. I. Priyadarsini, Free Rad. Res., 2011, 45, 317ꢀ
3
25.
7. J. S. Rathee, B. S. Patro, S. Mula, S. Gamre and S. Chattopadhyay, J. Agric. Food
Chem., 2006, 54, 9046ꢀ9054.
8. D. Huang, B. Ou and R. L. Prior, J. Agric. Food Chem., 2005, 53, 1841ꢀ1856.

New Journal of Chemistry
Page 22 of 22
View Article Online
DOI: 10.1039/C4NJ01968G
2
3
3
3
3
3
9. A. PérezꢀGonzález, A. Galano and J. R. AlvarezꢀIdaboy, New J. Chem., 2014, 38,
639ꢀ2652.
0. P. Yadav, M. S. Kulkarni, M. B. Shirdhonkar and B. S. M. Rao, Curr. Sci., 2007, 92,
99ꢀ605.
2
5
1. S. D. Phadatare, K. K. Sharma, B. S. M. Rao, S. Naumov and G. K. Sharma, J. Phys.
Chem. B, 2011, 115, 13650ꢀ13658.
2. J. S. Londhe, T. P. A. Devasagayam, L. Y. Foo and S. S. Ghaskadbi, Redox Report
008, 13, 199ꢀ207.
,
2
3. R. Joshi, P. N. Pathak, V. K. Manchanda, S. K. Sarkar and T. Mukherjee, Res. Chem.
Intermed., 2010, 36, 503–510.
4. J. S. Londhe, T. P. A. Devasagayam, L. Y. Foo and S. S. Ghaskadbi, J. Radiat. Res.
009, 50, 303–309.
,
2
3
3
3
5. S. Rustagi and P. Riesz, Int. J. Radiat. Biol., 1978, 33, 325ꢀ339.
6. G. H. Naik, K. I. Priyadarsini and H. Mohan, Curr Sci., 2006, 90, 1100–1105.
7. S. Ghosh, A. Derle, M. Ahire, P. More, S. Jagtap, S. D. Phadatare, A. B. Patil, A. M.
Jabgunde, G. K. Sharma, V. S. Shinde, K. Pardesi, D. D. Dhavale and B. A. Chopade,
Plos One, 2013, 8, e82529.
3
3
4
4
4
4
8. M.A.A. Rahman and S.S. Moon, Bull. Korean Chem. Soc., 2007, 28, 827ꢀ831.
9. F. Liu, V. E. C. Ooi and S. T. Chang, Life Sci., 1997, 60, 763ꢀ771.
0. R. Pulido, L. Bravo and F. SauraꢀCalixto, J Agric Food Chem., 2000, 48, 3396–3402.
1. E. G. Knapick, P. Ander and J. A. Hirsch, Synthesis, 1985, 58ꢀ60.
2. G. Thiollet and C. Musikas, Solv. Extr. Ion Exch., 1989, 7, 813ꢀ827.
3. K. I. Priyadarsini, Curr. Pharma. Des., 2013, 19, 2093ꢀ2100.