Synthesis and comparison of antioxidant properties of indole-based melatonin analogue indole amino acid derivatives

Article abstract of DOI:10.1111/j.1747-0285.2011.01216.x

Increased levels of reactive oxygen species attributed to oxidative stress have been found to be responsible for the development of some vital diseases such as cardiovascular, neurodegenerative and autoimmune diseases. Recently, it was observed that melatonin is a highly important antioxidant, and melatonin analogues are under investigation to find out improved antioxidant activity. In this study, 14 melatonin -based analogue indole amino acid and N-protected amino acid derivatives were synthesized and elucidated spectrometrically. To investigate the antioxidant activity of the synthesized compounds and to compare with melatonin, butylhydroxytoluene and vitamin E, lipid peroxidation inhibition and 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activities were tested. The results indicated that the synthesized new indole amino acid derivatives have similar activities to melatonin in 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity assay but more potent activities in lipid peroxidation inhibition assay. A series of indole-based melatonin analogue indole-amino acid and N-protected amino acid derivatives were synthesized to investigate antioxidant activity by different assays. The results indicated that the synthesized compounds have similar activities to melatonin in DPPH radical scavenging activity assay but more potent activities in lipid peroxidation inhibition assay.

Full text of DOI:10.1111/j.1747-0285.2011.01216.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01216.x
Chem Biol Drug Des 2012; 79: 76–83
Research Article
Synthesis and Comparison of Antioxidant
Properties of Indole-Based Melatonin Analogue
Indole Amino Acid Derivatives
Sibel Suzen1,*, Seyhan Sezen Cihaner1 and
Tulay Coban2
It is known that indole derivatives are significant substances for
their medicinal and biological features. Some 3-substituted indoles
(2), indolyl thiohydantoin derivatROSives (3) and indolyl triazoles (4)
have anticancer activity, melatonin has (5) antioxidant activity, indo-
1Faculty of Pharmacy, Department of Pharmaceutical Chemistry,
Ankara University, 06100 Tandogan, Ankara, Turkey
lin-2-one derivatives (6) and indol-5-carbonyl hydrazines (7) show
antirheumatoidal activity, and indolyl thiohydantoins have anti-HIV
activity (8). Series of 2-(hydrazinocarbonyl)-3-substituted-phenyl-
1H-indole-5-sulphonamide derivatives (9,10) are known as carbonic
anhydrase inhibitors.
2Faculty of Pharmacy, Department of Pharmaceutical Toxicology,
Ankara University, 06100 Tandogan, Ankara, Turkey
*Corresponding author: Sibel Suzen, sibel@pharmacy.ankara.edu.tr
Increased levels of reactive oxygen species attrib-
uted to oxidative stress have been found to be
responsible for the development of some vital dis-
eases such as cardiovascular, neurodegenerative
and autoimmune diseases. Recently, it was
observed that melatonin is a highly important anti-
oxidant, and melatonin analogues are under inves-
tigation to find out improved antioxidant activity.
In this study, 14 melatonin -based analogue indole
amino acid and N-protected amino acid derivatives
were synthesized and elucidated spectrometrical-
ly. To investigate the antioxidant activity of the
synthesized compounds and to compare with mel-
atonin, butylhydroxytoluene and vitamin E, lipid
peroxidation inhibition and 2,2-diphenyl-1-pic-
rylhydrazyl radical-scavenging activities were
tested. The results indicated that the synthesized
new indole amino acid derivatives have similar
activities to melatonin in 2,2-diphenyl-1-pic-
rylhydrazyl radical-scavenging activity assay but
more potent activities in lipid peroxidation inhibi-
tion assay.
Antioxidant effects of the indole ring-containing melatonin (MLT)
have been well described and evaluated by Tan et al. (11). It acts
as a free radical scavenger and has a broad-spectrum antioxidant
(12). Owing to its free radical scavenger and antioxidant properties,
MLT-related compounds such as MLT metabolites and synthetic ana-
logues are under investigation to determine which exhibit the high-
est activity with the lowest side-effects (13–15). Antioxidant activity
of synthetic indole derivatives such as indole-3-propionic acid (16),
indole amine-triazoles (17) and stobadine (18) was studied exten-
sively. Moreover, our group previously identified the antioxidant
activity of MLT analogue indole derivatives such as 2-phenylindole
derivatives (19), indole hydrazide and hydrazones (20,21) and indole-
amides (22). Recently, we observed the relationship between aldose
reductase and superoxide dismutase inhibition capacities of indole-
based analogues of MLT derivatives (23).
It is known that many amino acids have potential antioxidant activ-
ity. In a study, troloxyl-methionine and Troloxyl-cysteine showed sig-
nificant antioxidant activity (24,25). Several tryptophan derivatives
function as a free radical scavengers and antioxidants. Furthermore,
they stimulate a number of antioxidative enzymes and stabilize cell
membranes that help to resist free radical damage (26,27). N-(4-pyr-
idoxylmethylene)-L-serine was found as an antioxidant to suppress
iron-catalysed ROS generation (28). L-alanine stimulates expression
of the antioxidant defence proteins and ferritin in endothelial cells
(29). Fullerene-substituted phenylalanine and lysine derivatives were
determined to be significantly more potent than Trolox (30). Dehydro
amino acids and corresponding peptides can function as radical
scavengers (31). In our earlier studies (32,33), we showed that
substituted dehydroalanines scavenge ROS. In the first study (32),
novel N-acyl dehydroalanine derivatives were studied as antioxi-
dants on rat liver LP and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free
radical-scavenging activity. Most of the compounds showed strong
inhibitory effect on LP. In the second study (33), comparative effect
of N-substituted dehydroamino acids and alpha-tocopherol on rat
Key words: amino acid, antioxidant activity, indole, melatonin, syn-
thesis
Received 29 March 2011, revised 18 June 2011 and accepted for publi-
cation 2 August 2011
Reactive oxygen (ROS) and reactive nitrogen species can cause vital
damages to all cellular macromolecules, including nucleic acids, pro-
teins, carbohydrates and lipids. Membrane lipids are most important
targets of, and lipid peroxidation (LP) may lead to membrane
dysfunction and change the cell permeability. Oxidative stress is
associated in wide selection of disorders including ischaemia–
reperfusion injury, neurodegenerative diseases, diabetes,
inflammatory diseases and ageing (1).
76

Antioxidant Properties of Indole Amino Acids
liver LP activities was investigated. The results indicated that all the
synthesized compounds showed very good inhibitory effect on the LP.
the Central Laboratory of the Faculty of Pharmacy, Ankara Univer-
sity. Chromatography was carried out using Merck silica gel 60
(230–400 mesh; Merck KGaA, Darmstadt, Germany). The chemical
reagents used in synthesis were purchased from Sigma (Hamburg,
Germany) and Aldrich (Burbank, CA, USA).
Melatonin has a role in the regulation of many physiological pro-
cesses but therapeutic use is limited because of two major prob-
lems. The first is very short biological half-life, owing to its fast
metabolism to 6-hydroxymelatonin and N(1)-acetyl-N(2)-formyl-5-
methoxykynuramine and the second is the non-selectivity of MLT at
target sites (34,35). To increase the biological half-life, the acetyl
amino ethyl side chain was replaced with bulky amino acid deriva-
tives by making hydrazones. Hydrazone derivatives possess a range
of pharmacological activities including antitumoural, anti-microbial,
antimalarial, anti-convulsant and anti-inflammatory activity (36).
Recently, hydrazones were found as potential antioxidants (37–40).
It is possible that the synthesized compounds may undergo some
hydrolysis under the in vivo conditions because oxidative stress
induces a LP of cellular membranes, resulting in the generation of
reactive carbonyl compounds that involves in the 'carbonyl stress'
(41,42). Hydrazine derivatives also exhibit carbonyl scavenger activ-
ity by the reaction of ketones ⁄ aldehydes (43).
Experimental
Target compounds were derived from N-protected amino acid esters
(4a–i) and hydrazine hydrate using simple reaction strategies. The
hydrazide derivatives of N-protected amino acids (2a–h) were
reacted with 1-methyl-1H-indole-3-carboxaldehyde (3) to give the
resulting indole amino acid hydrazones (4a–i) using a methodology
similar to that of Kidwai et al. (44). All new compounds were char-
acterized on the basis of their spectral and analytical data.
General procedure for the synthesis of
compounds 2a–h
Compounds 1a–f (1 mmol) were treated with excess hydrazine
hydrate in MeOH (10 mL) by refluxing for 3–4 h on the hot water
bath. On cooling, the precipitate was collected, washed with cold
MeOH and recrystallized from MeOH to give 2a–h with 76–96%
yield.
There has been no research published related to antioxidant proper-
ties of indole amino acid derivatives. In this study, 14 MLT-based
analogue indole amino acid derivatives and N-protected amino acids
(Figure 1) were synthesized. Their antioxidant activity was investi-
gated in vitro by LP inhibition and DPPH radical-scavenging assays.
The results were compared with MLT, butylhydroxytoluene (BHT)
and vitamin E. All new MLT analogue compounds 4a–i and new
amino acid derivatives 2c and 2f were characterized on the basis
N-Benzoyl-D-serine hydrazide 2c. Yield 92.4%, m.p. 195–196 ꢀC;
1H-NMR: d 3.73 (2H, t, CH2) 4.29 (2H, s, NH2), 4.52 (1H, m, CH),
5.00 (1H, t, OH), 7.52–7.96 (5H, m, Ar-H), 8.30 (1H, d, NH), 9.24 (1H,
s, NH); ESI MS m ⁄ z 192 (M- CH2OH, 100%), 224 (M + 1).
1
of H- and 13C-NMR, mass and FT-IR spectra.
N-Benzoyl-L-proline hydrazide 2f. Yield 95.1%, m.p. oily compound;
1H-NMR: d 1.74 (4H, m, (CH2)2), 2.17–3.40 (2H, m, CH2), 3.57 (2H,
m, NH2), 4.41 (1H, t, CH), 7.32–7.56 (5H, m, Ar-H), 7.65 (1H, m, NH);
ESI MS m ⁄ z 202 (M- NH-NH2, 100%), 256 (M+Na), 234 (M + 1); FT-
IR (KBr) ⁄ cm 1687 (C-N stretch), 3338–3224 (N-H stretch).
Melatonin molecule was modified in the 5-methoxy and acylamino
groups showed in Scheme 1. These chemically significant modula-
tions of the lead structure were made at two different points: the
methoxy group at the fifth position of the indole ring and replace-
ment of acetyl with amino acids derivatives. Scientific rationales
and perspectives for the synthesis of new MLT analogues can be
summarized as combining indole aldehyde and amino acids which
have antioxidant properties to create synergistic antioxidant activity,
use of bulky amino acid derivatives by making hydrazones with
indole aldehyde to create longer biological half-life than MLT, to
have carbonyl scavenger activity as well as antioxidant activity from
hydrazine in case of the possibility of hydrolysis of the molecules
and to see the possibility of antioxidant activity of the molecules
without having methoxy on the fifth position of the indole ring.
O
II
HN
CH3
III
O
H3C
I
Melatonin
N
H
O
Methods and Materials
HN
Amino acid
General
NH-protection
with amino acid
N
N-Bz-Glycine
Uncorrected melting points were determined using a Bꢀchi SMP-20
apparatus (Bꢀchi Labortechnik, St. Gallen, Zurich, Switzerland). The
1H- and 13C- NMR spectra were measured with a Varian 400 MHz
instrument (Agilent Technologies, Santa Clara, CA, USA) using tetra-
methylsilane (TMS) internal standard and DMSO-d6 as solvent. ESI
Mass spectra were determined on a Waters Micromass ZQ. FT-IR
spectra were recorded on a Jasco 420 Fourier Transform apparatus
(Jasco Corp., Tokyo, Japan). All spectral analysis was performed at
N-Bz-Alanine
N-BOC-Alanine
N-Bz-Serine
N-BOC-Serine
N-Bz-Methionine
N-Bz-Tryptophan
N-Bz-Proline
N
H
N-BOC-Histidine
Figure 1: Modifications made on melatonin molecule.
Chem Biol Drug Des 2012; 79: 76–83
77

Suzen et al.
General procedure for the synthesis of
compounds 4a–i
66.92, 168.29, 173.30 167.36, 172.79; ESI MS m ⁄ z 450 (M + 1), 472
(M+Na, 100%); FT-IR (KBr) ⁄ cm 1641 (C=N, azomethine stretch),
3399 (NH-CO stretch).
N-protected amino acid hydrazines 2a–h (1.2 mmol) and 1H-indole-
3-carboxaldehyde 3 (1 mmol) in EtOH (10 mL) was refluxed until the
starting materials disappeared on TLC plate, on the hot water bath.
On cooling, the precipitate was collected, washed with cold EtOH
and recrystallized from EtOH to give 4a–i with 22–96% yield.
(E)-N'-((1H-indol-3-yl)methylene)-1-benzoylpyrrolidine-2-carbohydrazide
4f. Yield 25.6%, m.p. 105–106 ꢀC; 1H-NMR: d 1.73–2.12 (6H, m,
(CH2)3), 5.45 (1H, m, CH), 7.10–8.24 (10H, m, Ar-H), 8.40 (1H, s,
azomethine-CH), 11.09 and 11.21 (1H, 2s, NH), 11.69 (1H, brs,
indole-NH); 13C-NMR: d 112.08, 112.61, 121.31, 122.21, 123.32,
124.77, 127.94, 129.03, 131.05, 131.99, 134.87, 137.77, 141.53,
144.57 (azomethine-C), 165.44, 167.29, 170.12; ESI MS m ⁄ z 361
(M + 1), 383 (M+Na, 100%); FT-IR (KBr) ⁄ cm 1611 (C=N, azomethine
stretch), 3317 (NH-CO stretch).
(E)-N-(2-(2-((1H-indol-3-yl)methylene)hydrazinyl)-2-oxoethyl)benzamide
4a. Yield 38.3%, m.p. 239–240 ꢀC; 1H-NMR: d 3.98 (1H, d, NH),
4.48 (2H, d, CH2), 7.16–8.71 (10H, m, Ar-H), 8.40 (1H, s, azome-
thine-CH), 11.17 and 11.22 (1H, 2s, NH), 11.57 (1H, brs, indole-NH);
13C-NMR: d 112.08, 112.61, 121.31, 122.21, 123.32, 124.77, 127.94,
129.03, 131.05, 131.99, 134.87, 137.77, 141.53, 144.57 (azomethine-
C), 165.44, 167.29, 170.12; ESI MS m ⁄ z 321 (M + 1), 343 (M+Na,
100%). FT-IR (KBr) ⁄ cm 1611 (C=N, azomethine stretch), 3365 (NH-
CO stretch).
(E)-tert-butyl 1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-1-oxopropan-2-
ylcarbamate 4g. Yield 21.7%, m.p. 176–177 ꢀC; 1H-NMR: d 1.27
(3H, m, CH3), 1.38 (9H, s, (CH3)3), 4.02 (1H, m, NH), 4.90 (1H, m,
CH), 6.95–8.90 (5H, m, Ar-H), 8.36 (1H, s, azomethine-CH), 10.96
and 11.07 (1H, 2s, NH), 11.55 (1H, brs, indole-NH); 13C-NMR: d
17.49, 24.63, 28.93, 47.44, 56.50, 61.82, 709.70, 78.48, 112.08,
120.97, 121.28, 122.57, 123.27, 131.03, 135.99, 137.66, 141.34,
142.83, 145.41 (azomethine-C), 152.96, 155.79, 157.37; ESI MS m ⁄ z
331 (M + 1, 100%), 354 (M + 1 + Na); FT-IR (KBr) ⁄ cm 1658 (C=N,
azomethine stretch), 3396 (NH-CO stretch).
(E)-N-(1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-1-oxopropan-2-yl)ben-
1
zamide 4b. Yield 81.1%, m.p. 139–140 ꢀC; H-NMR: d 1.47 (3H, d,
CH3), 3.44 (1H, m, NH), 4.54 (1H, m, CH), 7.16–8.65 (10H, m, Ar-H),
8.40 (1H, s, azomethine-CH), 11.07 and 11.20 (1H, 2s, NH), 11.56
(1H, s, indole-NH); 13C-NMR: d 17.25, 46.93, 40.07, 56.73, 112.14,
120.99, 121.31, 122.02, 122.59, 123.30, 124.77, 128.17, 131.06,
131.92, 134.85, 137.79, 141.45, 144.73 (azomethine-C), 166.77,
168.93, 173.73; ESI MS m ⁄ z 335 (M + 1), 357 (M+Na, 100%); FT-IR
(KBr) ⁄ cm 1641 (C=N, azomethine stretch), 3249 (NH-CO stretch).
(E)-tert-butyl
1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-3-hydroxy-1-
oxopropan-2-ylcarbamate 4h. Yield 91%, m.p. 118–119 ꢀC; 1H-
NMR: d 1.38 (9H, s, (CH3)3), 3.60 (1H, m, NH), 3.78 (1H, brs, OH),
4.05 (1H, m, CH), 4.81–4.99 (2H, m, CH2), 6.58–8.22 (5H, m, Ar-H),
8.39 (1H, s, azomethine-CH), 11.04 and 11.10 (1H, 2s, NH), 11.56
(1H, brs, indole-NH); 13C-NMR: d 19.21, 28.88, 54.88, 56.64, 62.49,
78.89, 112.23, 112.47, 120.99, 122.14, 122.58, 123.29, 124.71,
130.88, 137.76, 141.53, 144.69 (azomethine-C), 151.88, 166.99,
171.25; ESI MS m ⁄ z 347 (M + 1), 369 (M+Na, 100%); FT-IR (KBr)
⁄ cm 1668 (C=N, azomethine stretch), 3297 (NH-CO stretch).
(E)-N-(1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-3-hydroxy-1-oxopro-
pan-2-yl)benzamide 4c. Yield 60.6%, m.p. 123–126 ꢀC; H-NMR: d
1
3.76 (1H, m, NH), 3.94 (1H, brs, OH), 4.56 (1H, m, CH), 4.99–5.04
(2H, m, CH2) 7.16–8.42 (10H, m, Ar-H), 8.42 (1H, s, azomethine-CH),
11.16 and 11.22 (1H, 2s, NH), 11.57 (1H, brs, indole-NH); 13C-NMR:
d 54.51, 56.19, 56.73, 61.40, 62.26, 112.107, 121.02, 122.26,
123.27, 124.74, 134.71, 137.68, 141.61, 144.90 (azomethine-C),
166.63, 167.02, 170.87; ESI MS m ⁄ z 351 (M + 1), 373 (M+Na,
100%); FT-IR (KBr) ⁄ cm 1642 (C=N, azomethine stretch), 3260 (NH-
CO stretch).
(E)-tert-butyl 1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-3-(1H-imidazol-
4-yl)-1-oxopropan-2-ylcarbamate 4i. Yield 95.9%, m.p. 169–170 ꢀC;
1H-NMR: d 1.36 (9H, s, (CH3)3), 2.82 (2H,m, CH2), 3.06 (1H, dd,
CH), 4.22 (1H, m, NH), 5.65 (1H, dd, NH), 6.83–8.36 (7H, m, Ar-H),
8.36 (1H, s, azomethine-CH), 10.99 and 11.12 (1H, 2s, NH), 11.54
(1H, brs, indole-NH); 13C-NMR: d 14.77, 17.02, 21.44, 28.90,
52.00, 54.36, 78.60, 112.25, 121.14, 122.76, 123.24, 124.96,
130.98, 135.38, 137.72, 141.36, 144.61 (azomethine-C), 156.05,
168.04, 171.81; ESI MS m ⁄ z 397 (M + 1, 100%), 398 (M+Na); FT-
IR (KBr) ⁄ cm 1666 (C=N, azomethine stretch), 3247 (NH-CO
stretch).
(E)-N-(1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-3-(methylthio)-1-oxo-
propan-2-yl)benzamide 4d. Yield 33.5%, m.p. 106–108 ꢀC; 1H-
NMR: d 1.04 (2H, m, S-CH2), 2.00 (3H, s, S-CH3), 2.53–2.74 (2H, m,
CH2), 4.37 (1H, t, CH), 4.60 (1H, m, NH), 7.16–8.68 (10H, m, Ar-H),
8.42 (1H, s, azomethine-CH), 11.12 and 11.27 (1H, 2s, NH), 11.58
(1H, brs, indole-NH); 13C-NMR: d 15.36, 30.74, 50.76, 112.06,
121.011, 122.43, 123.35, 124.73, 128.23, 130.98, 134.69, 137.68,
141.77, 144.99 (azomethine-C), 167.36, 172.79; ESI MS m ⁄ z 395
(M + 1), 417 (M+Na, 100%); FT-IR (KBr) ⁄ cm 1639 (C=N, azomethine
stretch), 3258 (NH-CO stretch).
In vitro antioxidant activities
(E)-N-(1-(2-((1H-indol-3-yl)methylene)hydrazinyl)-3-(indolin-3-yl)-1-oxo-
1
propan-2-yl)benzamide 4e. Yield 55.6%, m.p. 118 ꢀC (decomp); H-
DPPH free radical-scavenging activity
NMR: d 3.29 (2H, m, CH2), 4.36 (1H, m, CH), 4.79 (1H, m, NH),
6.76–8.73 (15H, m, Ar-H), 8.43 (1H, s, azomethine-CH), 10.83 (1H, d,
NH), 11.19 and 11.40 (1H, 2s, NH), 11.58 (1H, brs, indole-NH); 13C-
NMR: d 19.22, 56.74, 111.48, 112.22, 118.93, 121.04, 121.55,
122.44, 123.24, 124.05, 124.75, 128.09, 128.87, 131.00, 131.94,
134.84, 136.76, 137.69, 140.70, 142.05, 145.041 (azomethine-C),
The radical-scavenging assay was determined by the modified
method described previously (45). The stock solutions of the synthe-
sized compounds were prepared at 10)2 M in DMSO. A series of
stock solution in DMSO were diluted to varying concentrations in
96-well microplates. Then, methanolic DPPH solution (100 lM) was
added to each well.
78
Chem Biol Drug Des 2012; 79: 76–83

Antioxidant Properties of Indole Amino Acids
O
O
H
NH2
H
N
CH3
N
N
H
R
R
O
NH2-NH2.H2O/MeOH
2
R1
1
R1
. HCl
R
R1
NH
CHO
HN
N
O
N
H
3
EtOH
4
N
H
CHO
N
O
N
HN
N
H
H
N
O
O
3
. HCl
O
EtOH
NH2-NH2.H2O
MeOH
O
CH3
N
O
CH3
4f
N
H
O
Compound 1a-f
Compound 2a-h and 4a-i
No
1a
1b
1c
1d
R
R1
No
R
R1
2a, 4a
2b, 4b
2c, 4c
2d, 4d
H
H
H
H
-H
-Bz
-Bz
-Bz
-Bz
-H
-CH3
-CH2OH
-CH3
-CH2OH
-(CH2)2-S-CH3
-(CH2)2-S-CH3
CH2
1e
H
2e, 4e
-Bz
CH2
N
H
N
H
1f, 2f, 4f
Synthesis is given
2g, 4g
2h, 4h
4i
-BOC
-BOC
-BOC
-CH3
-CH2OH
N
CH2
N
Scheme 1: Synthetic pathway of indol-aminoacid derivatives.
The plate was shaken to ensure thorough mixing before being
wrapped with aluminium foil and placed into the dark. After 30 min,
the optical density of the solution was read at the wavelength
517 nm. The methanolic solution of DPPH served as a control. Per-
centage inhibition was calculated using the following formula:
compounds. Each experiment was performed in triplicate. Melatonin
and BHT were used as reference compounds (Table 1).
Assay of lipid peroxidation
The effect of the synthesized indol-amino acids on rat liver homoge-
nate induced with FeCl3-ascorbic acid, and LP was determined by the
method of modified Mihara et al. (46). Wistar rats (200–225 g) were
fed with standard laboratory rat chow and tap water. The animals
were starved for 24 h prior to sacrifice and then killed by decapitation
% Inhibition = [(Acontrol ) Asample) ⁄ Acontrol] · 100
where Acontrol is the absorbance of the control with DMSO and
Asample is the absorbance of the sample in the presence of the
Chem Biol Drug Des 2012; 79: 76–83
79

Suzen et al.
Table 1: DPPH radical-scavenging and LP effect of synthesized
(version 11.0 for Windows 98; SPSS Inc., Chicago, IL, USA).
One-way analysis of variance was performed by ANOVA procedures.
Significant differences between means were determined by
Duncan's multiple range tests. Values of p, 0.05 were regarded as
statistically significant.
compoundsa,b
No
DPPH assay
LP (10)3 M)
(10)3 M) inhibition, %
inhibition, %
1b
1c
10 € 1.2
13 € 1.0
2 € 1.2
NE
NE
NE
NE
1d
1e
Results
12 € 1.6
13 € 1.4
9.0 € 0.8
17 € 0.4
17 € 0.8
17 € 0.8
26 € 0.8
45 € 2.1
8.0 € 1.2
8 € 1.0
12 € 0.6
77 € 1.1
82 € 0.8
19 € 2.8
–
1f
4a
NE
N-protected amino acids and MLT-based analogue indole amino
acids derivatives were synthesized and tested for their antioxidant
activities using DPPH radical-scavenging and LP inhibitory activity
tests. All the results were compared with standard antioxidants
BHT, vitamin E and MLT. The results are shown in Table 1.
33 € 2.2
25 € 1.7
17 € 2.2
30 € 2.2
42 € 3.5
34 € 2.0
42 € 2.1
26 € 3.4
25 € 1.1
–
4b
4c
4d
4e
4f
4g
In DPPH radical-scavenging test, compound 4f showed better activ-
ity (approximately two times higher) than MLT at 10)3 M concentra-
tion (45%). Compound 4e also showed higher activity than MLT at
10)3 M concentration (26%). Compounds 4b–d possessed similar
scavenging activity against the DPPH radical like MLT. Percentages
of inhibition of rest of the tested compounds except 1d and 4h
were found similar to MLT. None of the compounds were found
more potent than BHT.
4h
4i
BHT 10)3
BHT 10)4
Melatonin
Vitamin E
M
M
–
21 € 2.1
75 € 2.2
aThe values represent the average of 2–4 determinations €SD.
bCompounds were diluted with DMSO (solvent showed no antioxidant activ-
ity).
In LP inhibition test, except non-active compounds 1b–f and com-
pound 4c, all the tested compounds showed very strong antioxidant
activity compared with MLT. The most potent compounds were 4e
and 4g at 10)3 M concentration with same inhibition value (42%).
The second potent compound was 4f (34%). This was followed by
compounds 4a, 4d, 4h, 4b and 4i with 33%, 30%, 26%, 25%
and 25%, respectively. The results of two assays were found simi-
lar to compare the most potent activities. None of the compounds
were found more potent than vitamin E.
NE, no effect; DPPH, 2,2-diphenyl-1-picrylhydrazyl; LP, lipid peroxidation.
under anaesthesia. The study was carried out in accordance with the
Guide for the Care and Use of Laboratory Animals. The livers were
removed immediately and washed in ice-cold distilled water and
homogenized straight away with teflon homogenizer in ice chilled.
Lipid peroxidation was measured spectrophotometrically by estima-
tion of thiobarbituric acid reactive substances (TBARS). Amounts of
TBARS were expressed in terms of mmol malondialdehyde ⁄ g tissue.
A typical optimized assay mixture contained 0.5 mL of liver homo-
genate, 0.1 mL of Tris–HCl buffer (pH 7.2), 0.05 mL of 0.1 mM ascor-
bic acid, 0.05 mL of 4 mM FeCl3 and 0.05 mL of various concentration
of synthesized compounds, BHT ⁄ a-tocopherol or MLT, were incubated
for 1 h at 37 ꢀC. After incubation, 3.0 mL of H3PO4 and 1 mL of 0.6%
TBA were added and shaken vigorously. The mixture was boiled for
30 min. After cooling, n-butanol was added, and the mixture was sha-
ken vigorously. The n-butanol phase was separated by centrifugation
at 1006 g for 10 min. The absorbance of the supernatant was read at
532 nm against a blank, which contained all reagents except liver
homogenate. Melatonin and vitamin E were used as positive control.
Lipid peroxidation inhibitory activity (%) is expressed as follows:
Discussion
The present work was designed to synthesize, characterize and
investigate the potential antioxidant effects of indole-based MLT
analogue hydrazide ⁄ hydrazone derivatives.
This study offers a new approach to structure-antioxidant activity
relationship of five substituted indole rings.
It is likely that the synthesized indole derivatives may perhaps expe-
rience some hydrolysis under the in vivo conditions. On the other
hand, in our previous studies in vitro electrochemical data showed
that oxidation starts on the nitrogen atom in the indole ring, which
leads finally to the hydroxylation of the benzene ring (47–50). Hy-
drazones can be chemically hydrolysed to relevant hydrazines and
carbonyl compounds under acidic conditions. However, the biological
system metabolizes hydrazones via the more complex NAD+-depen-
dent oxidation reaction (51). This is a remarkable difference
between the chemical and biological systems for degrading hydraz-
ones. It is clear that synthesized compounds have free radical-scav-
enging activity, and they might have carbonyl scavenging activity if
they metabolize into hydrazine and aldehyde. This gives synthesized
compounds advantage over MLT.
Lipid peroxidation inhibitory activity (%): (Acontrol ) Asample) ⁄
(Acontrol ) Ablank) · 100
where Acontrol is the absorbance of the control, Asample is the absor-
bance of the sample and Ablank is the absorbance of the blank, to
which the sample and the free radical-generating system
(Fe+2 ⁄ ascorbate) were not added (Table 1).
Statistical analysis
All data are the average of duplicate analyses. The data were
recorded as mean € standard deviation and analysed by SPSS
80
Chem Biol Drug Des 2012; 79: 76–83

Antioxidant Properties of Indole Amino Acids
In general, all the synthesized indole derivatives 4a–i were found
to have reasonable antioxidant activity according to the results of
LP inhibition assay against MLT. Interestingly, no significant antioxi-
dant activity was observed in compounds 1b–f. These compounds
are the N-protected amino acid esters, and they have no indole ring
in their structure. Structure activity investigation of the rest of the
active compounds showed that presence of indole ring in the mole-
cule increases the antioxidant activity (except 4c). This finding is in
the same direction of the literature. It is noteworthy that the most
potent antioxidant and free radical scavenger amino acids are
attached to a known molecule such as trolox (24), fullerene (30) or
Pyridoxal (28).
activity tests. This result is very similar to our earlier findings
(13,14) and the data in literature (11) and confirmed that for antiox-
idant activity, not only the indole-type aromatic ring is important,
but so is the side chain especially at the third position to have bet-
ter antioxidant activity than MLT.
The hydroxyl radical is considered to be the most reactive, harmful
of all free radicals (56). The high LP inhibition results of the new
indole amino acid derivatives in particular, comprises of the reactiv-
ity towards OH radical. Our results have shown that the combina-
tion of indole and amino acid groups is, as we expected, decisive
for melatonin analogue compound's antioxidant activity.
Compounds 4a–i (except 4c) are the most promising compounds
that should be kept in mind for designing new MLT-based indole
derivatives for our ongoing study. These results suggest a new
approach for the in vitro antioxidant activity properties and struc-
ture activity relationships of 3-substituted indole rings. Lack of a
methoxy group in the fifth position did not affect the antioxidant
capacity of the new indole derivatives. In fact, the in vitro assays
showed that a lack of a methoxy group in the indole ring and an
amino acid side chain resulted in much more active compounds
than MLT itself. This may be due to increased stability of the indole
ring and delocalization of the electrons to help to scavenge free
radicals by forming stable indolyl cation radicals. The real function
of methoxy group in fifth position of the indole ring of MLT is still
not clear. Despite the removal of the methoxy group, the antioxi-
dant capacity of the molecule may be enhanced (15); interestingly,
all the synthesized indole derivatives (H in the fifth position of the
indole ring) showed more potent antioxidant activity in LP inhibition
assay.
References
1. Maiese K., Morhan S.D., Chong Z.Z. (2007) Oxidative stress biol-
ogy and cell injury during type 1 and type 2 diabetes mellitus.
Curr Neurovasc Res;4:63–71.
2. Rao V.K., Chhikara B.S., Shirazi A.N., Tiwari R., Parang K., Kumar
A. (2011) 3-Substituted indoles: one-pot synthesis and evaluation
of anticancer and Src kinase inhibitory activities. Bioorg Med
Chem Lett;21:3511–3514.
3. Suzen S., Buyukbingol E. (2000) Anti-cancer activity studies of
indolalthiohydantoin (PIT) on certain cancer cell lines. Farma-
co;55:246–248.
4. Kumar D., Narayanam M.K., Chang K.H., Shah K. (2011) Synthe-
sis of novel indolyl-1,2,4-triazoles as potent and selective anti-
cancer agents. Chem Biol Drug Des;77:182–188.
5. Kadoma Y., Fujisawa S. (2011) Radical-scavenging activity of
melatonin, either alone or in combination with vitamin E, ascor-
bate or 2-mercaptoethanol as co-antioxidants, using the induc-
tion period method. In Vivo;25:49–53.
In our previous studies, lack of methoxy group and introduction of
Cl (52) or Br (33) in the fifth position did not make a dramatic
change on the antioxidant capacity of the new indole derivatives
infect the in vitro assays showed that many of the compounds were
much more active than MLT itself. Literature data also suggest that
melatonin shows antioxidant activity without methoxy group.
Regarding the structure-antioxidant activity relationship, the 5-meth-
oxy group of melatonin do not seem to significantly affect capacity
of radical trapping. Related indoles being very similar to that
obtained for melatonin (53).
6. Lai Y., Ma L., Huang W., Yu X., Zhang Y., Ji H., Tian J. (2010)
Synthesis and biological evaluation of 3-[4-(amino ⁄ methylsulfo-
nyl)phenyl]methylene-indolin-2-one derivatives as novel COX-1 ⁄ 2
and 5-LOX inhibitors. Bioorg Med Chem Lett;20:7349–7353.
7. Mokale V.J., Raut M.K., Burange P.J., Dhoot N.S., Bhandari S.V.,
Bothara K.G., Naik J.B. (2010) Synthesis and pharmacological
evaluation of novel 1-(2-(benzoyl-substituted-2-phenyl-1H-indol-5-
carbony) hydrazinyloxy) vinyl nitrate derivatives as potent non-
ulcerogenic, analgesic and anti-inflammatory agents. Med
Chem;6:211–218.
8. Suzen S., Buyukbingol E. (1998) Evaluation of anti-HIV activity of
5-(2-phenyl-3¢- Indolyl)-2-thiohydantoin. Farmaco;53:525–527.
9. Guzel O., Maresca A., Hall R.A., Scozzafava A., Mastrolorenzo
A., Muhlschlegel F.A., Supuran C.T. (2010) Carbonic anhydrase
inhibitors. The beta-carbonic anhydrases from the fungal patho-
gens Cryptococcus neoformans and Candida albicans are
strongly inhibited by substituted-phenyl-1H-indole-5-sulfona-
mides. Bioorg Med Chem Lett;20:2508–2511.
Conclusion
Several experimental reports show that high concentrations of mel-
atonin are needed to effectively scavenge free radicals, suggesting
that these are unlikely mechanisms of action of physiological levels
of melatonin in vivo (54,55). More research is needed to develop
MLT analogues to find out improved antioxidant activity.
10. Guzel O., Innocenti A., Vullo D., Scozzafava A., Supuran C.T.
(2010) 3-phenyl-1H-indole-5-sulfonamides: structure-based drug
design of a promising class of carbonic anhydrase inhibitors.
Curr Pharm Des;16:3317–3326.
11. Tan D.X., Chen L.D., Poeggeler B., Manchester L.C., Reiter R.J.
(1993) Melatonin: a potent endogenous hydroxyl radical scaven-
ger. Endocr J;1:57–60.
The results indicated significant strong antioxidant activity for most
of the compounds, when compared to melatonin. Lack of a methoxy
group in the fifth position did not affect the antioxidant capacity of
the new indole derivatives. Structural investigation of the active
compounds showed that having tryptophan (4e) and proline (4g) as
side chains increase the antioxidant activity in two antioxidant
Chem Biol Drug Des 2012; 79: 76–83
81

Suzen et al.
12. Sreejith P., Beyo R.S., Divya L., Vijayasree A.S., Manju M., Oom-
men O.V. (2007) Triiodothyronine and melatonin influence antioxi-
dant defense mechanism in a teleost Anabas testudineus
(Bloch): in vitro study. Indian J Biochem Biophys;44:164–168.
13. Suzen S. (2006) Recent developments of melatonin related anti-
oxidant compounds. Comb Chem High Throughput Screen;9:409–
419.
14. Suzen S. (2007) Topics in heterocyclic chemistry, bioactive het-
erocycles V. In: Khan M.T.H., editor. Antioxidant Activities of
Synthetic Indole Derivatives and Possible Activity Mechanisms,
Vol 11. Berlin, Heidelberg: Spinger-Verlag; p. 145–178.
15. Tan D.X., Reiter R.J., Manchester L.C., Yan M.T., El-Sawi M.,
Sainz R.M., Mayo J.C., Kohen R., Allegra M., Hardeland R.
(2002) Chemical and physical properties and potential mecha-
nisms: melatonin as a broad spectrum antioxidant and free radi-
cal scavenger. Curr Top Med Chem;2:181–197.
27. Christen S., Peterhans E., Stocker R. (1990) Antioxidant activities
of some tryptophan metabolites: possible implication for inflam-
matory diseases. Proc Natl Acad Sci USA;87:2506–2510.
28. Kitazawa M., Ishitsuka Y., Kobayashi M., Nakano T., Iwasaki K.,
Sakamoto K., Arakane K., Suzuki T., Kligman L.H. (2005) Protec-
tive effects of an antioxidant derived from serine and vitamin b6
on skin photoaging in hairless mice. Photochem Photobiol
;81:970–974.
29. Grosser N., Oberle S., Berndt G., Erdmann K., Hemmerle A.,
Schrçder H. (2004) Antioxidant action of L-alanine: heme oxygen-
ase-1 and ferritin as possible mediators. Biochem Biophys Res
Commun;314:351–355.
30. Yang J., Alemany L.B., Driver J., Hartgerink J.D., Barron A.R.
(2007) Fullerene-derivatized amino acids: synthesis, characteriza-
tion, antioxidant properties, and solid-phase peptide synthesis.
Chem Eur J;13:2530–2545.
16. Cano A., Alcaraz O., Arnao M.B. (2003) Free radical-scavenging
activity of indolic compounds in aqueous and ethanolic media.
Anal Bioanal Chem;376:33–37.
31. Stadtman E.R., Levine R.L. (2003) Free radical-mediated oxidation
of free amino acids and amino acid residues in proteins. Amino
Acids;25:207–218.
17. Andreadou I., Tsantili-Kakoulidou A., Spyropoulou E., Siatra T.
(2003) Reactions of indole derivatives with cardioprotective
activity with reactive oxygen species. Comparison with melato-
nin. Chem Pharm Bull;51:1128–1131.
32. Ferreira P.M.T., Monteiro L.S., Coban T., Suzen S. (2009) Compar-
ative effect of N-substituted dehydroamino acids and a-tocoph-
erol on rat liver lipid peroxidation activities. J Enzym Inh Med
Chem;24:967–971.
18. Misik V., Ondrias K., Stasko A. (1999) EPR spectroscopy of free
radical intermediates of antiarrhythmic-antihypoxic drug stoba-
dine, a pyridoindole derivative. Life Sci;65:1879–1881.
19. Suzen S., Bozkaya P., Coban T., Nebioglu D. (2006) Investigation
of the in vitro antioxidant behaviour of some 2-phenylindole
derivatives: iscussion on possible antioxidant mechanisms and
comparison with melatonin. J Enzyme Inhib Med Chem;21:405–
411.
20. Gurkok G., Coban T., Suzen T. (2009) Melatonin analogue new
indole hydrazide ⁄ hydrazone derivatives with antioxidant behav-
ior: synthesis and structure-activity relationships. J Enzyme Inhib
Med Chem;24:506–515.
21. Shirinzadeh H., Eren B., Gurer-Orhan H., Suzen S., Ozden S.
(2010) Novel indole-based analogs of melatonin: synthesis and
in vitro antioxidant activity studies. Molecules;15:2187–2202.
22. Ates-Alagoz Z., Coban T., Suzen S. (2005) A comparative study:
evaluation of antioxidant activity of melatonin and some indole
derivatives. Med Chem Res;14:169–179.
23. Das-Evcimen N., Yıldırım O., Suzen S. (2009) Relationship
between aldose reductase and superoxide dismutase inhibition
capacities of indole-based analogs of melatonin derivatives.
Arch Biol Sci;61:675–681.
24. Taylor M.J., Richardson T., Jasensky R.D. (1981) Antioxidant
activity of amino acids bound to Trolox-C. J Am Oil Chemists'
Soc;58:622–626.
25. Elias R.J., McClements D.J., Decker E.A. (2005) Antioxidant
activity of cysteine, tryptophan, and methionine residues in con-
tinuous phase b-lactoglobulin in oil-in-water emulsions. J Agric
Food Chem;53:10248–10253.
26. Reiter R.J., Tan D.X., Cabrera J., D'Arpa D. (1999) Advance in
experimental medicine and biology. In: Huether G., Kochen W.,
Simat T.J., Steinhart H., editors. Tryptophan, Serotonin and MLT,
Basic Aspects and Applications. MLT and Tryptophan Derivatives
as Free Radical Scavengers and Antioxidants, Vol 467. New
York: Springer; p. 379–387.
33. Suzen S., Gurkok G., Coban T. (2006) Novel N-acyl dehydroala-
nine derivatives as antioxidants: studies on rat liver lipid peroxi-
dation levels and DPPH free radical scavenging activity. J
Enzyme Inh Med Chem;21:179–185.
34. Depreux P., Lesieur D., Mansour H.A., Morgan P., Howell H.E.,
Renard P., Caignard D.H., Pfeiffer B., Delagrange P., Guardiola
B., Yous S., Demarque A., Adam G., Andrieux J.J. (1994) Synthe-
sis and structure-activity relationships of novel naphthalenic and
bioisosteric related amidic derivatives as melatonin receptor
ligands. Med Chem;37:3231–3239.
35. Mor M., Silva C., Vacondio F., Plazzi P.V., Bertoni S., Spadoni
G., Diamantini G., Bedini A., Tarzia G., Zusso M., Franceschini
D., Giusti P. (2004) Indole-based analogs of melatonin: in vitro
antioxidant and cytoprotective activities. J Pineal Res;36:95–
102.
36. Rajitha G., Saideepa N., Praneetha P. (2011) Synthesis and eval-
uation of N-(a-benzamidocinnamoyl) aryl hydrazone derivatives
for anti-inflammatory and antioxidant activities. Indian
Chem;50B:729–733.
J
37. Prathapa J., Himajaa M., Malib S.V., Ranjithaa A., Karigarc A.,
Sikarwar M. (2010) Synthesis and antioxidant activity of novel
ketone hydrazones bearing 5-(Pyridine-3-yl)-1,3,4-thiadiazole. J
Pharm Res;3:2460–2462.
38. Kumar P.P., Rani B.L. (2011) Synthesis and characterization of
new Schiff bases containing pyridine moiety and their deriva-
tives as antioxidant agents. Int J ChemTech Res;3:155–160.
39. Thangadurai T.D., Gowri M., Natarajan K. (2002) Synthesis and
characterization of ruthenium(III)complexes containing monobasic
bidentate Schiff bases and their biological acivities. Synth React
Inorg Met-Org Chem;32:329–343.
40. Hruskova K., Kovarikova P., Bendova P., Haskova P., Mackova E.,
Stariat J., Vavrova A., Vavrova K., Simunek T. (2011) Synthesis
and initial in vitro evaluations of novel antioxidant aroylhydraz-
one iron chelators with increased stability against plasma hydro-
lysis. Chem Res Toxicol;24:290–302.
82
Chem Biol Drug Des 2012; 79: 76–83

Antioxidant Properties of Indole Amino Acids
41. Belkheiri N., Bouguerne B., Bedos-Belval F., Duran H., Bernis C.,
Salvayre R., Nꢁgre-Salvayre A., Balta M. (2010) Synthesis and
antioxidant activity evaluation of a syringic hydrazones family.
Eur J Med Chem;45:3019–3026.
42. Negre-Salvayre A., Coatrieux C., Ingueneau C., Salvayre R.
(2008) Advanced lipid peroxidation end products in oxidative
damage to proteins. Potential role in diseases and therapeutic
prospects for the inhibitors. Br J Pharmacol;153:6–20.
43. Galvani S., Coatrieux C., Elbaz M., Grazide M.H., Thiers J.C., Pa-
rini A., Uchida K., Kamar N., Rostaing L., Baltas M., Salvayre R.,
Nꢁgre-Salvayre A. (2008) Carbonyl scavenger and antiatherogen-
ic effects of hydrazine derivatives. Free Radic Biol
Med;45:1457–1467.
49. Shirinzadeh H., Yilmaz A.D., Gumustas M., Suzen S., Ozden S.,
Ozkan S.A. (2010) Electrochemical behavior of indole-3-carbox-
aldehyde izonicotinoyl hydrazones: discussion on possible bio-
logical behavior. Comb Chem High Throughput Screen;13:619–
627.
50. Bozkaya P., Dogan B., Suzen S., Nebioglu D., Ozkan S.A. (2006)
Determination and investigation of electrochemical behaviour of
2-phenylindolederivatives: discussion on possible mechanistic
pathways. Can J Anal Sci Spec;51:125–139.
51. Itoh H., Suzuta T., Hoshino T., Takaya N. (2008) Novel dehy-
drogenase catalyzes oxidative hydrolysis of carbon-nitrogen
double bonds for hydrazone degradation. J Biol Chem;283:
5790–5800.
44. Kidwai M., Negi N., Gupta S.D. (1994) Synthesis and antifertility
activity of 1,5-diaryl-3-(30-indolyl)formazans. Chem Pharm Bull
(Tokyo);42:2363–2364.
45. Blois M.S. (1958) Antioxidant determinations by the use of a
free radical. Nature;181:1199–1200.
46. Mihara M., Uchiyama M., Fukuzawa K. (1980) Thiobarbituric
acid value on fresh homogenate of rat as a parameter of lipid
peroxidation in aging, CCl4 intoxication, and vitamin E deficiency.
Biochem Med;23:302–311.
47. Suzen S., Demircigil T., Buyukbingol E., Ozkan S.A. (2003) Elec-
troanalitical evaluation and determination of 5-(3¢-indolyl)-2-thi-
ohydantoin derivatives by voltammetric studies: possible
relevance to in vitro metabolism. New J Chem;27:1007–1011.
52. Yılmaz A.D., Coban T., Suzen S. (2011) Synthesis and antioxidant
activity evaluations of melatonine based analogue indole-hydra-
zide ⁄ hydrazone derivatives. J Enzyme Inhib Med Chem; [Epub
ahead of print].
53. Turjanski A.G., Rosenstein R.E., Estrin D.A. (1998) Reactions of
melatonin and related indoles with free radicals: a computa-
tional study. J Med Chem;41:3684–3689.
54. Matuszak Z., Reszka K.J., Chignell C.F. (1997) Reaction of mela-
tonin and related indoles with hydroxyl radicals: EPR and spin
trapping investigations. Free Radic Biol Med;3:367–372.
55. Marshall K.A., Reiter R.J., Poeggeler B., Aruoma O.I., Halliwell
B. (1996) Evaluation of the antioxidant activity of melatonin in
vitro. Free Radic Biol Med;3:307–315.
48. Suzen S., Ates
¸
-Alagoz Z., Demircigil T., Ozkan S.A. (2001) Syn-
56. Poeggeler B., Reiter R.J., Tan D.X., Chen L.D., Manchester L.C.
(1993) Melatonin, hydroxyl radical-mediated oxidative damage
and aging: a hypothesis. J Pineal Res;14:151–168.
thesis and analytical evaluation by voltammetric studies of some
new indole-3-propionic acid derivatives. Farmaco;56:835–840.
Chem Biol Drug Des 2012; 79: 76–83
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