Antioxidant activity of indole-based melatonin analogues in erythrocytes and their voltammetric characterization

Article abstract of DOI:10.3109/14756366.2012.717223

Melatonin (MLT) is a strong free-radical scavenger, which protects the body from the effects of oxidants. In recent years, MLT have been described resulting in much attention in the development of synthetic compounds possessing. As a part of our ongoing study a series of indole-based MLT analogue hydrazide/hydrazone derivatives were synthesized, characterized and in vitro antioxidant activity was investigated by evaluating their reducing effect against oxidation of a redox sensitive fluorescent probe. Membrane stabilizing effect of all compounds was also investigated by lactate dehydrogenase leakage assay. Furthermore voltammetric methods have been applied to the synthesized compounds to characterize oxidation potentials to get insight into their metabolism owing to the oxidation mechanisms taking place at the electrode and in the body share similar principles.

Full text of DOI:10.3109/14756366.2012.717223

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; Early Online: 1–13
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.717223
ReseaRch aRtIcle
Antioxidant activity of indole-based melatonin analogues in
erythrocytes and their voltammetric characterization
Sibel Suzen1, Betul Tekiner-Gulbas1, Hanif Shirinzadeh1, Duysal Uslu2, Hande Gurer-Orhan2,
Mehmet Gumustas3, and Sibel Aysil Ozkan3
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara, Turkey,
2Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Ege University, Izmir, Turkey, and
3Department of Analytical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara, Turkey
abstract
Melatonin (MLT) is a strong free-radical scavenger, which protects the body from the effects of oxidants. In recent years,
MLT have been described resulting in much attention in the development of synthetic compounds possessing. As a
part of our ongoing study a series of indole-based MLT analogue hydrazide/hydrazone derivatives were synthesized,
characterized and in vitro antioxidant activity was investigated by evaluating their reducing effect against oxidation
of a redox sensitive fluorescent probe. Membrane stabilizing effect of all compounds was also investigated by lactate
dehydrogenase leakage assay. Furthermore voltammetric methods have been applied to the synthesized compounds
to characterize oxidation potentials to get insight into their metabolism owing to the oxidation mechanisms taking
place at the electrode and in the body share similar principles.
Keywords: indole, hydrazone/hydrazide, melatonin, synthesis, antioxidant activity, voltammetry
Introduction
rhythm and immune functions6. In recent years, many
physiological properties of MLT have been described
resulting in much attention in the development of syn-
thetic compounds possessing the indole ring. Synthetic
indole derivatives such as indole-3-propionic acid7,
indole amine-triazoles8, and stobadine9 have shown
significant antioxidant activity. Our group identified the
antioxidant activity of MLT analogue indole derivatives
such as 2-phenylindoles10, indole-3-propionamides11,
5-bromoindole hydrazide/hydrazones derivatives12, and
N-methylindole hydrazide/hydrazones13. In these stud-
ies most of the compounds showed significant antioxi-
dant activity at concentrations comparable with or much
higher than that of MLT.
Oxidants and antioxidants have a significant function
in maintaining a balance between free radicals and the
antioxidant system of the body1. Reactive oxygen species
(ROS) and reactive nitrogen species are well known as
both harmful and beneficial species. Overproduction of
ROS results in oxidative stress that can cause fatal dam-
age to cell structures, including lipids and membranes,
proteins, and DNA2. It is known that antioxidants could
be suitable for preventive in several diseases related with
oxidative stress such as atherosclerosis, inflammatory
injury, cancer, cardiovascular diseases, and neurodegen-
erative disorders3,4
.
It is experimentally and clinically well established
that melatonin (MLT) and its metabolites can function
as endogenous free-radical scavengers and broad-spec-
trum antioxidants5. Studies also showed the role of MLT
and its derivatives in many physiological processes and
therapeutic functions, such as the regulation of circadian
Hydrazone derivatives possess
a
range of
pharmacological activities including antitumoral, anti-
microbial, anti-malarial, anti-convulsant, and anti-
inflammatory activity13. Recently hydrazones were found
as potential antioxidants14–17. It was shown that oxidative
Address for Correspondence: Prof. Sibel Suzen, Department of Pharmaceutical Chemistry, Ankara University, Faculty of Pharmacy, 06100
Tandogan, Ankara, Turkey,. Tel: +90 312 2033074. Fax: +90 312 2131081. E-mail: sibel@pharmacy.ankara.edu.tr
(Received 28 July 2012; accepted 28 July 2012)
1

2
S. Suzen et al.
stress induces lipid peroxidation of cellular membranes
resulting in the generation of reactive carbonyl
was investigated in vitro by evaluating their reducing
effect against oxidation of a redox sensitive fluorescent
probe, 2′,7′-dichlorofluorescin (DCFH)-diacetate (DA),
in the presence or absence of H2O2. e results were com-
pared with a well-known antioxidant ascorbic acid and
MLT, as their parent compound. Membrane stabilizing
effect of all compounds was also investigated via lactate
dehydrogenase (LDH) leakage assay in Chinese Hamster
Ovary (CHO) cells. All the analogue compounds were
compounds that involves in the “carbonyl stress”18,19
.
Hydrazine derivatives also exhibit carbonyl scavenger
activity by the reaction of ketones/aldehydes20.
e use of electrochemical techniques for the determina-
tion of compounds of pharmaceutical interest is continually
gaining in importance21. e obtained results from the redox
properties of drugs and biomolecules might have profound
effects on our understanding of their in vivo redox behavior
or pharmaceutical activity22–24. Voltammetric techniques
are most suitable for investigating the redox properties of
new drug candidates to obtain information for its metabolic
fate25. Electrode mechanisms might mimic enzyme reac-
tions; therefore, electrochemistry may be of value in the
study of enzyme reactions in biological systems24. Due to
the existence similarity between electrochemical and bio-
logical reactions it can be assumed that the oxidation mech-
anisms taking place at the electrode and in the body share
similar principles. We have previously reported the electro-
analytical evaluation and determination of indole-3-propi-
onic acid derivatives26, indolylthiohydantoin derivatives27,
2-phenylindole derivatives28, and indole-3-carboxaldehyde
izonicotinoyl hydrazones29 by voltammetric studies in order
to enlighten the possible relevance to in vitro metabolism of
these compounds.
1
characterized on the basis of H- and 13C-NMR, mass,
Fourier transform infrared spectroscopy (FT-IR) spec-
tra and elemental analysis. Furthermore voltammetric
studies were established for the electrochemical oxida-
tion and determination of synthesized MLT analogues
using a glassy carbon electrode and investigated the pos-
sible oxido-reduction mechanisms that gave ideas about
in vitro metabolism of these compounds.
Material and methods
With this study based on MLT, series of new indole imines
were developed. ree parts of the MLT molecule were
modified in order to find out the antioxidant behavior and
structure activity relationship of the new indole analogue
compounds. emainmodificationsofMLTmoleculewere
shown in Figure 1. e target imines derived from 1-methy-
lindole-2-carboxaldehyde and appropriate hydrazine or
hydrazide derivatives using simple reaction strategies. For
the synthesis of compounds 1a-p a similar methodology
has been adopted from Kidwai et al. 30. Phenyl hydrazine
derivatives and 1-methylindole-2-carboxaldehyde were
heated in the presence of ethanol. e hydrazones 1t, 1u
were prepared from the reaction of equimolar amounts of
hydrazide with 1-methylindole-2-carboxaldehyde in the
In new drug development studies, combination of dif-
ferent pharmacophores in the same structure may lead to
new compounds having higher biological activity. It was
thought the combination of indole-and hydrazone-type
compounds might provide new effective drugs against
free radicals. As a part of our ongoing study 21 new
indole-based MLT analogue hydrazide/hydrazone deriv-
atives were synthesized and their antioxidant activity
Figure 1. Parts of the MLT molecule modified to develop new indole-based MLT analogue compounds. MLT, melatonin.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antioxidant activity of indole-based melatonin analogues
3
presence of ethanol. Finally compound 1v was synthe-
sized using equimolar amounts of hydrazine hydrate with
1-methylindole-2-carboxaldehyde and compound 1y was
synthesized using one equimolar of hydrazine hydrate
with two equimolars 1-methylindole-2-carboxaldehyde in
the presence of ethanol (Figure 2). All the compounds were
characterized on the basis of spectral data.
mmol) in 10 ml of ethanol in the presence of 0.5g
CH3COONa for 30 min on the hot water bath. On cooling,
the precipitate was collected washed with cold ethanol to
give 1a-p with 22–91% yield.
1-Methylindole-2-carboxaldehyde
phenylhydrazone
1
(1a) Yield 41.1%; m.p. 185–186°C; H-NMR: 4.06(3H, s),
6.70(1H, s), 6.76(1H, m), 7.04(3H, m), 7.21(3H, m), 7.50(2H,
dd), 8.03(1H, s, azomethine CH), 1038(1H, s, hydrazine
NH); 13C-NMR: 32.55, 104.41, 110.31, 112.56, 119.50, 120.32,
120.94, 122.87, 127.91, 129.93, 131.03, 135.79, 139.58, 145.82
(azomethine CH); ESI MS m/z 250 (M + 1, %100), 251 (M
+ 2); Anal. Calcd. For C16H15N3: C, 77.07%; H, 6.07%; N,
16.86%. Found: C, 76.11%; H, 6.35%; N, 16.28%. FT-IR (KBr)
cm−1 1603 (C=N azomethine stretch), 3302 (N–H stretch).
1-Methylindole-2-carboxaldehyde (2- fluorophenyl)
e chemical reagents used in synthesis were purchased
from Sigma (Taufkirchen, Germany) and Aldrich (St. Louis,
MO, USA). Uncorrected melting points were determined
1
with a Buchi SMP-20 apparatus. e H- and 13C-NMR
spectra were measured with a Varian 400MHz using TMS
internal standard and DMSO-d6 as solvent. ESI Mass spec-
tra were determined on a Waters micromass ZQ.FT-IR
spectra were recorded on Jasco 420Fourier. Elemental anal-
yses were performed using CHNS-932 (LECO). All spectral
analysis was performed at Ankara University, Faculty of
Pharmacy, Central Laboratory. Voltammetric measure-
ments were performed using a Bioanalytical Systems (BAS
100W, USA) electrochemical analyzer.
1
hydrazone (1b) Yield 54.3%; m.p. 157–158°C; H-NMR:
4.06(3H,s), 6.728(1H, s), 6.78(1H, m), 7.12(4H, m),
7.49(3H, m), 8.32(1H, s, azomethine), 10.29(1H, s, hydra-
zine); 13C-NMR: 32.53, 104.94, 110.39, 114.33, 115.66,
115.84, 119.32, 120.36, 121.09, 123.09, 125.80, 127.82,
133.88, 134.04, 135.47, 139.63, 148.61 (azomethine C),
150.98; ESI MS m/z 268 (M + 1, %100), 269 (M + 2); Anal.
Calcd. For C16H14N3F: C, 71.88%; H, 5.28%; N, 15.73%.
Found: C, 71.22%; H, 5.60%; N, 15.29%. FT-IR (KBr) cm−1
1623 (C=N azomethine stretch), 3320 (N–H stretch).
General procedure for the synthesis
of compounds 1a-p.
1-Methylindole-2-carboxaldehyde (0.1 mmol) was
reacted with phenyl hydrazine or its derivatives (0.13
Figure 2. Synthetic pathways of new indole-based MLT analogue compounds. MLT, melatonin.
© 2012 Informa UK, Ltd.

4
S. Suzen et al.
1-Methylindole-2-carboxaldehyde (3- fluorophenyl)
1-Methylindole-2-carboxaldehyde
(3-chlorophenyl)
1
hydrazone (1c) Yield 48.9%; m.p. 153–154°C; H-NMR:
4.07(3H, s), 6.79(3H, m), 7.05(1H, t), 7.23(2H, m), 7.51(2H,
dd), 8.05(1H, s, azomethine CH), 10.61(1H, s, hydrazine
NH); 13C-NMR: 31.75, 98.14, 98.41, 104.28, 104.65, 104.87,
107.98, 109.58, 119.57, 120.28, 122.30, 127.01, 130.73,
131.49, 134.59, 138.85, 147.01(azomethine C), 161.99,
162.09, 164.48; ESI MS m/z 268 (M + 1, %100), 269 (M + 2);
Anal. Calcd. For C16H14N3F: C, 71.88%; H, 5.28%; N, 15.73%.
Found: C, 70.99%; H, 5.46%; N, 15.12%. FT-IR (KBr) cm−1
1609 (C=N azomethine stretch), 3305 (N–H stretch).
1-Methylindole-2-carboxaldehyde (4- fluorophenyl)
hydrazone (1i) Yield 28.1%, m.p. 172–173°C; 1H-NMR:
4.05(3H, s), 6.78(2H,m), 6.96(1H,m), 7.05(2H,m), 7.22(2H,
m), 7.51(2H, dd), 8.05(1H, s, azomethine-CH), 10.59(1H,
s, hydrazine-NH). 13C-NMR: 32.49, 105.05, 110.37, 111.29,
111.82, 118.83, 120.36, 121.07, 123.09, 127.78, 131.54,
132.45, 134.56, 135.33, 139.62, 147.25 (azomethine C); ESI
MS m/z 284.41(M + 1, %100), 286.56 (M + 3); Anal. Calcd.
For C16H14N3Cl: C, 67.71%; H, 4.98%; N, 14.81%. Found: C,
67.24%; H, 5.27%; N, 14.77%. FT-IR (KBr) cm−1 1599 (C=N
azomethine stretch), 3297 (N–H stretch).
1-Methylindole-2-carboxaldehyde
(4-chlorophenyl)
1
1
hydrazone (1d) Yield 51.1%; m.p. 174–175°C; H-NMR:
hydrazone (1j) Yield 91.5%, m.p. 198–199°C; H-NMR:
4.05(3H, s), 6.73(1H, s), 7.04(3H, m), 7.23(3H, m),
7.51(2H, dd), 8.04(1H, s, azomethine-CH), 10.53(1H,s,
hydrazine-NH); 13C-NMR: 32.52, 104.80, 110.34, 113.99,
120.33, 121.00, 122.65, 127.82, 129.69, 131.86, 135.49,
139.60, 144.73 (azomethine C); ESI MS m/z 284.7(M +
1, %100), 286.8 (M + 3); Anal. Calcd. For C16H14N3Cl: C,
67.71%; H, 4.98%; N, 14.81%. Found: C, 67.51%; H, 5.26%;
N, 14.37%. FT-IR (KBr) cm−1 1599 (C=N azomethine
stretch), 3304 (N–H stretch).
4.05(3H, s), 6.70(1H, s), 7.11(6H, m), 7.50(2H, dd),
8.02(1H, s, azomethine CH), 10.39(1H, s, hydrazine NH);
13C-NMR: 32.51, 104.41, 110.29, 113.51, 116.30, 116.53,
120.29, 120.92, 122.85, 127.86, 131.08, 135.70, 139.54,
142.51 (azomethine C), 155.39, 157.72; ESI MS m/z 268
(M + 1, %100), 269 (M + 2); Anal. Calcd. For C16H14N3F: C,
71.88%; H, 5.28%; N, 15.73%. Found: C, 71.22%; H, 5.40%;
N, 15.22%. FT-IR (KBr) cm−1 1609 (C=N azomethine
stretch), 3298 (N–H stretch).
1-Methylindole-2-carboxaldehyde (2,4-difluorophenyl)
hydrazone (1e) Yield 27.4%; m.p. 159–160°C; H-NMR:
1-Methylindole-2-carboxaldehyde (2,4-dichlorophenyl)
hydrazone (1k) Yield 24.7%, m.p. 143–144°C; H-NMR:
1
1
4.05(3H, s), 6.73(1H, s), 7.04(2H, m), 7.23(2H, m), 7.47(3H,
m), 8.30(1H, s, azomethine CH), 10.26(1H, s, hydrazine
CH); ESI MS m/z 286 (M + 1, %100), 287 (M + 2); Anal.
Calcd. For C16H13N3F2: C, 67.34%; H, 4.60%; N, 14.73%.
Found: C, 66.62%; H, 4.74%; N, 14.40%. FT-IR (KBr) cm−1
1602 (C=N azomethine stretch), 3317 (N–H stretch).
1-Methylindole-2-carboxaldehyde (2,5-difluorophenyl)
4.04(3H, s), 6.78(1H, s), 7.06(1H, m), 7.21(1H, m), 7.34(1H,
dd), 7.25(4H, m), 8.51(1H, s, azomethine), 10.09(1H, s,
hydrazine); 13C-NMR: 32.49, 105.39, 110.48, 115.43, 117.28,
120.42, 121.22, 122.81, 123.30, 127.74, 128.87, 129.32,
135.15, 135.59, 139.64, 141.11 (azomethine C); ESI MS m/z
318(M + ,%100), 320 (M + 2); Anal. Calcd. For C16H13N3Cl2:
C, 60.37%; H, 4.12%; N, 13.21%. Found: C, 60.20%; H,
4.16%; N, 13.00%. FT-IR (KBr) cm−1 1593 (C=N azomethine
stretch), 3309 (N–H stretch).
1-Methylindole-2-carboxaldehyde (3,4-dichlorophe-
nyl)hydrazone (1l) Yield 40.9%; m.p. 220–221°C; 1H-
NMR: 4.04(3H, s), 6.79(1H, s), 7.02(2H, m), 7.21(2H, m),
7.50(3H, m), 8.06(1H, S, azomethine), 10.68(1H, s, hydra-
zine); 13C-NMR: 31.70, 104.56, 109.64, 112.12, 112.64,
119.31, 119.61, 120.35, 122.42, 126.96, 130.94, 131.52,
132.34, 134.34, 138.88, 145.06 (azomethine); ESI MS m/z
318(M +, %100), 320 (M + 2); Anal. Calcd. For C16H13N3Cl2:
C, 60.37%; H, 4.12%; N, 13.21%. Found: C, 60.05%; H,
4.19%; N, 12.84%. FT-IR (KBr) cm−1 1589 (C=N azome-
thine stretch), 3300 (N–H stretch).
1-Methylindole-2-carboxaldehyde (3,5-dichlorophe-
nyl)hydrazone (1m) Yield 22.0 %; m.p. 173.5–174.5°C;
1H-NMR: 4.04(3H, s), 6.82(1H, s), 6.87(1H, t), 6.98(2H, d),
7.06(1H,t), 7.21(1H, m), 7.52(2H, dd), 8.07(1H, s, azome-
thine), 10.76(1H, s, hydrazine); 13C-NMR: 32.46, 105.67,
110.45, 110.75, 118.02, 120.43, 121.22, 123.34, 127.71,
133.83, 134.93, 139.70, 147.97(azomethine); ESI MS m/z
318(M+, %100), 320 (M + 2); Anal. Calcd. For C16H13N3Cl2:
C, 60.37%; H, 4.12%; N, 13.21%. Found: C, 60.34%; H,
4.38%; N, 13.01%. FT-IR (KBr) cm−1 1580 (C=N azome-
thine stretch), 3306 (N–H stretch).
1
hydrazone (1f) Yield 61.9%; m.p. 168–169°C; H-NMR:
4.05(3H, s), 6.55(1H, m), 6.78(1H, s), 7.14(4H, m), 7.53(2H,
dd), 8.35(1H, s, azomethine CH), 10.35(1H, s, hydrazine
NH); ESI MS m/z 286 (M + 1, %100), 287 (M + 2); Anal.
Calcd. For C16H13N3F2: C, 67.34%; H, 4.60%; N, 14.73%.
Found: C, 67.10%; H, 4.58%; N, 14.30%. FT-IR (KBr) cm−1
1631 (C=N azomethine stretch), 3325 (N–H stretch).
1-Methylindole-2-carboxaldehyde (3,5-difluorophe-
nyl)hydrazone (1g) Yield 59.9%; m.p. 183–184°C; 1H-
NMR: 4.02(3H, s), 6.54(1H, m), 6.61(2H, dd), 6.77(1H, s),
7.03(1H,t), 7.18(1H, m), 7.49(2H, dd), 8.05(1H, s, azome-
thine CH), 10.78(1H, s, hydrazine NH); ESI MS m/z 286
(M + 1, %100), 287 (M + 2); Anal. Calcd. For C16H13N3F2: C,
67.34%; H, 4.60%; N, 14.73%. Found: C, 67.03%; H, 4.88%;
N, 14.30%. FT-IR (KBr) cm−1 1631 (C=N azomethine
stretch), 3305 (N–H stretch).
1-Methylindole-2-carboxaldehyde
(2-chlorophenyl)
hydrazone (1h) Yield 56.3%, m.p. 136.5-137.5°C; 1H-
NMR: 4.03(3H, s), 6.77(2H, m), 7.03(1H,t), 7.25(3H, m),
7.47(3H, d. dd), 8.47(1H, s, azomethine-CH), 9.23(1H, s,
hydrazine-NH); 13C-NMR: 32.49, 105.04, 110.43, 114.44,
116.82, 120.29, 120.39, 121.14, 123.17, 127.78, 128.86,
130.09, 134.86, 135.37, 139.60, 141.91 (azomethine C); ESI
MS m/z 284.7 (M + 1, %100), 286.7 (M + 3); Anal. Calcd.
For C16H14N3Cl: C, 67.71%; H, 4.98%; N, 14.81%. Found: C,
67.37%; H, 5.16%; N, 14.64. FT-IR (KBr) cm−1 1591 (C=N
azomethine stretch), 3316 (N–H stretch).
1-Methylindole-2-carboxaldehyde
hydrazone (1n) Yield 34.8%; m.p. 134–135°C; H-NMR:
(2-bromophenyl)
1
4.05(3H, s), 6.75(2H, m), 7.05(1H, t), 7.21(1H, m), 7.32
Journal of Enzyme Inhibition and Medicinal Chemistry

Antioxidant activity of indole-based melatonin analogues
5
(1H, m), 7.52(4H, m), 8.53(1H, s, azomethine CH), 9.72(1H,
s, hydrazine NH); 13C-NMR: 32.46, 105.00, 106.68, 110.44,
114.91, 120.39, 121.00, 121.16, 123.17, 127.78, 129.40,
133.32, 135.04, 135.38, 139.60, 142.96(azomethine C); ESI
MS m/z 328 (M+, %100), 287 (M + 2), 330 (M + 2), 331 (M
+ 3); Anal. Calcd. For C16H14N3Br: C, 58.53%; H, 4.30%; N,
12.80%. Found: C, 58.36%; H, 4.51%, N, 12.69%. FT-IR (KBr)
cm−1 1602 (C=N azomethine stretch), 3305 (N–H stretch).
acid hydrazide (for 1u) (0.05 mmol) in 50 mL of ethanol
was heated for 2.5 h on the hot water bath. On cooling,
the precipitate was collected washed with cold ethanol to
give 1t and 1u with 12–16% yield.
1-Methylindole-2-carboxaldehyde
izonicotynoilhy-
drazone (1t) Yield 16.5%; m.p. 190–191°C; 1H-NMR:
4.08(3H, s), 6.95(1H, s), 7.07(1H, t), 7.25(1H, t), 7.55(2H,
m), 7.82(2H, d), 8.59(1H, s, azomethine CH), 8.75(2H,
d), 12.06(1H, s, hydrazine NH); ESI MS m/z 279 (M + 1,
%100), 280 (M + 2); Anal. Calcd. For C16H14N4O: C, 69.04%;
H, 5.07%; N, 20.14%. Found: C, 66.80%; H, 5.65%; N,
17.84%. FT-IR (KBr) cm−1 1649 (C=N azomethine stretch),
3320 (N–H stretch).
1-Methylindole-2-carboxaldehyde
(3-bromophenyl)
1
hydrazone (1o) Yield 44.5%; m.p. 177–178°C; H-NMR:
4.05(3H, s), 6.76(1H, s), 6.91(1H, t), 7.03(2H, m), 7.19(3H,
m), 7.51(2H, dd), 8.05(1H, s, azomethine CH), 10.56(1H,
s, hydrazine NH); 13C-NMR: 32.48, 105.04, 110.38, 111.65,
114.72, 120.36, 121.73, 123.10, 123.15, 127.79, 131.85,
132.48, 135.33, 139.63, 147.37 (azomethine C); ESI MS m/z
328 (M+, %100), 287 (M + 2), 330 (M + 2), 331 (M + 3); Anal.
Calcd. For C16H14N3Br: C, 58.53%; H, 4.30%; N, 12.80%.
Found: C, 57.88%; H, 4.49%; N, 12.33%. FT-IR (KBr) cm−1
1590 (C=N azomethine stretch), 3296 (N–H stretch).
N-(4-methoxybenzoyl)-N′-(1-methylindolyl-2-
methylene)-hydrazine (1u) Yield 12.4%; m.p. 199–200°C;
1H-NMR: 3.82(3H, s), 4.07(3H, s), 6.88(1H, s), 7.06(3H,
m), 7.23(1H, t), 7.53(2H, dd), 7.91(2H, d), 8.57(1H, s,
azomethine CH), 11.71(1H, s, hydrazine NH); ESI MS
m/z 308 (M + 1, %100), 309 (M + 2); Anal. Calcd. For
C18H17N3O2: C, 70.33%; H, 5.58%; N, 13.68%. Found: C,
68.40%; H, 6.42%, N, 12.42%. FT-IR (KBr) cm−1 1647 (C=N
azomethine stretch), 3327 (N–H stretch).
1-Methylindole-2-carboxaldehyde (4-bromophenyl)
1
hydrazone (1p) Yield 87.2%; m.p. 207–208°C; H-NMR:
4.05(3H, s), 6.73(1H, s), 6.91(1H, t), 7.02(2H, m), 7.19(3H,
m), 7.46(4H, m), 8.04(1H, s, azomethine CH), 10.53(1H,
s, hydrazine NH); 13C-NMR: 32.52, 104.83, 110.22, 110.34,
114.51, 120.34, 121.01, 123.01, 127.82, 131.95, 132.51,
135.48, 139.61, 145.10 (azomethine C); ESI MS m/z 328
(M + ,%100), 287 (M + 2), 330 (M + 2); Anal. Calcd. For
C16H14N3Br: C, 58.53%; H, 4.30%; N, 12.80%. Found: C,
58.49%; H, 4.51%; N, 12.62%. FT-IR (KBr) cm−1 1590 (C=N
azomethine stretch), 3306 (N–H stretch).
General procedure for the synthesis
of compounds 1v,1y
1-Methylindole-2-carboxaldehyde (0.1 mmol) and
hydrazine hydrate (0.1 mmol for 1v, 0.2 mmol for 1y) in
25 mL of ethanol was heated for 4 h on the hot water bath.
On cooling, the precipitate was collected washed with
cold ethanol to give 1v and 1y with 56–57% yield.
N,N′-bis-(1-methylindole-2-ylmethylene)hydrazine
1-Methylindole-2-carboxaldehyde (2,3-dimethylphe-
nyl)hydrazone (1r) Yield 73.9%, m.p. 151–152°C; 1H-NMR:
2.14(3H, s), 2.24(3H, s), 4.06(3H, s), 6.67(2H, t), 7.03(2H,
m), 7.50(2H,dd), 8.34(1H, s, azomethine-CH), 9.59(1H,
s, hydrazine-NH); 13C-NMR: 12.62, 20.11, 31.68, 103.36,
109.50, 110.17, 119.04, 119.50, 120.12, 120.85, 122.02,
125.93, 127.13, 131.58, 135.174, 136.28, 138.73, 142.92
(azomethine-C); ESI MS m/z 278 (M + 1, %100), 279 (M
+ 2); Anal. Calcd. For C18H19N3:C, 77.39%; H, 6.91%; N,
15.16%. Found: C, 76.83%; H, 6.78%; N, 13.98. FT-IR (KBr)
cm−1 1586 (C=N azomethine stretch), 3338 (N–H stretch).
1-Methylindole-2-carboxaldehyde (2,4- dimethylphe-
nyl)hydrazone (1s) Yield 50.7%, m.p. 146–147°C; 1H-
NMR: 2.20(3H, s), 3.35(3H, s), 4.05(3H, s), 6.69(1H,s),
6.92(2H,t), 7.04(1H, m), 7.20(2H, m), 7.50(2H, dd),
8.30(1H, s, azomethine-CH), 9.52(1H, s, hydrazine-NH);
13C-NMR: 18.12, 20.87, 32.43, 103.96, 110.26, 112.62,
120.27, 120.87, 121.32, 122.74, 127.91, 128.00, 131.67,
131.92, 135.99, 139.48, 141.46 (azomethine-C); ESI MS
m/z 278 (M + 1, %100), 279 (M + 2); Anal. Calcd. For
C18H19N3:C, 77.39%; H, 6.91%; N, 15.16%. Found: C,
77.50%; H, 7.13%; N, 14.59%. FT-IR (KBr) cm−1 1615 (C=N
azomethine stretch), 3307 (N–H stretch).
1
(1v) Yield 56.0%; m.p. 297–298°C; H-NMR: 4.16(6H, s),
7.13(4H, m), 7.31(2H, t), 7.62(2H, dd), 8.90(2H, s, azome-
thine CH); ESI MS m/z 315 (M + 1, %100), 316 (M + 2), 317
(M + 3); Anal. Calcd. For C20H18N4: C, 76.4%; H, 5.58%; N,
13.68%. Found: C, 68.40%; H, 6.42%; 12.42%. FT-IR (KBr)
cm−1 1627 (C=N azomethine stretch).
1-Methylindole-2-carboxaldehyde hydrazone (1y)
Yield 57.1%; m.p. 124–125°C; 1H-NMR: 4.69(3H,
s), 7.30(1H, s), 7.66(2H, s, hydrazine NH), 7.90(1H,
t), 7.92(1H, s), 8.19(1H, d), 8.27(1H, d), 8.67(1H, s,
azomethine CH); 13C-NMR: 32.80, 103.11, 110.95,
120.91,121.49,123.10, 128.71, 133.43, 137.31, 139.89(azo-
methine C); ESI MS m/z 174 (M + 1, %100); Anal. Calcd.
For C10H11N3: C, 69.33%; H, 6.40%; N, 24.27%. Found: C,
69.34%; H, 6.79%; N, 23.34%. FT-IR (KBr) cm−1 1463 (C=N
azomethine stretch), 3355 (N–H stretch).
Biological activity studies
Antioxidant activity on ROS-induced DCFH-DA oxidation
For estimation of ROS inside cells DCFH-DA was used as
a fluorescent probe. In cellular systems non fluorescent
DCFH-DA readily crosses the cell membrane and under-
goes hydrolysis by intracellular esterases to nonfluores-
cent DCFH. DCFH is then rapidly oxidized in the presence
of reactive oxygen species to highly fluorescent DCFH31. In
the present study, human erythrocytes were used to evalu-
ate the potential antioxidant effect of test compounds.
General procedure for the synthesis
of compounds 1t,1u
A solution of 1-methylindole-2-carboxaldehyde (0.05
mmol) and izonicotinic acid hydrazide (for 1t) or anisic
© 2012 Informa UK, Ltd.

6
S. Suzen et al.
e assay is performed as detailed in Shirinzadeh et al.13.
Effects of the compounds (100 μM) on DCFH oxidation
were investigated after incubation of the cells with test
compounds alone or after co-incubation of the cells with
test compounds and H2O2 (10 μM) as a known reactive
oxygen species. Antioxidant effects of the compounds
were compared to the effect of ascorbic acid (100 μM) as a
well-known antioxidant and melatonin (MLT) (100 μM) as
their parent compound.
solution of the selected synthesized indoles was prepared
daily by direct dissolution in methanol. e working solu-
tions were prepared by the dilution of the stock solution
with selected supporting electrolytes contained differ-
ent percent of methanol. Four different supporting elec-
trolytes, namely sulfuric acid (0.1M), phosphate buffer
(0.2M, pH 2.0–8.0), acetate buffer (0.2M, pH 3.7–5.7), and
Britton–Robinson buffer (0.04M, pH 2–12) were prepared
in double distilled water. All solutions were protected from
light and were used within 24h to avoid decomposition.
Membrane stabilizing effect; LDH leakage
CHO-K1 cells were cultured in FC-1: Dulbecco’s Modified
Eagles Medium (1:1), supplemented with 10% foetal
bovine serum, 1% of a 100 U/ml penicillin-streptomycin
solution, 2 mM (final concentration) l-glutamine and
1 mM (final concentration) sodium pyruvate at 37°C,
5% CO2. is medium was used in all cell incubations.
Cytotoxic effects of the compounds were evaluated via
LDH activity assay according to the method of Hassoun
et al.32 with a minor modification. e activity of LDH in
100 μL of media was determined by direct calculation
based on the decrease in absorbance33.
Results and discussion
Antioxidant effects on ROS-induced
DCFH-DA oxidation
e protective effect of newly synthesized MLT analogues
against DCFH-DA oxidation was determined in human
erythrocytes that were preloaded with the fluorescent
probe. Oxidation of the probe was screened at 10min
intervals up to 60min. DCFH oxidation at 60min incu-
bation is given for cells exposed to the test compound
alone (Figure 3) and for cells co-exposed to H2O2 and the
test compounds (Figure 4). e aim of the second model
(Figure 4) is to evaluate the antioxidant capacity of the syn-
thesized MLT analogs in a ROS challenge situation. Almost
all compounds were found to exhibit higher antioxidant
activity when they are challenged with H2O2. Comparing
to the results from our previous study13, N-methylation of
the indole ring decreased the antioxidant capacity of all
MLT derivatives. Similar to our previous results13 among
mono-halogenated derivatives p-halogenation was found
to decrease antioxidant activity compared to o- and
m-substitution of the same halogen atom. 1t, 1u, and 1y
had the weakest antioxidant activity in both cases (with
or without H2O2). is can be explained by having no aro-
matic halogenations or a carbonyl group on the side chain.
Electrochemical measurements
e cyclic, linear sweep, differential pulse voltammetric
(DPV) and square wave voltammetric (SWV) measure-
ments at a glassy carbon electrode were performed using a
BAS 100W (Bioanalytical System, Los Angeles, California,
USA) electrochemical analyzer. e three electrode sys-
tem a glassy carbon working electrode (BAS; Ø: 3mm
diameter), with a platinum wire counter electrode and Ag/
AgCl reference electrode (KCl 3M, BAS) and a standard
one-compartment three-electrode cell of 10mL capacity.
For analytical application, the following parameters were
employed: SWV amplitude, 25 mV; frequency, 15 Hz; scan
increment, 4 mV. DPV parameters were: pulse amplitude,
50 mV; pulse width 50ms; scan rate, 20 mV s−1. e pH val-
ues were measured using a pH meter Model 538 (WTW,
Wien, Austria) using a combined electrode (glass electrode
reference electrode) with an accuracy of 0.05 pH. A stock
Membrane stabilizing effect; LDH leakage
Lipid peroxidation is known to change membrane fra-
gility, motility and permeability34. LDH is a cytoplasmic
Figure 3. Oxidation of DCFH via reactive oxygen species in erythrocytes after incubation with ascorbic acid, MLT and newly synthesized
analogs for 60 min. Control group represents cells incubated with PBS without any compound addition. Values are mean SD of three
individual experiments. *p < 0.05, compared to control group. **p < 0.005, compared to control group. #p < 0.0005, compared to control group.
MLT, melatonin.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antioxidant activity of indole-based melatonin analogues
7
enzyme and its leakage from injured cells into the culture
medium has been shown to be useful as an indicator
of cellular membrane damage. In the present study, we
determined LDH activity in the culture medium in order
to assess the membrane stabilizing effect of MLT ana-
logues. No increases in LDH activity were observed with
any of the tested compounds. Even several compounds
(1b, 1g, and 1t) were found to decrease LDH leakage
indicating membrane stabilizing effect. is assay is
also an indicator of a possible cytotoxic effect of the test
compounds by means of inducing membrane damage.
From this point of view, as possible drug candidates, we
can conclude that none of the synthesized MLT analogs
are found to have cytotoxic effect. In accordance with the
antioxidant assay (Figures 3 and 4) 1d and 1r were found
to have the highest, but statistically not significant, LDH
activity in media (Figure 5). is finding can be explained
by the increased ROS formation with 1d and 1r analogs
resulted in membrane damage and leakage of the LDH
enzyme into the media.
1f, 1g, and structurally different compounds 1y and 1u.
erefore, several electrochemical measurements using
cyclic, linear sweep, differential pulse, and square wave
voltammetric methods were performed with various
supporting electrolytes and buffers in order to obtain
such information.
As first step, compounds 1f, 1g, 1y, and 1u were sub-
jected to a cyclic and linear sweep voltammetric studies
with the aim to characterize of their electrochemical oxi-
dation behavior in details on the glassy carbon electrode
over a wide pH range (1.5–12.0). Voltammograms exhib-
ited one distinct and well defined main anodic peak (Ox1)
and/or one ill-defined anodic wave at different potential
values depending on pH values and supporting electro-
lyte composition (Figures 6 and 7). In addition to this
first oxidation step, the second oxidation response was
obtained when working on compound 1u depending on
the methoxy benzene oxidation (Ox2). CV measurements
showed an irreversible nature of the oxidation process.
In acidic media, the anodic oxidation did not occur
until about +0.50 V for all selected compounds on glassy
Electrochemical measurements
In this study the measurements from the selected indole
derivatives are two-dimensional, with the potential being
related to qualitative properties and current related
properties. In order to characterize the electrochemical
process that occurs on the glassy carbon electrode, both
pH, nature of the buffer and scan rate studies were car-
ried out in the potential range from −0.25 to 1.6 V. Cyclic
voltammetry (CV) experiments were achieved over a
range from acidic (0.1M H2SO4) to alkaline (pH 12.00) in
acidic solutions and different buffer aqueous media. For
the quantitative determination, DPV and SWV methods
were applied to the selected synthesized derivatives [35–37]
.
For the synthesized compounds, the peak currents and
peak potentials were determined in different support-
ing electrolytes such as H2SO4, phosphate, acetate, and
Britton-Robison buffers containing 40, 50, 20, and 30%
methanol (v/v) to maintain solubility for 1f, 1g, 1y, and
1u, respectively. e compounds were selected as two
of the most active and structurally similar compounds
Figure 5. Effect of MLT analogs on LDH leakage from CHO cells
into the culture media. Control group represents cells incubated
in regular media without any compound addition. % 2.5 DMSO is
used to cause membrane damage where ascorbic acid is used as an
antioxidant. *p < 0.05, **p < 0.005 compared with control.
Figure 4. Antioxidant effect of indole-analogs on H2O2-induced oxidation of DCFH in erythrocytes. Control group represents cells incubated
with PBS without any compound addition. Values are mean SD of three individual experiments. §p < 0.005, compared with control. *p < 0.05,
compared with H2O2 group. **p < 0.005, compared with H2O2 group. #p < 0.0005, compared with H2O2 group.
© 2012 Informa UK, Ltd.

8
S. Suzen et al.
Figure 6. CV voltammograms of 20 µg/mL 1f and 10 µg/mL 1g, 1y, and 1u in 0.1 M H2SO4. (A,B,C, and D): (A) 20 µg/mL 1f, 40% MeOH (B) 10
µg/mL 1g, 50% MeOH(C) 10 µg/mL 1y 20% MeOH (D) 10 µg/mL 1u 30% MeOH.
Figure 7. CV voltammograms of 1f, 1g, 1y, and 1u. (A,B,C, and D): (A) 20 µg/mL 1f, pH 6 FT (B) 10 µg/mL 1g, pH 4.7 AT (C) 10 µg/mL 1y, pH
7.0 BRT (D) 10 µg/mL 1u, pH 10 BRT.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antioxidant activity of indole-based melatonin analogues
9
carbon electrode (Figure 6). By reversing at +1.80 V no
reduction signal corresponding to the anodic response
was observed on the cathodic branch in all pH values
(Figure 7). e anodic oxidation peaks of the selected
indolic compounds decreased to the second or higher
cycles (Figures 6 and 7). is phenomenon may be partly
attributed to the consumption of adsorbed indolic com-
pounds on the electrode surface.
Ep (mV) = 664.25 − 55.67 pH; r = 0.998, n = 11 (for 1f,
between pH 2 and 12)
Ep (mV) = 497.02 − 50.78 pH; r = 0.997, n = 10 (for 1g,
between pH 2 and 12)
Ep (mV) = 632.68 − 42.40 pH; r = 0.999, n = 6 (for 1y,
between pH 1 and 12)
Ep (mV) = 915.82 − 45.03 pH; r = 0.981, n = 8 (for 1u, Ox1,
between pH 2 and 12)
e effect of the potential scan rate varied between 5
and 1000 mV s−1 on the peak current and potential of the
selected indolic compounds were evaluated. e different
amounts shifts in the peak potentials confirmed the irre-
versibility of the oxidation processes of these compounds
(Table 1). Scan rate studies were carried out to assess
whether the processes at the glassy carbon electrode was
under diffusion or adsorption control for the first and/or
second peaks. When the scan rate was varied between 5
and 1000 mV s−1 in 20 µg/mL solution of compound 1f,
and 10 µg/mL solution of compounds 1g, 1y, and 1u a
linear dependence of the peak intensity ip (µA) upon the
Ep (mV) = 1293.90 − 18.63 pH; r = 0.994, n = 5 (for 1u,
Ox2, between pH 1 and 6)
e peak potential of the anodic process moved to
less positive potential values and an ill-defined oxida-
tion wave disappeared by increasing pH. In all instances
and techniques, the indole compounds undergoes one
main irreversible oxidation process and/or one addi-
tional ill-defined wave, which shifted towards less posi-
tive potentials as the pH was increased. When working
on compound 1u, one additional oxidation step was
obtained depending on the apart from indole oxidation,
which is occurred by the methoxy benzene oxidation
(Ox2).
According to the slopes of the above equations are
found between 42.40 and 55.67 mV/pH for the main
oxidation process (Ox1) of all studied compounds. ese
slopes are found close to the expected theoretical value of
59 mV/pH. For the second oxidation step of compound
1u, the slope value was obtained as 18.63 mV/pH. ese
slopes are found close to the expected theoretical value
of 30 mV/pH. According to the obtained slope values of
these equations, same amount of electrons and protons
are involved in the rate-determining steps. e experi-
mental results showed that shapes of the curves and
maximum peak current were obtained in different sup-
porting electrolytes and pH values with constant amount
of methanol, which indicated in Table 2. e main peak
1
square root of the scan rate ν /2 (mV s−1) was found in all
studied supporting electrolytes, demonstrating a diffu-
sional behavior. e equation is noted in Table 1 for all
compounds and all studied media. All the equations gave
a straight line with a slope values between 0.36 and 0.66,
very close to the theoretical value of 0.5, which is expressed
for an ideal reaction the diffusion controlled electrode pro-
cess38. Nonetheless, the peak potentials shifted to the more
positive potentials to the anodic direction when the scan
rate was increased (Table 1) in all supporting electrolyte.
e effect of pH on the peak potential and intensity
were investigated between pH 2.0 and 12.0 for all selected
indole compounds using CV, DPV, and SWV techniques.
All obtained results from CV, DPV, and SWV were found
similar. For this reason, only DPV results for the obtained
oxidation steps were given as below for Ep–pH equations:
Table 1. e results of the scan rates experiments on 1f, 1g, 1y, and 1u.
Scan rate
∆Ep/
Equation of
Correlation
coefficient.
Equation of
log υ vs. log ip
Correlation
coefficient
½
Step
Ox1
Medium
range (mV s−1) (mV)
υ
vs. Ip
1f
0.1M H2SO4 %40 methanol
10–750
5–100
53.0
54.0
y = 0.0355x − 0.0595
y = 0.0261x − 0.0417
y = 0.0783x − 0.0806
y = 0.0637x + 0.0003
0.999
0.952
0.999
0.996
y = 0.619x − 1.8005
y = 0.5504x − 1.9032
y = 0.5344x − 1.2369
y = 0.5634x − 1.3248
0.998
0.960
0.996
0.990
Ox1 pH 10 BR buffer %40 methanol
1g
Ox1
0.1M H2SO4 %50 methanol
5–1000
5–500
110.0
101.0
pH 4.7 acetate buffer %50
methanol
Ox1
pH 7 phosphate buffer %50
methanol
5–500
152.0
y = 0.0234x + 0.0722
0.990
y = 0.3603x − 1.2067
0.994
pH 10 BR buffer %50 methanol
0.1M H2SO4 %20 Methanol
10–750
10–1000
25–750
90.0
27.0
72.0
y = 0.098x − 0.2867
y = 0.0379x − 0.0542
y = 0.0627x − 0.1948
0.993
0.998
0.989
y = 0.6541x − 1.5226
y = 0.664x − 1.8913
y = 0.7281x − 1.874
0.990
0.981
0.980
1y
1u
Ox1
Ox1
pH 3 phosphate buffer %20
methanol
Ox1
Ox1
Ox1
pH 3.7 acetate buffer %20
methanol
10–1000
10–750
5–1000
75.0
52.0
99.0
y = 0.094x − 0.2216
y = 0.2081x − 0.2692
y = 0.1354x − 0.351
0.999
0.996
0.995
y = 0.6608x − 1.5158
y = 0.5393x − 0.827
y = 0.6651x − 1.4151
0.998
0.998
0.990
pH 7 BR buffer %20
methanol
pH 5.7 acetate buffer %30
methanol
Ox1 pH 2 BR buffer %30 methanol
Ox2 0.1M H2SO4 %30 methanol
10–100
10–500
113.0
17.0
y = 0.0576x − 0.0197
y = 0.1282x − 0.3656
0.994
0.986
y = 0.4864x − 1.2225
y = 0.6284x − 1.3465
0.994
0.992
© 2012 Informa UK, Ltd.

10 S. Suzen et al.
that appeared with less positive potential was the best
developed and became sharper and occurred as without
shoulder in these supporting electrolytes. For this rea-
son, the indicated supporting electrolytes in Table 2 were
chosen with respect to have sharp response and better
peak shape for the calibration equation.
ring36–39,42. e electrochemical oxidation of indole deriva-
tives with a substituent presented similar features to the
indole electroactive centre. It is a well-established fact
that the benzene ring of indoles is less reactive than the
pyrrole ring. Since all indole derivatives are substituted,
the oxidation reaction taking place at the main peak (Ox1)
corresponds to the oxidation at nitrogen on the pyrrole
ring43,44. e cyclic voltammograms of the indole library
and model compounds demonstrated that the oxidation
potential peak values were close to those of the nonsub-
stituted indole nucleus, but higher than those described
for the antioxidant melatonin. In addition, no reversibility
was observed in the obtained voltammograms. e lack
of reversibility is an advantage, meaning that once oxi-
dized these species do not tend to receive electrons.
Although the exact oxidation mechanism was not
determined, some conclusions about the potentially
electroactive centers under working conditions could
be reached. From the CV curves, the main voltammetric
behavior of indole and methoxy benzene derivatives,
which are structurally related to the mechanism of oxida-
tion of synthesized indole compounds, may be postulated
by the oxidation of these groups26–29,35–41. Our results on
model compounds show similar behavior that the electro-
active center corresponding to the main anodic response
was the nitrogen atom on the indole ring. In addition to,
the methoxy benzene anodic response in compound 1u
was obtained as the second oxidation step. Indole and
In addition to this step for 1u, second oxidation step
was obtained because of the methoxy group on the ben-
zene moiety of the molecule. Nearly in all pH values, both
indole and methoxy benzene rings responses of 1u was
clearly and separately obtained. e study on verapamil,
tamsulosin, and other synthesized indole compounds was
realized by CV at GC electrode as a function of pH in order
to identify the responsible atom for the second oxidation
process of 1u. Our results revealed a good agreement with
the redox mechanism postulated for similar compounds
such as 4-methoxyphenol, tamsulosin and verapamil sug-
gested that tamsulosin can be determined electrochemi-
its derivatives are extensively metabolized in vivo26–29,36,37
.
To support the working hypothesis that indole moiety
(similar oxidation pathway with indol-3-substituted
derivative) and methoxy benzene moiety (similar to
phenolic group oxidation step) of compounds 1f, 1g,
1y, and 1u that undergoes oxidation, behavior of Step I
(related to indole moiety) and Step II (related to methoxy
benzene moiety) of the compounds were compared with
similar model compounds. Comparative study on indole,
synthesized indole compounds, indole-3-acetic acid,
etodolac, fluvastatine, and ziprasidon were realized by
CV at a function of pH in order to identify the oxidation
process of the synthesized compounds. It was assumed
that oxidation occurred firstly on the nitrogen atom of
indole ring, which is electroactive in both acidic and basic
media, leading finally to hydroxylation of the benzene
cally by oxidation of methoxybenzene groups40,41,45,46
.
Analytical applications and methods validation
In order to develop a voltammetric methodology for
determining compound 1f, g, y, and u, DPV and SWV
modes were selected. In this study SWV were used as an
alternative technique. Various electrolytes such as sul-
phuric acid, Britton–Robinson, phosphate, and acetate
Table 2. Calibration parameters of 1f, 1g, 1y, and 1u.
1f
1g
1y
1u
pH:10 BR buffer
(%40 methanol)
pH: 10 BR buffer
(%50 methanol)
pH:3 phosphate buffer
(%20 metanol)
0.1M H2SO4 buffer
(%30 methanol)
pH:5.7 cetate uffer
(%30 metanol)
Medium
Technique
Potential/V
DPV
0.100
1–8
DPV
0.100
1–8
DPV
0.400
1–10
DPV
0.400
1–10
DPV
0.400
1–10
SWV
0.412
1–8
DPV
1.200
1–10
SWV
1.300
1–10
DPV
0.648
1–8
SWV
0.700
1–8
Linearity/range
(µg/mL)
Slope
0.0814
0.0237
0.997
0.0814
0.0237
0.997
0.0988
0.0218
0.999
0.0988
0.0218
0.999
0.0988
0.0218
0.999
0.0999
0.0113
0.999
0.1033
0.0209
0.999
0.1177
0.0073
0.999
0.1156
0.0169
0.999
0.1059
0.0261
0.999
Intercept
Correlation
coefficient
SE of slope
3.180 × 10−3 3.180 × 10−3 1.914 × 101 1.914 × 101 1.914 × 101 2.425 × 101 1.354 × 10−2 5.848 × 10−4 1.340 × 10−3 1.102 × 10−2
SE of intercept 1.569 × 10−2 1.569 × 10−2 1.231 × 102 1.231 × 102 1.231 × 102 1.574 × 102 7.983 × 10−2 1.832 × 10−3 4.716 × 10−3 6.369 × 10−2
Limit of
detection
0.125
0.381
0.555
1.686
0.125
0.381
0.555
1.686
0.038
0.038
0.038
0.110
0.061
0.186
0.545
0.820
0.033
0.100
0.412
0.497
0.083
0.254
0.246
1.271
0.135
0.410
0.372
1.975
Limit of
quantification
0.115
0.115
0.115
0.334
Repeatability
(RSD %)
0.5846
0.4925
0.5846
0.4925
0.5846
0.4925
0.3472
1.5586
Reproducibility
(RSD %)
BR, Britton–Robinson; DPV, differential pulse voltammetric measurement; RSD, relative standard deviation; SWV, square wave voltammetric measurement.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antioxidant activity of indole-based melatonin analogues 11
buffer were examined as a supporting electrolyte. e
best condition for analytical applications proved to be
indifferent supporting electrolytes, which are indicated
in Table 2. ese supporting electrolytes with a constant
amount of methanol were chosen for the subsequent
experiments. e effects of methanol amount on peak
current and potential were also studied. As expected,
the peak current decreased with changing ionic strength
and viscosity of the medium. Good correlations were
obtained in different concentration ranges using both
techniques, which were reported in Table 2. Above the
linearity high limit concentration, a loss of linearity was
probably due to the adsorption of the compounds on
the electrode surface. e characteristics of the calibra-
tion plots are summarized in Table 2. e detection limit
and quantification limit of the procedures (Table 2) were
calculated according to the 3 s/m and 10 s/m criterions,
respectively, where s is the standard deviation of the peak
currents (five runs) and m is the slope of related calibra-
tion graph45–47. e low values of SE of slope and intercept
and greater than 0.999 correlation coefficient nearly in all
media established the precision of the proposed method.
e developed methods were validated according to the
standard procedures and the results obtained are shown
in Table 2. Accuracy, precision and reproducibility of the
proposed method were assessed by performing replicate
analysis of the standard solutions in supporting electro-
lytes within the calibration curves. e within day and
between day precision, accuracy and reproducibility
were determined as the RSD% (relative standard devia-
fluorescence at the same excitation and emission wave-
lengths (which can cause false positive results). None
of the compounds were found to have any. Finally in
order to make sure that added compounds do not have a
quenching effect we examined the absorbance spectrum
of the compounds and determined that the absorbance
peaks were not overlapping with either the excitation
and emission peak of the oxidized dye.
Although we have thought about the possible esterase
inhibition because of the following reasons such an inter-
action for the present compounds is not possible. First of
all two known esterases were identified in RBC cytosol;
acetylcholinesterase and arylesterase. e esterases are
reported to exhibit specificities for certain substrates and
inhibitors but a drug is often hydrolysed by more than
one esterase at different sites. Among the most common
acetylcholinesterase inhibitors are phosphorus-based
compounds, which are designed to bind to the active site
of the enzyme. e structural requirements are a phos-
phorus atom bearing two lipophilic groups, a leaving
group (such as a halide or thiocyanate), and a terminal
oxygen50,51. As a conclusion none of the compounds that
we tested are structurally thought to be an inhibitor of
the esterases as well as even one of the isozyme or the
type of the esterase in RBC cytosol is inhibited by any of
our compound we think that there should still be enough
activity to hydrolyse DCFH-DA.
Structural investigation of the rest of the active com-
pounds showed that having o- and m-halogenated aro-
matic side chain increase the antioxidant activity (such as
compounds 1f, 1k, and 1m). ese are the most promis-
ing compounds that should be kept in mind for designing
new MLT-based indole derivatives for our ongoing study.
Lack of a methoxy group in the five positions did
not affect the antioxidant capacity of the new indole
derivatives. In fact, the in vitro assays showed that lack
of a methoxy group, introduction of a methyl group at the
nitrogen in the indole ring and a halogenated aromatic
side chain resulted in much more active compounds
than MLT itself. is 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.
tion) and the results are shown in Table 2 31,47,48
.
conclusions
e present work aimed to synthesize, characterize inves-
tigate the potential antioxidant effects, and electrochemi-
cal evaluation of indole-based MLT analogue hydrazide/
hydrazone derivatives. In general all the synthesized
indole derivatives were found to have potent antioxi-
dant activity, even higher than MLT itself, according to
the results of in vitro antioxidant experiments revealing
differences in their relative potencies probably related
to electronic distribution. Having halogenated aromatic
side chain increase the antioxidant activity. Antioxidant
effect of indole-analogs on H2O2-induced oxidation of
DCFH in erythrocytes showed that all analogues except
1d and 1r were effective.
e use of electrochemical techniques to study
mechanisms directly with the electrodes and the deter-
mination of compounds of pharmaceutical interest is
becoming a mature field23.
We considered several possible interactions during
the fluorescence measurements and performed several
negative controls. We examined the fluorescence of
untreated loaded cells. e healthy cells exhibited lower
levels of fluorescence that was relatively stable during the
experiment (with a slight gradual increase possibly due
to auto oxidation) to make sure that the cells are not pro-
gressing to death or another oxidative event is not taking
place. e fluorescence intensity of the compounds in
the same test environment without DCFH was analyzed
to assure that the added compounds are not giving any
Electrochemical mechanisms are important to all
redox chemistry including biological systems usually
concerning electron transport chains. Such reactions are
most often studied with electrode techniques such as
CV35. In this study electrochemical behavior of selective
MLT analogues through mechanistic studies was investi-
gated. It was observed that oxidation occurred firstly on
the nitrogen atom of indole ring leading finally to hydrox-
ylation of the benzene ring36–39,42. is investigation gives
some suggestions about the possible in vivo metabolism
of newly synthesized compounds.
© 2012 Informa UK, Ltd.

12 S. Suzen et al.
hydrazones bearing 5-(Pyridine-3-yl)-1,3,4-thiadiazole. J Pharm
Res 2010;3:2460–2462.
is study presents supportive data and will contrib-
ute for the design of indole-related drugs with improved
antioxidant activity.
16. Kumar PP, Rani BL. Synthesis and characterization of new
Schiff bases containing pyridine moiety and their derivatives as
antioxidant agents. Int J Chem Tech Research 2011;3:155–160.
17. angadurai TD, Gowri M, Natarajan K. Synthesis and
characterisation of ruthenium(III) complexes containing
monobasic bidentate Schiff bases and their biological activities.
Synth React Inorg Met-org Chem 2002;32:329–343.
18. Belkheiri N, Bouguerne B, Bedos-Belval F, Duran H, Bernis C,
Salvayre R et al. Synthesis and antioxidant activity evaluation of a
syringic hydrazones family. Eur J Med Chem 2010;45:3019–3026.
19. Negre-Salvayre A, Coatrieux C, Ingueneau C, Salvayre R. Advanced
lipid peroxidation end products in oxidative damage to proteins.
Potential role in diseases and therapeutic prospects for the
inhibitors. Br J Pharmacol 2008;153:6–20.
Declaration of interest
is work was supported by e Scientific and
Technological Research Council of Turkey (TÜBİTAK)
Research and Development Grant 109S099. Cell culture
laboratory where the antioxidant activity studies per-
formed was established with the TÜBİTAK Research and
Development Grant 108S202.
e authors report no conflicts of interest.
20. Yılmaz AD, Coban T, Suzen S. JSynthesis and antioxidant activity
evaluations of melatonin-based analogue indole-hydrazide/
hydrazonederivatives.JEnzymeInhibMedChem2012;27:428–436.
21. Suzen S, Karaaslan C. Electrochemical behavior of biologically
important indole derivatives. J Int Electrochem 2011;2011:1–10.
22. Kissinger PT, Heineman WR, eds. (1996). Laboratory techniques in
electroanalytical chemistry. 2nd ed. New York: Marcel Dekker.
23. Wang J, ed. (1988). Electroanalytical techniques in clinical
chemistry and laboratory medicine. New York: VCH Publisher Inc.
24. Hart HP. (1990). Electroanalysis of biologycally important
compounds. England: Ellis Horwood Ltd.
References
1. Singh RP, Sharad S, Kapur SJ. Free radicals and oxidative stress in
neuro degenerative diseases: Relevance of dietary antioxidants.
Ind Acad Clin Med 2004;4:218–225.
2. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free
radicals and antioxidants in normal physiological functions and
human disease. Int J Biochem Cell Biol 2007;39:44–84.
3. Fresco P, Borges F, Diniz C, Marques MP. New insights on the
anticancer properties of dietary polyphenols. Med Res Rev
2006;26:747–766.
4. Suzen S. (2007). Topics in Heterocyclic Chemistry, Bioactive
Heterocycles V. In: Khan MTH, ed. Antioxidant activities of
synthetic indole derivatives and possible activity mechanisms.
Berlin, Heidelberg: Spinger-Verlag, ;11, 145–178.
5. Reiter RJ, Tan DX, Cabrera J, D’Arpa D, Sainz RM, Mayo JC et al.
e oxidant/antioxidant network: role of melatonin. Biol Signals
Recept 1999;8:56–63.
25. Vire JC, Kauffmann JM. Curr. Top. Trends in electrochemistry in
drug analysis. Electrochem 1994;3:493–515.
26. Süzen S, Ates-Alagöz Z, Demircigil BT, Ozkan SA. Synthesis and
analytical evaluation by voltammetric studies of some new indole-
3-propionamide derivatives. Farmaco 2001;56:835–840.
27. Suzen S, Demircigil T, Buyukbingol E, Ozkan SA. Electroanalitical
evaluation and determination of 5-(3′-indolyl)-2-thiohydantoin
derivatives by voltammetric studies: possible relevance to in vitro
metabolism. New J Chem 2003;27:1007–1011.
28. Bozkaya P, Dogan B, Suzen S, Nebioglu D, Ozkan SA. Determination
and investigation of electrochemical behaviour of 2-phenylindole
derivatives: discussion on possible mechanistic pathways. Can J
Anal Sci Spec 2006;51:125–139.
6. Korkmaz A, Reiter RJ, Tan DX, Manchester LC. Melatonin; from
pineal gland to healthy food. Spatula 2011;1:33–36.
7. Cano A, Alcaraz O, Arnao MB. Free radical-scavenging activity of
indolic compounds in aqueous and ethanolic media. Anal Bioanal
Chem 2003;376:33–37.
29. Shirinzadeh H, Yılmaz AD, Gumustas M, Suzen S, Ozden S,
Ozkan SA. Electrochemical behavior of indole-3-carboxaldehyde
izonicotinoyl hydrazones: discussion on possible biological
behavior. Chem High T Synt 2010;13:619–627.
8. Andreadou I, Tsantili-Kakoulidou A, Spyropoulou E, Siatra T.
Reactions of indole derivatives with cardioprotective activity
with reactive oxygen species. Comparison with melatonin. Chem
Pharm Bull 2003;51:1128–1131.
30. Kidwai M, Negi N, Gupta SD. Synthesis and antifertility activity
of 1,5-diaryl-3-(3’-indolyl)formazans. Chem Pharm Bull
1994;42:2363–2364.
9. Misik V, Ondrias K, Stasko A. EPR spectroscopy of free radical
intermediates of antiarrhythmic-antihypoxic drug stobadine, a
pyridoindole derivative. Life Sci 1999;65:1879–1881.
31. Lautraite S, Bigot-Lasserre D, Bars R, Carmichael N. Optimisation
of cell-based assays for medium throughput screening of oxidative
stress. Toxicol In Vitro 2003;17:207–220.
32. Hassoun EA, Roche VF, Stohs JS. Release of enzymes by ricin from
macrophages and Chinese hamster ovary cells in culture. Toulouse
Med 1993;3:119–129.
33. Moss DW, Henderson AR, Kochmar JR. (1986). Enzymes. In:
Tietz NW, ed. Textbook of clinical chemistry. Philadelphia: WB
Saunders, 619–663.
34. Barros SB, Simizu K, Junqueira VB. Liver lipid peroxidation-
related parameters after short-term administration of
hexachlorocyclohexane isomers to rats. Toxicol Lett
1991;56:137–144.
35. Suzen S, Ozkan SA. (2011). Medicinal and pharmaceutical
applications and prospects of electrochemistry. In: Ozkan SA, ed.
Electroanalytical methods in pharmaceutical analysis and their
validation. NHB Publishing, ISBN: 978-0-9664286-7-4.
36. Kul D, Gumustas M, Uslu B, Ozkan SA. Electroanalytical
characteristics of antipsychotic drug ziprasidone and its
determination in pharmaceuticals and serum samples on solid
electrodes. Talanta 2010;82:286–295.
10. Suzen S, Bozkaya P, Coban T, Nebiogu D. Investigation of
the in vitro antioxidant behaviour of some 2-phenylindole
derivatives: discussion on possible antioxidant mechanisms
and comparison with melatonin. J Enzyme Inhib Med Chem
2006;21:405–411.
11. Ates-Alagoz Z, Coban T, Suzen S. A Comparative study: evaluation
of antioxidant activity of melatonin and some indole derivatives.
Med Chem Res 2005;14:169–179.
12. Gürkök G, Coban T, Suzen S. Melatonin analogue new indole
hydrazide/hydrazone derivatives with antioxidant behavior:
synthesis and structure-activity relationships. J Enzyme Inhib Med
Chem 2009;24:506–515.
13. Shirinzadeh H, Eren B, Gurer-Orhan H, Suzen S, Ozden S.
Novel indole-based analogs of melatonin: synthesis and in vitro
antioxidant activity studies. Molecules 2010;15:2187–2202.
14. Rajitha G, Saideepa N, Praneetha P. Synthesis and evaluation
of N-(α-benzamidocinnamoyl) aryl hydrazone derivatives
for anti-inflammatory and antioxidant activities. Ind J Chem
2011;50B:729–733.
15. Prathapa KJ, Himajaa M, Malib SV, Ranjithaa A, Karigarc A,
Sikarwar M. Synthesis and antioxidant activity of novel ketone
Journal of Enzyme Inhibition and Medicinal Chemistry

Antioxidant activity of indole-based melatonin analogues 13
37. Altun Y, Dogan-Topal B, Uslu B, Ozkan SA. Anodic behavior
of sertindole and its voltammetric determination in
pharmaceuticals and human serum using glassy carbon
and boron-doped diamond electrodes. Electrochim Acta
2009;54:1893–1903.
38. Laviron E, Roullier C, Degrand J. A multilayer model for the study
of space distributed redox modified electrodes: Part II. eory and
application of linear potential sweep voltammetry for a simple
reaction. Electroanal Chem 1980;112:11–23.
39. Goyal RN, Kumar RN, Signhal NK. Oxidation chemistry and
biochemistry of indole and effect of its oxidation product in albino
mice. Bioelectrochem Bioenerg 1998;45:47–53.
40. Ozkan SA, Uslu B, Aboul-Enein HY. Voltammetric investigation of
tamsulosin. Talanta 2003;61:147–156.
42. Jennings P, Jones AC, Mount AR, omson AD. Electrooxidation of
5-substitutedindoles.JChemSocFaradayTrans1997;93:3791–3797.
43. Enache TA, Oliveira-Brett AM. Pathways of electrochemical
oxidationofindoliccompounds.Electroanalysis2011;23:1337–1344.
44. Somers KRF, Kryachko ES, Ceulemans A. eoretical study of
indole: protonation, indolyl radical, tautomers of indole, and its
interaction with water. Ceulemans Chem Phys 2004;301:61–79.
45. Grimshow J, ed. (2000). Electrochemical reactions and
mechanisms in organic chemistry. 1st ed. Amsterdam: Elsevier.
46. Lund H, Hammerich O. (2001). Organic electrochemistry. 4th ed.
New York: Marcel Dekker.
47. Dekker M. (1997). Analytical Method Development and Validation.
In: Swartz ME, Krull IS, eds. New York: Marcel Dekker, Inc.
48. Ermer J, Miller JHMcB. (2005) Method validation in pharmaceutical
analysis. 1st ed. Weinheim, Germany: Wiley-VCH.
49. Cossum PA. Role of the red blood cell in drug metabolism.
Biopharm Drug Dispos 1988;9:321–336.
41. Demircan S, Ozkan SA, Kır S. Electroanalytical characterization of
verapamil and its voltammetric determination in pharmaceuticals
and human serum. Anal Lett 2007;40:1177–1195.
© 2012 Informa UK, Ltd.