Synthesis and antioxidant activity evaluations of melatonin-based analogue indole-hydrazide/hydrazone derivatives

Article abstract of DOI:10.3109/14756366.2011.594048

Melatonin (MLT) is a hormone synthesized from the pineal gland. It is a direct scavenger of free radicals, which is related to its capability to defend cells from oxidative stress. Recently MLT-related compounds are under investigation to establish which exhibit the maximum activity with the lowest side effects. In this study 5-chloroindole hydrazide/hydrazone derivatives were synthesized from 5-chloroindole-3-carboxaldehyde and phenyl hydrazine derivatives. All the compounds characterized and in vitro antioxidant activity was investigated against MLT and BHT. Most of the compounds showed strong inhibitory effect on the superoxide radical scavenging assay at 1mM concentration (79 to 95%). Almost all the tested compounds possessed strong scavenging activity against the DPPH radical scavenging activity with IC ₅₀ values (2 to 60 μM). Lastly, compound 1j revealed stronger inhibitory activity against MLT in the LP inhibitory assay at 0.1mM concentration (51%) while the rest of the compounds showed moderate inhibition.

Full text of DOI:10.3109/14756366.2011.594048

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(3): 428–436
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.594048
ReseaRch aRtIcle
Synthesis and antioxidant activity evaluations of melatonin-
based analogue indole-hydrazide/hydrazone derivatives
Ayse Didem Yılmaz1, Tulay Coban2, and Sibel Suzen1
1Department of Pharmaceutical Chemistry and 2Department of Pharmaceutical Toxicology, Faculty of Pharmacy,
Ankara University 06100 Tandogan, Ankara Turkey
abstract
Melatonin (MLT) is a hormone synthesized from the pineal gland. It is a direct scavenger of free radicals, which is
related to its capability to defend cells from oxidative stress. Recently MLT-related compounds are under investigation
to establish which exhibit the maximum activity with the lowest side effects. In this study 5-chloroindole hydrazide/
hydrazone derivatives were synthesized from 5-chloroindole-3-carboxaldehyde and phenyl hydrazine derivatives.
All the compounds characterized and in vitro antioxidant activity was investigated against MLT and BHT. Most of the
compounds showed strong inhibitory effect on the superoxide radical scavenging assay at 1mM concentration (79
to 95%). Almost all the tested compounds possessed strong scavenging activity against the DPPH radical scavenging
activity with IC50 values (2 to 60 µM). Lastly, compound 1j revealed stronger inhibitory activity against MLT in the LP
inhibitory assay at 0.1mM concentration (51%) while the rest of the compounds showed moderate inhibition.
Keywords: Indole, hydrazone, melatonin, synthesis, antioxidant activity
Introduction
species including hydroxyl radical, hydrogen peroxide,
singlet oxygen, nitric oxide and peroxynitrite anion.
Synthesis of MLT-related compounds such as MLT
metabolites and synthetic analogues are under inves-
tigation to determine which exhibit the highest activity
with the lowest side effects4,5. Many antioxidant activity
studies of synthetic indole derivatives such as indole-3-
propionic acid6, indole amine-triazoles7, stobadine8 are
present. Moreover, our group formerly identified the
antioxidant activity of MLT anaologue indole derivatives
such as 2-phenylindoles9, and indole-3-propionamides10.
Recently, we observed the relationship between aldose
reductase and superoxide dismutase inhibition capaci-
ties of indole-based analogs of MLT derivatives11.
Research has proved that the indole ring in the MLT
molecule is the reactive centre dealing with oxidants due
to its high resonance stability and very low activation
energy barrier towards free radical reactions3,12,13. In our
earlier studies, new MLT-based analogues with changes
in the 5-methoxy and 2-acylaminoethyl groups of MLT
were synthesized and tested for their in vitro antioxidant
Harmful effects of free radicals to the human body have
been studied over the last decade. Overproduction of
the free radicals can be responsible for tissue injuries
that cause many health problems which include cancer,
aging, heart diseases, neurological disorders, Alzheimer’s
disease, Huntington disease and so on. Free radicals are
involved in normal physiological functions in required
concentrations but excess formation of free radicals
or/and decrease in antioxidant level leads to oxidative
stress. e human body has several systems to eliminate
the effect of oxidative stress by producing antioxidants,
which are either naturally produced, or externally sup-
plied through foods or supplements. Increasing the anti-
oxidant intake can prevent diseases and lower the health
problems1,2
.
Melatonin (MLT), the main secretory product of the
pineal gland is a well-known antioxidant and free radi-
cal scavenger. It is a neurohormone produced from the
amino acid tryptophan. Tan et al.3 described that MLT
scavenges a variety of reactive oxygen and nitrogen
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 23 May 2011; revised 01 June 2011; accepted 01 June 2011)
428

Antioxidant melatonin analogues 429
potencyintheDPPH, superoxidedismutaseandlipidper-
oxidation (LP) assays14. With a few exceptions, most of the
compoundstestedshowedsignificantantioxidantactivity
at concentrations comparable with or much higher than
that of MLT. We also found that N-methylindole deriva-
tives of MLT analogue hydrazide/hydrazone derivatives15
have protective effect against membrane lipid peroxida-
tion (LP) and determining inhibitory effect hemolysis
of human erythrocytes and showed potent antioxidant
activity, even higher than MLT.
Twenty-one MLT analogue indole hydrazide/hydra-
zone derivatives were synthesized (Figure 2) and anti-
oxidant activity was investigated in vitro by measuring
DPPH, superoxide radical scavenging and LP inhibitory
activities (Table 1). e results were compared with MLT
and BHT. All the analogue compounds except previously
synthesized 1a18 and 1u19, were characterized on the basis
1
of H and 13C NMR, Mass, FT-IR spectra and elemental
analysis.
Since the modifications on the MLT molecule resulted
in a set of compounds having different physical property
and different substitution at the indole nucleus, it was
hoped to have better SAR discussion in view of the effect
of substituents with different electronic properties on the
antioxidant activity. On the other hand, carbonyl scav-
enger activity of hydrazine/hydrazone derivatives gave
the idea that antioxidant MLT and hydrazine-containing
analogues are good candidates against LP in coopera-
tion with antioxidant and carbonyl scavenger activities.
It is known that oxidative stress induces LP of cellular
membranes resulting in the generation of reactive car-
bonyl compounds (RCC) that react rapidly with free
amino groups resulting with oxidative stress-associated
diseases16. Some of the suggested metabolism products of
synthesized MLT analogue compounds might be hydra-
zine and aldehyde derivatives. Galvani et al.17 showed that
Phenylhydrazine reacts with RCCs which facilitates the
break of the covalent bond RCC–protein, and the bind-
ing of RCCs on hydrazine, thus forming hydrazone. e
chemical combination between hydrazine and ketones or
aldehydes and the formation of hydrazones is a character-
istic of the carbonyl scavenger effect of these molecules.
All previous results showed that halogenated aro-
matic side chain resulted in much more active com-
pounds than MLT. 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. ese results prompted us to synthe-
size more indol-3-aldehyde hydrazone and hydrazide
derivatives. With this study based on MLT, new indole
imines were developed. ese chemically significant
modulations of the lead structure were made at mainly
two different points (Figure 1): the methoxy group at
the 5-position of the indole ring (modification I) and
2-N-acetylaminoethyl side chain including formation of
imine (Modification II).
experimental
Chemistry
Melting points of the compounds were determined with a
Buchi SMP-20 apparatus and uncorrected. e 1H and 13
C
NMR spectra were measured with a Varian 400MHz using
TMS internal standard and DMSO-d6 as solvent. ESI Mass
spectraweredeterminedonaWatersmicromassZQ.FT-IR
spectra were recorded on Jasco 420Fourier. Elemental
analyses were performed using CHNS-932 (LECO). HPLC
studies were determined on Waters LC Module Plus. All
spectral analysis was performed at Ankara University,
Faculty of Pharmacy, Central Laboratory. 2,2-Diphenyl-1-
picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT),
thiobarbituric acid (TBA), ascorbic acid (AA), xanthine,
xanthine oxidase, nitroblue tetrazolium (NBT), sitocrom
c, malondialdehyde (MDA) were purchased from Sigma
Chemical Co. (St Louis, MO, USA). e chemical reagents
used in synthesis were purchased from Sigma (Germany)
and Aldrich (US).
e target imines derived from 5-chloro-1H-indole-
3-carboxaldehyde
1 and appropriate hydrazine or
hydrazide derivatives using simple reaction strategies.
For the synthesis of compounds 1a-t, a similar method of
Kidwai et al.20 has been used by heating phenyl hydrazine
derivatives and compound 1 in the presence of ethanol.
e hydrazones 1u and 1v were also prepared from the
reaction of equimolar amounts of hydrazide (isonico-
tinic hydrazide for 1u, anisic acid hydrazide for 1v) with
compound 1 in ethanol. Finally, N,N’-bis-(5-chloro-1H-
indole-3-ylmethylene)hydrazine (1y) was synthesized
using equimolar amounts of hydrazine hydrate with
compound 1 in the presence of ethanol. All the new
compounds were characterized on the basis of spectral
data except 1a18 and 1u19. Compound 1u was previously
described in view of its electrochemical behaviour by our
group. 13C spectra of compounds 1g and 1h showed some
Figure 1. Modifications made on MLT molecule.
© 2012 Informa UK, Ltd.

430 A. D. Yılmaz et al.
difficulty of interpreting fluorine magnetic shielding.
ese spectra are not given.
(1H, d), 7.71 (1H, d), 8.23 (1H, d), 8.31 (1H, s), 8.98 (1H, s,
hydrazine-NH) 11.53 (1H, s, indole-NH); 13C NMR: 17.38,
20.06, 111.14, 112.54, 113.18, 120.21, 120.57, 122.03,
124.31, 125.16, 126.15, 127.04, 128.53, 130.78, 135.37,
141.63 (azomethine-C): ESI mass m/z 298 (M+1, %100),
300 (M+2); Analysis for C17H16ClN3; Calcd: C; 68.57, H;
5.42, N; 14.11. Found: C; 68.87, H; 5.08, N; 13.51. FT-IR
(KBr) cm−1 1609 C=N (azomethine) stretch band, 3316
N-H stretch band.
General procedure for the synthesis of 5-Chloro-1H-indole-3-
carboxaldehyde phenylhydrazones (1a-t)
5-Chloro-1H-indole-3-carboxaldehyde
(0.1mmol)
was reacted with phenyl hydrazine or its derivatives
(0.13mmol) in 10 ml of EtOH in the presence of 0.5 g
CH3COONa for 30 min on the hot water bath. On cooling,
the precipitate was collected and washed with cold EtOH
to give 1a-t with 10–92% yield.
5-Chloro-1H-indole-3-carboxaldehyde (2,3-dimethylphenyl)
hydrazone 1c
1
5-Chloro-1H-indole-3-carboxaldehyde (2,4-dimethylphenyl)
hydrazone 1b
Yield 80.5%, m.p 145–146°C; H NMR (400 MHz): 2.12
(3H, s), 2.23 (3H, s), 6.60 (1H, d), 7.04 (1H, t), 7.18-7.21
(1H, dd), 7.26 (1H, dd), 7.45 (1H, d), 7.72 (1H, s), 8.23 (1H,
d), 8.35 (1H, d), 9.07 (1H, s, hydrazine-NH) 11.55 (1H, s,
1
Yield 77.8%, m.p 148–149°C; H NMR (400 MHz): 2.19
(6H, d), 6.86 (1H, s), 6.96 (1H, d), 7.18-7.21 (2H, dd), 7.44
Figure 2. Synthetic pathway of melatonin analogues.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antioxidant melatonin analogues 431
Table 1. Antioxidant activity results of synthesized compounds.
Superoxide radical inhibition (%)
DPPH scavenging activity
IC50 µM
LP (% inhibition)
0.1 mM
Compoundsa
1 mM
64 3.0
P
0.1 mM
1a
P
42
60
60
19
21
55
22
20
22
2
2
20.68 0.55
31.03 0.71
34.48 0.36
37.93 0.25
37.93 0.33
20.68 0.44
41.38 0.29
44.83 0.36
41.37 0.28
51.72 0.22
37.93 0.12
41.37 0.19
37.93 0.84
27.58 0.29
27.58 0.52
31.03 0.55
31.03 0.61
34.48 0.54
34.48 0.32
44.82 0.54
27.58 0.60
41.38 0.56
46 1.4
1b
1c
P
P
P
1
1d
1e
39 7.5
7.0 1.5
NA
P
2
P
1
1f
NA
4
1g
P
P
3
1h
1i
93 2.4
45 2.0
79 2.5
88 4.8
46 1.7
93 3.0
91 2.0
89 5.2
95 1.0
86 4.5
81 5.7
88 3.5
P
P
2
NA
1
1j
NA
2
0.4
2
1k
P
24
23
15
28
20
20
30
19
24
1l
P
1
1m
1n
1o
P
2
P
NA
NA
P
1
4
1p
1r
2
5
1s
P
3
1t
P
1
1u
1v
P
650 19
—
P
25 1.2
NA
20 1.4
—
1y
P
—
Melatonin
BHT
80 1.5
—
780 25
15
2
—
P= prooxidant, NA=no activity.
aCompounds were diluted with DMSO (solvent showed no antioxidant activity).
indole-NH); 13C NMR: 12.61, 20.16, 109.42, 112.51, 113.19,
118.56, 119.93, 120.56, 122.06, 124.34, 125.15, 125.85,
128.67, 135.37, 135.71, 136.05, 143.79 (azomethine-C):
ESI mass m/z 298 (M+1, %100), 300 (M+2); Analysis for
C17H16Cl N3; Calcd: C; 68.57, H; 5.42, N; 14.11. Found:
C; 69.00, H; 5.73, N; 13.84. FT-IR (KBr) cm−1 1582 C=N
(azomethine) stretch band, 3410 N-H stretch band.
108.17, 112.83, 114.05, 121.24, 122.93, 125.26, 125.84,
130.09, 131.32, 131.42, 136.14, 136.22, 148.57, 148.68
(azomethine-C), 162.94, 165.32: ESI mass m/z 288 (M+1,
%100), 290 (M+2); Analysis for C15H11FClN3; Calcd: C;
62.62, H; 3.85, N; 14.60. Found: C; 62.71, H; 3.85, N; 13.37.
FT-IR (KBr) cm−1 1616 C=N (azomethine) stretch band,
3675 N-H stretch band.
5-Chloro-1H-indole-3-carboxaldehyde (2-fluorophenyl)
hydrazone 1d
5-Chloro-1H-indole-3-carboxaldehyde (4-fluorophenyl)
hydrazone 1f
Yield 35.4%, m.p 153–154°C; H NMR (400 MHz): 6.95-
Yield 48%, m.p 198–199°C; 1H NMR (400 MHz): 6.69-6.74
(1H, m), 7.10-7.18 (2H, m), 7.19 (1H, d), 7.22 (1H, d), 7.41-
7.47 (1H, m), 7.75 (1H, d), 8.22 (1H, s), 8.34 (1H, s), 9.82
(1H, s, hydrazine-NH) 11.59 (1H, s, indole-NH); 13C NMR:
112,94, 113,51, 114,04, 115,50, 115,68, 118,09, 118,16,
121,31, 122,94, 125,27, 125,75, 125,86, 130,08, 134,84,
134,94, 136,16, 137,94 (azomethine-C): ESI mass m/z 288
(M+1, %100), 290 (M+2); Analysis for C15H11FClN3; Calcd:
C; 62.62, H; 3.85, N; 14.60. Found: C; 62.70, H; 3.96, N;
14.11. FT-IR (KBr) cm−1 1622 C=N (azomethine) stretch
band, 3751 N-H stretch band.
1
6.98 (2H,m), 7.04-7.09 (2H, t), 7.15-7.18 (1H, dd), 7.42
(1H, s), 7.70 (1H, d), 8.06 (1H, s, -CH), 8.17 (1H, d), 9.85
(1H, s, hydrazine-NH) 11.51 (1H, s, indole-NH); 13C NMR:
112.75, 112.81, 113.06, 113.99, 116.21 116.43, 121.25,
122.87, 125.14, 125.85, 129.58, 135.29, 136.12, 143.47
(azomethine-C), 154.82, 157.13: ESI mass m/z 288 (M+1,
%100), 290 (M+2); Analysis for C15H11FClN3; Calcd: C;
62.62, H; 3.85, N; 14.60. Found: C; 62.46, H; 3.70, N; 14.44.
FT-IR (KBr) cm−1 1507 C=N (azomethine) stretch band,
3462 N-H stretch band.
5-Chloro-1H-indole-3-carboxaldehyde (3-fluorophenyl)
hydrazone 1e
Yield 35.1%, m.p 176–177°C; H NMR (400 MHz): 6.48
5-Chloro-1H-indole-3-carboxaldehyde (2,4-difluorophenyl)
hydrazone 1g
Yield 87.2%, m.p 128–129°C; H NMR (400 MHz): 7.03-
1
1
(1H, t), 6.79 (2H, d), 7.23 (2H, t), 7.46 (1H, d), 7.77 (1H,
s, -CH), 8.16 (2H, d), 10.18 (1H, s, hydrazine-NH) 11.59
(1H, s, indole-NH); 13C NMR: 98.17, 98.43, 104.25, 104.46,
7.08 (1H, m), 7.14-7.20 (2H, m), 7.33-7.43 (2H, m), 7.72
(1H, d), 8.15 (1H, d), (1H, s), 8.30 (1H, s), 9.72 (1H, s,
hydrazine-NH) 11.57 (1H, s, indole-NH); ESI mass m/z
© 2012 Informa UK, Ltd.

432 A. D. Yılmaz et al.
306 (M+1, %100), 308 (M+3); Analysis for C15H10F2ClN3;
Calcd: C; 58.93, H; 3.30, N; 13.75. Found: C; 58.87, H;
3.07, N; 13.35. FT-IR (KBr) cm−1 1517 C=N (azomethine)
stretch band, 3452 N-H stretch band.
(KBr) cm−1 1594 C=N (azomethine) stretch band, 3444
N-H stretch band.
5-Chloro-1H-indole-3-carboxaldehyde (4-chlorophenyl)
hydrazone 1l
1
5-Chloro-1H-indole-3-carboxaldehyde (2,5-difluorophenyl)
hydrazone 1h
Yield 76.8%, m.p 185–186°C; H NMR (400 MHz): 7.00
(2H, m), 7.18-7.21 (3H, m), 7.27 (1H, d), 7.45 (1H, d), 7.75
(1H, s), 8.10 (1H, s, -CH), 8.18 (1H, d), 10.06 (1H, s, hydra-
zine-NH) 11.58 (1H, s, indole-NH); 13C NMR: 112.11,
112.52, 113.24, 120.42, 120.67, 122.14, 124.42, 125.03,
128.82, 129.14, 135.20, 135.34, 144.78 (azomethine -C);
ESI mass m/z 304 (M+, %100), 306(M+2); Analysis for
C15H11N3Cl2; Calcd: C; 59.23, H; 3.65, N; 13.81. Found:
C; 59.03, H; 3.41 N; 13.62. FT-IR (KBr) cm−1 1595 C=N
(azomethine) stretch band, 3448 N-H stretch band.
1
Yield 10.4%, m.p 152–153°C; H NMR (400 MHz): 6.40-
6.45 (1H, m), 6.57-6.60 (2H, dd), 7.20-7.23 (1H, dd),
7.47 (1H, d), 7.81(1H, s, -CH), 8.14 (1H, d), 9.78 (1H, s,
hydrazine-NH) 10.40 (1H, s, indole-NH), 11.64 (1H, s,
indole-NH); ESI mass m/z 306 (M+1, %100), 308 (M+3);
Analysis for C15H10F2ClN3; Calcd: C; 58.93, H; 3.30, N;
13.75. Found: C; 58.78, H; 3.17, N; 13.48%. FT-IR (KBr)
cm−1 1517 C=N (azomethine) stretch band, 3407 N-H
stretch band.
5-Chloro-1H-indole-3-carboxaldehyde (2,4-dichlorophenyl)
hydrazone 1m
Yield 57%, m.p 197–1986°C; H NMR (400 MHz): 7.20-
5-Chloro-1H-indole-3-carboxaldehyde (3,5-difluorophenyl)
hydrazone 1i
1
Yield 90%, m.p 159–160°C; 1H NMR (400 MHz): 6.47-6.48
(1H, m), 7.07-7.23 (3H, m), 7.46 (1H, d), 7.79 (1H, s), 8.17
(1H, d), 8.36 (1H, s), 10.12 (1H, s, hydrazine-NH) 10.66
(1H, s, indolamine); 13C NMR: 92.49, 92.76, 93.02, 94.48,
94.76, 112.51, 114.13, 116.40, 121.17, 123.01, 125.39,
125.79, 130.68, 136.15, 137.33, 148.38, 148.74, 148.98,
149.14, 149.27 (azomethine -C), 150.69, 153.38, 162.92,
163.09, 165.33, 165.49; ESI mass m/z 306 (M+1, %100),
308 (M+3); Analysis for C15H10F2ClN3; Calcd: C; 58.93, H;
3.30, N; 13.75. Found: C; 59.12, H; 3.98, N; 13.47. FT-IR
(KBr) cm−1 1635 C=N (azomethine) stretch band, 3428
N-H stretch band.
7.23 (1H, dd), 7.37-7.48 (4H, m), 7.79 (1H, s, -CH), 8.15
(1H, d), 8.49 (1H, s), 9.63 (1H, s, hydrazine-NH) 11.67
(1H, s, indole-NH); 13C NMR: 112.60, 114.19, 114.51,
116.84, 121.15, 121.69, 123.09, 125.46, 125.81, 128.88,
129.27, 130.81, 136.22, 139.84, 141.80 (azomethine -C);
ESI mass m/z 338 (M+, %100), 340 (M+2, %100), 342
(M+4); Analysis for C15H10Cl3N3; Calcd: C; 53.20, H; 2.98,
N; 12.42. Found: C; 54.34, H; 2.82 N; 12.12. FT-IR (KBr)
cm−1 1596 C=N (azomethine) stretch band, 3442 N-H
stretch band.
5-Chloro-1H-indole-3-carboxaldehyde (2,5-dichlorophenyl)
hydrazone 1n
5-Chloro-1H-indole-3-carboxaldehyde (2-chlorophenyl)
hydrazone 1j
Yield 62.3%, m.p 144–145°C; H NMR (400 MHz): 6.73-
Yield 68%, m.p 171–172°C; 1H NMR (400 MHz): 6.76-
6.79 (1H, dd), 7.21-7.24 (1H, dd), 7.35 (1H, d) 7.43-7.49
(2H, m), 7.82 (1H, s), 8.18 (1H, d), 8.52 (1H, s), 9.77 (1H,
s, hydrazine-NH) 11.68 (1H, s, indole-NH); 13C NMR:
112.54, 112.93, 114.22, 114.95, 118.41, 121.21, 123.07,
125.52, 125.86, 130.95, 131.43, 133.43, 136.22, 140.15,
143.74 (azomethine -C); ESI mass m/z 338 (M+, %100),
340 (M+2, %100), 342 (M+4); Analysis for C15H10Cl3N3;
Calcd: C; 53.20, H; 2.98, N; 12.42. Found: C; 52.50, H; 2.78
N; 12.48. FT-IR (KBr) cm−1 1596 C=N (azomethine) stretch
band, 3428 N-H stretch band.
1
6.77 (1H, m), 7.20-7.23 (1H, dd), 7.31 (2H, t), 7.48 (2H,
t), 7.77 (1H, s, -CH), 8.20 (1H, d), 8.48 (1H, s), 9.48 (1H,
s, hydrazine-NH) 11.64 (1H, s, indole-NH); 13C NMR:
112.78, 113.59, 114.10, 116.40, 119.23, 121.23, 122.99,
125.34, 125.85, 128.80, 130.01, 130.38, 136.17, 138.96,
142.63 (azomethine -C); ESI mass m/z 304 (M+, %100),
306 (M+2).; Analysis for C15H11Cl2N3; Calcd: C; 59.23, H;
3.65, N; 13.81. Found: C; 59.29, H; 3.42, N; 13.67. FT-IR
(KBr) cm−1 1598 C=N (azomethine) stretch band, 3323
N-H stretch band.
5-Chloro-1H-indole-3-carboxaldehyde (3,4-dichlorophenyl)
hydrazone 1o
5-Chloro-1H-indole-3-carboxaldehyde (3-chlorophenyl)
hydrazone 1k
Yield 57.2%, m.p 123–124°C; H NMR (400 MHz): 6.69-
Yield 65%, m.p 142–143°C; 1H NMR (400 MHz): 66.89–6.92
(1H, dd), 7.15-7.20 (2H, m), 7.42 (2H, t), 7.77 (1H, d), 8.10
(2H, s, CH), 8.14 (1H, d), 10.24 (1H, s, hydrazine-NH),
11.59 (1H, s, indole-NH); 13C NMR: 112.29, 112.63, 112.77,
114.11, 118.82, 121.20, 123.00, 125.36, 125.80, 130.47,
131.64, 132.24, 136.15, 137.12, 146.63 (azomethine -C);
ESI mass m/z 338 (M+, %100), 340 (M+2, %100), 342
(M+4); Analysis for C15H10Cl3N3; Calcd: C; 53.20, H; 2.98,
N; 12.42. Found: C; 52.90, H; 2.92 N; 12.24. FT-IR (KBr)
cm−1 1593 C=N (azomethine) stretch band, 3426 N-H
stretch band.
1
6.72 (2H, tt), 6.90-6.92 (1H, dd), 7.04 (1H, s), 7.19-7.26
(2H, m), 7.77 (1H, s), 8.11 (1H, s), 8.18 (1H, d), 10.16 (1H,
s, hydrazine-NH) 11.59 (1H, s, indole-NH); 13C NMR:
110.69, 111.27, 112.83, 114.09, 117.68, 121.27, 122.96,
125.30, 125.86, 130.16, 131.46, 134.54, 136.15, 136.43,
148.08 (azomethine -C); ESI mass m/z 304 (M+, %100),
306(M+2); Analysis for C15H11Cl2N3; Calcd: C; 59.23, H;
3.65, N; 13.81. Found: C; 59.29, H; 3.48 N; 13.52. FT-IR
Journal of Enzyme Inhibition and Medicinal Chemistry

Antioxidant melatonin analogues 433
1y; 0.1 mmol) in 50 mL of EtOH was heated for 2.5 h (4h
for 1y) on the hot water bath. On cooling, the precipitate
was collected and washed with cold EtOH to give 1u-y
with 20–45% yield.
5-Chloro-1H-indole-3-carboxaldehyde (3,5-dichlorophenyl)
hydrazone 1p
Yield 61%, m.p 168–169°C; 1H NMR (400 MHz): 6.79
(1H, t), 6.95 (2H, d), 7.20-7.23 (1H, dd), 7.47 (1H, d), 7.82
(1H, s), 8.15 (2H, t), 10.37 (1H, s, hydrazine-NH), 11.64
(1H, s, indole-NH); 13C NMR: 109.36, 111.71, 113.38,
116.03, 120.40, 122.24, 124.64, 125.03, 129.96, 134.54,
135.37, 136.95, 147.90 (azomethine-C); ESI mass m/z
338 (M+, %100), 340 (M+2, %100), 342 (M+4); Analysis
for C15H10Cl3N3; Calcd: C; 53.20, H; 2.98, N; 12.42. Found:
C; 52.90, H; 2.92 N; 12.24. FT-IR (KBr) cm−1 1594 C=N
(azomethine) stretch band, 3453 N-H stretch band.
N-(4-methoxybenzoyl)-N’-(5-chloro-1H-indolyl-3-methylene)
hydrazone 1v
Yield 42%, m.p 249–250°C; 1H NMR (400 MHz): 3.04
(3H, s), 7.07 (2H, d), 7.21-7.23 (1H, dd), 7.47 (1H, s),
7.92 (3H, t), 8.33 (1H, d), 8.59 (1H, s, -CH), 11.49 (1H, s,
hydrazine-NH), 11.77 (1H, s, indole-NH); 13C NMR: 56.10,
112.26, 114.10, 114.37, 121.87, 123.28, 125.69, 126.04,
126.61, 130.02, 132.34, 136.21, 144.53 (azomethine -C),
162.45 (C=O), 162.71; ESI mass m/z 328 (M+1, %100), 330
(M+3); Analysis for C17H14ClN3O2; Calcd: C; 62.30, H; 4.31,
N; 12.82. Found: C; 62.82, H; 4.60 N; 12.09. FT-IR (KBr)
cm−1 1647 C=N (azomethine) stretch band, 3330 N-H
stretch band.
5-Chloro-1H-indole-3-carboxaldehyde (2-bromophenyl)
hydrazone 1r
Yield77.3%, m.p183–184°C;1HNMR(400 MHz):6.64-6.68
(1H, m), 7.18-7.21 (1H, dd), 7.30-7.34 (1H, m), 7.44-7.47
(3H, m), 7.74 (1H, s, -CH), 8.19 (1H, d), 8.48 (1H, s), 9.21
(1H, s, hydrazine-NH), 11.5 (1H, s, indole-NH); 13C NMR:
106.41, 112.77, 114.08, 114.12, 119.99, 121.24, 123.02,
125.40, 125.87, 129.34, 130.39, 133.24, 136.18, 139.14,
143.62 (azomethine-C); ESI mass m/z 348 (M+, %100),
350 (M+2, %100), 352 (M+4); Analysis for C15H11ClBrN3;
Calcd: C; 51.68, H; 3.18, N; 12.05. Found: C; 51.32, H; 3.22
N; 11.85. FT-IR (KBr) cm−1 1588 C=N (azomethine) stretch
band, 3447 N-H stretch band.
N,N’-bis-(5-chloro-1H-indole-3-ylmethylene)hydrazine 1y
1
Yield 45.4%, m.p 285–286°C; H NMR (400 MHz): 7.23-
7.26 (2H, dd), 7.51 (2H, d), 8.02 (2H, s, -CH), 8.35 (2H, d),
8.93 (2H, s), 11.90 (2H, brs, indole-NH); 13C NMR: 112.34,
114.27, 121.85, 123.29, 125.89, 126.41, 134.20, 136.33,
155.95 (azomethine -C); ESI mass m/z 355 (M+, %100),
357 (M+2, %100), 359 (M+4); Analysis for C18H12Cl2N4;
Calcd: C; 60.86, H; 3.41, N; 15.77. Found: C; 60.75, H; 3.28
N; 13.48. FT-IR (KBr) cm−1 1646 C=N (azomethine) stretch
band, 3420 N-H stretch band.
5-Chloro-1H-indole-3-carboxaldehyde (3-bromophenyl)
hydrazone 1s
Yield 92%, m.p 133–134°C; 1H NMR (400 MHz): 6.82-6.85
(1H, dd), 6.93-6.96 (1H, m), 7.15-7.22 (3H, m), 7.46 (1H,
d), 7.77 (1H, d), 8.10 (1H, s, -CH), 8.18 (1H, d), 10.14 (1H,
s, hydrazine-NH), 11.59 (1H, s, indole-NH); 13C NMR:
111.02, 112.81, 114.07, 114.18, 120.53, 121.26, 122.94,
123.18, 125.28, 125.83, 130.14, 131.75, 136.13, 136.43,
148.18 (azomethine-C); ESI mass m/z 348 (M+, %100),
350 (M+2, %100), 352 (M+4); Analysis for C15H11ClBrN3;
Calcd: C; 51.68, H; 3.18, N; 12.05. Found: C; 51.34, H; 3.03
N; 11.84. FT-IR (KBr) cm−1 1591 C=N (azomethine) stretch
band, 3446 N-H stretch band.
Measurement of e/Z isomer ratio
Mass spectra showed that compounds 1d, 1j and 1r which
have o-halogenated phenyl ring in the molecule exist as E
and Z isomers. e isomers could not separated by chro-
matographic methods. However the E/Z isomer ratio was
measured by HPLC. Literature data21,22 indicated that the
major product might be (E)-isomer. erefore (E)-isomer
was assumed as major product (Compound 1d 99.5%, 1j
99.9% and 1r 91.71); the minor (Z)-isomer was detected
in small amounts (Compound 1d 0.5%, 1j 0.09% and 1r
8.29).
5-Chloro-1H-indole-3-carboxaldehyde (4-bromophenyl)
hydrazone 1t
A
high-performance liquid chromatographic
Yield 80.3%, m.p 167–168°C; 1H NMR (400MHz): 6.93
(2H, d), 7.16-7.19 (1H, dd), 7.34-7.43 (3H, m), 7.72 (1H,
d), 8.08 (1H, s, -CH), 8.15 (1H, d), 10.05 (1H, s, hydra-
zine-NH), 11.55 (1H, s, indole-NH); 13C NMR: 108.96,
112.90, 113.87, 114.04, 121.22, 122.94, 125.24, 129.97,
132.43, 136.09, 136.13,145.92 (azomethine -C); ESI mass
m/z 348 (M+, %100), 350 (M+2, %100), 352 (M+4); Analysis
for C15H11ClBrN3; Calcd: C; 51.68, H; 3.18, N; 12.05. Found:
C; 51.35, H; 3.04 N; 11.99. FT-IR (KBr) cm−1 1594 C=N
(azomethine) stretch band, 3422 N-H stretch band.
(HPLC) method was established for the enantio-
meric determination21,23 of compounds 1d, 1j and 1r.
Experimentation was achieved under normal-phase
chiral stationary phase liquid chromatography using the
Chiralpak AS chiral column (250mm×4.6mm). e opti-
mizedchromatographicconditionsweren-hexane-isopro-
pyl alcohol (87:13:1) as the mobile phase with a flow rate of
1mL/min, and detection at the wavelength of 254nm. e
column temperature was set at room temperature.
In vitro antioxidant activity studies
General procedure for the synthesis of compounds (1u-y)
A solution 5-chloro-1H-indole-3-carboxaldehyde (0.05
mmol) and izonicotinic acid hydrazide (for 1u) or anisic
acid hydrazide (for 1v; 0.05 mmol), hydrazine hydrate (for
All the MLT analogue indole hydrazide/hydrazone deriv-
atives were subjected to test DPPH, superoxide radical
scavenging and anti LP activities. All the results were
compared with MLT and BHT.
© 2012 Informa UK, Ltd.

434 A. D. Yılmaz et al.
the Guide for the Care and Use of Laboratory Animals.
e livers were removed immediately and washed in ice-
cold distilled water, and homogenized straight away with
teflon homogenizer in ice chilled. LP was measured spec-
trophotometrically by estimation of thiobarbituric acid
reactive substances (TBARS). Amounts of TBARS were
expressed in terms of mmol malondialdehyde (MDA)/g
tissue. A typical optimized assay mixture contained
0.5 mL of liver homogenate, 0.1mL of Tris-HCl buffer (pH
7.2), 0.05 mL of 0.1 mM ascorbic acid, 0.05 mL of 4 mM
FeCl2 and 0.05 mL of various concentration of synthesized
compounds, or MLT, were incubated for 1 h at 37°C. After
incubation, 3.0 mL of H3PO4 and 1mL of 0.6% TBA were
added and shaken vigorously. e mixture was boiled for
30 min. After cooling, the absorbance of the supernatant
was read at 532 nm against a blank, which contained
all reagents except liver homogenate. MLT was used as
positive control. Lipid peroxidation inhibitory activity is
expressed as follows:
DPPH free radical scavenging activity
e free radical scavenging activities of MLT analogues
were tested by their ability to bleach the stable radical
2,2,diphenyl-1-picrylhydrazyl (DPPH)24. e stock solu-
tions of the compounds were prepared at 10−2 M concen-
tration in DMSO. A series of stock solution in DMSO were
diluted to varying concentrations in 96-well microplates.
en, methanolic DPPH solution (100 µM) was added
to each well. e plate was shaken and placed into the
dark. After 30 min, the optical density of the solution was
read at the wavelength 517 nm. e methanolic solution
of DPPH served as a control. Percentage inhibition was
calculated using the following formula:


Radical scavenging
activity %
A
Control − ASample
AControl






=
× 100






Where: Acontrol: absorption of blank sample; Asample
absorption of tested solution
:
A dose response curve was plotted to determine the
IC50 values. IC50 is defined as the concentration sufficient
to obtain 50% of a maximum scavenging capacity. All
tests and analyses were run in triplicate and averaged.
e standard used in this assay was MLT and BHT.
A
Control − ASample
Lipid peroxidation
(
)
=
× 100
inhibitory activity (%)
A
Control − Ablank
(
)
Where, Acontrol is the absorbance of the control, Asample is the
absorbance of the sample and Ablank is the absorbance of
the blank, to which the sample and the free radical gener-
ating system (Fe+2/ascorbate) were not added. MLT was
used as positive controls.
Superoxide radical scavenging activity
e capacity of the examined MLT analogues to scavenge
superoxide anion formation was determined spectro-
photometrically. Superoxide was generated by xanthine/
xanthine oxidase and measured by the inhibition of cyto-
chrome c reduction25,26. 100 μl of 4 mM xanthine, 400 μl
sitocrom c, 50mL of 50mM phosphate buffer (pH 7.8,
1mM EDTA) and 10 mL of the test compounds were pre-
pared in a 96 well plate, and 40 μl of xanthine oxidase was
added to each mixture. e absorbance of each reaction
mixture was monitored at 550 nm. Superoxide radical
scavenging activity (%) was expressed as the degree of
sitocrom c reduction decrease of the test group versus the
control group after 1 min. Superoxide radical scavenging
capacity was calculated as follows:
Results
e synthesized compounds were tested for their anti-
oxidant activities using DPPH and superoxide radical
scavenging and LP inhibitory activity tests. All the results
were compared with standard antioxidant MLT and BHT.
e results are shown in Table 1.
Ten of the synthesized compounds showed strong
inhibitory effect on the superoxide radical scavenging
assay at 1 mM concentration (79 to 95%), with the excep-
tion of compounds 1d, 1e, 1i, 1l and the ones that have
pro-oxidant activity (1b, 1c, 1g, 1u, 1v and 1y). Rest of the
tested compounds displayed higher antioxidant activity
then that of MLT. Compound 1p demonstrated the best
inhibitory activity with 95%. Compounds 1m, 1h, 1n,
1o, 1k, 1t, 1r, 1s and 1j followed compound 1p with the
inhibitory concentrations of 93, 93, 91, 89, 88, 88, 86 and
79%, respectively. Most of the compounds showed pro-
oxidant activity at 0.1 mM concentration.
Almost all the tested compounds possessed strong
scavenging activity against the DPPH radical scaveng-
ing activity with IC50 values (2 to 60 µM). Compound
that have anisic acid hydrazide 1v in the 3rd position
of indole ring showed no free radical scavenging effect
while Compound that have nicotinic acid hydrazide 1u
in the 3rd position showed the lowest scavenging activ-
ity. Compound 1j established the best inhibitory activity
with 2 µM IC50 value. Rest of the compounds showed very
strong antioxidant activity compare to MLT. Compound
A
Control − ASample
Superoxide radical
(
)
=
× 100
scavenging activity(%)
A
Control − Ablank
(
)
Where, A control is the absorbance of the control, in which
the sample was not treated, A is the absorbance of
sample
test sample which the sample was treated, and A
is
the absorbance of blank, to which the sample anbdlantkhe
Each experiment was triplicated. MLT was used as posi-
tive controls.
Assay of lipid peroxidation
e effect of synthesised MLT analogues on rat liver
homogenate induced with FeCl2-ascorbic acid and LP
was determined by the method of modified Mihara et al.27
Wistar rats (200–225 g) were fed with standard laboratory
rat chow and tap water. e animals were starved for 24 h
prior to sacrifice and then killed by decapitation under
anaesthesia. e study was carried out in accordance with
Journal of Enzyme Inhibition and Medicinal Chemistry

Antioxidant melatonin analogues 435
1j showed stronger antioxidant activity compare while
compound 1m exhibited similar activity to BHT. Rest
of the compounds which showed significantly good
activity against MLT showed similar DPPH scavenging
activity to BHT a well-known antioxidant with 15 µM
IC50 value.
Lastly, compound 1j revealed stronger inhibitory
activity against MLT in the LP inhibitory assay at 0.1mM
concentration (51%). Compounds 1h, 1u, 1g, 1l, 1i, 1m,
1e, 1d, 1s and 1t followed compound 1j with the moder-
ate inhibitory concentration of 44, 44, 41, 41, 41, 37, 37,
37, 34 and 23%, respectively.
MLT has redox properties because of the presence
of an electron-rich aromatic ring system, which allows
the indoleamine to easily function as an electron
donor12,13,31. It is possible that making the indole ring
more stable electronically helped to act as a better
electro donor. MLT scavenges the radicals via nitrogen
centred radical, the indolyl (or melatonyl) cation radi-
cal32. Introduction of an imine group in to the side chain
increased the stability of the indole molecule by helping
the delocalization of the electrons. is might help to
have high free radical scavenging activity in the synthe-
sized compounds.
Discussion
conclusions
is study was performed to investigate the potential
in vitro antioxidant effects of MLT-based analogues of
indole hydrazide/hydrazone derivatives. A series of
indole derivatives, with changes in the 5-methoxy and
2-acylaminoethyl groups of MLT were synthesized and
tested for their in vitro antioxidant potency in the DPPH,
superoxide dismutase and LP assays.
The present work aimed to synthesize, characterize
and investigate the potential antioxidant effects of
indole-based MLT analogue hydrazide/hydrazone
derivatives.
is study proposes a new approach for the in vitro
antioxidant activity properties and structure activity
relationship of 3- and 5-substituted indole ring as MLT
analogue.
Most of the compounds tested showed significant
antioxidant activity at concentrations comparable with
or much higher than that of MLT in DPPH scaveng-
ing and LP assays. e only exception was in DPPH
assay, compounds that have anisic acid hydrazide
1v and 5-chloro-1H-indole 1y in the 3rd position of
indole ring. ese compounds were also found pro-
oxidant in superoxide radical scavenging activity assay.
is finding was found in agreement with our earlier
It is possible that the synthesized compounds may
undergo some hydrolysis under the in vivo conditions
of the LP assay. However, in our earlier 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 ring12,13,19,31. Regardless
of their medicinal importance, few biological systems
metabolize hydrazides and hydrazones and not much
is known about those that attack the double bond of
the hydrazone groups. Peptidylglycine hydroxylase (EC
1.14.17.3)33 and glutamine transaminase (EC 2.6.1.15)34
are the recognized enzymes that react with hydrazones.
Since hydrazones are not their physiological substrates,
how biological systems employ hydrazones remains
unclear. Hydrazones can be chemically hydrolyzed to
relevant hydrazines and carbonyl compounds under
acidic conditions. However, the biological system
metabolizes hydrazones via the more complex NAD+-
dependent oxidation reaction35. is is a remarkable dif-
ference between the chemical and biological systems for
degrading hydrazones.
In conclusion, majority of synthesized indole deriva-
tives related to MLT showed significant antioxidant
activity in three in vitro assays. In general, lack of meth-
oxy group and introduction of Cl in the 5-position did
not effect 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. Introduction of hydrazide or hydrazone side
chain containing aromatic halogenated ring increased
the antioxidant activity of indoles comparing to MLT.
is may be due to increased stability of the indole ring
and delocalization of the electrons to help to scavenge
free radicals. For the antioxidant activity of melatonin
analogue indole derivatives, not only the indole ring is
studies14,15
.
Replacement the 5-methoxy group of MLT by Cl which
have different electronic and lipophilic properties helped
to investigate the role of structure antioxidant activity
relationships of the synthesized compounds. e real
function of methoxygroup in 5-position of the indole ring
of MLT is still under investigation. Although by replace-
ment of the methoxy group, the antioxidant capacity of
the molecule may be enhanced3, we did not observed
significant difference between compounds bearing Br
or H in the 5th position of the indole ring in our earlier
studies14,15. In this study, addition of Cl in the 5th posi-
tion did not change the earlier suggestions. Similar to our
findings, Poeggeler et al.29,30 showed that the MLT deriva-
tives bearing Cl in the 5th position performed parallel
activity to MLT.
According to the DPPH scavenging and LP assays
results, although the most active compound was 1j (o-Cl
substitution on phenyl), in generally the active com-
pounds were established as di-flouro substituted (1g-1i)
in LP assay, di-flouro and di-chloro substituted (1g-1p)
in DPPH assay. e results of two assays were found simi-
lar but the superoxide dismutase assay showed some dif-
ferences. e best activity was observed with compound
1p (3,5 dichloro substitution on phenyl) but there was
no significant activity obtained with compound 1a-1g in
superoxide dismutase assay.
© 2012 Informa UK, Ltd.

436 A. D. Yılmaz et al.
15. Shirinzadeh H, Eren B, Gurer-Orhan H, Suzen S, Ozden S. Novel
indole-based analogs of melatonin: Synthesis and in vitro anti-
oxidant activity studies. Molecules 2010;15:2187–2202.
16. 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.
17. Galvani S, Coatrieux C, Elbaz M, Grazide MH, iers JC, Parini A
et al. Carbonyl scavenger and antiatherogenic effects of hydrazine
derivatives. Free Radic Biol Med 2008;45:1457–1467.
18. Bulatova NN, Suvorov NN. Indole derivatives XXXVI. Reaction
of 3-beta-nitrovinylindoles with nucleophilic reagents. Khim.
Geterosikli. Soedin 1969;5:813–817.
19. Shirinzadeh H, Yilmaz AD, Gumustas M, Suzen S, Ozden S,
Ozkan SA. Electrochemical behavior of indole-3-carboxaldehyde
izonicotinoyl hydrazones: Discussion on possible biological
behavior. Com Chem High T Synt 2010;13:619–627.
important, but so is the side chain containing the amide
group.
acknowledgements
is work was supported by e Scientific and
Technological Research Council of Turkey (TÜBİTAK)
Research and Development Grant 109S099.
Declaration of interest
e authors report no conflicts of interest. e authors
alone are responsible for the content and writing of the
paper.
20. 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.
21. Soltani Rad MN, Khalafi-Nezhad A, Behrouz S. Synthesis of some
novel hydrazono acyclic nucleoside analogues. Beilstein J Org
Chem 2010;6:49.
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