Novel indole-based melatonin analogues substituted with triazole, thiadiazole and carbothioamides: studies on their antioxidant, chemopreventive and cytotoxic activities

Article abstract of DOI:10.3109/14756366.2015.1132209

Melatonin (MLT) is a well-known free-radical scavenger, involving in the prevention of cellular damage that can lead to cancer, ageing and a variety of neurodegenerative diseases. Research on MLT-related compounds has been required to optimise the maximum

Full text of DOI:10.3109/14756366.2015.1132209

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Novel indole-based melatonin analogues
substituted with triazole, thiadiazole and
carbothioamides: studies on their antioxidant,
chemopreventive and cytotoxic activities
Hanif Shirinzadeh, Elif Ince, Andrew D. Westwell, Hande Gurer-Orhan & Sibel
Suzen
To cite this article: Hanif Shirinzadeh, Elif Ince, Andrew D. Westwell, Hande Gurer-
Orhan & Sibel Suzen (2016): Novel indole-based melatonin analogues substituted with
triazole, thiadiazole and carbothioamides: studies on their antioxidant, chemopreventive
and cytotoxic activities, Journal of Enzyme Inhibition and Medicinal Chemistry, DOI:
10.3109/14756366.2015.1132209
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1132209
Published online: 08 Jan 2016.
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
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2016 Taylor & Francis. DOI: 10.3109/14756366.2015.1132209
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RESEARCH ARTICLE
Novel indole-based melatonin analogues substituted with triazole,
thiadiazole and carbothioamides: studies on their antioxidant,
chemopreventive and cytotoxic activities
Hanif Shirinzadeh1, Elif Ince2, Andrew D. Westwell3, Hande Gurer-Orhan2, and Sibel Suzen4
2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Erzincan University, Yalnizbag Yerleskesi, Erzincan, Turkey, Department of
3
Pharmaceutical Toxicology, Faculty of Pharmacy, Ege University, Izmir, Turkey, School of Pharmacy and Pharmaceutical Sciences, Cardiff
4
University, Cardiff, Wales, UK, and Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara, Turkey
Abstract
Keywords
Melatonin (MLT) is a well-known free-radical scavenger, involving in the prevention of cellular
damage that can lead to cancer, ageing and a variety of neurodegenerative diseases. Research
on MLT-related compounds has been required to optimise the maximum pharmaceutical
activity with the lowest side effects. In our ongoing research, we have synthesized new indole-
based MLT analogues as potential antioxidant agents by modifying the MLT molecule. In this
study, we build on previous findings, through the synthesis, characterization and in vitro
antioxidant profiling of a series of new indole-based MLT analogues which possess triazole,
thiadiazole and carbothioamides on the third position on the indole ring. In vitro antioxidant
activity was investigated by evaluating their reducing effect against oxidation of a redox
sensitive fluorescent probe and their radical scavenging activity was assessed via the DPPH
assay. In addition, in vitro cytotoxic effects of newly synthesized compounds were investigated
in CHO-K1 cells using the MTT assay.
Antioxidant, antioxidant activity, cytotoxicity,
indole, melatonin
History
Received 20 September 2015
Revised 2 December 2015
Accepted 2 December 2015
Published online 7 January 2016
Introduction
high concentrations of ROS. Also MLT acts in the fixation of
oxidant-antioxidant balance in mitochondria19,20. However, MLT
has very short half-life in the human body, of approximately 1 h in
blood following oral administration. Therefore, the synthesis of
MLT analogues with longer half-lives is an interesting topic for
Reactive oxygen species (ROS), and reactive nitrogen species
(RNS), are products of cellular metabolism, and are well known
for having both a harmful and beneficial role1,2. Overproduction
of reactive species causes oxidative stress3 which may lead to
researchers20,21
.
serious damage to vital cell structures like proteins and DNA4,5
.
Recent research has shown that compounds with an indole ring
such as indol-3-propionic acid22, indole amine triazole23 and
indolehydrazide/hydrazone derivatives24,25 have significant anti-
oxidant effects. The indole ring of these compounds is the reactive
centre dealing with oxidants due to its high resonance stability
and low activation energy barrier towards free radical reac-
tions24,26. In addition to these compounds, there are some studies
that demonstrate the antioxidant effect of 1,2,4-triazoles and
1,3,4-thiadiazoles27–29. The triazole molecule is one of the most
significant and well recognized heterocycles which is a common
feature of a selection of natural products as well as pharmaceut-
ical agents. Triazoles and their derivatives were investigated in
many studies and established to be associated with a variety of
biological activities like antibacterial30, antifungals31, antitumor,
anti-inflammatory, analgesic32 and antioxidant33. In addition this
1,3,4-thiadiazoles have also been found to be key group of
molecules with biological activity particularly antibacterial and
antioxidant34. The broad spectrum activities of 1,2,4-triazoles
derivatives have attracted the attention of researchers in recent
years. Until now, studies showed that modifications of the triazole
derivatives have proven highly efficient with better potency and
lesser toxicity. Based on the above findings it was planed and
Oxidative stress has been associated with various diseases such as
cancer6, heart diseases7, neurological disorders such as
Alzheimer’s disease8, Huntington’s disease9,10 and ageing11.
Melatonin (MLT) is a hormone primarily secreted by the pineal
gland along with many other parts of the human body11–13 and has
a significant anti-oxidant property14. MLT (Figure 1) and its
metabolites are able to function as endogenous free-radical
scavengers and broad-spectrum antioxidants15,16. In addition,
MLT and its derivatives have been shown to exhibit the regulation
of circadian rhythm and immune functions in many physiological
processes and therapeutic functions17,18. In recent years, the many
physiological properties of MLT have stimulated much interest in
the development of synthetic compounds possessing the indole
ring. In addition MLT is a highly lipophilic compound that easily
passes through cell membranes to reach intracellular compart-
ments, especially mitochondria which are well-known for having
Address for correspondence: Assist. Prof. Hanif Shirinzadeh, Department
of Pharmaceutical Chemistry, Faculty of Pharmacy, Erzincan University,
Yalnizbag Yerleskesi 24100, Erzincan, Turkey. E-mail: hanif.shirinzade@
gmail.com

2
H. Shirinzadeh et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
O
S
Figure 1. Modifications on MLT molecule to
develop new indole-based analogues.
NH NH
NH
R
(A)
N
H
1a-h
N
N
CH
H
N
3
N
R
SH
CH O
3
(B)
O
N
H
N
H
3a-h
Melatonin
N
N
S
NH
(C)
R
N
H
2a-h
synthesized some indole derivatives with 1,2,4-triazole and 1,3,4- the 5-position of the indole ring as modification 1 and the
thiadiazole with different side chains to improve biological acetylaminoethyl side chain was replaced by hydrazinecarbothioa-
activity of indole derivatives.
mide (1a–h), thiadiazole (2a–h) and triazole (3a–h) groups as
In the present study, some modifications were made on the modification 2. Finally, S-alkylation on the compounds (3a–h)
MLT molecule to improve its antioxidant effect. The acetylami- provided analogues (4a–g) (Table 1). These modifications
noethyl side chain in the third position of the indole ring was resulted in a new series of compounds having different hetero-
removed and substituted with hydrazinecarbothioamide, triazole cyclic groups as side chain in the indole nucleus. These
and thiadiazole groups. As a part of our ongoing study 31 new modifications helped to investigate the effect of substituents
compounds were synthesized using four different routes. with different electronic and lipophilic properties of heterocyclic
Synthesis of 1a–h and 3a–h compounds demonstrated high groups on the antioxidant activity and biological half-life of new
yield with average of 87%. However, synthesis of 4a–g and 2a–h indole derivatives. Synthetic routes to new MLT analogues are
compounds showed low yield, respectively, with 28% and 37%. outlined in Scheme 1. Reaction of indole 3-methylhydrazine with
Biological activity of new synthesis compounds was investigated isothiocyanates in ethanol under reflux gave the corresponding
in vitro. The MTT assay was performed to evaluate their cytotoxic hydrazinecarbothioamides (1a–h) in high yields. Treatment of
profiles, the DCFH-DA (20,70-dichlorodihydrofluorescein diace- 1a–h under strongly acidic conditions proceeded with full
tate) assay was carried out for revealing the antioxidant activity of regiochemical control to gives the corresponding 2-aminothia-
the newly synthesized compounds, and the DPPH (2,2-diphenyl- diazoles (2a–h), albeit in low to moderate yields. Conversely,
1-picrylhydrazyl) free radical scavenging activity test. All the treatment of 1a–h under basic conditions (aq. NaOH) with heating
synthesized compounds except those formerly synthesized (2d35, produced the 2-thiotriazoles (3a–h) in high yields with full
1
2h36, 3c36, 3d36) were characterized on the basis of H and 13C control of cyclization regiochemistry. Triazoles (3a–h) could be
NMR, mass spectra and elemental analysis.
further alkylated under basic conditions to produce substituted
triazole 4a–g in low to moderate yield.
Materials and methods
Chemistry – experimental
The present study aimed to synthesize, characterize and
investigate the potential antioxidant and cytotoxic effects of Uncorrected melting points were determined with a Bu¨chi melting
indole-based MLT analogues containing hydrazinecarbothioa- point B-540 apparatus (Buchi Labortechnik, Flawil, Switzerland).
mide, triazole and thiadiazoles as side chains attached to the The 1H and 13C NMR spectra were measured with a Varian
3-position of the indole ring. Two parts of the MLT molecule 400 MHz instrument (Palo Alto, CA) using TMS as an internal
were modified to develop new indole-based MLT analogue standard and DMSO-d6 as solvent. ESI mass spectra were
compounds as shown in Figure 1. These modifications were done determined on a Waters Micromass ZQ. Elemental analyses
mainly on the MLT acylamino group (Figure 1).
were performed using
a
CHNS-932 instrument (Leco
These modulations of the lead structures were made at two Corporation, St. Joseph, MI). All spectral analysis was performed
different points: the methoxy group was replaced with hydrogen at at Central Laboratory of the Faculty of Pharmacy, Ankara

DOI: 10.3109/14756366.2015.1132209
Novel indole-based melatonin analogues
3
Table 1. Chemical structures of new indole-based MLT analogues.
N
N
N
N
N
N
S
O
R1
NH
R
SH
S
N
S
NHNH
NH
R
N
R
R
N
H
N
H
N
H
(1a-h)
N
(2a-h)
(3a-h)
(4a-g)
H
R
R
R
R
R1
O
CH -CH -
CH -CH -
CH -CH -
CH -CH -
1a
2a
3a
4a
3
2
3
2
3
2
3
2
NH
CH
CH
2
CH -CH -CH -
CH -CH -CH -
CH -CH -CH -
CH -CH -CH -
O
1b
1c
2b
2c
3b
3c
4b
4c
3
2
2
3
2
2
3
2
2
3
2
2
NH
NH
2
O
H
CH
2
F
O
1d
1e
1f
2d
2e
2f
3d
3e
3f
4d
4e
4f
NH
NH
NH
NH
CH
2
F
F
F
F
O
O
CH
CH
2
F
F
F
F
F
F
F
F
2
O
Cl
Cl
Cl
Cl
1g
2g
3g
4g
CH
2
Cl
Cl
Cl
Cl
Cl
Cl
Cl
1h
2h
3h
University. Chromatography was carried out using Merck silica
2-(2-(1H-Indol-3-yl)acetyl)-N-ethylhydrazincarbothioamide
gel 60 (230–400 mesh ASTM). The chemical reagents used in (1a). Yield 95%, m.p. 132 ꢀC; 1H NMR: ꢀ 1.01 (t, 3H,
synthesis were purchased from Sigma (Germany) and Aldrich J ¼ 7.6 Hz, CH3); 3.42 (q2H, CH2); 3.55 (s, 2H, Ar–CH2); 6.96
(USA).
(m, 1H, H-5); 7.05 (m, 1H, H-6); 7.21 (d, 1H, J ¼ 2.4 Hz, H-2);
7.32 (d, 1H, J ¼ 8 Hz, H-7); 7.55 (d, 1H, J ¼ 7.6 Hz, H-4); 7.78,
9.16, 9.84, 10.88 (s, 4H, NH–NH–CS–NH and NH indole); 13C
NMR: ꢀ 14.87, 31.07, 38.85, 108.44, 110.00, 111.73, 118.76,
119.19, 121.42, 124.38, 127.65, 136.46, 170.84 (C¼O); ESI
MS m/z 277 (M + H, 100%); Anal. calcd. for C13H16N4OS: C,
55.59%; H, 5.92%; N, 19.95%. Found: C, 55.21%; H, 6.24%;
N, 19.66%.
2-(2-(1H-Indol-3-yl)acetyl)-N-propylhydrazincarbothioamide
(1b). Yield 86%, m.p. 143.5 ꢀC; 1H NMR: ꢀ 0.80 (t, 3H,
J ¼ 7.2 Hz, CH3); 1.46 (m, 2H, CH2); 3.37 (t, 2H, J ¼ 7.6 Hz,
NH–CH2); 3.57 (s, 2H, COCH2); 6.98 (t, 1H, J ¼ 7.6 Hz, H-5);
General procedure for the synthesis of hydrazinecarbothioamide
compounds 1a–h
Equal amounts of 2-(1H-indol-3-yl) acetohydrazide (5 mmol) and
aryl or alkylisothiocyanates (5 mmol) were dissolved in absolute
ethanol (20 ml). The mixture was heated under reflux for 4–5 h at
80–85 ꢀC. After completion of the reaction, the reaction mixture
was concentrated on the rotary evaporator under reduced pressure
and kept overnight at room temperature. The crystals thus
obtained were purified by washing with petroleum ether35.

4
H. Shirinzadeh et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
O
NHNH
2
N
H
Abs.EtOH
80-85°C / 5h
R N C S
NH
N
S
S
N
R
O
Aq.NaOH
80-85°C / 4h
NH
NHNH
R
N
H
N
H
(1a-h)
N
N
SH
N
R
Conc.H SO
0-5°C / 2h
2
4
N
H
N
N
(3a-h)
NH
S
R
Abs.EtOH
KOH / 3h
X R
N
H
1
N
(2a-h)
N
R
1
S
N
R
N
H
(4a-g)
Scheme 1. Synthetic route to obtain new indole-based melatonin analogue compounds.
7.07 (t, 1H, J ¼ 8.4 Hz, H-6); 7.23 (d, 1H, J ¼ 2.4 Hz, H-2); 7.34 124.45, 125.44, 126.04, 127.71, 128.55, 136.50, 139.57, 167.71,
(d, 1H, J ¼ 8 Hz, H-7); 7.57 (d, 1H, J ¼ 7.6 Hz, H-4); 7.75; 9.18; 171.01 (C¼O), 181.33 (S¼C); ESI MS m/z 325 (M + H, 100%);
9.87; 10.90; (s, 4H, NH-NH-CS-NH and NH-indole); 13C NMR: ꢀ Anal. calcd. for C17H16N4OS: C, 62.94%; H, 4.97%; N, 17.27%.
11.60, 22.40, 31.10 45.72 (NH–C); 108.46, 111.75, 118.77, Found: C, 63.05%; H, 5.33%; N, 17.05%.
119.19, 121.44, 124.40, 127.65, 136.49, 170.86 (C¼O), 175.28
2-(2-(1H-Indol-3-yl)acetyl)-N-(2,4-difluorophenyl)hydrazin-
1
(C¼S); ESI MS m/z 291 (M + H, 100%); Anal. calcd. for carbothioamide (1e). Yield 81%, m.p. 169 ꢀC; H NMR: ꢀ 3.64
C14H18N4OS: C, 57.91%; H, 6.24%; N, 19.29%. Found: C, (s, 2H, Ar–CH2); 6.98–7.61 (m, 8H, Ar–H); 9.60 (s, 1H, NH
58.19%; H, 6.75%; N, 19.14%.
indole); ESI MS m/z 361 (M + H, 100%); Anal. calcd. for
2-(2-(1H-Indol-3-yl)acetyl)-N-benzylhydrazincarbothioamide C17H14F2N4OS: C, 56.66%, H, 3.92%, N, 15.55%. Found: C,
(1c). Yield 78%, m.p. 142.5 ꢀC; 1H NMR: ꢀ 3.58 (s, 2H, COCH2); 56.68%, H, 4.14%, N, 15.66%.
4.74 (d, 2H, J ¼ 5.6 Hz, benzyl-CH2); 6.96 (m, 1H, H-5); 7.06 (m,
2-(2-(1H-Indol-3-yl)acetyl)-N-(3-fluorophenyl)hydrazincar-
1
1H, H-6); 7.23 (d, 1H, J ¼ 2.4 Hz, H-2); 7.21–7.35 (m, 6H, Ar– bothioamide (1f). Yield 92%, m.p. 183.5 ꢀC; H NMR: ꢀ 3.63
H); 7.56 (d, 1H, J ¼ 8 Hz, H-4); 8.44, 9.35, 9.94, 10.88 (s, 4H, (s, 2H, COCH2); 6.97 (t, 1H, J ¼ 6.8 Hz, H-5); 7.07 (t, 1H,
NH–NH–CS–NH and NH-indole); 13C NMR: ꢀ 30.50; 46.54 J ¼ 7.2 Hz, H-6); 7.23–7.61 (m, 7H, Ar-H); 9.60, 9.78, 10.13,
(NH-C); 107.84; 111.15; 118.17; 118.68; 120.84; 123.80; 126.50; 10.90, (s, 4H, NH–NH–CS–1NH and NH indole); 13C NMR: ꢀ
126.88, 127.15, 127.94, 135.91, 139.20, 170.36 (C¼O) ESI MS 31.15, 108.38, 111.76, 118.75, 119.25, 121.41, 124.46, 127.68,
m/z 339 (M + H, 100%); Anal. calcd. for C18H18N4OS: C, 62.55%; 130.10, 136.50, 141.33, 141.44, 160.76, 163.15, 164.568, 165.87,
H, 5.49; N, 16.21%. Found: C, 63.07%; H, 6.01%; N, 16.68%.
167.30, 168.76, 170.22 (C¼O); 175.49, 181.17 (C¼S); ESI MS
2-(2-(1H-Indol-3-yl)acetyl)-N-phenylhydrazincarbothioamide m/z 343 (M + H, 100%); Anal. calcd. for C17H15FN4OS: C,
(1d). Yield 90%, m.p. 178.5 ꢀC; 1H NMR: ꢀ 3.63 (s, 2H, COCH2); 59.63%; H, 4.42%; N, 16.36%. Found: C, 59.91%; H, 4.51%; N,
6.98 (m, 1H, H-5); 7.07 (m, 1H, H-6); 7.26 (d, 1H, J ¼ 2.4 Hz, H- 16.51%.
2); 7.60 (d, 1H, J ¼ 7.6, H-4); 7.14–7.41 (m, 6H, Ar–H); 9.54,
2-(2-(1H-Indol-3-yl)acetyl)-N-(2,4-dichlorophenyl)hydrazin-
9.64, 10.11, 10.91 (s, 4H, NH–NH–CS–NH and NH indole); 13C carbothioamide (1g). Yield 91%, m.p. 172.5 ꢀC; 1H NMR: ꢀ 3.62
NMR: ꢀ 31.17, 108.45, 110.00, 111.75, 118.80, 119.27, 121.44, (s, 2H, COCH2); 6.97 (t, 1H, J ¼ 7.2 Hz, H-5); 7.07 (t, 1H,

DOI: 10.3109/14756366.2015.1132209
Novel indole-based melatonin analogues
5
J ¼ 6.8 Hz, H-6); 7.59 (d, 1H, J ¼ 8 Hz, H-4); 7.25–7.68 (m, 5H, (M + H + CH3CN, 44%); Anal. calcd. for C17H12 F2N4S: C,
Ar–H); 9.47, 9.87, 10.20, 10.90 (s, 4H, NH–NH–CS–NH and NH 58.11%, H, 3.73%, N, 15.94%. Found: C, 57.98%, H, 3.63%, N,
indole); 13C NMR: ꢀ 31.01, 108.31, 111.71, 118.72, 119.29, 15.80%.
121.41, 124.45, 127.73, 129.23, 132.34, 136.46, 171.07 (C¼O);
5-((1H-Indol-3-yl)methyl)-2-(3-fluorophenyl)amino-1,3,4-thia-
1
182.14 (C¼S); ESI MS m/z (M+, 100%); 395 (M + 2, 85%); 397 diazole (2f). Yield 22%, m.p. 183 ꢀC; H NMR: ꢀ 4.36 (s, 2H,
(M + 4, 38%); Anal. calcd. for C17H14Cl2N4OS: C, 51.92%; H, Ar-CH2); 6.73–7.63 (m, 9H, Ar-H); 10.36 (s, 1H, NH); 11.01 (s,
3.59%; N, 14.25%. Found: C, 52.30%; H, 3.92%; N, 14.51%.
1H, NH indole); 13C NMR: ꢀ 26.36; 104.38; 104.64; 108.28;
2-(2-(1H-Indol-3-yl)acetyl)-N-(3-chlorophenyl)hydrazincar- 110.94; 112.06; 113.51; 118.76; 119.18; 121.77; 124.34; 126.97;
bothioamide (1h). Yield 84%, m.p. 168 ꢀC; 1H NMR: ꢀ 3.64 131.02; 136.77; 124.75; 161.88; 164.25 (S–C–N); ESI MS m/z
(s, 2H, COCH2); 6.98 (t, 1H, J ¼ 6.8 Hz, H-5); 7.07 (t, 1H, 325 (M + H, 100%), 366 (M + H + CH3CN, 33%); Anal. calcd. for
J ¼ 7.2 Hz, H-6); 7.21 (d, 1H, J ¼ 7.2 Hz, H-2); 7.60 (d, 1H, C17H13FN4S: C, 61.24%, H, 4.23%, N, 16.81%. Found: C,
J ¼ 1.6 Hz, H-4); 7.26–7.44 (m, 5H, Ar–H); 9.63, 9.81, 10.13, 61.16%, H, 4.07%, N, 16.69%.
10.91 (s, 4H, NH–NH–CS–NH and NH indole); 13C NMR: ꢀ
5-((1H-Indol-3-yl)methyl)-2-(2,4-dichlorophenyl)amino-1,3,4-
25.81, 31.15, 108.35, 111.74, 118.78, 119.24, 121.44, 124.45, thiadiazole (2g). Yield 50%, m.p. 112 ꢀC; 1H NMR: ꢀ 4.35 (s, 2H,
125.18, 127.68, 130.13, 136.49, 141.12, ESI MS m/z 359.5 Ar–CH2); 6.97 (m, 1H, H-5); 7.07 (m, 1H, H-6); 7.39 (d, 1H,
(M + H, 72%), 361 (M + H + 2, 28%); Anal. calcd. for J ¼ 2.4 Hz, H-7); 7.48 (d, 1H, J ¼ 7.6 Hz, H-4); 7.21–7.59 (m, 4H,
C17H15N4SOCl: C, 56.90%; H, 4.21%; H, 15.61%. Found: C, Ar-H); 8.33 (d, 1H, J ¼ 9.2 Hz, NH); 11.02 (s, 1H, NH indole);13C
57.26%; H, 4.41%; N, 15.31%.
NMR: ꢀ 25.78; 110.38; 111.51; 118.22; 118.61; 121.21; 121.89;
122.74; 123.81; 126.08; 126.38; 127.74; 128.76; 136.21; 136.48;
162.66; 164.11 (S–C–N); ESI MS m/z 375 (M+, 100%), 377
(M + 2, 68%), 379 (M + 4, 13%), 416 (M++CH3CN, 77%); Anal.
calcd. for C17H12Cl2N4S: C, 49.43%, H, 3.95%, N, 13.56%.
Found: C, 49.03%, H, 3.54%, N, 14.03%.
General procedure for the synthesis of compounds 2a–h
Conc. sulfuric acid (3 ml) was placed in a conical flask, and
2-(1H-Indol-3-yl-acetyl)-N-(aryl or alkyl)hydrazincarbothioamide
(1 mmol) was added in small portions over a period of 2 h under
stirring while maintaining the temperature at 0–5 ꢀC. When the
reaction was completed the mixture was poured into crushed ice
General procedure for the synthesis of compounds 3a–h
and neutralized with 2 N NH4OH dropwise until the pH was 2-(1H-Indol-3-yl-acetyl)-N-(aryl or alkyl)hydrazincarbothioamide
adjusted to 7. The formed precipitate was filtered, washed with (2 mmol) and 2 N NaOH solution (25 ml) were placed in conical
water, dried at room temperature and recrystallized from absolute flask. The mixture was heated under reflux for 4–5 h. After
ethanol35.
completion of the reaction, the reaction mixture was neutralized
5-((1H-Indol-3-yl)methyl)-2-ethylamino-1,3,4-thiadiazole (2a). with 2 N HCl dropwise till pH was adjusted to 7. The mixture was
Yield 42%, m.p. 125 ꢀC; 1H NMR: ꢀ 1.12 (t, 3H, J ¼ 6.8 Hz, kept aside for a few minutes. The precipitate thus obtained was
CH3); 3.21 (q, 2H, CH2); 4.26 (s, 2H, Ar–CH2);6.98 (m, 1H, H-5); filtered, washed with water and recrystallized from a mixture of
7.09 (m, 1H, H-6); 7.30 (d, 1H, J ¼ 2.4, H-2); 7.36 (d, 1H, ethanol/water (4:1)35.
J ¼ 8 Hz, H-7); 7.46 (d, 1H, J ¼ 8 Hz, H-4); 7.73 (s, 1H, NH);
5-((1H-Indol-3-yl)methyl)-4-ethyl-4H-1,2,4-triazole-3-thiol (3a).
11.01 (s, 1H, NH indole); 13C NMR: ꢀ 14.62, 26.51, 39.31(NH– Yield 91%, m.p. 191.5 ꢀC; H NMR: ꢀ 0.92 (t, 3H, J ¼ 7.6 Hz,
C); 110.94, 112.02, 118.76, 119.11, 121.73, 124.25, 126.99, CH3); 3.90 (q, 2H, CH2); 4.20 (s, 2H, Ar–CH2); 6.98 (dt, 1H,
136.75, 159.29, 168.89 (S–C–N); ESI MS m/z 259 (M + H, J1 ¼7.2, J2 ¼0.8 Hz, H-5); 7.09 (dt, 1H, J1 ¼7.2, J2 ¼1.2 Hz H-
100%), 300 (M + H + CH3CN, 76%); Anal. calcd. for C13H14N4S: 6); 7.33 (d, 1H, J ¼ 2.4 Hz, H-2); 7.37 (d, 1H, J ¼ 8 Hz, H-7); 7.49
C, 57.43%, H, 5.75%, N, 20.61%. Found: C, 57.55%, H, 5.66%, N, (d, 1H, J ¼ 7.6 Hz, H-4); 11.05 (s, 1H, NH indole); 13.54 (s, 1H,
1
20.23%.
SH); 13C NMR: ꢀ 13.31; 22.34; 38.72; 107.70; 112.057; 118.73;
5-((1H-Indol-3-yl)methyl)-2-propylamino-1,3,4-thiadiazole (2b). 119.16; 121.76; 124.61; 127.14; 136.70; 151.70; 166.67 (N–C–S);
Yield 44%, m.p. 148 ꢀC; 1H NMR: ꢀ 0,86 (t, 3H, J ¼ 7.6 Hz, ESI MS m/z 259 (M + H, 100%), 300 (M + H + CH3CN, 8%);
CH3); 1.52 (m, 2H, CH2); 3.15 (q, 2H, NH–CH2); 4.26 (s, 2H, Ar– Anal. calcd. for C13H14N4S: C, 57.63%, H, 5.73%, N, 20.68%.
CH2); 6.98 (t, 1H, J ¼ 8 Hz, H-5); 7.09 (t, 1H, J ¼ 8 Hz, H-6); 7.31 Found: C, 57.93%, H, 5.96%, N, 20.51%.
(d, 1H, J ¼ 2.4 Hz, H-2); 7.36 (d, 1H, J ¼ 8 Hz, H-7); 7.46 (d, 1H,
5-((1H-Indol-3-yl)methyl)-4-propyl-4H-1,2,4-triazole-3-thiol
J ¼ 8.4 Hz, H-4); 7,82 (s, 1H, NH); 11.00 (s, 1H, NH-indole); 13C (3b). Yield 76%, m.p. 151.5 ꢀC; 1H NMR: ꢀ 0.73 (t, 3H,
NMR: ꢀ 11.78, 22.16, 26.51, 47.05 (NH–C); 110.91, 112.03, J ¼ 7.6 Hz, CH3); 1.35 (m, 2H, CH2); 3.78 (t, 2H, J ¼ 7.6 Hz, NH–
118.77, 119.12, 121.74, 124.29, 127.01, 136.76, 159.22, 169.06 CH2); 4.19 (s, 2H, Ar–CH2); 6.98 (t, 1H, J ¼ 7.2 Hz, H-5); 7.09
(N–C–S); ESI MS m/z (M + H, 100%), 314 (M + H + CH3CN, (t, 1H, J ¼ 7.2 Hz, H-5); 7.33 (d, 1H, J ¼ 2 Hz, H-2); 7.37 (d, 1H,
100%); Anal. calcd. for C14H16N4S: C, 51.51%; H, 6.79%; N, J ¼ 8 Hz, H-7); 7.47 (d, 1H, J ¼ 7.6 Hz, H-7); 11.047 (s, 1H, NH
17.16%. Found: C, 51.23%; H, 5.57%; N, 16.78%.
indole); 13.54 (s, 1H, SH); 13C NMR: ꢀ 10.59; 20.51; 21.77;
5-((1H-Indol-3-yl)methyl)-2-amino-1,3,4-thiadiazole
(2c). 44.42 (N–C); 107.12; 111.50; 118.14; 118.58; 121.20; 124.00;
Yield 39%, m.p. 177 ꢀC; 1H NMR: ꢀ 4.22 (s, 2H, Ar–CH2); 126.57; 136.13; 151.27; 166.40 (N–C–S); ESI MS m/z 273
6.94 (bs, 2H, NH2); 6.97 (dt, 1H, J1 ¼8.4, J2 ¼1.2 Hz, H-5); 7.07 (M + H, 100%); Anal. calcd. for C14H16N4S: C, 60.73% H, 6.01%,
(dt, 1H, J1 ¼8.4, J2 ¼1.2 Hz H-6); 7.27 (d,1H, J ¼ 2.4 Hz, H-2); N, 20.23%. Found: C, 60.25%, H, 5.79%, N, 19.77%.
7.35 (d, 1H, J ¼ 8 Hz, H-7); 7.45 (d, 1H, J ¼ 8 Hz, H-4); 10.97 (s,
5-((1H-Indol-3-yl)methyl)-4-(2,4-difluorophenyl-4H-1,2,4-tri-
1H, NH-indole); 13C NMR: ꢀ 25.98, 110.98, 111.42, 118.24, azole-3-thiol (3e). Yield 85%, m.p. 242.5 ꢀC; 1H NMR: ꢀ 3.96 (q,
118.48, 121.11, 123.52, 126.47, 136.18, 159.21, 168.41 (N–C–S); 2H, Ar–CH2); 6.70–7.48 (m, 8H, Ar–H); 10.86 (s, 1H, NH
ESI MS m/z (M + H, 37%), 272 (M + H + CH3CN, 100%); Anal. indole); 13.85 (s, 1H, SH); ESI MS m/z 343 (M + H, 100%); Anal.
calcd. for C11H10N4S: C, 55.21%, H, 4.63%, N, 23.41%. Found: C, calcd. for C17H12 F2N4S: C, 59.63%, H, 3.53%, N, 16.36%. Found:
54.90%, H, 4.58%, N, 23.35%.
C, 59.50%, H, 3.77%, N, 16.21%.
5-((1H-Indol-3-yl)methyl)-2-(2,4-difluorophenyl)amino-1,3,4-
5-((1H-Indol-3-yl)methyl)-4-(3-fluorophenyl)-4H-1,2,4-tri-
thiadiazole (2e). Yield 26%, m.p. 141 ꢀC; 1H NMR: ꢀ 4.34 (s, 2H, azole-3-thiol (3f). Yield 94%, m.p. 134 ꢀC; 1H NMR: ꢀ 3.96
Ar–CH2); 6.70–8.33 (m,8H, Ar–H); 9.89 (s, 1H, NH); 11.01 (s, (s, 2H, Ar–CH2); 6.67–7.49 (m, 9H, Ar-H); 10.85 (bs, 1H, NH
1H, NH indole); ESI MS m/z 343 (M + H, 100%), 384 indole); 13C NMR: ꢀ 22.76; 107.56; 111.84; 116.08; 116.24;

6
H. Shirinzadeh et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
116.32; 116.45; 118.58; 118.89; 121.51; 124.07; 124.98; 127.04; benzyl-CH2); 5.18 (s, 2H, amide-CH2); 6.92–7.88 (m, 14H,
130.92; 131.02; 136.28; 136.43; 151.38; 160.98; 163.41; 167.94 Ar-H); 10.16 (s, 1H, amide-NH); 10.91 (s, 1H, NH-indole); 13C
(N–C–S); ESI MS m/z 325 (M + H, 100%); Anal. calcd. For NMR: ꢀ 22.20; 37.64 (S–C); 47.02 (N–C); 108.48; 111.89;
C17H13FN4S: C, 55.54%, H, 4.88%, N, 15.24%. Found: C, 115.87; 116.07; 118.98; 121.67; 124.09; 124.25; 124.87; 125.86;
55.03%, H, 3.96%, N, 15.04%.
126.27; 126.38; 127.02; 127.22; 128.20; 129.08; 135.832; 136.76;
5-((1H-Indol-3-yl)methyl)-4-(2,4-dichlorophenyl)-4H-1,2,4- 149.86; 152.56; 155.00; 155.46; 166.82 (C¼O); ESI MS
triazole-3-thiol (3g). Yield 89%, m.p. 254.5 ꢀC; 1H NMR: ꢀ 3.91 m/z 472.2 (M + H, 100%); Anal. calcd. for C26H22 FN5OS:
(q, 2H, Ar–CH2); 6.92 (t, 1H, J ¼ 8 Hz, H-5); 7.05 (t, 1H, C, 66.22%, H, 4.70%, N, 14.85%. Found: C, 65.73%, H, 5.08%
J ¼ 8 Hz, H-6); 7.497 (d, 1H, J ¼ 6.4 Hz, H-7); 7.52 (d, 1H, N, 14.36%.
_
J ¼ 2.4 Hz, H-4); 6.77–7.78 (m, 4H, Ar-H); 10.91 (s, 1H, NH
2-(5-((1H-Indol-3-yl)methyl)-4-phenyl-4H-1,2,4-triazole-3-
indole); 13.86 (s, 1H, SH); 13C NMR: ꢀ 22.01; 105.87; 111.30; ylthio)-N-propylacetamide (4d). Yield 17%, m.p. 165.5 ꢀC; H
117.80; 118.39; 120.99; 123.90; 126.52; 128.28; 129.60; 130.13; NMR: ꢀ 0.89 (t, 3H, J ¼ 7.6 Hz, CH3); 1.53 (m, 2H, CH2); 3.20
132.05; 133.22; 135.23; 135.91; 151.11; 167.735 (N–C–S); ESI (q, 2H, CO–NH–CH2); 3.73 (s, 2H, Ar–CH2); 4.16 (s, 2H,
MS m/z 375 (M+, 100%), 377 (M + 2, 58%), 379 (M + 4, 13%), S–CH2–CO); 6.61–7.50 (m, 10H, Ar–H); 7.96 (bs, 1H, amide-
426 (M + CH3CN, 11%); Anal. calcd. for C17H12Cl2N4S: C, NH); 8.10 (s, 1H, NH indole); 13C NMR: ꢀ 11.39; 22.05; 22.56;
53.76%, H, 3.32%, N, 14.75%. Found: C, 53.57%, H, 3.29%, N, 34.56; 41.56; 109.34; 110.00; 111.10; 118.78; 119.71; 122.28;
1
14.69%.
122.69; 126.63; 127.02; 129.78; 130.19;132.72; 136.02; 152.17;
5-((1H-Indol-3-yl)methyl)-4-(3-chlorophenyl)-4H-1,2,4- 155.39 (N–C–S); 168.68 (C¼O); ESI MS m/z 406 (M + H, 100%);
triazole-3-thiol (3h). Yield 83%, m.p. 140 ꢀC; 1H NMR: ꢀ 3.96 Anal. calcd. for C22H23 N5OS: C, 64.44%, H, 5.78%, N, 17.08%.
(s, 2H, Ar–CH2); 6.91 (t, 1H, J ¼ 7.2 Hz, H-5); 7.028 (t, 1H, Found: C, 64.21%, H, 5.88%, N, 16.85%.
J ¼ 7.6 Hz, H-6); 7.19 (d, 1H, J ¼ 7.6 Hz, H-2); 6.71–7.58 (m, 6H,
2-(5-((1H-Indol-3-yl)methyl)-4-(2,4-difluorophenyl)-4H-1,2,4-
Ar–H); 10.86 (s,1H, NH indole); 13.77 (s, 1H, SH); 13C NMR: ꢀ triazole-3-ylthio)-N-propylacetamide (4e). Yield 45%, m.p.
1
22.73; 107.03; 111.88; 118.50; 118.97; 121.57; 124.26; 126.99; 140 ꢀC; H NMR: ꢀ 0.81 (t, 3H, J ¼ 7.6 Hz, CH3); 1.37 (m, 2H,
127.56; 128.74; 129.78; 131.09; 133.57; 135.56; 136.43; CH2); 2.99 (q, 2H, CO-NH-CH2); 3.85 (s, 2H, Ar–CH2);
151.72;168.14 (S–C–N); ESI MS m/z 341.2 (M+, 100%); 343.3 4.08 (s, 2H, S–CH2–CO); 6.69–7.51 (m, 8H, Ar–H); 8.16
(M + 2, 30%), 382 (M + CH3CN, 20%); Anal. calcd. for (t, 1H, J ¼ 5.2 Hz, amide-NH); 10.81 (s, 1H, NH indole); ESI
C17H13ClN4S: C, 58.36%, H, 4.03%, N, 16.01%. Found: C, MS m/z 442 (M + H, 100%); Anal. calcd. for C22H21F2N5OS:
58.54%, H, 4.01%, N, 15.70%.
C, 59.85%, H, 4.79%, N, 15.86%. Found: C, 59.70%, H, 4.97%,
N, 15.66%.
2-(5-((1H-Indol-3-yl)methyl)-4-(3-fluorophenyl)-4H-1,2,4-tri-
azole-3-ylthio)-N-propylacetamide (4f). Yield 40%, m.p. 145 ꢀC;
1H NMR: ꢀ 0.81 (t, 3H, J ¼ 7.6 Hz, CH3); 1.37 (m, 2H, CH2); 2.98
(q, 2H, CO-NH-CH2); 3.84 (s, 2H, Ar–CH2); 4.11 (s, 2H, S-CH2-
CO); 6.69–7.55 (m, 9H, Ar-H); 8.16 (t, 1H, J ¼ 5.2 Hz, amide-
NH); 10.80 (s, 1H, NH indole); 13C NMR: ꢀ 11.75; 22.025; 22.61;
36.69; 41.16; 108.36; 111.77; 115.19; 115.43; 117.18; 117.38;
118.63; 118.87; 121.53; 123.84; 124.03; 124.06; 127.02; 131.66;
131.75; 134.87; 134.97; 136.45; 150.10; 155.134; 161.13; 163.58
(N–C–S); 166.85 (C¼O); ESI MS m/z 424 (M + H, 100%); Anal.
calcd. for C22H22FN5OS: C, 62.39%, H, 5.24%, N, 16.54%.
Found: C, 62.32%, H, 5.28%, N, 16.35%.
General procedure for the synthesis of compounds 4a–g
5-((1H-Indol-3-yl) methyl)-4-(substituted aryl/alkyl)-4H-1,2,4-tri-
azole-3-thiol (1 mmol) was dissolved in a solution of 2 N KOH
(2 ml) and absolute ethanol (20 ml) in a conical flask. R-X
(X ¼Cl) (1 mmol) was added in to the mixture and stirred at room
temperature for 2–3 h. After completion of the reaction, the
reaction mixture was poured onto crushed ice. The formed
precipitate was filtered, washed with water, dried at room
temperature and recrystallized from absolute ethanol33.
2-(5-((1H-Indol-3-yl)methyl)-4-ethyl-4H-1,2,4-triazole-3-ylthio)-
N-phenylacetamide (4a). Yield 17%, m.p. 120 ꢀC; 1H NMR: ꢀ 0.96
(t, 3H, J ¼ 6.8 Hz, CH3); 3.91 (q, 2H, CH2); 4.09 (s, 2H, Ar–CH2);
4.25 (s, 2H, S–CH2–CO); 6.92–7.55 (10H, m, Ar–H); 10.32 (s,
1H, amide-NH); 10.97 (s,1H, NH indole); 13C NMR: ꢀ 14.55;
21.43; 28.90; 37.51; 108.30; 111.37; 118.40; 118.45; 119.01;
121.11; 123.42; 123.61; 126.65; 128.69; 136.16; 138.66; 148.32;
154.44 (N–C–S); 165.62 (C¼O); ESI MS m/z 392 (M + H, 100%);
Anal. calcd. for C21H21N5OS: C, 56.04%, H, 6.15%, N, 15.56%.
Found: C, 55.68%, H, 5.60%, N, 15.22%.
2-(5-((1H-Indol-3-yl)methyl)-4-propyl-4H-1,2,4-triazole-3-
ylthio)-N-propylacetamide (4b). Yield 32%, m.p. 79 ꢀC; 1H
NMR: ꢀ 0.71 (t, 3H, CH3); 0.79 (t, 3H, J ¼ 7.2 Hz, CH3); 1.34
(m, 2H, CH2); 1.43 (m, 2H, CH2); 2.97 (q, 2H, CO–NH–CH2);
3.78 (t, 2H, J ¼ 7.6, triazole–NH–CH2); 3.82 (s, 2H, Ar–CH2);
4.23 (s, 2H, S–CH2–CO); 6.94 (t,1H, J ¼ 8 Hz, H-5); 7.06
(t,1H, J ¼ 8.4 Hz, H-6); 7.25 (d, 1H, J ¼ 2 Hz, H-2); 7.34
(d, 1H, J ¼ 8.4 Hz, H-7); 7.50 (d, 1H, J ¼ 8 Hz, H-4); 8.16 (bs,
1H, amide–NH); 10.960 (s,1H, NH indole); 13C NMR: ꢀ 11.08;
11.72; 22.06; 22.59; 22.99; 37.29; 43.12; 45.26; 79.63; 108.95;
111.90; 118.95; 121.65; 124.07; 127.21; 136.71; 149.14; 155.04;
167.12 (C¼O); ESI MS m/z 372.3 (M + H, 100%); Anal. calcd.
for C19H25N5OS: C, 58.18%, H, 7.02%, N, 17.85%. Found: C,
58.77%, H, 6.96%, N, 17.13%.
2-(5-((1H-Indol-3-yl)methyl)-4-(2,4-dichlorophenyl)-4H-1,2,4-
triazole-3-ylthio)-N-phenylacetamide (4g). Yield 15%, m.p.
1
219 ꢀC; H NMR: ꢀ 4.03 (s, 2H, Ar–CH2); 4.12(s, 2H, S–CH2–
CO); 6.72–7.80 (m, 13H, Ar–H); 10.27 (s,1H, amide-NH); 10.82
(s, 1H, NH indole); 13C NMR: ꢀ 21.47; 37.30; 107.15; 111.19;
117.96; 118.28; 118.99; 120.93; 123.43; 123.47; 126.56; 128.51;
128.69; 129.36; 129.76; 131.13; 132.53; 135.68; 135.90; 138.61;
149.66; 154.80 (N–C–S); 165.26 (C¼O); ESI MS m/z 508 (M+,
100%), 510 (M + 2, 75%), 512 (M + 4, 16%); Anal. calcd. for
C25H19Cl2N5OS: C, 59.06%, H, 3.77%, N, 13.77%. Found: C,
59.60%, H, 4.27%. N, 13.47%.
Experimental – biological activity
Cell culture and reagents
CHO-K1 cells were purchased from ATCC and maintained at
37 ꢀC in 5% CO2 atmosphere. Dulbecco’s modified Eagle’s
medium/F12 (Sigma, St. Louis, MO) (1:1) nutrient mixture
medium supplemented with 10% foetal bovine serum (FBS), 1%
of a 100 U/ml penicillin–streptomycin solution, 2 mM (final
concentration) L-glutamine and 1 mM (final concentration)
sodium pyruvate was used in all cell incubations. Hyclone
characterized FBS is purchased from Thermo Scientific
(Waltham, MA). 20,70-Dichlorofluorescin diacetate (DCFH-DA),
DPPH, sodium pyruvate and L-glutamine were obtained from
2-(5-((1H-Indol-3-yl)methyl)-4-benzyl-4H-1,2,4-triazole-3-
ylthio)-N-(2-fluorophenyl)acetamide (4c). Yield 30%, m.p.
182.5 ꢀC; 1H NMR: ꢀ 4.13(s, 2H, indole-CH2); 4.20 (s, 2H,

DOI: 10.3109/14756366.2015.1132209
Novel indole-based melatonin analogues
7
Sigma (St. Louis, MO). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphe- readily crosses the cell membrane and undergoes hydrolysis by
nyl-2H-tetrazolium bromide (MTT) was purchased from intracellular esterases to non-fluorescent 20,70-dichlorofluorescin
Invitrogen (Waltham, MA). Test materials were dissolved in (DCFH). In the presence of ROS, DCFH is oxidized to highly
dimethyl sulfoxide (DMSO) and the final concentration of DMSO fluorescent dichlorofluorescein (DCF)37 which can be detected by
never exceeded 0.1% v/v in cell culture medium.
a fluorescent microplate reader. The DCF fluorescence intensity
is believed to be parallel to the amount of ROS formed
intracellularly.
Cytotoxic effects via the MTT assay
Cells were seeded in black 96-well plates at a density of
5 ꢁ 103 cells/well and incubated at 37 ꢀC in a humid atmosphere
containing 5% CO2 for 24 h for cell attachment. The medium was
removed and then cells were incubated with DCFH-DA (20 mM)
containing medium for 30 min. Cells were washed with PBS to
remove excess DCFH-DA. Ten micromolar synthesized com-
pounds and 10 mM cumene hydroperoxide (CMHP) were added
into medium. The production of fluorescent DCF was evaluated
by monitoring fluorescence intensity at 488 nm excitation, 530 nm
emission wavelengths for 60 min.
CHO-K1 cells were seeded in 96-well plates (5 ꢁ 103 cells/well)
and incubated at 37 ꢀC in a humid atmosphere containing 5% CO2
for 24 h. Cells were treated with the newly synthesized com-
pounds (10 mM) for 24 h. Control (medium only), vehicle control
and positive control (15 mM Triton X-100) were included in every
experiment. Following the exposure period the medium was
removed, cells were washed with phosphate buffered saline (PBS)
and then incubated with MTT (1 mg/ml) for 4 h at 37 ꢀC. MTT
solution was removed and formazan crystals were dissolved in
DMSO. The absorbance was recorded at 550 nm on a microplate
reader. The ratio of the absorbance of treated samples to the
absorbance of control (taken as 100%) was expressed as % cell DPPH free radical scavenging activity
viability.
The free radical scavenging activities of newly synthesized
compounds were tested by their ability to bleach the stable
radical DPPH38. DPPH presents a maximum of absorbance at
515 nm; when DPPH reacts with an antioxidant compound, which
Measurement of intracellular ROS (antioxidant activity on
ROS-induced DCFH-DA oxidation)
For estimation of intracellular ROS, DCFH-DA was used as a can donate hydrogen, this absorbance diminishes and can be
probe. In cellular systems, the non-fluorescent probe DCFH-DA measured on a visible spectrophotometer.
Figure 2. Effect of samples (10 mM) on CHO-K1 cell viability evaluated by MTT assay. Bars represent ‘‘medium standard deviation’’ values
from five individual experiments. *p50.05, **p50.005, ***p50.0005, groups compared to vehicle control group. Triton X-100 used as a positive
control group.
Figure 3. DCFH oxidation in CHO cells after 60 min incubation with compounds (10 mM) in the presence of CMHP. Bars represent
‘‘medium standard deviation’’ values obtained from five individual experiments. Statistical comparisons of samples with CMHP were compared
to CMHP alone control group. *p50.05, **p50.005 (AA, ascorbic acid).

8
H. Shirinzadeh et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure 4. DCFH oxidation in CHO cells after
60 min incubation with MLT, compounds 1b
and 2e in the presence of CMHP. Bars
represent ‘‘medium standard deviation’’
values from five individual experiments. The
values above the bars represent percentage of
the absorbance values comparing with
CMHP group used as control. All the statis-
tical analysis of samples with CMHP was
performed by comparing to the CMHP alone
group. *p50.05, **p50.005, ***p50.0005
(MLT, melatonin).
Figure 5. Radical scavenging activity of compounds (100 mM) determined by DPPH assay. Bars represent ‘‘medium standard deviation’’ values from
four different experiments. BHT and MLT were used as a reference (BHT, butylated hydroxytoluene; MLT, melatonin).
Twenty microlitres of the samples (100 mM) was added to in triplicate. MLT and BHT were used as reference compounds.
180 ml of DPPH solution (150 mM) in methanol–water (80:20, v/v) The radical scavenging activities were expressed as the inhibition
in 96-well plates, incubated for 30 min at room temperature then percentage and were calculated using the formula: Radical
DPPH reduction was estimated at 517 nm. Percentage inhibition scavenging activity (%) ¼ [(A0ꢂA1/A0) ꢁ 100], where A0 is the
by the sample treatment was determined by comparison with a absorbance of the control (blank, without compound) and A1 is the
DMSO-treated control group. All experiments were carried out absorbance of the compound.

DOI: 10.3109/14756366.2015.1132209
Novel indole-based melatonin analogues
9
Results and discussion
This finding indicates that the antioxidant effect of 1b is probably
is a result of its radical scavenging activity. However, compound
2e which was also found to have antioxidant activity according to
Cytotoxic effects of the synthesized compounds
The cytotoxic effect of the synthesized compounds in CHO-K1 the DCFH assay, and was found to have very low scavenging
cells was evaluated by the MTT assay (Figure 2). CHO-K1 cells activity in the DPPH assay which suggests an alternative
are used because many studies as well as our previous study mechanism for its antioxidant activity. However, almost all
suggested them as a relevant model for in vitro antioxidant hydrazincarbothioamide and 1,2,4-triazole-3-thiol derivatives
screening studies39. Furthermore, CHO cells are advantageous were found to have radical scavenging activity in the DPPH
since they have active CYP enzymes that make them a good assay where no antioxidant activity was observed in the cell based
in vitro model for detecting the toxicity of chemicals as well as DCFH assay. This result could be the result of limited availability
some of their metabolites without adding S9 fraction.
of the compounds in cell cytosol because of the possible
membrane passage. This speculation needs to be further verified
experimentally.
Antioxidant effects of the synthesized compounds
The protective effect of newly synthesized MLT analogues against
CMHP induced DCFH-DA oxidation was determined in CHO-K1
cells that were preloaded with the fluorescent probe. Oxidation of
the probe that locates in the cytosol was screened at various time
intervals up to 60 min (Figure 3). Several compounds were found
to have antioxidant activity, whereas 1b and 2e were found to be
the most potent two compounds among all. Therefore, their
reducing activity against DCFH oxidation was further investigated
at their various concentrations (Figure 4).
Declaration of interest
The authors report no conflicts of interest. The chemical synthesis and
characterization section of this work were supported by The Scientific
and Technological Research Council of Turkey (TUBITAK) Research and
Development Grant 112S599. The cell culture facility was established by
the support of a TUBITAK 108S202 Grant.
¨
_
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