Behaviour of 9-Ethyl-9H-carbazole hydrazone derivatives against oxidant systems: Protective effect on amyloid β-induced damage

Article abstract of DOI:10.5562/cca3481

Antioxidants are helpful in prevention of several diseases related with oxidative stress including neurodegenerative disorders. In recent studies, carbazoles were given proof of promising antioxidant activities. In this article, 9-ethyl-9H-carbazole hydra

Full text of DOI:10.5562/cca3481

O R I G I NA L S C I E N T I F I C P A P E R
Croat. Chem. Acta 2019, 92(1)
Published online: June 19, 2019
DOI: 10.5562/cca3481
Behaviour of 9-Ethyl-9H-carbazole Hydrazone
Derivatives Against Oxidant Systems: Protective
Effect on Amyloid β-Induced Damage
Cigdem Karaaslan,1,* Elif Ince,2 Hande Gurer-Orhan,2 Marjan Tavakkoli,3 Omidreza Firuzi,3 Luciano Saso,4 Sibel Suzen1
1
2
3
4
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara, Turkey
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Ege University, Izmir, Turkey
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University of Rome, P. le Aldo Moro 5, 00185 Rome, Italy
Corresponding author’s e-mail address: karaslan@pharmacy.ankara.edu.tr
*
RECEIVED: February 15, 2019
REVISED: May 9, 2019
ACCEPTED: May 13, 2019
Abstract: Antioxidants are helpful in prevention of several diseases related with oxidative stress including neurodegenerative disorders. In
recent studies, carbazoles were given proof of promising antioxidant activities. In this article, 9-ethyl-9H-carbazole hydrazone derivatives were
synthesized, characterized and their in vitro antioxidant activity and possible cytotoxic effects were investigated. Furthermore, protective effect
of the synthesized derivatives against amyloid β-induced damage in PC12 neuronal cells was examined by using MTT assay. The newly synthe-
sized carbazoles were found to have radical scavenging activity with a varying potency both in cell-free and cell-based in vitro assays. Several
compounds, especially such as 3d and 3e, 3m and 3n bearing two halogen groups on the phenyl ring, were found to have cytotoxic activity.
However, their cytotoxic activities were not higher than that of melatonin. Several compounds also significantly protected neuronal PC12 cells
against amyloid β-induced damage, which can be defined as neuroprotective agents. (4-(2-((9-Ethyl-9H-carbazol-3-yl)methylene)-
hydrazinyl)benzonitrile) 3r was found as the most active compound with both radical scavenging activity and neuroprotective effects against
amyloid β-induced damage. These findings might provide an alternative strategy for developing novel carbazole derivatives for management
of neurodegenerative diseases, such as Alzheimer's disease.
Keywords: carbazole, antioxidant, cytotoxicity, alzheimer’s disease, β-amyloid.
N-substituted carbazoles have been also well known for
their variety of pharmacological activities, such as
antioxidant[6] and antitumor[7] properties.
INTRODUCTION
ARBAZOLES represent a family of tricyclic compounds
which are widely found in nature. Several studies have
C
Cl
been conducted to reveal biological effects of these com-
pounds since last decade. Therefore, synthesis of substi-
tuted carbazoles have attracted considerable attention.[1]
Carbazole derivatives have been reported to demonstrate
diverse biological activities such as antimicrobial, anti-
tumor, antiepileptic, antihistaminic, antioxidative, anti-in-
flammatory, analgesic, neuroprotective and pancreatic
lipase inhibition properties.[2] Carbazole structure is already
present in many drugs such as carvedilol,[3] an
antihypertensive; carprofen,[4] a non-steroidal anti-inflam-
matory agent, carazostatin,[5] an antioxidant (Figure 1).
O
O
N
H
OH
O
O
N
OH
N
H
H
Carvedilol
Carprofen
OH
N
H
H
C7
15
Carazostatin
Figure 1. Biologically active compounds in carbazole scaffold.
This work is licensed under a Creative Commons Attribution 4.0 International License.

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2
C. KARAASLAN et al.: 9-Ethyl-9H-carbazole Hydrazone Derivatives …
Antioxidant effects of carbazole derivatives are spe-
Due to the plaque formation caused by cytosolic
accumulation of amyloid β-peptide (Aβ), many studies
have focused on designing new molecules that are able to
inhibit Aβ aggregation. Saturnino et al.[18] found out two
promising N-alkyl carbazole derivatives by docking
simulations which also exhibited the higher activity in
vitro. N-substituted carbazoles have been reported as
neuroprotective agents with potent anti-oxidative activity
performing good neuroprotective effects on neuronal
cells HT22 against cell injury induced by glutamate or
homocysteic acid.[9]
In the light of these observations, it was taken an
interest in synthesizing a new class of carbazole-hydra-
zones in order to study the possible antioxidant activity
and protection of neuronal PC12 cells against amyloid β-
induced damage. Herein, we report the synthesis of 9-
ethyl-9H-carbazole hydrazone derivatives starting from 9-
ethyl-9H-carbazol-3-carboxaldehide and phenyl hydra-
zine derivatives. Their potential antioxidant activity was
investigated by evaluating their reducing effect against
oxidation of a redox sensitive fluorescent probe, 2′,7′-
dichlorofluorescin diacetate (DCFH-DA), in a cell-based in
vitro model and their radical scavenging activity was
assessed via the 2,2-diphenyl-1-picrylhydrazyl (DPPH)
assay in a cell-free in vitro model. MTT (3-(4,5-dimethyl-
thiazolyl-2)-2,5-diphenyltetrazolium bromide) assay was
performed to evaluate cytotoxic activity of the
compounds and neuroprotective effect was examined in
PC12. All the analogue compounds were characterized on
the basis of 1H and 13C NMR and mass spectra and
elemental analysis.
cially focused in many studies. Thus, 1,3-dihydroxy-2-car-
boxycarbazole analogues exhibited antioxidative proper-
ties, promising neuroprotective activities in a cellular
model of Alzheimer's disease (AD) and inhibitory activity
against Aβ aggregation.[8] In another study 1- and 3-substi-
tuted carbazole derivatives were observed with significant
neuroprotective effects on neuronal cells HT22 against cell
injury. Substituents introduced to the carbazole ring sys-
tem play crucial roles in their biological activities. It was
found that bulky groups favour the neuroprotective activ-
ity.[9] 3-Oxygenated and 3,4-dioxygenated carbazole
alkaloids and their related carbazoles such as
3,8-dihydroxycarbazoles carbazomadurin A and B showed
higher antioxidant activities.[10] Bandgar et al.[11] examined
radical scavenging activity, cytotoxicity and antimicrobial
activities of novel carbazole chalcones (Figure 2). Most of
the compounds showed good 2,2-diphenyl-1-picrylhydrazyl
(DPPH) and hydroxyl radical scavengers activity.
A series of carbazole-based sulfonamide and carba-
mate derivatives exhibited antioxidant activities from mod-
erate to good levels. According to the results, the most
potent antioxidant activities were observed in the com-
pounds having strong electron withdrawing groups such as
NO2, CCl3, and Cl. This might be related with their high an-
tioxidant activity.[12] Similar to this example, we have deter-
mined that combination of two different pharmacophores
carbazole and hydrazine derivatives led to series of new
compounds having antioxidant activity. Similar to carba-
zoles, hydrazone derivatives were also reported to possess
various pharmacological activities including antioxidant
activity.[13,14]
Many in vitro, in vivo and epidemiological studies
suggested involvement of oxidative damage in neuro-
degenerative diseases such as Parkinson’s and Alzheimer
Diseases[15] Elevated DNA oxidation products (such as 8-
OHdG and other DNA base oxidation products), protein ox-
idation products (such as protein carbonyls and nitrotyro-
sine) and lipid peroxidation products (such as 4-HNE) in the
brain of animals (in vivo studies) suggested as evidences for
the role of oxidative damage in Alzheimer Diseases.[16,17]
Therefore, antioxidants seem to be beneficial in prevention
and/or treatment of Alzheimer Disease.
EXPERIMENTAL
The chemical reagents used in synthesis were purchased
from Sigma (Germany) and Aldrich (USA). Uncorrected
melting points were determined with a Buchi SMP-20
1
apparatus. The H and 13C NMR spectra were measured
with a Varian 400 MHz using TMS internal standard and
DMSO-d6 as solvent. ESI Mass spectra were determined on
a Waters micromass ZQ. FT-IR spectra were recorded on
Jasco 420 Fourier. Elemental analyses were performed us-
ing CHNS-932 (LECO). All spectral analyses were performed
at Ankara University Faculty of Pharmacy Central Labora-
tory.
O
R
NH
RPMI 1640 was obtained from Atocel (Austria).
Amyloid-β peptide (residues 25 to 35, Aβ25–35) was purchased
from Tocris Bioscience (USA). Penicillin / streptomycin,
sterile phosphate-buffered saline (PBS), and trypsin EDTA
0.25 % were purchased from Shell Max. Fetal bovine serum
and horse serum were acquired from Thermo Scientific
(USA), while dimethyl sulfoxide was obtained from Merck
(Germany).
N
R
N
N
H3C
H3C
(a)
(b)
Figure 2. General formula of (a) Antioxidant 3-(substituted)-
aryl-5-(9-methyl-3-carbazole)-1H-2-pyrazolines and (b)
synthesised 9-ethyl-9H-carbazole hydrazone derivatives.
Croat. Chem. Acta 2019, 92(1)
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C. KARAASLAN et al.: 9-Ethyl-9H-carbazole Hydrazone Derivatives …
3
Chemistry
92.2, 92.5, 92.8, (94.3, 94.6), (109.2, 109.3), (118.9, 119.0),
120.6, (122.1, 122.3), 123.9, (125.9, 126.1), (139.8, 139.9),
140.4, 148.1, 148.3, 148.4, (162.2, 162.3), (164.5, 164.7).
ESI-MS: m / z = 350 [M+1] (100 %). Found, %: C 71.39; H
5.23; N 11.98. C21H17N3F2. 0.2 H2O Calcd, %: C 71.46; H 4.97;
N 11.90.
For the synthesis of new carbazole imine compounds (3a–
3r), phenyl hydrazine derivatives and 9-ethyl-9H-carbazole-
3-carboxaldehyde were heated in the presence of ethanol.
All the compounds were characterized on the basis of
spectral data. The characterization results of the novel
compounds 3e and 3m–r are given as follows. Compounds
3a–d and 3f–l were previously described by Aboul-Enein.[24]
3-((2-(4-METHOXYPHENYL)HYDRAZONO)METHYL)-9-
ETHYL-9H-CARBAZOLE (3o)
Yield 78.7 %; m.p.141–144 °C; H NMR (DMSO): δ = 1.32
1
General Procedure for the Synthesis of
Compounds
9-Ethyl-9H-carbazole-3-carboxaldehyde (0.1 mmol) was
reacted with phenyl hydrazine or its derivatives (0.13 mmol)
in 20 mL of EtOH for 3 h on the hot water bath. On cooling,
the precipitate was collected washed with cold EtOH to give
the final compounds with 45–87 % yield.
(3H, t, CH3), 3.70 (3H, s, OCH3), 4.43 (2H, q, CH2), 6.86 (2H,
d, J = 8.8 Hz, H-2’,6’), 7.07 (2H, d, J = 8.8 Hz, H-3’,5’), 7.22
(1H, t, J = 7.6 Hz, H-6), 7.46 (1H, t, J = 7.2 Hz, H-7), 7.59-7.62
(2H, m, H-5,8), 7.83(1H, dd, J = 8.4 Hz, J = 1.6 Hz, H-2), 8.02
(1H, s, -NH), 8.22 (1H, d, J = 7.6 Hz, H-1), 8.34 (1H, d, J =
1.2 Hz, H-4), 9.99 (1H, s, -CH=N). 13C NMR: δ = 13.7, 36.9,
55.2, 109.2, 109.3, 112.8, 114.5, 117.9, 118.8, 120.4, 122.1,
122.3, 123.3, 125.8, 127.2, 136.9, 139.3, 139.8, 139.9,
152.2. ESI-MS: m / z = 344 [M+1] (100 %). Found, %: C 75.45;
H 6.61; N 11.22. C22H21N3O. 0.25 H2O. 0.25 C2H6O Calcd, %:
C 75.18; H 6.45; N 11.69.
3-((2-(3,5-CHLOROPHENYL)HYDRAZONO)METHYL)-9-
ETHYL-9H-CARBAZOLE (3e)
1
Yield 58.6 %; m.p. 154–157 °C; H NMR (DMSO): δ = 1.30
(3H, t, CH3), 4.41 (2H, q, CH2), 6.79 (1H, d, J = 1.6 Hz, H-4’),
7.06 (1H, d, J = 1.2 Hz, H-2’,6’), 7.20 ( 1H, t, J = 7.6 Hz, H-6),
7.46 (1H, t, J = 8 Hz, H-7), 7.58-7.61 (2H, m, H-5,8), 7.85 (
1H, dd, J = 8.8 Hz, J = 1.6 Hz, H-2), 8.09 (1H, s, -NH), 8.21
(1H, d, J =7.2 Hz, H-1), 8.40 (1H, d, J =1.2 Hz, H-4), 10.69 (1H,
s, -CH=N). 13C NMR: δ = 13.7, 37.0, 109.3, 109.4, 109.8,
116.6, 118.9, 119.0, 120.6, 122.1, 122.3, 123.8, 125.9,
126.0, 134.6, 139.9, 140.9, 147.8. ESI-MS: m / z = 383[M+1]
(100 %), 385 [M+1+2] (65 %), 387 [M+1+4] (37%). Found, %:
C 64.47; H 4.58; N 10.73. C21H17N3Cl2. 0.5 H2O Calcd, %: C
64.46; H 4.64; N 10.74.
3-((2-(2,4-DIMETHYLPHENYL)HYDRAZONO)METHYL)-9-
ETHYL-9H-CARBAZOLE (3p)
Yield 44.6 %; m.p. 98–102 °C; 1H NMR (DMSO): δ = 1.30 (3H,
t, CH3), 2.18 (3H,s, CH3(phenyl)), 2.19 (3H,s, CH3(phenyl)), 4.42
(2H, q, CH2), 6.85 (1H, s, H-3’), 6.92 (1H, d, J = 8Hz, H-5’),
7.20 (1H, t, J = 7.6 Hz, H-6), 7.39 (1H, d, J = 8Hz, H-6’), 7.46
(1H, td, J = 8.4 Hz, J = 1.2 Hz, H-7), 7.57-7.61 (2H, m, H-5,8),
7.83 (1H, dd, J = 8.8Hz, J = 1.2 Hz, H-2), 8.22 (1H, d, J = 7.6
Hz, H-1), 8.27 (1H, s, -NH), 8.33 (1H, d, J = 1.6 Hz, H-4), 9.27
(1H, s, -CH=N). 13C NMR: δ = 13.7, 17.4, 20.1, 36.9, 109.2,
109.3, 112.1, 118.1, 118.9, 120.4, 120.5, 122.1, 122.3,
123.5, 125.8, 126.6, 127.0, 127.1, 130.8, 138.9, 139.4,
139.8, 141.3. ESI-MS: m / z = 342 [M+1] (100 %). Found, %:
C 78.18; H 6.91; N 9.27. C23H23N3. 0.5 H2O.0.25 C2H6O
Calculated, %: C 77.97; H 7.10; N 11.60.
3-((2-(2,5-FLUOROPHENYL)HYDRAZONO)METHYL)-9-
ETHYL-9H-CARBAZOLE (3m)
1
Yield 83.1 %; m.p.112–116 °C; H NMR (DMSO): δ = 1.31
(3H, t, CH3), 4.44 (2H, q, CH2), 6.46-6.51 (1H, m, H-6’), 7.11–
7.22 (2H, m, H-6, 3’), 7.29-7.34 (1H, m, H-4’), 7.45 (1H, td, J
= 8 Hz, J = 1.2 Hz, H-7), 7.59-7.63 (2H, m, H-5,8), 7.84 (1H,
dd, J = 8.4 Hz, J = 1.6 Hz, H-2), 8.25 (1H, d, J = 7.2 Hz, H-1),
8.32 (1H, s, -NH), 8.41 (1H, d, J = 1.2 Hz, H-4), 10.32 (1H, s,
-CH=N). ESI-MS: m / z = 350 [M+1] (100 %). Found, %: C
71.85; H 5.32; N 11.54. C21H17N3F2. 0.2 H2O Calcd, %: C
71.46; H 4.97; N 11.90.
3-((2-(3,4-DIMETHYLPHENYL)HYDRAZONO)METHYL)-9-
ETHYL-9H-CARBAZOLE (3q)
1
Yield 65.1 %; m.p. 152–156 °C; H NMR (DMSO): δ = 1.30
(3H, t, CH3), 2.12 (3H,s, CH3(phenyl)), 2.19 (3H,s, CH3(phenyl)),
4.42 (2H, q, CH2), 6.83 (1H, dd, J = 8.4 Hz, J = 2.4 Hz, H-6’),
6.91 (1H, d, J = 2Hz, H-2’), 6.96 (1H, d, J = 8.4 Hz, H-5’), 7.19
(1H, t, J = 7.2 Hz, H-6), 7.43 (1H, td, J = 8.4 Hz, J = 1.2 Hz, H-
7), 7.58 (2H, dd, J = 8.8 Hz, J = 2.4 Hz, H-5,8), 7.82 (1H, dd, J
= 8.8Hz, J = 1.2 Hz, H-2), 7.99 (1H, s, -NH), 8.20 (1H, d, J =
7.6 Hz, H-1), 8.32 (1H, d, J =1.6 Hz, H-4), 9.97 (1H, s, -CH=N).
13C NMR: δ = 13.7, 18.5, 19.7, 36.9, 109.2, 109.3, 113.1,
118.0, 118.9, 120.5, 122.1, 122.3, 123.4, 125.4, 125.8,
127.1, 129.9, 136.5, 137.1, 139.3, 139.8, 143.7. ESI-MS: m / z =
342 [M+1] (100 %). Found, %: C 80.08; H 7.12; N 12.02.
C23H23N3. 0.2 H2O.0.1 C2H6O Calcd, %: C 79.69; H 6.92; N 12.01.
3-((2-(3,5-FLUOROPHENYL)HYDRAZONO)METHYL)-9-
ETHYL-9H-CARBAZOLE (3n)
1
Yield 66.5 %; m.p. 148–151 °C; H NMR (DMSO): δ = 1.30
(3H, t, CH3), 4.43 (2H, q, CH2), 6.40-6.46 (1H, m, H-4’), 6.71
(2H, dd, J = 10.4 Hz, J = 2.4 Hz, H-2’,6’), 7.20 (1H, t, J =
7.6 Hz, H-6), 7.45 (1H, td, J = 7.2 Hz, J = 1.2 Hz, H-7), 7.58-
7.62 (2H, m, H-5,8), 7.85 (1H, dd, J = 8.4 Hz, J = 1.6 Hz, H-2),
8.07 (1H, s, -NH), 8.22 (1H, d, J = 7.6 Hz, H-1), 8.42 (1H, d, J
= 1.2 Hz, H-4), 10.63 (1H, s, -CH=N). 13C NMR: δ = 13.7, 37.0,
DOI: 10.5562/cca3481
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C. KARAASLAN et al.: 9-Ethyl-9H-carbazole Hydrazone Derivatives …
Triton X-100) were included in every experiment. Following
4-(2-((9-ETHYL-9H-CARBAZOL-3-YL)-
the exposure period, the medium was removed, cells were
washed with phosphate buffered saline (PBS) and then
incubated with MTT (1 mg mL–1) for 4 h at 37 °C. MTT solu-
tion 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 viability.
METHYLENE)HYDRAZINYL)BENZONITRILE (3r)
1
Yield 86.9 %; m.p. 195–199 °C; H NMR (DMSO): δ = 1.30
(3H, t, CH3), 2.12 (3H, s, CH3(phenyl)), 2.19 (3H,s, CH3(phenyl)),
4.42 (2H, q, CH2), 7.17–7.23 (3H, m, H-6,2’,6’), 7.45 (1H, td,
J = 8.4 Hz, J = 1.2 Hz, H-7), 7.59-7.63 (4H, m, H-5,8,3’,5’),
7.87 (1H, dd, J = 8.8Hz, J = 1.6 Hz, H-2), 8.15 (1H, s, -NH),
8.21 (1H, d, J = 7.6 Hz, H-1), 8.42 (1H, d, J =1.2 Hz, H-4),
10.89 (1H, s, -CH=N). 13C NMR: δ = 13.7, 37.0, 98.4, 109.3,
109.4, 111.8, 119.1, 120.2, 120.5, 122.1, 122.3, 123.9,
125.9, 126.0, 133.5, 139.8, 139.9, 141.5, 148.8. ESI-MS: m /
z = 339 [M+1] (100 %). Found, %: C 77.02; H 5.65; N 16.29.
C22H18N4. 0.2 H2O.0.1 C2H6O Calcd., %: C 76.92; H 5.25; N
16.16.
ANTIOXIDANT ACTIVITY ON ROS-INDUCED DCFH-DA
OXIDATION
For estimation of intracellular ROS, DCFH-DA was used as a
probe. In cellular systems the non-fluorescent probe DCFH-
DA readily crosses the cell membrane and undergoes hy-
drolysis by intracellular esterases to non-fluorescent 2',7'-
dichlorofluorescin (DCFH). In the presence of ROS, DCFH is
Biological Activity
[25]
oxidized to highly fluorescent dichlorofluorescein (DCF)
CELL CULTURE AND REAGENTS
which can be detected by a fluorescent microplate reader.
Cells were seeded in black 96-well plates at a density
of 5 × 103 cells / well and incubated at 37 ˚C in a humid at-
mosphere containing 5 % CO2 for 24 h for cell attachment.
The medium was removed and then cells were incubated
with DCFH-DA (20μM) containing medium for 30 minutes.
Cells were washed with PBS to remove excess DCFH-DA.
10 µM synthesized compounds and 10 µM 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 minutes.
CHO-K1 cells were purchased from ATCC and maintained at
37 °C in 5 % CO2 atmosphere. Dulbecco’s modified Eagle’s
medium (DMEM) / F12 (Sigma, St. Louis, MO) (1 : 1) nutrient
mixture medium supplemented with 10 % foetal bovine
serum, 1 % of a 100 U / mL penicillin-streptomycin solution,
2 mM L-glutamine and 1 mM sodium pyruvate was used in
all cell incubations. Hyclone characterized fetal bovine se-
rum (FBS) was purchased from Thermo Scientific. 2′,7′-
Dichlorofluorescin diacetate (DCFH-DA), 2,2-diphenyl-1-
picrylhydrazyl (DPPH), 7-ethoxyresorufin, sodium pyruvate
and L-glutamine were obtained from Sigma (St. Louis, MO).
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) was purchased from Invitrogen (Oregon,
USA). Phenobarbital-induced rat liver microsomes were
kindly provided by Prof. Hilmi Orhan. Test materials were
dissolved in dimethyl sulfoxide (DMSO) and the final
concentration of DMSO never exceeded 0.1 % v / v in cell
culture medium.
DPPH FREE RADICAL SCAVENGING ACTIVITY
Free radical scavenging activity of newly synthesized
compounds was tested by their ability to bleach the stable
radical DPPH.[26] DPPH presents a maximum of absorbance
at 515 nm; when DPPH reacts with an antioxidant
compound, which can donate hydrogen, this absorbance
PC12 cells were a generous gift from Professor Lloyd
A. Greene (Department of Pathology and Cell Biology,
Columbia University, New York, USA). They were maintained
in RPMI 1640 supplemented with 10 % horse serum, 5 % fetal
bovine serum, 100 U / mL penicillin G and 100 µg mL–1
streptomycin. The cells were cultured on plates coated with
collagen obtained from rat tail and were grown at 37 °C in
humidified air containing 5 % CO2. Two-thirds of the
growing medium was changed every 2–3 days.
diminishes and can be measured on
a
visible
spectrophotometer. 20 μL of the samples (100 μM) were
added to 180 μL of DPPH solution (150 μM) in methanol–
water (80 : 20, v / v) in 96-well plates, incubated for 30
minutes at room temperature then DPPH reduction was
estimated at 517 nm. Percentage inhibition by the sample
treatment was determined by comparison with a DMSO-
treated control group. All experiments were carried out in
triplicate. MLT and butylated hydroxytoluene (BHT) were
used as a reference compounds. The radical scavenging
activities were expressed as the percent of inhibition and
were calculated by using the below formula:
CYTOTOXIC EFFECTS VIA THE MTT ASSAY
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 compounds (10 µM) for 24 h. Control
(medium only), vehicle control and positive control (15 µM
Radical scavenging activity (%) = [(A0 – A1 / A0) × 100]
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C. KARAASLAN et al.: 9-Ethyl-9H-carbazole Hydrazone Derivatives …
5
R
1
R
R
where A0 is the absorbance of the control (blank) and A1 is
the absorbance of the test compound.
O
1
2
NH
R
R
NH
N
H
N
2
2
R
+
3
R
N
N
3
R
H
C
H
C
1
2
3 a-r
3
3
INHIBITION OF NEUROTOXICITY INDUCED BY Aβ IN PC12
CELLS
Cell viability after exposure to Aβ25–35 was determined by
MTT assay, which was performed as previously described
with some modifications. [27] Briefly, the cells were seeded
in collagen-coated 96-well plates at a density of 5 × 104 cells
/ well and were allowed to adhere at 37 °C for 48 h. Three
different concentrations of carbazole derivatives (5, 25 and
100 µM) were added in duplicate and incubated for 3 h.
Maximum concentration of DMSO in the wells was kept
below 0.5 %. Aβ25–35 was then added at a final concentra-
tion of 5 µM for an additional 24 h in the presence or
absence of test compounds. After 24 h, the medium was
replaced with 20 µL of MTT 0.5 mg mL–1 dissolved in RPMI
and incubated for an additional 2 h at 37 °C. Afterwards,
formazan crystals were solubilized in 200 µl DMSO. The
optical density was measured at 570 nm with background
correction at 655 nm using a Bio-Rad microplate reader
(Model 680, Bio-Rad). Each experiment was repeated 3–5
times. The percent inhibition of cell damage was calculated
with reference to the absorbance of control wells not
treated with Aβ (assumed as 100 % protection) and the
absorbance of wells treated with Aβ in the absence of any
test compound (assumed as 0 % protection).
Scheme 1. Synthesis of 9-ethyl-9H-carbazole hydrazone
derivatives.
using a tetrozolium salt, MTT. The assay based on the re-
duction of MTT only by metabolically active cells to a col-
ored formazan product that can be quantified
spectrophotometrically.[20] Almost all compounds (10 µM)
were found to have no cytotoxic effect more than melato-
nin (MLT), an endogenous indole compound with known
antioxidant and anticarcinogenic activity (Figure 2) on CHO-
K1 cells when incubated for 24 hours. Only 3e and 3k
decreased cell viability more than MLT. Among halogen-
ated carbazole derivatives chloro- and fluoro-substituted
ones were found to be more cytotoxic than bromo-substi-
tuted compounds. The substances without any halogen
group and with methoxy substitution on aromatic ring 3p,
3q, 3r, did not have any effects on cell viability (Figure 3).
Statistical Analysis
Unpaired Student t-test was performed to evaluate the
significance of the differences between groups. Differences
with p < 0.05 were considered as statistically significant.
RESULTS AND DISCUSSION
Antioxidant Effects of the Synthesized
Compounds
Chemistry
The protective effect of synthesized carbazole-hydrazone
derivatives (10 µM) against cumene hydroperoxide induced
DCFH-DA oxidation was determined in CHO-K1 cells that
were preloaded with the fluorescent probe. Oxidation of
the probe located in the cytosol was detected after 60 min
of incubation (Figure 4). All of the carbazole derivatives,
except 3b, 3c, 3g and 3j had potent reducing activity against
DCFH oxidation, even higher than MLT. Compounds 3o, 3p,
3q and 3r bearing no halogen substituent, were found to
have reducing activity where 3r was found to be the most
potent antioxidant among all tested compounds. Among
the halogenated derivatives fluoro-substituted ones were
found to have better antioxidant effects compared to
chloro- and bromo-halogenated derivatives.
Synthesis of all described carbazole hydrazone derivatives
were carried out according to the method described in lit-
erature[24] (Scheme 1). 9-Ethyl-9H-carbazole-3-carboxalde-
hyde was heated with several phenyl hydrazine derivatives
in the presence of ethanol to obtain carbazole hydrazone
derivatives.
Cytotoxic Effects of the Synthesized
Compounds
The failure of early identification of toxic effects of com-
pounds resulted in attrition of new drug candidates,[19]
which cause important losses for pharmaceutical industry.
Therefore, screening for cytotoxicity has become one of the
most important steps in early phases of drug discovery. In
the present study cytotoxicity of the synthesized carbazole-
hydrazone derivatives were evaluated in CHO-K1 cells by
Free radical scavenging activity of the newly
synthesized compounds was further investigated in a cell
free in vitro model where an active radical, DPPH was used.
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C. KARAASLAN et al.: 9-Ethyl-9H-carbazole Hydrazone Derivatives …
The radical scavenging activity of the compounds was
detected via decrease in the absorbance of DPPH. All of the
synthesized compounds had radical scavenging activity in
tested concentration (100 µM) where MLT was found to
have the lowest effect (Figure 5). Among mono-halogen-
ated derivatives m-substituted derivatives were found to
have higher activity and p-halogenation was found to
decrease antioxidant activity which was similar to our
previous findings.[21,22] 3r was found to be the most potent
radical scavenger among all tested compounds similar to its
reducing effect found in DCFH assay (Figure 4).
Antioxidant molecules have proved to be effective in
AD mouse models and small clinical studies. Clinical trials
for AD prevention and treatment by antioxidants are still
under discussion. Hence, advance studies are necessary to
determine if antioxidants may decrease the risk or slow the
progression of the disease for AD patients.[23]
Figure 3. Effect of samples (10µM) on cell viability evaluated
by MTT assay. Bars represent ‘medium ± standard deviation’
values from five individual experiments. *p < 0.05,
** p < 0.005, *** p < 0.0005, groups compared to vehicle
control group. TritonX-100 used as positive control group
(MLT: Melatonin).
Protection of Neuronal PC12 Cells
Against Aβ-Induced Damage
Neuroprotective capacity of synthesized derivatives was
assessed in PC12 neuronal cells challenged with Aβ, which
is a well-established cell model of neurodegeneration.
None of the compounds showed any cytotoxicity against
the cells when incubated with the cells alone (data not
shown). Several carbazole derivatives showed significant
protection against Aβ at 25 and 100 µM including 3d, 3e,
3h and 3n. It was remarkable to note that compound 3r
showed a high protection against Aβ damage at the lower
dose of 25 µM (Figure 6).
Figure 4. DCFH oxidation in CHO cells after 60 minutes
incubation with compounds (10 µM) in the presence of
cumene hydroperoxide (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. *p < 0.05,
** p < 0.005.(MLT: Melatonin).
Figure 6. Protection of PC12 neuronal cells against Aβ-
induced damage by synthesized carbazole derivatives
measured by MTT assay. PC12 cells seeded in collagen-
coated 96-well microplates were pre-incubated with
different concentrations of synthesized derivatives for 3 h
and then challenged with Aβ 5 µM. MTT assay was
performed to measure cell viability after 24 h. Each
experiment was repeated 3–5 times. Concentrations of
analogues are expressed as µM. Data are expressed as
mean ± S.E.M. *Significantly different from control wells
treated with Aβ (p < 0.05).
Figure 5. Radical scavenging activity of compounds
(100 µM) determined by DPPH assay. Bars represent
‘medium ± standard deviation’ values from four different
experiments. BHT and MLT were used as reference (BHT:
Butylated hydroxytoluene, MLT: Melatonin).
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7
Compounds with electron withdrawing moieties on
the phenyl ring including those bearing Cl (3–3f), Br (3–3i)
and F (3–3n) showed much better protective effects in
PC12 cells compared to derivatives bearing electron
donating groups including the ones containing methoxy
(3o) and methyl moieties (3p–3q) on the phenyl ring.
Among halogen containing synthesized compounds, the
ones bearing two halogen groups on the phenyl ring,
including 3d and 3e with 3,4-dichlorophenyl and 3,5-
dichlorophenyl rings, respectively, as well as 3m and 3n,
with 2,5-dibromophenyl and 3,5-dibromophenyl rings,
respectively, were more effective than those bearing only
one halogen group.
spectrophotometric analysis were performed at Ege
University Faculty of Pharmacy, Pharmaceutical Science
Research Center (FABAL). The authors also wish to thank for
the support of Vice-Chancellor for Research, Shiraz
University of Medical Sciences, Iran.
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