Novel indole hydrazide derivatives: Synthesis and their antiproliferative activities through inducing apoptosis and DNA damage

Article abstract of DOI:10.1002/ardp.202000059

A series of novel indole hydrazide derivatives was synthesized and evaluated for their anticancer activities. Compound 12 exhibited the highest antiproliferative activity against the MCF-7 cell line, with an IC₅₀ value of 3.01 μM. Treatment of MCF-7 cells with compound 12 led to cell cycle arrest at the G0/G1 phase and also displayed a significant annexin V binding pattern, indicating that compound 12 is effective in apoptotic cell death. The Western blot analysis showed that compound 12 increased the expression of proapoptotic Bax and decreased the levels of the antiapoptotic Bcl-2 protein. It was also observed that MCF-7 cells treated with compound 12 showed reduced levels of procaspase-3 and -9 proteins. Moreover, compound 12 treatment induced a significant DNA damage in MCF-7 cells by increasing H2AX and ATM phosphorylation.

Full text of DOI:10.1002/ardp.202000059

Received: 26 February 2020
Revised: 23 April 2020
Accepted: 25 April 2020
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DOI: 10.1002/ardp.202000059
F U L L P A P E R
Novel indole hydrazide derivatives: Synthesis and their
antiproliferative activities through inducing apoptosis and
DNA damage
Zühal Kilic‐Kurt1
| Cemre Acar1
| Mustafa Ergul2
| Filiz Bakar‐Ates3
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Tunca G. Altuntas1
1Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Ankara University,
Ankara, Turkey
Abstract
A series of novel indole hydrazide derivatives was synthesized and evaluated for
their anticancer activities. Compound 12 exhibited the highest antiproliferative
activity against the MCF‐7 cell line, with an IC50 value of 3.01 µM. Treatment of
MCF‐7 cells with compound 12 led to cell cycle arrest at the G0/G1 phase and
also displayed a significant annexin V binding pattern, indicating that compound
12 is effective in apoptotic cell death. The Western blot analysis showed that
compound 12 increased the expression of proapoptotic Bax and decreased
the levels of the antiapoptotic Bcl‐2 protein. It was also observed that MCF‐7
cells treated with compound 12 showed reduced levels of procaspase‐3 and
‐9 proteins. Moreover, compound 12 treatment induced a significant DNA
damage in MCF‐7 cells by increasing H2AX and ATM phosphorylation.
2Department of Biochemistry, Faculty of
Pharmacy, Sivas Cumhuriyet University, Sivas,
Turkey
3Department of Biochemistry, Faculty of
Pharmacy, Ankara University, Ankara, Turkey
Correspondence
Zühal Kilic‐Kurt, Department of
Pharmaceutical Chemistry, Faculty of
Pharmacy, Ankara University, Tandogan,
06100 Ankara, Turkey.
Email: zkurt@ankara.edu.tr
Funding information
Ankara University
K E Y W O R D S
anticancer activity, apoptosis, cell cycle, DNA damage, synthesis
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1
INTRODUCTION
synthesized indole‐based analogs, which were evaluated for
their antiproliferative activity against leukemia, breast, and
The indole scaffold is a privileged structure in medicinal chem-
istry due to its versatile pharmacological activities such as anti-
bacterial, antifungal, anti‐inflammatory, antioxidant, antiviral,
and anticancer activities. In the last decade, numerous indole
derivatives have been reported to demonstrate anticancer
activities through different targets.[1] Camalexin (Figure 1), an
indole phytoalexin, was reported as a potent antitumor agent
against human leukemia cell lines and primary human AML
cells through the reactive oxygen species (ROS)–endoplasmic
reticulum (ER) stress–mitochondrial apoptosis pathway.[2]
Zhang et al. reported 5‐chloro‐3‐phenyl‐indole‐2‐carboxylic
acid (4‐nitrobenzylidene)‐hydrazide (I) (Figure 1) as a potent
apoptosis inducer through cell‐based high‐throughput screening
assay. Compound I showed caspase activation with an EC50 value
of 0.1 µM in T47D breast cancer cells and growth inhibition with
a GI50 value of 0.9 µM in T47D cells.[3] The Spallarossa group
renal cancer cell lines. Among them, compound II (Figure 1)
showed strong proapoptotic potential and a partial depolymer-
izing effect on tubulin.[4] El‐Nakkady et al.[5] reported a series of
2‐phenylindole derivatives as potential anticancer agents. Com-
pound III (Figure 1) exhibited a remarkable antitumor activity,
with an IC50 value of 1.6 nM, against the MCF‐7 breast carcinoma
cell line. Moreover, a series of indole‐2‐carbohydrazide deriva-
tives was synthesized, with some of them displaying potent cy-
totoxic activities against HCT116 and SW480 cell lines with GI50
values in sub‐micromolar concentrations.[6] Haddach et al.[7]
identified 3‐(5‐((2‐oxoindolin‐3‐ylidene)methyl)furan‐2‐yl)amide
derivative (CX‐6258) (Figure 1) as a pan‐Pim kinase inhibitor
with excellent antiproliferative potency in cellular assays. More
recently, panobinostat (Figure 1) has been approved as an oral
pan‐histone deacetylase inhibitor for the treatment of relapsed
multiple myeloma. It can also inhibit tumor growth and
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Arch Pharm. 2020;e2000059.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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FIGURE 1 Chemical structures of some indole derivatives showing anticancer activity
metastasis.[8,9] Sreenivasulu et al. reported the synthesis and
anticancer evaluation of a series of hydrazide–hydrazone‐linked
indole and indazole moieties, and N‐methyl and N,N‐dimethyl bis
(indolyl)hydrazide–hydrazone derivatives.[10,11] Among them,
compound IV[10] exhibited strong cytotoxicity, with an IC50 value
of 1.93 µM, against the MCF‐7 breast cancer cell line, as com-
pared with the standard drug doxorubicin (IC50 value: 0.98 µM).
Compound V[11] induced apoptosis via a caspase‐independent
pathway through the participation of mitogen‐activated protein
kinases such as extracellular signal‐regulated kinase and p38 as
well as p53 pathways.
2
RESULTS AND DISCUSSION
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2.1
Chemistry
The general methods for preparing indole‐2‐carbohydrazide deriva-
tives are shown in Scheme 1. First, the methyl esters of indole‐2‐
carboxylic acid derivatives (1a–c) were prepared in a quantitative
yield.[12] Hydrazide derivatives of indoles (2a–c) were obtained by
the reaction of indole esters with hydrazine hydrate in ethanol.[13]
Hydrazide derivatives were reacted with appropriate aromatic al-
dehydes or ketones in the presence of the catalytic amount of glacial
acetic acid[14] to afford the target compounds (3–17).
In light of the abovementioned observations regarding indole
compounds, it is understood that substitution on 2‐, 3‐, and
5‐positions of the indole scaffold is useful for anticancer
activity. Therefore, in this study, we synthesized a series of
5‐substituted indole hydrazide derivatives and evaluated their
anticancer activities.
It is well known that N‐acylhydrazones may exist as geometrical
isomers (E/Z) and as cis/trans rotamers due to the assemblage of
imine functions (C═N) and amide bond (CO–NH), respectively. The
acylhydrazones of aromatic aldehydes are reported to be prevailingly
or completely in the Ε‐configuration.[15–17] In 1H nuclear magnetic
SCHEME 1 The synthesis method for
the target compounds. Reagents and
conditions: (a) H2SO4, methanol, reflux,
overnight; (b) hydrazine hydrate,
methanol, reflux; (c) appropriate aldehyde
or ketone derivatives, absolute ethanol,
glacial acetic acid, reflux

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Apoptosis assay
resonance (NMR) spectra of aldehyde‐derived compounds, one set of
singlet belongs to N═CH and CO–NH protons, indicating the pre-
sence of a single isomer of the compounds. The appearance of du-
plicate signals for amide and methyl protons in 1H NMR spectra of
compounds (7 and 16) derived from ketone revealed that these
compounds exist as a mixture of two possible rotamers or E/Z iso-
mers. Unlike ketone derivatives 7 and 16, in 1H NMR spectrum of
compound 11, one set of singlet arising from imine and amide pro-
tons was determined.
2.2.3
According to the XTT (2,3‐bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐
tetrazolium‐5‐carboxanilide) assay results, compound 12 showing the
strongest antiproliferative activity against MCF‐7 cells has been se-
lected for further assays. The apoptotic effects of compound 12 on
MCF‐7 cells were evaluated using the annexin V binding analyses
through flow cytometry. Treatment of MCF‐7 cells with IC50 value of
compound 12 for 48 hr significantly induced apoptosis, as shown in
Figure 4. The percentage of the early apoptotic and late apoptotic
cells in compound 12‐treated MCF‐7 cells increased to 29.13 2.99%
and 27.18 0.99%, respectively, compared with the control cells
(2.49 1.50% and 1.50 0.70%, respectively; p < .01).
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2.2
Biological activity
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2.2.1
Antiproliferative activity
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DNA damage analysis
Antiproliferative activities of all synthesized compounds (3–17) were
evaluated against MCF‐7, HepG2, and PC3 cell lines. Initially, the
cancer cells were treated with compounds 3–17 at 20‐µM con-
centration. The cell viability % (CV%) values were calculated after
treating cells for 48 hr. The CV% results are presented in Table 1, and
it has been shown that all compounds generally have better anti-
proliferative activity against MCF‐7 than HepG2 and PC3 cell lines.
Compounds 5, 12, and 14 demonstrated higher cytotoxicity than
other compounds against MCF‐7 cells. However, almost all com-
pounds demonstrated moderate, low, or no antiproliferative activity
against HepG2 and PC3 cell lines.
2.2.4
To demonstrate whether compound 12 treatment induced DNA da-
mage in MCF‐7 cells, the cells were treated with an IC50 con-
centration of compound 12 for 48 hr and then DNA damage assay
was performed. Treatment of MCF‐7 cells with compound 12 sig-
nificantly induced DNA damage response in MCF‐7 cells, as shown in
Figure 5. After compound 12 exposure, the level of H2AX and ATM
phosphorylation markedly increased as compared with control cells.
The percentage of DNA double‐strand breaks and total single‐strand
breaks (pATM) in compound 12‐treated cells increased to
24.12 2.19% and 38.44 2.30%, respectively, compared with the
control cells (5.40 1.40% and 6.8 1.69%, respectively; p < .01).
Meanwhile, as shown in Figure 2, the three active compounds
(5, 12, and 14) significantly decreased the cell viability in a dose‐
dependent manner (p < .01). The IC50 values of compounds 5, 12,
and 14 were determined as 7.32, 3.01, and 5.48 µM, respectively,
for 48 hr in MCF‐7 cells. Regarding the activity against MCF‐7
cells, 5‐methoxyindole derivatives (12–17) were found to be more
potent than the fluoro (3–8) and chloro (9–11) derivatives.
Replacement of the 3,4‐dichlorobenzylidene moiety (14) with
4‐chlorobenzylidene (12) increased the anticancer activity. How-
ever, the five‐ring scaffold such as pyrrole (8, 10, and 17) or imi-
dazole (9) instead of benzene ring at the side chain of the indole
scaffold decreased the anticancer activity. Structure–activity re-
lationship results suggested that a methoxy group at the 5‐position
of the indole ring and chloro substitution on the benzylidene moiety
are important for the anticancer activity.
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2.2.5
Western blot analysis
The Western blot analysis was performed to evaluate the alteration
in the expression of apoptosis‐related proteins. In this context, the
proapoptotic Bax and antiapoptotic Bcl‐2 protein expression levels
were determined by sodium dodecyl sulfate‐polyacrylamide gel
electrophoresis (SDS‐PAGE). As shown in Figure 6, after the treat-
ment of MCF‐7 cells with compound 12, the proapoptotic Bax ex-
pression significantly increased as compared with untreated control
(p < .05), whereas there was a decrease in the expression of anti-
apoptotic Bcl‐2. Although the apoptotic potential of compound 12
has been demonstrated by annexin V binding analysis, its effect on
the levels of proapoptotic caspases has also been evaluated. Cas-
pases are a group of endoprotease enzymes that hydrolyze peptide
bonds in a reaction that depends on catalytic cysteine residues in the
caspase and aspartic acid residues in the substrate.[18] Initially, cas-
pases are produced as inactive monomeric procaspases, and they
become active after a dimerization or cleavage process.[19] In the
present study, we evaluated the alteration in procaspase‐3 and ‐9
expression after the treatment of MCF‐7 cells with compound 12.
The results showed that caspase‐3 expression significantly reduced
as compared with control (p < .01), confirming the caspase‐activating
effect of compound 12.
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2.2.2
Cell cycle assay
The cell cycle assay was performed for further evaluation of the
cytotoxic activity of compound 12 against MCF‐7 cells. The cells
were treated with IC50 concentration of compound 12 for 48 hr.
As shown in Figure 3, the cell population percent at the G0/G1
phase increased to 45.6 1.13% in compound 12‐treated group,
whereas it was 1.7 0.14% in the control group, suggesting that
compound 12 induced a significant cell cycle arrest at the G0/G1
phase (p < .0001).

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TABLE 1 The percentage cell viability (CV%) of the cancer cell lines after 48 hr of exposure to synthesized compounds at 20 µM
Compounds
3
R1
R2
Ar
MCF‐7
HepG2
PC3
F
H
42.04 2.13
78.86 3.45
71.47 1.18
4
5
F
F
H
H
99.84 3.12
98.19 4.17
49.81 3.79
101 2.16
15.09 1.37 (7.32 µM)
40.63 3.26
6
7
F
F
H
49.66 2.88
53.92 1.13
66.30 3.11
60.88 4.56
71.49 1.08
74.19 1.17
CH3
8
F
H
107.95 1.49
58.41 2.93
79.56 1.09
92.63 4.13
73.22 2.79
70.65 1.13
63.64 1.11
69.34 2.07
98.32 2.54
62.1 1.56
64.61 4.12
62.11 4.02
9
Cl
H
10
11
12
Cl
H
68.72 4.19
Cl
CH3
31.97 1.29
OCH3
H
23.03 3.51 (3.01 µM)
13
14
OCH3
OCH3
H
H
38.45 3.13
68.38 2.31
67.54 3.09
78.9 1.32
22.15 4.59 (5.48 µM)
50.76 1.04
15
OCH3
H
25.50 1.09
62.09 1.13
54.71 3.19
16
17
OCH3
OCH3
CH3
26.15 1.91
42.86 4.01
54.52 5.47
69.57 4.57
46.13 3.12
53.13 2.18
H
Note: Data are presented as mean standard deviation. The three most active compounds are represented in bold.

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4
EXPERIMENTAL
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4.1
Chemistry
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4.1.1
General
Commercial reagents were used without further purification. The
NMR spectra (400 MHz) were recorded on Varian Mercury
a
400 NMR spectrometer (Varian Inc., Palo Alto, CA) using tetra-
methylsilane as the internal standard. Thin‐layer chromatography
was performed on F254 (Merck) silica gel plates. The electrospray
ionization (ESI) spectra were recorded on a Waters ZQ Micromass
liquid chromatography–mass spectrometry (LC–MS) spectrometer
(Waters Corporation, Milford, MA) with ESI source as ionization.
The elemental analysis was performed using a Leco‐932 CHNS‐O
Analyzer (Leco, St. Joseph, MI) within 0.4% of the theoretical
values.
FIGURE 2 The antiproliferative activity of compounds 5, 12, and
14 against the most sensitive cell line MCF‐7. The cells were treated
with the compounds at different concentrations (2.5–40 μM) for
48 hr, and the cell viability was assessed using the XTT assay. All data
are expressed as mean SD of three replicates. All the cell viability
values at 2.5‐, 5‐, 10‐, 20‐, and 40‐μM concentrations are significantly
different from control (p < .01). SD, standard deviation; XTT, 2,3‐bis‐
(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide
The original spectra of the investigated compounds as well as
their InChI codes, together with some biological activity data, are
provided as Supporting Information Data.
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3
CONCLUSION
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General procedure for the synthesis of
methyl esters of indole‐2‐carboxylic acids 1a–c
In this study, we synthesized a series of novel 5‐substituted indole
hydrazide derivatives and evaluated their anticancer activities.
Compound 12 exhibited the highest cytotoxic activity against
MCF‐7 human breast cancer cells with IC50 value of 3.01 µM. Ex-
posure of MCF‐7 cells to compound 12 significantly induced
apoptosis. Preliminary mechanism of action studies showed that
compound 12 increased the expression of proapoptotic Bax and
decreased the expression of antiapoptotic Bcl‐2 protein. Also,
treatment of MCF‐7 cells with compound 12 resulted in reduced
levels of procaspase‐3 and ‐9 proteins, indicating that compound
4.1.2
Indole‐2‐carboxylic acids (1 mmol) were refluxed in MeOH (5 ml) in
the presence of concentrated sulfuric acid (0.1 ml) overnight. Then,
the mixture was concentrated and saturated NaHCO3 (aq) was ad-
ded. The precipitate was filtered, washed with water, and dried to
afford the target compounds (1a,b,[20] 1c[21]).
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General procedure for the synthesis of
12 induced apoptosis through
a
caspase‐dependent pathway.
4.1.3
Moreover, in compound 12‐treated experiments, the level of
H2AX and ATM phosphorylation, the percentage of DNA double‐
strand breaks, and total single‐strand breaks increased, confirming
DNA damage effect of compound 12.
indole‐2‐carbohydrazide derivatives 2a–c
Hydrazine hydrate (10 mmol) was added to a suspension of methyl
esters of indole‐2‐carboxylic acids (1a–c, 1 mmol) in methanol (15 ml).
FIGURE 3 The effect of compound 12 on cell cycle inhibition. The MCF‐7 cells were treated with compound 12 with an IC50 value of
3.01 μM for 48 hr, and the cell population was detected by Muse Cell Analyzer (Millipore, Germany). The nontreated cells were used as
control. The results indicate mean standard deviation of two independent experiments. The difference is expressed as #p < .0001, compared
with control

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FIGURE 4 Apoptotic effects of compound 12 on MCF‐7 cells. The cells were treated with the IC50 concentration of compound 12 and
incubated for 48 hr, and the apoptosis was detected by Muse Cell Analyzer (Millipore). All experiments were carried out in triplicate, thereby
obtaining similar results. Statistically significant differences are expressed as *p < .01, compared with the control
The reaction mixture was refluxed for 3 hr, cooled to room tempera-
ture, and filtered to afford hydrazide derivatives (2a,[22] 2b,[6] 2c[13]).
145.91, 155.99, 157.30, 158.31. MS (ESI) m/z: 316.08 [M + H].
Anal. calcd. for C16H11ClFN3O·0.2H2O (%): C, 60.17; H, 3.59; N,
13.15. Found (%): C, 59.92; H, 3.58; N, 13.87.
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4.1.4
General procedures for the synthesis of
5‐Fluoro‐N′‐(4‐fluorobenzylidene)‐1H‐indole‐2‐carbohydrazide (4)
Yield: 70%; Mp: 251–253°C. 1H NMR (400 MHz, DMSO‐d6) δ: 7.087 (td,
1H, J = 13.6 Hz, 2.4 Hz), 7.29 (t, 3H), 7.43–7.47 (m, 2H), 7.80 (t, 2H), 8.44
(s, 1H, –N═CH), 11.92 (s, 2H, NH). 13C NMR (100 MHz, DMSO‐d6) δ:
103.55, 105.89, 112.626, 113.594, 115.90, 127.03, 129.26, 130.87,
131.71, 133.56, 146.22, 156.06, 157.34, 158.38, 161.86, 164.32. MS
(ESI) m/z: 300.52 [M + H]. Anal. calcd. for C16H11F2N3O (%): C, 64.21; H,
3.70; N, 14.04. Found (%): C, 64.18; H, 3.71; N, 14.25.
compounds 3–17
Indole‐2‐carbohydrazide derivatives (2a–c, 1 mmol), appropriate
aromatic aldehyde or ketone derivatives (1 mmol), and the catalytic
amount of glacial acetic acid in absolute ethyl alcohol were refluxed
for 24 hr. The precipitate was filtered and washed with ethanol. The
crude products were recrystallized from ethanol to afford the pure
target compounds 3–17.
N′‐(3,4‐Dichlorobenzylidene)‐5‐fluoro‐1H‐indole‐2‐
N′‐(4‐Chlorobenzylidene)‐5‐fluoro‐1H‐indole‐2‐carbohydrazide (3)
Yield: 93%; Mp: 263–265°C. 1H NMR (400 MHz, dimethyl
sulfoxide [DMSO]‐d6) δ: 7.09 (td, 1H, J = 8.8 Hz, 2.4 Hz), 7.31
(s, 1H, H‐3), 7.44–7.49 (m, 2H), 7.53 (d, 2H, J = 8.4 Hz), 7.78
(d, 2H, J = 8.4 Hz), 8.44 (s, 1H, –N═CH), 11.94 (s, 1H, NH), 12.01
(s, 1H, NH). 13C NMR (100 MHz, DMSO‐d6) δ: 103.59, 105.84,
112.62, 113.54, 126.94, 128.76, 131.55, 133.16, 133.52, 134.40,
carbohydrazide (5)
Yield: 68%; Mp: 257–260°C. 1H NMR (400 MHz, DMSO‐d6) δ: 7.10 (t,
1H), 7.33 (s, 1H), 7.45–7.48 (m, 2H), 7.70–7.77 (m, 2H), 7.97 (d, 1H,
J = 1.6 Hz), 8.42 (s, 1H), 11.92 (s, 1H, NH), 12.10 (s, 1H, NH). 13C NMR
(100 MHz, DMSO‐d6) δ: 103.89, 105.90, 112.75, 113.59, 126.87,
127.01, 128.47, 131.04, 131.69, 132.16, 133.60, 135.10, 144.52,
156.05, 158.36. MS (ESI) m/z: 350.22 [M + H]. Anal. calcd. for

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FIGURE 5 Compound 12 induced DNA damage response in MCF‐7 cells. The cells were treated with compound 12 at IC50 concentration for
48 hr to induce DNA damage, and activation of ATM and H2AX was determined using Muse™ Multi‐Color DNA Damage Kit (Merck Millipore).
Results are expressed as mean standard deviation; *p < .01
C16H10Cl2FN3O·0.025H2O (%): C, 54.80; H, 2.88; N, 11.98. Found
(%): C, 54.41; H, 2.94; N, 12.30.
8.01 (d, 1H, J = 3.2 Hz), 8.63 (s, 1H), 11.94 (s, 1H, NH), 12.05
(s, 1H, NH). 13C NMR (100 MHz, DMSO‐d6) δ: 103.99, 104.62,
105.974, 112.76, 118.76, 127.048, 127.97, 131.51, 133.66,
139.25, 156.09, 157.36, 158.41, 159.74, 162.03, 164.43. MS
(ESI) m/z: 340.51 [M + Na]. Anal. calcd. for C16H10F3N3O·1.2H2O
(%): C, 56.70; H, 3.68; N, 12.39. Found (%): C, 56.47; H, 3.77;
N, 12.65.
N′‐(2,4‐Difluorobenzylidene)‐5‐fluoro‐1H‐indole‐2‐
carbohydrazide (6)
Yield: 54%; Mp: 233–235°C. 1H NMR (400 MHz, DMSO‐d6) δ:
7.09 (t, 1H), 7.21 (s, 1H), 7.30–7.39 (m, 2H), 7.47 (d, 2H, J = 7.6 Hz),
FIGURE 6 Effects of compound 12 on apoptotic protein expression in MCF‐7 cells. The cells were treated with 3.01 μM of compound 12 for
48 hr, and total cell lysates were prepared and immunoblotted to detect Bax, Bcl‐2, and caspase‐3/‐9. β‐Actin served as a loading control.
Results were expressed as the mean standard deviation; *p < .05, **p < .01, as compared with control

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N′‐[1‐(2,4‐Dichlorophenyl)ethylidene]‐5‐fluoro‐1H‐indole‐2‐
carbohydrazide (7)
N′‐(4‐Chlorobenzylidene)‐5‐methoxy‐1H‐indole‐2‐
carbohydrazide (12)
Yield: 72%; Mp: 204–206°C. 1H NMR (400 MHz, DMSO‐d6) δ: 2.30 (s,
3H, CH3), 7.03–7.11 (m, 1H), 7.37–7.47 (m, 2H), 7.52 (s, 1H), 7.55 (dd,
1H, J = 8.0 Hz, 2.0 Hz), 7.72 (t, 1H), 7.75 (d, 1H, J = 2.0 Hz), 10.30 (s, 1H,
NH), 11.89 (s, 1H, NH). 13C NMR (100 MHz, DMSO‐d6) δ: 18.32, 105.94,
112.63, 113.56, 126.97, 127.56, 128.06, 129.35, 130.78, 131.59,
132.19, 133.28, 133.95, 134.62, 137.99, 155.92, 158.29. MS (ESI) m/z:
364.68 [M + H], 386.54 [M + Na]. Anal. calcd. for C17H12Cl2FN3O (%): C,
56.06; H, 3.32; N, 11.53. Found (%): C, 55.77; H, 3.64; N, 11.62.
Yield: 84%; Mp: 243–245°C. 1H NMR (400 MHz, DMSO‐d6) δ: 3.75
(s, 3H, CH3), 6.86 (dd, 1H, J = 9.2 Hz, 2.4 Hz), 7.12 (s, 1H), 7.21 (d, 1H,
J = 3.2 Hz), 7.33 (d, 1H, J = 8.8 Hz), 7.51 (d, 2H, J = 8.0 Hz), 7.75 (d, 2H,
J = 8.4 Hz), 8.42 (s, 1H), 11.66 (s, 1H, NH), 11.89 (s, 1H, NH). 13C NMR
(100 MHz, DMSO‐d6) δ: 55.18, 102.01, 103.34, 113.14, 115.17,
127.21, 128.57, 128.85, 130.08, 132.11, 133.24, 134.28, 145.51,
153.80, 157.54. MS (ESI) m/z: 328.50 [M + H], 350.46 [M + 23]. Anal.
calcd. for C17H14ClN3O2·0.02H2O (%): C, 62.22; H, 4.31; N, 12.80.
Found (%): C, 61.83; H, 4.40; N, 12.88.
N′‐[(1H‐Pyrrol‐2‐yl)methylene]‐5‐fluoro‐1H‐indole‐2‐
carbohydrazide (8)
N′‐(4‐Fluorobenzylidene)‐5‐methoxy‐1H‐indole‐2‐
Yield: 50%; Mp: 243–245°C. 1H NMR (400 MHz, DMSO‐d6) δ: 6.13
(s, 1H), 6.49 (s, 1H), 6.90 (s, 1H), 7.04 (t, 1H), 7.23 (s, 1H), 7.40–7.44
(m, 2H), 8.27 (s, 1H, –N═CH), 11.52 (s, 1H, NH), 11.58 (s, 1H, NH),
11.86 (s, 1H, NH). 13C NMR (100 MHz, DMSO‐d6) δ: 102.91, 105.674,
109.20, 112.182, 113.374, 122.48, 126.89, 127.0, 132.09, 133.32,
140.47, 155.96, 156.77, 158.27. MS (ESI) m/z: 271.54 [M + H]. Anal.
calcd. for C14H11FN4O·0.3H2O (%): C, 60.99; H, 4.24; N, 20.32.
Found (%): C, 60.90; H, 4.08; N, 20.63.
carbohydrazide (13)
Yield: 75%; Mp: 213–215°C. 1H NMR (400 MHz, DMSO‐d6) δ: 3.76
(s, 3H, CH3), 6.87 (dd, 1H, J = 9.2 Hz, 2.4 Hz), 7.13 (s, 1H), 7.22 (s, 1H),
7.28–7.36 (m, 3H), 7.78–7.82 (m, 2H), 8.45 (s, 1H), 11.65 (s, 1H, NH),
11.85 (s, 1H, NH). 13C NMR (100 MHz, DMSO‐d6) δ: 55.74, 102.56,
103.77, 113.69, 115.65, 116.38, 127.79, 129.66, 130.70, 131.44,
132.65, 146.30, 154.35, 158.08, 162.28, 164.74. MS (ESI) m/z: 312.61
[M + H], 334.55 [M + Na]. Anal. calcd. for C17H14FN3O2·0.05H2O (%):
C, 65.39; H, 4.55; N, 13.45. Found (%): C, 65.00; H, 4.83; N, 13.42.
N′‐[(1H‐Imidazol‐2‐yl)methylene]‐5‐chloro‐1H‐indole‐2‐
carbohydrazide (9)
N′‐(3,4‐Dichlorobenzylidene)‐5‐methoxy‐1H‐indole‐2‐
carbohydrazide (14)
Yield: 80%; Mp: 318–320°C. 1H NMR (400 MHz, DMSO‐d6) δ:
7.14–7.28 (m, 4H), 7.45 (d, 1H, J = 8.8 Hz), 7.76 (s, 1H), 8.36 (s, 1H,
–N═CH), 12.25 (bs, 3H, NH). 13C NMR (100 MHz, DMSO‐d6) δ:
103.13, 113.92, 120.77, 123.91, 124.40, 127.95, 131.49, 135.17,
139.28, 142.34, 157.24. MS (ESI) m/z: 288.39 [M + H]. Anal. calcd. for
C13H10ClN5O·0.15H2O (%): C, 53.76; H, 3.57; N, 24.11. Found (%): C,
53.50; H, 3.72; N, 23.86.
Yield: 78%; Mp: 274–276°C. 1H NMR (400 MHz, DMSO‐d6) δ: 3.77
(s, 3H, CH3), 6.89 (dd, 1H, J = 8.4 Hz, 2.4 Hz), 7.14 (s, 1H), 7.26 (s, 1H),
7.36 (d, 1H, J = 9.2 Hz), 7.70–7.76 (m, 2H), 7.96 (s, 1H), 8.41 (s, 1H),
11.69 (s, 1H, NH), 12.04 (s, 1H, NH). 13C NMR (100 MHz, DMSO‐d6)
δ: 55.26, 102.09, 103.69, 113.26, 115.40, 126.81, 127.28, 128.43,
130.0, 131.08, 131.71, 132.09, 132.26, 135.24, 144.15, 153.91,
157.75. MS (ESI) m/z: 362.62 [M + H], 384.37 [M + Na]. Anal. calcd.
for C17H13Cl2N3O2 (%): C, 56.37; H, 3.61; N, 11.60. Found (%): C,
56.00; H, 3.75; N, 11.87.
N′‐[(1H‐Pyrrol‐2‐yl)methylene]‐5‐chloro‐1H‐indole‐2‐
carbohydrazide (10)
Yield: 38%; Mp: 253–254°C. 1H NMR (400 MHz, DMSO‐d6) δ: 6.14
(s, 1H), 6.50 (s, 1H), 6.91 (s, 1H), 7.18–7.23 (m, 2H), 7.43 (d, 1H,
J = 8.8 Hz), 7.74 (s, 1H), 8.27 (s, 1H, –N═CH), 11.54 (s, 1H, NH), 11.64
(s, 1H, NH), 11.98 (s, 1H, NH). 13C NMR (100 MHz, DMSO‐d6) δ:
102.54, 109.31, 113.46, 113.89, 120.68, 122.62, 123.66, 124.37,
126.97, 128.07, 131.97, 135.07, 140.61, 156.80. MS (ESI) m/z: 287.50
[M + H]. Anal. calcd. for C14H11ClN4O·0.2H2O (%): C, 57.91; H, 3.95;
N, 19.29. Found (%): C, 57.70; H, 4.03; N, 19.61.
N′‐(2,4‐Difluorobenzylidene)‐5‐methoxy‐1H‐indole‐2‐
carbohydrazide (15)
Yield: 70%; Mp: 245–247°C. 1H NMR (400 MHz, DMSO‐d6) δ: 3.77
(s, 3H, CH3), 6.89 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 7.14 (d, 1H, J = 2.4 Hz),
7.19–7.23 (m, 2H), 7.36 (d, 1H, J = 8.8 Hz), 7.40 (d, 1H, J = 2.0 Hz),
7.98–8.04 (m, 1H), 8.64 (s, 1H), 11.68 (s, 1H, NH), 11.97 (s, 1H, NH).
13C NMR (100 MHz, DMSO‐d6) δ: 55.26, 102.10, 103.44, 104.48,
112.63, 113.24, 115.33, 118.81, 127.30, 127.90, 130.09, 132.23,
138.79, 153.90, 157.65, 159.62, 162.51, 164.46. MS (ESI) m/z: 330.58
[M + H], 352.48 [M + Na]. Anal. calcd. for C17H13F2N3O2·0.01H2O (%):
C, 61.97; H, 3.98; N, 12.75. Found (%): C, 61.65; H, 4.31; N, 12.71.
5‐Chloro‐N′‐[1‐(2,4‐dichlorophenyl)ethylidene]‐1H‐indole‐2‐
carbohydrazide (11)
Yield: 90%; Mp: 216–218°C. 1H NMR (400 MHz, DMSO‐d6) δ: 2.36
(s, 3H, CH3), 7.20 (dd, 1H, J = 8.8 Hz, 2.0 Hz), 7.35 (s, 1H),
7.43–7.50 (m, 3H), 7.70 (d, 2H, J = 1.2 Hz), 10.90 (s, 1H, NH), 11.95
(s, 1H, NH). 13C NMR (100 MHz, DMSO‐d6) δ: 18.32, 113.94,
120.88, 124.04, 124.34, 127.57, 127.97, 129.24, 131.60, 132.19,
133.95, 135.06, 137.97. MS (ESI) m/z: 402.43 [M + Na]. Anal. calcd.
for C17H12Cl3N3O·0.07H2O (%): C, 53.46; H, 3.20; N, 11.00. Found
(%): C, 53.08; H, 3.22; N, 11.44.
N′‐[1‐(2,4‐Dichlorophenyl)ethylidene]‐5‐methoxy‐1H‐indole‐2‐
carbohydrazide (16)
Yield: 60%; Mp: 196–197°C. 1H NMR (400 MHz, DMSO‐d6) δ: 2.29
(s, 3H, CH3), 3.72 (s, 3H, CH3), 6.85 (td, 1H, J = 9.6 Hz, 2.4 Hz), 7.08
(s, 1H), 7.28–7.35 (m, 2H), 7.44 (d, 1H, J = 8.4 Hz), 7.51–7.56 (m, 1H),
7.74 (d, 1H, J = 15.2 Hz), 10.12 (s, 1H, NH), 11.52 (s, 1H, NH). 13C

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KILIC‐KURT ET AL.
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NMR (100 MHz, DMSO‐d6) δ: 24.31, 55.20, 102.02, 113.66, 115.17,
127.18, 127.58, 128.10, 129.36, 130.79, 131.61, 131.90, 132.20,
133.23, 133.90, 134.62, 138.07, 153.76. MS (ESI) m/z: 376.48
[M + H], 398.48 [M + Na]. Anal. calcd. for C18H15Cl2N3O2 (%): C,
57.46; H, 4.01; N, 11.16. Found (%): C, 57.19; H, 4.37; N, 11.41.
measured using an Enzyme‐Linked Immunosorbent Assay Micro-
plate Reader (Thermo Fisher Scientific, Germany) at 450 nm. All
experiments were performed in triplicate, and the cell viability was
expressed as a viable cell amount percentage, compared with
control as untreated cells. The IC50 values of compounds 15, 12,
and 14 in MCF‐7 cells were calculated by GraphPad Prism 7
software (GraphPad Software Inc.).
N′‐[(1H‐Pyrrol‐2‐yl)methylene]‐5‐methoxy‐1H‐indole‐2‐
carbohydrazide (17)
Yield: 55%; Mp: 126–128°C. 1H NMR (400 MHz, DMSO‐d6) δ: 3.74 (s,
3H, CH3), 6.13 (s, 1H), 6.48 (s, 1H), 6.84 (d, 1H, J = 8.4 Hz), 6.90
(s, 1H), 7.10 (d, 1H, J = 2.0 Hz), 7.15 (s, 1H), 7.31 (d, 1H, J = 8.4 Hz),
8.27 (s, 1H), 11.51 (s, 2H, NH), 11.60 (s, 1H, NH). 13C NMR (100 MHz,
DMSO‐d6) δ: 55.18, 101.95, 102.63, 109.17, 113.05, 113.17, 114.71,
122.38, 126.97, 127.27, 130.62, 131.92, 140.14, 153.75, 157.08. MS
(ESI) m/z: 283.60 [M + H]. Anal. calcd. for C15H14N4O2·0.6-
H2O·0.6C2H5OH (%): C, 60.66; H, 5.90; N, 17.46. Found (%): C, 60.38;
H, 5.93; N, 17.59.
|
4.2.3
Cell cycle assay
The cell cycle analysis was performed by Muse Cell Cycle Assay Kit
(Merck Millipore, Germany). The MCF‐7 cells were treated with
3.01 μM of compound 12 for 48 hr, and the nontreated cells were
used as control. The cells were then fixed with ethanol and prepared
for the analysis according to the instructions. The cell population at
G0/G1, S, and G2/M stages of the cell cycle was measured by Muse
Cell Analyzer (Millipore, Germany).
|
4.2
Biological evaluation
|
4.2.4
Annexin V binding assay
|
4.2.1
Cell culture
MCF‐7 cells were treated with IC50 concentration of compound 12,
which is obtained from XTT analysis, and the extent of apoptosis was
assessed using the Muse Annexin V/Dead Cell (Merck Millipore)
assay. Shortly before treatment, the cells were seeded in six‐well
plates and were allowed to attach overnight. The next day, the cells
were treated with compound 12 at its IC50 concentration and in-
cubated for 48 hr. After incubation, the cells were collected, diluted
with PBS containing 1% FBS, and incubated with Annexin V and Dead
Cell reagent for 20 min at room temperature in the dark. The events
for live, dead, early, and late apoptotic cells were counted by Muse
Cell Analyzer (Millipore).
MCF‐7 (HTB‐22), human breast cancer cell line, PC3 (CRL‐1435),
human prostate cancer cell line, and HepG2 (HB‐8065), human he-
patocellular carcinoma cell line, were purchased from American Type
Culture Collection and grown in Dulbecco's modified Eagle's medium
(DMEM; Gibco, Thermo Fisher Scientific), supplemented with
10% fetal bovine serum (FBS; Sigma‐Aldrich), 1% L‐glutamine
(Sigma‐Aldrich), and 1% penicillin/streptomycin (Sigma‐Aldrich). The
cultured cells were incubated at 37°C in a humidified atmosphere
containing 5% CO2. All newly synthesized compounds were dissolved
in DMSO, and stock solutions were diluted with DMEM as the final
concentration of DMSO did not exceed 0.5%.
|
4.2.5
DNA damage assay
|
4.2.2
Cell viability assay
ATM and H2AX activation was assessed using the Muse Multi‐Color
DNA Damage Kit (Merck Millipore) according to the manufacturer's
instructions. First, the cells were treated with the IC50 concentration
of compound 12 and incubated for 48 hr at 37°C. The cells were then
centrifuged, washed once with PBS, and resuspended in 1× assay
buffer. Then, the cells were fixed in a fixation buffer and incubated
for 10 min on ice. Next to fixation, the cells were washed and per-
meabilized in the ice‐cold buffer. Then, 10 µl antibody working
cocktail solution was added to each tube that contained the cell
suspension and incubated for 30 min in the dark at room tempera-
ture. Finally, the cells were centrifuged and washed, and then the
cells were resuspended in 200 µl 1× assay buffer, and percentages of
negative cells (no DNA damage), percentages of ATM‐activated cells,
percentages of H2AX‐activated cells, and percentages of DNA
double‐strand breaks were evaluated using Muse Multi‐Color DNA
Damage software module (Millipore).
Metabolically active cells were evaluated using the XTT colori-
metric assay (Roche Diagnostic, Germany). The MCF‐7, HepG2,
and PC3 cells were plated in 96‐well plates at a density of
1 × 104 cells/well in 100‐µl regular culture media, and they were
allowed to attach overnight before treatment. Then, the cells were
treated with 20‐µM constant concentration of the compounds to
determine CV% for 48 hr. Additionally, the cells were treated with
selected three compounds, which were found to be most effective
against MCF‐7 cells, at 2.5‐, 5‐, 10‐, 20‐, 40‐µM final concentra-
tions for 48 hr. After incubation, DMEM, which contained the
compounds, was removed and the wells were washed twice with
200 μl phosphate‐buffered saline (PBS). Afterward, 100 μl DMEM
without phenol red and a mixture of 50 μl XTT labeling solution
were added to each well, and then the plates were incubated at
37°C for 4 hr. The plates were shaken and the absorbances were

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4.2.6
Western blot assay
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The MCF‐7 cells treated with compound 12 at 3.01‐μM concentra-
tion were lysed on ice in lysis buffer with proteinase inhibitor cocktail
(Cell Signal Technology, Germany). The protein concentration
was measured by bicinchoninic acid assay (Pierce Biotechnology,
Rockford, IL), and 20 μg of denaturated protein samples were
resolved by SDS‐PAGE and transferred to a nitrocellulose membrane.
After the transfer process, the membranes were incubated overnight
at 4°C with primary antibodies (Bax, Bcl‐2, procaspase‐3, procaspase‐
9, 1:1,000 [CST, Germany]) diluted in 1× PBS. The membranes were
then incubated with IRDye secondary antibody diluted (1:1,000) in
1× PBS with 0.1% Tween‐20 for 1 hr. After the final wash,
the membranes were visualized with the CLX System (LI‐COR
Biosciences) by using IR screening. The band intensity was quantified
by densitometric analysis using Image Studio Lite software (LI‐COR
Biosciences). The values were normalized to β‐actin, and these re-
sults were expressed as a fold change.
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CONFLICT OF INTERESTS
The authors declare that there are no conflicts of interests.
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ORCID
[19] R. C. Taylor, S. P. Cullen, S. J. Martin, Nat. Rev. Mol. Cell Biol. 2008,
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Zühal Kilic‐Kurt
Cemre Acar
http://orcid.org/0000-0002-7491-4089
http://orcid.org/0000-0001-8965-3267
http://orcid.org/0000-0003-4303-2996
http://orcid.org/0000-0003-2809-8946
[20] Z. Zhu, J. Yuan, Y. Zhou, Y. Qin, J. Xu, Y. Peng, Eur. J. Org. Chem. 2014,
2014, 511.
Mustafa Ergul
Filiz Bakar‐Ates
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Tunca G. Altuntas
http://orcid.org/0000-0003-1194-8588
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Narayana, Acta Crystallogr., Sect. E: Crystallogr. Commun. 2006, E62,
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
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Bakar‐Ates F, Altuntas TG. Novel indole hydrazide
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