Anti-cancer, anti-oxidant and molecular docking studies of thiosemicarbazone indole-based derivatives

Article abstract of DOI:10.1007/s11164-019-03765-9

Based on the structural elements of bioactive 3-substituted indoles, a new series of indole–thiosemicarbazone hybrid derivatives were designed, synthesized, and well-characterized using different spectral techniques. The intended scaffolds were screened for their in vitro anti-proliferative activities against breast cancer (MCF-7), lung cancer (A-549), and liver cancer (Hep-G2) cell lines, as well as their anti-oxidant properties. Cytotoxicity studies revealed that compound 6n was the most potent, at least threefold more potent than the commercially available reference drug etoposide, against A-549. In addition, morphological analysis by the acridine orange/ethidium bromide double staining test and flow cytometry analysis confirmed induction of apoptosis in the A-549 cells by compound 6n. In order to validate the experimental results, molecular studies were performed to achieve the possible binding interactions of the most potent compound (6n) and colchicine with tubulin as well as ANP with ATPase domain of topoisomerase IIα active sites. Moreover, the radical scavenging potential of the final derivatives was found to be excellent with the range of 0.015–0.630?μM, comparable to the standard ascorbic acid (0.655?μM).

Full text of DOI:10.1007/s11164-019-03765-9

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-019-03765-9
Anti‑cancer, anti‑oxidant and molecular docking studies
of thiosemicarbazone indole‑based derivatives
Zohreh Bakherad1 · Maliheh Safavi2 · Afshin Fassihi1 · Hojjat Sadeghi‑Aliabadi1 ·
Mohammad Bakherad3 · Hossein Rastegar4 · Jahan B. Ghasemi5 ·
Saghi Sepehri6 · Lotfollah Saghaie1 · Mohammad Mahdavi7
Received: 13 December 2018 / Accepted: 30 January 2019
© Springer Nature B.V. 2019
Abstract
Based on the structural elements of bioactive 3-substituted indoles, a new series
of indole–thiosemicarbazone hybrid derivatives were designed, synthesized, and
well-characterized using diferent spectral techniques. The intended scafolds were
screened for their in vitro anti-proliferative activities against breast cancer (MCF-
7), lung cancer (A-549), and liver cancer (Hep-G2) cell lines, as well as their anti-
oxidant properties. Cytotoxicity studies revealed that compound 6n was the most
potent, at least threefold more potent than the commercially available reference drug
etoposide, against A-549. In addition, morphological analysis by the acridine orange/
ethidium bromide double staining test and fow cytometry analysis confrmed induc-
tion of apoptosis in the A-549 cells by compound 6n. In order to validate the experi-
mental results, molecular studies were performed to achieve the possible binding
interactions of the most potent compound (6n) and colchicine with tubulin as well as
ANP with ATPase domain of topoisomerase IIα active sites. Moreover, the radical
scavenging potential of the fnal derivatives was found to be excellent with the range
of 0.015–0.630 µM, comparable to the standard ascorbic acid (0.655 µM).
Keywords Indole · Thiosemicarbazone · Anti-oxidant · Anti-cancer · Apoptosis ·
Flow cytometry
This paper is dedicated to the memory of our wonderful teacher in Chemistry and Medicinal
Chemistry, Professor Abbas Shafee (1937–2016).
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1116
4-019-03765-9) contains supplementary material, which is available to authorized users.
*
*
Lotfollah Saghaie
saghaie@pharm.mui.ac.ir
Mohammad Mahdavi
momahdavi@tums.ac.ir
Extended author information available on the last page of the article
Vol.:(0123456789)
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Z. Bakherad et al.
Introduction
Cancer is a class of diseases in which a group of cells demonstrate incontrolla-
ble growth, infux, and sometimes metastasis [1]. After cardiovascular disease,
cancer is the second leading cause of mortality [2]. Although chemotherapy is
among the main remedy options for cancer treatment, the use of available chem-
otherapeutics has limitations due to undesirable adverse efects and multi-drug
resistance [3]. Therefore, the design and discovery of more efective and safer
anti-cancer drug candidates have recently received great attention [4]. Molecu-
lar hybridization by a combination of diferent pharmacophores is a strategy for
targeting two or more diferent receptors or enzymes to increase the efcacy [5].
Thus, a single molecule with more than one pharmacophore displaying diverse
modes of action could be more useful in cancer treatment [6]. These drugs have
the potential to overcome the fast progress of resistance, elevate patient admis-
sion, and reduce both the cost and risk of drug–drug interactions [7]. Heterocy-
clic compounds are an exceedingly important class of compounds, and they have
attracted increasing attention for diverse biological studies [8–14].
Indole is a heterocyclic scafold with various valuable biologic activities such
as anti-infammatory [15], anti-viral [16], anti-depressant, anti-hypertensive, anti-
hypoglycemic [17], and notably anti-cancer properties [18–21]. The indole ring
system is present in various marketed drugs [22]. Angerer’s research group have
designed and reported some analogs of 3-formyl-2-phenylindoles with the most
potent one, A, which displayed IC50 values of 35 nm and 1.5 µm for cell growth
and tubulin polymerization inhibition, respectively (Fig. 1) [23]. Prompted by
these results, Kaufmann et al. synthesized a number of 2-phenyl indole-3-car-
baldehydes with lipophilic substituents in both aromatic rings, which showed
good anti-proliferative activities. The imine B (Fig. 1) strongly inhibited tubulin
polymerization with an IC50 value of 1.2 µm, which was lower than that of colchi-
cine (5 µm) in this assay [24].
On the other hand, thiosemicarbazones exhibit diverse biological activities,
such as anti-plasmoid [5], anti-leishmanial [25], anti-microbial [26], anti-tumor
[27, 28], and anti-oxidant [29] properties. Huang et al. synthesized another series
of thiosemicarbazones, C, which exhibited a broad anti-proliferative activity in a
panel of human tumor cells (Fig. 2). It blocked topoisomerase IIα-catalyzed ATP
hydrolysis by competing with ATP for binding to the ATPase domain of topoi-
somerase IIα [30].
CH3
N
O
H
OCH3
OCH3
N
H3CO
N
H
n-Bu
H
A
B
Fig. 1 Some potent anti-cancer 2-phenylindole-3-carbaldehyde analogs
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Anti-cancer, anti-oxidant and molecular docking studies…
S
S
HN
N
N
N
R
N
O
N
R
H
CH3
N
H
N
N
CH3
N
H
S
N
H
N
E
D
C
Fig. 2 Thiosemicarbazone derivative demonstrating profound anti-cancer activity
In 2017, de Oliveira et al. reported some thiosemicarbazone derivatives, as
well as their in vitro antiproliferative activity against eight human tumor cell lines
via inhibition of topoisomerase IIα (derivatives of D and E, Fig. 2) [4]. It has
been revealed that a series of indole thiosemicarbazones, D derivatives, has been
much more active than indole-thiazolidinone series (E derivatives). The molecu-
lar structures of C and D and E are provided in Fig. 2.
In order to fnd some novel multitarget indole anti-tumor and anti-oxidant
agents, we focused on the design and synthesis of some novel chemical hybrids of
indole and thiosemicarbazone pharmacophores. Diferent substituted aryl groups
were incorporated into the structure to investigate the importance of substitu-
tions at the C-2 position of the indole ring. Substitution of the thiosemicarbazone
moiety with a methyl group have increased the antiproliferative activities against
four human tumor cell lines [30]. Here, in search for the same efect, we sub-
stituted the thiosemicarbazone pharmacophore with a methyl group. Substituted
phenyl ring was incorporated in this position as well. The general structure of the
designed compounds is provided in Fig. 3.
N
H
N
H
N
N
OCH3
N
H
S
n-Bu
S
N
HN
N
R1
N
H
R2
N
H
Designed compounds
Fig. 3 General structure of the designed compounds
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Z. Bakherad et al.
Results and discussion
Chemistry
The synthesis of thiosemicarbazone derivatives (6a–p) was performed in three steps
as shown in Scheme 1. First, a nucleophilic addition reaction between hydrazine
and isothiocyanates produced a thiosemicarbazide [30]. In another reaction, sub-
stituted indole 2-aryl was generated by Fischer reaction of substituted acetophe-
none and phenyl hydrazine [31]. Subsequently, the formyl group was introduced in
position 3 of the indole by a Vilsmeier reaction with DMF and POCl3 [25]. In the
third step, the synthesized substituted indole 2-aryl 3-carboxaldehyde reacted with
substituted thiosemicarbazide in the presence of a catalytic amount of CH3COOH,
via a condensation reaction, leading to thiosemicarbazone derivatives [32]. Crys-
tallization of the products was performed in ethylacetate/petroleum ether, and the
reaction yields ranged from 71 to 85%. Finally, the thiosemicarbazone structures
1
(6a–p) were determined by H and 13CNMR, IR, ESI–MS and elemental analyses.
The IR spectra of compounds 6n–p display the expected absorption bands for NH at
3211–3441 cm−1. In addition, these compounds show stretching of C=N and C=S
bonds around 1593–1609 and 1196–1238 cm−1, respectively. As exemplifed by the
1HNMR analysis of 1-((2-phenyl-1H-indol-3-yl)methylene)-4-p-tolylthiosemicar-
bazide (6h), the singlet at δ 2.90 corresponds to the para-methyl group at the phenyl
ring. The phenyl protons substituted at 2-indole position appeared at 7.52, 7.59, and
7.66 ppm. Moreover, the para-methyl phenyl group at the thiosemicarbazone moiety
Scheme 1 Reagents and conditions: (a) isopropanol, 30 min; (b) (i) CH3COOH, EtOH, refux (ii)
polyphosphoric acid, 120 °C; (c) POCl3, DMF, 70 °C; (d) EtOH, CH3COOH, refux
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Anti-cancer, anti-oxidant and molecular docking studies…
displayed two doublets at 6.73 and 7.35 ppm. Also, the indole protons occurred as
two triplets at 7.16 and 7.24 ppm and two doublets at 7.44 and 8.35 ppm. The sin-
glet at 8.59 ppm related to imine proton (H–C=N) confrms the success of the reac-
tion between para-tolyl thiosemicarbazide and 2-phenyl indole carbaldehyde. In
addition, the signal related to N–H appeared at 9.31 ppm. The N–H connected to
phenyl group (HN–Ph) showed a singlet at 11.42 ppm. Moreover, the N–H indole
ring signal appeared at 11.94 ppm. Similarly, in the 13CNMR spectra, the azo meth-
ane group (H–C=N) occurred at 141.5 ppm, while the C=S group appeared at
174.6 ppm. Also, the E confguration was confrmed by the 2D NMR NOESY spec-
trum of compound 6h. In this respect, there was a spatial correlation between hydro-
gen of =N–NH at 11.42 ppm and azomethine (–CH=N–) at 9.31 ppm. These results
suggest the E confguration, which corroborates the NOESY spectrum of E thio-
semicarbazone presented in the literature [5, 33]. In addition, ESI–MS confrmed
the identity of all the synthesized compounds.
Biology
Anti‑proliferative activity
The prepared derivatives 6a–p were classifed into three categories based on
the diverse groups substituted on thiosemicarbazone moiety and C-2 indole ring.
Then their inhibitory activities were evaluated against the MCF-7 (breast), A-549
(lung), and Hep-G2 (liver) cell lines as summarized in Table 1. Compound 6g with
4-dimethylaminophenyl moiety substituted on thiosemicarbazone and 4-methylphe-
nyl group at C-2 position of the indole ring, exhibited the highest inhibitory activity
against MCF-7 and HepG2 with IC50 values of 23.39 μm and 24.46 μm, respectively.
For etoposide as the reference drug IC50 values were =34.25 µm against MCF-7 and
33.17 µm against HepG2. In addition, compound 6n, with the methyl group substi-
tuted on thiosemicarbazone and 4-methoxyphenyl substitute at C-2 position of the
indole ring, revealed the highest inhibitory activity against A-549 cell line with IC50
of 12.5 µm. This result also indicated that the methoxy group at para phenyl ring of
indole C-2 position and methyl group substituted on thiosemicarbazone moiety had
important roles in the inhibition activity of 6n against A-594 cell line. Remarkably,
those compounds bearing 4-dimethylaminophenyl group substituted on the thio-
semicarbazone moiety and substitutes of 4-hydroxylphenyl (6b) and 3,4,5-trimeth-
oxyphenyl (6c) at C-2 position of the indole ring exhibited a high MCF-7 inhibi-
tory activity, compared with the other compounds. Replacement of 3,4,5 trimethoxy
moieties (6c) with 4-OH group (6b), at C-2 position of the indole ring improved the
activity (Table 1).
Moreover, compounds 6m–p in the third class that have methyl group substi-
tuted to thiosemicarbazone moiety did not show any activity against MCF-7 cell line
(IC50 >100 µm), whereas these compounds exhibited the highest cytotoxic activ-
ity against A-549 and Hep-G2 cell lines compared with others. This shows that the
presence of small groups (e.g. methyl) at the thiosemicarbazone moiety is possibil-
ity signifcant for activity against both A-549 and Hep-G2 cell lines, a result that is
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Anti-cancer, anti-oxidant and molecular docking studies…
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Z. Bakherad et al.
in accordance with the literature [30]. In the second series (6h–l), compounds 6h
(IC50 =42.83 µm against Hep-G2) and 6i (IC50 =24.97 µm against MCF-7) showed
strong activities, whereas other compounds in the series were weak with IC50 val-
ues more than 100 µm. This modifcation revealed that the para hydroxylphenyl ring
substituted at the C-2 position of indole ring was ideal for inhibitory activity against
MCF-7. However, unsubstituted phenyl ring incorporated to the C-2 position of
indole of 6h is optimum for cytotoxic activity against Hep-G2 cell line.
In the frst series (6a–g), compounds 6d showed good activity against Hep-G2
cell line (IC50s=36.43 µm). Replacement of methoxy (6d) with chloro group (6e) in
para position of the phenyl ring substituted at indole C-2 position led to a decrease
in the cytotoxicity. Compounds 6a, 6f, 6j, 6k, and 6l demonstrated no cytotoxicity
against three cell lines (IC50 >100).
Interestingly, the presence of methyl substituent on the phenyl ring at the C-2
position of indole ring increased the cytotoxicity of compound 6g in the frst series
(6a–g) with IC50 values of 23.39 µm, 80.84 µm, and 24.46 µm against MCF-7, A-
594, and Hep-G2 cell lines, respectively. Compounds with other substituents in
this position showed less activities. The presence of hydroxyl, methoxy, chloro
and bromo groups on the para phenyl ring at the indole C-2 position of the second
series compounds (6i–l) did not display any cytotoxic activity against A-594 and
Hep-G2 cell lines (IC50 >100). Para chloro and para bromophenyl substituents at
indole C-2 position in the third group (compounds 6o and 6p), showed the high-
est activities against Hep-G2 (IC50s=48.94 µm and 49.97 µm). The assay results
revealed that probably the presence of halogens (Cl and Br) on para phenyl ring at
C-2 position of indole increased the activity. However, incorporation of hydroxyl
and methoxy substituents at the same position led to a much more reduction in cyto-
toxicity (IC50s=51.7 µm and 56.0 µm, respectively). Finally, it should be noted that
compounds in the 6m–p series showed a higher activity against A-549 and Hept-G2
compared with other series and that compounds in the 6a–g series exhibited better
activities against MCF-7.
Morphological evaluation by fuorescence microscopy
The most active compound 6n was selected to determine the apoptotic or necrotic
cell death in A-549 cells morphologically by the acridine orange/ethidiumbromide
(AO/EB) double staining test [34]. AO penetrates the living as well as the dead cells
and emits green fuorescence as a result of intercalation into double-stranded DNA,
while ethidium bromide (EB) staining precedes the loss of membrane integrity and
intercalates in DNA of cells with an altered cell membrane and emits red fuores-
cence. The AO/EB staining that is sensitive to DNA was used to access changes in
nuclear morphology. Analysis of the AO/EB staining revealed that the selected most
potent compound, 6n, clearly can be the inducer of apoptosis as the appearance of
chromatin condensation and nuclear fragmentation are evident in Fig. 4. As shown,
the viable cells are observed green, but the apoptotic cells with chromatin condensa-
tion or fragmentation show orange-stained nuclei. The lung cancer cell treated with
the most potent test compound showed extended apoptosis relative to the control.
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Anti-cancer, anti-oxidant and molecular docking studies…
Fig. 4 AO/EB double staining of cancer cells with characteristic symptoms of apoptosis of A-549 a
DMSO 1% as control, b, c cells treated for 24 h with IC50 concentrations of etoposide and 6n, respec-
tively
Flow cytometry analysis of apoptotic cells with Annexin V‑FITC/PI staining
Apoptosis induction of the prepared compounds was also confrmed by fow cytom-
etry analysis. Annexin V-FITC/PI followed by fow cytometric analysis detects the
externalization of phosphatidylserine in apoptotic cells using recombinant annexin
V conjugated to green-fuorescent FITC dye and dead cells using propidium iodide
(PI). The results indicated that the novel compounds induced apoptosis of A-549
cancer cells. Flow cytometric analysis revealed that the cells underwent apoptosis
after treatment with the test compounds. As illustrated in Fig. 5, the apoptotic index
of compound 6n was compared with the negative control and etoposide in A-549
cells. Double staining followed by fow cytometric analysis revealed the percentage
of apoptotic cells as 27.5% resulting from treatment with compound 6n after a 24-h
incubation. In addition, the corresponding value obtained after treatment with etopo-
side was 24.5% following a 24-h incubation (Fig. 5).
Anti‑oxidant assay
All the synthesized compounds 6a–p were subjected to in vitro 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH) assay in order to assess their radical scavenging properties.
DPPH is a stable free radical with violet color. When a compound donates a radical
Fig. 5 Flow cytometric analysis of A-549 cells treated with the prepared compound 6n. Cells were
stained with Annexin V-FITC/PI and quantitated by fowcytometry. Cells were treated with DMSO 1%
(negative control) or with IC50 values of etoposide (positive control) and compound 6n
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Z. Bakherad et al.
Table 2 IC50 values for DPPH
radical scavenging activity
of indole–thiosemicarbazide
derivatives 6
Compound
DPPH ICa50 (µM)
Compound
DPPH ICa50 (µM)
6a
6b
6c
6e
6f
6g
6h
6i
0.117 0.01
0.041 0.003
0.227 0.05
0.121 0.007
0.241 0.01
0.148 0.006
0.043 0.001
0.016 0.001
6j
0.204 0.008
0.210 0.02
0.228 0.03
0.160 0.005
0.336 0.04
0.567 0.01
0.630 0.05
0.655 0.02
6k
6l
6m
6n
6o
6p
Ascorbic
aThe IC50 values represent an average of three independent experi-
ments (mean SD)
Fig. 6 DPPH radical scavenging activities of indole-thiosemicarbazone derivatives 6
hydrogen species to a DPPH molecule, DPPH is decolorized, and thus the absorb-
ance of DPPH is decreased. In brief, lighter violet color accounts for higher anti-
oxidant activity. The ratio of anti-oxidant/DPPH required to decrease the concen-
tration of DPPH to 50% of its initial value, is denoted as IC50 or is an indicator of
the radical scavenging activity [35]. The anti-oxidant activities of the compounds
were screened at the concentrations of 125–2000 µg mL−1 at 517 nm for the DPPH
assay, and the obtained results are presented in Table 2 and Figs. 6 and 7. Ascorbic
acid was used as the standard. Based on the experimental results, it was found that
all compounds showed potent anti-oxidant activities in the range of 0.016–0.630 µm
compared with ascorbic acid (0.655 µm). A higher anti-oxidant activity is refected
in a lower IC50 value.
In DPPH assay, antioxidants react with free radicals by single electron transfer
(SET) mechanism. In SET mechanisms, the antioxidant provides an electron to the
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Anti-cancer, anti-oxidant and molecular docking studies…
Fig. 7 IC50 for compounds (6a–p) using DPPH assay
free radical and itself then becomes a radical cation [36]. It seemed that existence of
a good electron transfer group such as phenyl at thiosemicarbazide moiety, increases
antioxidants activity. Thus, compounds 6a–g and 6h–l having 4-dimethylaminophe-
nyl and 4-methylphenyl groups substituted to thiosemicarbazide revealed higher
antioxidant activity than compounds 6m–p having methyl group. Antioxidant activ-
ity studies displayed that bulky substitution on the phenyl ring incorporated on the
indole C-2 position makes steric hindrance that avoids from near DPPH molecule,
thus these compounds showed weak antioxidant activity compared with the unsub-
stituted compound. Therefore, compounds 6a and 6h displayed high antioxidant
activity. Moreover, compounds 6b, 6i and 6m having 4-hydroxyphenyl at C-2 indol
ring exhibited good antioxidant activity because of OH group.
Molecular docking study
Molecular docking as an important method in structure-based computer-assisted
drug design, predicts the main binding mode(s) of a ligand with a protein of known
3D structure [37]. Molecular docking simulations and analysis of the binding modes
of the designed compounds within tubulin and ATPase domain of topoisomerase IIα
binding active sites were performed by Gold 5.3.0 in order to rationalize the anti-
cancer activity results. At frst, the binding sites of ANP and colchicine were pre-
cisely characterized in the active sites of ATPase domain of topoisomerase IIα and
tubulin, respectively. As presented in Fig. 8a, formation of hydrogen bonds between
some parts of ANP and Gly166, Tyr165, Gly164, Asn163, Ser148, Ser149, Gln376,
Asn150, Asn91, Lys168, Arg162, Ala167, and Asn120 residues was distinguished.
In addition, hydrophobic interactions of ANP with Ile125, Ile141, Gly161, Asn95,
Lys378 and Ala92 were fully recognized, as indicated which can also be observed
in Fig. 8.
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Z. Bakherad et al.
Fig. 8 Binding sites of a ANP and b colchicine in the active sites of ATPase domain of topoisomerase
IIα and tubulin. Green dashed lines indicate hydrogen bonds
Figure 8b also clearly represents the hydrogen bond formation of methoxy
group and Cys241 residue. Moreover, the hydrophobic interactions of difer-
ent parts of colchicine with Ala316, Lys352, Val315, Met259, Ala180, Ala250,
Val238, and Leu248 were well established.
Docking studies revealed that the binding modes of the most active compounds
are coherent with co-crystallized colchicine and ANP. Compounds 6j and 6l
showed the highest goldscore ftness values at the topoisomerase IIα and tubulin
active sites (Table 1). It seems that para methyl phenyl substituted to the thiosem-
icarbazone moiety of 6j and 6l might be responsible for additional hydrophobic
interactions with topoisomerase IIα and tubulin. These two compounds showed
weak anti-cancer activities (Table 1). A lower anti-cancer activity might be attrib-
uted to the improper geometric orientation of compounds in the topoisomerase
IIα and tubulin binding sites. This can be seen in Figs. 9 and 10 by superimposing
6j and 6l with ANP and colchicine in the active sites of both targets.
Fig. 9 Interactions of 6j (a) and 6l (b) in topoisomerase IIα active site and superimposition of both with
ANP (dark blue)
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Anti-cancer, anti-oxidant and molecular docking studies…
Fig. 10 Interactions of 6j (a) and 6l (b) in the tubulin active site and superimposition of both with colchi-
cine (blue)
According to the cLogP data, 6l and 6j are lipophilic (Table 1). Thus, these
compounds increase hydrophobic interactions in the hydrophobic pocket of both
enzymes and show better goldscore ftness values. Compound 6n was the most
potent compound with IC50 equal to 12.5 μm against A-549 cell line. Docking stud-
ies demonstrated that 6n participated in the key interactions with the binding site
residues of both targets (Goldscore ftness=52.70 and 59.61 for tubulin and topoi-
somerase IIα, respectively). Detailed analysis of hydrophobic binding patterns in
tubulin active site show that two interactions exist between indole ring of 6n and
Leu248 and Lys254 (Fig. 11a). Moreover, the methyl substituent of thiosemicarba-
zone is responsible for further hydrophobic interactions with Ala317 and Lys352 in
the hydrophobic pocket. The methyl group of para methoxyphenyl substituent at the
C-2 position of indole ring formed hydrophobic interactions with the Val315 and
Met259 residues. In addition, analysis of the hydrophobic binding patterns in the
Fig. 11 Compound 6n at active sites of tubulin (a) and topoisomerase IIα (b). Green dashed line indi-
cates hydrogen bond
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Z. Bakherad et al.
topoisomerase IIα active site revealed that the methyl group of thiosemicarbazone
interacted with Asn120 (Fig. 11b). Thiosemicarbazone substituent at the C-3 indole
ring formed a hydrophobic interaction with Asn91 amino acid. Other parts of the
molecule had hydrophobic contacts with Ile141, Ser148, Ala167, and Lys168 resi-
dues in topoisomerase IIα hydrophobic packet. Furthermore, 6n was able to form a
hydrogen bond between the oxygen atom of the methoxy group and amino group of
Lys168 residue. Thus, the existence of this hydrogen bond led to an increase in the
afnity of 6n to topoisomerase IIα (Goldscore ftness=59.61) compared with tubu-
lin (Goldscore ftness=52.70).
Replacement of the methyl substituent of the thiosemicarbazone moiety in com-
pound 6n with para methylphenyl (6j) led to a weak activity against the A-549 cell
line, and the para N,N-dimethylphenyl analogue (6d) showed a good activity against
the Hep-G2 cell line. Docking results obtained for compound 6d displayed more
afnity to topoisomerase IIα than tubulin (Goldscore ftness=59.48 and 62.64 for
tubulin and topoisomerase IIα, respectively). In addition, reduction in lipophilic
moieties (methyl vs. N,N-dimethylphenyl ring) at the thiosemicarbazone substituent
increased the activity. Compound 6d contributed in no hydrogen bond to the tubulin
active site amino acids. This compound only formed hydrophobic interactions with
the Leu242, Cys241, Ala354, Ala250, Lys254, Asn101, Leu255, Val318, Ala354,
and Ala316 residues. Similar to tubulin, compound 6d did not show any hydrogen
bond with topoisomerase IIα active site amino acids. Furthermore, investigation of
hydrophobic binding patterns showed that these interactions were formed between
the N,N-dimethyl amino group at the para position of the phenyl ring and Thr215
and Asn120 residues. The phenyl ring of thiosemicarbazone interacted with Ile125.
Also, other moieties of the molecule formed hydrophobic interactions with the
Ser149, Ile141, Ser148, and Thr147 amino acids. Interactions of 6d with the binding
site amino acids of tubulin and topoisomerase IIα are shown in Fig. 12.
Replacement of the methoxy group at the para position of the phenyl ring of
C-2 indole ring in 6d with the methyl group in 6g increased the activity against
three cell lines. Based on the docking results, analogues with increased hydro-
philic character were equal to or slightly improved in potency, but addition of
polar groups had a negative efect. Thus, the 4-methylphenyl group provided
additional hydrophobic contacts with residues at the active site. Moreover,
Fig. 12 Compound 6d at active sites of tubulin (a) and topoisomerase IIα (b)
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Anti-cancer, anti-oxidant and molecular docking studies…
docking observations for compound 6g showed that it had more afnity to tubu-
lin than topoisomerase IIα (goldscore ftness = 61.31 vs. 51.20). The docking
results revealed that compound 6g formed hydrogen bond with Thr147 in the
topoisomerase IIα active site, while compound 6g did not participate in hydrogen
bonding with the tubulin active site. Compound 6g showed hydrophobic contacts
with Ala354, Val318, Leu255, Lys254, Leu252, Ala250, Ala316, Cys241, and
Leu242 tubulin residues and Ala167, Asn91, Lys157, Arg98, and Ile125 topoi-
somerase IIα amino acids. The hydrogen bond and hydrophobic interactions of 6g
in both proteins are shown in Fig. 13.
Substitution of 3,4,5-trimethoxyphenyl moiety at the para position of the phe-
nyl ring of C-2 indole ring reduced the goldscore ftness values compared with
para hydroxyphenyl and para chlorophenyl substitutions at the same position.
Docking studies displayed that the presence of two methoxy groups in meta posi-
tions of 6c caused steric hindrance that changed its position in the active site, thus
the interactions of compound 6c decreased with the nearby amino acids of both
proteins.
Replacing para chlorophenyl (6e) with para hydroxyphenyl (6b) at the C-2
position of the indole ring decreased the goldscore ftness value in interac-
tion with tubulin. According to the cLogP data, 6e is more lipophilic than 6b
(cLogP = 5.92 vs. 4.76). This shows that the tubulin active site is lipophilic, and
an extra lipophilic group (methyl vs. hydroxyl) increases hydrophobic contacts
with lipophilic amino acids. Docking results of compounds 6h and 6i were the
same in the two proteins (Table 1). The presence of a hydroxyl group or hydro-
gen atom at the para position of the phenyl ring substituted on the indole C-2
position is not important for the afnity. Finally, it was found that compound 6n
was the most potent compound against the A-549 cell line, and compound 6g
was the most potent compound against all three cell lines. Compounds 6j and 6l
showed the highest goldscore ftness values in molecular docking studies. In addi-
tion, compounds of the third series (6m–p) indicated higher activity than those
in the other series. Based on the cLogP data, this series has the least cLogP val-
ues (3.60–4.89), thus this range of cLogP is suitable for the cytotoxicity activity
against A-549 and Hep-G2 cell lines.
Fig. 13 Interactions of compound 6g at active sites of tubulin (a) and topoisomerase IIα (b). Green
dashed line indicates hydrogen bond
1 3

Z. Bakherad et al.
Conclusion
A new class of indole-thiosemicarbazons hybrids were designed, synthesized,
and evaluated as cytotoxic agents against MCF-7, A-549, and Hep-G2 cell lines.
Etoposide and colchicine were the reference drugs. The synthesized compounds
were generally categorized into three classes based on the substituted groups at
the thiosemicarbazone moiety, as well as C-2 position of the indole moiety. Com-
pound 6n revealed the highest inhibitory activity against A-549 cell line. More-
over, compound 6g displayed a high anti-proliferative activity with IC50 values
between 23.39 and 80.84 µm against three studied cell lines. Interestingly, intro-
duction of a methyl group at the thiosemicarbazone moiety (6m–p), showed a
higher activity against A-549 and Hep-G2 compared with other series. In addi-
tion, morphological analysis by the acridine orange/ethidium bromide double
staining test and fow cytometry analysis confrmed induction of apoptosis in
A-549 cells by compound 6n. Moreover, all of the compounds displayed excel-
lent anti-oxidant activities in the range of 0.015-0.630 µm compared with ascorbic
acid (0.655 µm). Compound 6h bearing a methyl group on the para position of
the phenyl thiosemicarbazone moiety and para hydroxyl phenyl at C-2 position of
the indole ring exhibited a strong free radical scavenging ability compared with
other compounds.
Experimental
Chemistry
All reagents and solvents used in this work were commercially available (from
Merck Chemical) and were used without further purifcation. Melting points were
determined on a Kofer hot-stage apparatus (Reichert, Vienna, Austria) and uncor-
rected. TLC was conducted on silica gel 250 µm, F254 plates. The IR and NMR
spectra were recorded using a Nicolet Magna FTIR 550 spectrophotometer (potas-
sium bromide disks) and Bruker 500 MHz NMR instruments, respectively. Chemi-
cal shifts were reported in parts per million (ppm, δ), down flled from tetramethyl-
silane coupling constant (J) values are presented in Hz and spin multiplicities are
given as s (singlet), d (double), t (triplet) and m (multiplet). Elemental analyses were
carried out by a CHN-Rapid Heraeus elemental analyzer. The results of elemental
analyses (C, H, N) were within 0.4% of the calculated values. The atoms number-
ing of the target compound used for 1HNMR are shown in Scheme 1.
General method for synthesis of substituted thiosemicarbazides (2)
Isothiocyanate 1 (2 mmol) was added dropwise to a stirred solution of hydrazine
hydrate (24 mmol, 80%) in 20 mL of isopropanol. Precipitate was formed imme-
diately. Stirring was continued for 30 min. Then the mixture was fltered and the
1 3

Anti-cancer, anti-oxidant and molecular docking studies…
precipitate was washed with isopropanol three times to give substituted thiosemicar-
bazids 2 [30].
General method for synthesis of substituted 2‑phenyl‑1H‑indoles (4a–g)
Appropriate amounts of substituted acetophenone 3 (1 mmol) and phenyl hydrazine
(1 mmol) were mixed in ethanol (20 mL), and a few drops of glacial acetic acid were
added. The solution was heated under refux at 80 °C for 1–2 h. The solvent was
evaporated in vacuo to give a solid that was added to polyphosphoric acid (PPA)
(30 mL), and the mixture was slowly heated to 120 °C and kept at this temperature
for a few hours until the reaction was complete (TLC monitoring). The mixture was
allowed to cool and then poured into cold water (50 mL). The acidic solution was
neutralized by the slow addition of NaOH (1 M), and the solid precipitate of the
crude product was collected. Purifcation by column chromatography (hexane/ethyl
acetate) gave the substituted 2-aryl indoles 4a–g [31].
General procedure for synthesis of 2‑arylindole‑3‑carbaldehydes (5a–g)
Under nitrogen gas, phosphorous oxychloride (10 mmol) was added dropwise to dry
dimethylformamide (DMF) (10 mmol) while cooling in an ice bath, and the reaction
mixture was stirred for 1 h. A solution of compound 4 (1 mmol) in DMF (50 ml)
was added dropwise to the mixture with continuous stirring, which was then heated
to 70 °C. The mixture was poured onto ice cold water (200 mL), naturalized with
40% NaOH, and extracted with chloroform. The chloroform extract was washed
with water and dried over Na2SO4. The solvent was removed under vacuum. The
residue was crystalized from an ethanol/water mixed solvent system [24].
2‑phenyl‑1H‑indole‑3‑carbaldehyde (5a) Cream powder; Mp: 249–250 °C; Yield:
1
89%; H NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 7.24 (t, J=7.9 Hz,
1H, H6), 7.29 (t, J=7.9 Hz, 1H, H7), 7.51 (d, J=7.9 Hz, 1H, H8), 7.56–7.62 (m,
3H, H3′,5′,4′), 7.78 (d, J=9.3 Hz, 2H, H2′,6′), 8.21 (d, J=7.9 Hz, 1H, H5), 9.96 (s, 1H,
CHO), 12.40 (s, 1H, NH-indole). 13C NMR (125 MHz, DMSO-d6): (δ, ppm) 112.1,
113.5, 121.2, 122.5, 123.7, 125.8, 128.9, 129.2, 129.8, 130.1, 135.9, 149.1, 185.5.
Anal. Calcd. for C15H11NO: C, 81.43; H, 5.01; N, 6.33. Found: C, 81.53; H, 4.85; N,
6.45; IR (KBr, cm−1): 3144 (N–H stretching), 3058 (Aromatic C–H stretching) 2858
(H–CO), 1625 (C=O), 1455 (C=C); ESI–MS (m/z): 222 [M+H]+.
2‑(4‑hydroxyphenyl)‑1H‑indole‑3‑carbaldehyde (5b) Cream powder; Mp: 225–
227 °C; Yield: 92%; 1H NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 6.97 (d,
J=7.6 Hz, 2H, H3′,5′), 7.19–7.26 (m, 2H, H5,6), 7.45 (d, J=7.5 Hz, 1H, H7), 7.60 (d,
J=7.6 Hz, 2H, H2′,6′), 8.17 (d, J=7.5 Hz, 1H, H8), 9.92 (s, 1H, CHO), 10.04 (s, 1H,
OH), 12.20 (s, 1H, NH-indole). 13C NMR (125 MHz, DMSO-d6): (δ, ppm) 111.6,
112.7, 115.8, 120.4, 120.9, 122.2, 123.3, 125.9, 131.2, 135.8, 149.8, 159.1, 185.5.
Anal. Calcd. for C15H11NO2: C, 75.94; H, 4.67; N, 5.93. Found: C, 76.04; H, 4.71;
N, 5.72; IR (KBr, cm−1): 3211 (N–H stretching), 3151 (OH), 3074 (Aromatic C–H
1 3

Z. Bakherad et al.
stretching), 2862 (H–CO), 1619 (C=O), 1455 (C=C), 1177 (C–O); ESI–MS (m/z):
238 [M+H]+.
2‑(3,4,5‑trimethoxyphenyl)‑1H‑indole‑3‑carbaldehyde (5c) Cream powder, Mp:
1
191–194 °C; Yield: 83%; H NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm):
3.81 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 4.01 (s, 3H, OCH3), 6.83 (d, J=9.5 Hz,
1H, H2′), 7.23 (t, J=7.9 Hz, 1H, H5), 7.30 (t, J=7.9 Hz, 1H, H6), 7.43 (d, J=9.5 Hz,
1H, H6′), 7.62 (d, J=7.9 Hz, 1H, H7), 7.71 (s, 1H, CHO), 8.14 (d, J=7.9 Hz, 1H,
H8), 10.49 (s, 1H, NH-indole). 13C NMR (125 MHz, DMSO-d6): (δ, ppm) 54.4,
55.3, 105.0, 105.6, 110.1, 121.8, 122.4, 123.9, 126.0, 127.5, 135.9, 141.8, 151.4,
155.2, 185.4. Anal. Calcd. for C18H17NO4: C, 69.44; H, 5.50; N, 4.50. Found: C,
69.65; H, 5.61; N, 4.59; IR (KBr, cm−1): 3200 (N–H stretching), 3057 (Aromatic
C–H stretching), 2844 (H–CO), 1617 (C=O), 1473 (C=C), 1120 (C–O); ESI–MS
(m/z): 312 [M+H]+.
2‑(4‑methoxyphenyl)‑1H‑indole‑3‑carbaldehyde (5d) Cream powder, Mp: 207–
1
209 °C; Yield: 90%; H NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 3.87
(s, 3H, OCH3), 7.16 (d, J=8.7 Hz, 2H, H3′,5′), 7.20–7.28 (m, 2H, H5,6), 7.47 (d,
J=7.6 Hz, 1H, H7), 7.72 (d, J=8.7 Hz, 2H, H2′,6′), 8.18 (d, J=7.6 Hz, 1H, H8), 9.94
(s, 1H, CHO), 12.28 (s, 1H, NH-indole). 13C NMR (125 MHZ, DMSO-d6): (δ, ppm)
55.8, 111.8, 113.2, 114.7, 120.9, 122.0, 122.4, 123.7, 125.9, 131.4, 135.9, 149.3,
160.6, 185.4. Anal. Calcd. for C16H13NO2: C, 76.48; H, 5.21; N, 5.57. Found: C,
76.60; H, 5.29; N, 5.48; IR (KBr, cm−1): 3147 (N–H stretching), 3056 (Aromatic
C–H stretching), 2837 (H–CO), 1621 (C=O), 1454 (C=C), 1179 (C–O); ESI–MS
(m/z): 252 [M+H]+.
2‑(4‑chlorophenyl)‑1H‑indole‑3‑carbaldehyde (5e) Cream Powder, Mp > 50 °C;
1
Yield: 93%. H NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 7.25 (t,
J=7.6 Hz, 1H, H6), 7.30 (t, J=7.6 Hz, 1H, H7), 7.51 (d, J=7.6 Hz, 1H, H8), 7.74
(d, J=8.1 Hz, 2H, H3′,5′), 7.80 (d, J=8.1 Hz, 2H, H2′,6′), 8.21 (d, J=7.6 Hz, 1H, H5),
9.95 (s, 1H, CHO), 12.46 (s, 1H, NH-indole). 13C NMR (125 MHZ, DMSO-d6): (δ,
ppm) 112.1, 113.6, 121.1, 122.6, 123.5, 123.9, 125.8, 129.0, 131.8, 132.0, 135.9,
147.4, 185.4. Anal. Calcd. for C15H10ClNO: C, 70.46; H, 3.94; N, 5.48. Found: C,
70.69; H, 3.85; N, 5.51; IR (KBr, cm−1): 3168 (N–H stretching), 3036 (Aromatic
C–H stretching) 2866, 1625, 1450; ESI–MS (m/z): 256 [M+H]+.
2‑(4‑bromophenyl)‑1H‑indole‑3‑carbaldehyde (5f) Cream powder, Mp> 250 °C;
1
Yield: 92%. H NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 7.25 (t,
J=7.6 Hz, 1H, H6), 7.30 (t, J=7.6 Hz, 1H, H7), 7.51 (d, J=7.6 Hz, 1H, H8), 7.74
(d, J=8.3 Hz, 2H, H3′,5′), 7.80 (d, J=8.3 Hz, 2H, H2′,6′), 8.20 (d, J=7.6 Hz, 1H, H5),
9.95 (s, 1H, CHO), 12.47 (s, 1H, NH-indole). 13C NMR (125 MHZ, DMSO-d6): (δ,
ppm) 112.1, 113.6, 121.1, 122.6, 123.5, 123.9, 125.5, 128.8, 131.9, 132.0, 136.2,
147.4, 185.2. Anal. Calcd. for C15H10BrNO: C, 60.02; H, 3.36; N, 4.67. Found: C,
60.21; H, 3.48; N, 4.60; IR (KBr, cm−1): 3167 (N–H stretching), 3058 (Aromatic
1 3

Anti-cancer, anti-oxidant and molecular docking studies…
C–H stretching), 2840 (H–CO), 1623 (C=O), 1452 (C=C); ESI–MS (m/z): 300
[M+H]+.
2‑p‑tolyl‑1H‑indole‑3‑carbaldehyde (5g) Cream powder, Mp: 239–241 °C, Yield:
88%. 1H NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 2.41 (s, 3H, CH3), 7.23
(t, J=7.5 Hz, 1H, H6), 7.28 (t, J=7.5 Hz, 1H, H7), 7.41 (d, J=7.9 Hz, 2H, H3′,5′),
7.49 (d, J=7.5 Hz, 1H, H8), 7.67 (d, J=7.9 Hz, 2H, H2′,6′), 8.19 (d, J=7.5 Hz, 1H,
H5), 9.95 (s, 1H, CHO), 12.34 (s, 1H, NH-indole). 13C NMR (125 MHz, DMSO-d6):
(δ, ppm) 21.0, 112.0, 113.3, 121.1, 122.4, 123.6, 125.8, 126.9, 129.6, 129.9, 135.9,
139.7, 149.1, 184.9. Anal. Calcd. for C16H13NO: C, 81.68; H, 5.57; N, 5.97. Found:
C, 81.78; H, 5.48; N, 5.79; IR (KBr, cm−1): 3213 (N–H stretching), 3061 (Aromatic
C–H stretching), 2863 (H–CO), 1625 (C=O), 1452 (C=C); ESI–MS (m/z): 236
[M+H]+.
General procedure for synthesis of compounds (6a–p)
The substituted 2-arylindole-3-carbaldehyde derivative 5a–g (1 mmol) and a few
drops of acetic acid were added to a solution of substituted thiosemicarbazide
derivative 2 (1.2 mmol) in ethanol (10 mL) . The mixture was stirred at refux for
1–3 h and then cooled to room temperature. Then the solid separated was fltered
and recrystallized from the mixture of ethyl acetate and n-hexane to give compounds
6a–p [32].
(E)‑4‑(4‑(dimethylamino)phenyl)‑1‑((2‑phenyl‑1H‑indol‑3‑yl)methylene)thio‑
1
semicarbazide (6a) Yellow to green powder; Mp: 210–212 °C; Yield: 75%; H
NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 2.90 (s, 6H; NCH3); 6.73 (d,
J=8.2 Hz; H2″, 6″), 7.17 (t, J=7.4 Hz, 1H; H6), 7.24 (t, J=7.4 Hz, 1H; H7), 7.35
(d, J=8.2 Hz, 2H; H3″,5″), 7.44 (d, J=7.4 Hz, 1H; H8), 7.52 (t, J=7.0 Hz, 1H; H4′),
7.59 (t, J=7.0 Hz, 2H; H3′,5′), 7.66 (d, J=7.0 Hz, 2H; H2′,6′), 8.34 (d, J=7.4 Hz, 1H;
H5), 8.57 (s, 1H; H–C=N), 9.29 (s, 1H; NH–Ph), 11.40 (s, 1H; NH–C=S), 11.92 (s,
1H; NH-indole); 13C NMR (DMSO-d6, 25 °C, TMS) (δ, ppm): 40.5, 107.4, 111.5,
112.0, 121.1, 122.6, 123.2, 125.2, 126.6, 128.6, 128.9, 129.0, 129.3, 130.9, 136.5,
141.5, 142.5, 148.4, 174.8; IR (KBr, cm−1): 3305, 3211 (N–H stretching), 3136
(Aromatic C–H stretching), 1607 (C=N), 1536 (C=C), 1273 (C–N), 1199 (C=S);
Anal. Calcd. for C24H23N5S: C 69.71, H 5.61,N 16.94, Found: C 69.51, H 5.49, N
17.06; ESI–MS (m/z): 414 [M+H]+.
(E)‑4‑(4‑(dimethylamino)phenyl)‑1‑((2‑(4‑hydroxyphenyl)‑1H‑indol‑3‑yl)
meth1yl‑
ene) thiosemicarbazide (6b) Yellow powder; Mp: 220–221 °C; Yield: 81%; H
NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 2.89 (s, 6H; NCH3), 6.72 (d,
J=8.0 Hz, 2H; H3″,5″), 6.96 (d, J=8.0 Hz, 2H; H3′,5′), 7.13 (t, J=7.6 Hz, 1H; H6),
7.19 (t, J=7.6 Hz, 1H; H7), 7.36 (d, J=8.0, 2H; H2″,6″), 7.40 (d, J=7.6 Hz, 1H;
H8), 7.48 (d, J=8.0 Hz, 2H; H2′,6′), 8.29 (d, J=7.6 Hz, 1H; H5), 8.52 (s, 1H; H–
C=N), 9.26 (s, 1H; NH–Ph), 9.88 (s, 1H; OH), 11.35 (s, 1H; NH–C=S), 11.74 (s,
1H, NH-indole); 13C NMR (DMSO-d6, 25 °C, TMS) (δ, ppm): 40.4, 106.4, 112,
1 3

Z. Bakherad et al.
115.7, 120.9, 121.6, 122.6, 122.6, 125.3, 126.3, 126.4, 128.6, 130.4, 130.7, 136.3,
141.8, 143.2, 148.3, 158.2, 174.6; IR (KBr, cm−1): 3425, 3312 (N–H stretching),
3243 (OH), 3136 (Aromatic C–H stretching), 1608 (C=N), 1536 (C=C), 1271
(C–N), 1204 (C=S), 1177 (C–O); Anal. Calcd. for C24H23N5OS: C 67.11, H 5.40, N
16.30, Found: C 67.24, H 5.45, N 16.25; ESI–MS (m/z): 430 [M+H]+.
(E)‑4‑(4‑(dimethylamino)phenyl)‑1‑((2‑(3,4,5‑trimethoxyphenyl)‑1H‑indol‑3‑yl)
methylene) thiosemicarbazide (6c) Light yellow powder; Mp: 205–207 °C; Yield:
1
71%; H NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 2.91 (s, 6H; NCH3);
3.81 (s, 3H; OCH3), 3.95 (s, 3H; OCH3), 3.98 (s, 3H; OCH3), 6.73 (d, J=8.7 Hz,
2H; H3′,5′), 6.94 (s, 1H; H2′), 7.17 (d, J=7.5 Hz, 1H; H6), 7.27–7.37 (m, 4H;
H7, 2″,6″), 7.37 (s, 1H; H6′), 7.47 (d, J=7.8 Hz, 1H; H8), 8.36 (d, J=7.8 Hz, 1H;
H5), 8.92 (s, 1H; H–C=N), 9.49 (s, 1H; NH–Ph), 11.80 (s, 1H; NH–C=S), 11.89
(s, 1H; NH-indole); 13C NMR (DMSO-d6, 25 °C, TMS) (δ, ppm): 40.7, 54.7, 55.3,
107.1, 111.3, 114.4, 122.1, 122.4, 124.2, 125.2, 125.4, 127.2, 127.7, 128.6, 129.0,
130.7, 133.0, 142.5, 143.2, 153.5, 174.3; IR (KBr, cm−1): 3441, 3312 (N–H stretch-
ing), 3144 (Aromatic C–H stretching), 1609 (C=N), 1533 (C=C), 1267 (C–N),
1196 (C=S), 1119 (C–O); Anal. Calcd. for C27H29N5O3S: C 64.39, H 5.80, N 13.91,
Found: C 64.46, H 5.89, N 14.03; ESI–MS (m/z): 504 [M+H]+.
(E)‑4‑(4‑(dimethylamino)phenyl)‑1‑((2‑(4‑methoxyphenyl)‑1H‑indol‑3yl)
methyl‑
ene) thiosemicarbazide (6d) Dark cream powder; Mp: 211–212 °C; Yield: 81%;
1H NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 2.90 (s, 6H; NCH3), 3.85
(s, 3H; OCH3), 6.73 (d, J=7.7, 2H; H3″,5″), 7.14–7.22 (m, 4H; H3′,5′,6,7), 7.36 (d,
J=7.7 Hz, 2H; H2″,6″), 7.42 (d, J=7.2 Hz, 1H; H8), 7.60 (d, J=7.2 Hz, 2H, H2′,6′),
8.31 (d, J=7.2 Hz, 1H, H5); 8.54 (s, 1H, H–C=N), 9.27 (s, 1H, NH–Ph), 11.38 (s,
1H; NH–C=S), 11.82 (s, 1H; NH-indole); 13C NMR (DMSO-d6, 25 °C, TMS) (δ,
ppm): 40.4, 55.3, 106.7, 112, 114.3, 114.4, 121.0, 122.8, 123.2, 125.2, 126.3, 126.4,
128.5, 130.6, 136.3, 141.7, 142.6, 148.4, 159.8, 174.7; IR (KBr, cm−1): 3307, 3239
(N–H stretching), 3147 (Aromatic C–H stretching), 1607 (C=N), 1531 (C=C), 1278
(C–N), 1205 (C=S), 1181 (C–O); Anal. Calcd. for C25H25N5OS: C 67.69, H 5.68, N
15.79, Found: C 67.80, H 5.74, N, 15.86; ESI–MS (m/z): 444 [M+H]+.
(E)‑1‑((2‑(4‑chlorophenyl)‑1H‑indol‑3‑yl)methylene)‑4‑(4‑(dimethylamino)
phe‑
1
nyl) thiosemicarbazide (6e) Yellow powder; Mp: 230–231 °C; Yield: 85%; H
NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 2.89 (s, 6H, NCH3), 6.72 (d,
J=8.0 Hz, 2H; H3″,5″), 7.17 (t, J=7.6, 1H; H6), 7.24 (t, J=7.6 Hz, 1H; H7), 7.34 (d,
J=8.0 Hz, 2H; H2″,6″), 7.44 (d, J=7.6 Hz, 1H; H8), 7.60 (d, J=8.0 Hz, 2H; H3′,5′),
7.78 (d, J=8.0, 2H; H2′,6′), 8.34 (d, J=7.6 Hz, 1H; H5), 8.52 (s, 1H; H–C=N), 9.30
(s, 1H; NH–Ph), 11.35 (s, 1H; NH–C=S), 11.98 (s, 1H; NH-indole); 13C NMR
(DMSO-d6, 25 °C, TMS) (δ, ppm): 40.8, 107.9, 111.6, 112.2, 121.2, 122.3, 122.7,
123.5, 125.0, 125.8, 126.7, 129.4, 130.1, 131.3, 131.9, 136.5, 141.0, 141.1, 174.8;
IR (KBr, cm−1): 3310, 3211 (N–H stretching), 3148 (Aromatic C–H), 1608 (C=N),
1538 (C=C), 1273 (C–N), 1200 (C=S); Anal. Calcd. for C24H22ClN5S: C 64.35, H
4.95, N 15.63, Found: C 64.24, H 4.87, N 15.55; ESI–MS (m/z): 448 [M+H]+.
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Anti-cancer, anti-oxidant and molecular docking studies…
(E)‑1‑((2‑(4‑bromophenyl)‑1H‑indol‑3‑yl)methylene)‑4‑(4‑(dimethylamino)phe‑
1
nyl) thiosemicarbazide (6f) Dark cream powder; Mp: 228–229 °C; Yield: 85%; H
NMR (500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 2.87 (s, 6H, NCH3), 6.72 (d,
J=8.7 Hz, 2H; H3″,5″), 7.16 (t, J=7.7, 1H; H6), 7.24 (t, J=7.7 Hz, 1H; H7), 7.34 (d,
J=8.7 Hz, 2H; H2″,6″), 7.44 (d, J=7.7 Hz, 1H; H8), 7.60 (d, J=8.3 Hz, 2H; H3′,5′),
7.78 (d, J=8.3 Hz, 2H; H2′,6′), 8.36 (d, J=7.7 Hz, 1H; H5), 8.53 (s, 1H; H–C=N),
9.32 (s, 1H; NH–Ph), 11.39 (s, 1H; NH–C=S), 12.00 (s, 1H, NH-indole); 13C NMR
(DMSO-d6, 25 °C, TMS) (δ, ppm): 40.8, 107.9, 111.6, 112.6, 121.2, 122.3, 122.7,
123.4, 125.1, 126.6, 128.10, 130.1, 131.3, 131.9, 134.7, 136.6, 141.0, 141.12, 174.8;
IR (KBr, cm−1): 3325, 3246 (N–H stretching), 3173 (Aromatic C–H), 1609 (C=N),
1534 (C=C), 1275 (C–N), 1205 (C=S); Anal. Calcd. for C24H22BrN5S: C 58.54, H
4.50, N 14.22, Found: C 58.68, H 4.57, N 14.27; ESI–MS (m/z): 494 [M+H]+.
(E)‑4‑(4‑(dimethylamino)phenyl)‑1‑((2‑p‑tolyl‑1H‑indo l‑3‑yl)methylene) thiosemi‑
carbazide (6g) Yellow powder; Mp: 225–227 °C; Yield: 75%; 1H NMR (500 MHz,
DMSO-d6, 25 °C, TMS) (δ, ppm): 2.41 (s, 3H; CH3), 2.90 (s, 6H; NCH3), 6.73 (d,
J=8.2 Hz, 2H; H3″,5″), 7.15 (t, J=7.5 Hz, 1H; H6), 7.22 (t, J=7.5 Hz, 1H; H7),
7.35 (d, J=8.2 Hz, 2H; H2″,6″), 7.39 (d, J=7.5 Hz, 2H; H3′,5′), 7.43 (d, J=7.5 Hz,
1H; H8), 7.55 (d, J=7.5 Hz, 2H; H2′,6′), 8.32 (d, J=7.5, 2H; H5), 8.55 (s, 1H; H–
C=N), 9.28 (s, 1H; NH–Ph), 11.38 (s, 1H; NH–C=S), 11.86 (s, 1H; NH-indole);
13C NMR (DMSO-d6, 25 °C, TMS) (δ, ppm): 31.2, 40.6, 107.2, 111.2, 111.5, 121.0,
122.5, 123.0, 125.2, 126.7, 127.0, 127.7, 128.1, 129.2, 129.6, 136.4, 138.5, 141.9,
142.8, 174.5; IR (KBr, cm−1): 3310, 3212 (N–H stretching), 3132 (Aromatic C–H),
1607 (C=N), 1539 (C=C), 1272 (C–N), 1200 (C=S); Anal. Calcd. for C23H20N4S:
C 71.85, H 5.24, N 14.57, Found: C 71.96, H 5.30, N 14.66; ESI–MS (m/z): 428
[M+H]+.
(E)‑1‑((2‑phenyl‑1H‑indol‑3‑yl)methylene)‑4‑p‑tolylthiosemicarbazide (6h) Yellow
1
to green powder; Mp: 225–227 °C; Yield: 81%; H NMR (500 MHz, DMSO-d6,
25 °C, TMS) (δ, ppm): 2.90 (s, 3H; CH3), 6.73 (d, J=8.5 Hz, 2H; H3″,5″), 7.16 (t,
J=7.7 Hz, 1H; H6), 7.24 (t, J=7.7 Hz, 1H; H7), 7.35 (d, J=8.5 Hz, 2H; H2″,6″), 7.44
(d, J=7.7 Hz, 1H; H8), 7.52 (t, J=7.5 Hz, 1H; H4′), 7.59 (t, J=7.5 Hz, 2H; H3′,5′),
7.66 (d, J=7.5 Hz, 2H; H2′,6′), 8.35 (d, J=7.7 Hz, 1H; H5), 8.59 (s, 1H; H–C=N),
9.31 (s, 1H; NH–Ph), 11.42 (s, 1H; NH–C=S), 11.94 (s, 1H; NH-indole); 13C NMR
(DMSO-d6, 25 °C, TMS) (δ, ppm): 21.0, 108.8, 110.5, 121.6, 122.1, 123.3, 124.0,
125.1, 125.8, 126.3, 127.9, 130.0, 131.9, 134.2, 136.1, 136.9, 141.2, 141.5, 174.6;
IR (KBr, cm−1): 3310, 3209 (N–H stretching), 3159 (Aromatic C–H), 1607 (C=N),
1537 (C=C), 1273 (C–N), 1202 (C=S). C23H20N4S: C 71.85, H 5.24, N 14.57,
Found: C 71.65, H 5.19, N 14.60; ESI–MS (m/z): 385 [M+H]+.
(E)‑1‑((2‑(4‑hydroxyphenyl)‑1H‑indol‑3‑yl)methylene)‑4‑p‑tol1ylthiosemicarbazide
(6i) Cream to yellow powder; Mp: 234–236 °C; Yield: 75%; H NMR (500 MHz,
DMSO-d6, 25 °C, TMS) (δ, ppm): 2.32 (s, 3H; CH3), 6.96 (d, J=8.0 Hz, 2H;
H3′,5′), 7.13–7.21 (m, 4H; H6,7,3″,5″), 7.40 (d, J=7.7 Hz, 1H; H8), 7.47–7.52 (m, 4H;
H2′,6′,2″,6″), 8.29 (d, J=7.7 Hz, 1H; H5), 8.54 (s, 1H; H–C=N), 9.44 (s, 1H; NH–Ph),
1 3

Z. Bakherad et al.
9.89 (s, 1H; OH), 11.48 (s, 1H; NH–C=S), 11.77 (s, 1H; NH-indole); 13C NMR
(DMSO-d6, 25 °C, TMS) (δ, ppm): 20.7, 106.3, 111.3, 115.8, 121.0, 121.6, 122.3,
122.7, 125.0, 125.3, 128.7, 130.7, 134.1, 136.4, 136.7, 142.3, 143.5, 158.3, 174.2;
IR (KBr, cm−1): 3362 (N–H stretching), 3243 (OH), 3159 (Aromatic C–H), 1606
(C=N), 1553 (C=C), 1270 (C–N), 1206 (C=S), 1179 (C–O); Anal. Calcd. for
C23H20N4OS: C 68.98, H 5.03, N 13.99, Found: C 68.86, H 4.96, N 13.90; ESI–MS
(m/z): 430 [M+H]+.
(E)‑1‑((2‑(4‑methoxyphenyl)‑1H‑indol‑3‑yl)methylen1e)‑4‑p‑tolylthiosemicarbazide
(6j) Cream powder; Mp: 244–246 °C; Yield: 77%; H NMR (500 MHz, DMSO-
d6, 25 °C, TMS) (δ, ppm): 2.31 (s, 3H; CH3), 3.85 (s, 3H; OCH3), 7.14-7.23 (m,
6H; H3′,5′,3″,5″,6, 7), 7.43 (d, J=7.7 Hz, 1H; H8), 7.52 (d, J=7.5 Hz, 2H; H2″,6″), 7.60
(d, J=8.0 Hz, 2H; H2′,6′), 8.31 (d, J=7.7 Hz, 1H; H5), 8.55 (s, 1H; H–C=N), 9.45
(s, 1H; NH–Ph), 11.50 (s, 1H; NH–C=S), 11.84 (s, 1H; NH-indole). 13C NMR
(DMSO-d6, 25 °C, TMS) (δ, ppm): 20.3, 55.3, 106.7, 111.3, 111.4, 114.3, 114.4,
123.2, 124.7, 124.8, 125.2, 128.5, 128.6, 130.6, 134.0, 136.7, 142.0, 142.8, 159.8,
174.3; IR (KBr, cm−1): 3360, 3307 (N–H stretching), 3186 (Aromatic C–H), 1607
(C=N), 1544 (C=C), 1273 (C–N), 1202 (C=S), 1182 (C–O); Anal. Calcd. for
C24H22N4OS: C 69.54, H 5.35, N 13.52, Found: C 69.39, H 5.28, N 13.47; ESI–MS
(m/z): 444 [M+H]+.
(E)‑1‑((2‑(4‑chlorophenyl)‑1H‑indol‑3‑yl)methylene)‑4‑p‑tolylthiosemicarbazide
(6k) Yellow to light green powder; Mp: 239–242 °C; Yield: 82%; 1H NMR
(500 MHz, DMSO-d6, 25 °C, TMS) (δ, ppm): 2.31 (s, 3H; CH3), 7.17–7.18 (m, 3H;
H3″,5″,6), 7.25 (t, J=7.8, 1H; H7), 7.45 (d, J=7.8 Hz, 1H; H8), 7.49 (d, J=8.0 Hz,
2H; H3′, 5′), 7.65–7.69 (m, 4H; H2′,6′,2″,6″), 8.36 (d, J=7.8 Hz, 1H; H5), 8.54 (s, 1H;
H–C=N), 9.49 (s, 1H; NH–Ph), 11.51 (s, 1H; NH–C=S), 12.03 (s, 1H; NH-indole);
13C NMR (DMSO-d6, 25 °C, TMS) (δ, ppm): 21.6, 106.9, 111.3, 116.8, 123.4,
124.5, 126.1, 128, 129.6, 130.4, 132.7, 133.5, 135.4, 135.7, 137.6, 138.0, 138.1,
140.8, 175.3; IR (KBr, cm−1): 3346, 3315 (N–H stretching), 3142 (Aromatic C–H),
1607 (C=N), 1544 (C=C), 1272 (C–N), 1203 (C=S); Anal. Calcd. for C23H19ClN4S:
C 65.94, H 4.57, N 13.37, Found: C 65.91, H 4.61, N 13.35; ESI–MS (m/z): 448
[M+H]+.
(E)‑1‑((2‑(4‑bromophenyl)‑1H‑indol‑3‑yl)methylene)‑4‑p‑tolylthiosemicarbazide
(6l) Yellow to light green powder; Mp> 250 °C; Yield: 80%; 1H NMR (500 MHz,
DMSO-d6, 25 °C, TMS) (δ, ppm): 2.31 (s, 3H; CH3), 7.17–7.18 (m, 3H; H3″,5″,6),
7.25 (t, J=7.8 Hz, 1H; H7), 7.45 (d, J=7.8 Hz, 1H; H8), 7.49 (d, J=8.1 Hz, 2H;
H3′,5′), 7.61 (d, J=8.2 Hz, 2H; H2″,6″), 7.79 (d, J=8.1 Hz, 2H; H2′,6′), 8.36 (d,
J=7.8 Hz, 1H; H5), 8.54 (s, 1H; H–C=N), 9.48 (s, 1H; NH–Ph), 11.50 (s, 1H;
NH–C=S), 12.02 (s, 1H; NH-indole); 13C NMR (DMSO-d6, 25 °C, TMS) (δ, ppm):
20.9, 107.8, 111.6, 121.2, 122.4, 122.7, 123.4, 125.1, 125.2, 128.7, 130.1, 131.3,
131.9, 134.2, 136.6, 136.7, 141.2, 141.5, 174.50; IR (KBr, cm−1): 3431, 3342 (N–H
stretching), 3141 (Aromatic C–H), 1593 (C=N), 1542 (C=C), 1273 (C–N), 1202
1 3

Anti-cancer, anti-oxidant and molecular docking studies…
(C=S); Anal. Calcd. for C23H19BrN4S: C 59.61, H 4.13, N 12.09, Found: C 59.72, H
4.21, N 12.18; ESI–MS (m/z): 494 [M+H]+.
(E)‑1‑((2‑(4‑hydroxyphenyl)‑1H‑indol‑3‑yl)methylene)‑4‑methylthiosemicarbazide
1
(6m) Cream powder; Mp> 250 °C; Yield: 83%; H NMR (500 MHz, DMSO-d6,
25 °C, TMS) (δ, ppm): 3.09 (d, J=4 Hz, 3H; CH3), 6.94 (d, J=8.3 Hz, 2H; H3′,5′),
7.14 (t, J=7.6 Hz, 1H; H6), 7.20 (t, J=7.6 Hz, 1H; H7), 7.38 (d, J=7.6 Hz, 1H; H8),
7.44 (d, J=8.3 Hz, 2H; H2′,6′), 7.80–7.86 (m, 1H; NHCH3), 8.33 (d, J=7.6 Hz, 1H;
H5), 8.44 (s, 1H; H–C=N), 9.86 (s, 1H; OH), 11.11 (s, 1H; NH–C=S), 11.68 (s, 1H;
NH-indole); 13C NMR (DMSO-d6, 25 °C, TMS) (δ, ppm): 31.2, 106.5, 111.2, 115.7,
120.8, 121.7, 122.6, 122.7, 125.3, 130.7, 136.2, 141.5, 142.9, 158.2, 176.6; IR (KBr,
cm−1): 3359 (N–H stretching), 3243 (OH), 3141 (Aromatic C–H), 1605 (C=N),
1553 (C=C), 1271 (C–N), 1234 (C=S), 1180 (C–O); Anal. Calcd. for C17H16N4OS:
C 62.94, H 4.97, N 17.27, Found: C 62.83, H 5.05, N 17.19; ESI–MS (m/z): 325
[M+H]+.
(E)‑1‑((2‑(4‑methoxyphenyl)‑1H‑indol‑3‑yl)methylene)‑4‑methylthiosemicarbazide
1
(6n) Dark cream; Mp: 229–230 °C; Yield: 79%; H NMR (500 MHz, DMSO-d6,
25 °C, TMS) (δ, ppm): 3.08 (d, J=4.4 Hz, 3H; CH3), 3.85 (s, 3H; OCH3), 7.12–7.17
(m, 3H; H3′,5′,6), 7.21 (t, J=7.7 Hz, 1H; H7), 7.40 (d, J=7.7 Hz, 1H; H8), 7.56 (d,
J=8.5 Hz, 2H; H2′,6′), 7.83–7.87 (m, 1H; NHCH3), 8.35 (d, J=7.7 Hz, 1H; H5),
8.45 (s, 1H; H–C=N), 11.13 (s, 1H; NH–C=S), 11.76 (s, 1H; NH-indole); 13C NMR
(DMSO-d6, 25 °C, TMS) (δ, ppm): 31.3, 55.6, 106.9, 111.5, 114.4, 121.0, 122.9,
123.3, 125.1, 127.8, 130.6, 136.3, 141.5, 142.4, 159.7, 176.5; IR (KBr, cm−1): 3345,
3201 (N–H stretching), 3159 (Aromatic C–H), 1609 (C=N), 1544 (C=C), 1275
(C–N), 1242 (C=S), 1176 (C–O); Anal. Calcd. for C18H18N4OS: C 63.88, H 5.36, N
16.56, Found: C 63.97, H 5.41, N 16.61; ESI–MS (m/z): 339 [M+H]+.
(E)‑1‑((2‑(4‑chlorophenyl)‑1H‑indol‑3‑yl)methylene)‑4‑methylthiosemicarbazide
1
(6o) Yellow powder; Mp: 235–237 °C; Yield: 84%; H NMR (500 MHz, DMSO-
d6, 25 °C, TMS) (δ, ppm): 3.08 (d, J=4.3 Hz, 3H; CH3), 7.18 (t, J=7.7 Hz, 1H;
H6), 7.25 (t, J=7.7 Hz, 1H; H7), 7.43 (d, J=7.7 Hz, 1H; H8), 7.60–7.67 (m, 4H;
H3′,5′ 2′,6′), 7.85–7.93 (m, 1H; NHCH3), 8.39 (d, J=7.7 Hz, 1H; H5), 8.45 (s, 1H;
,
H–C=N), 11.14 (s, 1H; NH–C=S), 11.94 (s, 1H; NH-indole); 13C NMR (DMSO-d6,
25 °C, TMS) (δ, ppm): 31.2, 107.9, 111.5, 121.1, 123.0, 123.3, 125.0, 128.9, 129.8,
130.9, 133.5, 136.5, 140.6, 140.9, 176.9; IR (KBr, cm−1): 3383, 3305 (N–H stretch-
ing), 3161 (Aromatic C–H), 1597 (C=N), 1546 (C=C), 1269 (C–N), 1238 (C=S);
Anal. Calcd. for C17H15ClN4S: C 59.56, H 4.41, N 16.34, Found: C 59.47, H 4.36,
N, 16.28; ESI–MS (m/z): 343 [M+H]+.
(E)‑1‑((2‑(4‑bromophenyl)‑1H‑indol‑3‑yl)methylene)‑4‑methylthiosemicarbazide
1
(6p) Dark yellow powder; Mp: 242–244 °C; Yield: 83%; H NMR (500 MHz,
DMSO-d6, 25 °C, TMS) (δ, ppm): 3.08 (d, J=4.4 Hz, 3H; CH3), 7.18 (t, J=7.8 Hz,
1H; H6), 7.25 (t, J=7.8 Hz, 1H; H7), 7.43 (d, J=7.8 Hz, 1H; H8), 7.57 (d,
J=8.3 Hz, 2H; H3′,5′), 7.77 (d, J=8.3 Hz, 2H; H2′,6′), 7.86–7.91 (m, 1H; NHCH3),
1 3

Z. Bakherad et al.
8.39 (d, J=7.8 Hz, 1H; H5), 8.45 (s, 1H; H–C=N), 11.13 (s, 1H; NH–C=S), 11.93
(s, 1H; NH-indole); 13C (DMSO-d6, 25 °C, TMS) (δ, ppm): 31.1, 108.0, 111.5,
121.1, 122.2, 122.9, 123.0, 123.3, 125.0, 130.1, 131.2, 131.8, 136.5, 140.7, 176.7;
IR (KBr, cm−1): 3387, 3303 (N–H stretching), 3161 (Aromatic C–H), 1597 (C=N),
1552 (C=C), 1272 (C–N), 1237 (C=S); Anal. Calcd. for C17H15BrN4S: C 52.72, H
3.90, N 14.47, Found C 52.65, H 3.86, N 14.41; ESI–MS (m/z): 389 [M+H]+.
Biology
Anti‑proliferative assay
The cytotoxic activity of synthesized compounds 6a–p was assessed against three dif-
ferent cancer cell lines [38]. Tumor cell lines including lung cancer cell line (A-549),
liver cancer cell line (Hep-G2), and breast cancer cell line (MCF-7) were obtained
from the National Cell Bank of Iran and grown in RPMI-1640 medium (Gibco, UK).
Exponentially growing cells (1×104 cells/well) were seeded in 96-well plates in
RPMI1640 medium at 37 °C under 5% CO2 supplemented with 10% FBS, 1% l-glu-
tamine, and penicillin-streptomycin and incubated overnight. The cells were treated
by diferent concentrations of test compounds and allowed to incubate for 48 h in a
humidifed atmosphere. All compounds were initially dissolved in DMSO, and the
fnal concentration of DMSO was less than 1% in all concentration of the applied
compounds. Etoposide and colchicine were used as the positive control in this experi-
ment. After 48 h further incubation, the medium was replaced with MTT (1 mg/mL)
and followed by 4-h incubation. After formation of blue formazan crystals, the culture
medium was replaced with 100 µL of DMSO and absorbance values were measured
using a multi-well plate reader (Gen5, Epoch, BioTek, America) at 492 nm wave-
lengths. The IC50 values were compared with the control and expressed in mean SD
from the dose–response logarithmic curves of at least three independent experiments.
Acridine orange/ethidium bromide double staining
Apoptosis in treated cancer cells was determined morphologically after staining with
acridine orange/ethidium bromide (AO/EB) using fuorescence microscopy [37]. A-
549 cell grown in 12 well plates (5×105 cells/well) were treated with and without
IC50 concentrations of compound 6n for 24 h. Then the cells were washed for three
times with PBS. Finally, ethidium bromide/acridin orange (1:1, 100 mg/mL) solution
was added to the cell suspension, and the nuclear morphology was evaluated by fuo-
rescence microscopy (Zeiss, Germany). All experiments were repeated three times.
Flow cytometry analysis of the apoptotic cells with Annexin V‑FITC/PI staining
Flow cytometry analyses for 6n were performed to confrm apoptosis induced in
comparison with standard drug etoposide. In brief, 5×105 cells/well of A-549
cells was treated with IC50 dose of most potent compound 6n. After 24 h, the cells
were washed twice with cold PBS, collected by centrifugation and resuspended in
1 3

Anti-cancer, anti-oxidant and molecular docking studies…
1×annexin V binding bufer (0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM
CaCl2). Then, the cells were double stained with Annexin V-FITC and 5 µL of PI,
and the cells were gently vortex and incubated at room temperature for 15 min in the
dark before fow cytometry. Finally, 400 μL of 1x annexin binding bufer was added
into the suspension, and the cells were analyzed using a fow cytometer within 1 h.
Antioxidant activity
The DPPH antioxidant activities of 6a–p were evaluated according to the literature
[39, 40]. The DPPH solution (6.25×10−5 M) was prepared in methanol. Then difer-
ent sample solutions of compounds 6a–p (2000, 1000, 500, 250, and 125 µg mL−1)
were obtained in methanol. Subsequently, 0.1 mL of each compound solution was
added to 3.9 mL of DPPH solution and was shaken vigorously. The mixture was
allowed to stand at room temperature in dark for 30 min, and their absorbance was
measured at 517 nm. Ascorbic acid was used as the reference standard. The control
sample contained all the reagents except the test compound, and methanol was used
as the blank. The percentage of radical scavenging activity was calculated as follows:
Radical scavenging activity (%)=[(Acontrol −Asample)/Acontrol]×100, where Acontrol
is the absorbance of the control reaction, and Asample is the absorbance in the pres-
ence of the compounds or standard absorbance of the test compounds. The IC50 val-
ues for the sample compounds were determined by plotting the radical scavenging
activity percentage versus the concentration of the sample compound.
Molecular docking
A docking study by using the Discovery Studio 4.1 (DS4.1) were performed to
understand the binding modes of the compounds with tubulin and ATPase domain
of topoisomerase IIα crystal structure.
For ligand preparation, all the molecular structures were sketched in the Chem-
Draw ultra 8.0 program, and were shifted into the Discovery Studio 4.1 (DS4.1)
for minimization. The compounds were then typed with a CHARMm force feld,
the partial charges were computed by the Momany–Rone option [41]. A minimizer
algorithm was used to minimize the resulting structures, that carried out 1000 steps
of the steepest descent with a RMS gradient tolerance of three followed by a conju-
gate gradient minimization. The intended crystal protein structures were extracted
from the protein data bank (www.rcsb.org) (PDB ID: 1S0 [21] and 1ZXM [30]
for tubulin and topoisomerase II, respectively). All protein preparations and mini-
mizations were done using the protocols in DS4.1. The region of interest used for
GOLD docking was defned as when all the protein residues were within the 8.96
Å and 10 Å of the reference ligands of tubulin and topoisomerase IIα, respectively.
The C and D chains were eliminated from the structure of tubulin and chain A was
selected for the docking study In the structure of topoisomerase IIα. All bound water
molecules and ligands were removed from the proteins, non-polar hydrogen atoms
were merged and the polar hydrogen atoms were added. At the beginning of the
docking, it is essential to validate the docking reliability. The ligands colchicine and
1 3

Z. Bakherad et al.
ANP were re-docked to the bonding site of tubulin and topoisomerase, respectively.
The docked conformation of colchicine and ANP using Gold showed similar bind-
ing poses with the original X-ray structure with the root mean of square deviation
(rmsd) of 0.51 and 0.89 for colchicine and ANP, respectively. Therefore, the docking
protocol could be extended to inspect the binding modes of the corresponding com-
pound into the tubulin and ATPase domain of topoisomerase II using GOLD 5.3.0.
The annealing parameters of the Van der Waals and hydrogen bonding interactions
were 6.0 and 3.0 Å, respectively. All the other parameters were saved at their default
values for the docking study. Gold fexible ligand docking generated 10 positions of
each ligand, which were ranked using the Goldscore function. The top ranked pose
with highest Goldscore ftness was analyzed using Accelrys Discovery studio to
reveal the hydrogen bond interaction and binding mode within the binding domain.
Calculation of logP
The cLogP values for the synthesized series six compounds as well as etoposide and
colchicine were calculated using Molinspiration webME Editor 1.16 [42] in order to
measure their lipophilicity (Table 1).
Acknowledgements This work was supported by Grants from Isfahan university of Medical Sciences and
Tehran University of Medical Sciences.
Compliance with ethical standards
Confict of interest The authors declare that they have no confict of interests.
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Z. Bakherad et al.
Afliations
Zohreh Bakherad1 · Maliheh Safavi2 · Afshin Fassihi1 · Hojjat Sadeghi‑Aliabadi1 ·
Mohammad Bakherad3 · Hossein Rastegar4 · Jahan B. Ghasemi5 ·
Saghi Sepehri6 · Lotfollah Saghaie1 · Mohammad Mahdavi7
1
Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan
University of Medical Sciences, Isfahan 81746-73461, Iran
2
Department of Biotechnology, Iranian Research Organization for Science and Technology,
Tehran 33535-111, Iran
3
School of Chemistry, Shahrood University of Technology, Shahrood, Iran
4
Food and Drug Control Laboratories, Food and Drug Laboratory Research Center, MOE
and ME, Tehran, Iran
5
Drug Design in Silico Lab, Chemistry Faculty, School of Sciences, University of Tehran,
Teheran, Iran
6
Department of Medicinal Chemistry, School of Pharmacy, Ardabil University of Medical
Sciences, Ardabil 5618953141, Iran
7
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Research
Institute, Tehran University of Medical Sciences, Tehran, Iran
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