Design and Synthesis of Novel Cytotoxic Indole-Thiosemicarbazone Derivatives: Biological Evaluation and Docking Study

Article abstract of DOI:10.1002/cbdv.201800470

In this work, two novel series of indole-thiosemicarbazone derivatives were designed, synthesized, and evaluated for their cytotoxic activity against MCF-7, A-549, and Hep-G2 cell lines in comparison to etoposide and colchicine as the reference drugs. Generally, the synthesized compounds showed better cytotoxicity towards A-549 and Hep-G2 than MCF-7. Among them, (2E)-2-{[2-(4-chlorophenyl)-1H-indol-3-yl]methylidene}-N-(4-methoxyphenyl)hydrazinecarbothioamide (8l) was found to be the most potent compound against A-549 and Hep-G2, at least three times more potent than etoposide. The morphological analysis by the acridine orange/ethidium bromide double staining test and flow cytometry analysis indicated that compound 8l induced apoptosis in A-549 cells. Moreover, molecular docking methodology was exploited to elucidate the details of molecular interactions of the studied compounds with putative targets.

Full text of DOI:10.1002/cbdv.201800470

Title: Design and synthesis of novel cytotoxic indole-
thiosemicarbazone derivatives: biological evaluation and docking
study
Authors: lotfoallah saghaie, Zohreh Bakherad, Maliheh Safavi, Hojjat
Sadeghi-Aliabadi, Afshin Fassihi, Mohammad Bakherad,
Hossein Rastegar, Mina Saeedi, Jahan B Ghasemi, Lotfollah
Saghaie, and Mohammad Mahdavi
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201800470
Link to VoR: http://dx.doi.org/10.1002/cbdv.201800470

10.1002/cbdv.201800470
Chemistry & Biodiversity
Design and synthesis of novel cytotoxic indole‐thiosemicarbazone derivatives:
biological evaluation and docking study
Zohreh Bakherad1, Maliheh Safavi2, Afshin Fassihi1, Hojjat Sadeghi‐Aliabadi1, Mohammad Bakherad3, Hossein
Rastegar4, Mina Saeedi5,6, Jahan B Ghasemi7 ,Lotfollah Saghaie1*and Mohammad Mahdavi8 *
1Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan
University of Medical Sciences, 81746‐73461, Isfahan, Iran.
2Department of Biotechnology, Iranian Research Organization for Science and Technology, 33535‐111, Tehran, Iran
3School of Chemistry, Shahrood University of Technology, Shahrood, Iran
4Food and drug control laboratories, food and drug Laboratory Research center, MOE and ME, Tehran, Iran.
5Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
6Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran
7Drug Design in Silico Lab, Chemistry Faculty, School of Sciences, University of Tehran, Teheran, Iran
8Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences, Tehran, Iran
Email: saghaie@pharm.mui.ac.ir
Email: momahdavi@tums.ac.ir
1
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10.1002/cbdv.201800470
Chemistry & Biodiversity
Abstract
In this work, two novel series of indole‐thiosemicarbazone derivatives were designed, synthesized, and evaluated for their cytotoxic activity against MCF‐7, A‐
549, and Hep‐G2 cell lines in comparison with etoposide and colchcine as the reference drug. Generally, the synthesized compounds showed better cytotoxicity
towards A‐549 and Hep‐G2 than MCF‐7. Among them, (E)‐1‐((2‐(4‐chlorophenyl)‐1H‐indol‐3‐yl)methylene)‐4‐(4‐methoxyphenyl) hydrazinecarbothioamide (8l)
was found to be the most potent compound compared with etoposide, at least three times more potent, against A‐549 and Hep‐G2. The morphological analysis
by the acridine orange/ethidium bromide double staining test and flow cytometry analysis indicated that compound 8l induced apoptosis in A‐549 cells.
Moreover, molecular docking methodology was exploited to elucidate the details of molecular interactions of the studied compounds with putative targets.
Key words: Apoptosis • Cancer • Indole • Thiosemicarbazone
This paper is dedicated to the memory of our wonderful teacher in Chemistry and Medicinal Chemistry, Professor Abbas Shafiee (1937‐2016).
Introduction
Cancer has been characterized as the main public health problem worldwide and known as the second cause of death in the United States.[1,2] American Cancer
Society has estimated 1,688,780 new cancer cases and 600,920 cancer deaths in the US.[3,4] Among the current tools for the treatment of cancer, chemotherapy
is still one of the key therapeutic options, though, there are possible restrictions such as various side‐effects and drug resistance. [5] In this regard, development
of novel anti‐cancer compounds targeting the hallmarks of tumor cells is highly in demand.[6‐8] Considering the process of proliferation, DNA has been emerged
as one of the molecular targets for cancer therapy leading to well‐known medications applied in the current clinical cancer therapy.[9] DNA targeting agents may
act through inter‐ or intrastrand crosslinking of DNA, examples are cyclophosphamide, melphalan, mitomycin C, bizelesin, and cisplatin. Stabilization of
topoisomerase‐I‐DNA cleavable complex by SN‐38 and topoisomerase‐II‐DNA cleavable complex by doxorubicin, etoposide, mitoxantrone is the second
mechanism of action in DNA targeting. Finally, the inhibition of DNA‐directed RNA synthesis by actinomycin D is another way of DNA targeting.[9] In addition,
antimitotic drugs such as paclitaxel, cabazitaxel, ixabepilone, and eribulin which are generally categorized into two classes of microtubule‐destabilizing and
microtubule‐stabilizing agents have attracted great attention in cancer treatment.[10‐12]
Indole is a bicyclic heterocyclic scaffold with various valuable biological activities such as anti‐inflammatory, [13] antimicrobial, [14] anti‐viral, [15] anti‐
depressant, anti‐hypertensive, anti‐hypoglycemic, [16] and notably anti‐cancer, [17‐21] properties. Indole system have been found as a privileged scaffold in anti‐
cancer drug discovery since it can efficiently mimic peptide structures to bind to an specific enzyme. [22,23] Potent tubulin polymerization inhibitors such as taxol
and colchicine have been successfully studied as anti‐cancer agents. In fact, microtubule‐based anti‐cancer drugs have occupied a special place in cancer
chemotherapy and various indole‐based compounds have shown inspiring activities in preclinical studies. [24] Molecular modeling of microtubule‐interacting
indoles focusing on the colchicine binding domain has been extensively explored. [25] Gastpar et al. synthesized some novel indole derivatives as a new class of
potent microtubule destabilizing agents acting through binding with the colchicine binding site in the microtubule structure. [26] One of these agents, 6‐methoxy‐
2‐(4‐methoxyphenyl)‐1H‐indole‐3‐carbaldehyde, compound A in Fig. 1, has shown an inhibitory activity towards MDA‐MB 231 (IC50 = 35 µM) and tubulin
polymerization inhibitory activity (IC50 = 1.5 µM). [26] In addition, Giueppe La Regina et al. introduced some new 2‐ phenyl indole derivatives as potential anti‐
cancers which inhibited colchicine binding with a mean value 70%. [18]
The anti‐cancer activity of indole derivatives through inhibition of topoisomerase have also been the center of attention. The 4‐thiazolidinones
indole‐based derivatives reported by Lima et al. have shown anti‐proliferative activities against eight human tumor cell lines via inhibition of topoisomerase IIα
(derivatives of B and C, Fig. 1). [27] It has been revealed that a series of indole‐thiosemicarbazones, B derivatives, has been much higher active than indole‐
thiazolidinone series (C derivatives) in which (E)‐2‐(1H‐indol‐3ylmethylene)‐N‐(naphthalen‐1‐yl) hydrazinecarbothioamide has depicted the best activity against
the colorectal adenocarcinoma (HT‐29) and leukemia (K‐562) cell lines with GI50 = 0.01 mM for both. A series ofnew thiosemicarbazone derivatives were designed
2
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10.1002/cbdv.201800470
Chemistry & Biodiversity
and synthesized by Huang and co‐workers, among them compound D indicated broad anticancer activity in a panel of human tumor cells. Further evaluations
showed that this compound induced antiproliferative activity through inhibition of topoisomerase IIα due to direct interaction with the ATPase domain of this
enzyme and inhibition of topoisomerase IIα‐mediated ATP hydrolysis. [28] The anti‐cancer activity of oxindole‐thiosemicarbazones (derivatives of E, Fig. 1) with
selective potency toward multidrug‐resistant cells has also been considered in the literature, and the efficacy of this pharmacophore has been highlighted by
aromatic/hydrophobic features at the N4 position of the thiosemicarbazone. [29]
S
S
HN
N
O
R
N
N
N
H
O
N
H
R
OCH3
N
H3CO
N
H
H
N
H
A
B
C
R
S
S
NH
HN
N
R1
CH3
H
NH
O
N
N
N
N
H
N
CH3
S
N
N
N
R2
H
H
Designed compounds
E
D
Figure 1. Structures of indole‐based and thiosemicarbazone‐based anti‐cancer agents A‐E and designed compounds.
Focusing on the efficacy of indoles as well as thiosemicarbazide moiety, [30‐32] as anti‐cancer agents and in continuation of our efforts to design and
synthesize cytotoxic heterocycles, [33‐37] herein, we report synthesis of novel indole‐thiosemicarbazone derivatives as potent cytotoxic agents. Looking for a
synergistic effect which may make the designed compounds successful anticancer agents we exploited the concept of multi‐target compounds in the design of
the structures. Three structural motives were considered in the designed scaffold. The indole moiety for the inhibition of tubulin polymerization and the
thiosemicarbazone structure due to its inhibitory activity on the ATPase domain of topoisomerase IIα.[28] The fact that a closed ring containing the
thiosemicarbazone moiety such as the ring observed in derivatives of C led to a decrease in antiproliferative activity fully considered in the design of these
compounds. The third element which was implemented in the designed structures was a substituted phenyl ring that was proved to be efficient in the
antiproliferative activity of compound A.[26]
Results and discussion
Chemistry
Synthesis of the novel indole‐thiosemicarbazone derivatives 8a‐n was conducted as schematically described in Scheme 1.
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10.1002/cbdv.201800470
Chemistry & Biodiversity
H
H
SCN
N
N
NH2
1
H2N
a
H2N
+
R1
S
R1
2
3
H2N
O
CH3
NH
5
CHO
c
b
+
R2
N
H
R2
N
R2
H
4
6a-g
7a-g
3''
S
2''
R1
HN
N
O
N
H
5''
H
5
2'
3
6''
3'
H2N
N
6
2
d
4'
+
S
7
R2
N
R1
N
5' R2
H
H
6'
8
3
7
8a-n
R1= H, OMe
R2= H, 4-OH, 3,4,5-(OMe)3, 4-OMe, 4-Cl, 4-Br, 4-Me
Scheme 1. Reagents and conditions; (a) Iso‐propanol alcohol, 30 min; (b) i) CH3COOH, EtOH, reflux ii) Polyphosphoric acid, 120 C; (c) POCl3, DMF, 70 C; (d) EtOH, CH3COOH, reflux.
The reaction of hydrazine 1 with the aryl isothiocyanate derivatives 2 in iso‐propanol alcohol at room temperature for 30 min afforded the related aryl thiourea
derivative 3. [28] Also, the reaction of substituted acetophenone derivatives 4 and phenylhydrazine 5 in acetic acid and polyphosphoric acid at 120 ºC gave indole
[38]
derivatives 6a‐g via Fischer indole synthesis.
Subsequently, Vilsmeier reaction of compound 6a‐g provided the desired 2‐aryl‐1H‐indole‐3‐carbaldehyde
derivatives 7a‐g. [39] Finally, the reaction of compounds 3 and 7 in EtOH in the presence of a catalytic amount of acetic acid at reflux led to the formation of the
corresponding indole‐thiosemicarbazone derivatives 8a‐n. [40] All compounds were purified by recrystallization from ethyl acetate and n‐hexane.
1
The structures of the synthesized compounds 8a‐n were determined by H and 13C NMR, IR, Mass spectrometry and elemental analyses. The IR
spectrum of these compounds showed characteristic stretches of C=S and C=N bonds around 1196‐1242 and 1596‐1610 cm‐1, respectively. In addition, the
absence of bands around 2500‐2700 cm‐1 excluded the possibility of a thione‐thiol tautomerism (eg. H‐N‐C=S↔‐N=C‐SH). [41,42] All compounds displayed signals
around 6.94‐8.42 ppm corresponding to protons related to phenyl groups in 1H NMR spectra. Furthermore, a singlet was observed between 8.53‐8.97 ppm
[43,44]
related to imino proton (H‐C=N) which is in accordance with the reports involving thiosemicarbazones.
The signals of protons belonging to the NH
connected to the (substituted) phenyl ring and indole ring appeared around 9.40‐9.72 ppm and 11.78‐12.04 ppm, respectively. All compounds could exist in either
the E or Z isomeric forms of the imino bond (‐CH=N‐). Since in the 1H NMR spectrum analysis, the –N=CH signal was observed as a single peak, it indicates that
only one isomer was formed. Compound 8l was selected for elucidation of isomerism of the synthesized compounds. On the other side, in 2D NMR NOESY
spectrum, the spatial correlation was observed between the two hydrogens of azomethine (‐CH=N‐) at 8.58 ppm and HN‐C=S at 11.48 ppm. Furthermore, the
chemical shift values corresponding to E isomer of HN‐C=S appears around 11 ppm as reported in the literature, but higher ppm values (over 12 ppm) were
reported for the Z forms. [27] The 1H NMR spectra for all compounds (8a‐n) showed a singlet between 11.46‐11.58 ppm belonging to the NH signal which was in
good agreement with NOESY spectrum of (E)‐thiosemicarbazone. [27]
Biology
In vitro cytotoxicity
Cytotoxicity of all the synthesized compounds was evaluated against different cancer cell lines including MCF‐7 (breast), A‐549 (lung), and Hep‐G2 (liver) (Table
1). Series 8a‐n compounds could be classified into two subseries based on the substituted aryl group connected to the thiosemicarbazone moiety which may be
phenyl (Series I, compounds 8a‐g) or 4‐methoxyphenyl (Series II, compounds 8h‐n).
The cytotoxic activity of compounds 8a‐n against MCF‐7 revealed that compound 8b having a phenyl group connected to the thiosemicarbazone
moiety and 4‐hydroxyphenyl group at C2 position of the indole ring, showed the highest cytotoxic activity towards MCF‐7 (IC50 = 21.76 µM) compared with
etoposide (IC50 = 33.45 µM). It should be noted that replacement of phenyl by 4‐methoxyphenyl group diminished cytotoxicity in compound 8i (IC50>100 µM).
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10.1002/cbdv.201800470
Chemistry & Biodiversity
Compound 8j in the second category (Series II), having 3,4,5‐(OMe)3 at the C2 position of the indole ring, showed lower activity (IC50 = 31.72 µM) compared with
8b, however, it was a little more potent than etoposide. Its counterpart, compound 8c, showed a lower activity against MCF‐7 (IC50 = 44.20 µM). Compounds 8e
and 8h showed moderate‐to‐low activities against the breast cancer cell line (IC50s= 52.20 and 83.04 µM, respectively). To sum up, most of the studied
compounds were not active against MCF‐7 in such a manner that compounds 8a, 8d, 8f, 8g, 8i, and 8k‐n showed (IC50>100 µM).
As it can be seen in this table, compounds of both series demonstrated much higher activities against A‐549 and Hep‐G2 than MCF‐7. Generally, they
showed similar activities and compounds 8a, 8h, 8i, 8k, and 8n demonstrated no cytotoxicity against A‐549 and Hep‐G2 (IC50>100 µM). Compound 8l was found
to be the most potent against A‐549 and Hep‐G2 with IC50 = 11.77 and 11.91 µM, respectively. They were approximately three times more potent than etoposide
(IC50s = 39.88 and 31.38 µM, against A‐549 and Hep‐G2, respectively). Our results related to the cytotoxic activity of compounds 8 against Hep‐G2 revealed that
compounds 8d and 8l were the most potent compounds among the first and second category, respectively (IC50s =11.59 and 11.91 µM). Considering the
cytotoxicity of the compound series of 8a‐g, only compound 8a was inactive toward Hep‐G2, and the others showed good‐to‐moderate activities. In this series,
compound 8d having 4‐methoxyphenyl at the C2 position of indole as mentioned above showed the highest activity. Increasing the number of methoxy group
(compound 8c) reduced the cytotoxic activity (IC50 = 75.04 µM). Furthermore, replacement of the methoxy group by hydroxyl, halogens (Cl and Br), and methyl
on the indole moiety deteriorated cytotoxicity (entries 2, 5, 6, and 7). However, compound 8f possessing 4‐bromophenyl group showed cytotoxicity against Hep‐
G2 almost as potent as etoposide (IC50 = 39.34 µM). The results related to the compound series 8h‐n indicated the efficiency of chlorine on the aryl group at the
C2 position of indole to induce a desirable cytotoxicity in compound 8l. Replacement of Cl by Br (compound 8m) as well as electron‐rich substituent 3,4,5‐(OMe)3
(compound 8j) led to the reduction of activity (IC50s = 14.17 and 13.25 µM, respectively). Interestingly, the presence of 4‐OH, 4‐OMe and 4‐Me as well as 4‐H
substituents on the phenyl ring at the C2 position of indole moiety eliminated the cytotoxic ability from the compounds of the second series II (IC50>100 µM). As
mentioned earlier, compound 8l was the most potent compound against A‐549 (IC50 = 11.77 µM). Similar to the anti‐Hep‐G2 activity, replacement of Cl with
3,4,5‐(OMe)3 groups (8j) led to a sharp decrease in the cytotoxicity (IC50 = 33.78 µM). It should be noted that the presence of Br in the structure induced a much
lower cytotoxic effect in compound 8m (IC50 = 30.43 µM); however, this compound was more potent than etoposide (IC50 = 39.88 µM). Also, in the series II of
compounds 8h‐n, similar to the activity of compounds against Hep‐G2, introduction of 4‐OH, 4‐OMe and 4‐Me as well as 4‐H substituents on the phenyl ring at
the C2 position of indole moiety disappeared cytotoxicity against A‐549 (IC50>100 µM). Compounds 8a‐g in the first series showed a good‐to‐low activity against
A‐549. Compounds 8b and 8g having the 4‐hydroxy and 4‐methylphenyl groups were found to be potent cytotoxic agents with IC50s = 26.75 and 24.00 µM,
respectively and they were more potent than etoposide (IC50 = 39.88 µM). However, introduction of 4‐methoxyphenyl group (compound 8d) reduced the activity
against A‐549 (IC50 = 33.05 µM). The results obtained revealed that the presence of halogens (Cl and Br) on the aryl group at C2 position of the indole ring reduced
the activity (IC50s= 49.96 and 47.73 µM, respectively for Cl and Br containing compounds). Omission of Cl and incorporation of 3,4,5‐(OMe)3 into the structure led
to much more reduction of cytotoxicity (IC50 = 98.22 µM for 8c). At the end, it should be noted that most of the compounds belonging to the series I showed a
good cytotoxic activity against A‐549 compared with the compounds in series II (Table 1).
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10.1002/cbdv.201800470
Chemistry & Biodiversity
Table 1. Cytotoxic activities, Calculation of cLogP and Goldscore fitness values of indole‐thiosemicarbazone derivatives of 8.
3''
S
2''
R1
HN
N
N
H
5''
5
8
6''
2'
3
3'
6
2
4'
7
N
5' R2
H
6'
8a-n
bGold score
Entry
Compound
R1
R2
aMCF‐7
aHep‐G2
aA‐549
cLogP
cGold score fitness
fitness
1
2
8a
H
H
H
4‐OH
3,4,5‐(OMe)3
4‐OMe
4‐Cl
100
21.76±5.95
44.25±5.17
100
100
42.56±5.51
75.04±9.21
11.59±0.85
45.56±3.04
39.34±4.85
63.87±3.20
100
100
5.14
4.66
4.77
5.20
5.82
5.95
5.59
5.20
4.72
4.83
5.25
5.88
6.01
5.64
0.70
1.10
56
51.6
59
61.7
51
8b
26.75±0.59
98.22±6.47
33.05±2.69
49.96±3.04
47.73±6.94
24.00±0.31
100
3
8c
H
60
65
4
8d
H
61
5
8e
H
52.23±3.58
100
59
59.7
56
6
8f
H
4‐Br
58
7
8g
H
4‐Me
100
59.6
57
61
8
8h
OMe
OMe
OMe
OMe
OMe
OMe
OMe
‐‐‐‐
‐‐‐‐
H
83.04±3.07
100
67
9
8i
4‐OH
3,4,5‐(OMe)3
4‐OMe
4‐Cl
100
100
54
59
10
11
12
13
14
15
16
8j
8k
31.72±2.53
100
13.25±1.92
100
33.78±2.28
100
58
62
65
58
8l
100
11.91±1.27
14.17±0.94
100
11.77±0.27
30.43±2.57
100
62
65
8m
4‐Br
100
56
67
8n
4‐Me
100
58.5
‐‐‐‐
‐‐‐‐
69
‐‐‐‐
‐‐‐‐
Etoposide
Colchicine
‐‐‐‐
33.45±2.13
7.15±0.63
31.38± 1.23
6.1±0.51
39.88±1.67
1.8±0.26
‐‐‐‐
aThe IC50 (half maximal inhibitory concentration) values represent an average of three independent experiments (mean ± SD)
bThe binding affinity of ligand with tubulin
c The binding affinity of ligand with ATPase domain of topoisomerase IIα
Morphological evaluation by fluorescence microscopy
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Chemistry & Biodiversity
The morphological changes due to apoptosis were determined in treated lung and liver cancer cells after staining with acridine orange/ethidium bromide using
fluorescence microscopy. Acridine orange penetrates living as well as dead cells and emits green fluorescence as a result of intercalation into the cell membrane
of the living cell and emits red fluorescence in dead ones. Analysis of the acridine orange/ethidium bromide double staining of the selected most potent
compound (8l) in A‐549 and 8d and 8l in Hep‐G2 cell lines is shown in Fig. 2. In this figure, the viable cells are observed green but apoptotic cells with chromatin
condensation or fragmentation are seen as orange‐stained nuclei. The lung and liver cancer cells treated with 8d and 8l showed higher apoptosis compared with
the control.
(A)
(B)
Figure 2. Acridine orange/ethidium bromide double staining of cancer cells with characteristic symptoms of apoptosis. (A) A‐549 a, DMSO 1% as control, b, c, cells treated with IC50
concentrations of etoposide and compound 8l, respectively, for 24 h. (B) Hep‐G2 a, DMSO 1% as control, b, c, and d, cells treated with IC50 concentrations of etoposide, compounds 8d
and 8l, respectively, for 24 h. White arrows indicate live cells, dash arrows show apoptosis. The images of cells were taken with a fluorescence microscope at 400 x.
Flow cytometry analysis of apoptotic cells with Annexin V‐FITC/PI staining
The apoptosis induction caused by the prepared compounds was also confirmed by flow cytometry analysis. Annexin V‐FITC/PI was used as a quantitative
method for determining apoptosis. As illustrated in Fig. 3, the apoptotic index of the test compounds was compared with the negative control in A‐549 cells.
Double staining followed by flow cytometric analysis revealing the percentage of apoptotic cells (49.5%) was resulted by the treatment of cells with compound
8l after 24 h incubation. Besides, the corresponding value obtained after treatment with etoposide was 35.8% following 24 h incubation.
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10.1002/cbdv.201800470
Chemistry & Biodiversity
Figure 3. Flow cytometric analysis of A‐549 cells treated with the compound 8l. Cells were stained with Annexin V‐FITC/PI and quantitated by flow cytometry. The cells were treated
with a) DMSO 1% (negative control); b) IC50 values of etoposide as the positive control; c) IC50 values of the compound 8l for 24 h.
cLogP
The logP value of a compound which is the logarithm of its partition coefficient between n‐octanol and water, log(Coctanol / C water), is a well‐established measure
ofthe compound's hydrophilicity. [45] Among the compounds 8a‐g, compound 8c possessing three electron‐donating substituted groups (OMe) atthe C2 position
of the indole ring was found to be the least lipophilic compound (cLogP = 4.77), and compound 8e having a chlorine substituent at the same position
demonstrated the highest lipophilicity (cLogP = 5.82). As it can be seen in Table 1, compound 8c demonstrated the lowest cytotoxicity against Hep‐G2 and A‐
549. Furthermore, compound 8e exhibited a moderate activity against Hep‐G2. Compound 8d, known as the most potent compound against Hep‐G2 in this
category, had a cLogP of 5.20. In the case of the second category of compounds, 8h‐n, the most potent compound against Hep‐G2 and A‐549, 8l, indicated a
high lipophilicity (cLogP = 5.88). Moreover, the least lipophilic compound, 8j with a cLogP of 4.83, and the most hydrophobic one, 8m cLogP = 6.01, also showed
good cytotoxic activity toward both cell lines. It is clear that there is not a direct relationship between the in vitro cytotoxicity and the lipophilicity of compounds
and other factors such as the rotameric forms of the compounds and spatial hindrance of the substituents attached to the aryl rings may play remarkable roles.
In‐silico molecular modelling and docking studies
Computer‐simulated docking studies were performed using the GOLD (Genetic Optimization for Ligand Docking) 5.3.0 program as one of the most commonly
and appropriately used programs in diverse docking studies. [46] Gold performs genetic algorithm for docking of flexible ligands to the receptor binding sites in
order to investigate the full area of conformational flexibility of ligands with partial flexibility of receptor. It utilizes the Gold score fitness function to evaluate
the binding affinities of the ligands with protein. The docking performance and accuracy of this program was obtained by the flexible docking of the native
co‐crystallized ligands, colchicine and ANP extracted from the crystal structures of tubulin and ATPase binding site of topoisomerase IIα, respectively. The
docked conformation of colchicine and ANP showed similar binding poses with the original X‐ray structures with the root mean of square deviation (rmsd) of
0.44 Å for colchicine (Goldscore fitness=41.9) and 0.97 Å for ANP (Goldscore fitness=138.2), respectively as shown in Fig. S1. After validation of the docking
protocol, the corresponding compounds were docked into the tubulin and ATPase domain of topoisomerase IIα. A maximum of 10 docking GA runs per molecule
were used to provide 1.5 Å RMS deviations. The annealing parameters of the van der Waals and hydrogen bonding interactions were 6.0 Å and 3.0 Å, respectively.
All the other parameters were saved at their default values for the docking study.
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10.1002/cbdv.201800470
Chemistry & Biodiversity
Molecular docking study was performed to predict the interactions of the synthesized compounds of the series 8 in the tubulin and ATPase domain of
topoisomerase IIα binding active sites and compared with colchicine and ANP as the reference ligands. Docking results consisting of hydrophobic interactions,
hydrogen bonds, and predicted docking affinities of the best docked pose of molecules are summarized in Tables S1 and S2.
At first, the binding elements of colchicine and ANP were precisely characterized in the active site of tubulin and ATPase domain of topoisomerase
IIα, respectively (Fig. 4). As can be seen in Fig. 4(a), formation of hydrogen bond between oxygen of methoxy group and Cys241 was distinguished. In addition,
the hydrophobic interactions of different parts of colchicine with Lys352, Val315, Ala316, Ala180, Met259, Leu255, Leu248, Ala250, and Val238 were well‐
established. Fig. 4(b) also clearly represented the hydrogen bonding interactions of some parts of ANP with Gln376, Tyr165, Gly166, Gly164, Asn163, Arg162,
Ser148, Ser149, Asn150, Gly161, Asn91, Lys168, Ala167, and Asn120 amino acid residues. Moreover, the hydrophobic interactions of ANP with Ala92, Ile125,
Lys378, and Asn95 were fully recognized.
(a)
(b)
Figure 4. Docking poses of (a) colchicine and (b) ANP in the active sites of tubulin and ATPase domain of topoisomerase IIα. Green dashed lines indicate hydrogen bonds.
As presented in Fig. 5, the orientations of compound 8b in the active sites of tubulin and ATPase domain of topoisomerase IIα were the same as
colchicine and ANP, respectively.
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10.1002/cbdv.201800470
Chemistry & Biodiversity
(a)
(b)
Figure 5. a) Superimposition of compounds 8b (yellow) and colchicine (blue) in the tubulin binding active site b) Superimposition of compounds 8b (yellow) and ANP (blue) in ATPase
domain of topoisomerase IIα active site. Green dashed lines indicate hydrogen bonds.
As can be seen in Fig. 5(a), formation of hydrogen bonds between two NH groups and Thr179 in the tubulin active site is distinguished. In addition, hydrophobic
interaction of different parts of colchicine with Ala180, Val181, Lys 254, Met259, Ala316, Lys352, Leu255, and Ala250 have been well established. Fig. 5(b) also
clearly represented hydrogen bonding interaction of OH substituent on the aryl ring at the C2 position of the indole moiety of 8b with Asn150, Lys157, and NH
indole with Asn95 amino acid residues, respectively. Moreover, hydrophobic interactions of 8b with Arg98, Ala167, Asn91 and Lys168 has been fully recognized
as indicated in Fig. 5. As indicated in Fig. 6, docking of compound 8d in the active site of tubulin confirmed two hydrogen bond interactions of two NHs of
thiosemicarbazone with Thr179. Furthermore, compound 8d indicated a hydrophobic interaction with Val181, Met259, Leu248, Ala180, Ala316, and Lys352
which mimicked the desired interaction of colchicine in the active site of tubulin, as depicted in Fig. 4(a).
Figure 6. Docking pose of compound 8d in the active site of tubulin. Green dashed lines indicate hydrogen bonds.
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Besides, molecular docking of compounds 8j (a potent compound against Hep‐G2) and 8l (the most potent compound against A‐549 and Hep‐G2) in
the tubulin and ATPase domain of topoisomerase IIα active sites (Figs. 7 and 8) confirmed the desired interactions with the same residues. As it can be seen in
Fig. 7(a), the hydrophobic interactions of Leu255, Met259, Val181, Ala316, and Lys352 with different moieties of 8j in the tubulin active site were fully conserved
for this compound. In addition, it is worthwhile to pay attention to the hydrogen bonding interaction between methoxy group and significant residue Cys241 in
the tubulin active site. [47] Furthermore, the desirable interactions such as hydrogen bonding interaction of methoxy groups with Lys168 in the topoisomerase
IIα active site were distinguished.
Fig. 8 demonstrated hydrophobic interactions of compound 8l with Val238, Cys241, Ala316, Lys352, Ala250, Leu255, Met259, Lys254, Ala180, and
Val181 in the tubulin active site. Moreover, hydrogen bonding interaction of methoxy group and NH of thiosemicarbazone moiety with Asn163, Tyr165, Gly164
and Thr147 residues as well as hydrophobic interaction of different parts of 8l with Ala167, Lys168, Arg98, and Ile125 in the topoisomerase IIα active site was
detected in Fig. 8b. These interactions confirmed the potency of compound 8l as a cytotoxic agent presumably through the inhibition of tubulin polymerization
and topoisomerase function. As depicted in Fig. 8, compound 8l indicated no hydrogen bonding in the tubulin active site.
(a)
(b)
Figure 7. Docking poses of compound 8j in the active sites of (a) tubulin and (b) ATPase domain of topoisomerase IIα. Green dashed lines indicate hydrogen bonds.
(a)
(b)
Figure 8. Docking poses of compound 8l (a) superimposition of 8l (green) and colchicine (yellow) in the tubulin active site (b) interaction of 8l in the ATPase domain of topoisomerase
IIα active site. Green dashed lines indicate hydrogen bonds.
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Finally, Goldscore fitness values of the docked compounds are provided in Table 1 for a best predicted ligand binding poses. As can be seen in this table,
compounds 8l and 8d showed favorable and high Goldscore fitness as well as high cytotoxicity of the corresponding compounds against A‐549 and Hep‐G2
which is probably related to the inhibition of tubulin polymerization and topoisomerase function. However, some compounds such as 8k and 8n showed no
cytotoxic activity in spite of high Goldscore fitness values, which can be associated with other factors such as the rotameric forms of the compounds and spatial
hindrance of the substituents attached to the aryl rings may play remarkable roles.
Conclusion
Some novel indole‐thiosemicarbazons 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 prepared compounds of series 8 were generally categorized into two classes based on the aryl substituted groups
attached to the thiosemicarbazone moiety as well as the C2 position of the indole moiety. The cLogP and Goldscore fitness values revealed that cytotoxicity of
the title compounds was not affected only by these factors; it seems that some other factors such as the rotameric forms of the compounds and spatial hindrance
of the substituents attached to the aryl rings may play remarkable roles. In this regard, compound 8l was found to be the most potent compound against A‐549
and Hep‐G2. Moreover, compound 8d showed a high activity against Hep‐G2. In addition, morphological analysis by the acridine orange/ethidium bromide
double staining test and flow cytometry analysis confirmed the induction of apoptosis in A‐549 cells by compound 8l. Finally, the molecular docking study of
compounds 8j and 8l indicated desirable hydrogen bonding and hydrophobic interactions in the tubulin and ATPase domain of topoisomerase IIα binding sites.
Experimental section
Chemistry
All reagents and solvents were purchased from Merck and used without further purification. Melting points were determined on a Kofler hot‐stage apparatus
(Reichert, Vienna, Austria) and were uncorrected. TLC was conducted on silica gel 250 µm, F254 plates. The IR and NMR spectra were recorded using a Nicolet
Magna FTIR 550 spectrophotometer (potassium bromide disks) and Bruker 500 MHz NMR instruments, respectively. Chemical shifts were reported in parts per
million (ppm, δ) down filed from tetramethylsilane coupling constant (J) values are presented in Hz and spin multiplicities are given as s (singlet), d (double), t
(triplet) and m (multiplet). Mass spectra were recorded on an API 3200 LC/MS/MS system, AB Sciex. Elemental analysis were carried out with a CHN‐Rapid
Heraeus elemental analyzer.
General procedure for the synthesis of substituted thiosemicarbazides (3)
To a stirred solution of hydrazine hydrate 80% of 1 (24 mmol) in iso‐propanol (20 mL), aryl iso‐thiocyanate derivative 2 (2 mmol) was added dropwise and the
product precipitated immediately and stirring was continued for further 30 min at room temperature. Then, the precipitate was filtered off and washed with
iso‐propanol three times to give substituted thiosemicarbazides 3. [28]
General procedure for the synthesis of 2‐aryl‐1H‐indoles (6a‐g)
Appropriate amounts of phenyl hydrazine 4 (1 mmol) and substituted acetophenone 5 (1 mmol) were mixed in ethanol (20 mL), and a few drops of glacial acetic
acid were added. The solution was heated under reflux 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. Purification by column chromatography (hexane/ethyl acetate) gave the substituted 2‐aryl indoles 6a‐g. [38]
General procedure for the synthesis of 2‐arylindole‐3‐carbaldehydes (7a‐g)
Phosphorous oxychloride (10 mmol) was added dropwise to dry DMF (10 mmol) under nitrogen with ice cooling and the mixture was stirred for 1 h. Then, a
solution of compound 6 (1mmol) in DMF (50 mL) was added dropwise to the mixture with continuous stirring which was then heated at 70 C. After completion
of the reaction (checked by TLC), 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 and the residue was recrystallized from a mixture of
ethanol and water. [39]
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2‐Phenyl‐1H‐indole‐3‐carbaldehyde (7a)
1
Cream Powder; Mp: 249‐250 C; Yield: 89%; IR (KBr, cm‐1): 3144 (NH), 2858 (H‐CO), 1625 (C=O), 1455 (C=C). H NMR (500 MHz ‐DMSO‐d6) (, 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) (, ppm): 112.1 (C3), 113.5 (C8), 121.2 (C5), 122.5 (C7), 123.7 (C6), 125.8 (C2), 128.9 (C4), 129.2
(C2’,C6’), 129.8 (C4’), 130.1(C3’,C5’), 135.9 (C1’), 149.1 (C9), 185.5 (CHO). Anal. Calcd. for C15H11NO: C, 81.43; H, 5.01; N, 6.33. Found: C, 81.53; H, 4.85; N, 6.45.
2‐(4‐Hydroxyphenyl)‐1H‐indole‐3‐carbaldehyde (7b)
Cream Powder; Mp: 225‐227 C; Yield: 86%; IR (KBr, cm‐1): 3151 (OH), 3074 (NH), 2862 (H‐CO), 1619 (C=O), 1455 (C=C), 1177 (C‐O) 1H NMR (500 MHz ‐DMSO‐d6)
(, ppm): 6.97 (d, J = 7.6 Hz, 2H, H3',5'), 7.19‐7.26 (m, 2H, H6,7), 7.45 (d, J = 7.5 Hz, 1H, H8), 7.60 (d, J = 7.6 Hz, 2H, H2',6'), 8.17 (d, J = 7.5 Hz, 1H, H5), 9.92 (s, 1H, CHO),
10.04 (s, 1H, OH), 12.20 (s, 1H, NH‐indole). 13C NMR (125 MHZ, DMSO‐d6) (, ppm): 111.6 (C3), 112.7 (C8), 115.8 (C3’,C5’), 120.4 (C5), 120.9 (C7), 122.2 (C6), 123.3 (C2),
125.9 (C1’), 131.2 (C4), 135.8 (C2’,C6’), 149.8 (C9), 159.1 (C4’), 185.5 (CHO).
Anal. Calcd. for C15H11NO2: C, 75.94; H, 4.67; N, 5.93. Found: C, 76.04; H, 4.71; N, 5.72.
2‐(3,4,5‐Trimethoxyphenyl)‐1H‐indole‐3‐carbaldehyde (7c)
Cream Powder, Mp: 191‐194 C; Yield: 74%. IR (KBr, cm‐1): 3200 (NH), 2844 (H‐CO), 1617 (C=O), 1473 (C=C), 1120 (C‐O). 1H NMR (500 MHz, DMSO‐d6) (, 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 = 7.7 Hz, 1H, H6), 7.30 (t, J = 7.7 Hz, 1H, H7), 7.43 (d, J = 9.5 Hz, 1H, H6'),
7.62 (d, J = 7.7 Hz, 1H, H8), 7.71 (s, 1H, CHO), 8.14 (d, J = 7.7 Hz, 1H, H5), 10.49 (s, 1H, NH‐indole)13C NMR (125 MHZ, DMSO‐d6) (, ppm): 54.4 (C3’‐OCH3, C5’‐OCH3),
55.3 (C4’‐OCH3 ), 105.0 (C3), 105.6 (C2’,C6’), 110.1 (C8), 121.8 (C5), 122.4 (C7), 123.9 (C6), 126.0 (C2), 127.5 (C4), 135.9 (C1’), 141.8 (C9), 151.4 (C4’), 155.2 (C3’,C5’), 185.4
(CHO). Anal. Calcd. for C18H17NO4: C, 69.44; H, 5.50; N, 4.50. Found: C, 69.65; H, 5.61; N, 4.59.
2‐(4‐Methoxyphenyl)‐1H‐indole‐3‐carbaldehyde (7d)
Cream powder, Mp: 207‐209 C; Yield: 90%. IR (KBr, cm‐1): 3147 (NH), 2837 (H‐CO), 1621 (C=O), 1454 (C=C), 1179 (C‐O). 1H NMR (500 MHz, DMSO‐d6) (, ppm):
3.87 (s, 3H, OCH3), 7.16 (d, J = 8.7 Hz, 2H, H3',5'), 7.20‐7.28 (m, 2H, H6,7), 7.47 (d, J = 7.6 Hz, 1H, H8), 7.72 (d, J = 8.7 Hz, 2H, H2',6'), 8.18 (d, J = 7.6 Hz, 1H, H5), 9.94
(s, 1H, CHO), 12.28 (s, 1H, NH‐indole). 13C NMR (125 MHZ, DMSO‐d6) (, ppm): 55.8 (OCH3), 111.8 (C3), 113.2 (C8), 114.7 (C3’,C5’), 120.9 (C5), 122.0 (C7), 122.4 (C6),
123.7 (C2), 125.9 (C4), 131.4 (C1’), 135.9 (C2’,C6’), 149.3 (C9), 160.6 (C4’), 185.4 (CHO). Anal. Calcd. for C16H13NO2: C, 76.48; H, 5.21; N, 5.57. Found: C, 76.60; H, 5.29;
N, 5.48.
2‐(4‐Chlorophenyl)‐1H‐indole‐3‐carbaldehyde (7e)
Cream Powder, Mp 250C; Yield: 88%. IR (KBr, cm‐1): 3168 (NH), 2866 (H‐CO), 1625 (C=O), 1450 (C=C). 1H NMR (500 MHz, DMSO‐d6) (, 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) (, ppm): 112.1 (C3), 113.6 (C8), 121.1 (C5), 122.6 (C7), 123.5 (C6), 123.9 (C2), 125.8 (C4), 129.0 (C2’,C6’),
131.8 (C3’,C5’), 132.0 (C1’), 135.9 (C4’), 147.4 (C9), 185.4 (CHO). Anal. Calcd. for C15H10ClNO: C, 70.46; H, 3.94; N, 5.48. Found: C, 70.69; H, 3.85; N, 5.51.
2‐(4‐Bromophenyl)‐1H‐indole‐3‐carbaldehyde (7f)
1
Cream powder, Mp 250 C; Yield: 92%. IR (KBr, cm‐1): 3167 (NH), 2840 (H‐CO), 1623 (C=O), 1452 (C=C). H NMR (500 MHz, DMSO‐d6) (, 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) (, ppm): 112.1 (C3), 113.6 (C8), 121.1 (C5), 122.6 (C7), 123.5 (C6), 123.9 (C2), 125.5 (C4),
128.8 (C2’,C6’),131.9 (C3’,C5’), 132.0 (C1’), 136.2 (C4’), 147.4 (C9), 185.2 (CHO). Anal. Calcd. for C15H10BrNO: C, 60.02; H, 3.36; N, 4.67. Found: C, 60.21; H, 3.48; N, 4.60.
2‐p‐Tolyl‐1H‐indole‐3‐carbaldehyde (7g)
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Cream Powder, Mp: 239‐241C, Yield: 88%. IR (KBr, cm‐1): 3213(NH), 2863(H‐CO), 1625(C=O), 1452(C=C). 1H NMR (500 MHz, DMSO‐d6) (, 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) (, ppm): 21.0 (CH3), 112.0 (C3), 113.3 (C8), 121.1 (C5), 122.4 (C7), 123.6 (C6), 125.8
(C2), 126.9 (C4), 129.6 (C2’,C6’), 129.9 (C3’,C5’), 135.9 (C1’), 139.7 (C4’), 149.1 (C9), 184.9 (CHO). Anal. Calcd. for C16H13NO: C, 81.68; H, 5.57; N, 5.97. Found: C, 81.78;
H, 5.48; N, 5.79.
General procedure for the synthesis of compounds (8a‐n )
To a solution of substituted thiosemicarbazide derivative 3 (1.2 mmol) in ethanol (10 mL), the substituted 2‐arylindole‐3‐carbaldehyde derivative 7 (1
mmol) and a few drops of acetic acid was added. The mixture was stirred at reflux for 1‐3 h and then cooled to room temperature. After, the solid separated was
filtered and recrystallized from the mixture of ethyl acetate and n‐hexane to give compounds 8a‐n [40]
.
(E)‐4‐Phenyl‐1‐((2‐phenyl‐1H‐indol‐3‐yl)methylene)thiosemicarbazide (8a)
Yellow Powder, Mp: 210‐211 C; Yield: 78%. IR (KBr, cm‐1): 3433, 3374, 3313 (N‐H stretching), 3141 (Aromatic C‐H stretching), 1596 (C=N), 1548 (C=C),
1267
(C‐N), 1196 (C=S). 1H NMR (500 MHz, DMSO‐d6) (, ppm): 7.18‐7.21 (m, 2H, H6,7), 7.25 (t, J = 7.2 Hz, 1H, H4''), 7.38 (t, J = 7.2 Hz, 2H, H3'',5''), 7.46 (d, J = 7.4 Hz, 1H,
H8), 7.53 (t, J = 7.2 Hz, 1H, H4'), 7.59 (t, J = 7.2 Hz, 2H, H3',5'), 7.66‐7.67 (m, 4H, H2',6',2'',6''), 8.36 (d, J = 7.4 Hz, 1H, H5), 8.59 (s, 1H, H‐C=N), 9.56 (s, 1H, NH‐Ph), 11.58
(s, 1H, NH‐C=S), 11.96 (s, 1H, NH‐indole). 13C NMR (125 MHZ, DMSO‐d6) (, ppm): 107.5 (C3), 111.6 (C8), 121.2 (C5), 122.6 (C7), 123.2 (C6), 125.0 (C2), 125.2 (C4’’),
127.04 (C2’’, C6’’), 128.2 (C4), 128.9 (C2’, C6’), 129.0 (C4’), 129.4 (C3’’, C5’’), 130.9 (C3’, C5’), 136.6 (C1’), 139.2 (C9), 142.1 (C1’’), 142.8 (C=N), 174.3 (C=S). ESI‐MS (m/z):
371.14 ([M + H]+, C22H18N4S; calc. 371.13). Anal. Calcd. for C22H18N4S: C, 71.32; H, 4.90; N, 15.12; Found C, 71.10; H, 5.05; N, 15.25. ).
(E)‐1‐((2‐(4‐Hydroxyphenyl)‐1H‐indol‐3‐yl)methylene)‐4‐phenylthiosemicarbazide (8b)
White Powder, Mp: 226‐229 C; Yield: 81%. IR (KBr, cm‐1): 3362, 3300 (N‐H stretching), 3243 (O‐H stretching), 3150 (Aromatic C‐H stretching), 1612 (C=N), 1555
(C=C), 1269 (C‐N), 1241(C=S), 1176 (C‐O). 1H NMR (500 MHz, DMSO‐d6) (, ppm): 6.96 (d, J = 8.4 Hz, 2H, H3',5'), 7.13‐ 7.21 (m, 3H, H6,7,4''), 7.36‐7.41 (m, 3H, H3'',5'',8),
7.48 (d, J = 8.4 Hz, 2H, H2',6'), 7.66 (d, J = 7.7 Hz, 2H, H2'',6''), 8.31(d, J = 7.9 Hz, 1H, H5), 8.55 (s, 1H, H‐C=N), 9.54 (s, 1H, NH‐Ph), 9.93 (s, 1H, OH), 11.55 (s, 1H, NH‐
C=S), 11.80 (s, 1H, NH‐indole). 13C NMR (125 MHZ, DMSO‐d6) (, ppm): 106.7 (C3), 107.4 (C8), 121.51 (C3’, C5’), 124.1 (C5), 125.1 (C7), 125.2 (C6), 128.4 (C2), 129.4
(C4’’), 129.9 (C1’), 135.3 (C2’’, C6’’), 135.7 (C4), 136.8 (C2’, C6’), 137.1 (C3’’, C5’’), 139.2 (C9), 141.7 (C1’’), 142 (C=N), 156.8 (C4’), 174.5 (C=S). ESI‐MS (m/z): 387.17 ([M + H]+,
C22H18N4OS; calc. 387.12). Anal. Calcd. for C22H18N4OS: C, 68.37; H, 4.69; N, 14.50; Found C, 68.59; H, 4.61; N, 14.60.
(E)‐N‐Phenyl‐2‐((2‐(3,4,5‐trimethoxyphenyl)‐1H‐indol‐3‐yl)methylene)‐4‐phenylthiosemicarbazide (8c)
Light yellow powder, Mp: 196‐198 ºC; Yield: 77%. IR (KBr, cm‐1): 3446 (N‐H stretching), 3147 (Aromatic C‐H stretching), 1612 (C=N), 1539 (C=C), 1268 (C‐N), 1197
(C=S), 1176 (C‐O). 1H NMR (500 MHz, DMSO‐d6) (, ppm): 3.81 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 6.94 (s, 1H, H2'), 7.17‐7.22 (m, 2H, H6,4''),
7.29 (t, J = 7.5 Hz, 1H, H7), 7.37‐7.40 (m, 3H, H6',3'',5''), 7.48 (d, J =7.9 Hz, 1H, H8), 7.61 (d, 2H, J = 7.8 Hz, H2'', 6''), 8.34 (d, 1H, J = 7.9 Hz, H5), 8.94 (s, 1H, H‐C=N),
9.72 (s, 1H, NH‐Ph), 11.57 (s, 1H, NH‐C=S), 11.89 (s, 1H, NH‐indole). 13C NMR (125 MHZ, DMSO‐d6) (, ppm): 54.6 (C3’‐OCH3 , C5’‐OCH3), 55.3 (C4’‐OCH3), 106.4 (C3),
106.8 (C2’, C6’) , 111.1 (C8), 120.1(C5), 121.8 (C7), 122.7 (C6), 122.9 (C2), 124.8 (C4’’), 125.2 (C4), 127.7 (C2’’, C6’’), 128.3 (C1’), 130.8 (C3’’, C5’’), 136.40 (C9), 140.3 (C1’’), 141.9
(C4’), 142.8 (C=N), 150.6 (C3’, C5’) , 174.9 (C=S).
Anal. Calcd for C25H24N4O3S: C, 65.20; H, 5.25; N, 12.17; Found C, 65.39; H, 5.08; N, 12.32.
(E)‐1‐((2‐(4‐Methoxyphenyl)‐1H‐indol‐3‐yl)methylene)‐4‐phenylthiosemicarbazide (8d)
Cream Powder, Mp: 210‐211 C; Yield: 81%. IR (KBr, cm‐1): 3347, 3202 (N‐H stretching), 3180 (Aromatic C‐H stretching), 1612 (C=N), 1542 (C=C), 1283 (C‐N), 1242
(C=S), 1176 (C‐O). 1H NMR (500M Hz, DMSO‐d6) (, ppm): 3.85 (s, 3H, OCH3), 7.14‐7.20 (m, 5H, H3',5',3'',5'',4''), 7.37 (t, J = 7.7 Hz, 2H, H6,7), 7.42 (d, J = 7.7 Hz, 1H, H8),
7.60 (d, J = 8.3 Hz, 2H, H2',6'), 7.65 (d, J = 8.3 Hz, 2H, H2'',6''), 8.33 (d, J = 7.7 Hz, 1H, H5), 8.56 (s, 1H, H‐C=N), 9.55 (s, 1H, NH‐Ph), 11.57 (s, 1H, NH‐C=S), 11.87 (s, 1H,
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Chemistry & Biodiversity
NH‐indole). 13C NMR (125 MHz, DMSO‐d6) (, ppm): 55.41(OCH3), 106.7(C3), 108.9 (C3’, C5’) , 110.9 (C8), 114.4 (C5), 121.0 (C7), 122.4 (C6), 122.9 (C2), 123.2 (C4’’),
125.0 (C1’), 125.3 (C4), 128.2 (C2’’, C6’’), 130.8 (C2’, C6’), 136.4 (C3’’, C5’’), 139.3 (C9), 142.2 (C1’’), 143.0 (C=N), 159.9 (C4’), 174.5 (C=S). ESI‐MS (m/z): 401.18 ([M + H]+,
C23H20N4OS; calc. 401.14). Anal. Calcd. For C23H20N4OS: C, 68.98; H, 5.03; N, 13.99; Found C, 69.10; H, 5.19; N, 14.05.
(E)‐1‐((2‐(4‐Chlorophenyl)‐1H‐indol‐3‐yl)methylene)‐4‐phenylthiosemicarbazide (8e)
Cream Powder, Mp: 229‐231 C, Yield: 83%. IR (KBr, cm‐1): 3423, 3388 (N‐H stretching), 3161 (Aromatic C‐H stretching), 1598 (C=N), 1551 (C=C), 1272 (C‐N), 1237
(C=S). 1H NMR (500M Hz, DMSO‐d6) (, ppm): 7.17‐7.20 (m, 2H, H6,7), 7.25 (t, J = 7.7 Hz, 1H, H4''), 7.38 (t, J = 7.7 Hz, 2H, H3'',5''), 7.45 (d, J = 7.9 Hz, 1H, H8), 7.64‐7.69
(m, 6H, H2',6',3',5',2'',6''), 8.37 (d, J = 7.9 Hz, 1H, H5), 8.55 (s, 1H, H‐C=N), 9.57 (s, 1H, NH‐Ph), 11.56 (s, 1H, NH‐C=S), 12.03 (s, 1H, NH‐indole). 13C NMR (125 MHz,
DMSO‐d6) (, ppm): 107.8 (C3), 111.6 (C8), 121.2 (C5), 122.7 (C7), 123.4 (C6), 125.0 (C2), 125.1 (C4’’), 128.2 (C4), 129.0 (C2’’, C6’’), 129.7 (C2’, C6’), 131.1 (C3’’, C5’’), 133.7
(C3’, C5’), 134.1 (C1’), 136.6 (C4’), 139.3 (C9) , 141.2 (C1’’) , 141.6 (C=N), 174.4 (C=S). Anal. Calcd. for C22H17ClN4S: C, 65.26; H, 4.23; N, 13.84; Found: C, 65.03; H, 4.10;
N, 13.92.
(E)‐1‐((2‐(4‐Bromophenyl)‐1H‐indol‐3‐yl)methylene)‐4‐phenylthiosemicarbazide (8f)
Light green Powder, Mp: 232‐235 C; Yield: 82%. IR (KBr, cm‐1): 3433, 3374, 3313 (N‐H stretching), 3141 (Aromatic C‐H stretching), 1596 (C=N), 1548 (C=C), 1267
(C‐N), 1196 (C=S). 1H NMR (500 MHz, DMSO‐d6) (, ppm): 7.17‐7.20 (m, 2H, H6,7), 7.26 (t, J = 7.8 Hz, 1H, H4''), 7.38 (t, J = 7.8 Hz, 2H, H3'',5''), 7.45 (d, J = 7.9 Hz, 1H,
H8), 7.60‐7.65 (m, 4H, H3',5',2',6'), 7.79 (d, J = 7.8 Hz, 2H, H2'',6''), 8.37 (d, J =7.9 Hz, 1H, H5), 8.55 (s, 1H, H‐C=N), 9.58 (s, 1H, NH‐Ph), 11.56 (s, 1H, NH‐C=S), 12.04
(s, 1H, NH‐indole). 13C NMR (125 MHz, DMSO‐d6) (, ppm): 107.8 (C3), 111.3 (C8), 121.3 (C5), 122.5 (C7), 122.7 (C6), 123.4 (C2), 125.0 (C4’’), 125.1 (C4), 128.2 (C2’’, C6’’),
130.1 (C2’, C6’), 131.2 (C3’’, C5’’), 131.9 (C3’, C5’), 136.6 (C1’), 139.2 (C4’), 139.3 (C9), 141.4 (C1’’), 141.6 (C=N), 174.4 (C=S). Anal. Calcd. for C22H17BrN4S: C, 58.80; H, 3.81;
N, 12.47; Found C, 58.95; H, 3.90; N, 12.59.
(E)‐4‐Phenyl‐1‐((2‐p‐tolyl‐1H‐indol‐3‐yl)methylene)thiosemicarbazide (8g)
Light yellow powder, Mp: 212‐215bC; Yield: 78%. IR (KBr, cm‐1): 3399, 3300, 3235 (N‐H stretching), 3149 (Aromatic C‐H stretching), 1592 (C=N), 1553 (C=C), 1269
(C‐N), 1198 (C=S).1H NMR (500 MHz, DMSO‐d6) (, ppm): 2.41 (s, 3H, CH3), 7.15‐7.25 (m, 3H, H6,7,4''), 7.36‐7.40 (m, 4H, H3',5',3'',5''), 7.44 (d, J = 7.7 Hz, 1H, H8), 7.55
(d, J = 7.8 Hz, 2H, H2',6'), 7.65 (d, J = 7.7 Hz, 2H, H2'',6''), 8.34 (d, J =7.7 Hz, 1H, H5), 8.58 (s, 1H, H‐C=N), 9.55 (s, 1H, NH‐Ph), 11.57 (s, 1H, NH‐C=S), 11.92
(s, 1H, NH‐indole). 13C NMR (125. MHz, DMSO‐d6) (, ppm): 21.1 (CH3), 107.1 (C3), 111.5 (C8) , 121.1 (C5), 122.5 (C7), 123.0 (C6), 124.8 (C2), 124.9 (C4’’), 125.2 (C4),
128.0 (C2’’, C6’’), 128.2 (C2’, C6’), 129.2 (C3’’, C5’’), 129.5 (C3’, C5’), 136.5 (C1’), 138.6 (C1’’), 139.3 (C4’), 142.1 (C9), 142.9 (C=N), 174.3 (C=S). ESI‐MS (m/z): 401.15 ([M + H]+,
C23H20N4S; calc. 401.14). Anal. Calcd. for C23H20N4S: C, 71.85; H, 5.24; N, 14.57; Found C, 72.00; H, 5.16; N, 14.70.
(E)‐4‐(4‐Methoxyphenyl)‐1‐((2‐phenyl‐1H‐indol‐3‐yl)methylene)thiosemicarbazide (8h)
Yellow powder, Mp: 209‐211 C; Yield: 76%. IR (KBr, cm‐1): 3347 (N‐H stretching), 3143 (Aromatic C‐H stretching), 1601 (C=N), 1549 (C=C), 1275 (C‐N), 1242 (C=S)
1176 (C‐O). 1H NMR (500 MHz, DMSO‐d6) (, ppm): 3.77 (s, 3H, CH3), 6.94 (d, J = 7.0 Hz, 2H, H3'',5''), 7.17 (t, J = 7.3 Hz, 1H, H6), 7.25 (t, J = 7.3 Hz, 1H, H7), 7.44‐7.54
(m, 4H, H2'',6'',8,4'), 7.59 (t, J = 7.3 Hz, 2H, H3',5'), 7.66 (d, J = 7.3 Hz, 2H, H2',6'), 8.38 (d, J = 7.3 Hz, 1H, H5), 8.58 (s, 1H, H‐C=N), 9.42 (s, 1H, NH‐Ph), 11.48 (s, 1H, NH‐
C=S), 11.94 (s, 1H, NH‐indole). 13C NMR (125 MHz, DMSO‐d6) (, ppm): 55.4 (OCH3), 107.3 (C3), 111.3 (C8), 113.3 (C3’’, C5’’), 121.0 (C5), 122.6 (C7), 123.0 (C6), 125.1
(C2), 127.0 (C4), 128.8 (C2’’, C6’’), 129.3 (C2’, C6’), 130.9 (C3’, C5’), 132.2 (C4’), 136.3 (C1’’), 136.4 (C1’), 141.7 (C9), 142.5 (C=N), 156.7 (C4’’), 174.7 (C=S). Anal. Calcd. For
C23H20N4OS: C, 68.98; H, 5.03; N, 13.99; Found: C, 68.85; H, 4.95; N, 13.91.
(E)‐1‐((2‐(4‐Hydroxyphenyl)‐1H‐indol‐3‐yl)methylene)‐4‐(4‐methoxyphenyl) thiosemicarbazide (8i)
White powder, Mp: 231‐233 C; Yield: 78%. IR (KBr, cm‐1): 3393 (OH), 3309 (N‐H stretching), 3175 (Aromatic C‐H stretching), 1609 (C=N), 1543 (C=C), 1274 (C‐N),
1199 (C=S), 1171 (C‐O). 1H NMR (500 MHz, DMSO‐d6) (, ppm): 3.76 (s, 3H, OCH3), 6.92‐6.96 (m, 4H, H3',5',3'',5''), 7.13 (t, 1H, J = 7.3 Hz, H6), 7.19 (t, J = 7.3 Hz, 1H, H7),
7.39 (d, J = 7.3 Hz, 1H, H8), 7.45‐7.48 (m, 4H, H2',6',2'',6''), 8.33 (d, J = 7.3 Hz, 1H, H5), 8.53 (s, 1H, H‐C=N), 9.41 (s, 1H, NH‐Ph), 9.93 (s, 1H, OH), 11.46 (s, 1H, NH‐C=S),
11.78 (s, 1H, NH‐indole). 13C NMR (125 MHz, DMSO‐d6) (, ppm): 55.5 (OCH3), 106.4 (C3), 111.3 (C8), 113.3 (C3’’, C5’’), 115.7 (C3’, C5’), 120.9 (C5), 121.7 (C7), 122.4 (C6),
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Chemistry & Biodiversity
122.8 (C2), 125.2 (C4), 127.2 (C1’), 130.9 (C2’’, C6’’), 132.3 (C2’, C6’), 136.3 (C1’’), 142.3 (C9) , 143.6 (C=N), 156.9 (C4’’), 158.4 (C4’), 174.8 (C=S). Anal. Calcd. For C23H20N4O2S:
C, 66.33; H, 4.84; N, 13.45; Found: C, 66.41; H, 4.92; N, 13.51.
(E)‐4‐(4‐Methoxyphenyl)‐1‐((2‐(3,4,5‐trimethoxyphenyl)‐1H‐indol‐3‐yl)methylene) thiosemicarbazide (8j)
Pale yellow powder, Mp: 201‐203 C; Yield: 79%. IR (KBr, cm‐1): 3452, 3326 (N‐H stretching), 3136 (Aromatic C‐H stretching), 1606 (C=N), 1535 (C=C), 1261 (C‐N),
1196 (C=S), 1172 (C‐O). 1H NMR (500 MHz, DMSO‐d6) (, ppm): 3.81 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 6.78
(d, J = 8.4 Hz, 2H, H3'',5''), 6.90 (s, 1H, H2'), 7.17 (t, J = 8.9 Hz, 1H, H6), 7.27‐7.32 (m, 3H, H2'',6'',7), 7.39 (s,1H, H6'), 7.52 (d, J = 8.9 Hz , 1H, H8), 8.41(d, 1H, J = 8.9 Hz, H5),
8.97 (s, 1H, H‐C=N), 9.54 (s, 1H, NH‐Ph), 11.47 (s, 1H, NH‐C=S), 12.03 (s, 1H, NH‐indole). 13C NMR (125 MHz, DMSO‐d6) (, ppm): 54.2 (C4’’‐OCH3), 54.7
(C3’‐OCH3 , C5’‐OCH3), 55.5 (C4’‐OCH3), 105.0 (C3), 106.9 (C2’, C6’), 110.8 (C8), 120.2 (C3’’, C5’’), 121.9 (C5), 124.0 (C7), 124.9 (C6), 126.2 (C2), 126.9 (C4), 128.6 (C1’), 128.8
(C2’’, C6’’), 130.9 (C1’’), 131.5 (C9), 138.6 (C4’), 141.9 (C=N), 143.1 (C3’, C5’), 153.6 (C4’’), 174.2 (C=S). Anal. Calcd for C26H26N4O4S: C, 63.66; H, 5.34; N, 11.42; Found
C, 63.47; H, 5.22; N, 11.38.
(E)‐4‐(4‐Methoxyphenyl)‐1‐((2‐(4‐methoxyphenyl)‐1H‐indol‐3‐yl)methylene)thiosemicarbazide (8k)
Cream powder, Mp: 200‐202 C, Yield: 77%. IR (KBr, cm‐1): 3339 (N‐H stretching), 3130 (Aromatic C‐H stretching), 1601 (C=N), 1549 (C=C), 1275 (C‐N), 1237 (C=S),
1
1171 (C‐O). H NMR (500 MHz, DMSO‐d6) (, ppm): 3.77 (s, 3H, CH3), 4.03 (s, 3H, OCH3), 6.94 (d, J = 8.4 Hz, 2H, H3'',5''), 7.17 (t, J = 7.4, 1H, H6), 7.25
(t, J = 7.4 Hz, 1H, H7), 7.45‐7.47 (m, 3H, H3',5',8), 7.65‐7.69 (m, 4H, H2',6',2'',6''), 8.39 (d, J = 7.4 Hz, 1H, H5), 8.54 (s, 1H, H‐C=N), 9.44 (s, 1H, NH‐Ph), 11.46 (s, 1H, NH‐
C=S), 12.01 (s, 1H, NH‐indole). 13C NMR (DMSO‐d6) (, ppm): 55.2 (C4’‐OCH3), 55.4 (C4’’‐OCH3), 106.5 (C3), 111.3 (C8), 113.2 (C3’’, C5’’), 114.2 (C3’, C5’), 123.3 (C5), 124.9
(C7), 125.4 (C6), 126.4 (C2), 127.1 (C1’), 128.9 (C4), 130.7 (C2’’, C6’’), 136.3 (C2’, C6’), 137.0 (C1’’), 142.1 (C9), 142.9 (C=N), 158.7 (C4’’), 159.9 (C4’), 174.8 (C=S). Anal. Calcd.
For C24H22N4O2S: C, 66.96; H, 5.15; N, 13.01 Found: C, 67.10; H, 5.26; N, 12.90.
(E)‐1‐((2‐(4‐Chlorophenyl)‐1H‐indol‐3‐yl)methylene)‐4‐(4‐methoxyphenyl)thiosemicarbazide (8l)
Cream powder, MP 250 C, Yield: 81%. IR (KBr, cm‐1): 3339 (N‐H stretching), 3126 (Aromatic C‐H stretching), 1601 (C=N), 1542 (C=C), 1276 (C‐N), 1233 (C=S),
1175 (C‐O). 1H NMR (500 MHz, DMSO‐d6) (, ppm): 3.81 (s, 3H, OCH3), 6.98 (d, J = 7.9 Hz, 2H, H3'',5''), 7.21 (t, J = 7.0 Hz, 1H, H6), 7.29 (t, J = 7.0 Hz, 1H, H7), 7.48‐7.51
(m, 3H, H2'',6'',8), 7.68‐7.71 (m, 4H, H2',6',3',5'), 8.42 (d, J = 7.0 Hz, 1H, H5), 8.58 (s, 1H, H‐C=N), 9.46 (s, 1H, NH‐Ph), 11.48 (s, 1H, NH‐C=S), 12.03 (s, 1H, NH‐indole).
13C NMR (125 MHZ, DMSO‐d6) (, ppm): 54.9 (OCH3), 107.8 (C3), 111.2 (C8), 113.3 (C3’’, C5’’), 121.1 (C5), 122.8 (C7), 123.3 (C6), 125.2 (C2), 127.1 (C4), 128.9 (C2’’, C6’’),
129.8 (C2’, C6’), 131.0 (C3’, C5’), 132.2 (C1’’), 133.8 (C1’), 136.5 (C4’), 141.0 ( C9), 141.3 (C=N), 156.8 (C4’’), 174.9 (C=S). Anal. Calcd. for C23H19ClN4OS: C, 63.51; H, 4.40;
N, 12.88; Found: C, 63.45; H, 4.35; N, 12.75.
(E)‐1‐((2‐(4‐Bromophenyl)‐1H‐indol‐3‐yl)methylene)‐4‐(4‐methoxyphenyl)thiosemicarbazide (8m)
Pale yellow powder, Mp: 220‐222 C; Yield: 83%. IR (KBr, cm‐1): 3337 (N‐H stretching), 3128 (Aromatic C‐H stretching), 1600 (C=N), 1540 (C=C), 1272 (C‐N), 1234
(C=S), 1179 (C‐O). 1H NMR (500 MHz, DMSO‐d6) (, ppm): 3.77 (s, 3H, OCH3), 6.94 (d, J = 8.6 Hz, 2H, H3'',5''), 7.17 (t, J = 7.6 Hz, 1H, H6), 7.25 (t, J = 7.6 Hz, 1H, H7),
7.44‐7.47 (m, 3H, H2'',6'',8), 7.60 (d, J = 8.2 Hz, 2H, H3',5'), 7.79 (d, J = 8.2 Hz, 2H, H2',6'), 8.39 (d, J = 7.6 Hz, 1H, H5), 8.54 (s, 1H, H‐C=N), 9.44 (s, 1H, NH‐Ph), 11.46
(s, 1H, NH‐C=S), 12.02 (s, 1H, NH‐indole). 13C NMR (125 MHZ, DMSO‐d6) (, ppm): 55.2 (OCH3), 107.7 (C3), 111.6 (C8), 113.3 (C3’’, C5’’), 121.2 (C5), 122.4 (C7), 122.8
(C6), 123.4 (C2), 125.1 (C4), 127.2 (C2’’, C6’’), 130.1 (C2’, C6’), 131.3 (C3’, C5’), 131.9 (C1’’), 132.3 (C1’), 136.6 (C4’), 141.2 (C9), 141.4 (C=N), 156.8 (C4’’), 174.9 (C=S).
ESI‐MS (m/z): 480.08 ([M + H]+, C23H19BrN4OS; calc. 480.05). Anal. Calcd. for C23H19BrN4OS: C, 57.62; H, 3.99; N, 11.69 Found: C, 57.45; H, 4.20; N, 11.51.
(E)‐4‐(4‐Methoxyphenyl)‐1‐((2‐p‐tolyl‐1H‐indol‐3‐yl)methylene)thiosemicarbazide (8n)
Cream powder, Mp: 233‐234 C, Yield: 75%. IR (KBr, cm‐1): 3344 (N‐H stretching), 3130 (Aromatic C‐H stretching), 1602 (C=N), 1543 (C=C), 1276 (C‐N), 1233 (C=S),
1173 (C‐O). 1H NMR (500 MHz, DMSO‐d6): (, ppm) 2.41 (s, 3H, CH3), 3.77 (s, 3H, OCH3), 6.94 (d, J = 7.4 Hz, 2H, H3'',5''), 7.16 (t, J = 7.1 Hz, 1H, H6),
7.23 (t, J = 7.1 Hz, 1H, H7), 7.39‐7.44 (m, 3H, H3',5',8), 7.48 (d, J = 7.4 Hz, 2H, H2'',6''), 7.55 (d, J = 7.1 Hz, 2H, H2',6'), 8.35 (d, J = 7.1 Hz, 1H, H5), 8.57 (s, 1H, H‐C=N), 9.40
(s, 1H, NH‐Ph), 11.46 (s, 1H, NH‐C=S), 11.87 (s, 1H, NH‐indole). 13C NMR (125 MHZ, DMSO‐d6) (, ppm): 20.6 (CH3), 55.4 (OCH3), 107.1 (C3), 111.5 (C8), 113.3
16
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Chemistry & Biodiversity
(C3’’, C5’’), 120.9 (C5), 122.9 (C7), 125.2 (C6), 126.8 (C2), 128.1 (C4), 129.1 (C2’’, C6’’), 129.4 (C2’, C6’), 131.0 (C3’, C5’), 132.2 (C1’’), 136.3 (C1’), 138.4 (C4’), 141.8 (C9), 142.6
(C=N), 156.7 (C4’’),174.7 (C=S). Anal. Calcd. for C24H22N4OS: C, 69.54; H, 5.35; N, 13.52; Found: C, 69.65; H, 5.43; N, 13.50.
Biology
In vitro cytotoxicity assay
The cytotoxic activity of the synthesized compounds 8a‐n on cancer cell viability was assessed using MTT (3‐(4,5‐ dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium
bromide) assay against three human different cancer cell lines. [33, 36] 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 RPMI medium supplemented with 10% FBS, 1% L‐Glutamine, and Penicilline‐Streptomycin and were
incubated overnight. Then cells were treated by different concentrations of test compounds for 48 h at 37 °C in a humidified atmosphere with 5% CO2. The final
concentration of DMSO, as compounds’ solvent, in the highest concentration of the applied compounds was 1%. Etoposide and colchicine was used as positive
control in this experiment. After 48 h incubation, the culture medium was replaced with MTT (1 mg/mL) and plates were incubated for further 4h. Then, the
supernatant was removed and the formazan crystals were dissolved using 100 µL DMSO. The absorbance of each well was measured using a microplate reader
(Gen5, Epoch, BioTek, America) at 492 nm wavelengths. The data were expressed in IC50 as the mean±SD from the dose‐response logarithmic curves of at least
three independent experiments.
Morphological evaluation by fluorescence microscopy
Apoptosis was determined morphologically by staining with acridine orange/ethidium bromide and using fluorescence microscopy. [33] A‐549 and Hep‐G2 cells
were grown in 6‐well plates (5×105 cells/well) and incubated overnight at 37 °C in a humidified atmosphere. A‐549 Cells were treated with and without IC50
concentration of compound 8l and Hep‐G2 were treated with 8d and 8l for 24 h. After treatment, cells were harvested and washed 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 fluorescence
microscopy (Zeiss, Germany).
Flow cytometry analysis of the apoptotic cells with Annexin V‐FITC/PI staining
The annexin V apoptosis detection kit FITC/PI (BD Pharmingen™) was used to allow identifying apoptotic and necrotic cells as per manufacturer’s instructions.
In brief, 5×105 cells/well of A‐549 cells was treated with IC50 dose of most potent compounds, 8l, and etoposide as a reference drug. After 24h incubation, the
cells were harvested and washed twice with cold PBS. The cell suspension was centrifuged, and resuspended in 1x annexin V binding buffer (0.1 M Hepes/NaOH
(pH 7.4), 1.4 M NaCl, 25 mM CaCl2). Then 5 µl of FITC Annexin V and 5 µl PI were added and the cells were gently mixed with a vortex and incubated at room
temperature for 15 minutes in the dark. Finally, 400 μL of 1x annexin binding buffer was added into the suspension and cells were analyzed using a flow cytometer
within 1h.
Calculation of logP
The cLogP values for the synthesized series 8 compounds as well as etoposide and colchicine were calculated using Molinspiration webME Editor 1.16, [49] in
order to predict their lipophilicity (Table 1). The accuracy of the data obtained from this server is reliable. For etoposide the calculated logP was 0.7, quite close
to the experimental value of 0.6 and for colchicine the calculated logP was 1.10, close to the experimental value of 1.30. [50]
Molecular docking modeling
In order to understand the inhibition and binding modes of the synthesized compounds with tubulin and ATPase domain of topoisomerase IIα binding sites, in
silico docking studies were performed.
Ligand structure preparation
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Chemistry & Biodiversity
All the molecular structures were sketched in ChemDraw ultra 8.0 program, and for minimization, they were transferred into the Discovery Studio 4.1 (DS4.1).
The compounds were then typed with a CHARMm force field, and the partial charges were calculated by the Momany–Rone option. [48] A smart minimizer
algorithm was used to minimize the resulting structures, which performs 1000 steps of the steepest descent with a RMS gradient tolerance of three followed by
a conjugate gradient minimization.
Protein structure preparation
[51]
[52]
Two protein structures obtained from X‐ray diffraction experiment were extracted from protein data bank,
PDB ID: 1SA0 for tubulin,
and 1ZXM for
topoisomerase II. [28] All protein preparations and minimizations were performed using the tools and protocols in DS4.1 (AccInc, San Diego, CA, USA).
The region of interest used for GOLD docking was defined as 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. In the structure of topoisomerase IIα, there were
two identical subunits each containing AMP‐PNP (ANP) and Mg2+ at the ATPase domain and chain A was selected for the docking study. All bound water
molecules and ligands were removed from the proteins, Non‐polar hydrogens of compounds were merged and polar hydrogen atoms were added.
GOLD allows for partial protein flexibility in all dockings. Specifically GOLD rotates the torsion angles of serine, threonine and tyrosine hydroxyl
groups during docking in order to optimise hydrogen bonding interactions of these residues with the ligand. The positions of lysine NH3+ groups are similarly
optimised. According to this protocol the residues inside the selected active site of tubulin which considered flexible are Thr 314, , Lys 352, Thr 353, Ser 178, Lys
254, Thr 179. Furthermore, the flexible residues of ATPase domain of topoisomerase II consist of the residues allowed for changing of rotatble bonds by the
GOLD protocol are Thr 215, Lys 168, Ser 148, Ser 149, Tyr 151, Lys 157, Thr 159, Lys 378, Tyr 165. The fitness function was set to the GOLD Score fitness function
with the default input and annealing parameters. The best docking poses were selected based on the gold fitness scores and the critical interactions reported in
the literatures. Gold Score ‘‘Allow early termination” option was employed to allow maximal exploration of ligand conformation. When the top three solutions
attained root mean square deviation (RMSD) values within 1.5 Å, docking was terminated. The annealing parameters of the van der Waals and hydrogen bonding
interactions were 6.0 Å and 3.0 Å, respectively. We used 10 genetic algorithm (GA) docking runs with internal energy offset. All other parameters accepted the
default settings. Gold flexible ligand docking generated 10 poses of each ligand, which were ranked using the Gold score scoring function. The top ranked pose
with highest Gold Score fitness was analyzed using Accelrys Discovery studio to reveal the hydrogen bond interaction and binding mode within the binding
domain.
Supplementary Material
Supporting information for this article is available on the on the WWW under http://dx.doi.org/10.1002/cbdv.2018xxxxx’.
Acknowledgment
This work was supported by grants from Isfahan university of Medical Sciences and Tehran University of Medical Sciences.
Conflict of interest
There is no Conflict of interest by others
Author Contribution Statement
Zohreh Bakherad designed, synthesized, and characterized compounds. Maliheh Safavi and Hojjat Sadeghi‐Aliabadi supervised biological tests. Hossein
Rastegar contributed reagents/materials/analysis tools. Mohammad Bakherad contributed to the synthesis and characterization of compounds. Afshin Fassihi
contributed to synthesis compounds and edited manuscript. Jahan B Ghasemi supervised docking study. Mina Saeedi contributed to the preparation of
manuscript. Lotfollah Saghaie and Mohammad Mahdavi supervised the project.
18
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10.1002/cbdv.201800470
Chemistry & Biodiversity
References
[1]
[2]
[3]
[4]
[5]
[6]
X. Ma, H. Yu ,‘Global Burden of Cancer’, Yale J. Biol. Med. 2006, 79, 85‐9.
American Cancer Society, ‘Breast Cancer Facts & Figures 2017‐2018’, Atlanta: American Cancer Society, Inc. 2017.
R. L. Siegel, D. K. Miller, A. Jemal, ‘Cancer statistics’, Ca‐ Cancer J. Clin. 2017, 67, 7–30.
A. Jemal, R. Siegel, E. Ward, T. Murray, J. Xu, M.J. Thun, ‘Cancer statistics’, Ca‐ Cancer J. Clin. 2007 , 57, 43‐66.
J.B. Gibbs, ‘Mechanism‐based target identification and drug discovery in cancer research’ , Science 2000, 287, 1969‐1971.
N. Bailon‐Moscoso, J.C. Romero‐Benavides, P. Ostrosky‐Wegman, ‘Development of anticancer drugs based on the hallmarks of tumor cells’ ,Tumor
Biol. 2014 , 35, 3981‐3995.
[7]
A. Rana, J. M. Alex, M. Chauhan, G. Joshi, R. Kumar, ‘A review on pharmacophoric designs of antiproliferative agents’, Med. Chem. Res. 2015, 24, 903‐
920.
[8]
Y. A. Fouad, C. Aanei, ‘Revisiting the hallmarks of cancer’, Am. J. Cancer Res. 2017, 7(5), 1016‐1036.
[9]
L. H. Hurley, ‘DNA and its associated processes as targets for cancer therapy’, Nat. Rev. Cancer 2002, 2, 188–200.
R. J. Vuuren, M. H. Visagie, A. E. Theron, A. M. Joubert, ‘Antimitotic drugs in the treatment of cancer’, Cancer Chemother. Pharmacol. 2015, 76, 1101–
1112.
[10]
[11]
[12]
[13]
M. Kavallaris, ‘Microtubules and resistance to tubulin binding agents’, Nat. Rev. Cancer 2010, 10, 194–204.
C. Dumontet, M. A. Jordan,' Microtubule‐binding agents: a dynamic field of cancer therapeutics’, Nat. Rev. Drug Discovery 2010, 9(10), 790‐803.
A. Ozdemir, M. D. Altıntop, G. Turan‐Zitouni, G. A. Çiftçi, I. Ertorun, O. Alatas, Z. A. Kaplancıklı, ‘Synthesis and evaluation of new indole‐based
chalcones as potential antiinflammatory agents’, Eur. J. Med. Chem. 2015, 89, 304‐309.
[14]
A. V. Bogdanov, I. F. Zaripova, A. D. Voloshina, A. S. Sapunova, N. V. Kulik, J. K. Voronina, V. F. Mironov, ‘Synthesis and antimicrobial study of novel
1‐1‐benzylated water soluble isatin‐3‐hydrazones’, Chem. Biodiversity 2018, 15, 1800088.
[15]
[16]
M. Z. Zhang, Q. Chen, G. F. Yang, ‘A review on recent developments of indole‐containing antiviral agents’, Eur. J. Med. Chem. 2015, 89, 421‐441.
N. T. Tzvetkov, S. Hinz, P. Küppers, M. Gastreich, C. E Müller, ‘Indazole‐ and Indole‐5‐carboxamides: selective and reversible monoamine oxidase B
Inhibitors with subnanomolar potency’, J. Med. Chem. 2014, 57(15), 6679‐6703.
[17]
[18]
S. Kumar, S. Mehndiratta, K. Nepali, M. K. Gupta, S. Koul, P. R. Sharma, A. K. Saxena, K. L. Dhar, ‘Novel indole‐bearing combretastatin analogues as
tubulin polymerization inhibitors’, Org. Med. Chem. Lett. 2013, 3, 3.
G. L. Regina, R. Bai, A. Coluccia, V. Famiglini, S. Pelliccia, S. Passacantilli, C. Mazzoccoli, V. Ruggieri, A. Verrico, A. Miele, L. Monti, M. Nalli, R. Alfonsi,
L, D. Marcotullio, A. Gulino, B. Ricci, A. Soriani, ‘New indole tubulin assembly inhibitors cause stable arrest of mitotic progression, enhanced
stimulation of natural killer cell cytotoxic activity and repression of hedgehog‐dependent cancer’, J. Med. Chem. 2015, 58 (15), 5789–580.
G. D. Martino, M. C. Edler, G. L. Regina, A. Coluccia, M. C. Barbera, D. Barrow, R. I. Nicholson, G. Chiosis, A. Brancale, E. Hamel, M. Artico, R. Silvestri,
‘New Arylthioindoles: Potent inhibitors of tubulin polymerization. 2. Structure‐Activity Relationships and molecular modeling studies’, J. Med. Chem.
2006, 49, 947‐954.
[19]
[20]
[21]
[22]
S. N. Baytas, N. Inceler, A. Yılmaz, A. Olgac, E. Banoglu, E. Hamel, R. Bortolozzi, G. Viola, S. Menevse,’ Synthesis, biological evaluation and molecular
docking studies of trans‐indole‐3‐acrylamide derivatives, a new class of tubulin polymerization inhibitors’, Bioorg. Med. Chem. 2014, 22, 3096–3104.
Po. R. Kamath, D. Sunil, A. A. Ajees, K. S. R. Pai, S. Das, ’Some new indole–coumarin hybrids; Synthesis, anticancer and Bcl‐2 docking studies’, Bioorg.
Chem. 2015, 63, 101–109.
J. S. Sidhu, R. S. Mayank, V. Jaitak, ‘Indole Derivatives as Anticancer Agents for Breast Cancer Therapy: A Review’, Anti‐Cancer Agents Med. Chem.
2015, 16 (2), 160 – 173.
19
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201800470
Chemistry & Biodiversity
[23]
[24]
M. T. El Sayed, N. A. Hamdy, D. A. Osman, K. M. Ahmed, ‘Indoles as anti‐cancer agents, Adv. Mod. Oncol. Res. 2015, 1, 20‐35.
R. Patil, S. A. Patil, K. D. Beaman, S. A. Patil, ‘Indole molecules as inhibitors of tubulin polymerization: potential new anticancer agents, an update
(2013–2015)’, Future Med. Chem. 2016, 8(11), 1291‐1316.
[25]
[26]
[27]
S. A. Patil, R. Patil, D. D. Miller, ‘Indole molecules as inhibitors of tubulin polymerization: potential new anticancer agents’, Future Med. Chem. 2012,
4(16), 2085–2115.
R. Gastpar, M. Goldbrunner, D. Marko, E. von Angerer, ‘Methoxy‐substituted 3‐formyl‐2phenylindoles inhibit tubulin polymerization’, J. Med. Chem.
1998, 41, 4965–4972.
J. F. de Oliveira, T. S. Lima, D. B. Vendramini‐Costa, S. C. B. de Lacerda Pedrosa, E. A. Lafayette, R. M. F. da Silva, S. M. V. de Almeida, R. O. de Moura,
A. L. T. G. Ruiz, J. E. de Carvalho, M. C. A. de Lima, ‘Thiosemicarbazones and 4‐thiazolidinones indole‐based derivatives: Synthesis, evaluation of
antiproliferative activity, cell death mechanisms and topoisomerase inhibition assay’, Eur. J. Med. Chem. 2017, 136, 305‐314.
H. Huang, Q. Chen, X. Ku, L. Meng, L. Lin, X. Wang, C. Zhu, Y. Wang, Z. Chen, M. Li, H. Jiang, K. Chen, J. Ding, H. Liu, ‘A series of α‐heterocyclic
carboxaldehyde thiosemicarbazones inhibit topoisomerase IIα catalytic activity’, J. Med. Chem. 2010, 53, 3048‐3064.
M. D. Hall, N. K. Salam, J. L. Hellawell, H. M. Fales, C. B. Kensler, J. A. Ludwig, G. Szaka´cs, D. E. Hibbs, M. M. Gottesman, ‘Synthesis, activity, and
pharmacophore development for isatin‐β‐thiosemicarbazones with selective activity toward multidrug‐resistant cells’, J. Med. Chem. 2009, 52, 3191‐
3204.
[28]
[29]
[30]
[31]
[32]
[33]
A. H. Mouayed, A. I. Muhammad, M. Asif, R. A. Haque, M. B. Khadeer Ahamed, A. M. S. Abdul Majid, T. S. Guan, ‘Asynthesis, crystal structures and
in vitro anticancer studies of new thiosemicarbazone derivatives’, Phosphorus, Sulfur Silicon Relat. Elem. 2015, 190, 1498‐1508.
B. Hu, B. Wang, B. Zhao, Q. Guo, Z. H. Li, X. H. Zhang, G. Y. Liu, Y. Liu, Y. Tang, F. Luo, Y. Du, Y. X. Chen, L. Y. Ma, H. M. Liu, ‘Thiosemicarbazone‐
based selective proliferation inactivators inhibit gastric cancer cell growth, invasion, and migration’, Med. Chem. Comm. 2017, 2, 30‐38.
Ş. Güniz Küçükgüzel, G. P. Coşkun, ‘Macromolecular drug targets in cancer treatment and thiosemicarbazides as anticancer agents’, Anti‐Cancer
Agents in Medicinal Chemistry 2016, 10, 1288 – 1300.
M. Safavi, A. Ashtari, F. Khalili, S. S. Mirfazli, M. Saeedi, S. Kabodian‐ Ardestani, P. R. Ranjbar, M. B. Tehrani, B. Larijani, M. Mahdavi, ‘Novel
quinazolin‐4(3H)‐one linked to1,2,3‐triazoles: Synthesis and anticancer activity Running title: Quinasoline as anti cancer agent’, Chem. Biol. Drug Des.
2018, https://doi.org/10.1111/cbdd.13203.
[34]
[35]
M. Mahdavi, K. Pedrood, M. Safavi, M. Saeedi, M. Pordeli, S. K. Ardestani, S. Emami, M. Adib, A. Foroumadi, A. Shafiee, ‘Synthesis and anticancer
activity of N‐substituted 2‐arylquinazolinones bearing trans‐stilbene scaffold’, Eur. J. Med. Chem. 2015, 95, 492‐499.
M. Mahdavi, S. Dianat, B. S. Khavari Moghimi, M. Abdollahi, M. Safavi, A. Mouradzadegun, S. Kabodian‐Ardestani, R. Sabourian, S. Emami, T.
Akbarzadeh, A. Shafiee, A. Foroumadi, ‘Synthesis and biological evaluation of novel imidazopyrimidin‐3‐amines as anticancer agents’, Chem. Biol.
Drug Des. 2016, 89(5), 797‐805.
[36]
[37]
M. Mohammadi‐Khanaposhtani, M. Safavi, R. Sabourian, M. Mahdavi, M. Pordeli, M. Saeedi, S. Kabodian‐Ardestani, A. Foroumadi, A. Shafiee, T.
Akbarzadeh, ‘Design, synthesis, in vitro cytotoxic activity evaluation, and apoptosis‐induction study of new 9 (10H)‐acridinone‐1, 2, 3‐triazoles’, Mol.
Diversity 2015, 19, 787‐795.
M. Mohammadi‐Khanaposhtani , M. Mahdavi , M. Saeedi, R. Sabourian, S. Maliheh, M. Khanavi, A. Foroumadi, A. Shafiee, T. Akbarzadeh, ‘Design
synthesis, biological evaluation, and docking study of acetylcholinesterase inhibitors: new acridone‐1,2,4‐oxadiazole‐1,2,3‐triazole hybrids’, Chem.
Biol. Drug Des 2015, 86, 1425–1432.
[38]
[39]
[40]
C. Karaaslan, H. Kadri, T. Coban, S. Suzen, ‘ Synthesis and antioxidant properties of substituted 2‐phenyl‐1H‐indoles’, Bioorg. Med. Chem. Lett.
2013, 23, 2671‐2674.
D. Kaufmann, M. Pojarova, S. Vogel, R. Liebl, R. Gastpar, D. Gross, T. Nishino, T. P. faller, E. von Angerer, ‘Antimitotic activities of 2‐phenylindole‐3‐
carbaldehydes in human breast cancer cells’, Bioorg. Med. Chem. 2007, 15, 5122‐5136.
V. Mashayekhi, K. H. M. E. Tehrani, P. Azerang, S. Sardari, F. Kobarfard, ‘Synthesis, antimycobacterial and anticancer activity of novel indole‐based
thiosemicarbazones’, Arch. Pharm. Res. 2013, https://doi.org/10.1007/s12272‐013‐0242‐z.
20
This article is protected by copyright. All rights reserved.

10.1002/cbdv.201800470
Chemistry & Biodiversity
[41]
[42]
[43]
[44]
N. Bharti, Shailendra, S. Sharma, F. Naqvi, A. Azam, ‘New palladium (II) complexes of 5‐nitrothiophene‐2‐carboxaldehyde thiosemicarbazones:
synthesis,spectral studies and in vitro anti‐amoebic activity’, Bioorg. Med. Chem. 2003, 11, 2923‐2929.
I. Antonini, F. Claudi, P. Franchetti, M. Grifantini, S. Marteli, ‘Elucidation of the structure of the antineoplastic agents, 2‐formylpyridine and formyl
isoquinoline thiosemicarbazones’, J. Med. Chem. 1977, 20, 447‐449.
R. Pingaew, S. Prachayasittikul, S. Ruchirawat, V. Prachayasittikul, ‘Synthesis and cytotoxicity of novel N‐sulfonyl‐1,2,3,4‐tetrahydroisoquinoline
thiosemicarbazone derivatives’, Med. Chem. Res. 2013, 22, 267‐277.
I. Dilovic, M. Rubcic, V. Vrdoljak, S.K. Pavelic, M. Kralj, I. Piantanida, M. Cindric, ‘Novel thiosemicarbazone derivatives as potential antitumor agents:
synthesis, physicochemical and structural properties, DNA interactions and antiproliferativeactivity’, Bioorg. Med. Chem. 2008, 16, 5189‐5198.
F. A. Alvarez Nuas, S. H. Yalkowsky, ’Correlation between log P and clogP for some steroids’, J. Pharm. Sci. 1997, 86(10), 1187‐1189.
M. L. Verdonk, J. C. Cole, M.J. Hartshorn, C. W. Murray, R. D. Taylor, ‘Improved protein ligand docking using GOLD’, Proteins 2003, 52, 609–623.
A. Dorleans, B. Gigant, R. B. Ravelli, P. Mailliet, V. Mikol, M. Knossow, ‘Variations in the colchicine‐binding domain provide insight into the structural
switch of tubulin’, Proc. Natl. Acad. Sci. USA 2009, 106(33), 13775–13779.
[45]
[46]
[47]
[48]
[49]
F.A. Momany, R. Rone, ‘Validation of the general purpose QUANTA® 3.2/CHARMm® force field’, J. Comput. Chem. 1992, 13, 888–900.
http://www.molinspiration.com/cgi‐bin/properties
[50]
[51]
[52]
C. Hansch, A. Leo, D Hoekman, 'Exploring QSAR Hydrophobic, Electronic and Steric Constants’, 1995, ACS, Wash‐ington, DC.
https://www.rcsb.org/
M. R. Yang, T. B. Liu, Y. J. Qin, C. Chen, Y. L. Zhang, B. Y. Li, H. B. Gong, B. Z. Wang, H. L. Zhu, ‘Synthesis, biological evaluation and molecular
docking studies of novel 1‐(4,5‐dihydro‐1H‐pyrazol‐1‐yl)ethanone‐containing 1‐methylindol derivatives as potential tubulin assembling inhibitors’,
RSC Advances 2016, 6(36), 30412‐30424.
21
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Graphical Illustration
Scheme 1. Reagents and conditions; (a) Isopropanol alcohol, 30 min; (b) i) CH3COOH, EtOH, reflux ii) Polyphosphoric acid, 120 C; (c) POCl3, DMF, 70 C; (d)
EtOH, CH3COOH, reflux.
Figure 1. Structures of indole‐based and thiosemicarbazone‐based anti‐cancer agents A‐E and designed compounds.
Figure 2. Acridine orange/ethidium bromide double staining of cancer cells with characteristic symptoms of apoptosis. (A) A‐549 a, DMSO 1% as control, b, c,
cells treated with IC50 concentrations of etoposide and compound 8l, respectively, for 24 h. (B) Hep‐G2 a, DMSO 1% as control, b, c, and d, cells treated with IC50
concentrations of etoposide, compounds 8d and 8l, respectively, for 24 h. White arrows indicate live cells, dash arrows show apoptosis. The images of cells were
taken with a fluorescence microscope at 400 x.
Figure 3. Flow cytometric analysis of A‐549 cells treated with the compound 8l. Cells were stained with Annexin V‐FITC/PI and quantitated by flow cytometry.
The cells were treated with a) DMSO 1% (negative control); b) IC50 values of etoposide as the positive control; c) IC50 values of the compound 8l for 24 h.
Figure 4. Docking poses of (a) colchicine and (b) ANP in the active sites of tubulin and ATPase domain of topoisomerase IIα. Green dashed lines indicate
hydrogen bonds.
Figure 5. a) Superimposition of compounds 8b (yellow) and colchicine (blue) in the tubulin binding active site b) Superimposition of compounds 8b (yellow) and
ANP (blue) in ATPase domain of topoisomerase IIα active site. Green dashed lines indicate hydrogen bonds.
Figure 6. Docking pose of compound 8d in the active site of tubulin. Green dashed lines indicate hydrogen bonds.
Figure 7. Docking poses of compound 8j in the active sites of (a) tubulin and (b) ATPase domain of topoisomerase IIα. Green dashed lines indicate hydrogen
bonds.
Figure 8. Docking poses of compound 8l (a) superimposition of 8l (green) and colchicine (yellow) in the tubulin active site (b) interaction of 8l in the ATPase
domain of topoisomerase IIα active site. Green dashed lines indicate hydrogen bonds.
22
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