In silico and in vitro antioxidant and anticancer activity profiles of urea and thiourea derivatives of 2,3-dihydro-1H-inden-1-amine

Article abstract of DOI:10.1080/10799893.2019.1710848

Synthesis of a series of new urea and thiourea compounds have been accomplished by the reaction of 2,3-dihydro-1H-inden-1-amine with various phenyl isocyanates and isothiocyanates. These compounds were evaluated for their antioxidant activity by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and nitric oxide (NO) radical scavenging assay methods including IC₅₀ values. Some of the compounds exhibited potential activity in the two tested methods. Among the series of compounds, urea derivative linked with 4-bromo phenyl ring (4b), and thiourea derivatives bonded with phenyl ring (4e), 4-fluoro phenyl ring (4f) and 4-nitro pheyl ring (4h) were found to exhibit promising anti oxidant activity with low IC₅₀ values. Where four of the title comounds exhibited higher bindig energies than the reference compound (Imatinib) in in silico molecular docking studies with Aromatase. All the synthesized compounds were characterized by IR, ¹H, ¹³C NMR and mass spectral data.

Full text of DOI:10.1080/10799893.2019.1710848

Journal of Receptors and Signal Transduction
ISSN: 1079-9893 (Print) 1532-4281 (Online) Journal homepage: https://www.tandfonline.com/loi/irst20
In silico and in vitro antioxidant and anticancer
activity profiles of urea and thiourea derivatives of
2,3-dihydro-1H-inden-1-amine
Mandala Chandrasekhar, Gandavaram Syam Prasad, Chintha
Venkataramaiah, Kollu Umapriya, Chamarthi Naga Raju, Kalluru Seshaiah &
Wudayagiri Rajendra
To cite this article: Mandala Chandrasekhar, Gandavaram Syam Prasad, Chintha
Venkataramaiah, Kollu Umapriya, Chamarthi Naga Raju, Kalluru Seshaiah & Wudayagiri Rajendra
(2020): Inꢀsilico and in vitro antioxidant and anticancer activity profiles of urea and thiourea
derivatives of 2,3-dihydro-1H-inden-1-amine, Journal of Receptors and Signal Transduction, DOI:
10.1080/10799893.2019.1710848
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JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION
https://doi.org/10.1080/10799893.2019.1710848
RESEARCH ARTICLE
In silico and in vitro antioxidant and anticancer activity profiles of urea and
thiourea derivatives of 2,3-dihydro-1H-inden-1-amine
Mandala Chandrasekhara, Gandavaram Syam Prasada, Chintha Venkataramaiahb, Kollu Umapriyaa,
Chamarthi Naga Rajua , Kalluru Seshaiaha and Wudayagiri Rajendrab
b
aDepartment of Chemistry, Sri Venkateswara University, Tirupati, India; Departments of Zoology, Sri Venkateswara University, Tirupati, India
ABSTRACT
ARTICLE HISTORY
Received 8 November 2019
Revised 20 December 2019
Accepted 23 December 2019
Synthesis of a series of new urea and thiourea compounds have been accomplished by the reaction
of 2,3-dihydro-1H-inden-1-amine with various phenyl isocyanates and isothiocyanates. These com-
pounds were evaluated for their antioxidant activity by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) rad-
ical and nitric oxide (NO) radical scavenging assay methods including IC50 values. Some of the
compounds exhibited potential activity in the two tested methods. Among the series of compounds,
urea derivative linked with 4-bromo phenyl ring (4b), and thiourea derivatives bonded with phenyl
ring (4e), 4-fluoro phenyl ring (4f) and 4-nitro pheyl ring (4h) were found to exhibit promising anti
oxidant activity with low IC50 values. Where four of the title comounds exhibited higher bindig ener-
gies than the reference compound (Imatinib) in in silico molecular docking studies with Aromatase. All
KEYWORDS
Antioxidant activity (DPPH/
NO); aromatase; inden-1H-
1-amine; molecular docking;
thiourea derivatives; urea
derivatives
1
the synthesized compounds were characterized by IR, H, 13C NMR and mass spectral data.
Introduction
inhibit chitin synthesis which is responsible for the formation
of insect cuticle, N-(1,2,4-triazol-3-yl)-N0arylthiourea acts as an
effective uncoupler of oxidative phosphorylation in mito-
chondria. Due to the important applications of urea and thio-
urea derivatives, many research groups have been interested
to develop the possibility of next generation urea and thio-
urea molecules which could be more potent as chemothera-
peutic agents.
In continuation our work [21–23] on synthesis of bioactive
urea and thiourea derivatives, new urea and thiourea deriva-
tives of 2,3-dihydro-1H-inden-1-amine 4a–j has been synthe-
sized by reacting 2,3-dihydro-1H-inden-1-amine (2) with various
substituted phenyl isocyanates 3a–d and isothiocyanates 3e–j
and evaluated their antioxidant activity. The synthesized mole-
cules were also studied for evaluation of breast cancer activity
molecular docking was done with Aromatase enzyme.
Inden is a non-heterocyclic compound and this is a fused ring
of benzene ring with penta-cyclic ring. It is well-known that
indene chemistry has a very long history due to these deriva-
tives have found in numerous biologically active molecules
[1–14]. The inden derivatives having the substitutions on
either benzene ring or on five membered ring are valuable
synthetic targets in organic and medicinal chemistry because
of their important biological activities and applications in func-
tional materials [15]. For example, Resagiline, Braziline and
Brx-019 are important inden drug candidates (Figure 1).
Inden scaffolds have found a prompt place in the carbo-
cyclic products because of the presence of these moieties in
various natural products and in many drug candidates pos-
sessing potential estrogenic bioactivity [16]. Also, these deriv-
atives have been exhibited potential biological activities such
as analgesic [17], myorelaxation, immunomodulatory proper-
ties [18], type-I diabetic activities [19] and luminescent prop-
erties. In addition, bromoindenones are important precursors
for synthesis of ninhydrin derivatives and aminoindanes are
useful building blocks for many biologically important com-
pounds such as HIV (human immunodeficiency virus) prote-
ase inhibitors and neuroprotective agents [20].
Materials and methods
Chemistry
All the solvents and chemicals were purchased from Merck
and Sigma-Aldrich respectively and were used without fur-
ther purification. Melting points of the compounds were
determined in open capillary tube on Guna melting point
apparatus and are uncorrected. Infrared spectra were
Further, urea and thiourea derivatives are the most multi-
purpose bioactive molecules and have been reported as
antibacterial, antifungal, antitubercular, anti-inflammatory,
1
recorded on Bruker ALPHA interferometer instrument. H and
antithyroid, antihelmintic, rodenticidal, insecticidal, herbicidal 13C NMR were recorded on Bruker 400 MHz instrument. TMS
and plant growth regulatory properties. Benzoylphenyl urea was used as a standard in CDCl3 solutions, in the recording
compounds are one class of insect growth regulators, which of proton and carbon 13 NMR. Chemical shift and coupling
CONTACT Chamarthi Naga Raju
rajuchamarthi10@gmail.com
Department of Chemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh
517502, India
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
M. CHANDRASEKHAR ET AL.
OH
O
OAc
O
1-(2,3-Dihydro-1H-inden-1-yl)-3-(4-nitrophenyl)urea
(4c).
Greenish yellow solid, Yield (%): 88; mp: 338–340 ꢀC; IR
(cmꢂ1): 3356(N–H), 3227 (N–H), 1583 (C¼O); 1H NMR
(400 MHz, DMSO-d6): d 9.72 (1H, s, NH), 8.54 (1H, s, NH),
7.72–7.67 (2H, m, HAr), 7.60–7.55 (2H, m, HAr), 7.46–7.37 (3 H,
m, HAr), 6.49 (1H, d, J ¼ 8.0 Hz, HAr), 5.28 (1H, d, J ¼ 8.0 Hz,
CH–NH), 2.95–2.86 (1H, m, CH2), 2.82–2.76 (1H, m, CH2),
2.46–2.40 (1H, m, CH2), 1.84–1.76 (1H, m, CH2); 13C NMR
(100 MHz, DMSO-d6): d 153.9, 146.2, 144.4, 143.9, 142.6, 127.2,
126.4, 124.5, 123.8, 122.9, 120.4, 54.5, 33.9, 29.6; ESI-MS (m/z):
298 [M þ H]þ, 182, 117.
HN
H
H
HO
HO
AcO
AcO
OH
OAc
Resagiline
Figure 1. Inden derivatives.
Braziline
Brx-019
constant were expressed in ppm and Hz respectively. Mass
spectra were recorded on MLP 2103 mass spectrometer.
1-(3,4-Dichlorophenyl)-3-(2,3-dihydro-1H-inden-1-yl)urea
(4d). White solid, Yield (%): 82; mp: 198–200 ꢀC; IR (cmꢂ1):
3298 (N–H), 3109 (N–H), 1589 (C¼O); 1H NMR (400 MHz,
DMSO-d6): d 9.13 (1H, s, NH), 8.69 (1H, s, NH), 7.87 (1H, dd,
J ¼ 2.4, 5.2Hz, HAr), 7.52 (1H, d, J ¼ 8.8 Hz, HAr), 7.46 (1H, d,
J ¼ 8.8 Hz, HAr), 7.35 (1H, dd, J ¼ 2.4, 6.4 Hz, HAr), 7.28–7.16
(2H, m, HAr), 6.66 (1H, d, J ¼ 8.0 Hz, HAr), 5.18 (1H, dd, J ¼ 7.6,
8.0 Hz, CH–NH), 2.93–2.88 (1H, m, CH2), 2.83–2.75 (1H, m,
CH2), 2.47–2.39 (1H, m, CH2), 1.82–1.73 (1H, m, CH2); 13C NMR
(100 MHz, DMSO-d6): d 154.7, 144.1, 142.8, 139.5, 131.0, 130.6,
130.4, 127.5, 126.4, 124.6, 123.8, 122.5, 121.0, 54.4, 33.6, 29.6;
General procedure for the synthesis of urea and thiourea
derivatives of 2,3-dihydro-1H-inden-1-amine (4a-j)
A mixture of 2,3-dihydro-1H-inden-1-amine hydrochloride (1)
(250 mg, 1.48 mmol) and triethylamine (Et3N) (0.25 mL,
1.78 mmol) were taken into a round bottom flask containing
THF (10 mL). The mixture was stirred for 3.0 h at 60 ꢀC. The
solid, triethylamine hydrochloride (Et3NꢁHCl) salt was
removed by filtration as a residue and washed the bed with
3 mL of THF. The combined filtrate containing free base of
2,3-dihydro-1H-inden-1-amine (2) was transferred into a clean
round bottom flask. Triethyl amine (0.24 mL, 1.77 mmol) and
1-isocyanato-4-nitrobenzene (3c) (241 mg, 1.47 mmol) were
added to the reaction mass. The reaction mixture was stirred
at 45–50 ꢀC for 2 h. After completion of the reaction as
checked by TLC, the reaction mixture was concentrated
under vacuum to obtain crude 1-(2,3-dihydro-1H-inden-1-yl)-
3-(4-nitrophenyl)urea (4c). The pure desired product, 4c was
isolated by column chromatography using 10–30% ethyl
acetate: n-hexane as an eluent. The same procedure was
adopted for the synthesis of the remaining title compounds.
ESI-MS
(m/z):
321
[M þ H]þ,
323
[M þ H þ 2]þ,
325 [M þ H þ 4]þ.
1-(2,3-Dihydro-1H-inden-1-yl)-3-phenylthiourea
(4e).
Brown solid, Yield (%): 76; mp: 118–120 ꢀC; IR (cmꢂ1): 3363
1
(N–H), 3151 (N–H), 1373 (C¼S); H NMR (400 MHz, DMSO-d6):
d 9.43 (1H, s, NH), 8.07 (1H, d, J ¼ 8.0 Hz, HAr), 7.50–7.44 (2H,
m, HAr), 7.36–7.33 (1H, m, HAr), 7.26–7.17 (3 H, m, HAr),
7.16–7.10 (2H, m, HAr), 5.88 (1H, d, J ¼ 7.2Hz, CH–NH),
2.95–2.89 (1H, m, CH2), 2.85–2.77 (1H, m, CH2), 2.53–2.45 (1H,
m, CH2), 1.92–1.82 (1H, m, CH2); 13C NMR (100 MHz, DMSO-
d6): d 180.9, 144.3, 142.8, 136.7, 130.3, 127.6, 126.4, 126.2,
1-(2,3-Dihydro-1H-inden-1-yl)-3-(4-fluorophenyl)urea (4a). 124.6, 124.0, 123.8, 58.6, 33.9, 29.6. ESI-MS (m/z): 269
White solid, Yield (%): 73; mp: 210–212 ꢀC; IR (cmꢂ1): 3298 [M þ H]þ, 153, 102.
1
(N–H), 3152 (N–H), 1629 (C¼O); H NMR (400 MHz, DMSO-d6):
d 9.62 (1H, s, NH), 8.66 (1H, s, NH), 7.45–7.38 (2H, m, HAr),
7.28–7.16 (3 H, m, HAr), 7.12–7.02 (2H, m, HAr), 6.46 (1H, d,
J ¼ 8.0 Hz, HAr), 5.18 (1H, dd, J ¼ 7.6, 8.0 Hz, CH–NH),
2.95–2.88 (1H, m, CH2), 2.83–2.75 (1H, m, CH2), 2.45–2.39 (1H,
m, CH2), 1.80–1.70 (1H, m, CH2); 13C NMR (100 MHz, DMSO-
d6): d 158.2 (d, J ¼ 189.5 Hz), 155.1, 144.4, 142.8, 136.7, 127.4,
126.4, 124.6, 123.8, 119.2, 115.3, 54.4, 33.9, 29.6; ESI-MS (m/z):
271 [M þ H]þ, 249, 155.
1-(2,3-Dihydro-1H-inden-1-yl)-3-(4-fluorophenyl)thiourea
(4f). White solid, Yield (%): 73; mp: 164–166 ꢀC; IR (cmꢂ1):
1
3297 (N–H), 1388 (C¼S); H NMR (400 MHz, DMSO-d6): d 9.46
(1H, s, NH), 8.06 (1H, d, J ¼ 8.0 Hz, HAr), 7.55–7.46 (2H, m,
HAr), 7.38–7.30 (2H, m, HAr), 7.27–7.19 (2H, m, HAr), 7.15–7.08
(2H, m, HAr), 5.86 (1H, dd, J ¼ 7.2, 7.6 Hz, CH–NH), 3.02–2.95
(1H, m, CH2), 2.90–2.84 (1H, m, CH2), 2.52–2.44 (1H, m, CH2),
1.89–1.80 (1H, m, CH2); 13C NMR (100 MHz, DMSO-d6): d
180.8, 158.9 (d, J ¼ 191.8 Hz), 143.4, 143.9, 142.9, 135.7, 127.6,
126.3, 125.5, 124.6, 124.0, 115.1, 58.8, 32.7, 29.6; ESI-MS (m/z):
287 [M þ H]þ.
1-(4-Bromophenyl)-3-(2,3-dihydro-1H-inden-1-yl)urea (4b).
White solid, Yield (%): 82; mp: 304–306 ꢀC; IR (cmꢂ1): 3288
1
(N–H), 3198 (N–H), 1630 (C¼O); H NMR (400 MHz, DMSO-d6):
d 9.72 (1H, s, NH), 8.54 (1H, s, NH), 7.72–7.67 (2H, m, HAr),
7.60–7.55 (2H, m, HAr), 7.46–7.37 (3 H, m, HAr), 6.49 (1H, d,
J ¼ 8.0 Hz, HAr), 5.28 (1H, d, J ¼ 8.0 Hz, CH–NH), 2.95–2.86 (1H,
1-(3-Chlorophenyl)-3-(2,3-dihydro-1H-inden-1-yl)thiourea
(4g). White solid, Yield (%): 85; mp: 245–247 ꢀC; IR (cmꢂ1):
1
3255 (N–H), 1334 (C¼S); H NMR (400 MHz, DMSO-d6): d 9.46
m, CH2), 2.82–2.76 (1H, m, CH2), 2.46–2.40 (1H, m, CH2), (1H, s, NH), 8.06 (1H, d, J ¼ 8.0 Hz, HAr), 7.55–7.46 (2H, m,
1.84–1.76 (1H, m, CH2); 13C NMR (100 MHz, DMSO-d6): d HAr), 7.38–7.30 (2H, m, HAr), 7.27–7.19 (2H, m, HAr), 7.15–7.08
154.5, 144.3, 142.8, 137.4, 132.1, 127.3, 126.4, 124.5, 123.8, (2H, m, HAr), 5.88 (1H, dd, J ¼ 7.2, 7.6 Hz, CH–NH), 3.02–2.95
122.3, 121.7, 54.4, 33.8, 29.6; ESI-MS (m/z): 330 [M]þ, 332 (1H, m, CH2), 2.90–2.84 (1H, m, CH2), 2.52–2.44 (1H, m, CH2),
[M þ 2]þ, 198.
1.89–1.80 (1H, m, CH2); 13C NMR (100 MHz, DMSO-d6): d

JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION
3
180.6, 144.4, 142.8, 139.8, 135.4, 131.2, 129.4, 127.5, 126.4,
Ligand selection
125.9, 124.4, 123.7, 58.6, 33.8, 29.6; ESI-MS (m/z): 303 The chemical structures of compounds were prepared using
[M þ H]þ, 305 [M þ H þ 2]þ.
ChemBioDraw and all the ligands were converted into Pdbqt
file format and atomic coordinates were generated using
Pyrx 2010.12
1-(2,3-Dihydro-1H-inden-1-yl)-3-(4-nitrophenyl)thiourea
(4h). White solid, Yield (%): 89; mp: 190-192 ꢀC; IR (cmꢂ1):
3300 (N–H), 3192 (N–H), 1329 (C¼S); 1H NMR (400 MHz,
DMSO-d6): d 9.36 (1H, s, NH), 8.12 (1H, d, J ¼ 8.0 Hz, HAr),
8.08–8.02 (1H, m, HAr), 7.45–7.34 (3 H, m, HAr, NH), 7.25–7.18
(2H, m, HAr), 7.14–7.08 (2H, m, HAr), 5.86 (1H, dd, J ¼ 7.2,
7.6 Hz, CH–NH), 3.00–2.86 (2H, m, CH2), 2.52–2.48 (1H, m,
CH2), 1.91–1.85 (1H, m, CH2); 13C NMR (100 MHz, DMSO-d6): d
180.2, 144.9, 144.3, 143.4, 142.8, 127.4, 126.5, 125.1, 124.5,
124.1, 123.7, 58.7, 33.8, 29.6; ESI-MS (m/z): 314 [M þ H]þ.
Analysis of target active binding sites
The active sites are the coordinates of the ligand in the ori-
ginal target protein grids, and these active binding sites of
target protein were analyzed using the Drug Discovery
Studio version 3.0 and 3D Ligand Site virtual tools [25].
Molecular docking studies were carried against Aromatase
protein with compounds 4a–j, the reference drug Imatinib,
using the docking module implemented in Pyrx 2010.12.
Initially the protein structures were protonated with the add-
ition of polar hydrogens, followed by energy minimization
with the MMFF94x force field, in order to get the stable con-
former of the proteins. Flexible docking was employed, the
1-(3-Bromophenyl)-3-(2,3-dihydro-1H-inden-1-yl)thiourea
(4i). Pale brown solid, Yield (%): 80; mp: 140–142 ꢀC; IR
(cmꢂ1): 3303 (N–H), 1348 (C¼S); 1H NMR (400 MHz, DMSO-
d6): d 9.66 (1H, s, NH), 8.32 (1H, d, J ¼ 8.0 Hz, HAr), 7.94 (1H, s, inhibitor binding site residues were softened and highlighted
through the ‘Site Finder’ module implemented in the Pymol
HAr), 7.42–7.18 (7 H, m, HAr, NH), 5.87 (1H, d, J ¼ 7.6 Hz,
software [26]. The grid dimensions were predicted as X:
28.27, Y: 27.13, Z: 28.51 for Aromatase respectively. The dock-
ing was carried out with the default parameters i.e. place-
ment: triangle matcher, recording 1: London dG, refinement:
force field and a maximum of 10 conformations of each
compound were allowed to be saved in a separate database
file in a .mdb format. After the docking process, the binding
energy and binding affinity of the protein-ligand complexes
were calculated using Pymol viewer tool (www.pymol.org).
CH–NH), 3.08–2.78 (2H, m, CH2), 2.54–2.51 (1H, m, CH2),
1.92–1.83 (1H, m, CH2); 13C NMR (100 MHz, DMSO-d6): d
180.3, 144.4, 142.8, 138.4, 129.6, 128.0, 127.4, 126.5, 126.0,
124.5, 124.1, 123.8, 122.8, 58.8, 33.9, 29.6; ESI-MS (m/z): 347
[M þ H]þ, 349 [M þ H þ 2]þ.
1-(2,4-Difluorophenyl)-3-(2,3-dihydro-1H-inden-1-yl)th-
iourea (4j). White solid, Yield (%): 80; mp: 242–244 ꢀC; IR
(cmꢂ1): 3274 (N–H), 3191 (N–H), 1321 (C¼S); 1H NMR
(400 MHz, DMSO-d6): d 9.35 (1H, s, NH), 8.22 (1H, d, J ¼ 8.0 Hz,
HAr), 7.45–7.28 (5 H, m, HAr), 7.25–7.20 (2H, m, HAr), 7.17 (1H,
s, HAr), 5.88 (1H, d, J ¼ 7.2, 7.6 Hz, CH–NH), 3.05–2.80 (2H, m,
CH2), 2.49–2.45 (1H, m, CH2), 1.93–1.88 (1H, m, CH2); 13C NMR
(100 MHz, DMSO-d6): d 179.8, 165.4 (d, J ¼ 239.5 Hz), 159.2 (d,
J ¼ 180.2Hz), 144.3, 142.8, 130.2, 127.5, 126.4, 124.4, 124.1,
123.7, 116.4, 112.1 (d, J ¼ 47 Hz), 105.4, 58.7, 33.9, 29.6; ESI-
MS (m/z): 289 [M þ H]þ.
Structural analysis and visualization
Protein and ligand interactions were analyzed and visualized
through Pymol viewer tool (www.pymol.org).
In vitro anti cancer activity
The Human Breast Cancer cell line, MCF-7 was procured from
King Institute of PreventiveMedicine, Chennai. The cells were
grown in culture flask using Minimum Essential Medium sup-
plemented with 3% L-Glutamine, 10% Fetal bovine serum,
Molecular docking studies
The three-dimensional structure of Aromatase and Imatinib Penicillin (100 IU/mL), Streptomycin (100 lg/mL) and
Amphotericin B along with 7.5% sodium bicarbonate in a T
25 mL cultured vented flask and incubated at 37 ꢀC in 5%
CO2 incubator. After 3 days, about 80–90% confluent mono-
layer (adherent) formation was confirmed by inverted micro-
scope. Then it was sub cultured by using TPVG solution
along with minimum essential medium and used for fur-
ther study.
(Figure 2) were downloaded from the RCSB protein Data
Bank. The atomic coordinates of the protein was estranged
and geometry optimization was done using Argus Lab
4.0.1 [24].
Growth inhibition of MCF-7 cells by title compounds were
determined by MTT assay. The cells were harvested and
seeded in a 96 well plates and the plates were incubated for
24 h at 37 ꢀC in 5% CO2 for attachment of cells. After 12 h
different concentrations of title compounds such as 10 mg/
mL, 20 mg/mL, 30 mg/mL were added to the cells and incu-
bated for 24 h. After incubation, the medium was replaced
with phenol red and FBS free medium and 15 mL of MTT
(5 mg/mL) dye was added per well and wrapped with
Figure 2. Three-dimensional structure of Aromatase and Imatinib.

4
M. CHANDRASEKHAR ET AL.
aluminum foil and the plate was incubated again for 4 h. (in the presence of compounds). All tests were run in tripli-
cates (n ¼ 3), and average values were calculated. Inhibition
After incubation medium was aspirated and 100 mL of DMSO
concentration (IC50) parameter was used for the interpret-
ation of the results from DPPH method. The discoloration of
the sample was plotted against the sample concentration in
order to calculate the IC50 value. It is defined as the amount
of the sample necessary to decrease the absorbance of DPPH
by 50%.
were added to each well to solubilize the formazan crystals
[27]. The optical density (OD) was measured at the wave-
length of 570 nm. The percentage of cell inhibition was
determined by following formula:
OD of test
% of cell viability ¼
ꢃ 100
OD of control
NO radical scavenging assay
NO radical scavenging assay can be estimated by the use of
Griess IlIosvoy reaction [29]. The compound sodium nitro-
prusside is known to decompose in aqueous solution at
physiological pH (7.2) producing NO-. Under aerobic condi-
tions, NOꢂ reacts with oxygen to produce stable products
(nitrate and nitrite). The quantities can be determined using
Griess reagent. Scavengers of nitric oxide compete with oxy-
gen leading to reduced production of nitrite ions. 1.0 mL of
Antioxidant activity
DPPH radical scavenging assay
The DPPH radical scavenging method [28] was used to evalu-
ate the antioxidant property. The antioxidant activity was
compared with that of the natural antioxidant, ascorbic acid.
The concentrations of the synthetic compounds required to
scavenge DPPH showed a dose dependent response. The
antioxidant activity of each sample was expressed in terms various concentrations of the compound (25, 50, 75 and
100 mg/mL) in methanol was added to 4 mL of 0.004% (w/v)
methanol solution. The mixture was shaken vigorously and
incubated at room temperature for 30 min in the dark. After
the incubation period, 0.5 mL of Griess reagent was added.
The absorbance of the chromophore that formed during
diazotization of the nitrite with sulfanilamide and subsequent
coupling with Naphthylethylenediamine dihydrochloride was
immediately read at 550 nm. Inhibition of nitrite formation
by the plant extracts and the standard antioxidant ascorbic
acid were calculated relative to the control. Inhibition data
(percentage inhibition) were linearized against the concentra-
tions of each extract and standard antioxidant. IC50 which is
an inhibitory concentration of each extract required to
reduce 50% of the nitric oxide formation was determined.
of IC50, and was calculated from the graph after plotting
inhibition percentage against compound concentration.
1.0 mL of various concentrations of the compound (25, 50,
75 and 100 mg/mL) in methanol was added to 4 mL of
0.004% (w/v) methanol solution of DPPH. The mixture was
shaken vigorously and incubated at room temperature for
30 min in the dark. The reduction of the DPPH free radical
was measured by reading the absorbance at 517 nm by a
spectrophotometer. The solution without any compound and
with DPPH and methanol was used as control. The experi-
ment was replicated in three independent assays. Ascorbic
acid was used as positive control. Inhibition of DPPH free
radical in percentage was calculated by the formula:
ðAcontrolꢂAtest
Þ
% Inhibition ¼
ꢃ 100
% Inhibition
Acontrol
Absorbance of controlꢂAbsorbance of test sample
¼
ꢃ 100
where Acontrol is the absorbance of the control (L- Ascorbic
acid) and Atest is the absorbance of reaction mixture samples
Absorbance of control
R
OCN
R
O
N
3a-d
THF, Et3N
Reflux
N
H
THF, Et3N
60-65 oC, 2-3 h
H
HCl
NH2
NH2
4a-d
1
2
6
R = 3a/4a - 4-F
3b/4b - 4-Br
T
0
-
H
NCS
6
F
5
,
o
E
C
3c/4c
3d/4d - 3,4-Cl2
- 4-NO2
t
,
3
R
N
2
-
R = 3e/4e - H
3f 4f
3
h
3e-j
/
- 4-F
3g/4g - 3-Cl
3h/4h - 4-NO2
R
S
3i 4i
/
- 3-Br
3j/4j- 2,4-F2
N
N
H
H
4e-j
Scheme 1. Synthesis of urea and thiourea derivatives of 2,3-dihydro-1H-inden-1-amine 4a–j.

JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION
5
Table 1. In vitro anticancer activity of the compounds against MCF-7
cell lines.
Results and discussion
Chemistry
% of Inhibition
Compound
10 mg/mL
20 mg/mL
30 mg/mL
IC50 (mg)
Initially, 2,3-dihydro-1H-inden-1-amine hydrochloride (1) was
made as a free base on treating with Et3N to obtain 2,3-dihy-
dro-1H-inden-1-amine (2) and used without further purifica-
tion to the next process. 2,3-Dihydro-1H-inden-1-amine (2)
was treated with various substituted phenyl isocyanates
3a–d and isothiocyanates 3e–j in the presence of Et3N under
heating conditions (10–40 ꢀC) to afford substituted 1-(2,3-
dihydro-1H-inden-1-yl)-3-phenyl urea derivatives 4a–d and 1-
(2,3-dihydro-1H-inden-1-yl)-3-phenylthiourea derivatives 4e–j
in moderate yields (Scheme 1). The structures of all the
4a
4b
4c
4d
4e
4f
4g
4h
4i
58.24 1.28
33.25 3.22
52.12 1.22
48.48 2.12
26.28 4.23
30.66 2.46
30.12 1.16
48.56 1.86
26.48 2.66
32.28 3.26
20.88 3.22
100
68.36 2.32
46.28 4.12
66.24 2.22
60.22 1.14
48.88 2.38
54.86 1.86
50.16 2.46
61.22 4.24
49.18 3.24
51.22 2.86
38.12 1.96
100
77.24 3.32
69.32 1.28
78.26 3.12
68.22 1.14
60.12 4.22
58.24 1.12
61.84 2.84
68.12 1.36
62.54 1.28
59.36 1.68
51.28 1.48
100
5.24 0.12
21.46 0.22
6.12 0.16
17.46 0.72
21.22 0.46
19.17 0.40
19.66 0.60
11.86 0.16
23.18 0.42
19.66 0.46
28.66 0.66
–
4j
2
Control
Figure 3. Diagrammatic representation of 3D modeled binding modes of the compounds 4a–j and standard with the binding domain of Aromatase.

6
M. CHANDRASEKHAR ET AL.
Table 2. Bonding characterization of synthesized compounds 4a–j and Imatinib (Reference drug) against human placental aromatase protein.
Compound
Binding energy (kcal/mol)
Binding interaction
Bond Length (Å)
Bond Angle (ꢀ)
Bond Type
Imatinib
4a
4b
–9.2
–9.3
–7.9
ꢂ10.2
Arg 115 CB … HN
Met 311 CZ … .HN
Arg 115 CB … .HC
Met 149 CA … .OC
Gly 439 CZ … .OC
Arg 145 CZ … ..ON
Trp 141 CB … .ON
Arg 435 CZ … .ON
Arg 435 CZ … .ON
Arg 115 CB … .ON
Arg 115 CB … .ON
Ala 443 CA … ..ON
Arg 115 CZ … .ON
Ser 478CZ … ..ON
Ile 133 CB … ..HN
Ser 199 CZ … ..HN
Ile 133 CB … .HN
Asp 309 CA … .ON
2.2
2.7
2.4
2.7
2.2
2.4
2.2.
2.6
1.9
2.7
2.1
2.8
2.1
2.4
2.1
2.8
2.9
1.9
109.9
121.8
108.7
83.7
104.8
121.6
136.0
107.3
121.3
106.3
89.7
106.7
101.8
89.9
104.7
76.8
H-don
H-don
H-acc
H-acc
H-acc
H-acc
H-acc
H-acc
H-acc
H-acc
H-acc
H-acc
H-acc
H-acc
H-don
H-don
H-don
H-acc
4c
4d
4e
4f
4g
4h
4i
–9.8
–7.5
–7.6
ꢂ7.6
–9.5
–7.6
–7.8
78.9
123.3
4j
Table 4. Nitric oxide scavenging activity of the title compounds 4a–j.
Table 3. DPPH free radical scavenging activity of the synthesized com-
pounds 4a–j.
Compound
25 mg/mL
50 mg/mL
75 mg/mL
100 mg/mL
Compound
25 mg/mL
50 mg/mL
75 mg/mL
100 mg/mL
4a
4b
4c
4d
4e
4f
4g
4h
4i
24.95
27.36
25.62
26.28
29.93
30.43
28.60
29.02
27.94
28.02
25.37
38.14
47.77
37.72
30.84
34.07
41.62
37.39
38.05
38.39
44.82
36.81
28.44
47.01
66.81
55.02
57.47
51.45
51.57
47.59
50
50.91
62.62
45.60
47.81
57.96
82.12
70.08
68.83
63.44
61.60
57.37
56.71
57.04
72.23
50.41
68.75
67.74
4a
4b
4c
4d
4e
4f
4g
4h
4i
20.22
31.30
16.76
9.24
35.77
33.84
22.56
29.06
23.88
26.21
11.07
46.54
56.82
40.14
22.35
16.56
47.76
48.88
36.81
40.85
38.71
51.97
45.54
66.66
60.79
51.62
51.09
49.81
55.38
57.31
52.17
50.81
46.74
63.13
62.35
73.78
87.70
63.71
61.80
68.45
67.78
62.90
60.54
65.65
60.16
78.41
79.67
79.47
4j
2
Std
4j
2
Std
Std: Ascorbic Acid.
Std: Ascorbic Acid.
docking results of ligands against Aromatase showed that
newly synthesized compounds were characterized by IR,
the compounds 4a–j have significant binding modes, with
NMR (1H and 13C) and mass spectral data. The compounds dock scores of –9.3, –9.9, –10.2, –9.8, –7.5, –7.6, –7.6, –9.5,
4a–j showed characteristic IR stretching absorptions for N–H, –7.6 and –7.8 respectively, among all four compounds were
C¼O and C¼S in the region [30] 3363–3248, 1630–1583 and
shown higher dock scores when compared with the control
1388–1321 cmꢂ1 respectively. The aromatic protons of ben- drug Imatinib (–9.2) except 4b, 4e, 4f, 4g, 4i and 4j. The H-
zene rings of 4a–j showed signals in the region d 8.69–6.49. bonds, binding affinities and energy profiles of compounds
The protons of N–H resonated at d 9.72–9.13. The C-H proton 4a–j toward the active site amino acids of the enzyme are
attached to nitrogen showed a signal as doublet (d)/doblet summarized in Table 1 and their 3D modeled interactions of
of doublet (dd) in the region d 5.88–5.18, due to coupling the title compounds with aromatase protein were shown in
with proton(s) of neighboring carbon in five membered ring Figure 3. Thus these interactions provide support for the sig-
of inden moiety. The aliphatic protons of CH2-CH2 showed nificant decrease in Aromatase activity. Hence, the present
signal(s) as multiplet in the region d 3.08–1.70. The aromatic investigation demonstrate that the synthesized compounds
carbons resonated in the region d 158.2–105.4 based on the will be the promising next generation chemotherapeutic
functionalities attached to phenyl ring for all the compounds drugs, which can be effectively used in the treatment of
4a–j. The carbon chemical shifts for carbonyl group of 4a–d breast cancer and other related disorders. Compounds
and thiocarbonyl group of 4e–j appeared in the region d except 4c, all the title compounds have showed hydrophobic
155.1–153.9 and 180.9–179.8 respectively.
interactions against 3S7S protein.
Molecular docking studies
In vitro anti cancer activity
In order to provide strength to the synthesized compounds, The results obtained from this study, it is observed that all
docking analysis was carried out for compounds 4a–j with the title compounds have shown promising anti cancer activ-
selective pharmacological targets such as Aromatase protein ity on MCF-7 cell lines (Table 2). All the title compounds
of breast cancer which is involved in the pathogenesis and inhibited the cell viability significantly on dose dependent
induction of cancer. The crystal structure of aromatase (PDB manner. The maximum of 78% of viability reduction was
id: 3S7S) was retrieved from the protein data bank. The observed in the compound, 4c treated with the

JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION
7
nature of the title compounds was investigated using DPPH
and NO methods including IC50 values. Some of the com-
pounds, urea derivative linked with 4-bromo phenyl ring 4b,
and thiourea derivatives bonded with phenyl ring 4e, 4-flu-
oro phenyl ring 4f, and 4-nitro pheyl ring 4h exhibited
potential activity in all the tested methods. The overall
screening revealed that thiourea derivatives are acted as
antioxidants promisingly as compared with urea derivatives.
As an addition molecular docking studies of the synthesized
compounds against Aromatase enzyme revealed that major-
ity of the compounds showed higher binding affinities and
many more interactions when compared with reference
drug, Imatinib. These results suggested that a few of the syn-
thesized compounds can be considered as promising thera-
peutic candidates for further optimization and development
of potential anti-cancer drugs in near future.
Figure 4. Half inhibitory concentration of compounds by DPPH method.
Disclosure statement
All the authors declare that no conflict of interest in this work.
ORCID
Chamarthi Naga Raju
http://orcid.org/0000-0001-6566-2118
References
Figure 5. Half inhibitory concentration of title compounds by NO method.
[1]
[2]
[3]
Antonio DS, Piera S, Ivana C, et al. Preparation and pharmaco-
logical characterization of trans-2-Amino-5(6)-fluoro-6(5)-hydroxy-
1-phenyl-2,3-dihydro-1H-indenes as D2-like dopamine receptor
agonists. J Med Chem. 2005;48:2646–2654.
Ward SE, Harries M, Aldegheri L, et al. Discoveryof N-[(2S)-5-(6-flu-
oro-3-pyridinyl)-2,3-dihydro-1H-inden-2-yl]-2-propanesulfonamide,
a novel vlinical AMPA receptor positive modulator. J Med Chem.
2010;53(15):5801–5812.
concentration of 30 mg/mL. Whereas the majority of the com-
pounds exhibited potent anti cancer activity on MCF-7 cell
lines. Amongst the compounds 4a, 4c, 4d and 4h have
shown prominent anti cancer activity 77.24%, 78.26%,
68.22% and 68.12% and the remaining compounds also
exhibited good anti cancer activity than that of the com-
pound 2.
Yao W, Wasserman ZR, Chao M, et al. Design and Synthesis of
a
Series of (2R)-N4-Hydroxy-2-(3-hydroxybenzyl)-N1-[(1S,2R)-2-
hydroxy-2,3-dihydro-1H-inden-1-yl]butane-diamide derivatives as
potent, selective, and orally bioavailable aggrecanase inhibitors.
J Med Chem. 2001;44(21):3347–3350.
Antioxidant activity
The newly synthesized urea and thiourea compounds 8a–j
were screened for their antioxidant activity. DPPH radical
scavenging assay and NO radical scavenging assay were
used to examine the antioxidant nature of compounds. The
antioxidant nature of newly synthesized compounds were
investigated at different concentrations such as 25, 50, 75
and 100 mg/mL, (Tables 3 and 4) and ascorbic acid was used
as a standard to compare the activity. As well as, half max-
imal concentration (IC50) of the compounds was tested using
DPPH and NO method (Figures 4 and 5). The experimental
results disclosed that all the compounds exhibited moderate
to good antioxidant activity. Some of the compounds, urea
derivative 4b and thiourea derivatives 4e, 4f and 4h exhib-
ited potential activity, in conclusion the two tested methods
and IC50 values were evaluated in both the methods.
[4]
[5]
Lee T, Gong YD, Min KH. Synthesis and biological activity of ben-
zopyranyl urea and benzopyranyl thiourea derivatives as MDR
reversal agents. Bull Korean Chem Soc. 2012;33(11):3857–3860.
Sudhamani H, Thaslim Basha SK, Venkateswarlu N, et al. Synthesis
and characterization of new thiourea and urea derivatives of 6-
fluoro-3-(piperidin-4-yl)benzo[d] isoxazole: in vitro Antimicrobial
and Antioxidant activity. J Chem Sci. 2015;127(10):1739–1746.
Miller NR, Daniels RN, Bridges TM, et al. Synthesis and SAR of
analogs of the M1 allosteric agonist TBPB. Part II: amides, sulfona-
mides and ureas-The effect of capping the distal basic piperidine
nitrogen. Bioorg Med Chem Lett. 2008;18(20):5443–5447.
Upadhayaya RS, Kulkarni GM, Vasireddy NR, et al. Design, synthe-
sis and biological evaluation of novel triazole, urea and thiourea
derivatives of quinoline against Mycobacterium tuberculosis.
Bioorg Med Chem. 2009;17(13):4681–4692.
Rajesh HT, Rodge AH, Hatnapure GD, et al. The novel 3,4-dihydro-
pyrimidin-2(1H)-one urea derivatives of N-aryl urea: synthesis,
anti-inflammatory, antibacterial and antifungal activity evaluation.
Bioorg Med Chem Lett. 2011;21:4648–4651.
Zhang H, Zhang YA, Wu GZ, et al. Synthesis and biological evalu-
ation of sulfonylurea and thiourea derivatives substituted with
benzenesulfonamide groups as potential hypoglycemic agents.
Bioorg Med Chem Lett. 2009;19(6):1740–1744.
[6]
[7]
[8]
[9]
Conclusion
A series of new urea and thiourea derivatives of 3-dihydro-
1H-inden-1-amine were synthesized. The antioxidant activity

8
M. CHANDRASEKHAR ET AL.
€
ꢀ
[10]
Gulkok Y, Bic¸er T, Kaynak Onurdag F, et al. Synthesis of some
new urea and thiourea derivatives and evaluation of their anti-
microbial activities. Turk J Chem. 2012;36:279–291.
rasagiline mesylate using lipases. Appl Catal A. 2015;492:
76–82.
[21]
[22]
Sudhamani H, Thaslim Basha SK, Adam S, et al. Synthesis and
evaluation of urea and thiourea derivatives of lopinavir inter-
mediate as potent antimicrobial, antioxidant agents and molecu-
lar docking studies against Staphopain. Res Chem Intermed.
2017;43(1):103–120.
Sudhamani H, Thaslim Basha SK, Adam S, et al. Synthesis, charac-
terization, and bio-activity evaluation of thiourea derivatives of
epinephrine as antimicrobial and antioxidant agents: molecular
docking studies. Monatsh Chem. 2017;148(8):1525–1537.
Vedavathi P, Sudhamani H, Naga Raju C. Synthesis and antimicro-
bial activity of new urea and thiourea derivatives of (20-(1H-tetra-
zol-5-yl)biphenyl-4-yl)methanamine. Res Chem Intermed. 2017;
43(5):3251–3263.
[11]
[12]
[13]
Faidallah HM, Khan KA, Asiri AM. Synthesis and biological evalu-
ation of new 3- trifluoromethylpyrazolesulfonyl-urea and thiourea
derivatives as antidiabetic and antimicrobial agents. J Fluorine
Chem. 2011;132(2):131–137.
Saeed S, Rashid N, Jones PG, et al. Synthesis, characterization and
biological evaluation of some thiourea derivatives bearing benzo-
thiazole moiety as potential antimicrobial and anticancer agents.
Eur J Med Chem. 2010;45(4):1323–1331.
Esteves-Souza A, Pissinate K, Nascimento Mda G, et al. Synthesis, [23]
cytotoxicity, and DNA-topoisomerase inhibitory activity of new
asymmetric ureas and thioureas. Bioorg Med Chem. 2006;14(2):
492–499.
[14]
[15]
Ziessel R, Bonardi L, Retailleau P, et al. Isocyanate-, isothiocyan- [24]
ate-, urea-, and thiourea-substituted boron dipyrromethene dyes
as fluorescent probes. J Org Chem. 2006;71(8):3093–3102.
Morris GM, Huey R, Lindstrom W, et al. AutoDock4 and
AutoDockTools4: automated docking with selective receptor flexi-
bility. J Comput Chem. 2009;30(16):2785–2791.
Ter Haar E, Coll JT, Austen DA, et al. Structure of GSK3 beta
reveals a primed phosphorylation mechanism. Nat Struct Biol.
2001;8(7):593–596.
Pettersen EF, Goddard TD, Huang CC, et al. UCSF Chimera-A visu-
alization system for exploratory research and analysis. J Comput
Chem. 2004;25(13):1605–1612.
Denizot F, Lang R. Rapid colorimetric assay for cell growth and
survival: modifications to the tetrazolium dye procedure giving
improved sensitivity and reliability. J Immunol Methods. 1986;
89(2):271–277.
Vaijanathappa J, Badami S, Bhojraj S. In vitro antioxidant activity
of enicostemma axillare. J Health Sci. 2008;54(5):524–528.
Marcocci L, Maguire JJ, Droy-Lefaix MT, et al. The nitric oxide-
scavenging properties of Ginkgo biloba extract EGb 761. Biochem
Biophys Res Commun. 1994;201(2):748–755.
Thomas LC. Interpretation of the Infrared Spectra of
Organophosphorus Compounds. London (UK): Hyden and Son;
1974.
Yang J, Lakshmikantham MV, Cava MP, et al. Synthesis and char-
acterization of 5,10-bis(2-thienyl)indeno[2,1-a]indene derivatives:
The first examples of conducting polymers containing a rigid bis
(thienyl)butadiene core. J Org Chem. 2000;65(20):6739–6742.
Lee BH, Choi YL, Shin S, et al. Stereoselective palladium-catalyzed
a-arylation of 3-aryl-1-Indanones: an asymmetric synthesis of
(þ)-pauciflorol F. J Org Chem. 2011;76(16):6611–6618.
[25]
[26]
[27]
[16]
[17]
[18]
[19]
[20]
Bachar SC, Nahar L, Sarker SD. Synthesis and structure-activity-
relationships of inden acid derivatives as analgesic and anti-
inflammatory agents. Ref J Chem. 2016;6(2):125–128.
Choi SY, Yang KM, Jeon SD, et al. Brazilin modulates immune
[28]
[29]
function mainly by augmenting
T cell activity in halothane
administered mice. Planta Med. 1997;63(05):405–408.
Yang KM, Jeon SD, So DS, et al. Brazilin augments cellular
immunity in multiple low dose streptozotocin (MLD-STZ) induced
type I diabetic mice. Arch Pharm Res. 2000;23(6):626–632.
Thiago de Sousa F, Marcos Reinaldo da S, Maria da
[30]
~
Conceic¸ao Ferreirade O, et al. Chemoenzymatic synthesis of