The inhibitory potency of isoxazole-curcumin analogue for the management of breast cancer: A comparative in vitro and molecular modeling investigation

Article abstract of DOI:10.1007/s11696-021-01775-9

Curcumin, a potent phytochemical derived from the spice element turmeric, has been identified as a herbal remedy decades ago and has displayed promise in the field of medicinal chemistry. However, multiple traits associated with curcumin, such as poor bio

Full text of DOI:10.1007/s11696-021-01775-9

Chemical Papers
https://doi.org/10.1007/s11696-021-01775-9
ORIGINAL PAPER
The inhibitory potency of isoxazole‑curcumin analogue
for the management of breast cancer: A comparative in vitro
and molecular modeling investigation
Fiona C. Rodrigues1 · N. V. Anil Kumar2 · Gangadhar Hari3 · K. S. R. Pai3 · Goutam Thakur1
Received: 11 February 2021 / Accepted: 4 July 2021
© The Author(s) 2021
Abstract
Curcumin, a potent phytochemical derived from the spice element turmeric, has been identifed as a herbal remedy decades
ago and has displayed promise in the feld of medicinal chemistry. However, multiple traits associated with curcumin, such
as poor bioavailability and instability, limit its efectiveness to be accepted as a lead drug-like entity. Diferent reactive
sites in its chemical structure have been identifed to incorporate modifcations as attempts to improving its efcacy. The
diketo group present in the center of the structural scafold has been touted as the group responsible for the instability of
curcumin, and substituting it with a heterocyclic ring contributes to improved stability. In this study, four heterocyclic cur-
cumin analogues, representing some broad groups of heterocyclic curcuminoids (isoxazole-, pyrazole-, N-phenyl pyrazole-
and N-amido-pyrazole-based), have been synthesized by a simple one-pot synthesis and have been characterized by FTIR,
1H-NMR, 13C-NMR, DSC and LC–MS. To predict its potential anticancer efcacy, the compounds have been analyzed by
computational studies via molecular docking for their regulatory role against three key proteins, namely GSK-3β—of which
abnormal regulation and expression is associated with cancer; Bcl-2—an apoptosis regulator; and PR which is a key nuclear
receptor involved in breast cancer development. One of the compounds, isoxazole-curcumin, has consistently indicated a
better docking score than the other tested compounds as well as curcumin. Apart from docking, the compounds have also
been profled for their ADME properties as well as free energy binding calculations. Further, the in vitro cytotoxic evalu-
ation of the analogues was carried out by SRB assay in breast cancer cell line (MCF7), out of which isoxazole-curcumin
(IC50–3.97 µM) has displayed a sevenfold superior activity than curcumin (IC50–21.89 µM). In the collation of results, it can
be suggested that isoxazole-curcumin behaves as a potential lead owing to its ability to be involved in a regulatory role with
multiple signifcant cancer proteins and hence deserves further investigations in the development of small molecule-based
anti-breast cancer agents.
* Goutam Thakur
goutam.thakur@manipal.edu
1
Department of Biomedical Engineering, Manipal Institute
of Technology, Manipal Academy of Higher Education,
Manipal, India
2
Department of Chemistry, Manipal Institute of Technology,
Manipal Academy of Higher Education, Manipal, India
3
Department of Pharmacology, Manipal College
of Pharmaceutical Sciences, Manipal Academy of Higher
Education, Manipal, India
Vol.:(0123456789)
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Chemical Papers
Graphic abstract
Keywords Curcumin · Heterocyclic · Analogues · Molecular docking · Breast cancer
displayed by turmeric such as antibacterial (Moghad-
amtousi 2014), anti-infammatory (Menon and Sudheer
2007) (Chainani-Wu 2003), antithrombotic (Srivastava
et al. 1985), antitumor (Ravindran et al. 2009), neuropro-
tective (Cole et al. 2007), etc. Apart from playing a role
in multiple activities, curcumin indicates minimal toxicity
even on the administration of doses as high as 10 g/day in
humans (Aggarwal et al. 2003). With respect to antican-
cer activity, curcumin has an edge over other compounds
because of its efectiveness in various types of cancers
owing to its chemotherapeutic potential. Furthermore, it is
also known to have a pleiotropic modulatory behavior for
several molecular targets in turn regulating gene expres-
sions that play key roles in cell proliferation pathways
(Shishodia et al. 2007). These molecular targets range
from infammatory cytokines, growth factors, apoptosis-
related proteins, cell cycle proteins, transcription factors,
etc. (Shishodia 2013). Irrespective of the numerous posi-
tive traits exhibited by curcumin, a larger number of limi-
tations hinder its ability to be exploited to its full poten-
tial. These limitations are usually enlisted as poor aqueous
solubility, poor bioavailability and rapid elimination from
the systemic circulation (Priyadarsini 2014). One of the
approaches adopted to improve its pharmacological profle
is an exhaustive structure activity relationship of the mol-
ecule so as to understand the contributing aspect of each
reactive site in the skeletal structure of curcumin. This
allows for the identifcation of the sites that are respon-
sible for the instability of the molecule and retention of
the sites which attribute to the superlative activity of the
molecule to facilitate lead optimization. The privileged
Introduction
The quest for antitumor compounds is an ongoing pro-
cess that necessitates combinatorial chemistry and high-
throughput screening for the identification of potent
leads. Cancer, being a major health concern, has intrigued
researchers to intensively investigate drugs targeting the
various receptors in cell proliferation pathways. In the
recent past, the pharmaceutical industry has shifted its
focus to the repurposing of existing drug moieties and
recognizing natural products as bioactive molecules
(Demain and Vaishnav 2011). Natural product drug dis-
covery constitutes the identifcation of bioactivity stud-
ies, structure–activity relationship studies for derivatives/
analogues, along with the synchronized mechanism of
action directed synthesis, isolation and characterization for
rational drug design-based modifcations (Dholwani et al.
2008). Leading the research of bioactive natural products
at the forefront of this quest is curcumin, which is isolated
from turmeric and made from the rhizomes of Curcuma
longa L. turmeric, apart from being a primary spice in the
South East Asian cuisine, has also had the repute of being
a herbal remedy and has been mentioned in Ayurveda,
Siddha, Unani and Traditional Chinese Medicine (Prasad
S. 2011).
Curcumin was frst isolated in 1815 and was identifed
as 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadien-
3,5-dione and is the major constituent of turmeric (Vogel
and Pelletier 1815)(Goel et al. 2008). It is attributed to
be the entity that contributes to the multiple activities
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Chemical Papers
scafold of curcumin can be categorically divided as four
major reactive sites, namely the aryl side ring, the car-
bonyl chain/olefn bonds, the diketo moiety and the active
methylene site (Fig. 1). Numerous analogues and deriva-
tives have been designed targeting each of these sites to
analyze the corresponding change in activity and stability
of the molecule (Rodrigues et al. 2019) (Vyas et al. 2013).
Multiple papers have indicated that the diketo moiety has
contributed to the instability of curcumin and its rapid
metabolism in vivo and hence must be targeted by incor-
porating scafolds which can add to the potency of the
compound. In a structure–activity relationship analysis of
the β-diketone chain of curcumin, Reddy et al. postulated
that the introduction of hydrazine to curcumin leads to
derivatives wherein the central 1,3-diketo-enol is masked
or made rigid, which in turn improves the antitumor activ-
ity (Reddy et al. 2013). Furthermore, Jankun et al. have
also postulated by SAR studies that cyclizing the central
part of the compound could lead to improved antiangio-
genic and antitumor activity of curcumin (Jankun 2016).
Heterocyclic moieties such as isoxazole and pyrazoles
have had a great impact in the improvement of the thera-
peutic potential of natural products and act as ideal scaf-
folds for natural products in medicinal chemistry (Zhang
et al. 2018) (Kumar et al. 2013) (Naim 2016) (Jain, et al.
2006). It is postulated that the β-diketone site is an ideal
scafold for the integration of the heterocyclic moiety and
based on previously ordered studies isoxazole, pyrazole
and N-phenyl pyrazole and N-amido-pyrazoles are among
the most commonly synthesized heterocyclic curcumi-
noids that have been evaluated for a plethora of activities
(Mishra et al. 2008) (Sahu 2012) (Ahsan et al. 2015) (Sahu
et al. 2016) (Ahmed 2017) (Rodrigues et al. 2021). Such
modifcations allow for improved stability and bioavail-
ability as compared to curcumin adding to the chance of
retention of curcumin-like efcacy.
a simple and cost-efective one-pot synthesis protocol and
were further characterized. As a predictive auxiliary study to
understand the regulatory behavior of these compounds, the
compounds were screened for their ADME profle and were
docked in silico against major key proteins, i.e. GSK-3β,
Bcl-2 and PR, which are actively involved in the cell prolif-
eration pathway. To confrm the predictive model study, the
compounds were further evaluated for their cytotoxic capa-
bilities in vitro against human breast cancer cell line MCF 7
and were compared with curcumin to identify their potency.
Experimental section
Materials and methods
To facilitate the present work, analytical-grade chemicals
were obtained from Aldrich and Merck and were used with-
out any purifcation. The purity of the substrates and moni-
toring of the reaction was carried out by using thin-layer
chromatography (TLC) on TLC silica gel 60 F254 aluminum
sheets, Merck. The FTIR (Fourier-transform infrared) spec-
tra were determined on FTIR Spectrometer 6300, JASCO-
UK. For nuclear magnetic resonance (NMR) evaluation, all
1H NMR and 13C NMR data were recorded using a Bruker
Ascend 400 MHz. To record the melting point of the synthe-
sized compounds, diferential scanning colorimetry (DSC)
was recorded on DSC-60 plus (Shimadzu) for a tempera-
ture range of 20°–250 °C with 10 °C/min heating rate, and
LC–MS spectra were recorded on Waters, Synapt G2 High
Detection Mass spectrometer.
General procedure for the synthesis
of the heterocyclic curcumin analogues
As depicted in Scheme 1, to a solution of curcumin (1 mmol,
1 eq.) and glacial acetic acid (10 ml), appropriate amounts of
hydrazine derivatives (1 mmol, 1 eq.) were added and stirred
at 90 °C for 12–14 h to enable the synthesis of products in
good yields. The progress of the reaction was monitored
In this study, four representatives of heterocyclic cur-
cumin analogues, i.e., isoxazole-, pyrazole-, N-phenyl pyra-
zole- and N-amido-pyrazole-based curcuminoids, have been
identifed and the four analogues have been synthesized via
Fig. 1 Structure of curcumin representing the diferent reactive sites
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Chemical Papers
Scheme 1 General scheme for the synthesis of heterocyclic analogues of curcumin
by TLC. Once the reaction was complete, the product was
extracted by a two solvent system—hexane and ethyl acetate.
The extracted solution was passed through a bed of anhy-
drous sodium acetate, and the fltrate was stripped of the
solvent in the rotor vapor. The extract so obtained after the
evaporation was then subjected to ethanol recrystallization.
The fltrate was then left to dry and then scraped through
after a petroleum ether wash to give a resultant powder. All
the synthesized derivatives were further characterized by
FTIR, 1H NMR, 13C NMR, DSC and LC–MS for purity.
1H), 9.42–9.36 (2H). 13C NMR (400 MHz, DMSO) 168.8,
162.7, 148.6, 148.4, 148.3, 136.9, 135.2, 127.8, 127.4, 122.1,
121.7, 116.08, 116.02, 115.7, 113.1, 110.86, 110.81, 110.6,
98.3, 56.1. M.p.—182 °C; LC–MS for C21H19O5N—366.09.
C3, Curcumin‑2,4DNPH
4,4′-((1E,1′E)-(1-(2,4-Dinitrophenyl)-1H-pyrazole-3,5-diyl)
bis(ethene-2,1-diyl))bis(2-methoxy-phenol). Dark orange
powder. A mixture of the curcumin compound (1 mmol,
0.368 g) and 2, 4-dintrophenylhydrazine (2 mmol, 0.396 ml)
was taken in the presence of solvent glacial acetic acid
(10 ml) in a round-bottom fask. The mixture was continu-
ously stirred at refux conditions for 12 h at 90 °C. IR (Vmax,
cm−1): 3510 (C–OH), 3013, 1680 (C=N), 1521 (N–O for
C1, Curcumin‑pyrazole
4,4’-[(1E)-1H-pyrazole-3,5-diyldi-2,1-ethenediyl]bis[2-
methoxy-phenol] Dark yellow-orange colored powder.
To curcumin (1.2 mmol, 0. 441 g), hydrazine hydrate
(1.5 mmol, 0.7 ml) was taken in a round-bottom fask in the
solvent glacial acetic acid (7 ml). The mixture was continu-
ously stirred under refux conditions for 14 h at 85 °C in
an oil bath. IR (Vmax, cm−1): 3560 (C–OH), 3020, 1660
1
nitro group), 1260 (aromatic C=C), 1028 (C–O–C). H-
NMR (400 MHz, DMSO) δ ppm: 3.79–3.84 (s, 6H, OCH3),
6.77–7.28 (m, 12H, Ar–H), 8.02–8.04 (d, 1 H, J=8.8 Hz,
Ar–H containing NO2), 8.688–8.665 (d, 1 H, J = 9.2 Hz,
Ar–H containing NO2), 9.25–9.37 (s, 2H, OH). 13C NMR
(400 MHz, DMSO)—56.24, 56.07, 102.07, 110.05, 110.91,
111.32, 115.98, 116.12, 121.35, 121.81, 123.66, 127.87,
128.41, 128.88, 130.22, 130.50, 133.03, 136.68, 137.05,
144.51, 145.46, 146.62, 147.66, 148.24, 148.28, 148.39,
153.99. M.p.—120 °C; LC–MS for C27H22O8 N4—531.05.
1
(C=N), 1516 (C–C), 1010 (C–O–C). H-NMR (400 MHz,
DMSO) δ ppm: d 3.88 (s, 6H, 2 OCH3), 6.67 (s, 1H, C4-H),
6.85–6.83 (d, 2H, J=8 Hz), 6.99–6.97 (d, 2H, J=7.6 Hz),
7.03–7.19 (m, 6H, Ar–H) 7.43–7.41 (d, 2H, J=8 Hz, Ar–H).
13C NMR (400 MHz, DMSO)—173.3, 147.8, 146.9, 129.5,
128.3, 127.6, 120.0, 115.8, 115.6, 115.2, 109.5, 99.1, 55.5.
M.p.—216 °C; LC–MS for C21H20O4N2—365.08.
C4, Curcumin‑semicarbazide
C2, Curcumin‑isoxazole
3,5-Bis[(E)-2-(4-hydroxy-3-methoxyphenyl)vinyl]-1H-pyra-
zole-1-carboxamide. Yellow powder. A mixture of the cur-
cumin compound (1 mmol, 0.368 g) and semicarbazide
hydrochloride (2 mmol, 0.396 ml) was taken in the presence
of solvent glacial acetic acid (10 ml) in a round-bottom fask.
The mixture was continuously stirred at refux conditions for
12 h at 90 °C. IR (Vmax, cm−1): 3362 (C–OH), 3012, 1641
(C=N), 1520 (C–C), 1290 (aromatic C=C), 1100 (C–O–C).
1H-NMR (400 MHz, DMSO) δ ppm: 3.83 (s, 6H, OCH3),
6.62–7.38 (m, 11H, Ar–H), 9.112–9.235 (s, 2 H, OH),
12.814 (s, 1H, NH). 13C NMR (400 MHz, DMSO)—23.605,
38.883, 39.092, 39.301, 39.509, 39.718, 39.927, 40.135,
55.567, 99.186, 109.530, 115.257, 115.608, 115.861,
4-[2-[3-[2-(4-hydroxy-3-methoxyphenyl)ethenyl]-5-isoxa-
zolyl]ethenyl]-2-methoxy-phenol]. Orange-brown colored
powder. To curcumin (1 mmol, 0.368 g), hydroxylamine
hydrochloride (1 mmol, 0.69 ml) was taken in a round-
bottom fask in the solvent glacial acetic acid (10 ml). The
mixture was continuously stirred under refux conditions
for 14 h at 85 °C in an oil bath. IR (Vmax, cm−1): 3550
(C–OH), 3035, 1597 (C=N), 1518 (C–C), 1090 (C–O–C),
1270 (C–O, of fve-membered ring). 1H-NMR (400 MHz,
DMSO) δ ppm: d 3.84 (s, 6H, OCH3), 6.38 (s, 1H, C4-H),
6.61–6.96 (m, 4H), 7.00–7.28 (m, 4H, Ar–H), 7.51–7.30 (d,
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Chemical Papers
120.030, 127.602, 128.145, 129.524, 146.991, 147.894,
173.383. M.p.—210 °C; LC–MS for C22H21O5N3—409.02.
dimensions fxed around the centroid of the active site was
generated for each protein.
Molecular docking protocol
In silico analysis
The analogues were screened using extra precision (XP)
mode flexible ligand docking which is implemented in
GLIDE. Van der Waals scaling factor and partial charge
cutof were selected to be 0.80 and 0.15, respectively, for
ligand atoms. The optimized ligands were utilized for this
purpose. The best-docked pose with Glide score values was
recorded for each ligand.
ADME profling
To predict and analyze the ADME (absorption, distribution,
metabolism and excretion) characteristics of the synthesized
compounds, the QikProp module by Schrӧdinger 2019-1
was used. Selected descriptors were analyzed to obtain an
understanding of the solubility and absorption levels of the
compounds. Descriptors like QPlogPo/w, QPlogS, QPPCaco,
rule of 5 and % human oral absorption were calculated using
this module.
Free energy calculations
To calculate the free energy of the protein–ligand complex
of the titular compounds, MM-GBSA (molecular mechan-
ics energies combined with generalized Born and surface
area continuum solvation) method was implemented via the
prime module of Schrödinger’s molecular modeling pack-
age. Prime employs the VSGB 2.0 solvation model and the
OPLS2005 force feld to simulate these interactions (Li et al.
2011)(Shivakumar 2010).
Ligand preparation
The structures of the synthesized curcumin analogues were
sketched via the 2DSketcher tool. For the ligand optimiza-
tion, LigPrep tool was employed using OPLS 2005 force
feld. Default parameters of retaining specifc chiralities and
desalting were selected.
Cytotoxic evaluation
Protein optimization
Cell culture
Protein structure X-ray crystal structure of the proteins
GSK-3β (PDB ID: 4ACC), with resolution 2.21 Å; R-value:
0.191 (obs.)(Arfeen et al. 2015), Bcl-2 (PDB ID: 2W3L)
with resolution 2.10 Å; R-value: 0.218 (obs.) (Ponnulak-
shmi 2020)(Murray 2019) and PR (PDB ID: 4OAR) with
resolution 2.41 Å; R-value: 0.211 (obs.) (Acharya 2019)
were retrieved from the Protein Data Bank (Research Col-
laboratory for Structural Bioinformatics (RCSB) (http://
www.rcsb.org/pdb) (Berman 2000).
The human breast cancer cell line (MCF 7) was obtained
from National Centre of Cell Science, Pune, India. The cells
were maintained at 37 °C in a humidifed atmosphere (90%)
containing 5% CO2 and then cultured in DMEM (Dulbecco’s
modifed Eagle’s medium) with 10% (v/v) FBS (fetal bovine
serum), with 2% antibiotic–antimycotic solution. The cells
were seeded in 96-well plates for 24 h and then incubated
with diferent concentrations of the synthesized analogues.
Protein preparation In the Maestro interface of Schrödinger
2019-1, the ligand–protein complex was subjected to dock-
ing, wherein the proteins had to undergo prior pre-pro-
cessing using the Protein Preparation Wizard Tool. At this
step, optimization using ProtAssign was allotted, water
that made fewer hydrogen bonds than the specifed number
was deleted, hydrogens were added, and heavy atoms were
removed.
Cell viability
The synthesized analogues were evaluated for their antican-
cer potential by the SRB (sulforhodamine B) assay method
using the cancer cell line MCF7 to obtain their IC50 val-
ues. SRB assay is a highly sensitive in vitro cytotoxicity
screening assay with an ability to detect densities as low as
1000–2000 cells/well (Skehan 1990). All the compounds
were tested at concentrations ranging from 500 to 7.81 µM.
The compounds were tested against curcumin and doxoru-
bicin as control and model drug, respectively. The assay was
executed in accordance with previously reported methods
along with minor modifcations (Houghton 2007)(Vichai
and Kirtikara 2006). The cells were harvested with trypsin
and seeded in 96-well plates in a concentration of 5000
cells/well. Simultaneously, the synthesized analogues were
Receptor Grid Generation
Following protein preparation, a receptor grid was gener-
ated with the GLIDE module of Schrödinger keeping the
van der Waals scaling factor as 1.00 and charge cutof of
0.25 subjected to OPLS 2005 force feld with default param-
eters and without any constraints. A cubic box with defned
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Chemical Papers
dissolved in DMSO, and diferent concentrations were added
into the designated wells. On completion of 48 h, ice-cold
solution of 100 µl of 10% TCA (trichloroacetic acid) was
added per well and incubated for 1 h at 4 °C. Following this,
the wells were washed three times with distilled water and
air-dried. A 100 µl aliquot of the sulforhodamine B solu-
tion (0.057% w/v in 1% v/v acetic acid) was added to each
well, and the plates were left for incubation in the dark for
30 min. On completion of the incubation period, the plates
were rinsed thrice with 1% v/v acetic acid to remove any
unbound dye. After sufciently drying the plates at room
temperature, the protein-bound dye was solubilized by add-
ing 200 µl of 10 mM Tris base to each well. The absorbance
was determined at 540 nm wavelength on a scanning multi-
plate reader (ELx800, BioTek Instruments Inc., Winooski,
VT, USA), and the percentage cell viability was calculated
using excel sheet and IC50 values were determined using
GraphPad Prism and Origin.
Results and discussion
Chemistry
The preparation of the heterocyclic representatives of the
four major groups of analogues, i.e., isoxazole-, pyrazole-,
substituted phenyl pyrazole- and amido-pyrazole-based
analogues of curcumin, is illustrated in accordance with
Scheme 1. The diketone moiety of curcumin was converted
into its isoxazole with hydroxylamine hydrochloride by
refuxing in glacial acetic acid for 14 h. Pyrazole and sub-
stituted phenyl pyrazole derivatives were synthesized by
refuxing curcumin with hydrazine hydrate and 2,4 dini-
trophenyl hydrazine, respectively, in glacial acetic acid for
12–14 h. The amido-pyrazole derivative was achieved by
refuxing curcumin with semicarbazide hydrochloride for
12 h. The completion of the reaction was monitored by TLC
using mobile phase hexane/ethyl acetate (3:2). The impuri-
ties were isolated by passing the product through a bed of
anhydrous sodium acetate to remove any insoluble salts, and
further purifcation was carried out by ethanol recrystalliza-
tion. The yield of the titular compounds was good, ranging
from 67 to 78%, and the melting points as recorded by DSC
are mentioned in Table 1. The structures of the synthesized
curcumin analogues were confrmed by analytical and spec-
tral studies (IR, 1H-NMR, 13C-NMR, DSC and LC–MS),
Statistical analysis
All experiments were carried out in triplicates to assess
reproducibility and robustness. Mean and standard deviation
were computed. T test was performed to determine p-value
and results, indicating p < 0.05 were deemed statistically
signifcant.
Table 1 Structure and physical constants of synthesized compounds
Compound no
C1
Structure
Time
12 h
Yield (%)
72
Melting point
216 °C
N
N
NH
O
H3CO
HO
OCH3
OH
C2
C3
12 h
14 h
74
78
182 °C
120 °C
H3CO
HO
OCH3
OH
O2N
NO2
N
N
N
H3CO
HO
OCH3
OH
C4
14 h
67
210 °C
O
NH2
N
H3CO
HO
OCH3
OH
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Chemical Papers
and the synthesized compounds were in full accordance with
the proposed structures. The spectral peaks and empirical
data have been explicitly mentioned in the Supplementary
Information.
descriptors, such as molecular weight, QPlogPo/w, QPlogS,
QPPCaco, rule of 5 and % human oral absorption, were cal-
culated and analyzed. The majority of the synthesized com-
pounds have indicated ADME characteristics that lie within
the recommended values and have obeyed the Lipinski rule
of fve. C3 or N-Phenyl pyrazole-curcumin has indicated
poor predicative permeability and poor aqueous solubility.
Also, isoxazole-curcumin (C2) has indicated the highest
human oral absorption percentage of 93.862% (QikProp
2012) (Table 2).
All of the synthesized compounds have indicated
the characteristic peak of the –OH group in the range of
3500–3650 cm−1 with respect to the FTIR data. The absorp-
tion bands in the 3010–3100 cm−1 and 1590–1680 cm−1
regions correspond to N–H stretching and bending peaks,
respectively. The –NH group of curcumin-pyrazole (C1)
was observed at 3020 cm−1. For curcumin-isoxazole (C2),
a shift in the peak was observed to 1597 cm−1 from cur-
cumin’s peak at 1627 cm−1, which is indicative of the change
of ketone group to an imine. Compound C3 indicates the
additional peaks of 1521 cm−1 correspond to the nitro group.
And curcumin-semicarbazide (C4) indicated the presence
of its C=N groups at 3012 and 1641 cm−1. On analysis of
Molecular docking
The binding sites of the proteins were located after the grid
generation, and the ligands were docked in the pockets by
extra precision ligand docking. For this study, each protein
was individually evaluated by the three parameters, i.e. Dock
score, XP score and Glide GScore, and the scores were com-
pared to that of curcumin to identify which ligand posed an
ideal ft. The selected proteins have been known to play key
roles in the cancer proliferation pathway. GSK3β (glycogen
synthase kinase 3-beta) has been linked to tumorigenesis
and is a regulator of apoptosis in tumor cells and modulating
centrosome function (Yoshino and Ishioka 2015). Studies
have also indicated that inhibition of GSK3β could lead to
the induction of apoptosis and restraining cell motility (Zeng
et al. 2014). GSK is known to be phosphorylated by AKT,
modulate NF-КB and interact with other crucial signaling
pathways which are vital to cancer cells (Duda 2020). On
comparing the scores generated, isoxazole-curcumin (C2)
has indicated an overall better scoring than other compounds
and curcumin. The synthesized compounds have also dis-
played interactions with key residues of GSK 3β and have
interacted with 6 amino acids which take part in enzyme
inhibitory activities (Fig. 2) (Table 3). Other studies have
carried out a residue wise energy decomposition analysis and
have enlisted interactions with Lys85 as an ideal selective
1
the H NMR spectra, all compounds indicated the OCH3
peak between the ranges of δ 3.79–3.89 ppm. The aromatic
protons could be attributed to δ 6.62–8.02 ppm. Sharp bands
at δ 6.5–9.5 ppm attributed to the N–H proton. The OH
proton could be accounted for at δ 9.11–9.37 ppm. At some
places, the presence of the NH proton could be observed at
δ 12.00–12.81 ppm. 13C NMR also accurately indicated the
presence of the accounted carbons (such as C=O and CH3),
and this could be refected in the mass spectra recorded. The
spectral data and melting points have been comparatively
analyzed along with previous reports and seem to be in con-
gruence with the same (Mishra et al. 2008)(Sahu et al. 2012)
(Changtam et al. 2010).
In silico analysis
ADME studies
ADME profling for the synthesized compounds was facili-
tated via the QikPrep module of Schrödinger. The selected
Table 2 Summary of ADME
SI No Compound Molecular weight QPlogPo/w QPlogS QPPCaco Rule of 5 % Human
profles for the 4 synthesized
oral absorp-
tion
analogues and curcumin
1
2
3
4
5
Curcumin
368.385
364.400
365.385
530.493
407.425
2.628
3.757
3.798
4.557
2.330
−4.249 152.786
−5.256 277.894
−5.302 313.489
0
0
0
2
0
81.424
92.686
93.862
50.314
72.070
C1
C2
C3
C4
−7.085
−4.537
18.313
57.043
QPlogPo/w—Predicted octanol/water partition coefcient logP (acceptable range from −2 to 6.5)
QPlogS—Predicted aqueous solubility logS (acceptable range from −6.5 to 0.5)
QPPCaco—Predicted Caco-2 cell permeability in nm/s (acceptable range:<25 is poor and>500 indicates
high permeability)
Rule of 5—Number of violations of Lipinski’s rule of fve (maximum 4)
% Human oral absorption—Percentage human oral absorption (<25% is poor and>80% is high)
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Chemical Papers
Fig. 2 a GSK-3β protein (PDB
ID—4ACC), b molecular
surface structure of protein, c
representation of docking of the
ligand within suitably identifed
pocket, d ligand interaction of
curcumin, e ligand interaction
of pyrazole-curcumin (C1), f
ligand interaction of isoxazole-
curcumin (C2), g ligand interac-
tion of N-phenyl pyrazole-cur-
cumin (C3), h ligand interaction
of N-amido pyrazole-curcumin
(C4)
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Chemical Papers
Table 3 Summary of scoring, free energy and key interacting residues of the ligands and proteins
Protein
Compounds Dock Score* XP Score* Glide GScore* dG bind
Key Interacting Residues
VAL 135, ASN 186
(MM-
GBSA)*
GSK 3β (PDB ID – 4ACC) Curcumin
C1
−6.181
−7.954
−7.599
−7.954
−7.599
−7.954
−49.40
−39.28
VAL 135, LYS 85, LYS 183, ASP 264,
PHE 67
C2
C3
C4
Curcumin
C1
C2
C3
C4
Curcumin
C1
−8.199
−5.266
−6.351
−1.474
−3.654
−4.596
−2.731
−3.950
−7.404
−7.787
−8.725
−7.153
−8.663
−8.199
−5.266
−6.351
−2.980
−3.654
−4.596
−2.731
−3.950
−8.637
−7.787
−8.725
−7.153
−8.663
−8.199
−5.266
−6.351
−2.980
−3.654
−4.596
−2.731
−3.950
−8.637
−7.787
−8.725
−7.153
−8.663
−45.37
−55.12
−41.02
−32.87
−50.37
−44.12
−46.32
−44.25
−60.36
−51.89
−52.26
−65.66
−63.10
VAL 135, LYS 85, LYS 183, ASP 264
ASP 200, LYS 85, ARG 144
VAL 135, ASP 200, ASP 264
GLU 95, ARG 88, ASP 99
ARG 105, ARG 88
Bcl-2 (PDB ID – 2W3L)
PR (PDB ID – 4OAR)
ARG 88, PHE 63
PHE 63, TYR 67, ARG 105
ALA 108, GLY 77, TYR 67, PHE 63
GLN 725
SER 898, MET 759
PHE 778, GLN 725, GLN 897
PHE 794, THR 894
C2
C3
C4
PHE 778, GLN 725, THR 894, SER 898
VAL valine; ASN asparagine; LYS lysine; ASP aspartic acid; PHE phenylalanine; ARG arginine; GLU glutamic acid; TYR tyrosine; ALA alanine;
GLY glycine; GLN glutamine; SER serine; MET methionine; THR threonine
*All values are expressed as kcal/mol
inhibitor which was seen in the ligand–protein interaction
in 3 (pyrazole-curcumin (C1), isoxazole-curcumin (C2) and
N-phenyl pyrazole-curcumin (C3)) of the synthesized com-
pounds (Arfeen et al. 2015)(Lescot 2005).
score performance than the other compounds screened. The
compounds have also indicated interactions with key resi-
dues such as Met759, Phe778 and Gln725 which has been
enlisted in previous reports (Acharya et al. 2019) (Kiani
2006) (Table 2). In an overall analysis, it has been noted
that most of the bonds formed between the ligands and the
residues are H-bonds or pi-pi stacking and pi-cation bonds
and a trend has been observed in which isoxazole-curcumin
has indicated promising binding scores to the selected pro-
teins which can be a predictive indication of its potential role
as a promising inhibitor. Furthermore, the free energy of the
binding of small ligands to the biological macromolecules
was calculated by the MM-GBSA (molecular mechanics/
generalized born and surface area) method via the Prime
module. The study resulted in indicating that the binding
energies of the tested compounds are comparable to that of
curcumin and sometimes the identifed ligands have more
negative dG bind values which is indicative of a tendency for
stronger binding and formation of a more stable ligand–pro-
tein complex (Basu Mallik et al. 2017).
Bcl-2 (B cell lymphoma 2) family of proteins plays a cen-
tral role in the regulation of cell death mechanisms such as
apoptosis, necrosis and autophagy and work closely along
the intrinsic mitochondrial pathway (Frenzel et al. 2009)
(Pentimalli 2019). Bcl-2 protein expression plays a signif-
cant role in the prognosis of cancers, and since it has the
ability to suppress cell death, it is identifed as a noteworthy
target for cancer drug discovery (Yip and Reed 2008). The
ligands were docked in the site generated by the grid and the
scores were recorded, based on which it was observed that
all the synthesized analogues have indicated a better dock-
ing profle than curcumin. Interactions have been observed
with 7 amino acids and 8 residues and some crucial residue
interactions are Phe63 and Ala108 owing to Bcl-2 highly
conservative amino acid sites (Xu et al. 2019). PR or proges-
terone receptor, belonging to the nuclear receptor family, is a
receptor of paramount importance in breast cancer and is an
important marker in breast cancer prognosis and subsequent
hormone therapy. The overexpression of PR is indicative of
the expression of estrogen and in turn plays a vital role in the
understanding of the initiation and progression of breast can-
cer (Lim et al. 2016). The four synthesized compounds have
indicated docking equivalent, if not better than curcumin and
C2 or isoxazole-curcumin, has indicated the most overall
Cytotoxicity evaluation
To investigate its cytotoxic potential, curcumin and its
synthesized representative heterocyclic analogues were
screened in vitro. Since the proteins selected for the molecu-
lar docking study play a vital role in the signaling pathways
of breast cancer, a breast cancer cell line was chosen for this
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Chemical Papers
study. Breast cancer is one among the leading causes of can-
cer deaths in females, and constant eforts have been made
by researchers in the quest for suitable drugs and therapies
to treat breast cancer. MCF7 cell line is the most ubiqui-
tously used xenograft model of breast cancer for in vitro
studies (Comşa, Cîmpean 2015). MCF7 cells are ideal for
research as they retain several characteristics to the mam-
mary epithelium and are considered to be the frst hormone
responding breast cancer cell line (Camarillo 2015). The
SRB assay was the chosen assay for cytotoxicity screening
because of its high sensitivity, efciency, reproducibility and
better signal-to-noise ratio (Houghton et al. 2007). The aver-
age IC50 (µM) values are presented for three independent
experiments in Table 4.
A dose-dependent analytical study was carried out for cur-
cumin, isoxazole-curcumin and doxorubicin ranging from
7.81 to 500 µM. Isoxazole-curcumin (C2) has indicated bet-
ter activity when compared to curcumin at smaller doses
and comparable activity in higher doses. The highest cell
death of isoxazole-curcumin (C2) could be seen at 125 µM at
85.93%. The study has also revealed that isoxazole-curcumin
displays cell death similar to that of doxorubicin over the
diferent concentrations tested between 31.25 and 250 µM
(Fig. 3).
This study hypothesized that incorporating a heterocyclic
scafold in the central part of curcumin’s structural skeleton
could stabilize the otherwise unsteady nature of curcumin
which is usually credited to the β-diketone moiety (Liang
2009)(Liao 2019). Previous studies have also postulated
that apart from contributing to the improved stability and
bioavailability of the compound it could also lead to better
antitumor and antiangiogenic activity (Jankun et al. 2016).
The selected synthesized compounds had incorporations
of the heterocyclic moiety in the center of the compound
and were screened for their ADME characteristics in silico,
and most of the compounds indicated comparable activity
with curcumin. In the collation of the ADME, molecular
docking and in vitro analysis, isoxazole-curcumin has indi-
cated consistent potency as an anticancer agent. The fve-
membered cyclic N–O analogue has previously indicated
superior activities multiple times. A study was conducted to
analyze the antioxidant potential of curcumin-isoxazole and
curcumin-pyrazole, and it resulted in the fndings that the
two compounds indicated a better antioxidant potential than
the model drug Trolox and the compounds showed better
COX-1/COX-2 selectivity (Selvam et al. 2005). The superior
antioxidant potential of these was further reiterated by other
groups (Lozada-García 2017)(Shaikh 2019). A study by
Narlawar et al. was carried out in which the improved neu-
roprotective activity by isoxazole-curcumin was attributed
to the decreased rotational freedom and absence of stereoi-
somers, along with minimization of metal-chelation proper-
ties as compared to curcumin (Narlawar et al. 2008). In an
antimycobacterial activity investigation, curcumin indicated
a minimum inhibitory concentration of 100 μg/ml against
M. tuberculosis H37Ra strain, whereas curcumin-isoxazole
indicated a value of 1.56 μg/ml. the study further studied
various other isoxazole analogues of curcumin and con-
cluded that the presence of an isoxazole group and its nitro-
gen functionality improves the antimycobacterial activity
by several fold (Changtam et al. 2010). In investigating the
relevance of heterocyclic analogues in anticancer activity,
the relevance of curcumin’s Michael acceptor properties and
its ability to act as an anticancer agent in MCF7 and SKBR3
cell line and the study revealed that isoxazole-curcumin indi-
cated a lower IC50 value than curcumin in MCF7 cancer cell
line (Amolins and Peterson 2009). The mechanistic approach
IC50 value of 0.0652 µM and are in correspondence with
previously reported studies (Zaidi et al. 2011) (Poma 2007).
Among the four synthesized compounds, two compounds
(i.e., isoxazole-curcumin, C2, and N-phenyl pyrazole-cur-
cumin, C3) have indicated a better activity than curcumin.
However, even though the compound N-phenyl pyrazole-
curcumin (C3) has specifed a potent activity of 1.48 µM,
it has indicated the formation of black precipitate on visual
representation. This could be attributed to the non-polar
nature of the compound, the presence of nitro groups and
DMSO solubility (Popa-Burke and Russell 2014) (Ismail
2017). Additionally, on correlation with the ADME stud-
ies, N-phenyl pyrazole-curcumin (C3) has indicated a
molecular weight of 530 which is higher than 500 and is
usually considered to be a disadvantage for pharmaceuti-
cal development purposes (Bos and Meinardi 2000). Hence,
N-phenyl pyrazole-curcumin (C3) has not been considered
as a potent anticancer lead. Further, pyrazole-curcumin (C1)
and N-amido-pyrazole-curcumin (C4) have indicated very
high IC50 values (<50 µM) and can be considered to be less
potent than curcumin. This study is suggestive of the poor
cytotoxic potency of pyrazole-curcumin and its derivatives
when paralleled to curcumin.
The compound isoxazole-curcumin (C2) has indicated
potential inhibitory activity and has displayed a sevenfold
better IC50 value (isoxazole-curcumin (C2), IC50–3.97 µM)
when compared to curcumin (curcumin, IC50–21.89 µM).
Table 4 IC50 values of synthesized compounds, curcumin and doxo-
rubicin obtained from SRB assay
SR. No
Compound name
MCF7, IC50 (µM) ( SD)
1
2
3
4
5
6
Curcumin
C1
C2
C3
21.89 0.18
62.86 0.19
3.97 0.08
1.48 0.11
>80
C4
Doxorubicin
0.0652 0.008
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Chemical Papers
Fig. 3 Efects of curcumin,
isoxazole-curcumin and
doxorubicin on cell death in a
dose-dependent manner. (*A
signifcant statistical diference
of p<0.05 was found for all
concentrations between 15.63
and 500 µM by performing t
test.)
of the interactions between the thiol groups of cells and cur-
cumin and its derivatives was done, and the study resulted in
the fnding that in HA22T/VGH cells, the IC50 of curcumin
and curcumin-isoxazole was 17.4 1.2 and 12.8 1.5 μM,
respectively, indicating isoxazole-curcumin’s better potency
than curcumin (Labbozzetta 2009). In a comparative study
carried out by Chakraborti et al., curcumin-isoxazole and
curcumin-pyrazole not only indicated better anticancer activ-
ity in A549 cells but also indicated a stable IC50 value over
the passage of time as compared to that of curcumin which
increased gradually. This study is indicative of the stability
of these analogues as compared to curcumin in the absence
of serum (Chakraborti et al. 2013). A more relevant study
to elucidate the efects of curcumin and curcumin-isoxazole
on breast cancer cell line MCF7 and its multi-drug-resistant
variant MCF7R was also carried out. The IC50 of curcumin
was 29.3 1.7 μM in MCF7 and 26.2 1.6 μM in MCF7R,
whereas the IC50 of curcumin-isoxazole was 13.1 1.6 μM
in MCF7 and 12.0 2.0 μM in MCF7R. In this study, cur-
cumin-isoxazole analogue has shown more potent antitumor
and molecular activities both in parental and in MDR tumor
cells (Poma et al. 2007).
of curcumin, i.e., isoxazole-curcumin, when tested for its
potential as breast cancer agent owing to its cytotoxic poten-
tial in MCF 7 cell line. To the best of our knowledge, this
is the frst study establishing the role of isoxazole-curcumin
as a potential modulator of key proteins such as GSK-3β,
Bcl-2 and PR which are crucial for the breast cancer pathway
cascade. Hence, to further establish this compound as a lead
molecule, it must be evaluated by gene expression analysis
which can then translate to in vivo studies.
Conclusion
In summary, a series of heterocyclic curcumin analogues
mainly isoxazole-curcumin and pyrazole-curcumin and its
variants were synthesized by a simple one-pot synthesis
method. The structures of these compounds were verifed
by spectrophotometric routine evaluations. The titular com-
pounds were analyzed for their pharmacokinetic profles
and their ability to bind to key proteins such as GSK-3β,
Bcl-2 and PR which are actively involved in the various
cascades of cancer proliferation pathways. The synthesized
compounds have indicated ADME characteristics that lie
within the recommended values. The in silico investigation
consistently identifed the potency of isoxazole-curcumin
across the three proteins when analyzed for its docking
scores and compared with curcumin. The study also revealed
the ligand binding with major amino acids which could lead
to potential regulatory and inhibitory activities when tested
further. The compounds also indicated comparable free
binding energy to curcumin indicative of strong and stable
Hence, the results of this study are in congruence with
previously reported studies in which isoxazole-curcumin has
shown better stability and growth inhibitory potential when
screened for cytotoxicity. In summary, this study speculates
the retention of curcumin’s key residues of its skeletal struc-
ture contributing to curcumin’s multiple potencies and modi-
fying at the site associated with the instability of curcumin.
On incorporation of such modifcations, this study also dis-
plays the improvement of the efcacy of one such analogue
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Chemical Papers
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established the efcacy of isoxazole-curcumin at lower doses
and comparable activity with standard drug doxorubicin at
higher concentrations. Thus, this study is indicative of the
potency of isoxazole-curcumin, which can further act as a
lead for the development of newer breast cancer agents or
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Funding Open access funding provided by Manipal Academy of
Higher Education, Manipal. One of the authors wishes to acknowl-
edge the Dr. TMA Pai PhD Fellowship and KSTePS, DST, Govt. of
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Declarations
Conflicts of interest There are no conficts of interest to declare.
Comşa Ş, Cîmpean AM, Raica M (2015) The Story of MCF-7 breast
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or human subjects performed by any of the authors.
Demain AL, Vaishnav P (2011) Natural products for cancer chemo-
therapy. Microb Biotechnol 4(6):687–699
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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