Curcumin-cinnamaldehyde hybrids as antiproliferative agents against women’s cancer cells

Article abstract of DOI:10.1007/s00044-021-02783-w

Curcumin and cinnamaldehyde are natural products whose antineoplastic activity has been well explored in biological evaluations. However, their poor chemical stability under physiological conditions has been an obstacle to their use as therapeutic agents. Herein, we designed and synthesized two series of curcumin-cinnamaldehyde hybrids by removing reactive functionalities, including β-diketone and aldoxyl moieties. All compounds were evaluated by the MTT assay to determine their antiproliferative activity against women’s cancer cells. Compound 5a (3′-hydroxychalcone) demonstrated potent antiproliferative activity against all cancer cell lines tested, with IC₅₀ values ranging from 2.7 to 36.5 μM. Compound 5a was more active and selective than curcumin and cinnamaldehyde (parent compounds) against the CaSki, SiHa, C33, and A431 cell lines, displaying a higher selectivity index (SI = 8.5) than curcumin (SI = 0.8) toward the non-tumorigenic HaCaT cell line. Clonogenic experiments indicated that compound 5a inhibited A431 colony formation in a concentration-dependent manner. In addition, 5a was more stable than its parent compounds in pH 7.4 at 37 °C. In silico investigations suggested that 5a has good drug-likeness properties. In conclusion, our results indicate the use of curcumin and cinnamaldehyde as parent compounds for the design of hybrids with attractive antiproliferative activity and chemical stability.

Full text of DOI:10.1007/s00044-021-02783-w

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-021-02783-w
ORIGINAL RESEARCH
Curcumin-cinnamaldehyde hybrids as antiproliferative agents
against women’s cancer cells
Daiane B. Anselmo1 Carlos R. Polaquini1 Beatriz C. Marques1 Gabriela M. Ayusso1 Letícia R. Assis1
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Guilherme S. Torrezan1 Paula Rahal2 Ana L. Fachin3 Marília F. Calmon2 Mozart A. Marins3 Luis O. Regasini
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Received: 29 September 2020 / Accepted: 9 August 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
Curcumin and cinnamaldehyde are natural products whose antineoplastic activity has been well explored in biological
evaluations. However, their poor chemical stability under physiological conditions has been an obstacle to their use as
therapeutic agents. Herein, we designed and synthesized two series of curcumin-cinnamaldehyde hybrids by removing
reactive functionalities, including β-diketone and aldoxyl moieties. All compounds were evaluated by the MTT assay to
determine their antiproliferative activity against women’s cancer cells. Compound 5a (3′-hydroxychalcone) demonstrated
potent antiproliferative activity against all cancer cell lines tested, with IC50 values ranging from 2.7 to 36.5 µM. Compound
5a was more active and selective than curcumin and cinnamaldehyde (parent compounds) against the CaSki, SiHa, C33, and
A431 cell lines, displaying a higher selectivity index (SI = 8.5) than curcumin (SI = 0.8) toward the non-tumorigenic HaCaT
cell line. Clonogenic experiments indicated that compound 5a inhibited A431 colony formation in a concentration-dependent
manner. In addition, 5a was more stable than its parent compounds in pH 7.4 at 37 °C. In silico investigations suggested that
5a has good drug-likeness properties. In conclusion, our results indicate the use of curcumin and cinnamaldehyde as parent
compounds for the design of hybrids with attractive antiproliferative activity and chemical stability.
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Keywords Curcumin Cinnamaldehyde Antiproliferative Hybridization Cancer
Introduction
die from cancer than men [1]. Breast cancer is the leading
cancer in women [3] and the triple-negative type is the most
severe because of the lack of targeted pharmacotherapy [4].
Cervical cancer is the fourth most common cancer among
women [3] and its main causative agent is human papillo-
mavirus (HPV). The two main types (HPV-16 and HPV-
18), which are classified as high risk, are responsible
for 70% of cervical cancers [5] and may also be associated
with vulvar cancer [6]. In 2018, there were 44,235 cases
of vulvar cancer and 15,222 deaths due to this cancer
worldwide [7].
Cancer is a leading cause of death worldwide [1] and its
incidence has increased to about 18.1 million new cases and
9.6 million deaths in 2018 [2]. Women are more likely to
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1007/s00044-
021-02783-w.
* Luis O. Regasini
Curcumin (from Curcuma longa) and cinnamaldehyde
(from Cinnamomum species) are used as food additives and
are classified as safe by the FDA [8, 9]. Curcumin and
cinnamaldehyde exert activity against breast [10, 11] and
cervical cancers [12, 13]. The mechanisms of action of
curcumin include the induction of apoptosis, regulation of
the p53 and Rb tumor suppressor genes, and suppression
of HPV E6 and E7 oncogene expression [14]. Cinna-
maldehyde exerts an antiproliferative effect and decreases
the number of breast cancer cells [15]. Although curcumin
and cinnamaldehyde have shown to be promising
luis.regasini@unesp.br
1
Laboratory of Antibiotics and Chemotherapeutics, Department of
Chemistry and Environmental Sciences, Institute of Biosciences,
Humanities and Exact Sciences, São Paulo State University
(Unesp), São José do Rio Preto, SP 15054-000, Brazil
2
Laboratory of Genomic Studies, Department of Biology, Institute
of Biosciences, Humanities and Exact Sciences, São Paulo State
University (Unesp), São José do Rio Preto, SP 15054-000, Brazil
3
Laboratory of Molecular Genetics and Bioinformatics,
Biotechnology Unit, University of Ribeirão Preto (Unaerp),
Ribeirão Preto, SP 14096-900, Brazil

Medicinal Chemistry Research
Scheme 1 Design of curcumin-
cinnamaldehyde hybrids of
series I and II
antineoplastic agents, their poor chemical stability has
impeded advances in clinical studies [16, 17]. In previous
work, we demonstrated the potent antiproliferative activity
of curcumin analogs in which the β-diketone moiety (che-
latogenic group) was replaced by a monoketone [18–20].
We also found that the replacement of the aldoxyl group of
cinnamaldehyde (reactive functionality) by an acet-
ophenone moiety resulted in more stable antibacterial and
antitubercular analogs [21].
In the present study, we selected curcumin and cinna-
maldehyde as parent compounds for molecular hybridiza-
tion (Scheme 1). Two series of curcumin-cinnamaldehyde
hybrids were designed by removing the undesirable func-
tionalities of the parent compounds, including the
β-diketone and aldoxyl moieties of curcumin and cinna-
maldehyde, respectively. We explored the number of
double bonds between aromatic rings A and B, as well as
the role of hydroxyl and methoxyl substituents on ring A.
Series I and II curcumin-cinnamaldehyde hybrids consisted
of α,β-unsaturated ketones, and α,β,γ,δ-unsaturated
ketones, respectively.
group with minor modifications [22]. Appropriate acet-
ophenone derivatives (5 mmol) and benzaldehyde (5 mmol)
were solubilized in ethanol (10 mL). A 5-mL aliquot of
NaOH ethanolic solution (1 mol L−1) was added dropwise
to the reaction medium, which was submitted to magnetic
stirring at room temperature. Series II hybrids (1b–6b) were
synthesized as previously described by our group with
minor modifications [21]. Cinnamaldehyde (6 mmol) was
added to a solution of respective acetophenone (5 mmol) in
ethanol (5 mL). The solution was stirred at room tempera-
ture for 30 min, followed by dropwise addition of 5 mL
NaOH ethanolic solution (1 mol L−1). The progress of the
reactions was monitored using thin-layer chromatography
plates examined under UV light at 254 and 365 nm. All
compounds were purified over a silica gel chromatography
column eluted with mixtures of hexane and ethyl acetate.
The structure of the compounds was identified by analysis
of NMR spectra, which were obtained with a Bruker
Avance III 14.095 T (600 MHz) spectrometer.
Cell lines and cell culture
The antiproliferative activity of the curcumin-
cinnamaldehyde hybrids was evaluated against a panel of
women’s cancer cells, including cervical, breast, and vulvar
cancers. The most active hybrid (5a) was investigated
regarding its toxicity and selectivity toward non-
tumorigenic keratinocytes. We also investigated whether
5a inhibits the formation of vulvar cell colonies. The che-
mical stability of hybrid 5a was evaluated under physio-
logical conditions. In silico analysis using the
Molinspiration® explorer, toolkit contributed to predicting
the drug-likeness properties of 5a.
A panel of human cell lines was used: breast cancer cell
lines MCF-7 (ATCC® HTB-22 TM) and MDA-MB-231
(ATCC® HTB-26 TM); cervical cancer cell lines C33—HPV-
negative (ATCC® HTB-31TM), HeLa—positive for HPV-18
(ATCC® CCL-2TM), CaSki—positive for HPV-16 (ATCC®
CRM-CRL-1550TM), and SiHa—positive for HPV-16
(ATCC® HTB-35TM); vulvar cancer cell line A431
(SIGMA-85090402); immortalized non-tumorigenic human
keratinocyte cells (HaCaT—CLS 300493). The MCF-7 cell
lines were maintained in Eagle’s minimum essential medium
(Gibco Life Technologies). The MDA-MB-231, C33, HeLa,
CaSki, SiHa, and A431 cells were maintained in Dulbecco’s
Modified Eagle Medium (Gibco Life Technologies). All cell
cultures were supplemented with 10% (v/v) heat-inactivated
fetal bovine serum (Cultilab), 100 U/mL penicillin, and
100 μg/mL streptomycin (Invitrogen) in plastic flasks
(Corning) at 37 °C in a humidified 5% CO2 atmosphere.
Experimental
Synthesis of curcumin-cinnamaldehyde hybrids
Curcumin, cinnamaldehyde, doxorubicin, starting reagents,
and solvents were purchased from Merck®. The series I and
II curcumin-cinnamaldehyde hybrids were synthesized by
base-catalyzed Claisen-Schmidt aldol condensation reac-
tions using ethanol as a protic solvent. Series I hybrids
(1a–6a) were synthesized as previously described by our
MTT assay
Cell viability was analyzed using the protocol described by
our group [22, 23]. An aliquot of 1 × 104 cells/well was

Medicinal Chemistry Research
seeded into 96-well cell culture plates in a total volume of
100 µL and incubated for 24 h. The cells were treated with
the parent compounds and their hybrids at concentrations
ranging from 200.0 to 1.56 µM. After 48 h, the medium was
removed, 100 µL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] (1 mg/mL) was added, and
the plates were incubated for 30 min at 37 °C. MTT was
removed, 100 µL DMSO was added, and the mixture was
maintained under agitation for 5 min at 60 rpm. Absorbance
was measured at 570 nm in a FLUOstar Omega microplate
reader (BMG LABTECH). DMSO (0.1%) was used as a
solvent for the compounds and as vehicle control. Doxor-
ubicin was used as the positive control. The results were
plotted as the percentage of cell viability inhibition (CVI)
calculated as follows: CVI (%) = [1 − (absorbance of cells
treated with compounds/absorbance of cells treated with
0.1% DMSO)] × 100. The IC50 values were calculated from
this plot by non-linear regression. The experiments were
carried out in triplicate and three independent events were
used for statistical analysis.
followed by Tukey’s test using the GraphPad Prism 7
(GraphPad Software, Inc.) and Microsoft Office Excel
2007 software. A p value < 0.0001 was considered statisti-
cally significant.
Chemical stability assay
The chemical stability of hybrid 5a (at λmax 314 nm), cur-
cumin (at λmax 426 nm), and cinnamaldehyde (at λmax
288 nm) was analyzed by monitoring their UV–visible
absorption (Perkin Elmer® Lambda 25 UV/Visible Spec-
trophotometer) in phosphate buffer (pH 7.4) at 37 °C. Minor
modifications in this protocol and the detailed procedures
are reported in our previous work [26].
Drug-likeness properties
In silico investigations of the drug-likeness properties of
hybrid 5a were performed using the free Molinspiration®
toolkit to explore Lipinski’s, Veber’s, Muegge’s, and
Ghose’s rules [27].
Clonogenic assay
The clonogenic assay using A431 and HaCaT cell lines was
performed according to the protocols of Franken et al. [24],
with minor modifications [25]. Briefly, 5 × 103 cells/well
were seeded into 12-well plates and incubated for 24 h at
37 °C in a humidified 5% CO2 atmosphere. After incuba-
tion, the cells were treated with hybrid 5a at concentrations
corresponding to their respective ¼ IC50 and IC50 values
obtained in the MTT assays. After 48 h, the cells were
recovered in the compound-free medium for 15 days,
allowing cell growth and the formation of colonies. The
colonies were counted for qualitative analysis. Images of
the colonies were obtained with an iPhone digital camera
(Apple). For quantitative analyses, cell viability was mea-
sured by the MTT assay. DMSO (0.1%) was used as vehicle
control. The experiments were carried out in triplicate and
three independent events were used for statistical analysis.
Results and discussion
Two series of curcumin-cinnamaldehyde hybrids were
synthesized by Claisen-Schmidt condensation reaction, with
yields ranging from 60 to 95% (Scheme 2). All NMR
parameters, including hydrogen and carbon chemical shifts
(δH and δc in ppm), integrations, multiplicities, and cou-
pling constants (J in Hz), corresponded to the designed
structures and agreed with former literature [21, 22]. NMR
spectra and their parameter values are presented in the
Supplementary Material.
The antiproliferative effects of curcumin, cinnamalde-
hyde, and their hybrids were evaluated against human breast
(MCF-7 and MDA-MB-231), cervix (C33, HeLa, CaSki,
and SiHa), and vulvar (A431) cancer cells (Table 1). All cell
lines were selected because of the high incidence of these
cancers in women around the world, highlighting breast and
cervix [3]. Two breast cancer cell lines were used, MCF-7
and MDA-MB-231, which are estrogen-receptor-positive
(ER) and triple-negative (TNBC), respectively [28]. The
chemotherapy of breast cancer depends on the type of cel-
lular receptors. The ER-positive cancer is treated with
Statistical analyses
The experiments were repeated three times and quantitative
data are expressed as the mean SD. Statistical analyses
were performed by one-way analysis of variance (ANOVA)
Scheme 2 Synthesis of
curcumin-cinnamaldehyde
hybrids of series I and II

Medicinal Chemistry Research

Medicinal Chemistry Research
estrogen-receptor modulators, such as tamoxifen or aro-
matase inhibitors [29]. The TNBC cancer treatment is
focused on cytotoxic agents, such as doxorubicin and tax-
anes [30]. Thus, the investigation using two types of breast
cancer cells can contribute to targeted treatments. We also
used four types of cervix cancer cells, which were malig-
nantly transformed by the effects of HPV (HeLa, CaSki,
and SiHa cells) or the non-viral process (C33 cells). The
HeLa cells are positive for HPV-18 and Caski and SiHa
cells are positive for HPV-16. In this context, the HPV-16
and HPV-18 are the most aggressive HPV types in the
induction of high-risk cervical intraepithelial neoplasia and
cervical carcinoma [31].
The antiproliferative potency was expressed as IC50
values (the concentration able to inhibit 50% of cell viabi-
lity), which were obtained by the MTT assay. In addition,
we calculated average IC50 values (mIC50) to analyze the
overall effect of the curcumin-cinnamaldehyde hybrids and
their parent compounds on the panel of women’s cancer
cells. A mIC50 > 100 µM was attributed to hybrids with IC50
values > 100 µM against at least one cancer cell line. Dox-
orubicin was used as a reference antineoplastic drug, dis-
playing IC50 values ranging from 0.02 to 1.87 μM and a
mIC50 value of 1.02.
B in 1a and 1b by α,β-unsaturated and α,β,γ,δ-unsaturated
ketones, respectively. Hybrids 1a and 1b displayed activity
against all cell lines, with IC50 values of 12.3‒66.9 and
27.1‒66.3 μM, respectively. The mIC50 values of hybrids 1a
and 1b were 53.0 and 52.9 µM, respectively. The anti-
proliferative activity of hybrids 1a and 1b encouraged us to
investigate other analogs in an attempt to elucidate the role
of substituents (hydroxyl and methoxyl) on ring A, deriving
structure-activity relationship data.
We compared the activity of 1a and 1b with their
unsubstituted analogs 2a and 2b, respectively. Compound
2b (mIC50 > 100 µM) was less active than 1b (mIC50
=
52.9 µM). Among the cancer cell lines, HeLa and CaSki
cells were not sensitive to 2b (IC50 > 100 µM). On the other
hand, 1a (mIC50 = 53.0 µM) was less active than 2a (mIC50
= 29.5 µM). Taken together, these results suggest that the
OMe and OH substituents at meta and para positions,
respectively, are important for antiproliferative activity.
For a separate analysis of the effects of meta-OMe and
para-OH substituents, we evaluated pairs of compounds 3a/
3b and 4a/4b, respectively. In general, hydroxy-hybrids 4a/
4b was more potent than methoxy-hybrids 3a/3b, demon-
strating mIC50 values of 33.0/49.7 µM and 45.9/100 µM,
respectively. These results suggest that the hydroxyl group
at para position is more important for antiproliferative
activity than the methoxyl group at meta position.
Curcumin (parent compound) displayed activity against
all cell lines, with IC50 values of 10.4–54.1 µM (mIC50
=
26.5 µM). Against MCF-7 (breast cancer cell line), curcu-
min exhibited an IC50 value of 14.9 µM after 48 h. These
results are similar to those reported by other authors. Sri-
vastava and Srivastava found an IC50 value of 18 µM for
curcumin against MCF-7 after 72 h of treatment [10]. Wang
and collaborators reported an IC50 value of 12.1 µM against
HeLa after 72 h of curcumin treatment [32]. Paulraj and co-
authors described IC50 values of 26.7 and 15.8 µM against
HeLa and CaSki cells, respectively, after 72 h of treatment
[12]. Wu and collaborators showed that curcumin (at
15 µM) inhibited A431 cell viability by 54% after 48 h of
treatment [33].
We evaluated the regioisomers of pairs 3a/3b and 4a/4b,
which were designed by replacement of positions of OMe
and OH groups, providing hybrids 6a/6b and 5a/5b,
respectively. Hydroxy-hybrids 5a/5b (mIC50 = 19.3 and
37.9 µM) displayed more potent antiproliferative activity
than methoxy-hybrids 6a/6b (mIC50 = 43.1 and >100 µM),
corroborating the importance of hydroxyl substituents for
bioactivity. Compound 5a exhibited the lowest IC50 value
among the series I and II curcumin-cinnamaldehyde
hybrids (IC50 = 2.7 µM against vulvar cancer cells), as
well as a mIC50 of 19.3 µM. Both values were lower than
those obtained for curcumin (IC50 = 10.4 µM and mIC50
=
On the other hand, cinnamaldehyde, another parent
26.5 µM).
compound of the hybridization design, exhibited IC50
>
In brief, series I hybrids (mIC50 = 19.3–53.0 µM) dis-
played higher antiproliferative activity than series II hybrids
(mIC50 = 37.9–>100 µM). The comparison of these mIC50
values indicated that one double carbon-carbon bond as the
spacer is more important for antiproliferative activity than
two double carbon-carbon bonds. Within this context, series
I hybrids contain an α,β-unsaturated ketone bridge with an
electrophilic β carbon, which can act as a Michael acceptor
in reactions with biological nucleophiles from cancer cells.
This moiety has demonstrated pharmacophoric contribution
to the therapeutic effect of antineoplastic drugs such as
afatinib, osimertinib, and rociletinib [34].
100 µM against all cell lines. Similarly, Wani and colla-
borators reported IC50 values of 160 and 80 µM against
MCF-7 and MDA-MB-231 cells, respectively, after 24 h
[11]. Singh and co-authors described a 53% reduction of
A431 cell viability after treatment with cinnamaldehyde
at 80 µM for 24 h. Treating SiHa cells with the same
concentration, cinnamaldehyde reduced cell viability by
65% [13].
Hybrids 1a (series I) and 1b (series II) were evaluated to
investigate the initial efficacy of the molecular hybridization
design. Both hybrids contain a guaiacol ring (meta-OMe
and para-OH on a benzene ring), which is also found in
curcumin. Guaiacol (ring A) is spaced to unsubstituted ring
Taken together, the bioactivity of curcumin-
cinnamaldehyde hybrids corroborated the use of curcumin

Medicinal Chemistry Research
Table 2 IC50 values (in µM) of parent compounds and 5a against A431
and HaCaT cell lines and respective selectivity index (SI)a
Compounds
HaCaT
A431
SI
Curcuminb
Cinnamaldehydeb
Doxorubicinc
5a
7.6 2.1
>100
10.4 0.5
>100
0.7
nc
0.88 0.23
23.0 3.9
0.05 0.009
2.7 0.2
17.6
8.5
nc not calculated
aSI: the ratio between IC50 for HaCaT and IC50 for A431
bParent compounds
cPositive control
and cinnamaldehyde as parent compounds in molecular
hybridization. This tool has been widely used for the dis-
covery of antineoplastic agents. Wang and co-authors used
isatin and curcumin as parent compounds to design a series
of hybrids with antineoplastic activities. The activity of
curcumin-isatin hybrids was attributed to the presence of an
α,β-unsaturated ketone side chain as Michael acceptor [35].
Thus, our results indicate the potential of natural products
containing Michael acceptor moieties such as zerumbone
[36], xanthohumol [37], withaferin A [38], and celastrol
[39] for the discovery of novel antineoplastic agents.
Hybrid 5a was the most potent series I and II curcumin-
cinnamaldehyde hybrid and was selected for the subsequent
assays. To determine the effect of 5a on non-tumorigenic
cells, we selected a spontaneously transformed human
keratinocyte cell line derived from histologically normal
skin (HaCaT) (Table 2). Hybrid 5a exhibited an IC50 of
23.0 µM against HaCaT cells, which was three times less
toxic than curcumin (IC50 = 7.6 µM). The effect of hybrid
5a and its parent compounds on tumorigenic and non-
tumorigenic cells was compared by determining their SI.
This index was calculated as the ratio between the IC50
values obtained for the HaCaT and A431 cell lines. Cur-
cumin and hybrid 5a exhibited SIs of 0.7 and 8.5, respec-
tively, suggesting that hybrid 5a was ninefold more
selective than curcumin (Table 2). Cinnamaldehyde showed
IC50 values > 100 µM against A431 and HaCaT cells,
which did not allow the calculation of its SI. Mai and col-
laborators suggested compounds with IC50 values ≤ 10 μM
against tumorigenic cells and SI values ≥5 to be selective
and promising hits for in vivo investigation. Hybrid 5a
exhibited IC50 and SI values within the range proposed by
Mai and collaborators and may thus be a promising anti-
neoplastic hit [40].
Fig. 1 Qualitative and quantitative data of the inhibitory effect of
hybrid 5a against cell colony formation of A431 and HaCaT lines,
after 15 days. Cells were treated with hybrid 5a at concentrations
corresponding to their IC50 (2.7 and 23 µM) and ¼ IC50 (0.7 and
6.2 µM) values. Significant differences were determined using
ANOVA followed by Tukey test for results ****p < 0.0001 vs. vehicle
control
hybrid 5a at concentrations corresponding to their IC50 (2.7
and 23 µM) and ¼ IC50 (0.7 and 6.2 µM) (Fig. 1). A431
colony formation was significantly reduced by hybrid 5a in
a concentration-dependent manner when compared to the
vehicle control (0.1% DMSO). The number of A431 colo-
nies decreased by 60% in samples treated with 5a (at ¼
IC50), while no colony formation was observed in samples
treated with the hybrid at IC50. Hybrid 5a reduced the
number of HaCaT colonies by 44% (at ¼ IC50) when
compared to the vehicle control. Similar to the inhibition of
A431 colonies, HaCaT colonies were completely inhibited
by 5a at IC50. Comparison of the inhibitory effect of 5a (at
¼ IC50) against A431 and HaCaT colony formation sug-
gested a higher antiproliferative activity against tumorigenic
than non-tumorigenic cells, demonstrating its selectivity
over a long period of time.
The inhibitory effect of hybrid 5a on the colony forma-
tion of A431 and HaCaT cells was evaluated by the clo-
nogenic assay (Fig. 1). This assay permits the evaluation of
the antiproliferative activity of compounds over long peri-
ods of time [24]. A431 and HaCaT cells were treated with
Compound 5a belongs to the class of chalcones, which
are recognized as privileged structures by medicinal chem-
istry because they have shown numerous interesting bioac-
tivities, including anti-cancer activity [41]. Mahapatra and

Medicinal Chemistry Research
co-authors reviewed the inhibitory potential of chalcones
against various anti-cancer targets, including 5α-reductase,
aromatase, histone deacetylases, sirtuins, proteasome, JAK/
STAT signaling pathway, Wnt signaling pathway, cathe-
psin-K, tubulin, topoisomerases, kinase, and NF-κB. In
addition, chalcones are able to act on multidrug resistance
channels, inhibiting ABCG2, BCRP, and P-glycoprotein
[42]. Moreover, our group has dedicated efforts to describe
other anti-cancer targets of chalcones, including protein p53,
DNAJB1, Sp1, and CRM1 [43–46].
The chemical stability of hybrid 5a and its parent com-
pounds was evaluated at pH 7.4 and 37 °C by UV–Vis
absorption decay (Fig. 2). Curcumin and cinnamaldehyde
were rapidly degraded after 24 h (reduction of 60 and 40%,
respectively). The values of UV–Vis absorption decay of
curcumin were previously described by our group [26]. On
the other hand, hybrid 5a was more stable than its parent
compounds (reduction of 21%). The enhanced stability of
5a under physiological conditions is key to the development
of curcumin-cinnamaldehyde hybrids because their parent
compounds have demonstrated poor chemical stability,
which has been an obstacle to in vivo assays [16, 17].
Drug-likeness properties may be defined as a set of
structural and physicochemical features that indicate whe-
ther or not a hit compound is similar to drugs [47], justi-
fying further chemical and biological studies. Several drug-
likeness sets to filter hit compounds have been described,
including Lipinski’s [48], Veber’s [49], Muegge’s [50, 51],
and Ghose’s rules [52]. Among these sets, the “rule-of-five”
established by Lipinski and collaborators is one of the best-
known and widely used filters [48]. Lipinski’s rule includes
the logarithm values of the compound partition coefficient
between n-octanol and water (log Po/w ≤ 5.0), molecular
weight (MW ≤ 500 Da), the number of hydrogen bond
acceptors (HBA ≤ 10), and the number of hydrogen bond
donors (HBD ≤ 5). Compound 5a demonstrated log Po/w
=
3.31, MW = 224.26 Da, HBD = 1, and HBA = 2. Veber
and collaborators [49] reported two main drug-likeness
properties: the sum of HBA and HBD (HBA + HBD ≤ 12)
and the number of rotatable bonds (NROBT ≤ 10). Hybrid
5a presented HBA + HBD = 3 and NROBT = 3. Muegge
and co-authors developed rapid and simple criteria to select
pharmacophore points. The central point is related to the
number of functionalities and rings on the drug structure,
which can display pharmacophoric contributions [50, 51].
Hybrid 5a exhibited two functional groups, a carbonyl
group (as part of the enone bridge) and a hydroxyl group (as
part of phenolic ring A), which may be pharmacophoric
motifs. Moreover, hybrid 5a displayed two rings, a phenol
(ring A) and an unsubstituted benzene ring (ring B), which
are the result of molecular hybridization between curcumin
and cinnamaldehyde. Ghose and collaborators analyzed the
Comprehensive Medicinal Chemistry (CMC) database and
classified ranges of molecular properties of drugs, including
antineoplastic drugs [52]. Among these, drugs displayed a
total number of atoms in the range of 20–70 (average
number of 48). Compound 5a possesses 29 atoms. Thus,
hybrid 5a did not violate the Lipinski, Veber, Muegge, or
Ghose rules, demonstrating its drug-likeness properties.
Conclusions
We evaluated two series of curcumin-cinnamaldehyde
hybrids as part of the ongoing search for antineoplastic
compounds derived from natural products using molecular
hybridization. Series I hybrids were more potent anti-
proliferative agents than series II hybrids, a finding that
might be related to the presence of the electrophilic β car-
bon. Hybrid 5a was found to be a hit, showing activity
against breast, cervical and vulvar cancer cells and inhi-
biting vulvar colony formation after 15 days. Investigations
using non-tumorigenic cells (HaCaT) indicated that 5a was
ninefold more toxic to cancer vulvar cells than to normal
cells. Chemical stability assays demonstrated 5a to be more
stable than curcumin and cinnamaldehyde under physiolo-
gical conditions. In addition, a brief in silico investigation
confirmed the drug-likeness properties of 5a. These results
indicate the use of curcumin and cinnamaldehyde as parent
compounds for the design of hybrids with attractive anti-
proliferative activity and chemical stability.
Fig. 2 Chemical stability of hybrid 5a and parent compounds (pH 7.4)
by monitoring UV–Vis absorption decay at their respective maximum
absorbance and 37 °C for 24 h. Data are represented as means SEMs
and expressed as a percentage (%)
Acknowledgements The authors gratefully acknowledge financial
support from the Coordination for the Improvement of Higher
Education Personnel (CAPES, Coordenação de Aperfeiçoamento de

Medicinal Chemistry Research
Pessoal de Nível Superior, Finance Code 001), Brazilian Council for
Scientific and Technological Development (CNPq, Conselho Nacional
de Desenvolvimento Científico e Tecnológico, Grants #471129/2013-
5, 306251/2016-7, 429322/2018-6 and 309957/2019-2), and São Paulo
Research Foundation (FAPESP, Fundação de Amparo à Pesquisa do
Estado de São Paulo, Grants #2014/18330-0 and 2018/15083-2). D.B.
A. thanks CAPES (Finance Code 001) for the scholarships granted. All
authors thank the Center for Biomolecular Innovation (FAPESP Grant
#2009/53989-4), National Institute of Science and Technology –
Biodiversity and Natural Products INCT-BioNat (FAPESP #2014/
50926-0 and CNPq #465637/2014-0), and National Institute of Sci-
ence and Genomics Technology for Citrus Improvement INCT-Citrus
(FAPESP #2014/50880-0 and CNPq #465440/2014- 2). The authors
thank Kerstin Markendorf, a native translator, for English translation
and scientific writing contributions.
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Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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