Curcumin-1,2,3-Triazole conjugation for targeting the cancer apoptosis machinery

Article abstract of DOI:10.3390/molecules25133066

The burden of neoplastic diseasesis widely recognized as a severe cause ofmortality. The clinical inadequacy of most anticancer therapeutics urgently prompted intense drug discovery efforts toward the identification of new chemical entities endowed with a

Full text of DOI:10.3390/molecules25133066

molecules
Article
Curcumin-1,2,3-Triazole Conjugation for Targeting
the Cancer Apoptosis Machinery
Francesca Seghetti 1,† , Rita Maria Concetta Di Martino 1,†,‡ , Elena Catanzaro 2,†
Alessandra Bisi 1 , Silvia Gobbi 1 , Angela Rampa 1 , Barbara Canonico 3
,
,
Mariele Montanari 3, Dmitri V. Krysko 4,5,6 , Stefano Papa 3 , Carmela Fimognari 2,
*
and Federica Belluti 1,
*
1
Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna,
Via Belmeloro 6 40126 Bologna, Italy; francesca.seghetti2@unibo.it (F.S.); rita.dimartino@iit.it (R.M.C.D.M.);
,
alessandra.bisi@unibo.it (A.B.); silvia.gobbi@unibo.it (S.G.); angela.rampa@unibo.it (A.R.)
Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Corso d’Augusto 237,
47921 Rimini, Italy; elena.catanzaro2@unibo.it
2
3
4
Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via Ca’ Le Suore, 2, 61029 Urbino, Italy
barbara.canonico@uniurb.it (B.C.); mariele.montanari@uniurb.it (M.M.); stefano.papa@uniurb.it (S.P.)
Cell Death Investigation and Therapy Laboratory, Department of Human Structure and Repair, Ghent
University, 9000 Ghent, Belgium; dmitri.krysko@ugent.be
;
5
6
Cancer Research Institute Ghent, 9000 Ghent, Belgium
Department of Pathophysiology, Sechenov First Moscow State Medical University (Sechenov University),
119146 Moscow, Russia
*
Correspondence: carmela.fimognari@unibo.it (C.F.); federica.belluti@unibo.it (F.B.);
Tel.: +39-0541434658 (C.F.); Tel.: +39-0512099732 (F.B.)
†
‡
These authors contributed equally to this work.
Present address: Department of Drug Discovery and Development, Istituto Italiano di Tecnologia,
via Morego n. 30, 16163 Genova, Italy.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: M. Helena Vasconcelos
Received: 27 May 2020; Accepted: 2 July 2020; Published: 5 July 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract:Theburdenofneoplasticdiseasesiswidelyrecognizedasaseverecauseofmortality. Theclinical
inadequacy of most anticancer therapeutics urgently prompted intense drug discovery efforts toward
the identification of new chemical entities endowed with a potent and safe antitumor profile. In this
scenario, targeting cancer cells apoptosis machinery has emerged as a relevant strategy, useful for
tackling the emergence of drug resistance. On this basis, a small library of naturally inspired hybrid
molecules was obtained by combining, through a click chemistry approach, “privileged” synthons
such as curcumin scaffold and 1,2,3-triazole building block. Compound 1, bearing a para-fluoro phenyl
moiety, showed low-micromolar potency against T acute lymphoblastic leukemia cell growth. More
in-depth biologic studies demonstrated, for this analog, cell death-inducing properties associated with
its capability to simultaneously activate both the receptor and the mitochondrial apoptosis cascades.
This peculiar behavior offers promises for achieving an expanded anticancer effect, namely intense
cytotoxic response coupled with reduced predisposition of chemoresistance insurgence. Altogether,
this study allowed the identification of compound
an anticancer drug candidate.
1 as a lead compound worth to be progressed as
Keywords: apoptosis; cell cycle; cell death; click chemistry; curcumin; flow cytometry; fluorine;
privileged structure; triazole
Molecules 2020, 25, 3066; doi:10.3390/molecules25133066
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3066
2 of 21
1. Introduction
Cancer is expected to become, in every country of the world in the 21st century, the leading
cause of death along with a remarkable obstacle for the life expectancy increasing [ ]. It ranks as one
1
of the principal life-threatening maladies. Among conventional cancer treatments, chemotherapy
is one of the most significant approaches, but severe unwanted effects, namely drug toxicity and
chemoresistance, cause a reduction in both clinical outcome and long-term survival rates. Despite
decades of research, effective therapeutic interventions for several neoplastic diseases still remain
elusive and the identification of new molecules endowed with a potent and safe antitumor effect is an
urgent need.
The induction of apoptosis represents a common mechanism through which several therapeutics
influence cancer cell death. Apoptosis is a very complex cellular phenomenon that can be triggered by
caspases activation by two diverse, but overlapping pathways, the intrinsic and the extrinsic one [2].
The intrinsic pathway, also known as mitochondrial or stress or apoptosis, initiates in response to
different stresses, such as microtubule-targeting and DNA-damaging agents and oncogenes activation.
It is characterized by disruption of the mitochondrial membrane, as a consequence of the liberation of
apoptogenic factors, with cell membrane electrochemical gradient disturbance. In contrast, the extrinsic
death receptor pathway originates upon specific interactions between protein ligands, such as tumor
necrosis factor (TNF) and FAS ligand, and their specific death receptor, followed by activation of
caspase-8. Both routes converge in the activation of common downstream effector caspases (caspase-3,
-6 and -7) leading to apoptosis execution [3].
The aptitude of malignant cells to evade apoptosis is a widely recognized hallmark also involved
in the emergence of drug resistance, so that, targeting apoptosis has emerged as a valuable antitumor
therapeutic strategy [4]. In particular, the capability of an anticancer therapeutic to activate both the
receptor and the mitochondrial apoptosis cascades is, on two grounds, a greatly desired effect. On one
hand, the simultaneous activation of both pathways promotes a more intense cytotoxic response,
if compared to the activation of just one of them. On the other, very often, mutations in cancer cells
make them unable to deliver one or the other pathway [3]. Indeed, apoptosis inducers offer promises
for achieving, in both normal and malignant cells, a decreased toxicity coupled with an improved
therapeutic outcome [5,6].
The anticancer potential of natural products (NPs) has attracted considerable attention in both
industry and academia. Many screening programs proved to be particularly fruitful for the identification
of lead molecules and promising scaffolds for drug discovery purposes, as documented by the current
number of drugs based on NPs in clinical trials or available on the market [
collections based on or inspired by NP structures are expected to be enriched in bioactivity, modulating
multiple biologic processes. Among NPs, the class of molecules bearing an -unsaturated carbonyl
7,8]. In particular, compound
α,β
function, a well-known Michael acceptor system, has been extensively studied and exploited due to
the distinctive capability of interacting with nucleophile sites, namely cysteine and lysine residues,
of different target proteins [9].
Curcumin (Figure 1), the main bioactive component isolated from the rhizome of Curcuma longa,
is one of the most intensely studied phytochemical. Its broad spectrum of beneficial biologic properties
including anti-inflammatory, antiproliferative, and antioxidant proved to be particularly suitable for
treating complex multifactorial diseases, including cancer [10]. The antitumor effect of curcumin is the
consequence of the modulation of a large variety of signaling pathways, likely due to the electrophilic
nature of its α,β-unsaturated carbonyl function. For instance, the induction of caspases, principally
caspase-3 and -8, endoplasmic reticulum and mitochondrial stress pathways, results in a pro-apoptotic
outcome [11].

Molecules 2020, 25, 3066
3 of 21
OH
O
N
O
O
HO
OH
N
N
R
1, 2
A
C-4 position
functionalitation
R =
R1
1, 3, 5: R1 = F
2, 4, 6
OH
O
: R1 = OCH3
O
O
4
HO
curcumin
OH
side aryl ring(s)
modification
B
C
OH
O
OH
O
O
N
N
N
N
N
O
O
HO
O
5, 6
3, 4
N
N
N
N
R
R
R
Figure 1. Chemical structure of curcumin and design strategy for obtaining analogs 1–6.
In spite of the notably favorable antitumor profile, several drawbacks have been reported for
curcumin among which poor chemical stability and aqueous solubility that negatively affect its
bioavailability and cut the therapeutic window [12].
Curcumin, together with a large number of bioactive NPs, such as epigallocatechin gallate, quinone
and rifampicin, has recently been referred to as frequently hitter or pan-assay interfering compound
(PAIN) [13
related to the structural environment. Thus, derivatives properly designed based on PAIN-based
synthons would perform as discriminating bioactive agents rather than reactive chemicals [15 16].
,14]. However, a large body of evidence supported that compound reactivity may be strictly
,
Extensive medicinal chemistry efforts have furnished support to this issue with the identification of a
large variety of successful curcumin-related molecules, obtained upon appropriate modifications of
its basic framework, also confirming the “privileged” character of this pharmacophore, intrinsically
suitable for an efficient interaction with a various biologic targets [17–21]. A fine-tuning of curcumin
covalent reactivity, by modulation of the target engagement rate, could represent a strategy through
which avoiding the drawback of potential promiscuous reactions [22].
In the light of these concepts the main diarylheptadienone curcumin backbone was suitably
modified for achieving specificity while retaining appropriate reactivity. By applying a click chemistry
(CC) approach the curcumin scaffold was combined with a 1,2,3-triazole-based synthon bearing a
para-fluorine and a para-methoxy benzyl moieties. In detail, this nitrogen-based heterocycle, obtained
through the copper-catalyzed Huisgen 1,3-dipolar cycloaddition, could indeed represent a “privileged”
fragment for drug discovery purposes, as it is expected to perform non-covalent interactions with
a number of selected targets, offering an opportunity to obtain lead compounds endowed with an
enriched efficacy profile. Certainly, this strategy has been largely exploited for the design of lead
compounds for therapeutic applications among which the anticancer agents [23,24]. It has been
acknowledged that design strategies based on “privileged sub-substructures” combination are a
powerful tool to generate promising lead compounds [25]. The fluorine functionalization strategy
demonstrated its pivotal role in providing valuable bioactive lead compound in several therapeutic
areas [26–28].
Based on these considerations, a properly substituted benzyl 1,2,3-triazole structural unit was
introduced into different positions of the curcumin scaffold and a small library of analogues was
designed and synthesized (routes A, B and C, Figure 1). This strategy encompassed three main structural

Molecules 2020, 25, 3066
4 of 21
rearrangements: as a first step, by retaining the whole curcumin structure, functionalization of the central
position of the heptatrienone tether was performed (route A, analogs and ); then, by maintaining the
unmodified central linker, modifications progressively involved the aryl side portions of the parent
synthon, i.e., in route B one vanillin-function was kept (analogs and ), whereas in route C both
the lateral moieties were decorated with the selected triazoles (compounds and ). These diversely
arranged curcumin analogs allowed exploring the chemical space around the main skeleton, in the
1
2
3
4
5
6
1–6
light of drawing the essential features for achieving an improvement of the pharmacological profile,
in terms of biologic and physicochemical properties.
The newly synthesized analogs 1–6 were first evaluated for their cytotoxic potential, in comparison
to curcumin. The best performing candidate identified in this assay was then in-depth investigated to
unravel the molecular basis of its anticancer effect in terms of proapoptotic and antiproliferative activities.
Particular attention was paid to disqualify a contingent PAIN behavior due to intrinsic autofluorescence.
2. Results
2.1. Synthesis
The curcumin-based analogs
while, to attain synthons conjugation, the copper-catalyzed azide–alkyne cycloaddition (CuAAC),
between the suitable azide ( and 10) and alkyne ( and 12) derivatives was performed (Schemes 1–3).
As depicted in Scheme 1, pentane-2,4-dione underwent an alkylation reaction with propargyl
bromide, in the presence of K2CO3 as base, to give as mixture of -keto-enol (KE) and diketo
(KK) tautomers; then, a Pabon reaction between and 3-hydroxy-4-methoxy-benzaldehyde (vanillin),
in 1:1.8 stoichiometric ratio, allowed to obtain the curcumin-based intermediate as a mixture of
KE and KK tautomers. Finally, reacted with the azides and 10 through the Huisgen reaction
affording and , respectively as tautomeric couples. Azide intermediates ( and 10) were prepared by
a nucleophilic substitution reaction of the appropriate benzyl halides with NaN3. For the synthesis of
asymmetrical analogs and (Scheme 2) two sequential Pabon reactions were conducted [20], so that
pentane-2,4-dione was first reacted with 4-hydroxy-3-methoxybenzaldehyde in a 1:0.9 stoichiometric
ratio, to give the semisynthetic intermediate 11 20] which was then reacted with the functionalized
aldehydes 13 and 14, to attain the desired asymmetrical compounds and in KE tautomeric
form. The aldehyde intermediates (13 and 14) were prepared via the CuAAC reaction between
the appropriate azides ( and 10) and 4-(prop-2-yn-1-yloxy)benzaldehyde (12) obtained by reacting
4-hydroxybenzaldehyde and propargyl bromide under Williamson ether synthesis conditions. Finally,
Scheme 3 reports the synthetic route for the symmetrical analogs and , starting from pentane-2,4-dione
and the selected aldehyde derivatives (13 and 14), respectively.
The functionalization of the central curcumin framework promoted the partial conversion of the
KE tautomer into the corresponding KK counterpart for derivatives and , (Scheme 1). In detail,
the propynyl-based compound , obtained as a mixture of KE/KK tautomers with a fairly different
chromatographic behavior, was carefully isolated by flash column chromatography, thus obtaining
8-KE in high purity grade. On the other hand, by inserting the benzyl 1,2,3-triazole moiety ( and
), it was not possible to obtain and isolate each single tautomer as chromatographically different
1–6 were synthetized by exploiting the classical Pabon reaction [29],
9
8
7
β
7
8
8
9
1
2
9
3
4
[
3
4
9
5
6
1,
2
8
8
1
2
1
compound. Thus, additional 2D-NMR experiments (1H–1H COSY, H–13C HSQC, NOESY) were
performed on the tautomeric couple of derivative to further confirm compound’s structure (Figures
1
S1–S5, Supplementary Materials). Finally, the introduction of the substituted 1,2,3-triazole ring system
on curcumin side aryl ring(s) (3–6) did not affect the tautomeric conversion, thus maintaining the KE
tautomeric form.

Molecules 2020, 25, 3066
5 of 21
Scheme 1. Synthetic strategy for obtaining clicked curcumin-based compounds
1 and 2. Reagents and
conditions: (i) propargyl bromide, K2CO3, acetone, 80 ◦C; (ii) B2O3, DMF; (iii) B(n- BuO)3; (iv) vanillin,
n-BuNH2, 80 ◦C; (v) HCl, 80 ◦C; (vi) NaN3, DMF, 60 ◦C; (vii) TEA, CuSO4, (+)-sodium L-ascorbate,
DMF, r.t.
Scheme 2. Synthetic strategy for obtaining asymmetric clicked curcumin-based analogs 3 and 4. Reagents
and conditions: (i) B2O3, DMF; (ii) B(n-BuO)3; (iii) n-BuNH2, 80 ◦C; (iv) HCl, 80 ◦C; (v) propargyl bromide,
K2CO3, acetone, reflux; (vi) selected azide (9, 10), TEA, CuSO4, (+)-sodium L-ascorbate, DMF, r.t.

Molecules 2020, 25, 3066
6 of 21
Scheme 3. Synthetic strategy for obtaining symmetric clicked curcumin-based analogs 5 and 6. Reagents
and conditions: (i) B2O3, DMF; (ii) B(n- BuO)3; (iii) n-BuNH2, 80 ◦C; (iv) HCl, 80 ◦C.
2.2. Biologic Evaluation
2.2.1. Cytotoxicity
The newly synthesized curcumin-based derivatives
on T acute lymphoblastic leukemia cell line (CCRF–CEM) after 24- and 48-h treatment and data
reported are in Table 1 and Figure 2. All derivatives, with the exception of , decreased cell viability
in a time-dependent manner (Figure 2). After 24 h treatment, compounds showed IC50 values
in the two-digit- M level, proving to be the most potent of the series, more active than curcumin,
for which an IC50 higher than 150 M was observed. Interestingly, after 48-h treatment, a high potency
was observed for curcumin and derivatives and (IC50 = 10.51, 3.13 and 3.95 M, respectively).
1–6 were evaluated for their cytotoxic effect
3
1–3
µ
µ
1
2
µ
Here, the conjugation of the 1,2,3-triazole-based synthon with the unmodified curcumin framework
proved to be particularly favorable for antiproliferative effect that resulted more than three times
higher than that of curcumin itself. On the other hand, a mild to low antiproliferative effect was
observed for the linearly arranged compounds
In detail, when compared to and , molecules
3
3
–
6
(IC50 values ranging from 38.00 to 93.40
4
µM).
1
2
and , characterized by a mixed vanillin–triazole
fragment combination (i.e., in which one vanillin-based ring was maintained while the other side
aryl function was decorated with the selected 1,2,3-triazole-based synthons), showed a substantial
decreased activity (IC50 = 38.00 and 69.07
best result. The decoration of both aryl moieties with the 1,2,3-triazoles–synthons turned out into
a further reduction of the antiproliferative effect (IC50 = 88.94 and 93.40 M), suggesting that the
µM, respectively) giving the fluorine functionalization the
µ
presence of the vanillin ring is of pivotal importance for inhibition of CCRF–CEM cell proliferation.
This results trend was helpful and supportive for the pursued design strategy.
Based on these data, derivative 1, due to its good low-µM cytotoxic effect was chosen as prototype
compound for deeper evaluations aimed at better understanding its mechanism of action. This analog
may be interesting from a pharmacokinetic perspective, due to the presence of a fluorine atom that was
well-acknowledged to favorably affect pKa, lipophilicity and metabolic stability [30]; moreover, this
substituent, could also improve both binding interactions and selective reactivity of the molecule [31],
thus providing an enhancement of the therapeutic outcome.
To investigate the anticancer potential of compound 1 on CCRF–CEM cells, a screening pipeline
was established in which, the effect on cell-cycle progression was first evaluated. Then, a possible
pro-apoptotic effect was determined by a preliminary study on cell membrane structure and integrity.
Moreover, mitochondrial potential variation and caspase-8 activity were analyzed to evaluate the
impact of
1 on intrinsic and extrinsic apoptosis pathways, respectively. Of note, concentrations that do
not exceed its IC50 value were employed for these studies, to avoid that non-programmed cell death
could take over on a regulated mechanism of action.

Molecules 2020, 25, 3066
7 of 21
Table 1. Cytotoxic effect of compounds
1
–
6
against T acute lymphoblastic leukemia cell line (CCRF–CEM).
CCRF–CEM Cell Line IC50 (µM) a
Compound
R
24 h
48 h
1
2
3
4
5
68.30
3.13
55.63
38.68
>150
>150
3.95
38.00
69.07
88.94
6
122.1
93.40
10.51
curcumin
/
>150
a
IC50 values were calculated by interpolation from the nonlinear dose–response curve of raw data.
Figure 2. Cytotoxic effects normalized on untreated cells after 48 h of CCRF–CEM treatment with all
derivatives and the reference compound curcumin. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
versus untreated cells.

Molecules 2020, 25, 3066
8 of 21
2.2.2. Cell-Cycle Progression
Since during the cytotoxicity assays a decrease in the number of cells was noticed for compound
1
, the ability of this derivative to affect cell proliferation was investigated, and its effect on cell-cycle
progression was analyzed. CCRF–CEM cells were treated with 1.5 and 3.0 µM concentrations of 1 for
24 h; at the end of the treatment, no cell-cycle block was recorded at any tested concentration. However,
as foretold from cytotoxicity experiments, cell death proved to be the primary mechanism of action of
the antitumor potential of analog
1. Indeed, a significant increase in the fraction of the subG0 phase,
which represents the fraction of cells with fragmented DNA, was recorded (Figure 3).
Figure 3. Cell-cycle distribution of CCRF–CEM cells after 24 h treatment with 1. * p < 0.05 versus
untreated cells. C-: negative control.
2.2.3. Phosphatidylserine Exposure and Cell Death Analysis
A loss in plasma membrane asymmetry has been recognized as an early event in dead cells,
accompanied by surface exposure of phosphatidylserine (PS) residues, whereas the integrity of
the membrane remains unhampered. Surface exposed PS can be detected by its affinity for the
phospholipid binding protein annexin V (AV) [32]. On the contrary, both late apoptotic and necroptotic
cells suffer the loss of membrane integrity, resulting thus permeable to vital dyes namely the DNA
intercalator 7-amino-actinomycin (7-AAD) regarded as marker of membrane integrity [33]. Accordingly,
to differentiate cells undergoing early cell death from late cell death (i.e., apoptotic and necroptotic ones),
AV/7-AAD binding assay, followed by flow cytometry analysis, was performed allowing to identify
cells in the early cell death stage (7AAD−/AV+), late cell death stage (7AAD+/AV+) and completely
dead cells (7AAD+/ AV−) [34]. CCRF–CEM cells were treated with 1.5 and 3
compound for 24 h and stained by using AV and 7-AAD. In general, an increase in the fraction of
µM concentrations of
1
AV-positive cells was observed. At the highest tested dose, the percentage of cells at the early stages
of cell death (AV+/7-AAD−) was 53.4. However, 22.3% were the cells in the late stages of cell death
together with necrotic cells (AV+/7-AAD+), compared to 8.0% and 3.3%, respectively, recorded for
untreated cells (Figure 4A).
The cytotoxic and proapoptotic potential of compound
1 was also tested and confirmed on
a different tumor cell line, the acute T cell leukemia Jurkat (Figure S6, Supplementary Materials).
Since PS exposure is an event that characterizes both caspase-dependent and -independent types
of cell death [35], to exclude that beside apoptosis different forms of regulated cell death concur to
compound 1 antitumor potential, we used specific cell death inhibitors: (zVAD-fmk, a pan-caspase
blocker), necroptosis [necrostatin-1 s (Nec1s), a RIPK1 inhibitor] or ferroptosis [ferrostatin-1 (fer-1),
an inhibitor of reactive oxygen species and lipid peroxidation and deferoxamine (DFO), an iron chelator
to characterize apoptosis, necroptosis or ferroptosis, respectively [36
,
37]. Only zVAD-fmk was able to
rescue cells after exposure to compound
mere apoptotic cell death.
1
. Thus, this outcome indicates that compound triggers
1

Molecules 2020, 25, 3066
9 of 21
Figure 4. (A) Percentage and (B
) representative dot plot of 7-AAD−/annexin V−, 7AAD+/annexin V+
and 7-AAD−/annexin V+ cells after 24 h treatment of CCRF–CEM cells with compound
** p < 0.01, **** p < 0.0001 versus untreated cells. C-: control.
1. * p < 0.05,
2.2.4. Mitochondrial Transmembrane Potential
In normal cells, the mitochondrial transmembrane potential (MTP, ∆Ψm) plays a key role in
preserving the function of the respiratory chain, aimed at ATP generation. During apoptosis, MTP
disruption occurs, leading to disturbance of the mitochondrial outer membrane permeabilization and
collapse of the electrochemical gradient across the membrane [38]. Thus, to evaluate the involvement
of the intrinsic pathway, the degree of MTP reduction on CCRF–CEM was determined. The cells,
after treatment with compound
1 at 3 µM, were stained by using the membrane permeable lipophilic
cation 1,10,3,3,30,30-hexamethylindodicarbo-cyanine iodide DiIC1(5) and its mitochondria level was
determined by a flow cytometry analysis. In apoptotic cells, a reduced fluorescence intensity is
the indication of MTP disruption, due to the localization of the dye into the cytoplasm. Figure 5A
shows the percentage of viable cells suffering a loss of MTP after 1.5 and 3
µM treatment (24 h) with
compound . In particular, at 3 M, 55.9% of -treated cells showed a loss in MTP, clearly demonstrating
1
µ
1
the involvement of mitochondria pathway-mediated cell death in its anticancer effect. In accordance,
around 95% of them were in the early cell death phase, as indicated by AV+/7-AAD− assay outcome
(Figure 5B).
2.2.5. Caspase-8
Our data on kinetics of PS exposure and MTP suggest that compound 1 induces apoptotic cell
death in leukemia cells. In order to identify the type of cell death, caspase-8 activity was measured [34].
The initiator caspase-8 could be considered a connection point between receptor and mitochondrial
pathways and its activity allows to discriminate apoptotic cell death from necroptosis [39]. Death
receptors stimulation results in caspase-8 activation, which, in turn, by directly cleaving downstream
effector caspases, can spread apoptosis signal. On the other side, activation of caspase-8 may result
in cleavage of Bid, which translocates to mitochondria to release cytochrome c, thereby initiating

Molecules 2020, 25, 3066
10 of 21
a mitochondrial amplification loop [40]. Thus, the intracellular effect of compound
1
on caspase-8
activity, as representative for the extrinsic apoptosis signaling pathways, was examined and results
were expressed as percentage of apoptotic cells with active caspase-8 (Figure 5C). Compound at
1
the highest tested concentration proved to promote a significant activation (26.7%) of caspase-8 in
apoptotic cells.
These data reveal that derivative
1 activates both apoptosis signaling pathways, potentially
inducing an optimal activation of the apoptotic machinery.
Figure 5.
with decreased mitochondrial potential of
V–phycoerythrin and 7-AAD; ( ) percentage of apoptotic cells with active caspase-8. *** p < 0.001
(
A
) Percentage of 7-AAD+ and 7-AAD− cells and (
B
) representative dot plot of 7-AAD− cells
1-treated CCRF–CEM cells stained with DiIC1(5) and annexin
C
;
**** p < 0.0001 versus untreated cells. CCCP: carbonyl cyanide 3-chlorophenylhydrazone. C-: control.
2.2.6. Physicochemical Properties Prediction
Taking curcumin burdens into account, and in order to preliminarily exclude the possibility that
the synthetized derivatives could act as promiscuous bioactive molecules [41], compounds 1–6 were
analyzed for their assay interference potential according to three different in silico tools: FAFDrugs4
(http://fafdrugs4.mti.univ-paris-diderot.fr/) [42], False Positive Remover (http://www.cbligand.org/

Molecules 2020, 25, 3066
11 of 21
PAINS/) [43] and Aggregator Advisor. (http://advisor.bkslab.org/) [44]. All derivatives were not
recognized as potential PAINS or aggregators (Tables S1–S3, Supplementary Materials).
2.2.7. FACS analysis to Study PAINS-Like Behavior
Autofluorescence is one cause of general assays interference and characterizes at least 3.1%
of PAINS [16]. As many biologic assays exploit fluorescent probes, autofluorescence is one of the
trickiest aspects that mislead about the activity of a compound. False-positive or -negatives are the
severe consequences. Aware of this, and since flow cytometry was used to perform all the biologic
experiments, compounds’ autofluorescence was taken into account and dealt with.
First, we had to get the lay of the land. In the FACS, unstained cell samples (i.e., with no additional
probe) treated with the same concentrations of compounds and time points used for the real biologic
assays were run. Two different lasers 488 nm (blue) or 642 nm (far-red), were employed to excite samples.
Empirically, it was remarked that all unstained compounds emit fluorescence only if excited with the
blue laser. After excitation with this laser, light was filtered and assigned into three emission channels:
green, yellow and red. Cells treated with compounds 1–6 showed high green fluorescence intensity and
low yellow and red ones. With this in mind and since each probe has specific characteristics such as
brightness and intensity, for each assay the setting had to be adjusted, in terms of voltage and the signal
was compensated in order to cancel the autofluorescence of the analyzed compound.
As expected, the green channel was the most laborious to manage and it was adjusted for caspase-8
activity detection since the caspase-8 substrate emits there. Specifically, with the Guava easyCyte
6HT-2L, it was not possible lower autofluorescence of compound 1 enough to allow performing the
experiment. To cope with this situation, the more performant BD FACS Canto cytometer was employed.
Figure S7 (Supplementary Materials) shows the gating strategy for that analysis and the successful
management of green autofluorescence of 1. Furthermore, a similar, very recent methodologic approach
was applied by Sala de Oyanguren and coworkers, who successfully managed the relevant green
autofluorescence of curcumin and the specific signal of a green-emitting dye, as YO-PRO-1 [45].
The interference between any other probe and the compounds’ autofluorescence was handled in the
same way. The only exception was that samples were recorded with the FACS Guava. For instance,
for the proliferation assay, the red-emitting dye 7-AAD was used. Compounds’ red-autofluorescence
was easily displaced and did not interfere with the 7-AAD ones. Over and above, to be sure that
artifacts were not recorded, in parallel, FACS results were compared with those obtained by the eosin
exclusion dye without observing any significant discordance (data not shown). Conducting cell-cycle
analysis was even easier. Indeed, after cell fixation and permeabilization, autofluorescence of derivative
1
was utterly gone (data not shown). Thus, no special adjust setting or compensation was necessary.
Last, for apoptosis analysis, 7-AAD and AV–phycoerythrin, which emits in the yellow channel, were
used. To combine the phycoerythrin signal to those of 7-AAD and compound resulted in perfectly
1
manageable compensation analysis. All probes used for the remaining biologic assays were excited
by 642 nm laser, so we did not experience any trouble. In conclusion, taking into considerations all
these aspects we avoided to produce false results and provided solid data demonstrating that as far as
autofluorescence is concerned, these derivatives do not behave like PAINS.
2.2.8. Chemical Stability Study
Conscious of the chemical stability liability that may affect the curcumin scaffold, the chemical
stability of
1 as mixture of β-keto-enol form and its diketo tautomeric counterpart were studied by
RP-HPLC. In detail, a previously reported methodology was applied, and in the working conditions,
no degradation products formation was observed (Figure S8, Supplementary Materials).
3. Discussion
Herein we reported the design, synthesis and biologic evaluation of a small library of curcumin-
triazole conjugates. The conjugation of the “privileged” curcumin framework and triazole-based

Molecules 2020, 25, 3066
12 of 21
para-fluorine or para-methoxy benzyl moieties, allowed obtaining analogs
1–
6
that were analyzed for
their cell growth inhibitory effects on T acute lymphoblastic leukemia cells. The data showed that
compounds and , both characterized by the unmodified curcumin–scaffold, significantly inhibited
cancer cell line growth with IC50 values in the low- M range. The potency of , proved to be more
1
2
µ
1
than three times higher than that of its precursor curcumin, and this compound was selected for
deeper evaluation in order to assess the molecular basis of its anticancer profile. The induction of
apoptotic process turned out to be the molecular mode by which compound
1 induced cell death. Its
peculiar ability to affect both the intrinsic and the extrinsic pathways represents a significant added
value, useful to achieve a strengthening in anticancer outcome in terms of cytotoxic response and
decreased predisposition of chemoresistance insurgence. However, this evidence would be worthless
if the compound under assessment is a PAIN, in other words if the favorable results are just artefacts.
Chelation, covalent modifications, redox effect, and autofluorescence are only some of the conditions
that push drug candidates into the PAINS qualification [16]. In this study, we focused our attention on
the autofluorescence of the derivatives and we efficiently prevented its interference with the biologic
assays. We analyzed just one part of the “PAIN puzzle” and our results suggest, but do not prove, that
compound 1 is not a PAIN. Certainly, our data represent an interesting premise to keep on exploring
antitumor potential of 1 while keeping our eyes wide open.
4. Materials and Methods
4.1. Chemistry
Chemical reagents and solvents, unless otherwise specified, were employed as commercial
products with a high-grade purity. Reaction courses were monitored by thin-layer chromatography
(TLC) performed on precoated TLC plates (Silica Gel 60 F254, layer 0.20 mm, Merck, Darmstadt,
Germany) and then visualized under a UV lamp (λ = 254 and 365 nm). Chromatographic separations
were performed on silica gel columns by using the flash method (flash column chromatography
(FCC), Kiesegel 40, particle size 0.040–0.063 mm, Merck, Darmstadt, Germany). Melting points were
determined in open glass capillaries, using a Büchi apparatus (Büchi Italia s.r.l., Cornaredo, MI, Italy)
1
and are uncorrected. H NMR and 13C NMR spectra were recorded on Varian INOVA spectrometer
(Scientific instruments, Palo Alto, CA, USA) operating at 400 MHz and 101 MHz, respectively; data are
reported as follows: chemical shift (ppm δ value), multiplicity (indicated as: br., broad signal; s, singlet;
d, doublet; t, triplet; q, quartet; p, quintet; m, multiplet and combinations thereof), coupling constants
(J) in Hertz (Hz) and integrated intensity. Mass spectra were recorded on a Waters ZQ 4000 apparatus
(Waters Alliance, San Diego, CA, USA) operating in electrospray mode (ES). All tested compounds
were found to have >95% purity, as determined by HPLC analysis, performed on a chromatograph
PU-1587 UV model equipped with a 20
Luna 5 m C18 column (150 4.60 mm) as stationary phase and a mixture of H2O/MeCN (50:50, v/v)
for and , H2O/MeCN (35:65, v/v) for as mobile phase; detection at = 254 nm, flow rate of
µL loop valve (Jasco Europe, Italy) by using a Phenomenex
µ
×
1
2
3–
6
λ
1.0 mL/min. Compounds name is in accord with the naming algorithm developed by CambridgeSoft
Corporation used in Chem-BioDraw Ultra 19.1 (Perkin Elmer, Waltham, MA, USA.
4.1.1. Huisgen 1,3-Dipolar Cycloaddition. General Procedure for the Synthesis of Curcumin-Triazole
Conjugates 1, 2 and Intermediates 13, 14
To a solution of the corresponding alkyne
8 or 12 (1.00 mmol) and azide 9 or 10 (1.3 molar equiv)
in DMF (7.50 mL), TEA (0.1 molar equiv) was added dropwise, followed by a water solution of CuSO4
(0.1 molar equiv) and (+)-sodium L-ascorbate (0.5 molar equiv). The resulting mixture was stirred for
2–18 h at rt and the reaction progress was monitored by TLC. Upon reaction completion, the solution
was poured into H2O and the formed solid was filtered off and dried under vacuum.

Molecules 2020, 25, 3066
13 of 21
(1E,4Z,6E)-4-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-5-hydroxy-1,7-bis(4-hydroxy-3-
methoxyphenyl)hepta-1,4,6-trien-3-one (1-KE); (1E,6E)-4-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)
methyl)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (1-KK).
According to the general procedure, 8-KE/KK (0.24 g, 0.60 mmol) and
9 (0.12 g, 0.78 mmol) were
allowed to react for 18 h. The crude product was purified by FCC on silica gel (EP/EtOAc 1:1) and
further crystallized from DCM/EP to obtain 1-KE/KK as a red solid (0.18 g, 1.0:1.1 mixture ratio of
KE/KK tautomer), 41% yield, mp 95–97 ◦C. 1H NMR (CDCl3)
δ 3.38 (d, J = 7.2 Hz, 2H, CH2, KK), 3.91
(s, 12H, OCH3, KE+KK), 4.06 (s, 2H, CH2, KE), 4.76 (t, J = 7.2 Hz, 1H, CH, KK), 5.40 (s, 4H, CH2 N,
KE+KK), 5.92 (br, 2H, OH, KE), 5.98 (br, 2H, OH, KK), 6.67 (d, J = 16.0 Hz, 2H, CH=CH, KK), 6.81–6.88
(m, 4H, Ar and CH=CH, KE), 6.91 (d, J = 8.0 Hz, 4H, H-5, KK+KE), 6.93–6.99 (m, 4H, Ar and H-2,
KE), 7.01 (s, 2H, H-2, KK), 7.03–7.11 (m, 6H, Ar and H-6, KK+KE), 7.14–7.18 (m, 2H, Ar), 7.26 (s, 2H,
CH-triazole, KE+KK), 7.58 (d, J = 15.6 Hz, 2H, CH=CH, KK), 7.67 (d, J = 15.2 Hz, 2H, CH=CH, KE).
13C NMR (CDCl3)
δ 23.4 (CH2, KE), 24.9 (CH2, KK), 53.4 (CH2 N), 53.6 (CH2 N), 56.2 (4C, OCH3), 63.1
(CH), 77.4, 107.8 (2C, CH), 109.8 (4C, CH), 115.0 (4C, CH), 116.2 (d, J = 21.8 Hz, 4C, CH), 117.9 (2C,
CH=CH, KE), 122.0 (2C), 122.1 (2C, CH=CH, KK), 122.2, 123.4 (2C, CH), 124.3 (2C, CH), 126.8 (2C),
127.9 (2C), 129.7 (d, J = 8.4 Hz, 2C, CH), 129.9 (d, J = 8.4 Hz, 2C, CH), 130.6 (d, J = 3.2 Hz), 130.7 (d,
J = 3.2 Hz), 142.5 (2C, CH=CH, KE), 145.3 (2C, CH=CH, KK), 145.4 (2C), 148.2 (d, J = 255.6 Hz, 2C),
147.0 (2C), 148.3 (2C), 148.9 (2C), 183.3 (2C), 194.6 (2C). ESI-MS (m/z) 580 (M + Na).
The same reaction was performed starting from 8-KE and 1-KE/KK was obtained as 1.3:1.0
mixture ratio of KE/KK tautomers.
(1E,4Z,6E)-5-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-4-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-
4-yl)methyl)hepta-1,4,6-trien-3-one (2-KE); (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-4-((1-(4-
methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)hepta-1,6-diene-3,5-dione (2-KK).
Following the general procedure, 8-KE/KK (0.36 g, 0.89 mmol) and 10 (0.19 g, 1.15 mmol) were
allowed to react for 18 h. Purification by FCC (EP/EtOAc 7:3) and further crystallization from DCM/EP
gave 2-KK/KE as a red solid (0.22 g, 1.0:1.3 mixture ratio of KE/KK tautomer), 43% yield, mp 98–100 ◦C.
1H NMR (CDCl3)
δ 3.35 (d, J = 7.2 Hz, 2H, CH2, KK), 3.68 (s, 3H, OCH3, KE), 3.72 (s, 3H, OCH3, KK),
3.90 (s, 12H, OCH3, KE+KK), 4.03 (s, 2H, CH2, KE), 4.74 (t, J = 7.2 Hz, 1H, CH, KK), 5.35 (s, 4H, CH2 N,
KE+KK), 5.88 (br, 2H, OH, KK), 5.92 (br, 2H, OH, KE), 6.65 (d, J = 6.8 Hz, 4H, H-5, KE+KK), 6.68 (s,
4H, H-2, KE+KK), 6.78 (d, J = 8.8 Hz, 4H, H-6, KE+KK), 6.83 (d, J = 15.2 Hz, CH=CH, 2H, KK), 6.89
(d, J = 8.0 Hz, 4H, Ar, KE+KK), 6.95 (s, 1H, CH-triazole, KE), 7.00 (s, 1H, CH-triazole, KK), 7.04 (d,
J = 8.4 Hz, 4H, Ar, KE+KK), 7.11 (d, J = 15.2 Hz, 2H, CH=CH, KE), 7.56 (d, J = 15.6 Hz, 2H, CH=CH,
KK), 7.66 (d, J = 15.6 Hz, 2H, CH=CH, KE). 13C NMR (CDCl3)
δ 23.4, 24.8, 29.8, 53.7, 53.9, 55.3, 55.4,
56.1 (2C), 56.2 (2C), 62.9, 107.8 (2C), 109.8 (2C), 109.9 (2C), 114.4 (2C), 114.5 (2C), 114.9 (2C), 115.0 (2C),
117.9, 121.9, 122.1 (2C), 123.5 (2C), 124.3 (2C), 127.6 (2C), 127.9 (2C), 129.3 (2C), 129.6 (2C), 129.7, 142.5,
145.1 (2C), 145.3 (2C), 147.0 (2C), 148.3 (2C), 149.0 (2C), 149.2 (2C), 159.9 (2C), 183.2 (2C), 194.6 (2C).
ESI-MS (m/z): 592 (M + Na).
The same reaction was performed starting from 8-KE and 2-KE/KK was obtained as 1.0:1.2
mixture ratio of KE/KK tautomers.
4-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (13).
In line with the general procedure, 12 (0.19 g, 1.20 mmol) and
to react for 2 h. The formed solid was collected by vacuum filtration to afford 13 (0.16 g) as pure solid,
43% yield, mp 88–90 ◦C. 1H NMR (CDCl3)
5.25 (s, 2H, OCH2), 5.51 (s, 2H, CH2 N), 7.04–7.09 (m, 4H,
Ar), 7.28 (d, J = 8.4 Hz, 2H, Ar), 7.51 (s, 1H, CH-triazole), 7.82 (d, J = 8.4 Hz, 2H, Ar), 9.88 (s, 1H, CHO).
9 (0.24 g, 1.55 mmol) were allowed
δ

Molecules 2020, 25, 3066
14 of 21
4-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)benzaldehyde (14).
In line with the general procedure, 12 (0.21 g, 1.28 mmol) and 10 (0.27 g, 1.67 mmol) were allowed
to react for 3 h. The formed solid was collected by vacuum filtration to afford 14 (0.25 g) as pure solid,
60% yield, mp 83–85 ◦C. 1H NMR (CDCl3)
δ 3.81 (s, 3H, OCH3), 5.25 (s, 2H, OCH2), 5.48 (s, 2H, CH2
N), 6.90 (d, J = 8.4 Hz, 2H, Ar), 7.08 (d, J = 8.4 Hz, 2H, Ar), 7.24 (d, J = 8.4 Hz, 2H, Ar), 7.51 (s, 1H,
CH-triazole), 7.83 (d, J = 8.4 Hz, 2H, Ar), 9.89 (s, 1H, CHO).
4.1.2. Pabon Reaction. General Procedure for the Synthesis of Curcumin-Triazole Conjugates 3–6 and
Intermediates 8 and 11
A suspension of 2,4-dicarbonyl compound (pentane-2,4-dione, intermediates
and B2O3 (1.0 molar equiv) in DMF (1.50 mL), was heated for 30 min at 80 ◦C, then B(n-BuO)3 (2.0 molar
equiv for and 11, or 4.0 molar equiv for and derivatives) was added and the resulting mixture
was heated at the same temperature for additional 30 min. Afterward, a solution of the suitable aldehyde/s,
(0.9 molar equiv for and 11 or 1.8 molar equiv for and ) in DMF (1.00 mL) was added, followed
by a n-BuNH2 solution (0.2 molar equiv for and 11, or 0.4 molar equiv for and , in 1.00 mL of
7 or 11 [20], 1.00 mmol)
3,
4
5,
6
8
3,
4
5,
6
8
3,
4
5,
6
8
DMF). After stirring at 80 ◦C for 6–8 h, the resulting mixture was cooled to rt, acidified with HCl (0.5 N,
8.0 mL) and stirred at 80 ◦C for further 30 min. On cooling, a solid product precipitates, which was
separated by vacuum filtration. The obtained crude was suspended in H2O, filtered and purified by FCC
and/or crystallization from suitable solvent or mixture of solvents, to afford the title pure compound.
(1E,4Z,6E)-1-(4-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-hydroxy-7-(4-hydroxy-
3-methoxyphenyl)hepta-1,4,6-trien-3-one (3).
Following the general procedure, reaction of intermediate 11 (0.07 g, 0.29 mmol) and the aldehyde
13 (0.08 g, 0.26 mmol) gave the crude which was purified by FCC (PE/EtOAc 6:4) and further
crystallization from EtOH to obtain
NMR (CDCl3) 3.95 (s, 3H, OCH3), 5.23 (s, 2H, CH2 N), 5.52 (s, 2H, OCH2), 5.80 (s, 1H, CH), 6.49 (d,
3
as an orange powder (0.05 g), 40% yield, mp 113–115 ◦C. 1H
δ
J = 15.6 Hz, 1H, CH=CH), 6.50 (d, J = 15.6 Hz, 1H, CH=CH), 6.94 (d, J = 8.4 Hz, 1H, H-5), 6.99 (d,
J = 7.6 Hz, 2H, Ar), 7.06 (s, 1H, H-2), 7.07 (d, J = 7.6 Hz, 1H, H-6), 7.08–7.14 (m, 2H, Ar), 7.24–7.27 (m,
2H, Ar), 7.50 (d, J = 8.0 Hz, 2H, Ar), 7.55 (s, 1H, CH-triazole), 7.60 (d, J = 15.6 Hz, 1H, CH=CH), 7.61 (d,
J = 16.0 Hz, 1H, CH=CH). 13C NMR (CDCl3)
δ 53.7, 56.1, 62.2, 101.5, 109.7, 114.9, 115.3 (2C), 116.4 (d,
J = 21.4 Hz, 2C), 121.9, 122.3, 122.7, 123.1, 127.8, 128.5, 129.9 (2C), 130.0, 130.2 (d, J = 8.1 Hz, 2C), 140.0,
140.8, 144.4, 146.9, 148.0, 159.9, 160.7 (d, J = 249.4 Hz), 183.2, 183.6. ESI-MS (m/z): 550 (M + Na).
(1E,4Z,6E)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-
4-yl)methoxy)phenyl)hepta-1,4,6-trien-3-one (4).
According to the general procedure, reaction of intermediate 11 (0.20 g, 0.86 mmol) and the
aldehyde 14 (0.25 g, 0.77 mmol) gave the crude which was purified by FCC (PE/EtOAc 6:4) and further
crystallization from EtOH to give
NMR (CDCl3) 3.82 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 5.22 (s, 2H, OCH2), 5.48 (s, 2H, NCH2), 5.80 (s,
4
as an orange-brown powder (0.19 g), 45% yield, mp 149–151 ◦C. 1H
δ
1H, CH), 5.88 (br, 1H, OH), 6.49 (d, J = 15.6 Hz, 1H, CH=CH), 6.50 (d, J = 16.0 Hz, 1H, CH=CH), 6.91 (d,
J = 8.4 Hz, 2H, Ar), 6.94 (d, J = 8.0 Hz, 1H, H-5), 6.99 (d, J = 8.8 Hz, 2H, Ar), 7.06 (d, J = 1.6 Hz, 1H, H-2),
7.13 (dd, J = 1.6 and 8.4 Hz, 1H, H-6), 7.25 (d, J = 8.8 Hz, 2H, Ar), 7.50 (d, J = 8.8 Hz, 2H, Ar), 7.51 (s, 1H,
CH-triazole), 7.60 (d, J = 16.0 Hz, 1H, CH=CH), 7.61 (d, J = 16.0 Hz, 1H, CH=CH). 13C NMR (CDCl3)
δ
53.9, 55.5, 56.1, 62.2, 101.5, 109.7, 114.6 (2C), 115.0, 115.3 (2C), 121.7, 122.2, 122.6, 123.1, 126.4, 127.8,
128.4, 129.9 (4C), 140.0, 140.7, 144.1, 147.0, 148.0, 159.9, 160.1, 183.2, 183.6. ESI-MS (m/z): 538 (M—H).

Molecules 2020, 25, 3066
15 of 21
(1E,4Z,6E)-1,7-bis(4-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-hydroxyhepta-1,4,6-
trien-3-one (5).
In line with the general procedure, reaction of pentane-2,4-dione (0.03 mL, 0.29 mmol) and the
aldehyde 13 (0.16 g, 0.52 mmol) gave the crude product which was purified by FCC (PE/EtOAc 1:1) and
further crystallization from DCM/PE to afford
1H NMR (CDCl3)
5.22 (s, 4H, OCH2), 5.52 (s, 4H, CH2 N), 5.79 (s, 1H, CH), 6.51 (d, J = 15.6 Hz, 2H,
CH=CH), 7.00 (d, J = 8.8 Hz, 4H, Ar), 7.06–7.10 (m, 4H, Ar), 7.27–7.30 (m, 4H, Ar), 7.51 (d, J = 8.4 Hz,
4H, Ar), 7.54 (s, 2H, CH-triazole), 7.62 (d, J = 15.6 Hz, 2H, CH=CH). 13C NMR (CDCl3):
53.7 (2C),
5
as a yellow powder (0.07 g), 35% yield, mp 134–136 ◦C.
δ
δ
62.2 (2C), 101.5, 115.3 (4C), 116.35 (d, J = 21.4 Hz, 4C), 121.9, 122.3, 122.7 (2C), 128.5 (2C), 129.9 (4C),
130.0 (2C), 130.2 (d, J = 8.1 Hz, 4C), 140.0, 140.8, 144.4 (2C), 160.0 (2C), 160.7 (d, J = 249.4 Hz, 2C), 183.2,
183.6. ESI-MS (m/z): 687 (M + H).
(1E,4Z,6E)-5-hydroxy-1,7-bis(4-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)hepta-
1,4,6-trien-3-one (6).
Following the general procedure, reaction of pentane-2,4-dione (0.09 mL, 0.86 mmol) and the
aldehyde 14 (0.50 g, 1.55 mmol) gave the crude product which was purified by FCC (PE/EtOAc 1:1)
affording
6 δ 3.82 (s,
as an orange-brown powder (0.18 g), 30% yield, mp 173–175 ◦C. 1H NMR (CDCl3)
6H, OCH3), 5.22 (s, 4H, OCH2), 5.48 (s, 4H, CH2 N), 5.79 (s, 1H, CH), 6.48 (d, J = 15.6 Hz, 2H, CH=CH),
6.88 (d, J = 8.0 Hz, 4H, Ar), 6.97 (d, J = 8.0 Hz, 4H, Ar), 7.25 (d, J = 8.0 Hz, 4H, Ar), 7.48 (d, J = 8.0 Hz,
4H, Ar), 7.48 (s, 2H, CH-triazole), 7.59 (d, J = 15.6 Hz, 2H, CH=CH). 13C NMR (CDCl3)
δ 54.0 (2C), 55.5
(2C), 62.3 (2C), 101.6, 114.7 (4C), 115.4 (4C), 122.3 (2C), 122.6 (2C), 126.5 (2C), 128.5 (2C), 129.9 (8C),
134.7, 140.1, 144.2 (2C), 160.2 (2C), 167.3 (2C), 183.4 (2C). ESI-MS (m/z): 733 (M + Na).
(1E,4Z,6E)-5-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-4-(prop-2-yn-1-yl)hepta-1,4,6-trien-3-one
(8-KE); (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-4-(prop-2-yn-1-yl)hepta-1,6-diene-3,5-dione (8-KK).
Following the general procedure, reaction of (0.36 g, 2.61 mmol) and 3-methoxy-4-
7
hydroxybenzaldehyde (0.71 g, 4.70 mmol) gave a crude product which was purified by FCC (PE/EtOAc
7:3) and further crystallization from EtOH. In this case, the desired compound was isolated as 8-KE
pure and as 1.0:1.7 mixture of 8-KK/KE tautomers (0.61 g), 58% yield, mp 144–147 ◦C. 8-KE: 1H NMR
(CDCl3)
δ
2.15 (t, J = 2.4 Hz, 1H, C
≡
CH), 3.44 (d, J = 2.4 Hz, 2H, CH2C ), 3.96 (s, 6H, OCH3), 5.89
≡
(br, 2H, OH), 6.95 (d, J = 8.0 Hz, 2H,H-5), 6.99 (d, J = 15.4 Hz, 2H, CH=CH), 7.07 (d, J = 2.0 Hz, 2H,
H-2), 7.20 (dd, J = 8.0 and 2.0 Hz, 2H, H-6), 7.73 (d, J = 15.4 Hz, 2H, CH=CH). 8-KK/KE: 1H NMR
(CDCl3)
J = 2.4 Hz, 2H, CH2C
δ
2.15 (t, J = 2.4 Hz, 2H, C
≡
CH, KK+KE), 2.91 (dd, J = 7.4 and 2.4 Hz, 2H, CH2C
≡
, KK), 3.44 (d,
≡
, KE), 3.96 (s, 12H, OCH3, KK+KE), 4.34 (t, J = 7.4 Hz, 1H, CH, KK), 5.89 (br, 2H,
OH, KE), 5.94 (br, 2H, OH, KK), 6.70 (d, J = 15.4 Hz, 2H, CH=CH, KK), 6.91 (d, J = 8.0 Hz, 2H,H-5, KK),
6.95 (d, J = 8.0 Hz, 2H,H-5, KE), 6.99 (d, J = 15.4 Hz, 2H, CH=CH, KE), 7.04 (d, J = 2.0 Hz, 2H, H-2, KK),
7.07 (d, J = 2.0 Hz, 2H, H-2, KE), 7.12 (dd, J = 8.0 and 2.0 Hz, 2H, H-6, KK), 7.20 (dd, J = 8.0 and 2.0 Hz,
2H, H-6, KE), 7.66 (d, J = 15.4 Hz, 2H, CH=CH, KK), 7.73 (d, J = 15.4 Hz, 2H, CH=CH, KE).
3Z,5E-4-hydroxy-6-(4-hydroxy-3-methoxyphenyl)hexa-3,5-dien-2-one (11) [20].
In line with the general procedure, reaction of pentane-2,4-dione (1.00 g, 10.00 mmol) and vanillin
(1.37 g, 9.00 mmol) gave a crude product which was purified by FCC (PE/EtOAc 9.75:0.25), affording
11 as a yellow solid (1.16 g), 55% yield, mp 144–146 ◦C. 1H-NMR (CDCl3)
δ 2.16 (s, 3H, CH3), 3.94 (s,
3H, OCH3), 5.40 (br, 1H, OH), 5.63 (s, 1H, CH), 6.33 (d, J = 16.0 Hz, 1H, CH=CH), 6.92 (d, J = 8.0 Hz,
1H, H-5), 7.02 (d, J = 1.8 Hz, 1H, H-2), 7.09 (dd, J = 8.0 and 1.8 Hz, 1H, H-6), 7.53 (d, J = 16.0 Hz, 1H,
CH=CH). The spectroscopic data of this intermediate are in good agreement with those reported in
the literature.

Molecules 2020, 25, 3066
16 of 21
General Procedure for the Synthesis of Azido Intermediates 9 and 10.
A mixture of the appropriate benzyl halide (1.00 mmol) and NaN3 (10.0 molar equiv) in DMF
(10.0 mL) was stirred at 60 ◦C for 10 h. After cooling to rt the reaction mixture was poured into H2O
and extracted with Et2O (3
over Na2SO4 and concentrated to dryness. The desired azide was employed in the next synthetic step
without any further purification.
×
20.0 mL); the combined organic layers were washed with brine, dried
1-(azidomethyl)-4-fluorobenzene (9)
Following the general procedure and starting from 4-fluorobenzyl bromide (0.66 mL, 5.29 mmol)
1
and NaN3 (3.44 g, 52.90 mmol), the title compound
9
was obtained as a yellow oil, 50% yield. H NMR
(CDCl3) δ 4.29 (s, 2H, CH2 N3), 7.03–7.07 (m, 2H, Ar), 7.24–7.29 (m, 2H, Ar).
1-(azidomethyl)-4-methoxybenzene (10)
Following the general procedure and starting from 4-methoxybenzyl chloride (0.86 mL, 6.38 mmol)
1
and NaN3 (4.15 g, 63.80 mmol), the title compound 10 was obtained as a yellow oil, 67% yield. H
NMR (CDCl3)
2H, Ar).
δ 3.82 (s, 3H, OCH3), 4.28 (s, 2H, CH2 N3), 6.92 (d, J = 8.4 Hz, 2H, Ar), 7.26 (d, J = 8.4 Hz,
4-(Prop-2-yn-1-yloxy)benzaldehyde (12) [46]
To a stirred suspension of 4-hydroxybenzaldehyde (1.00 g, 8.19 mmol) and anhydrous K2CO3
(1.36 g, 9.83 mmol) in acetone (100 mL), propargyl bromide solution (80 wt% in toluene, 1.09 mL,
9.83 mmol) was added dropwise. The reaction mixture was stirred under reflux for 8 h, monitoring
the reaction progress by TLC. The mixture was hot filtered, and the solvent was evaporated under
reduced pressure. The resulting crude product was purified by FCC (PE/EtOAc 4:1) to afford 12 as a
pale yellow solid, 78% yield, mp 75
−
77 ◦C. 1H-NMR (CDCl3)
δ
2.81 (t, J = 2.4 Hz, 1H,
≡
CH), 4.96 (d,
J = 2.4 Hz, 2H, CH2C ), 7.10 (d, 2H, J = 7.6 Hz, Ar), 7.83 (d, 2H, J = 7.6 Hz, Ar), 9.90 (s, 1H, CHO).
≡
The spectroscopic data of this intermediate are in good agreement with those reported in the literature.
(Z)-3-(1-hydroxyethylidene)hex-5-yn-2-one (7-KE); 3-(prop-2-yn-1-yl)pentane-2,4-dione (7-KK).
A stirred suspension of pentane-2,4-dione (2.59 mL, 25.17 mmol), propargyl bromide solution
(80 wt% in toluene, 1.87 mL, 16.86 mmol), K2CO3 (2.32 g, 16.86 mmol) in acetone (150 mL) was heated
at 80 ◦C for 6 h, monitoring the reaction progress by TLC. The mixture was hot filtered, and the solvent
was evaporated under reduced pressure. The resulting crude was purified by FCC (PE/DCM 4:1) to
isolate the desired compound as 7-KE pure and as 1.7:1.0 mixture of 7-KK/KE tautomers (1.40 g), 60%
yield, mp 42–45 ◦C. 7-KE: 1H NMR (CDCl3)
δ
2.03 (t, J = 2.4 Hz, 1H, C
≡
CH), 2.18 (s, 6H, CH3), 2.99 (d,
CH, KK+KE), 2.18 (s,
, KK), 2.99 (d, J = 2.4 Hz,
J = 2.4 Hz, 2H, CH2C
6H, CH3, KE), 2.26 (s, 6H, CH3, KK), 2.70 (dd, J = 2.8 and 7.2 Hz, 2H, CH2C
≡
). 7-KK/KE: 1H NMR (CDCl3)
δ
2.03 (t, J = 2.4 Hz, 2H, C
≡
≡
2H, CH2C≡, KE), 3.86 (t, J = 7.2 Hz, 1H, CH).
4.2. Cell Cultures and Treatments
Human CCRF–CEM acute lymphoblastic leukemia T cells and Jurkat acute T leukemia cells were
purchased from ATCC (Manassas, VA, USA) and propagated in RPMI 1640 (ATCC), supplemented
with 10% heat-inactivated fetal bovine serum (FBS, Biochrome, a division of Harvard Bioscience, Inc.,
Holliston, MA, USA), 1% L-glutamine (Sigma-Aldrich, Saint Louis, MO, USA) and 1% penicillin/
streptomycin solution 100 U/mL (Sigma-Aldrich, Saint Louis, MO, USA). Cells were maintained at
◦
37 C in a humidified atmosphere containing 5% CO2. For all experiments, to keep cells in their
logarithmic growth phase, they were seeded at a density of 0.25
increasing concentrations of the different compounds for different time points, depending on the assay.
×
106 cells/mL and treated with

Molecules 2020, 25, 3066
17 of 21
4.3. Analysis of Cell Viability
CCRF–CEM was treated with increasing concentrations of all compounds or the reference
compound curcumin for 24 and 48 h and analyzed with the Guava ViaCount Reagent (Merck KGaA,
Darmstadt, Germany), following the manufacturer instructions. Briefly, cells were diluted with the
reagent containing 7-AAD and incubated at room temperature in the dark for 5 min before been
recorded at the flow cytometer. Then, to confirm the solidity of these data, viability was also determined
by the eosin exclusion dye. Viable cells were counted using a Bürker chamber and light microscopy
(Nikon eclipse TS2, Amsterdam, Netherlands). The IC50 was calculated by interpolation from the
nonlinear dose–response curve.
Cell death analysis with or without specific inhibitors on Jurkat cells was performed as follow.
Briefly, cells were pretreated for 1 h with or without the pan-caspase inhibitor carbobenzoxy-valyl-
alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD-fmk, 10
the RIPK1 inhibitor necrostatin-1 s (Nec1s, 10 M, Abcam, Cambridge, UK), the inhibitor of ROS and
lipid peroxidation ferrostatin-1 (Fer-1, 1 M, Sigma-Aldrich, St. Louis, MO, USA) or the iron chelator
deferoxamine (DFO, 10 M, Sigma-Aldrich) to, respectively block apoptosis, necroptosis or ferroptosis.
Then, Jurkat were treated for 48 h with compound M and cell death was analyzed through the
µM, Bachem, Bubendorf, Switzerland),
µ
µ
µ
1
6 µ
fluorescent apoptosis/necrosis (FAN) assay [37] using the cell-impermeant fluorescent nuclear probe
Sytox Red (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence was measured on a Tecan
Spark® 20-M multimode microplate reader.
4.4. Analysis of Cell Cycle
After treatment with
1 for 24 h, cells were fixed with 70% ice-cold ethanol and, after washing,
suspended in 200 L of Guava cell cycle reagent (Merck KGaA), containing propidium iodide. At the
µ
end of incubation at room temperature for 30 min in the dark, samples were analyzed via flow cytometry.
4.5. Analysis of Cell Death
After 24 h treatments with
1
, aliquots of 2
×
104 CCRF–CEM were resuspended in 100
µL of RPMI
1640 containing at least 10% of FBS and stained with an equal volume of Guava Nexin Reagent (Merck
KGaA) containing annexin V-phycoerythrin and 7-amino-actinomycin D (7-AAD) and analyzed via
flow cytometry.
4.6. Analysis of Mitochondrial Transmembrane Potential
The mitochondrial transmembrane potential was assessed using MitoProbeTM DilC1(5) Assay kit
(Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA). Briefly, after 24 h from compound
1
treatment, 106 cells were washed and treated with 50-nM DilC1(5) (1,10,3,3,30,30-hexamethylindo
dicarbocyanine iodide) for 20 min at 37 ◦C, 5% CO2. Cells were washed with PBS 1X (Sigma-Aldrich) and
suspended for flow cytometric analysis in a solution composed of equal volumes of RPMI 1640 containing
at least 10% of FBS and Guava Nexin Reagent. CCCP (carbonyl cyanide 3-chlorophenylhydrazone) was
used as positive control.
4.7. Evaluation of Caspase-8 Activity
The activity of caspase-8 cells was assessed using the CaspGLOW Fluorescein Active Caspase
8 Staining Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Briefly, after 24 h from
compound
1 treatment, 106 cells were washed and incubated for 30 min with the reagent. Cells were
washed again, stained with 7-AAD (BD Bioscience, Franklin Lakes, NJ, USA) for 1 h and read flow
cytometrically. Results were expressed as percentage of apoptotic cells with active caspase 8. Apoptotic
cells were identified thanks to the staining with 7-AAD (Supplementary Figure S7D) [47].
On note, compound
1 green autofluorescence was recorded during this assay, but did not affect the
analysis. Gating strategy and autofluorescence handling are showed in Figure S7, Supplementary Materials.

Molecules 2020, 25, 3066
18 of 21
4.8. Flow Cytometry
All procedures except for the analysis of caspase 8 activity were performed with a Guava EasyCyte
6HT-2 L (Merck KGaA) while for caspase 8 FACSCanto cytometer (Becton Dickinson, BD, San Jose, CA,
USA) was used. At least 10,000 events (cells) were recorded for each sample. In all cytofluorimetric
analyses, cell debris and clumps were gated out.
4.9. Statistical Analysis
All biologic experiments were performed at least in triplicates. All results are expressed as mean
±
SEM. Differences among treatments were evaluated by repeated ANOVA, followed by Bonferroni
or Dunnett as posttest, using GraphPad InStat version 6.00 (GraphPad Prism, San Diego, CA, USA).
p < 0.05 was considered significant.
4.10. Chemical Stability Study
The tested derivative 1 was dissolved in DMSO (0.50 mg/mL) and the pH of the solution was adjusted
to 7.4 using 50-mM phosphate. The obtained solutions were then maintained at room temperature and at
70 ◦C (oven) for 48 h and analyzed by RP-HPLC under the conditions reported in the Section 2.2.8.
5. Conclusions
The obtained data reveal that derivative
1 activates both apoptosis signaling pathways, potentially
inducing an optimal activation of the apoptotic machinery. This peculiar property offers promises for
achieving an amplified anticancer potential. Further studies will be also necessary to draw a toxicological
profile of compound
toxicity of curcumin in terms of selectivity for tumor cells and lack of toxicity in vivo
compound to be not less. The complete pharmaco-toxicological profile will support assessing if this
1
in order to elaborate a risk/benefit analysis. Given the high tolerability and low
[
48 50], we expect
–
1
compound could be regarded as suitable for its development as an effective anticancer therapeutics.
Follow-up studies will be devoted to deeply investigate the molecular mode of its cell death induction.
Moreover, an extensive structure–activity relationship (SAR) study is now ongoing focused at a rational
optimization of this lead structure. The outcomes of these efforts will be reported in due time.
Supplementary Materials: The following are available online, Figure S1. 1H NMR (CDCl3, 400 MHz) copy of
compound
1H–13C HSQC copy of compound
Jurkat cells pretreated with zVAD-fmk, Nec1s, DFO or Fer-1 and then treated with compound
S7. Gating strategy (A, B, C, D) and green autofluorescence management (E) in caspase 8 analysis, Figure S8.
HPLC copy for compound ; Table S1 and S2. Physicochemical properties prediction for compounds ; Table S3.
PAINS filters for compounds 1–6.
1
, Figure S2. 13C NMR copy of compound
1
, Figure S3. 1H–1H COSY copy of compound
1
. Figure S4.
, Figure S6. Cell-death analysis of
for 48 h, Figure
1, Figure S5. 2D NOESY copy of compound 1
1
1
1–6
Author Contributions: Conceptualization: F.B. and C.F. (conception of the project: design of the molecules and
the experiments, supervision of all contributions and finalization of the draft manuscript); methodology: F.S.,
R.M.C.D.M., (synthesis and purification of the compounds); methodology: E.C. (antiproliferative and cell death
studies and data analysis); methodology: M.M. (FACS analysis); formal analysis: A.B., S.G., A.R. (structural
characterization of the synthetized compounds); draft preparation: F.S., R.D.M., E.C.; review and editing: D.V.K.,
S.P., B.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the University of Bologna (RFO funds).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018:
GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer
J. Clin. 2018, 68, 394–424. [CrossRef] [PubMed]
2.
Baig, S.; Seevasant, I.; Mohamad, J.; Mukheem, A.; Huri, H.Z.; Kamarul, T. Potential of apoptotic pathway-
targeted cancer therapeutic research: Where do we stand? Cell Death Dis. 2016, 7, e2058. [CrossRef] [PubMed]

Molecules 2020, 25, 3066
19 of 21
3.
4.
5.
6.
Hassan, M.; Watari, H.; AbuAlmaaty, A.; Ohba, Y.; Sakuragi, N. Apoptosis and molecular targeting therapy
in cancer. BioMed Res. Int. 2014, 2014, 150845. [CrossRef] [PubMed]
Fulda, S.; Debatin, K.M. Targeting apoptosis pathways in cancer therapy. Curr. Cancer Drug Targets 2004, 4,
569–576. [CrossRef]
Lim, B.; Greer, Y.; Lipkowitz, S.; Takebe, N. Novel Apoptosis-Inducing Agents for the Treatment of Cancer, a
New Arsenal in the Toolbox. Cancers 2019, 11, 1087. [CrossRef]
Catanzaro, E.; Seghetti, F.; Calcabrini, C.; Rampa, A.; Gobbi, S.; Sestili, P.; Turrini, E.; Maffei, F.; Hrelia, P.;
Bisi, A.; et al. Identification of a new tamoxifen-xanthene hybrid as pro-apoptotic anticancer agent. Bioorg. Chem.
2019, 86, 538–549. [CrossRef]
7.
8.
Harvey, A.L. Natural products in drug discovery. Drug Discov. Today 2008, 13, 894–901. [CrossRef]
Newman, D.J.; Cragg, G.M.; Snader, K.M. Natural products as sources of new drugs over the period
1981–2002. J. Nat. Prod. 2003, 66, 1022–1037. [CrossRef]
9.
Amslinger, S. The tunable functionality of alpha,beta-unsaturated carbonyl compounds enables their
differential application in biological systems. Chem. Med. Chem. 2010, 5, 351–356. [CrossRef]
10. Di Martino, R.M.; Luppi, B.; Bisi, A.; Gobbi, S.; Rampa, A.; Abruzzo, A.; Belluti, F. Recent progress on
curcumin-based therapeutics: A patent review (2012–2016). Part I: Curcumin. Expert Opin. Ther. Pat. 2017
27, 579–590. [CrossRef]
,
11. Willenbacher, E.; Khan, S.Z.; Mujica, S.C.A.; Trapani, D.; Hussain, S.; Wolf, D.; Willenbacher, W.; Spizzo, G.;
Seeber, A. Curcumin: New Insights into an Ancient Ingredient against Cancer. Int. J. Mol. Sci 2019, 20, 1808.
[CrossRef] [PubMed]
12. Anand, P.; Kunnumakkara, A.B.; Newman, R.A.; Aggarwal, B.B. Bioavailability of curcumin: Problems and
promises. Mol. Pharm. 2007, 4, 807–818. [CrossRef] [PubMed]
13. Nelson, K.M.; Dahlin, J.L.; Bisson, J.; Graham, J.; Pauli, G.F.; Walters, M.A. The Essential Medicinal Chemistry
of Curcumin. J. Med. Chem. 2017, 60, 1620–1637. [CrossRef] [PubMed]
14. Bahadori, F.; Demiray, M. A Realistic View on “The Essential Medicinal Chemistry of Curcumin”. ACS Med.
Chem. Lett. 2017, 8, 893–896. [CrossRef]
15. Bajorath, J. Activity artifacts in drug discovery and different facets of compound promiscuity. F1000Research
2014, 3, 233. [CrossRef]
16. Jasial, S.; Hu, Y.; Bajorath, J. How Frequently Are Pan-Assay Interference Compounds Active? Large-Scale
Analysis of Screening Data Reveals Diverse Activity Profiles, Low Global Hit Frequency, and Many
Consistently Inactive Compounds. J. Med. Chem. 2017, 60, 3879–3886. [CrossRef]
17. Di Martino, R.M.C.; Bisi, A.; Rampa, A.; Gobbi, S.; Belluti, F. Recent progress on curcumin-based therapeutics:
A patent review (2012–2016). Part II: Curcumin derivatives in cancer and neurodegeneration. Expert Opin.
Ther. Pat. 2017, 27, 953–965. [CrossRef]
18. Bisceglia, F.; Seghetti, F.; Serra, M.; Zusso, M.; Gervasoni, S.; Verga, L.; Vistoli, G.; Lanni, C.; Catanzaro, M.;
De Lorenzi, E.; et al. Prenylated Curcumin Analogues as Multipotent Tools To Tackle Alzheimer’s Disease.
ACS Chem. Neurosci. 2019, 10, 1420–1433. [CrossRef]
19. Zusso, M.; Mercanti, G.; Belluti, F.; Di Martino, R.M.C.; Pagetta, A.; Marinelli, C.; Brun, P.; Ragazzi, E.; Lo, R.;
Stifani, S.; et al. Phenolic 1,3-diketones attenuate lipopolysaccharide-induced inflammatory response by an
alternative magnesium-mediated mechanism. Br. J. Pharmacol. 2017, 174, 1090–1103. [CrossRef]
20. Di Martino, R.M.C.; De Simone, A.; Andrisano, V.; Bisignano, P.; Bisi, A.; Gobbi, S.; Rampa, A.; Fato, R.;
Bergamini, C.; Perez, D.I.; et al. Versatility of the Curcumin Scaffold: Discovery of Potent and Balanced Dual
BACE-1 and GSK-3 beta Inhibitors. J. Med. Chem. 2016, 59, 531–544. [CrossRef]
21. Tedesco, S.; Zusso, M.; Facci, L.; Trenti, A.; Boscaro, C.; Belluti, F.; Fadini, G.P.; Skaper, S.D.; Giusti, P.;
Bolego, C.; et al. Bisdemethoxycurcumin and Its Cyclized Pyrazole Analogue Differentially Disrupt
Lipopolysaccharide Signalling in Human Monocyte-Derived Macrophages. Mediat. Inflamm. 2018, 2018,
2868702. [CrossRef] [PubMed]
22. Lagoutte, R.; Patouret, R.; Winssinger, N. Covalent inhibitors: An opportunity for rational target selectivity.
Curr. Opin Chem. Biol. 2017, 39, 54–63. [CrossRef] [PubMed]
23. Xu, Z.; Zhao, S.J.; Liu, Y. 1,2,3-Triazole-containing hybrids as potential anticancer agents: Current developments,
action mechanisms and structure-activity relationships. Eur. J. Med. Chem. 2019, 183, 111700. [CrossRef]

Molecules 2020, 25, 3066
20 of 21
24. Uliassi, E.; Piazzi, L.; Belluti, F.; Mazzanti, A.; Kaiser, M.; Brun, R.; Moraes, C.B.; Freitas, L.H.; Gul, S.;
Kuzikov, M.; et al. Development of a Focused Library of Triazole-Linked Privileged-Structure-Based Conjugates
Leading to the Discovery of Novel Phenotypic Hits against Protozoan Parasitic Infections. Chemmedchem 2018
,
13, 678–683. [CrossRef] [PubMed]
25. Spear, K.L.; Brown, S.P. The evolution of library design: Crafting smart compound collections for phenotypic
screens. Drug Discov. Today Technol. 2017, 23, 61–67. [CrossRef] [PubMed]
26. Gillis, E.P.; Eastman, K.J.; Hill, M.D.; Donnelly, D.J.; Meanwell, N.A. Applications of fluorine in medicinal
chemistry. J. Med. Chem. 2015, 58, 8315–8359. [CrossRef]
27. Bonvicini, F.; Gentilomi, G.A.; Bressan, F.; Gobbi, S.; Rampa, A.; Bisi, A.; Belluti, F. Functionalization of the
Chalcone Scaffold for the Discovery of Novel Lead Compounds Targeting Fungal Infections. Molecules 2019
24, 372. [CrossRef]
,
28. Ortalli, M.; Ilari, A.; Colotti, G.; De Ionna, I.; Battista, T.; Bisi, A.; Gobbi, S.; Rampa, A.; Di Martino, R.M.C.;
Gentilomi, G.A.; et al. Identification of chalcone-based antileishmanial agents targeting trypanothione
reductase. Eur. J. Med. Chem. 2018, 152, 527–541. [CrossRef]
29. Pabon, H.J. A synthesis of curcumin and related compounds. Recl. Trav. Chim. Pays-Bas. 1964, 83, 379–386.
[CrossRef]
30. Agnati, L.F.; Guidolin, D.; Cervetto, C.; Borroto-Escuela, D.O.; Fuxe, K. Role of iso-receptors in receptor-
receptor interactions with a focus on dopamine iso-receptor complexes. Rev. Neurosci. 2016, 27, 1–25.
[CrossRef]
31. Hagmann, W.K. The many roles for fluorine in medicinal chemistry. J. Med. Chem. 2008, 51, 4359–4369.
[CrossRef] [PubMed]
32. Cornelissen, M.; Philippé, J.; De Sitter, S.; De Ridder, L. Annexin V expression in apoptotic peripheral blood
lymphocytes: An electron microscopic evaluation. Apoptosis 2002, 7, 41–47. [PubMed]
33. Krysko, O.; Aaes, T.L.; Kagan, V.E.; D’Herde, K.; Bachert, C.; Leybaert, L.; Vandenabeele, P.; Krysko, D.V.
Necroptotic cell death in anti-cancer therapy. Immunol. Rev. 2017, 280, 207–219. [CrossRef] [PubMed]
34. Krysko, D.V.; Diez-Fraile, A.; Criel, G.; Svistunov, A.A.; Vandenabeele, P.; D’Herde, K. Life and death of
female gametes during oogenesis and folliculogenesis. Apoptosis 2008, 13, 1065–1087. [CrossRef] [PubMed]
35. Kloditz, K.; Fadeel, B. Three cell deaths and a funeral: Macrophage clearance of cells undergoing distinct
modes of cell death. Cell Death Discov. 2019, 5, 65. [CrossRef] [PubMed]
36. Demuynck, R.; Efimova, I.; Lin, A.; Declercq, H.; Krysko, D.V. A 3D Cell Death Assay to Quantitatively
Determine Ferroptosis in Spheroids. Cells 2020, 9, 703. [CrossRef]
37. Grootjans, S.; Hassannia, B.; Delrue, I.; Goossens, V.; Wiernicki, B.; Dondelinger, Y.; Bertrand, M.J.; Krysko, D.V.;
Vuylsteke, M.; Vandenabeele, P.; et al. A real-time fluorometric method for the simultaneous detection of cell
death type and rate. Nat. Protoc. 2016, 11, 1444–1454. [CrossRef]
38. Tait, S.W.; Green, D.R. Mitochondria and cell death: Outer membrane permeabilization and beyond. Nat. Rev.
Mol. Cell Biol. 2010, 11, 621–632. [CrossRef]
39. Kaczmarek, A.; Vandenabeele, P.; Krysko, D.V. Necroptosis: The release of damage-associated molecular
patterns and its physiological relevance. Immunity 2013, 38, 209–223. [CrossRef]
40. Fulda, S.; Debatin, K.M. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene
2006, 25, 4798–4811. [CrossRef]
41. Aldrich, C.; Bertozzi, C.; Georg, G.I.; Kiessling, L.; Lindsley, C.; Liotta, D.; Merz, K.M.; Schepartz, A.; Wang, S.
The Ecstasy and Agony of Assay Interference Compounds. ACS Med. Chem. Lett. 2017, 8, 379–382. [CrossRef]
[PubMed]
42. Lagorce, D.; Sperandio, O.; Baell, J.B.; Miteva, M.A.; Villoutreix, B.O. FAF-Drugs3: A web server for
compound property calculation and chemical library design. Nucleic Acids Res. 2015, 43, W200–W207.
[CrossRef] [PubMed]
43. Baell, J.B.; Holloway, G.A. New substructure filters for removal of pan assay interference compounds (PAINS)
from screening libraries and for their exclusion in bioassays. J. Med. Chem. 2010, 53, 2719–2740. [CrossRef]
[PubMed]
44. Irwin, J.J.; Duan, D.; Torosyan, H.; Doak, A.K.; Ziebart, K.T.; Sterling, T.; Tumanian, G.; Shoichet, B.K.
An Aggregation Advisor for Ligand Discovery. J. Med. Chem. 2015, 58, 7076–7087. [CrossRef]

Molecules 2020, 25, 3066
21 of 21
45. Sala de Oyanguren, F.J.; Rainey, N.E.; Moustapha, A.; Saric, A.; Sureau, F.; O’Connor, J.E.; Petit, P.X.
Highlighting Curcumin-Induced Crosstalk between Autophagy and Apoptosis as Supported by Its Specific
Subcellular Localization. Cells 2020, 9, 361. [CrossRef]
46. Giguere, J.B.; Thibeault, D.; Cronier, F.; Marois, J.S.; Auger, M.; Morin, J.F. Synthesis of 2- and 3 rotaxanes
through Sonogashira coupling. Tetrahedron Lett. 2009, 50, 5497–5500. [CrossRef]
47. Riccardi, C.; Nicoletti, I. Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat. Protoc.
2006, 1, 1458–1461. [CrossRef]
48. Soleimani, V.; Sahebkar, A.; Hosseinzadeh, H. Turmeric (Curcuma longa) and its major constituent (curcumin)
as nontoxic and safe substances: Review. Phytother Res. 2018, 32, 985–995. [CrossRef]
49. Syng-Ai, C.; Kumari, A.L.; Khar, A. Effect of curcumin on normal and tumor cells: Role of glutathione and
bcl-2. Mol. Cancer Ther. 2004, 3, 1101–1108.
50. Rezaee, R.; Momtazi, A.A.; Monemi, A.; Sahebkar, A. Curcumin: A potentially powerful tool to reverse
cisplatin-induced toxicity. Pharm. Res. 2017, 117, 218–227. [CrossRef]
Sample Availability: Samples of the compound 1 is available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).