Targeting the MDM2-p53 protein-protein interaction with prenylchalcones: Synthesis of a small library and evaluation of potential antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2018.07.037

Prenylation of several bioactive scaffolds is a very interesting strategy used in Medicinal Chemistry in order to improve biological/pharmacological effects. A small library of prenylchalcones was synthesized and evaluated for the ability to inhibit the MDM2-p53 interaction using a yeast-based assay. The capacity of all synthesized prenylchalcones and their non-prenylated precursors to inhibit the growth of human colon tumor HCT116 cells was also evaluated. The obtained results led to the identification of a hit compound, prenylchalcone 2e, which behaved as potential inhibitor of the MDM2-p53 interaction in yeast, and showed improved cytotoxicity against human tumor cells expressing wild-type p53, including liver hepatocellular carcinoma HepG2, breast adenocarcinoma MCF-7, and malignant melanoma A375 cells. In colon cancer cells, it was also shown that the growth inhibitory effect of prenylchalcone 2e was associated with the induction of cell cycle arrest, apoptosis, and increased protein expression levels of p53 transcriptional targets. Moreover, computational docking studies were performed in order to predict docking poses and residues involved in the MDM2-p53 potential interaction.

Full text of DOI:10.1016/j.ejmech.2018.07.037

Accepted Manuscript
Targeting the MDM2-p53 protein-protein interaction with prenylchalcones: Synthesis
of a small library and evaluation of potential antitumor activity
Pedro Brandão, Joana B. Loureiro, Sylvie Carvalho, Meriem Hadjer Hamadou, Sara
Cravo, Joana Moreira, Daniela Pereira, Andreia Palmeira, Madalena Pinto, Lucília
Saraiva, Honorina Cidade
PII:
S0223-5234(18)30593-2
10.1016/j.ejmech.2018.07.037
EJMECH 10572
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 March 2018
Revised Date: 23 May 2018
Accepted Date: 15 July 2018
Please cite this article as: P. Brandão, J.B. Loureiro, S. Carvalho, M.H. Hamadou, S. Cravo, J. Moreira,
D. Pereira, A. Palmeira, M. Pinto, Lucí. Saraiva, H. Cidade, Targeting the MDM2-p53 protein-protein
interaction with prenylchalcones: Synthesis of a small library and evaluation of potential antitumor
activity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.037.
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Docking
Yeast
Human Tumor Cell lines
p53
MDM2
2e
MDM2
Compounds 1c, 1h, 2e, 2i, and 3e
G2/M-phase cell cycle arrest
apoptotic cell death
2e
activation of the p53 pathway
Human Tumor Cell lines

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Targeting the MDM2-p53 protein-protein interaction with prenylchalcones:
synthesis of a small library and evaluation of potential antitumor activity
Pedro Brandãoa,#, Joana B. Loureirob,#, Sylvie Carvalhoa, Meriem Hadjer Hamadoub,
Sara Cravoa,c, Joana Moreiraa, Daniela Pereiraa, Andreia Palmeiraa,c, Madalena Pintoa,c,
Lucília Saraivab,*, Honorina Cidadea,c,**
aLaboratório de Química Orgânica e Farmacêutica, Departamento de Ciências
Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo
Ferreira 228, 4050-313 Porto, Portugal
bLAQV/REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências
Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo
Ferreira 228, 4050-313 Porto, Portugal
cCentro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR), Universidade
do Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n
4450-208 Matosinhos, Portugal
Abstract
Prenylation of several bioactive scaffolds is a very interesting strategy used in
Medicinal Chemistry in order to improve biological/pharmacological effects. A small
library of prenylchalcones was synthesized and evaluated for the ability to inhibit the
MDM2-p53 interaction using a yeast-based assay. The capacity of all synthesized
prenylchalcones and their non-prenylated precursors to inhibit the growth of human
colon tumor HCT116 cells was also evaluated. The obtained results led to the
identification of a hit compound, prenylchalcone 2e, which behaved as potential
inhibitor of the MDM2-p53 interaction in yeast, and showed improved cytotoxicity
against human tumor cells expressing wild-type p53, including liver hepatocellular
carcinoma HepG2, breast adenocarcinoma MCF-7, and malignant melanoma A375
cells. In colon cancer cells, it was also shown that the growth inhibitory effect of
prenylchalcone 2e was associated with the induction of cell cycle arrest, apoptosis, and
increased protein expression levels of p53 transcriptional targets. Moreover,
computational docking studies were performed in order to predict docking poses and
residues involved in the MDM2-p53 potential interaction.

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Keywords: Prenylated chalcones; MDM2-p53 inhibitors; antitumor activity.
*Corresponding author email adress: lucilia.saraiva@ff.up.pt (L. Saraiva) and
hcidade@ff.up.pt (H. Cidade)
#Authors contributed equally to this work.

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1. Introduction
The tumor suppressor protein p53 acts as a transcription factor, inducing the
expression of several downstream targets that play a central role in regulation of cell
cycle, apoptosis, and DNA repair, among other mechanisms [1,2]. Upon cellular stress
signals, the activation of the p53 pathway may compromise the tumor development and
growth, preventing the proliferation of damaged cells with oncogenic potential. TP53 is
one of the most relevant tumor suppressor genes, which is frequently mutated in human
cancers. About half of all cancers retain wild-type (wt) p53, however the p53 pathway is
inactivated due to the overexpression of endogenous negative regulators, particularly
murine double minute 2 (MDM2) [3,4]. The oncoprotein MDM2 binds p53 and
negatively regulates its activity by inhibiting p53 transcriptional activity and
translocation from the nucleus to the cytoplasm, and by enhancing p53 degradation via
ubiquitin-proteasome pathway (Figure 1S). The disruption of the MDM2-p53
interaction with small molecules to activate the p53 pathway in cancer cells harboring
wt p53 constitutes an appealing strategy for cancer therapeutics [5,6]. The first
successful sustentation of such therapeutic approach came with the discovery of the cis-
imidazoline nutlin-3a (Figure 1S). Nutlin-3a potently binds to the hydrophobic cleft in
the N-terminus of MDM2, preventing its association with p53 [7]. It is highly effective
killing wt p53-expressing cancer cells, both in vitro and in vivo in preclinical models
[7]. Nutlin-3a opened the way to the development of an impressive number of MDM2-
p53 interaction inhibitors, from a huge variety of chemical families, entering some of
them into clinical trials [6].
Flavonoids represent an outstanding class of naturally occurring compounds with
interesting biological activities [8,9]. Chalcones represent one of the major classes of
flavonoids that have long been recognized for their antitumor activity through the
interference with several molecular targets [10,11]. Recently, our research group have
shown that the antitumor activity of chalcone derivatives can be related with activation
of a p53-dependent pathway [12]. Additionally, it has been demonstrated that some
chalcone derivatives can disrupt the MDM2-p53 complex with the subsequent release of
p53 [13,14].
Among flavonoids, natural prenylated derivatives have attracted the attention of the
scientific community because of their myriad biological activities [15-20]. In fact, it has

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been demonstrated that isoprenylation of flavonoids significantly increased their growth
inhibitory effect on human tumor cell lines [21,22]. In a recent work from our group, it
was shown that the prenylation of a natural occurring chalcone (chalcone 1a, scheme 1)
led to the O-prenyl derivative 2a (scheme 1) with notably increased cytotoxic activity
against tumor cells [22]. In addition, the investigation of the molecular mechanism
underlying the improved tumor cytotoxicity of prenylchalcone 2a revealed that
prenylation could be a determinant factor for the enhancement of this effect by
improving their ability to disrupt the MDM2-p53 interaction [13]. Together, these
studies support the idea that prenylchalcone 2a represents a starting basis for the design
of new MDM2-p53 interaction inhibitors with improved antitumor properties.
The present work reports the identification of five (1c, 1h, 2e, 2i, and 3e) potent
growth inhibitors of human colon adenocarcinoma HCT116 cell lines with promising
MDM2-p53 inhibitory effect. Among these compounds, prenylchalcone 2e revealed
potent growth inhibitory activity against human cancer cells expressing wt p53 through
potential activation of p53 by disrupting the interaction with MDM2.
2. Results and Discussion
2.1. Chemistry
Analogues of chalcone 2a were synthesized according to the strategy illustrated
on Scheme 1. Firstly, chalcones 1a-1j were prepared by base-catalyzed aldol reactions
of 2-hydroxy-4,6-dimethoxyacetophenone with appropriately substituted benzaldehydes
as building blocks by MW synthesis. Among these compounds, chalcone 1j was here
described for the first time. For derivatives 1a-1i, although the synthesis by base-
catalyzed aldol reactions have been previously reported, using room temperature or
conventional heating, the use of MW irradiation was never described before. Using MW
irradiation, better yields and shorter reaction times were achieved when compared with
the reactions already reported. The prenylation of these chalcones was then carried out
by the reaction of the building block (1a-1j) with prenyl bromide in the presence of
tetrabutylammonium hydroxide at room temperature (Scheme 1). Although the reaction
conditions are similar to those previously reported for the synthesis of compound 2a
[22], the prenylation was now carried out using a mixture of chloroform/toluene instead

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of dichlorometane/toluene as solvent. Moreover, the reactions were conducted using an
amount of non-prenylated chalcones five-fold higher compared to previously reported
[22].
Scheme 1. Synthesis of chalcones. The numbering used concerns the NMR
assignments.
As shown on Scheme 1, using these reaction conditions in addition to the 2’-O-
prenyl derivatives 2a-2j obtained as major products, compounds possessing a prenyl
group at C-3’ or C-5’ (compounds 3a-3e) have been obtained as by-products. The
synthesis of these C-prenylated derivatives 3a-3e could be explained by the
delocalization of phenoxide anion within the aromatic ring. However, for the
prenylation of chalcone 1i, two C-prenylated derivatives without an O-prenyl group at
C-2’ (chalcones 3d and 3e) were synthesized as by-products.
To improve the yields of the monoprenylated derivative 2i, the prenylation of
chalcone 1i was performed using conventional heating and MW irradiation as described
in Table 1. The reaction time was reduced from 25 h in the room temperature to 8 h in
the conventional heating and to 0.75 h with MW. Using conventional heating and MW
irradiation the yields of the monoprenylated compound 2i were fairly increased when
compared with the reaction at room temperature. Comparing MW irradiation with
conventional heating these results confirmed that MW irradiation improved yields and
selectivity in shorter reaction times. In fact, using this methodology the yield of the

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monoprenylated compound 2i was increased, and the yield of diprenylated compound
3d was moderately reduced, being derivative 3e not isolated (Table 1).
Table 1. Comparison of yields obtained for the prenylation of chalcone 1i using
different reaction conditions.
Yield (%)
Reaction Temperature
Solvent
time (h)
(ºC)
2i
4
3d
2
3e
2
chloroform/Toluene 10:7
chloroform/Toluene 10:7
chloroform /Toluene 10:7
25
8
rt
50
32
50
8
9
0.75
50, 200 W
3
-
The structure elucidation of compounds 1a-1e, 1g-1i and 2a was established by
1H and 13C NMR techniques and data were in accordance to the previously reported [22-
28]. The structure elucidation of new compounds 2b-2j and 3a-3e was established on
13
basis of HRMS and NMR techniques. C NMR assignments were determined by 2D
heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond
correlation (HMBC) experiments. The coupling constants of the vinylic system (JHα-Hβ
=
15.6-16.2 Hz) confirm their (E)-configuration.
2.2. Biological activity evaluation
2.2.1. Screening for potential inhibitors of the MDM2-p53 interaction using a
yeast-based assay
Our group has previously developed a yeast-based screening assay to search for
potential inhibitors of the MDM2-p53 interactions [29-32]. In this assay, the expression
of human wt p53 in yeast causes a marked growth arrest, which is abolished by co-
expressed human MDM2. In this approach, inhibitors of the MDM2-p53 interaction
abolish the MDM2-inhibitory effect with reestablishment of the p53-induced growth
arrest.
Using this phenotypic assay, all synthesized chalcones were tested for their
ability to inhibit the MDM2-p53 interaction. The analysis of the effect of 1-50 µM of

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compounds, and of 10 µM of the positive control nutlin-3a (a known inhibitor of the
MDM2-p53 interaction [7]), showed that some of the tested compounds behaved as
potential inhibitors of the MDM2-p53 interaction, reverting the MDM2-inhibitory effect
on p53-induced growth inhibition. In Figure 1, it is represented the maximal effect
achieved with compounds and nutlin-3a for the concentration of 10 µM. When
comparing the effect of prenylated derivatives (2b-2j and 3a-3e) with the correspondent
non-prenylated precursors (1b-1j) it can be seen that for most of derivatives the
prenylation was associated with an improvement of the MDM2-p53 interaction
inhibitory effect, namely for chalcones 1b, 1d, 1e, 1g, 1i, being this in accordance with
the results published before [13]. Among the tested compounds, 1c, 1h, 2e, 2i, and 3e
revealed to be the most potent, presenting a similar (or even higher) ability to release
p53 from the MDM2-inhibitory effect when compared to nutlin-3a (Figure 1). It should
be noted that, none of these five compounds interfered with the growth of control yeast
(transformed with the empty vectors) or yeast expressing human p53 or MDM2 alone
(data not shown). Altogether, these results indicated that compounds 1c, 1h, 2e, 2i, and
3e were potential p53-activating agents through inhibition of its interaction with
MDM2.
Figure 1. Effect of compounds 1b-1j, 2b-2j and 3a-3e on the MDM2-p53
interaction using a yeast-based assay. Effect of 10 µM of 1b-1j, 2b-2j and 3a-3e and
10 µM Nutlin-3a (positive control) on the reversion of MDM2 effect, by

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reestablishment of p53-induced growth inhibition in yeast cells co-expressing p53 and
MDM2, after 42 h of treatment; the ability of compounds to disrupt the MDM2-p53
interaction was evaluated considering the percentage of DMSO-treated cells expressing
wtp53 as 100%; data are mean ± SEM of 4-5 independent experiments.
2.2.2. Evaluation of the antitumor activity of 1b-1j, 2b-2j and 3a-3e in human
tumor cells
To study the in vitro antitumor potential of 1b-1j, 2b-2j and 3a-3e, the
antiproliferative activity in human colon tumor HCT116 cells was evaluated by
sulforhodamine B (SRB) assay. In general, this small chemical library revealed
compounds with potent antiproliferative activity (IC50 values lower than 10 µM; Table
2). Interestingly, as what observed for the yeast assay, the presence of a prenyl group
was associated with an increase of the growth inhibitory effect of HCT116 cells for
most of the derivatives, suggesting that the strategy of prenylation may play an
interesting role in the cell growth inhibitory activity caused by this class of compounds
in human tumor cell lines. Among the compounds revealed by the yeast assay as
potential p53-activating agents, the compound 2e exhibited the lowest IC50 value (2.1 ±
0.1 µM), after 48 h treatment. Although the compounds 1j, 2f and 2e presented similar
antiproliferative activity in the SRB assay (Table 2), the compound 2e showed to be the
most effective as putative inhibitor of the MDM2 effect on p53 in the yeast assay
(Figure 1). Therefore, the compound 2e was selected to further studies in human tumor
cells.
Table 2. IC50 values of compounds 1b-1j, 2b-2j and 3a-3e in human HCT116 tumor
cells.
Compound
IC50 (µM)
27.6 ± 0.9
10.6 ± 0.4
4.4 ± 0.5
14.0 ± 2.0
2.7 ± 0.3
1b
1c
1d
1e
1f

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3.1 ± 0.1
50.0 ± 4.0
3.5 ± 0.3
1.9 ± 0.1
5.7 ± 0.4
7.6 ± 0.5
3.2 ± 0.3
2.1 ± 0.1
1.9 ± 0.2
7.7 ± 0.1
3.5 ± 0,1
11.7 ± 1.7
24.5 ± 2.0
2.9 ± 0.9
4.2 ± 0.2
7.4 ± 0.1
15.3 ± 0.3
29.5 ± 2.2
1g
1h
1i
1j
2b
2c
2d
2e
2f
2g
2h
2i
2j
3a
3b
3c
3d
3e
Growth inhibition was studied by SRB assay, after 48
h treatment; values correspond to the IC50 values and
are mean ± S.E.M. of 3-4 independent experiments;
growth obtained with solvent was set as 100%.
2.2.3. Study of the mechanism of action of compound 2e in human tumor cells
expressing wt p53
The ability of compound 2e to inhibit the growth of human tumor cells
expressing wt p53, namely liver hepatocellular carcinoma HepG2, breast
adenocarcinoma MCF-7, and malignant melanoma A375 cells, was further investigated
by SRB assay (Figure 2A). The IC50 values obtained in these cells, 3.6 ± 0.2 µM in
HepG2 cells, 2.8 ± 0.4 µM in MCF-7 cells, 1.6 ± 0.1 µM in A375 cells (Figure 2A),
were similar to that of HCT116 cells. These results reinforced the potent
antiproliferative activity of compound 2e against wt p53-expressing tumor cells. Of note

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that compound 2e revealed lower cytotoxicity in non-tumor cells (IC50 value in human
HFF cells approximately three-fold higher compared to tumor cells).
The cytotoxicity of compound 2e against HCT116 cells was strengthened by the
colony formation assay after 11 days of treatment (Figure 2B). In fact, in this assay, 2e
presented a lower IC50 value of 0.17 ± 0.09 µM when compared to that obtained in SRB
assay. This indicated that 2e may potently interfere with the viability of HCT116 cells
(Figure 2B). Actually, it was observed that, in HCT116 cells, the growth inhibitory
activity of 2e was associated not only with a G2/M-phase cell cycle arrest (Figure 2C),
but also with an apoptotic cell death (Figure 2D), after 48h treatment.
As a redox-active transcription factor, a strong connection has been established
between generation of reactive oxygen species (ROS) and p53 activity. In fact, ROS act
as both an upstream signal that triggers p53 activation and a downstream factor that
mediates apoptosis [33]. As such, we next checked the involvement of mitochondrial
ROS production in 2e-induced apoptosis. The results showed that, after 48 h treatment,
2e markedly increased mitochondrial ROS production (Figure 2E).
Figure 2. Chalcone 2e inhibits the growth of human tumor cells through induction
of apoptosis, cell cycle arrest, and mitochondrial ROS production. (A)

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Concentration-response curves for 2e in HCT116, HepG2, MCF-7, and A375 cells after
48 h treatment; data are mean±SEM of 4-5 independent experiments; growth obtained
with solvent was set as 100%. (B) Colony formation assay for HCT116 cells treated
with 2e (or DMSO only) for 11 days; images correspond to a representative experiment
of three; graphs represent mean ± SEM of three independent experiments; values
significantly different from DMSO are indicated: **P < 0.01; ***P <0.001. (C) Effect
of 4.2 µM 2e on cell cycle progression of HCT116 cells, after 48 h treatment; cell cycle
phases were analyzed by flow cytometry using PI; data are mean ± SEM of three
independent experiments; values significantly different from DMSO are indicated: *P <
0.05. (D) Effect of 4.2 µM 2e on apoptotic cell death of HCT116 cells was evaluated by
flow cytometer using FITC-AnnexinV and PI, after 48 h treatment; values correspond to
the increase in the percentage of AnnexinV-positive cells (early and late apoptotic
cells); data are mean ± SEM of three independent experiments; values significantly
different from DMSO are indicated: **P < 0.01. (E) Effect of 4.2 µM 2e on
mitochondrial ROS production of HCT116 cells, after 48 h treatment, was investigated
by flow cytometry using MitoSOX Red dye; values correspond to the increase in the
percentage of MitoSOX-positive stained cells; data are mean ± SEM of three
independent experiments; values significantly different from DMSO are indicated: **P
< 0.01.
Based on the results from the yeast assay, the activation of the p53 pathway by
2e was also investigated in HCT116 cells, by checking the protein expression levels of
some p53 transcriptional targets (MDM2, p21, GADD45, PUMA, and Bcl-2). As
expected, 2e increased the MDM2 expression levels (Figure 3). Additionally, in
accordance with the induction of a G2/M-phase cell cycle arrest, it increased p21 and
GADD45 protein levels (Figure 3). Moreover, it increased the pro-apoptotic PUMA
and decreased the anti-apoptotic Bcl-2 protein levels (Figure 3). It should be noted that
the involvement of a caspase-3-apoptotic cell death by 2e can also be demonstrated by
the induction of cleaved PARP (a product of caspase-3 activation) (Figure 3).

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Altogether, the results obtained in human colon cells support a potent antitumor
activity of 2e through activation of the p53 pathway.
Figure 3. Chalcone 2e increases the protein expression levels of p53 transcriptional
targets and induces PARP cleavage in human colon tumor cells. Protein expression
levels was performed by Western blot after 24 h (for p21) and 48 h (for MDM2,
PUMA, GADD45, PARP and BCL-2) treatment with 4.2 µM 2e in HCT116 cells;
immunoblots are representative of three independent experiments; GAPDH was used as
loading control.
2.3. Docking studies
It is common knowledge that the alpha-helical p53 peptide binds to a deep
hydrophobic cleft within the amino-terminal domain of MDM2 consisting of a larger
and a smaller pocket [34]. Moreover, the methoxy-benzene rings of chalcones have
already been described as chemical moieties that perfectly mimic the hydrophobic and
hydrogen-bonding interactions between MDM2 and p53 [12]. Thus, in order to further
understand the different activity profile of the tested chalcones, a docking study on
MDM2 (PDB id: 4HG7) was performed. AutoDock Vina was the software chosen to
predict docking conformations and scores, as it has been described as being the best
software in predicting crystallographic MDM2-p53 inhibitor poses (RMSD < 1.0 Å) by
redocking tests [35]. Docking scores are presented on Table 3.

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Table 3. Docking scores (kcal/mol) of chalcones onto MDM2 target.
Docking scores (kcal/mol)
-5.6
-6.9
-6.4
-6
1b
1c
1d
1e
-6.4
-6.1
-6.2
-6.4
-6.5
-6.2
-6
1f
1g
1h
1i
1j
2b
2c
-6.5
-6.5
-6.3
-6.1
-5.7
-7.1
-6
2d
2e
2f
2g
2h
2i
2j
-6.4
-6.1
-6.2
-6.9
-7.2
-7.6
3a
3b
3c
3d
3e
Nutlin 3a (positive control)
From the 5 compounds (1c, 1h, 2e, 2i, and 3e) identified as the most effective as
MDM2-p53 interaction inhibitors in the yeast assay, three of them had the highest in
silico binding affinity (compounds 1c, 2i and 3e). In order to understand the different
activity profile of prenylated derivatives in comparison to their non prenylated building
blocks chalcones 1c, 1e, 1h, 1i, 2c, 2e, 2h, 2i, and 3e were further analyzed in terms of

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docking poses and residues evolved in the MDM2-p53 potential interaction (Figures 4
and 5).
Figure 4. A) MDM2 (solid surface) interacting with crystallographic nutlin-3A (yellow
sticks). B) Predicted binding poses of 1i (salmon sticks), 2i, (pink sticks), and 3e (purple
sticks) in the binding site of MDM2. C) Predicted binding poses of 1e (light green
sticks), and 2e (dark green sticks) in the binding site of MDM2. MDM2 is represented
as surface, where carbon, oxygen, nitrogen, and sulfur are represented in white, red,
blue, and yellow, respectively. Hydrogen interactions are depicted with a dashed yellow
line. Residues evolved on polar interactions are labelled.
Figure 4A shows the binding conformation of crystallographic nutlin-3A. Figure
4B shows the most stable binding poses of compounds 1i, 2i, and 3e as suggested by the
docking protocol. Compounds 2i and 3e (Figure 4B), the two most active prenylated
chalcones in the yeast assay, are also predicted to bind stably in silico to MDM2
binding pocket (docking scores of -7.1 and -7.2 kcal.mol-1). The non-prenylated 1i
establishes two hydrogen interactions with Leu-54 and Tyr-100, and parallel stacking
interactions with Hys-96. The correspondent prenylated derivatived 2i establishes two
hydrogen interactions with Gln-72 and Tyr-100, parallel stacking interactions with Hys-
96, as well as additional van de walls interactions between its prenyl substituent and the
Gly-58 pocket group and the Tyr-100 pocket. Chalcone 3e establishes three hydrogen
interactions with Gln-72, Gly-58, and Leu-54, as well as additional van der waals
interactions between the prenyl group and Tyr-100 pocket (Figure 4B). 1e (docking
score of -6kcal.mol-1) establishes hydrogen interactions with Gly-58 and His-96,

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whereas 2e (docking score of -6.5 kcal.mol-1) establishes an addition hydrogen
interactions with Val-93 (Figure 4C). Interestingly, the docking conformation of 2e
bromobenzene ring (Figure 4C) and the methoxybenzene ring of crystallographic
nutlin-3A (Figure 4A) are identical and superimposable. Nonetheless, docking scores
and poses do not justify the complete absence of activity of 1e.
However, prenylation does not always guarantee an increase in MDM2 inhibition
activity. For example, 1c (docking score of -6.9 kcal.mol-1), a non prenylated derivative,
establishes five hydrogen interactions with MDM2, while its prenyl derivative 2c
(docking score of -6kcal.mol-1) establishes only three (Figure 5A). 1h (docking score of
-6.2 kcal.mol-1) establishes three hydrogen interactions with Gly-58, Leu-54, and Gln-
24, and one pi-stacking interaction with His-96, whereas 2h (docking score of -5.7
kcal.mol-1), in spite of establishing the same number of hydrogen interactions (Lys-54,
Val-93, and Tyr-100), adopts a conformation due to the presence of the prenyl group
that departs the dimethoxybenzene ring from His-96, preventing the establishment of pi-
stacking interactions (Figure 5B).
Figure 5. A) Predicted binding poses of 1c (light grey sticks), and 2c (dark grey sticks)
in the binding site of MDM2. B) Predicted binding poses of 1h (light blue sticks), and
2h (dark blue sticks) in the binding site of MDM2. Structural data were obtained from
PDB structure 4HG7. MDM2 is represented as surface, where carbon, oxygen, nitrogen,
and sulfur are represented in white, red, blue, and yellow, respectively. Hydrogen

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interactions are depicted with a dashed yellow line. Residues evolved on polar
interactions are labelled.
From the docking results, chalcones with prenyl substituents are predicted to orient
the molecule in the binding site in such a way that the prenyl ring dives into the Gly-58
flanked subpocket (2e, 2i, Figure 4), establishing van der walls interactions with its
hydrophobic amino acid residues; the adopted conformation also benefits the
establishment of polar interactions between chalcone hydroxyl/methoxy groups and
amino acid residues located on another subpockets (Gln-72, Tyr-100, amongst others).
However, in some cases, the presence of a prenyl substituent is prejudicial for the
chalcone:MDM2 complex stability, as the prenyl ring is turned to the outside of the
MDM2 pocket or orienting the chalcone in such a way that it misses other relevant
interactions (2c, 2h, Figure 5).
3. Conclusions
In this work, from a small library of chalcone derivatives, five compounds (1c,
1h, 2e, 2i, and 3e) with promising antiproliferative activity in human colon
adenocarcinoma HCT116 cell lines were identified for the first time as MDM2-p53
interaction inhibitors, using a yeast-based screening assay. From these group of
compounds, the most potent HCT116 cells growth inhibitor (compound 2e) revealed to
inhibit the growth of other human tumor cells expressing wt p53, namely liver
hepatocellular carcinoma HepG2, breast adenocarcinoma MCF-7, and malignant
melanoma A375 cells, being this effect associated with cell cycle arrest and apoptosis
through activation of the p53 pathway. Based on the results from the yeast assay, 2e
may activate p53 trough potential inhibition of its interaction with MDM2. Further
studies are required to confirm the molecular mechanism of action of 2e, which may
conduct to the identification of a new inhibitor of the MDM2-p53 interaction with
potent antitumor activity, particularly against colon cancer with a high prevalence
worldwide. The results obtained from the molecular docking studies suggest that
prenylation may be associated to an increase of MDM2 inhibitory activity, depending

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on the substitution pattern of B ring of chalcone scaffold and will offer a perspective on
the design and development of new prenyl derivatives as potent MDM2-p53 inhibitors.
4. Experimental section
4.1. Chemistry
Microwave (MW) reactions were performed using a glassware setup for
atmospheric pressure reactions of 100 mL capacity (internal reaction temperature
measurements with a fiber-optic probe sensor) and were carried out using an Ethos
MicroSYNTH 1600 MW Labstation from Milestone (Sorisole, Italy). The reactions
were monitored by thin-layer chromatography (TLC). Purifications of compounds were
carried out by flash chromatography using Macherey-Nagel silica gel 60 (0.04-0.063
mm), and preparative thin-layer chromatography (TLC) using Macherey-Nagel silica
gel 60 (GF254) plates. Melting points were obtained in a Köfler microscope and are
1
uncorrected. H and 13C NMR spectra were taken in CDCl3 at room temperature, on
Bruker Avance 300 instruments (300.13 MHz for 1H and 75.47 MHz for 13C). Chemical
shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as an internal
reference; 13C NMR assignments were made by 2D (HSQC and HMBC) NMR
experiments (long-range C, H coupling constants were optimized to 7 Hz). HRMS mass
spectra were recorded at C.A.C.T.I.-University of Vigo, Spain. Experiments were
performed on an APEXQe FT-ICR MS (Bruker Daltonics, Billerica, MA), equipped
with a 7T actively shielded magnet. Ions were generated using a Combi MALDI-
electrospray ionization (ESI) source. Ionization was achieved by electrospray, using a
voltage of 4500 V applied to the needle, and a counter voltage of 300 V applied to the
capillary. Samples were prepared by adding a spray solution of 70:29.9:0.1 (v/v/v)
methanol/water/formic acid or 70:29.9:0.1 (v/v/v) acetonitrile/water/formic acid to a
solution of the sample at a v/v ratio of 1 to 5% to give the best signal-to-noise ratio.
Data acquisition was performed using the ApexControl software version 3.0.0, and data
processing was performed using the Data Analysis software, version 4.0 both from
Bruker Daltonics. 2-hydroxy-4,6-dimethoxyacetophenone and benzaldehydes were
purchased from Sigma Aldrich. The following materials were synthesized and purified
by the described procedures.

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Normal Phase High Performance Liquid Chromatography (NP-HPLC) analysis was
performed on a Dionex Ultimate 3000 (Thermo Fisher Scientific, USA) equipped with a
3000 quaternary pump, a 3000 autosampler, and a 3000 Variable UV/Vis detector.
Chromeleon software version 7.2 Ultimate (Thermo Fisher Scientific, USA) was used to
manage chromatographic data. Liquid chromatography analysis was performed in a
isocratic mode with using as mobile phase a mixture of n-hexane / ethyl acetate (8:2),
and the eluent was monitored in UV/Vis at 254 nm. Semi-preparative separation was
carried out on a Luna 5µ silica (2) 100Å 250x10mm from phenomenex, with a flow rate
set to 3 mL/min or 5 mL/min and a volume of injection of 50 µL or 90 µL of a 2mg/ml
sample solution. All solvents used were HPLC grade, and prior to use, solvents were
degassed in an ultrasonic bath for 15 minutes. The preparative NP-HPLC fractions were
collected and the reinjected in a lower concentration to determine their purity.
4.1.1. General procedure for the synthesis of chalcones 1a-1j
To a cooled solution of 2-hydroxy-4,6-dimethoxyacetophenone (2.000 mmol,
0.392 g) in methanol was added an aqueous/methanol solution (2:1) of 40% NaOH
(m/v) followed by the slowly addition of a cooled solution of 4 mmol of the
corresponding benzaldehyde in the same solvent. The reaction was submitted to
successive 15 min periods of MW irradiation of 180 W of power. Total irradiation time
was 30-60 min and the final temperature was 75 °C. After cooling, the reaction was
poured onto crushed ice and the pH was adjusted to 4-2, with 5 M HCl solution and
extracted with chloroform (3x50 mL). The organic layer was rinsed with brine and
water, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The
obtained residues were purified by recrystallization from ethanol (for chalcones 1a, 1b,
1e, 1h, and 1i) or from chloroform/methanol (for chalcones 1f and 1j) or by flash
column chromatography (SiO2; n-hexane/ ethyl acetate 7:3; for chalcones 1c, 1d, and
1g). Compounds (yields): 1a (71%), 1b (55%), 1c (40%), 1d (90%), 1e (61%), 1f
(28%), 1g (77%), 1h (72%), 1i (90%), 1j (28%). Compounds 1a [25], 1b [24], 1c [23],
1d [23], 1e [23], 1g [28], 1h [27] and 1i [26] were shown to possess spectroscopic and
analytical data according to those previously reported. NMR data of compound 1f was
here described for the first time, as indicated below. The new chalcone derivative 1j was
characterized by the following procedures.

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(E)-3-(2,3-dichlorophenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one
(1f): mp (cloroform): 175-177 ºC; 1H NMR (CDCl3, 300.13 MHz): δ 14.14 (s, 1H, 2’-
OH), 8.12 (d, J= 15.6, 1H, H-β), 7.83 (d, J=15.6, 1H, H-α), 7.58 (dd, J=7.9, 1.4, 1H, H-
4), 7.48 (dd, J= 7.9, 1.4, 1H, H-6), 7.24 (t, J=7.9, 1H, H-5), 6.12 (d, J=2.2, 1H, H-5’),
5.96 (d, J=2.2, 1H, H-3’), 3.90 (s, 3H, 4’-OCH3), 3.85 (s, 3H, 6’-OCH3); 13C NMR
(CDCl3, 75.47 MHz): δ 192.1 (CO), 168.5 (C-4’), 166.6 (C-6’), 162.5 (C-2’), 137.7 (C-
β), 136.3 (C-3), 134.0 (C-1), 133.3 (C-2), 131.3 (C-α), 131.1 (C-6), 127.3 (C-5), 125.9
(C-4), 106.3 (C-1’), 93.8 (C-5’), 91.4 (C-3’), 55.9 (4’-OCH3), 55.7 (6’-OCH3).
(E)-3-(3,5-dichlorophenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one
(1j). mp (cloroform/methanol): 174-176 ºC 1H NMR (CDCl3, 300.13 MHz): δ 14.08 (s,
1H, 2’-OH), 7.84 (d, J=15.6, 1H, H-α), 7.58 (d, J= 15.6, 1H, H-β), 7.43 (d, J=1.8, 2H,
H-2,-6), 7.36 (t, J=1.8, 1H, H-4), 6.11 (d, J=2.3, 1H, H-5’), 5.97 (d, J=2.3, 1H, H-3’),
3.93 (s, 3H, 4’-OCH3), 3.85 (s, 3H, 6’-OCH3); 13C NMR (CDCl3, 75.47 MHz): δ 191.9
(CO), 168.5 (C-2’), 166.6 (C-6’), 162.5 (C-4’), 138.8 (C-β), 138.7 (C-1), 135.5 (C-3,-5),
130.2 (C-α), 129.5 (C-4), 126.4 (C-2,-6), 106.2 (C-1’), 93.8 (C-3’), 91.4 (C-5’), 56.0
(4’-OCH3), 55.7 (6’-OCH3); ESI-TOF-HRMS (+) m/z: Anal. Calc. for C17H15Cl2O4
(M+H+): 353.03419; found: 353.03429.
4.1.2. General procedure for the synthesis of prenylated chalcones 2a-2j and
3a-3e
To a solution of chalcones 1a-1j (1.270 mmol) and TBAOH 30.H2O (2.680
mmol) in chloroform/toluene 10:7 (17 mL) was added prenyl bromide (224 µL, 1.920
mmol) and the reaction was allowed to proceed at room temperature for 17-25 h under
gentle stirring. The reaction mixture was then poured into a mixture of water and ice
and extracted with chloroform (3x50 mL). The combined organic layers were washed
with brine solution, rinsed with water, dried over anhydrous sodium sulfate and
evaporated under reduced pressure. The resulting crude product was purified and
characterized as indicated below to afford the newly synthesized compounds 2b-2j and

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3a-3e. Compound 2a was shown to possess spectroscopic and analytical data according
to those previously reported [22].
(E)-1-(2,4-dimethoxy-6-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(3,4-
dimethoxyphenyl)prop-2-en-1-one (2b): Purified by flash column chromatography
1
(SiO2, n-hexane/ethyl acetate 7:3). Yield: 21% as yellow gum; H NMR (CDCl3,
300.13 MHz): δ 7.28 (d, J=15.9, 1H, H-β), 7.08 (dd, J=8.1, 2.0, 1H, H-6), 7.06 (d,
J=2.0, 1H, H-2), 6.85 (d, J=8.1, 1H, H-5), 6.84 (d, J=15.9, 1H, H-α), 6.16 (brs, 1H, H-
3’,-5´), 5.33 (t, J=6.5, 1H, H-2’’), 4.50 (d, J=6.5, 2H, H-1’’), 3.91 (s, 3H, 4-OCH3), 3.90
(s, 3H, 3-OCH3), 3.85 (s, 3H, 4’-OCH3), 3.76 (s, 3H, 6’-OCH3), 1.68 (s, 3H, H-4’’),
13
1.64 (s, 3H, H-5’’); C NMR (CDCl3, 75.47 MHz): δ 194.5 (CO), 162.0 (C-4’), 158.7
(C-6’), 158.0 (C-2’), 151.0 (C-4), 149.0 (C-3), 144.4 (C-β), 137.5 (C-3’’), 128.0 (C-1),
127.3 (C-α), 122.8 (C-6), 119.6 (C-2’’), 112.3 (C-1’), 110.9 (C-5), 109.8 (C-2), 91.9 (C-
5’), 90.7 (C-3’), 65.7 (C-1’’), 55.9 (3-,4-OCH3), 55.8 (4’-OCH3), 55.4 (6’-OCH3), 25.7
(C-4’’), 18.2 (C-5’’); ESI-TOF-HRMS (+) m/z: Anal. Calc. for C24H29O6 (M+H+):
413.19587; found: 413.19659.
(E)-1-(2,4-dimethoxy-6-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one (2c): Purified by flash column chromatography
(SiO2, n-hexane/ ethyl acetate 7:3) and semi-preparative HPLC (3 mL/min flow, n-
hexane/ ethyl acetate 8:2). Yield: 70% as yellow gum; 1H NMR (CDCl3, 300.13 MHz):
δ 7.46 (d, J=8.8, 2H, H-2,-6), 7.30 (d, J= 15.9, 1H, H-β), 6.88 (d, J= 8.4, 2H, H-3,-5),
6.84 (d, J=15.9, 1H, H-α), 6.15 (sl, 2H, H-3’,-5’), 5.34 (t, J=6.6, 1H, H-2’’), 4.49 (d,
J=6.5, 2H, H-1’’), 3.84 (s, 3H, 4’-OCH3), 3.83 (s, 3H, 6’-OCH3), 3.75 (s, 3H, 4-OCH3),
13
1.68 (s, 3H, H-4’’), 1.63 (s, 3H, H-5’’); C NMR (CDCl3, 75.47 MHz): δ 194.5 (CO),
162.1 (C-4’), 161.3 (C-6’), 158.7 (C-2’), 158.0 (C-4), 144.1 (C-β), 137.5 (C-3’’), 130.0
(C-2,-6), 127.8 (C-1), 127.2 (C-α), 119.7 (C-2’’), 114.2 (C-3,-5), 112.5 (C-1’), 92.0 (C-
5’), 90.9 (C-3’), 65.8 (C-1’’), 55.9 (4’-OCH3), 55.4 (6’-OCH3), 55.3 (4-OCH3), 25.7 (C-
4’’), 18.2 (C-5’’); ESI-TOF-HRMS (+) m/z: Anal. Calc. for C23H27O5
(M+H+):383.18530; found: 383.18570.
(E)-1-(2,4-dimethoxy-6-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(4-
fluorophenyl)prop-2-en-1-one (2d): Purified by flash column chromatography (SiO2,
n-hexane/ ethyl acetate 7:3) and semi-preparative HPLC (5 mL/min flow, n-hexane/

ACCEPTED MANUSCRIPT
ethyl acetate 8:2). Yield: 65%; mp (ethyl acetate): 125-128 ºC; 1H NMR (CDCl3, 300.13
MHz): δ 7.50 (dd, J=8.0, 2.0, 2H, H-2,-6), 7.32 (d, J=16.0, 1H, H-β), 7.05 (dd, J=8.0,
2.0, 2H, H-3,-5), 6.89 (d, J=16.0, 1H, H-α), 6.16 (brs, 1H, H-5’), 6.15 (brs, 1H, H-3’),
5.35 (m, 1H, H-2’’), 4.50 (d, J=6.6, 2H, H-1’’) 3.84 (s, 3H, 4’-OCH3), 3.76 (s, 3H,6’-
OCH3), 1.68 (s, 3H, H-4’’), 1.64 (s, 3H, H-5’’); 13C NMR (CDCl3, 75.47 MHz): δ 194.0
(CO), 165.4 (C-4), 162.3 (C-4’), 158.9 (C-6’), 158.2 (C-2´), 142.4 (C-β), 137.7 (C-3’’),
131.4 (C-1), 130.2 (C-2), 130.1 (C-6), 129.0 (C-α), 119.5 (C-2’’), 116.1 (C-5), 115.8
(C-3), 112.2 (C-1’), 92.0 (C-3’), 90.9 (C-5’), 55.9 (6’-OCH3), 55.5 (4’-OCH3), 25.7 (C-
4’’), 18.2 (C-5’’); ESI-TOF-HRMS (+) m/z: Anal. Calc. for C22H24FO4 (M+H+):
371.16531; found: 371.16472.
(E)-3-(4-bromophenyl)-1-(2,4-dimethoxy-6-((3-methylbut-2-en-1-
yl)oxy)phenyl)prop-2-en-1-one (2e): Purified by flash column chromatography (SiO2,
n-hexane:ethyl acetate 7:3) and then by preparative TLC (SiO2, n-hexane: ethyl acetate
1
8:2). Yield: 21% as orange gum; H NMR (CDCl3, 300.13 MHz): δ 7.49 (dd, J= 8.5,
2.0, 2H, H-2,-6), 7.37 (dd, J=8.5, 2.0, 2H, H-3,-5), 7.30 (d, J= 16.0, 1H, H-β), 6.94 (d,
J=16.0, 1H, H-α), 6.15 (d, J=2.1, 1H, H-5’), 6.14 (d, J=2.1, 1H, H-3’), 5.34 (t, J=6.6,
1H, H-2’’), 4.51 (d, J=6.6, 2H, H-1’’), 3.84 (s, 3H, 4’-OCH3), 3.76 (s, 3H, 6’-OCH3),
1.68 (sl, 3H, H-4’’), 1.65 (sl, 3H, H-5’’); 13C NMR (CDCl3, 75.47 MHz): δ 193.7 (CO),
162.3 (C-4’), 158.9 (C-6’), 158.2 (C-2’), 142.0 (C-β), 137.6 (C-3’’), 134.0 (C-1), 131.9
(C-2, -6), 129.6 (C-3, -5), 129.5 (C-α), 124.1 (C-4), 119.4 (C-2’’), 111.9 (C-1’), 91.8
(C-5’), 90.7 (C-3’), 65.6 (C-1’’), 55.8 (6’-OCH3), 55.3 (4’-OCH3), 25.6 (C-4’’), 18.2
(C-5’’); ESI-TOF-HRMS (+) m/z: Anal. Calc. for C22H24BrO4 (M+H+):431.08525;
found: 431.08626.
(E)-3-(2,3-dichlorophenyl)-1-(2,4-dimethoxy-6-((3-methylbut-2-en-1-
yl)oxy)phenyl)prop-2-en-1-one (2f): Purified by flash column chromatography (SiO2,
n-hexane: ethyl acetate 7:3) and semi-preparative HPLC (5 mL/min flow, n-hexane/
1
ethyl acetate 8:2). Yield: 23% as yellow gum; H NMR (CDCl3, 300.13 MHz): δ 7.76
(d, J= 16.0, 1H, H-β), 7.55 (dd, J=7.9, 1.4, 1H, H-4), 7.45 (dd, J= 7.9, 1.4, 1H, H-6),
7.22 (t, J=7.9, 1H, H-5), 6.90 (d, J=16.0, 1H, H-α), 6.15 (sl, 2H, H-3’,5’), 5.35 (t, J=
6.5, 1H, H-2’’), 4.51 (d, J=6.5, 2H, H-1’’), 3.85 (s, 3H, 4’-OCH3), 3.78 (s, 3H, 6’-
OCH3), 1.69 (s, 3H, H-4’’), 1.65 (s, 3H, H-5’’); 13C NMR (CDCl3, 75.47 MHz): δ 193.5
(CO), 162.6 (C-4’), 159.1 (C-6’), 158.5 (C-2’), 139.1 (C-β), 137.6 (C-3’’), 135.9 (C-3),

ACCEPTED MANUSCRIPT
133.8 (C-1), 133.0 (C-2), 132.5 (C-α), 131.2 (C-6), 127.3 (C-5), 125.9 (C-4), 119.5 (C-
2’’), 111.8 (C-1’), 91.9 (C-5’), 90.8 (C-3’), 65.8 (C-1’’), 55.9 (6’-OCH3), 55.4 (4’-
OCH3), 25.7 (C-4’’), 18.24 (C-5’’); ESI-TOF-HRMS (+) m/z: Anal. Calc. for
C22H23Cl2O4 (M+H+):421.09679; found: 421.09600.
(E)-1-(2,4-dimethoxy-6-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(3-
methoxyphenyl)prop-2-en-1-one (2g): Purified by flash column chromatography
(SiO2, n-hexane:ethyl acetate 7:3) and semi-preparative HPLC (3 mL/min flow, n-
1
hexane/ ethyl acetate 8:2). Yield: 6% as yellow oil; H NMR (CDCl3, 300.13 MHz): δ
7.32 (d, J= 15.8, 1H, H-β), 7.26 (brt, J=7.6, 1H, H-5), 7.10 (brd, J= 7.6, 1H, H-6), 7.03
(brs, 1H, H-2), 6.95 (d, J=15.8, 1H, H-α), 6.90 (brd, J= 7.6, 1H, H-4), 6.15 (s, 2H, H-
3’,-5’), 5.35 (t, J= 6.5, 1H, H-2’’), 4.50 (d, J=6.5, 2H, H-1’’), 3.84 (s, 3H, 3-OCH3),
3.82 (s, 3H, 4’-OCH3), 3.76 (s, 3H, 6’-OCH3), 1.68 (s, 3H, H-4’’), 1.64 (s, 3H, H-5’’);
13C NMR (CDCl3, 75.47 MHz): δ 194.3 (CO), 162.3 (C-4’), 159.8 (C-6’), 158.9 (C-2’),
158.2 (C-3), 143.8 (C-β), 137.6 (C-3’’), 136.5 (C-1), 129.7 (C-5), 129.4 (C-α), 119.6
(C-2’’), 116.0 (C-4), 113.0 (C-2), 112.3 (C-1’), 92.0 (C-5’), 90.8 (C-3’), 65.8 (C-1’’),
55.9 (4’-OCH3), 55.4 (6’-OCH3), 55.3 (4-OCH3), 25.7 (C-4’’), 18.2 (C-5’’); ESI-TOF-
HRMS (+) m/z: Anal. Calc. for C23H27O5 (M+H+):383.18530; found: 383.18539.
(E)-1-(2,4-dimethoxy-6-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(3,5-
dimethoxyphenyl)prop-2-en-1-one (2h): Purified by flash column chromatography
(SiO2, n-hexane: ethyl acetate 7:3). Yield: 8% as yellow gum; 1H NMR (CDCl3, 300.13
MHz): δ 7.26 (d, J= 16.0, 1H, H-β), 6.91 (d, J=16.0, 1H, H-α), 6.65 (d, J=2.2, 2H, H-2,-
6), 6.46 (t, J= 2.2, 1H, H-4), 6.15 (d, J=2.1, 1H, H-5’), 6.14 (d, J=2.1, 1H, H-3’), 5.33 (t,
J=6.6, 1H, H-2’’), 4.48 (d, J=6.6, 2H, H-1’’), 3.83 (s, 3H, 4’-OCH3), 3.77 (s, 6H, 3-,5-
OCH3), 3.75 (s, 3H, 6’-OCH3), 1.68 (s, 3H, H-4’’), 1.67 (s, 3H, H-5’’); 13C NMR
(CDCl3, 75.47 MHz): δ 194.1 (CO), 162.1 (C-4’), 160.7 (C-3,-5), 158.7 (C-6’), 158.0
(C-2’), 143.7 (C-β), 137.5 (C-3’’), 136.8 (C-1), 129.4 (C-α), 119.4 (C-2’’), 111.9 (C-
1’), 105.9 (C-2,-6), 102.2 (C-4), 91.7 (C-5’), 90.6 (C-3’), 65.5 (C-1’’), 55.7 (6’-OCH3),
55.3 (4’-OCH3), 55.2 (3-,5-OCH3), 25.5 (C-4’’), 18.1 (C-5’’); ESI-TOF-HRMS (+) m/z:
Anal. Calc. for C24H29O6 (M+H+): 413.19587; found: 413.19614.
(E)-1-(2,4-dimethoxy-6-((3-methylbut-2-en-1-yl)oxy)phenyl)-3-(2,3-
dimethoxyphenyl)prop-2-en-1-one (2i): Purified by flash column chromatography

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(SiO2, n-hexane: ethyl acetate 7:3) and then by preparative TLC (SiO2, toluene: ethyl
1
acetate 6:4). Yield: 4% as yellow gum; H NMR (CDCl3, 300.13 MHz): δ 7.69 (d, J=
16.2, 1H, H-β), 7.18 (dd, J=8.0, 1.4, 1H, H-6), 6.98 (d, J=16.2, 1H, H-α), 6.92 (dd, J=
8.0, 1.4, 1H, H-4), 6.15 (brs, 2H, H-3’,-5’), 5.34 (t, J=6.5, 1H, H-2’’), 4.49 (d, J=6.5,
2H, H-1’’), 3.87 (s, 3H, 2-OCH3), 3.85 (s, 3H, 3-OCH3), 3.77 (s, 3H, 4’-OCH3), 3.76 (s,
3H, 6’-OCH3), 1.68 (s, 3H, H-4’’), 1.67 (s, 3H, H-5’’); 13C NMR (CDCl3, 75.47 MHz):
δ 194.7 (CO), 162.3 (C-4’), 158.9 (C-6’), 158.4 (C-2’), 153.2 (C-3), 148.6 (C-2), 138.8
(C-β), 137.5 (C-3’’), 130.6 (C-α), 129.6 (C-1), 124.3 (C-5), 119.8 (C-6), 119.5 (C-2’’),
113.8 (C-4), 112.5 (C-1’), 92.1 (C-3’), 90.9 (C-5’), 65.9 (C-1’’), 61.4 (2-OCH3), 56.0
(4’-OCH3), 55.7 (3-OCH3), 55.6 (6´-OCH3), 25.8 (C-4’’), 18.4 (C-5’’); ESI-TOF-
HRMS (+) m/z: Anal. Calc. for C24H29O6 (M+H+): 413.19587; found: 413.19570.
(E)-3-(3,5-dichlorophenyl)-1-(2,4-dimethoxy-6-((3-methylbut-2-en-1-
yl)oxy)phenyl)prop-2-en-1-one (2j): Purified by flash column chromatography (SiO2,
n-hexane:ethyl acetate 7:3) and semi-preparative HPLC (3 mL/min flow, n-hexane/
1
ethyl acetate 8:2). Yield: 52% as yellow gum; H NMR (CDCl3, 300.13 MHz): δ 7.37
(d, J=1.83, 2H, H-2,-6), 7.33 (t, J=1.8, 1H, H-4), 7.25 (d, J= 16.0, 1H, H-β), 6.95 (d,
J=16.0, 1H, H-α), 6.15 (d, J= 2.1, 1H, H-5’), 6.14 (d, J=2.1, 1H, H-3’), 5.35 (m, 1H, H-
2’’), 4.51 (d, J=6.63, 2H, H-1’’), 3.85 (s, 3H, 4’-OCH3), 3.78 (s, 3H, 6’-OCH3), 1.75 (s,
13
3H, H-4’’), 1.67 (s, 3H, H-5’’); C NMR (CDCl3, 75.47 MHz): δ 192.8 (CO), 162.7
(C-4’), 159.2 (C-6’), 158.5 (C-2’), 139.8 (C-β), 138.3 (C-1), 138.0 (C-3’’), 135.4 (C-3,-
5), 131.4 (C-α), 129.4 (C-4), 126.3 (C-2,6), 119.3 (C-2’’), 111.9 (C-1’), 91.9 (C-5’),
90.9 (C-3’), 65.7 (C-1’’), 55.9 (6’-OCH3), 55.5 (4’-OCH3), 25.7 (C-4’’), 18.3 (C-5’’);
ESI-TOF-HRMS (+) m/z: Anal. Calc. for C22H23Cl2O4 (M+H+): 421.09679; found:
421.09688.
(E)-1-(4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)-2-((3-methylbut-2-en-1-
yl)oxy)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3a) : Purified by flash
column chromatography (SiO2, n-hexane: ethyl acetate 7:3). Yield: 8% as yellow gum;
1H NMR (CDCl3, 300.13 MHz): δ 7.29 (d, J=15.9, 1H, H-β), 6.92 (d, J=15.9, 1H, H-α),
6.75 (s, 2H, H-2,-6), 6.32 (s, 1H, H-5’), 5.40 (t, J=7.1, 1H, H-2’’), 5.18 (t, J=6.5, 1H, H-
2’’’), 4.31 (d, J=7.1, 2H, H-1’’), 3.90 (s, 3H, 4’-OCH3), 3.89 (s, 6H, 3-,4-OCH3), 3.87
(s, 3H, 5-OCH3), 3.79 (s, 3H, 6´-OCH3), 3.32 (d, J=6.5, 2H, H-1’’’), 1.75 (s, 3H, H-
13
4’’’), 1.69 (s, 3H, H-4’’), 1.68 (s, 3H, H-5’’’), 1.61 (s, 3H, H-5’’); C NMR (CDCl3,

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75.47 MHz): δ 194.5 (CO), 159.9 (C-4’), 156.3 (C-6’), 156.0 (C-2’), 153.3 (C-3,-5),
144.5 (C-β), 139.9 (C-4), 138.0 (C-3’’), 131.3 (C-1,-3’’’), 128.3 (Cα), 123.3 (C-2’’’),
120.1 (C-2’’) 116.8 (C-3’), 116.4 (C-1’), 105.4 (C-2,-6), 91.4 (C-5’), 72.5 (C-1’’), 61.0
(4-OCH3), 56.1 (6’-,3-,5-OCH3), 56.0 (4’-OCH3), 25.8 (C-4’’,-4’’’), 22.6 (C-1’’’),
18.0 (C-5’’), 17.9 (C-5’’’); ESI-TOF-HRMS (+) m/z: Anal. Calc. for C30H39O7 (M+H+):
511.26903; found: 511.26815.
(E)-1-(2,4-dimethoxy-3-(3-methylbut-2-en-1-yl)-6-((3-methylbut-2-en-1-
yl)oxy)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3b): Purified by flash
column chromatography (SiO2, n-hexane:ethyl acetate 7:3). Yield: 8% as yellow gum;
1H NMR (CDCl3, 300.13 MHz): δ 7.27 (d, J=16.0, 1H, H-β), 6.92 (d, J=16.0, 1H, H-α),
6.76 (s, 2H, H-2,-6), 6.33 (s, 1H, H-3’), 5.35 (t, J= 6.5, 1H, H-2’’), 5.19 (t, J=6.8, 1H,
H-2’’’), 4.52 (d, J=6.5, 2H, H-1’’), 3.87 (s, 12H, 4’-,3-,4-,5-OCH3), 3.70 (s, 3H, 6’-
OCH3), 3.30 (d, J=6.8, 2H, H-1’’’), 1.76 (s, 3H, H-4’’’), 1.69 (s, 3H, H-4’’), 1.68 (s,
13
3H, H-5’’’), 1.67 (s, 3H, H-5’’); C NMR (CDCl3, 75.47 MHz): δ 194.5 (CO), 159.7
(C-4’), 157.0 (C-6’), 155.7 (C-2´), 153.3 (C-3,-5), 144.4 (C-β), 140.0 (C-4), 137.4 (C-
3’’), 131.2 (C-3’’’), 130.4 (C-1), 128.4 (Cα), 123.1 (C-2’’’), 119.7 (C-2’’), 116.9 (C-1’),
116.1 (C-5’), 105.4 (C-2,-6), 93.1 (C-3’), 66.0 (C-1’’), 62.8 (6’-OCH3), 60.9 (4-OCH3),
56.1 (3-,5-OCH3), 55.1 (4’-OCH3), 25.7 (C-4’’,-4’’’), 22.4 (C-1’’’), 18.2 (C-5’’), 17.9
(C-5’’’); ESI-TOF-HRMS (+) m/z: Anal. Calc. for C30H39O7 (M+H+): 511.26903;
found: 511.27058.
(E)-1-(2,4-dimethoxy-3-(3-methylbut-2-en-1-yl)-6-((3-methylbut-2-en-1-
yl)oxy)phenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (3c): Purified by flash
column chromatography (SiO2, n-hexane: ethyl acetate 7:3). Yield: 7% as orange gum;
1H NMR (CDCl3, 300.13 MHz): δ 7.30 (d, J=15.9, 1H, H-β), 7.09 (dd, J=8.3, 1.4, 1H,
H-6), 7.07 (d, J=1.4, 1H, H-2), 6.90 (d, J=15.9, 1H, H-α), 6.84 (d, J=8.3, 1H, H-5), 6.32
(s, 1H, H-3’), 5.34 (t, J=6.6, 1H, H-2’’), 5.18 (t, J=6.8, 1H, H-2’’’), 4.51 (d, J=6.6, 2H,
H-1’’), 3.91 (s, 3H, 4-OCH3), 3.90 (s, 3H, 3-OCH3), 3.86 (s, 3H, 4’-OCH3), 3.69 (s, 3H,
6’-OCH3), 3.29 (d, J=6.7, 2H, H-1’’’), 1.76 (s, 3H, H-4’’’), 1.70 (s, 3H, H-4’’), 1.68 (s,
13
3H, H-5’’’), 1.66 (s, 3H, H-5’’); C NMR (CDCl3, 75.47 MHz): δ 194.7 (CO), 159.6
(C-4’), 156.9 (C-6’), 155.6 (C-2’), 151.0 (C-4), 148.9 (C-3), 144.9 (C-β), 144.7 (C-5),
137.5 (C-3’’), 131.2 (C-3’’’), 127.8 (C-1), 127.1 (C-α), 123.1 (C-6), 123.0 (C-2’’’),
119.8 (C-2’’), 116.0 (C-5’), 114.2 (C-1’), 109.8 (C-2), 93.1 (C-3’), 65.9 (C-1’’), 62.8

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(6’-OCH3), 55.9 (4-OCH3), 55.8 (3-OCH3), 55.7 (4’-OCH3), 24.9 (C-4’’’), 22.4 (C-
1’’’), 18.2 (C-4’’,-5’’), 17.8 (C-5’’’); ESI-TOF-HRMS (+) m/z: Anal. Calc. for
C29H37O6 (M+H+): 481.25847; found: 481.25801.
(E)-3-(2,3-dimethoxyphenyl)-1-(2-hydroxy-4,6-dimethoxy-3,5-bis(3-methylbut-2-
en-1-yl)phenyl)prop-2-en-1-one (3d): Purified by flash column chromatography
(SiO2, n-hexane: ethyl acetate 7:3) and then by preparative TLC (SiO2, toluene:ethyl
1
acetate 6:4). Yield: 2% as yellow gum; H NMR (CDCl3, 300.13 MHz): δ 12.97 (s,2’-
OH), 8.19 (d, J= 15.9, 1H, H-β), 7.96 (d, J=15.9, 1H, H-α), 7.30 (dd, J=8.0, 1.3, 1H, H-
6), 7.10 (t, J= 8.0, 1H, H-5), 6.96 (dd, J= 8.0, 1.3, 1H, H-4), 5.24 (t, J=6.6, 1H, H-2’’),
5.18 (t, J=6.6, 1H, H-2’’’), 3.92 (s, 3H, 2-OCH3), 3.90 (s, 3H, 3-OCH3), 3.76 (s, 3H, 4’-
OCH3), 3.64 (s, 3H, 6’-OCH3), 3.36 (d, J=6.6, 2H, H-1’’), 3.32 (d, J=6.6, 2H, H-1’’’),
1.79 (s, 6H, H-4’’, 4’’’), 1.69 (s, 6H, H-5’’, 5’’’); 13C NMR (CDCl3, 75.47 MHz): δ
194.1 (CO), 163.3 (C-4’), 161.7 (C-2’), 159.1 (C-6’), 153.2 (C-3), 148.9 (C-2), 138.2
(C-β), 131.8 (C-3’’), 131.3 (C-3’’’), 129.3 (C-1), 127.7 (C-α), 124.2 (C-5), 123.9 (C-
5’), 122.7 (C-2’’), 120.1 (C-2’’’) 119.6 (C-6), 119.5 (C-3’), 113.9 (C-4), 112.3 (C-1’),
63.3 (6’-OCH3), 61.4 (4’,2-OCH3), 55.8 (3-OCH3), 25.7 (C-4’’, 4’’’), 23.1 (C-1’’), 23.0
(C-1’’’), 17.9 (C-5’’, 5’’’); ESI-TOF-HRMS (+) m/z: Anal. Calc. for C29H37O6 (M+H+):
481.25847; found: 481.25895.
(E)-3-(2,3-dimethoxyphenyl)-1-(6-hydroxy-2,4-dimethoxy-3-(3-methylbut-2-en-1-
yl)phenyl)prop-2-en-1-one (3e): Purified by flash column chromatography (SiO2, n-
hexane: ethyl acetate 7:3) and then by preparative TLC (SiO2, toluene:ethyl acetate
6:4). Yield: 2% as yellow gum; 1H NMR (CDCl3, 300.13 MHz): δ 13.52 (s, 1H, 2’-OH),
8.17 (d, J= 15.9, 1H, H-β), 8.00 (d, J=15.9, 1H, H-α), 7.30 (dd, J=8.0, 1.3, 1H, H-6),
7.10 (t, J=8.0, 1H, H-5), 6.96 (dd, J= 8.0, 1.3, 1H, H-4), 6.29 (s, 1H, H-3’), 5.15 (t,
J=6.9, 1H, H-2’’), 3.92 (s, 3H, 2-OCH3), 3.90 (s, 3H, 3-OCH3), 3.86 (s, 3H, 4’-OCH3),
3.66 (s, 3H, 6’-OCH3), 3.29 (d, J=6.9, 2H, H-1’’), 1.77 (s, 3H, H-4’’), 1.69 (s, 2H, H-
5’’); 13C NMR (CDCl3, 75.47 MHz): δ 193.1 (CO), 165.1 (C-2’) 164.5 (C-4’), 160.3 (C-
6’), 153.2 (C-3), 148.8 (C-2), 137.8 (C-β), 131.4 (C-3’’), 129.4 (C-1), 127.6 (C-α),
124.2 (C-5), 123.0 (C-2’’), 119.4 (C-6), 115.8 (C-5’), 113.8 (C-4), 109.0 (C-1’), 96.0
(C-3’), 63.1 (6’-OCH3), 61.4 (2-OCH3), 55.8 (4’-,3-OCH3), 25.5 (C-4’’), 18.1 (C-5’’);
ESI-TOF-HRMS (+) m/z: Anal. Calc. for C24H29O6 (M+H+):413.19587; found:
413.19526.

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4.1.3. Synthesis of prenylated chalcones 2i, 3d and 3e under conventional
heating
To a solution of chalcone 1i (1.270 mmol) and TBAOH 30.H2O (2.160 g, 2.680
mmol) in chloroform/toluene 10:7 (17 mL) was added prenyl bromide (224 µL, 1.92
mmol) and the reaction was maintained under stirring at 50 °C in an oil bath for 8 h.
The reaction mixture was then poured into a mixture of water and ice and extracted with
chloroform (3x50 mL). The combined organic layers were washed with brine solution,
rinsed with water, dried over anhydrous sodium sulfate and evaporated under reduced
pressure. The resulting crude product was purified by flash column chromatography
(SiO2; n-hexane/ethyl acetate 7:3) to afford 2i (32%), 3d (8%) and 3e (9%).
4.1.4. Synthesis of prenylated chalcones 2i, 3d and 3e under MW irradiation
To a solution of chalcone 1i (1.270 mmol) and TBAOH 30.H2O (2.160 g, 2.680
mmol) in chloroform/toluene 10:7 (17 mL) was added prenyl bromide (224 µL, 1.92
mmol). The mixture was submitted to successive 15 min periods of MW irradiation of
200 W of power. Total irradiation time was 45 min and the final temperature was 50 °C.
The reaction mixture was then poured into a mixture of water and ice and extracted with
chloroform (3x50 mL). The combined organic layers were washed with brine solution,
rinsed with water, dried over anhydrous sodium sulfate and evaporated under reduced
pressure. The resulting crude product was purified by flash column chromatography
(SiO2; n-hexane/ethyl acetate 7:3) to afford 2i (50%) and 3d (3%).
4.2. Biological activity evaluation
4.2.1. Compounds
Nutlin-3a was purchased from Sigma-Aldrich (Sintra, Portugal). All tested
compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich).
4.2.2. Yeast-based screening assay

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All compounds were tested in Saccharomyces cerevisiae cells expressing human wt
p53 alone or co-expressed with human MDM2, and on control yeast (transformed with
empty vectors) as described by Soares et al. [30]. Briefly, to promote the expression of
human proteins, yeast cells were cultivated in selective induction medium with 2%
(w/w) galactose and 2% (w/w) raffinose, in the presence of 1-50 µM compounds or
solvent (0.1% DMSO), for 42 hours at 30°C; cell growth was analyzed by colony-
forming unit counts, on Sabouraud Dextrose Agar plates from Liofilchem (Frilabo,
Porto, Portugal), after 48 hours incubation at 30°C. Results were estimated considering
the growth of non-treated yeast cells as 100%.
4.2.3. Human tumor cell lines and growth conditions
Human colon adenocarcinoma HCT116 cell lines were provided by B.
Vogelstein (The Johns Hopkins Kimmel Cancer Center, Baltimore, MD, USA), liver
hepatocellular carcinoma HepG2 cells, breast adenocarcinoma MCF-7, malignant
melanoma A375, and human fibroblasts HFF cell lines were purchased from ATCC.
Tumor cells were cultured in RPMI‐1640 with UltraGlutamine (Lonza, VWR),
supplemented with (5%) fetal bovine serum (FBS; Gibco, Alfagene). Cell lines were
maintained in a humidified incubator at 37 °C with 5% CO2.
4.2.4. Sulforhodamine B (SRB) assay
Human cell lines were seeded in 96 well plates at a density of 5.0 x 103 cell/well
(for HCT116, A375, and HFF cells) and 4.0x103 cell/well (for HepG2 and MCF7) and
allowed to adhere for 24 hours, followed by treatment with 0.625-10 µM C4P for 48
hours. The effect on cell proliferation was measured by sulforhodamine B (SRB), as
described [30], and IC50 (concentration that causes 50% growth inhibition) values were
determined for each cell line using the GraphPad Prism software (version 6.0). The
solvent (DMSO 0.1%) was included as control.

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4.2.5. Cell cycle and apoptosis
HCT116 cells were seeded at a density of 1.5 x105 cells/well in 6-well plates and
allowed to adhere for 24 hours. Cells were then treated with 4.2 µM 2e (or DMSO only)
for 48 hours. Cell cycle was analyzed by flow cytometry using propidium iodide (PI;
Sigma-Aldrich) as described [30]. For apoptosis analysis, cells were stained using
Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, Enzifarma, Porto,
Portugal) according to the manufacturer’s instructions, followed by flow cytometric
analysis [30].
4.2.6. Mitochondrial reactive oxygen species (ROS) detection
HCT116 cells were seeded at a density of 1.5 x 105 cells/well in 6-well plates and
allowed to adhere for 24 hours. Cells were then treated with 4.2 µM 2e (or DMSO only)
for 48 hours. Cells were then detached, washed and resuspended in PBS. A Final
concentration of 3 µM MitoSOX was added to the wells and allowed to load, for 30 min
at 37°C, and then washed with PBS. Flow cytometry was performed to detect
mitochondrial ROS.
4.2.7. Colony formation assay
HCT116 cells were seeded in 6-well plates at a density of 1.0 x103cells/well,
followed by treatment with 2e (or DMSO only) for 11 days. Formed colonies were fixed
with 10% methanol and 10% acetic acid for 10 min, and then stained using 0.5% crystal
violet (Sigma-Aldrich) in 1:1 methanol/H2O for 15 min. Colonies with more than 20
cells were counted.
4.2.8. Western blot
HCT116 cells were seeded in 6-well plates at a density of 1.5 X105 cells/well and
allowed to adhere for 24 hours, followed by treatment with 4.2 µM C4P (or DMSO
only) for 48 hours. Cells were lysed, and protein lysates were analyzed by Western blot,
as described [31]. Mouse monoclonal anti-MDM2, anti-PARP, anti-PUMA, anti-
GAPDH, anti-Bcl-2, and rabbit polyclonal anti-p21 were from Santa Cruz