A Diarylpentanoid with Potential Activation of the p53 Pathway: Combination of in silico Screening Studies, Synthesis, and Biological Activity Evaluation

Article abstract of DOI:10.1002/cmdc.202100337

In silico studies of a library of diarylpentanoids led us to the identification of potential new MDM2/X ligands. The diarylpentanoids with the best docking scores obeying the druglikeness and ADMET prediction properties were subsequently synthesized and e

Full text of DOI:10.1002/cmdc.202100337

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A Diarylpentanoid with Potential Activation of the p53
Pathway: Combination of in silico Screening Studies,
Synthesis, and Biological Activity Evaluation
Joana Moreira,[a, b] Joana Almeida,[c] Joana B. Loureiro,[c] Helena Ramos,[c]
Andreia Palmeira,[a, b] Madalena M. Pinto,[a, b] Lucília Saraiva,*[c] and Honorina Cidade*[a, b]
In silico studies of a library of diarylpentanoids led us to the
identification of potential new MDM2/X ligands. The diary-
lpentanoids with the best docking scores obeying the druglike-
ness and ADMET prediction properties were subsequently
synthesized and evaluated for their antiproliferative activity on
colon cancer HCT116 and fibroblasts HFF-1 cells. The effect on
p53-MDM2/X interactions was evaluated through yeast-based
assays for compounds showing potent antiproliferative activity
in HCT116 cells and low toxicity in normal cells, resulting in the
identification of a potential dual inhibitor. Moreover, its
antiproliferative effect was significantly reduced in the absence
of p53 and in MDA-MB-231 cells expressing a mutant p53 form.
The antiproliferative effect of this compound was associated
with induction of cell cycle arrest, apoptosis, PARP cleavage and
increased p53 and its transcriptional targets, p21 and PUMA, in
HCT116 cells. Docking poses and residues involved in the
inhibition of p53-MDM2/X interactions were predicted by
docking studies.
Introduction
of great efforts in drug discovery.[3] For example, nutlin-3a and
RG7112 were reported to be potent in vitro growth inhibitors of
cancer cell lines retaining wt p53 as well as to be effective in
tumor regression after oral administration in animals.[4] SJ-
172550 can compete for binding to MDMX and cause p53-
dependent cell death in retinoblastoma cells.[5] RO-5963 was
reported as new potential dual p53-MDM2/X interaction
inhibitor.[6]
Among the identified inhibitors of the interaction between
p53 and MDM2 or MDMX, some p53-MDM2 interaction
inhibitors are already under clinical trials, proving the relevance
of these compounds in cancer therapy.[4,7] Nevertheless, some of
these compounds have shown inadequate pharmacokinetic
profile (e.g. RG7388) and adverse side effects, namely hemato-
logical toxicity (e.g. RG7112).[4] Therefore, new and safe p53-
MDM2 inhibitors are still required as potential anticancer
agents. Nonetheless, p53 inactivation by interaction with MDMs
occurs in a distinct and cooperative inhibitory effect and many
of the inhibitors reported, as MDM2 inhibitors, are weak
inhibitors of MDMX (e.g. nutlin-3a).[8] Therefore, compounds
that block the inhibitory effect of both MDM2 and MDMX,
would represent an ideal strategy to achieve a full p53
activation.[3c] However, the availability of such compounds is still
limited and there is currently no small molecule targeting dual
p53-MDM2/X interaction inhibitors in clinical trials.[9]
TP53 is one of the most relevant tumor suppressor genes, which
is frequently mutated in human cancers. The p53 tumor
suppressor protein 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] 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. Nevertheless the p53
pathway is commonly inactivated in cancers retaining wild-type
(wt) p53 due to interaction with endogenous negative
regulators such as murine double minute (MDM)2 and MDMX.[2]
Therefore, in the last years, the reestablishment of p53 activity
by disruption of its interaction with MDM2/X by small molecules
has received great attention in cancer therapy, being the focus
[a] J. Moreira, Prof. A. Palmeira, Prof. M. M. Pinto, Prof. H. Cidade
Laboratório 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)
E-mail: hcidade@ff.up.pt
[b] J. Moreira, Prof. A. Palmeira, Prof. M. M. Pinto, Prof. H. Cidade
CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental
Terminal de Cruzeiros do Porto de Leixões
4450-208 Matosinhos (Portugal)
[c] J. Almeida, Dr. J. B. Loureiro, Dr. H. Ramos, Prof. L. Saraiva
LAQV/REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências
Biológicas
Diarylpentanoids are chalcone analogues with two aromatic
rings connected by a five carbon bridge. These compounds are
described as having several pharmacological properties, prob-
ably due to strong flexibility of the molecule and the presence
of carbonyl functional group. The biological activities of diary-
lpentanoids include antioxidant,[10] anti-inflammatory,[11] anti-
bacterial,[12] and antitumor.[13] Among these biological activities,
the in vitro growth inhibitory effect against cancer cell lines has
been well recognized, being described their interference with
Faculdade de Farmácia, Universidade do Porto
Rua de Jorge Viterbo Ferreira, 228
4050-313 Porto (Portugal)
E-mail: lucilia.saraiva@ff.up.pt
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100337
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the apoptotic cascade and p53 expression.[14] However, the
interference of these compounds with p53-MDM2/X interac-
tions was never explored. In fact, the underlying molecular
mechanism by which these new analogues suppress cancer cell
growth is still unclear.
and Leu-26, play a dominant role in the interaction.[18] This
library of virtual diarylpentanoids was submitted to a docking
study using MDM2 and MDMX as targets. The results are
represented on Table 1. Several test compounds presented
similar
(28 test
compounds,
À 6.9 kcal/mol<docking
In the early stages of the drug discovery process, prediction
of pharmacokinetic properties known as ADMET (absorption,
distribution, metabolism, excretion, and toxicity) of drug
candidates can significantly reduce the chance of their failure
during clinical trials.[15] Hence, in order to pursuit our research
work, focused on the discovery of new compounds with
antitumor activity by targeting p53,[16] a small library of diary-
lpentanoids has been envisioned and preliminary assessed in
silico studies to predict their p53-MDM2/X inhibitory potential,
druglikeness and ADMET profile. Afterward, potential novel
MDM2/X ligands identified by virtual screening with the best
docking scores were synthesized and evaluated for their growth
inhibitory effect on human cancer and non-malignant cell lines.
For the most potent and selective growth inhibitors, assays
were performed aiming to evaluate their potential effect on the
p53-MDM2/X interaction.
scores�À 6.4 kcal/mol), or better (72 test compounds, docking
scores�À 7.0 kcal/mol) docking scores than nutlin-3A, a known
p53-MDM2 inhibitor.[8] Considering MDMX as target, docking
studies were performed for this library of virtual diarylpenta-
noids along with SJ-172550, already described in the literature
as MDMX inhibitor.[19] From the tested compounds,121 pre-
sented docking scores similar or better than SJ-172550.
Predicting druglikeness
The designed compounds that simultaneously presented dock-
ing scores lower or equal to À 6.4 kcal/mol in MDM2 and
À 5.6 kcal/mol in MDMX targets (100 compounds) were
screened for druglikeness properties using in silico models. The
druglikeness was evaluated accordingly to the following
chemical features: i) flexibility, inferred by the number of
rotatable bonds (RB); ii) unsaturation, inferred by fraction of
carbon sp3 (Fsp3); iii) solubility, inferred by log S; iv) polarity,
inferred by polar surface area (PSA); and v) lipophilicity, inferred
by the log P, using SwissADME web server (SwissADME, http://
www.swissadme.ch/). For each designed compound, the above-
mentioned molecular descriptors/physicochemical properties
were calculated (Table S2–S3, Supporting Information). For each
chemical feature, the obtained mean values were compared
with the range of values preconized by the druglikeness
guidelines. Unsaturation is the only feature that was not within
the preconized limits in drug-like chemical space. However, the
high unsaturation degree is a characteristic of the diary-
lpentanoid scaffold and per si cannot invalidate the design of
this library.
Using the same web server (SwissADME, http://www.swis-
sadme.ch/), several druglikeness rules were evaluated, namely
Ghose, Veber, Egan and Muegge rules. Additionally, PreADMET
web server (PreADME, https://preadmet.bmdrc.kr/) featured
prediction of other druglikeness properties, including CMC-like,
Lead-like, WDI-like, and MDDR-like rules. The acceptable value
of violations for a drug-like molecule is 1. Thereby, the
structures that respected the selected rules or violated no more
than one rule (total of 33 out of 100 compounds) were selected
and subsequently submitted to additional in silico studies
(Figure S1, Supporting Information).
Results and Discussion
In silico studies
Structure-based virtual screening
Most of diarylpentanoids with antitumor activity have a cyclic
C5 bridge, being the compounds with a tetrahydro-4H-pyran-4-
one moiety one of the most potent.[13] Interestingly, some
diarylpentanoids interfere with p53 pathway,[14] suggesting that
these compounds can act as p53-MDM2/X inhibitors. Therefore,
we hypothesized that diarylpentanoids with tetrahydro-4H-
pyran-4-one moiety may be designed in order to block these
interactions to reactivate p53 in cells harboring wt p53.
Considering that diarylpentanoids are commonly synthesized
by base-catalyzed aldol reactions of appropriately substituted
benzaldehydes with ketones, a total of 1000 diarylpentanoids
with tetrahydro-4H-pyran-4-one moiety were designed and in
silico studies were performed to predict p53-MDM2/X inhibitory
effect as well as druglikeness. A Lipinski’s pre-filter, at the dawn
of the screening workflow, were applied to remove less relevant
ligands in the library. In fact, the RO5 filter evaluates the
potential druglike profile of compounds belonging to a given
library, helping to make an easier selection of molecules with
better physicochemical properties.[15a] Results displayed that
145 out of 1000 compounds fitted with the Lipinski’s
properties[17] and thus proved to possess satisfactory drug-like
profiles, which were kept proceeding to the next steps (the
145 diarylpentanoids are listed Table S1, Supporting Informa-
tion).
ADMET prediction
The identification of efficient drug candidates at an early stage
of drug discovery and development process is one of the major
objectives in a successful drug discovery process, since it allows
to disregard compounds with poor pharmacokinetic properties,
minimizing extremely expensive and time-consuming steps.
Therefore, current computationally ADMET prediction is consid-
After, virtual screening was performed on 145 newly
designed compounds. p53-MDM2/X interactions are mediated
by a well-defined hydrophobic surface pocket in MDM2/X and
at least three key hydrophobic residues in p53, Phe-19, Trp-23,
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Table 1. Docking scores of designed diarylpentanoids onto MDM2 and MDMX targets.
MDM2
Ligand
MDMX
Ligand
Docking Score
kcal/mol
Ligand
Docking Score
kcal/mol
Docking Score
kcal/mol
Ligand
Docking Score
kcal/mol
1
2
3
4
5
6
7
8
À 7.5
À 8.0
À 8.1
À 7.0
À 8.4
À 7.5
À 7.1
À 7.4
À 6.7
À 7.1
À 7.5
À 7.1
À 7.4
À 3.9
À 1.7
26.8
À 8.3
À 6.6
À 7.3
À 6.8
À 8.4
À 6.3
À 6.4
À 8.0
À 6.4
À 4.3
25.4
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
À 7.1
À 5.4
À 7.1
À 6.8
21.9
À 7.4
À 8.4
À 7.2
À 7.3
À 6.8
À 6.8
À 7.1
À 7.5
19.5
1
2
3
4
5
6
7
8
À 6.6
À 6.1
À 6.6
À 6.1
À 6.1
À 6.0
À 6.1
À 6.0
À 6.4
À 6.4
À 6.6
À 5.6
À 6.6
À 5.2
3.1
À 3.8
À 7.4
À 6.2
À 5.7
À 5.7
À 6.5
À 6.0
À 5.8
À 7.2
À 5.6
À 5.2
21.2
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
À 6.1
À 3.4
À 5.9
À 5.9
1.9
À 6.8
À 6.5
À 6.5
À 6.0
À 5.5
À 5.8
À 6.0
À 5.7
À 5.4
À 5.4
À 5.6
À 3.6
À 6.4
À 6.2
À 6.0
À 7.0
À 5.8
À 5.7
À 5.8
À 5.8
À 6.0
À 6.6
À 7.0
À 1.9
À 6.2
À 6.0
À 4.0
À 6.2
À 6.0
À 6.0
À 6.1
À 6.2
4.0
À 6.5
À 3.8
À 6.5
À 6.4
À 6.5
À 5.9
2.5
À 6.1
À 6.4
1.4
À 6.4
À 5.9
À 6.8
À 3.9
À 5.6
À 6.0
À 5.0
À 6.8
À 6.1
À 5.7
À 7.0
À 6.0
À 7.0
À 6.1
À 5.7
À 6.9
À 6.2
À 5.7
1.0
9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
À 6.1
À 7.1
À 0.7
À 6.0
À 7.7
À 7.0
À 8.8
À 6.9
À 6.3
À 6.1
À 5.7
À 7.5
À 8.7
À 8.3
À 4.9
À 2.7
À 6.3
À 5.8
À 7.2
À 7.1
À 7.1
À 6.0
À 7.3
À 1.5
À 8.3
À 5.9
À 8.1
À 7.4
À 7.3
À 7.9
À 2.7
À 8.0
À 8.5
À 2.4
À 8.6
À 7.0
0.4
À 5.9
À 6.5
À 6.4
À 5.9
À 7.8
À 7.1
9.2
À 8.9
À 8.0
À 2.0
À 4.8
À 7.9
À 1.9
À 6.7
À 6.1
À 2.0
À 7.6
À 1.4
98
99
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
18
À 5.8
À 6.5
À 6.5
À 6.2
15.6
À 6.5
À 5.9
15.4
À 8.2
À 3.3
À 9.2
28.1
À 8.3
22.6
26.6
À 8.8
À 6.8
39.3
19.2
24.0
8.7
À 7.6
À 8.1
23.4
À 5.6
À 6.1
12.3
À 8.5
À 8.7
À 8.7
À 5.4
À 7.8
À 6.0
À 7.2
À 6.8
À 6.7
À 6.9
À 7.2
À 6.4
À 7.0
À 6.6
À 7.0
À 6.8
À 8.8
À 7.8
À 7.5
16.3
À 6.8
À 4.0
À 6.8
À 6.8
À 7.4
À 6.8
À 7.0
À 6.9
À 6.5
À 6.4
À 6.4
À 6.5
À 5.8
À 5.9
À 6.5
À 6.3
À 5.6
À 5.8
À 6.3
À 5.6
À 6.1
À 5.6
À 6.0
À 5.8
À 6.7
À 6.3
À 6.1
5.8
À 6.5
À 7.2
À 6.2
À 6.1
À 5.9
À 6.5
À 6.1
À 5.9
À 5.6
2.1
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Table 1. continued
MDM2
Ligand
MDMX
Ligand
Docking Score
kcal/mol
Ligand
Docking Score
kcal/mol
Docking Score
kcal/mol
Ligand
Docking Score
kcal/mol
70
71
72
73
À 6.8
À 6.6
À 6.7
À 6.7
143
144
À 8.5
À 3.3
À 8.8
À 6.7
70
71
72
73
À 6.0
À 6.1
À 6.0
À 6.5
143
144
À 6.5
À 6.9
À 7.7
À 5.6
145
145
Nutlin-3a[a]
SJ-172550[a]
[a] Positive control.
ered a valuable tool for successful lead optimization. Con-
sequently, as a step of the developed screening workflow,
33 compounds selected from the last step were evaluated for
ADMET properties using PreADMET web server (PreADMET,
https://preadmet.bmdrc.kr/).
This step allowed to select only ligands possessing key
pharmacokinetic properties within permissible ranges, as
defined by PreADMET. In this step of filtering, Caco-2 cell
permeability in nm/sec (Caco-2>45 nm/sec), percentage of
human oral absorption (%HIA >80%), plasma protein binding
(PPB>90%), capability of blocking hERG K+ channels and
cardiotoxicity (no evidence of carcinogenic activity=positive
prediction) were considered (Table S4, Supporting Information).
A total of 22 out of 33 compounds with predicted appropriate
pharmacokinetic properties passed the screening of ADMET
prediction.
and data were in accordance with the previously reported.[20]
The structure elucidation of new compounds 5, 6, 10, 15–17,
20, 23–25, and 146–148 was established on basis of NMR
(Figure S6, S7, S11, S16–S18, S21, S24–S26 and S28–S30,
Supporting Information) and high-resolution mass spectrometry
(HRMS) (Figure S31–S43, Supporting Information) techniques.
13C NMR assignments were determined by 2D heteronuclear
single quantum correlation (HSQC) and heteronuclear multiple
bond correlation (HMBC) experiments.
Biological evaluation
Effect on human cancer cell lines
Growth inhibitory activity of 1–26and 146–148in human
cancer cells
Synthesis
To study the in vitro antitumor potential of compounds 1--26
and 146–148, their antiproliferative activity was evaluated in
human colon cancer HCT116+/+ cells expressing wt p53 by
sulforhodamine B (SRB) assay. Among the studied compounds,
diarylpentanoids 3--11, 17–24, and 26 revealed potent anti-
proliferative activity (Table 2). The antiproliferative effect of
these compounds was also tested in human fibroblasts HFF-1
cells in order to assess their cytotoxicity toward normal cells.
Compounds 10 and 26 proved to have lower cytotoxicity
toward HFF-1 cells (Table 2), with a selective index (SI) higher
than 5.
When comparing the antiproliferative effect in human colon
cancer HCT116 p53+/+ cells of all studied compounds (Table 2),
some structure activity relationship (SAR) considerations can be
inferred (Figure 1). The antiproliferative activity is influenced by
the number and/or position of substituents in phenyl groups.
Interestingly, the substitution of both benzene rings with
methyl, methoxy, and halogen groups can contribute to
improved antiproliferative effects, dependent on the position of
the substituent. Nevertheless, the presence of amine groups at
para-position (14, 15, and 16) is associated with a drastic
decrease of the growth inhibitory effect (GI50 >25 μM). This low
antiproliferative effect is observed not only for diarylpentanoids
14, 15, and 16 (GI50 >25 μM), but also for their analogues 146,
147, and 148 (GI50 >25 μM). Considering diarylpentanoids with
monohalogenated benzene rings, halogenation at para position
(9, 10, and 11, GI50 =0.71–10.1 μM) is associated with a high
antiproliferative activity. Nevertheless, although the presence of
The diarylpentanoids with the best docking scores complying
with the druglikeness and ADMET properties were subsequently
prepared by base-catalyzed aldol reactions. In addition, struc-
ture-related diarylpentanoids 14–16, and 26 with docking
scores lower than positive controls, were also synthesized, in
order to understand the relationship between the docking
scores and the in vitro growth inhibitory activity in human
cancer cell lines expressing wt p53,. Thus, a total of 26 new
compounds of the series of 3,5-di((E)-benzylidene)tetrahydro-
4H-pyran-4-one derivatives were synthesized according to the
strategy illustrated on Scheme 1. The synthesis of diarylpenta-
noids 1–26 followed a base-catalyzed aldol reaction of tetrahy-
dro-4H-pyran-4-one with appropriative substituted benzalde-
hydes as building blocks. The reaction of tetrahydro-4H-pyran-
4-one with most of benzaldehydes resulted in the synthesis of
symmetric diarylpentanoids 1–13 and 17–26 with moderate to
good yields (56–87%). Nevertheless, when the reaction was
conducted with aminobenzaldehydes, in addition to the
predicted symmetric diarylpentanoids 14–16, analogues 146–
148 resulting from the aldol reaction in one of the α carbons to
the carbonyl group were also obtained as by-products, being
for this reason these compounds obtained with low yields.
The structure elucidation of compounds 1–4, 7–9, 11–14,
18, 19, 21, 22, and 26 was established by H and 13C nuclear
magnetic resonance (NMR) techniques (Figure S2–S5, S8–S10,
S12–S15, S19, S20, S22, S23 and S27, Supporting Information)
1
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Scheme 1. Synthetic pathway for obtaining 3,5-di((E)-benzylidene)tetrahydro-4H-pyran-4-one derivatives 1–26 and analogues 146–148.
fluor at ortho (3, GI50 =0.69 μM), and bromine at meta (6, GI50 =
0.9 μM) positions is favorable for this activity, the presence of
bromine (1), or chlorine (2) at ortho position is associated with a
decrease of activity (GI50 >25 μM). Additionally, diarylpenta-
noids with monosubstituted benzene rings with methoxy or
methyl groups at ortho and meta positions are much more
potent (4, 5, 7, and 8, GI50 =0.99–3.8 μM) than those with para-
methoxy/methyl substituted benzene rings (12, and 13, GI50
>25 μM). This promising antiproliferative effect is also observed
for diarylpentanoid 24 with a pyrazole moiety at ortho position
in the benzene rings (GI50 =0.22 μM).
group clearly affects the activity, as for the diarylpentanoid 21
with this group at 3 and 4 positions, displaying lower activity
(GI50 =4.55 μM) than 17 with a methylenedioxy group at 2 and
3 positions (GI50 =0.64 μM).
Finally, when comparing the activity of compounds with
trimethoxylated benzene rings, such as 25 and 26, the presence
of 3’,4’,5’-trimethoxyphenyl groups (26, GI50 =0.17 μM) is more
favorable than 2’,4’,5’- trimethoxyphenyl groups (25, GI50 >
25 μM).
Regarding compounds with dimethoxyphenyl groups (18–
20, and 22–23), it seems that the presence of methoxy groups
is associated with a high antiproliferative effect independently
of their position, being compounds with dimethoxyphenyl
groups more potent (0.63<GI50 <1.75 μM) than those with
monomethoxylated benzene rings (4, 7, and 12, 0.99<GI50 <
25 μM). On the other hand, the position of a methylenedioxy
Effect of diarylpentanoids 10and 26on p53-MDM2/X
interactions analyzed using yeast-based assays
As compounds 10 and 26 showed potent growth inhibitory
effect on HCT116 p53+/+ cells (expressing wt p53) and low
cytotoxicity in HFF-1 cells, their mode of action was inves-
tigated. In particular, it was checked the potential activation of
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MDMX: À 6.4 kcal/mol, Table 1), proved to be effective as p53-
MDM2/X inhibitor in the yeast assay (Figure 2). As expected,
diarylpentanoid 26, which presented the lowest docking score
(MDM2: À 4.3 kcal/mol, Table 1), was found to be inactive in the
yeast assay (Figure 2).
Table 2. GI50 values of compounds 1–26 and 146–148 in human cancer
HCT116 p53+/+ and normal fibroblasts HFF-1 cells.
GI50 [μM]
SI[a]
Compound ID
HCT116 p53+/+
HFF-1
1
>25
–
–
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
146
147
148
>25
–
–
0.69�0.21
2.45�0.07
3.8�0.42
0.9�0.14
0.99�0.01
1.21�0.01
10.1�4.85
6.25�1.18
0.71�0.08
>25
>25
>25
>25
>25
0.64�0.03
0.68�0.02
1.65�0.64
0.87�0.03
4.55�0.95
1.75�0.10
0.63�0.01
0.22�0.02
>25
0.77�0.30
1.44�0.12
3.24�0.01
0.63�0.008
0.55�0.15
3.88�0.022
9.45�2.62
36.20�5.54
0.55�0.01
–
–
–
–
–
0.59�0.005
0.89�0.07
2.21�0.08
0.63�0.03
14.76�2.21
0.69�0.04
0.49�0.01
0.33�0.06
–
1.12
0.59
0.85
0.7
0.56
3.21
0.94
5.79
0.77
–
–
–
–
–
0.92
1.31
1.34
0.72
3.24
0.39
0.78
1.5
–
7.12
–
–
–
Study of the mechanism of action of 10in human cancer cells
expressing wt p53
Considering the results obtained in the yeast screening assay,
10 was further studied in HCT116 p53+/+ cells, using the colony
formation assay in order to confirm its antiproliferative activity
(Figure 3A and 3B). Compound 10 inhibited the formation of
colonies up to 100% relative to DMSO at the concentration of
2 μM, a value lower than the GI50 value determined for the
referred cell line. Moreover, 10 was tested in HCT116 p53À /À
and MDA-MB-231 cell lines, using the SRB assay (Figure 3C). In
the absence of p53, the inhibitory effect was significantly
reduced. Nevertheless, 10 was still able to inhibit the growth of
p53-null cells, showing a GI50 of 10.13�0.47 μM, suggesting
that this derivative may have an additional mechanism that is
independent of p53. On the other hand, the low cytotoxic effect
of compound 10 on MDA-MB-231 cells, which expresses
mutp53, suggests a potential selectivity of compound 10
toward the wt p53 pathway.
0.17�0.01
1.21�0.07
>25
–
–
–
>25
>25
Altogether, 10 revealed to be the most promising com-
pound, as it showed some selectivity toward the wt p53
pathway. Thereafter, the effect of 10 on cell cycle and apoptosis
of HCT116+/+ cells was analyzed. For that, cancer cells were
incubated with 12 μM 10 (approximately two-fold GI50 value) for
48 h treatment. It was observed that the growth inhibitory
activity of 10 was associated with an apoptotic cell death of
over 24% (Figure 4A) and cell cycle arrest at G0/G1 phase
(Figure 4B).
To better understand the underlying mechanisms of 10-
induced apoptosis and cell cycle arrest, the expression levels of
p53 and transcriptional targets (p21 and PUMA), as well as
PARP cleavage were explored by western blot. As expected, in
HCT116 p53+/+, 12 μM 10 increased the p53 expression levels
(Figure 4C). Additionally, it increased p21 and the pro-apoptotic
protein PUMA (Figure 4C). 10 also led to PARP cleavage
(Figure 4C). Altogether, the results obtained in human colon
cells support the hint concerning a potent antitumor activity of
10, which involves an activation of the p53 pathway.
[a] Selective index (GI50 of HFF-1/GI50 of HCT116 p53+/+). GI50 values were
determined by SRB assay after 48 h of treatment (growth obtained with
vehicle was set as 100%). Data are mean�SEM of three to five
independent experiments. Nutlin-3a was used as positive control: GI50
(HCT116 p53+/+)=3.20�0.11 μM; GI50 (HFF-1)=18.74�0.51 μM.
wtp53 by inhibiting its interaction with MDM2, using the yeast
approach as described in.[21] Briefly, in this assay, human p53
induces growth inhibition that is counteracted by co-expression
of the human interactor MDM2. Inhibitors of the p53-MDM2
interaction re-establish the p53-induced growth inhibition. The
results obtained showed that only diarylpentanoid 10 signifi-
cantly reverted the negative effect of MDM2 on p53, at 25 μM
(Figure 2A), without interfering with the growth of control yeast
(vectors) (Figure 2B) or yeast cells expressing p53 only (Fig-
ure 2C).
Based on these results, 10 was selected to be tested on
yeasts co-expressing p53 and MDMX in order to investigate
whether this derivative also inhibited the p53-MDMX interac-
tion (Figure 2D). In these cells, 25 μM of 10 triggered a
significant growth inhibition, when compared to control yeasts
(82.79%�4.06). In fact, the growth percentage of treated yeasts
was close to that obtained with yeasts expressing p53 only
(77.52%�4.93), which indicated that 10 was also able to inhibit
the p53-MDMX interaction by over 82.2% (data not shown).
Altogether, the results obtained in yeast suggested that 10
was a potential dual inhibitor of the p53-MDM2/X interactions.
Comparing the effect of diarylpentanoids 10 and 26 on the
p53–MDM2/X interactions with docking scores prediction, 10,
that had one of the highest affinities (MDM2: À 7.1 kcal/mol;
Docking studies
In order to further understand the different activity profile of
diarylpentanoids 10 and 26, these compounds were further
analyzed in terms of docking poses and residues involved in the
p53–MDM2/X potential interactions (Figures 5 and 6).
Figure 5A shows the binding conformation of crystallo-
graphic nutlin-3A into MDM2. Figure 5B shows the most stable
binding poses of compounds 10 and 26 into MDM2 as
suggested by the docking protocol. Compound 10, the active
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Figure 1. Putative SAR for synthesized diarylpentanoids with antiproliferative activity in HCT116 p53+/+ cells.
Figure 2. (A) Effect of 10 μM nutlin-3A and compounds 10 and 26 (10 μM and 25 μM) on the percentage of growth of yeasts expressing p53 and MDM2, after
42 h treatment. (B) Effect of 25 μM compound 10 on the growth of yeast cells transformed with the empty vector, after 42 h treatment. The results obtained
with yeast cells treated with DMSO were considered as 100%. (C) Effect of 25 μM compound 10 on the growth of yeast cells expressing p53, after 42 h
treatment. (D) Effect of 25 μM SJ-172550 and compound 10 on the growth of yeasts expressing p53 and MDMX, after 42 h treatment. Data are mean�SEM of
at least five independent experiments; values significantly different from DMSO are indicated (***p<0.001, **p<0.01, *p<0.05). The unpaired student t-test
was used.
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Figure 3. (A, B) Effect of 10 on the colony formation of HCT116 p53+/+ cells. Cells were treated with 10 or solvent for 24 h, and colonies were allowed to grow
for 11 days. (A) Images correspond to a representative experiment of three. (B) The DMSO-treated cells were considered as 100% growth; data are
mean�SEM of three independent experiments. (C) Dose-response curves for the growth inhibitory activity of 10 in p53+/+ and p53À /À HCT116 cells, HFF-1
and MDA-MB-231 cells, determined by SRB assay, after 48 h treatment. Data are mean�SEM of three independent experiments.
Figure 4. Effect of 10 on cell cycle, apoptosis, and protein levels of p53 and its transcriptional targets, in HCT116 p53+/+ cells. (A) Effect of 12 μM 10 on
apoptosis after 48 h treatment. Data are mean�SEM of three independent experiments; values significantly different from DMSO: *p<0.05. The unpaired
student t-test was used. (B) Effect of 12 μM 10 on cell cycle progression after 48 h treatment. Data are mean�SEM of three independent experiments; values
significantly different from DMSO: *p<0.05. The unpaired student t-test was used. (C) Protein levels of p53 and its transcriptional targets after 48 h treatment
with 12 μM 10 or DMSO. Immunoblots represent two independent experiments; GAPDH was used as a loading control.
diarylpentanoid in the yeast assay and also one that was
predicted to bind more stably in silico to MDM2 binding pocket
(À 7.1 kcal.molÀ 1), establishes three hydrogen interactions with
Leu-54, Gly-58, and Val-93 (Figure 5C). Moreover, compound 10
also establishes non-polar interactions with MDM2 (not shown).
These type of interactions are also described as being important
halogenated rings are predicted to orient the molecule in the
binding site by establishing more hydrogen interactions. This
information could lead to a new family of diarylpentanoids,
which may bind with more affinity to the MDM2/X protein.
for p53-MDM2 interaction.[4] The inactive diarylpentanoid 26 in Conclusion
the yeast assay, and also one that was predicted to bind less
stably in silico to MDM2 binding pocket (À 4.3 kcal.molÀ 1),
establishes only one hydrogen interaction with Gln-24 (Fig-
ure 5D) and also non-polar interactions (not shown).
In this work, 29 diarylpentanoids (1–26 and 146–148) were
synthesized based on in silico studies and their in vitro
antiproliferative activity was evaluated in human HCT116 cancer
and normal fibroblasts HFF-1 cells. Two of the most effective
compounds (10 and 26) were further studied in a yeast-based
screening assay to evaluate their potential as inhibitors of p53–
MDM2/Xs interactions, being diarylpentanoid 10 identified for
the first time as a potential p53–MDM2/X interaction inhibitor.
The growth inhibitory effect of compound 10 was associated
with induction of cell cycle arrest and apoptosis through
Figure 6A shows the binding conformation of crystallo-
graphic SJ-172250 into MDMX. Compound 10, the active
diarylpentanoid in the yeast assay and also one that was
predicted to bind more stably in silico to MDMX binding pocket
(À 6.4 kcal.molÀ 1), establishes one hydrogen interaction with
Met-53 (Figure 6B) and non-polar interactions (not shown).
From the docking results, it should be highlighted that
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Figure 5. (A) MDM2 (transparent solid surface) interacting with crystallographic nutlin-3A (orange sticks). (B) The most potent diarylpentanoids tested as p53-
MDM2 interaction inhibitors (10 and 26 represented in blue and magenta sticks, respectively) docked into MDM2 structure, as predicted by the docking
studies. (C) Predicted binding poses of 10 (represented in blue sticks) in the binding site of MDM2. (D) Predicted binding poses of 26 (represented in magenta
sticks) in the binding site of MDM2. MDM2 is represented as surface, where carbon, oxygen, nitrogen, and sulfur are represented in yellow, red, blue, and
orange, respectively. Hydrogen interactions are depicted with a dashed yellow line. Residues evolved in polar interactions are labelled and represented in
green. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Figure 6. (A) MDMX (transparent solid surface) interacting with crystallographic SJ-172550 (red sticks). (B) Predicted binding poses of 10 (represented in blue
sticks) in the binding site of MDMX. MDMX is represented as surface, where carbon, oxygen, nitrogen, and sulfur are represented in, yellow, red, blue, and
orange, respectively. Hydrogen interactions are depicted with a dashed yellow line. Residues evolved on polar interactions are labelled and represented in
green. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Experimental Section
activation of the wtp53 pathway. The results obtained from this
study will be valuable for the rational design of new and more
potent diarylpentanoids with p53–MDM2/X inhibitory effect.
In silico studies
Structure-based virtual screening
A Lipinski’s pre-filter was applied to remove less relevant ligands in
the library using SwissADME (http://www.swissadme.ch/) web
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server. The structures of diarylpentanoids were drawn using
ChemDraw 17.0. The structures of known p53-MDM2 inhibitor
nutlin-3A and known p53-MDMX inhibitor SJ-172550 were obtained
from PubChem.[22] The 3-Dimensional (3D) structures of the diary-
lpentanoids and controls were minimized using the Austin Model 1
parameterization of the MNDO method (AM1) implemented in
ArgusLab 4.0.1. The calculation was finished when the gradient
between any two successive steps in the geometry search was less
than 0.1 kcalÅÀ 1 molÀ 1; the maximum number of geometry steps
were set to 1000. The calculation run until the maximum steps
were reached, or the convergence criteria was met, whichever
came first. The 3D structures of MDM2 and MDMX were obtained
from Protein Data Bank (PDB id (MDM2): 4HG7; PBD id (MDMX):
3LBJ) and prepared for docking using AutoDockTools 1.5.7. Docking
simulations between the MDM2/X and small molecules were
undertaken in PyRx 0.8 using AutoDock Vina.[23] Docking was run
using an exhaustiveness of 8, engulfing the cavity occupied by the
crystallographic nutlin-3A (PDB id: 4HG7)[24] or WK298 (PBD id:
3LBJ).[25] Nine conformations for each ligand were obtained. The top
ranked conformations of 10 and 26 were further analyzed
concerning noncovalent interactions using PyMOL 2.2.4.[26]
University of Vigo, Spain. Ions were generated using a Combi
MALDI electrospray ionization (ESI) source. Tetrahydro-4H-pyran-4-
one and benzaldehydes were purchased from Sigma Aldrich. The
following materials were synthesized and purified by the described
procedures.
General procedure for the synthesis of diarylpentanoids 1–26
and 146–148
An aqueous solution of 40% sodium hydroxide was added to a
solution of tetrahydro-4H-pyran-4-one (100 mg, 0.99 mmol) in
methanol until pH 13–14. Then, a solution of appropriate benzalde-
hyde (360–568 mg, 2.99 mmol) in methanol was slowly added to
the reaction mixture. The reaction was left at room temperature for
22 h–7 days and was monitored by TLC. After, crushed ice was
added to the reaction mixture and neutralized with 5 M HCl
solution. For the synthesis of the diarylpentanoids 1, 2, 6–9, 11, 13,
17, 21–24, and 26 after the addition of crushed ice, the solution
was extracted with ethyl acetate or chloroform (3x50 mL), and the
organic layers were collected, washed with water, dried over with
anhydrous sodium sulfate and concentrated under reduced
pressure. The obtained residues were purified as indicated below
for the referred diarylpentanoids. For the synthesis of compounds
3–5, 10, 12, 14–16, 18–20, 25, and 146–148, the obtained solid was
filtrated, washed with water, dried, and purified as indicated below
for the referred diarylpentanoids.
Predicting Druglikeness
The selected compounds from virtual screening were further
investigated for druglikeness properties, namely Ghose, Veber,
Egan, Muegge, CMC-like, Lead-like, WDI-like and MDDR-like rules by
SwissADME (http://www.swissadme.ch/) and PreADMET (https://
preadmet.bmdrc.kr/) web servers. They provide a quick and de-
tailed method for the calculation of a wide range of principal
molecular descriptors that are useful in the prediction of phys-
icochemical properties.
Experimental description of compounds 1–4, 7–9, 11–14, 18, 19,
21, 22, and 26 is presented in the Supporting Information
(Figure S2–S5, S8–S10, S12–S15, S19, S20, S22, and S27). NMR and
HRMS data of compounds 5, 6, 10, 15–17, 20, 23–25, and 146–148
were here described for the first time, as indicated below.
3,5-bis((E)-2-methylbenzylidene)tetrahydro-4H-pyran-4-one (5):
Purified by crystallization from methanol. Yield: 85% as yellow solid;
1
ADMET properties prediction
°
mp (methanol): 144–146 C; H NMR (CDCl3 300.13 MHz) δ=8.03 (sl,
2H, H-1’’), 7.31–7.25 (m, 4H, H-3’, À 4’), 7.21 (dt, J=7.3; 2.3 Hz, 2H, H-
5’), 7.02 (brd, J=7.4 Hz, 2H, H-6’), 4.78 (d, J=1.8 Hz, 4H, H-3, À 5),
2.39 (s, 6H, 2’-CH3) ppm; 13C NMR (CDCl3, 75.47 MHz) δ=185.8
(C=O), 138.5 (C-2’), 135.9 (C-1’’), 133.8 (C-2, À 6), 133.4 (C-1’), 130.6
(C-3’), 129.4 (C-4’), 129.2 (C-6’), 125.8 (C-5’), 68.8 (C-3, À 5), 20.2 (2’-
CH3) ppm; HRMS (ESI+): m/z Anal. Calc. for C21H21O2 (M+H+)
305.1536; found: 305.1536.
Aiming to achieve druglike molecules, PreADMET web server was
applied as an extra filter for in silico assessment of ADMET
properties of the potential hit molecules selected from previous
filter. It provides prediction of membrane permeability using Caco-
2 cell model,[27] human intestinal absorption,[28] cardiotoxicity which
is built from the data of NTP (National Toxicology Program) and US
FDA, and potential interaction with hERG channels.
3,5-bis((E)-3-bromobenzylidene)tetrahydro-4H-pyran-4-one
(6):
Purified by crystallization from methanol. Yield: 65% as yellow solid;
Synthesis
1
°
mp (methanol): 174–177 C; H NMR (CDCl3 300.13 MHz) δ=7.75 (sl,
2H, H-1’), 7.52 (dt, J=7.8; 1.6 Hz, 2H, H-4’), 7.45 (brt, J=1.8 Hz, 2H,
H-2’), 7.30 (t, J=7.7 Hz, 2H, H-5’), 7.23 (brd, J=9.3 Hz, 2H, H-6’), 4.90
(d, J=2.0 Hz, 4H, H-3, À 5) ppm; 13C NMR (CDCl3, 75.47 MHz) δ=
185.1 (C=O), 136.7 (C-1’), 135.2 (C-1’’), 134.0 (C-2, À 6), 133.1 (C-2’),
132.5 (C-4’), 130.4 (C-5’), 129.0 (C-6’), 68.1 (C-3, À 5) ppm; HRMS
(ESI+): m/z Anal. Calc. for C19H15Br2O2 (M+H+) 432.9433; found:
432.9431.
General methods
All reactions were monitored by thin-layer chromatography (TLC).
The purification of compounds by flash column chromatography
(CC) and preparative TLC was performed using Macherey-Nagel
silica gel 60 (0.04–0.063 mm) and Macherey-Nagel silica gel 60
(GF254) plates, respectively. Melting Points were obtained in a
Köfler microscope and are uncorrected. 1H and 13C NMR spectra
were taken in CDCl3 at room temperature, on Bruker Avance 300
and 500 instruments (300.13 MHz or 500.13 MHz for 1H and
75.47 MHz or 125.77 MHz for 13C). Chemical shifts are expressed in δ
(ppm) values relative to tetramethylsilane (TMS) used as an internal
reference; 13C NMR assignments were made by 2D (HSQC and
3,5-bis((E)-4-chlorobenzylidene)tetrahydro-4H-pyran-4-one (10):
Purified by crystallization from chloroform. Yield: 80% as light
yellow solid; mp (chloroform): 211–214 C; 1H NMR (CDCl3
°
300.13 MHz) δ=7.77 (sl, 2H, H-1’’), 7.40 (d, J=8.5 Hz, 4H, H-3’, À 5’),
7.25 (d, J=8.8 Hz, 4H, H-2’, À 6’), 4.89 (d, J=1.9 Hz, 4H, H-3, À 5)
ppm; 13C NMR (CDCl3, 75.47 MHz) δ=185.7 (C=O), 136.2 (C-4’),
135.8 (C-1’’), 133.8 (C-1’), 133.6 (C-2, À 6), 132.2 (C-2’, À 6’), 129.6 (C-
3’, À 5’), 69.0 (C-3, À 5) ppm; HRMS (ESI+): m/z Anal. Calc. for
C19H15Cl2O6 (M+H+) 345.04436, found 345.04349.
HMBC) NMR experiments (long-range 13CÀ H coupling constants
1
were optimized to 7 Hz). The spectral treatment was executed
using the MestReNova v6.0.2-5475 software. HRMS mass spectra of
compounds 5, 6, 10, 15–17, 20, 23–25, and 146–148 were
performed on an APEXQe FT-ICR MS (Bruker Daltonics, Billerica, MA)
equipped with a 7T actively shielded magnet, at C.A.C.T.I. –
3,5-bis((E)-4-diethylaminobenzylidene)tetrahydro-4H-pyran-4-one
(15). Purified by preparative TLC (SiO2; n-hexane: ethyl acetate,
1
7:3). Yield: 33% as orange gum; H NMR (CDCl3, 300.13 MHz) δ=
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°
7.75 (sl, 2H, H-1’’), 7.24 (d, J=8.9 Hz, 4H, H-2’, À 6’), 6.67 (d, J=
9.0 Hz, 4H, H-3’, À 5’), 4.95 (d, J=1.6 Hz, 4H, H-3, À 5), 3.40 (q, J=
7.1 Hz, 8H, 4’-NCH2CH3), 1.20 (t, J=7.0 Hz, 12H, 4’-NCH2CH3) ppm;
13C NMR (CDCl3, 75.47 MHz) δ=185.1 (C=O), 149.7 (C-4’), 136.6 (C-
1’’), 133.4 (C-2’, À 6’), 129.1 (C-2, À 6), 123.2 (C-1’), 111.5 (C-3’, À 6’),
68.4 (C-3, À 5), 43.8 (4’-NCH2CH3), 13.0 (4’-NCH2CH3) ppm; HRMS (ESI
+): m/z Anal. Calc. for C27H35N2O2 (M+H+) 419.26930; found:
419.26852.
as yellow solid; mp (ethyl acetate): 194–195 C; 1H NMR (CDCl3,
500.13 MHz) δ=8.04 (sl, 2H, H-1’’), 6.62 (s, 2H, H-6’), 6.53 (s, 2H, H-
3’), 4.83 (d, J=1.8 Hz, 4H, H-3, À 5), 3.93 (s, 6H, 4’-OCH3), 3.85 (s, 6H,
2’-OCH3), 3.84 (s, 6H, 5’-OCH3) ppm; 13C NMR (CDCl3, 75.47 MHz) δ=
185.4 (C=O), 154.1 (C-2’), 151.4 (C-4’), 143.3 (C-5’), 131.6 (C-1’’), 131.4
(C-2, À 6), 115.5 (C-1’), 114.0 (C-6’), 96.9 (C-3’), 69.4 (C-3, À 5), 56.7
(5’-OCH3), 56.4 (2’-OCH3), 56.1 (4’-OCH3) ppm; HRMS (ESI+): m/z
Anal. Calc. for C25H29O8 (M+H+) 457.18569; found: 457.18496.
3,5-bis((E)-4-morpholinobenzylidene)tetrahydro-4H-pyran-4-one
(16): Purified by preparative TLC (SiO2; n-hexane: ethyl acetate,
(E)-3-(4-(dimethylamino)benzylidene)tetrahydro-4H-pyran-4-one
(146): Purified by preparative TLC (SiO2; n-hexane: ethyl acetate,
7:3). Yield: 30% as yellow gum; 1H NMR (CDCl3, 300.13 MHz) δ=
7.62 (t, J=2.2 Hz, 1H, H-1’’), 7.24 (d, J=9.0 Hz, 2H, H-2’, À 6’), 6.69
(d, J=9.0 Hz, 2H, H-3’, À 5’), 4.90 (d, J=2.1 Hz, 2H, H-3), 4.07 (t, J=
6.1 Hz, 2H, H-5), 3.03 (s, 6H, 4’-NCH3), 2.65 (t, J=6.1 Hz, 2H, H-6)
ppm; 13C NMR (CDCl3, 75.47 MHz) δ=195.6 (C=O), 150.9 (C-4’),
137.1 (C-1’’), 133.0 (C-2’, À 6’), 128.1 (C-2), 122.0 (C-1’), 111.6 (C-3’,
À 5’), 69.0 (C-3), 65.2 (C-5), 39.9 (4’- NCH3), 39.3 (C-6) ppm; HRMS
(ESI+): m/z Anal. Calc. for C14H18NO2 (M+H+) 232.13321; found:
232.13332.
°
7:3). Yield: 42% as orange solid; mp (ethyl acetate): 265–267 C;
1H NMR (CDCl3, 300.13 MHz) δ=7.77 (sl, 2H, H-1’’), 7.28 (d, J=
8.7 Hz, 4H, H-2’, À 6’), 6.91 (d, J=8.9 Hz, 2H, H-3’, À 5’), 4.94 (d, J=
1.7 Hz, 4H, H-3, À 5), 3.87 (t, J=4.8 Hz, 8H, H-8’, À 9’), 3.26 (t, J=
4.9 Hz, 8H, H-7’, À 10’) ppm; 13C NMR (CDCl3, 75.47 MHz) δ=185.0
(C=O), 135.6 (C-1’’), 132.1 (C-2’, À 6’), 130.0 (C-2, À 6), 125.4 (C-1’),
114.2 (C-3’, À 5’), 68.4 (C-3, À 5), 66.3 (C-8’, À 9’), 47.6 (C-7’, À 10’)
ppm; HRMS (ESI+): m/z Anal. Calc. for C27H31N2O2 (M+H+)
447.22783; found: 447.22738.
(3E,5E)-3,5-bis(benzo[d][1,3]dioxol-4-ylmethylene)tetrahydro-4H-
pyran-4-one (17): Purified by preparative TLC (SiO2; n-hexane: ethyl
acetate, 7:3). Yield: 56% as yellow solid; mp (ethyl acetate): 115–
(E)-3-(4-diethylaminobenzylidene)tetrahydro-4H-pyran-4-one
(147): Purified by preparative TLC (SiO2; n-hexane: ethyl acetate,
7:3). Yield: 31% as yellow gum; 1H NMR (CDCl3, 300.13 MHz) δ=
7.61 (t, J=2.0 Hz, 1H, H-1’’), 7.22 (d, J=9.0 Hz, 2H, H-2’, À 6’), 6.66
(d, J=9.0 Hz, 2H, H-3’, À 5’), 4.90 (d, J=2.0 Hz, 2H, H-3), 4.06 (t, J=
6.0 Hz, 2H, H-5), 3.40 (q, J=7.1 Hz, 4H, 4’-NCH2CH3), 2.64 (t, J=
6.0 Hz, 2H, H-5’), 1.19 (t, J=7.1 Hz, 6H, 4’-NCH2CH3) ppm. 13C NMR
(CDCl3, 75.47 MHz) δ=195.3 (C=O), 148.4 (C-4’), 137.0 (C-1’’), 133.3
(C-2’, À 6’), 127.3 (C-2’), 121.0 (C-1’), 113.3 (C-3’, À 5’), 68.9 (C-3), 65.0
(C-5), 44.1 (4’-NCH2CH3), 39.1 (C-6), 12.3 (4’-NCH2CH3) ppm; HRMS
(ESI+): m/z Anal. Calc. for C16H22NO2 (M+H+) 260.16451; found:
260.16441.
1
°
117 C; H NMR (CDCl3, 300.13 MHz) δ=7.80 (sl, 2H, H-1’’), 6.89–6.83
(m, 4H, H-4’, À 5’), 6.72–6.69 (m, 2H, H-6’), 6.02 (s, 4H, H-7’), 4.82 (d,
J=1.9 Hz, 4H, H-3, À 5) ppm; 13C NMR (CDCl3, 75.47 MHz) δ=185.1
(C=O), 147.9 (C-3’), 146.7 (C-2’), 134.1 (C-2, À 6), 129.7 (C-1’’), 122.9
(C-6’), 121.8 (C-5’), 117.3 (C-1’), 101.3 (C-7’), 69.0 (C-3, À 5) ppm;
HRMS (ESI+): m/z Anal. Calc. for C21H16O6 (M+H+) 365.1020, found
365.1018.
3,5-bis((E)-2,5-dimethoxybenzylidene)tetrahydro-4H-pyran-4-one
(20): Purified by preparative TLC (SiO2; n-hexane: ethyl acetate,
7:3). Yield: 56% as light yellow solid; mp (ethyl acetate): 125–
(E)-3-(4-morpholinobenzylidene)tetrahydro-4H-pyran-4-one
(148): Purified by preparative TLC (SiO2; n-hexane: ethyl acetate,
7:3). Yield: 26% as yellow gum; 1H NMR (CDCl3, 300.13 MHz) δ=
7.60 (t, J=2.0 Hz, 1H, H-1’’), 7.25 (d, J=8.1 Hz, 2H, H-2’, À 6’), 6.89
(d, J=8.9 Hz, 2H, H-3’, À 5’), 4.88 (d, J=2.1 Hz, 2H, H-3), 4.07 (t, J=
6.1 Hz, 2H, H-5), 3.86 (t, J=4.8 Hz, 4H, H-8’, À 9’), 3.26 (t, J=4.9, 4H,
H-7’, À 10’), 2.66 (t, J=6.1, 2H, H-6) ppm; 13C NMR (CDCl3,
75.47 MHz) δ=185.0 (C=O), 135.6 (C-1’’), 132.1 (C-2’, À 6’), 130.0 (C-
2, À 6), 125.4 (C-1’), 114.2 (C-3’, À 5’), 68.4 (C-3, À 5), 66.3 (C-8’, À 9’),
60.5 (C-6), 47.6 (C-7’, À 10’) ppm; HRMS (ESI+): m/z Anal. Calc. for
C16H20NO3 (M+H+) 274.14377; found: 274.14368.
127 C; 1H NMR (CDCl3, 300.13 MHz) δ=δ 8.02 (sl, 2H, H-1’’), 6.90
°
(dd, J=9.0, 2.8 Hz, 2H, H-4’), 6.85 (d, J=8.9 Hz, 2H, H-3’), 6.63 (d, J=
2.8 Hz, 2H, H-6’), 4.80 (d, J=1.9 Hz, 4H, H-3, À 5), 3.82 (s, 6H, 2’-
OCH3), 3.78 (s, 6H, 5’-OCH3) ppm; 13C NMR (CDCl3, 75.47 MHz) δ=
186.3 (C=O), 153.1 (C-5’), 152.9 (C-2’), 133.6 (C-2, À 6), 132.3 (C-1’’),
124.8 (C-1’), 116.4 (C-6’), 115.7 (C-4’), 111.4 (C-3’), 69.0 (C-3, À 5),
56.2 (2’-OCH3), 56.0 (5’-OCH3) ppm; HRMS (ESI+): m/z Anal. Calc. for
C23H25O6 (M+H+) 397.1646; found: 397.1650.
3,5-bis((E)-3,5-dimethoxybenzylidene)tetrahydro-4H-pyran-4-one
(23): Purified by crystallization from methanol. Yield: 68% as yellow
1
°
solid; mp (methanol): 142–145 C; H NMR (CDCl3, 300.13 MHz) δ=
7.75 (s, 2H, H-1’’), 6.49 (t, J=2.3 Hz, 4H, H-4’), 6.45 (d, J=2.2 Hz, H-2,
H-2’, À 6’), 4.91 (d, J=2.0 Hz, 4H, H-3, À 5), 3.81 (s, 12H, 3’-, 5’-OCH3)
ppm; 13C NMR (CDCl3, 75.47 MHz) δ=185.6 (C=O), 161.0 (C-3’, À 5’),
136.8 (C-1’), 136.7(C-1’’), 133.6 (C-2, À 6), 108.5 (C-2’, À 6’), 101.7 (C-
4’), 68.8 (C-3, À 5), 55.6 (3’-, 5’-OCH3) ppm; HRMS (ESI+): m/z Anal.
Calc. for C23H25O6 (M+H+) 397.1646; found: 397.1649.
Biological activity
Human cell lines assays
Human colon adenocarcinoma HCT116 cell lines expressing wt p53
(HCT116 p53+/+) and its p53-null isogenic derivative (HCT116
p53À /À ) were provided by B. Vogelstein (The Johns Hopkins Kimmel
Cancer Center, Baltimore, MD, USA); human breast adenocarcinoma
MDA-MB-231 and non-tumorigenic foreskin fibroblasts HFF-1 cell
lines were purchase from American Type Culture Collection (ATCC,
Rockville, MD, USA). All cancer cell lines were routinely cultured in
RPMI-1640 medium with glutamine from Corning (VWR, Carnaxide,
Portugal) supplemented with 10% fetal bovine serum (FBS) from
Biowest (Labclinics, Barcelona, Spain). All cells were maintained in a
3,5-bis((E)-5-fluoro-2-(1H-pyrazol-1-yl)benzylidene)tetrahydro-4H-
pyran-4-one (24): Purified by preparative TLC (SiO2; n-hexane :
ethyl acetate, 7:3). Yield: 54% as yellow solid; mp (ethyl acetate):
1
°
120–123 C; H NMR (CDCl3, 300.13 MHz) δ=7.74 (d, J=1.8 Hz, 2H,
H-9’), 7.59–7.54 (m, 6H, H-1’’, À 3’, À 7’), 7.20 (td, J=8.2; 2.8 Hz, 2H,
H-4’), 6.92 (dd, J=8.9; 2.9 Hz, 3H, H-6’), 6.46 (t, J=2.2 Hz, 2H, H-8’),
4.66 (d, J=1.9 Hz, 4H, H-3, À 5) ppm; 13C NMR (CDCl3, 75.47 MHz)
δ=184.1 (C=O), 161.2 (d, J=249.1 Hz, C-5’), 141.7 (C-9’), 136.8 (d,
J=3.0 Hz, C-2’), 134.8 (C-2, À 6), 131.5 (C-7’), 130.8 (d, J=8.3 Hz, H-
1’), 127.8 (d, J=8.8 Hz, C-3’), 117.3 (d, J=22.6 Hz, C-4’), 116.8 (d, J=
23.5, C-6’), 107.7 (C-8’), 68.8 (C-3, À 5) ppm; HRMS (ESI+): m/z Anal.
Calc. for C25H19F2N4O2 (M+H+) 445.1471; found: 445.1466.
°
humidified incubator at 37 C with 5% CO2.
Sulforhodamine B (SRB) assay
Human cell lines were seeded in 96-well plates at a density of 5.0×
103 (HCT116 and MDA-MB-231), and 1.0×104 (HFF-1) cells/well, for
3,5-bis((E)-2,4,5-trimethoxybenzylidene)tetrahydro-4H-pyran-4-
one (25): Purified by crystallization from ethyl acetate. Yield: 57%
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24 h. Cells were treated with serial dilutions of SYNAP (ranging
from 3–150 μM), for additional 48 h. Effect on cell proliferation was
measured by SRB assay, as described,[29] and the concentration of
compound that induces 50% growth inhibition (GI50) was then
determined for each cell line using the GraphPad Prism software
version 7.0 (La Jolla, CA, USA).
differences between means were tested for significance using the
unpaired Student’s t-test (*p<0.05; **p<0.01; ***p<0.001).
Acknowledgements
This research was partially supported by the Strategic Funding
UIDB/04423/2020 and UIDP/04423/2020 (Group of Natural Prod-
ucts and Medicinal Chemistry, CIIMAR) and UIDB/50006/2020
(LAQV/REQUIMTE), through national funds provided by the FCT
and ERDF, within the framework of the program PT2020 and the
project PTDC/SAUPUB/28736/2017. Joana Moreira and Joana
Almeida acknowledges their grants (SFRH/BD/135852/2018 and
2020.05026.BD, respectively). The authors thank Sara Cravo for all
the technical and scientific support.
NMRYeast screening assay
Saccharomyces cerevisiae cells expressing human wt p53 alone or
co-expressed with human MDM2 or MDMX were used, as
previously described in.[21,29] Briefly, cells were grown in galactose-
induction selective medium with 1–25 μM compounds 10 and 26
°
for 42 h, at 30 C. Yeast cell growth was analyzed by colony-forming
unit counts.
Colony formation assay
HCT116 p53+/+ cells were seeded in 6-well plates at a density of
1.0×103 cells/well, followed by incubation with 0.25, 0.5, 1 and
2 μM 10 for 11 days. Formed colonies were fixed with 10%
methanol and 10% acetic acid for 10 min, and then stained with
0.5% crystal violet (Sigma-Aldrich) in 1:1 methanol/H2O for 15 min.
Colonies containing more than 20 cells were counted.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: Antitumor agents · diarylpentanoids · in silico
studies · p53-MDM2/X inhibitors · Structure-activity relationship
Cell cycle and apoptosis analysis
The analyses were performed basically as described.[29] Briefly,
HCT116 p53+/+ cells were seeded in 6-well plates at a density of
1.5×105 cells/well for 24 h, followed by treatment with 12 μM 10
for additional 48 h. For cell cycle analysis, cells were stained with
propidium iodide (PI; Sigma-Aldrich); cells were analyzed by flow
cytometry and cell cycle phases were identified and quantified
using the FlowJo X 10.0.7 Software (Treestar, Ashland, OR, USA). For
apoptosis, cells were stained using the Annexin V-FITC Apoptosis
Detection Kit I from BD Biosciences (Enzifarma, Porto, Portugal),
according to the manufacturer’s instructions. The AccuriTM C6 flow
cytometer and the BD Accuri C6 software (BD Biosciences) were
used.
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Manuscript received: May 16, 2021
Revised manuscript received: June 21, 2021
Accepted manuscript online: June 25, 2021
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J. Moreira, J. Almeida, Dr. J. B.
Loureiro, Dr. H. Ramos, Prof. A.
Palmeira, Prof. M. M. Pinto, Prof. L.
Saraiva*, Prof. H. Cidade*
1 – 14
A Diarylpentanoid with Potential
Activation of the p53 Pathway:
Combination of in silico Screening
Studies, Synthesis, and Biological
Activity Evaluation
Tumour suppression: We identified a
new diarylpentanoid as a potential
p53–MDM2/X dual inhibitor, with low
cytotoxicity toward HFF-1 cells. The
inhibitory effect on cancer cell lines
was significantly reduced in the
a mutant p53 form, suggesting some
selectivity of this compound toward
the wt p53 pathway. These results will
offer a perspective on the design of
new potent dual p53–MDM2/X inhibi-
tors.
absence of p53 and in cells expressing