Synthesis, biological evaluation, and in silico studies of novel aminated xanthones as potential p53-activating agents

Article abstract of DOI:10.3390/molecules24101975

Xanthone scaffold has been regarded as an attractive chemical tool in the search for bioactive molecules with antitumor activity, and in particular two xanthone derivatives, 12- hydroxy-2,2-dimethyl-3,4-dihydro-2H,6H-pyrano [3,2-b]xanthen-6-one (4) and 3,4-dimethoxy-9- oxo-9H-xanthene-1-carbaldehyde (5), were described as a murine double minute 2 (MDM2)-p53 inhibitor and a TAp73 activator, respectively. The xanthone 5 was used as a starting point for the construction of a library of 3,4-dioxygenated xanthones bearing chemical moieties of described MDM2-p53 inhibitors. Eleven aminated xanthones were successfully synthesized and initially screened for their ability to disrupt the MDM2-p53 interaction using a yeast cell-based assay. With this approach, xanthone 37 was identified as a putative p53-activating agent through inhibition of interaction with MDM2. Xanthone 37 inhibited the growth of human colon adenocarcinoma HCT116 cell lines in a p53-dependent manner. The growth inhibitory effect of xanthone 37 was associated with the induction of G1-phase cell cycle arrest and increased protein expression levels of p53 transcriptional targets. These results demonstrated the potential usefulness of coupling amine-containing structural motifs of known MDM2-p53 disruptors into a 3,4-dioxygenated xanthone scaffold in the design of novel and potent p53 activators with antitumor activity and favorable drug-like properties. Moreover, in silico docking studies were performed in order to predict the binding poses and residues involved in the potential MDM2-p53 interaction.

Full text of DOI:10.3390/molecules24101975

molecules
Article
Synthesis, Biological Evaluation, and In Silico
Studies of Novel Aminated Xanthones as Potential
p53-Activating Agents
Agostinho Lemos 1 , Ana Sara Gomes 2 , Joana B. Loureiro 2, Pedro Brandão 1
,
Andreia Palmeira 1,3, Madalena M. M. Pinto 1,3 , Lucília Saraiva 2,
*
and
Maria Emília Sousa 1,3,
*
1
Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences,
Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal;
up201002662@ff.up.pt (A.L.); pedrocgbrandao@gmail.com (P.B.); andreiapalmeira@gmail.com (A.P.);
madalena@ff.up.pt (M.M.M.P.)
2
3
UCIBIO/REQUIMTE, Laboratory of Microbiology, Department of Biological Sciences, Faculty of Pharmacy,
University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal;
anasarag4@gmail.com (A.S.G.); up201407524@ff.up.pt (J.B.L.)
CIIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto,
Novo Edificio do Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos, S/N,
4450-208 Matosinhos, Portugal
*
Correspondence: lucilia.saraiva@ff.up.pt (L.S.); esousa@ff.up.pt (M.E.S.);
Tel.: +351-22-0428-584 (L.S.); +351-22-0428-689 (M.E.S.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Qiao-Hong Chen
Received: 7 May 2019; Accepted: 21 May 2019; Published: 22 May 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Xanthone scaffold has been regarded as an attractive chemical tool in the search for
bioactive molecules with antitumor activity, and in particular two xanthone derivatives,
12-hydroxy-2,2-dimethyl-3,4-dihydro-2H,6H-pyrano [3,2-b]xanthen-6-one (
oxo-9H-xanthene-1-carbaldehyde ( ), were described as a murine double minute 2 (MDM2)-p53
inhibitor and a TAp73 activator, respectively. The xanthone was used as a starting point for the
4) and 3,4-dimethoxy-9-
5
5
construction of a library of 3,4-dioxygenated xanthones bearing chemical moieties of described
MDM2-p53 inhibitors. Eleven aminated xanthones were successfully synthesized and initially
screened for their ability to disrupt the MDM2-p53 interaction using a yeast cell-based assay. With
this approach, xanthone 37 was identified as a putative p53-activating agent through inhibition
of interaction with MDM2. Xanthone 37 inhibited the growth of human colon adenocarcinoma
HCT116 cell lines in a p53-dependent manner. The growth inhibitory effect of xanthone 37 was
associated with the induction of G1-phase cell cycle arrest and increased protein expression levels
of p53 transcriptional targets. These results demonstrated the potential usefulness of coupling
amine-containing structural motifs of known MDM2-p53 disruptors into a 3,4-dioxygenated xanthone
scaffold in the design of novel and potent p53 activators with antitumor activity and favorable
drug-like properties. Moreover, in silico docking studies were performed in order to predict the
binding poses and residues involved in the potential MDM2-p53 interaction.
Keywords: antitumor activity; computational docking; MDM2-p53 interaction; xanthones;
yeast-based assays
1. Introduction
The tumor suppressor p53 acts as a transcription factor, triggering the expression of multiple
downstream target genes that play a pivotal role in the regulation of cell cycle arrest, apoptosis,
Molecules 2019, 24, 1975; doi:10.3390/molecules24101975
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senescence, and DNA repair [
1–
6]. Upon cellular stress stimuli, the activation of the p53 pathway
may compromise the tumorigenesis, circumventing the proliferation of damaged cells with oncogenic
potential. Inactivation of the p53 function represents one of the most common events in human
cancers [
within the p53 gene (TP53) or by post-translational modifications of its gene product [
tumors retaining the wild type (wt) p53 function, the tumor suppressor activity of p53 can be abolished
by multiple mechanisms [ ], mainly by direct interaction between p53 and its primary endogenous
inhibitor, the murine double minute 2 (MDM2) protein [10 11]. The oncoprotein MDM2 physically
interacts with the amino-terminal domain of p53 and negatively regulates its activity by blocking
p53-mediated transactivation [12 13], by inducing the export of p53 from nucleus to the cytoplasm [14],
and by promoting E3 ubiquitin ligase-mediated p53 degradation via the proteasomal pathway [15 16].
Targeting the MDM2-p53 interaction with small molecules has been regarded an attractive approach
for the reactivation of the p53 pathway in wt p53-expressing tumors [17 18]. Since the seminal work of
Vassilev et al. reporting the discovery of nutlin-3A ( , Figure 1) as the first inhibitor of MDM2-p53
7]. In approximately half of all human cancer cells, the p53 inactivation is caused by mutations
8]. In those
9
,
,
,
,
1
interaction [19], an impressive number of MDM2-p53 disruptors from a huge diversity of chemical
families have been identified in recent years, entering some of them into clinical trials [20,21].
Figure 1. Chemical structures of nutlin-3A (1), gambogic acid (2 α-mangostin (3), 12-hydroxy-2,2-
),
dimethyl-3,4-dihydro-2H,6H-pyrano[3,2-b]xanthen-6-one (4), and 3,4-dimethoxy-9-oxo-9H-xanthene-
1-carbaldehyde (5).
Xanthones represent a privileged class of compounds with a multiplicity of biological activities,
in particular with antitumor activity against several human tumor cell lines [22 36]. Intriguingly,
–
our research group has demonstrated that the antitumor activity of some derivatives can be
correlated to the disruption of the MDM2-p53 complex and the activation of a p53-dependent
pathway [30,31]. In fact, implementing a yeast-based assay, the naturally occurring xanthones gambogic
acid (
on p53-induced growth inhibition and on p53 transcriptional activity [30]. The synthetic xanthone
12-hydroxy-2,2-dimethyl-3,4-dihydro-2H,6H-pyrano [3,2-b]xanthen-6-one ( , Figure 1) was identified as
2, Figure 1) and α-mangostin (3, Figure 1) were found to revert the inhibitory effect of MDM2
4
a potential MDM2-p53 disruptor, using an in silico virtual screening strategy against MDM2 protein and
a yeast-based assay [31]. Its molecular mechanism was corroborated in MDM2-overexpressing human
tumor cell lines with wt p53. In fact, xanthone
increasing the protein expression levels of p53-target genes in HCT116 human colon adenocarcinoma
cells [31]. Nevertheless, xanthone is highly hydrophobic and poorly water soluble, which could limit
its in vivo use. Interestingly, Chen and co-workers explored the antitumor activity of xanthone either
4 selectively upregulated the p53 transcriptional activity,
4
4
in combination or hybridized with 5,6-dimethylxanthone-4-acetic acid (DMXAA) [37], a xanthone
derivative with tumor-vascular disrupting activity. A very recent study reported the discovery of

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3,4-dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde (
5, Figure 1) as a new TAp73 activator via disruption
of its interaction with both MDM2 and mutant p53 [36].
Herein, based on the 3,4-oxygenated pattern of substitution of xanthones
4
and
5
(Figure 2A), and
19], 38],
40] (Figure 2B), we followed a molecular hybridization approach for the construction of
novel 3,4-oxygenated xanthone derivatives (Figure 2D). In the present work, we opted to synthesize a
library of aminated xanthones (Figure 2D) via a strategy of reductive amination of the xanthone with
a set of commercially available amine precursors ( 19) containing structural motifs of MDM2-p53
on chemical moieties derived from known MDM2-p53 disruptors, namely the molecules
39], and
1
[
6 [
7
[
8 [
5
9–
disruptors (Figure 2C). The introduction of amine moieties in the xanthone scaffold may contribute
to a potential enhancement of drug-like properties, a critical parameter for the identification of
promising drug candidates. The ability of the synthesized ligands to disrupt the MDM2-p53 interaction
and to activate a p53-dependent pathway was further investigated using the previously established
yeast-based assay and in human cancer cell lines. In silico docking studies were performed in order to
predict the binding poses and residues involved in the potential MDM2-p53 interaction.
2. Results and Discussion
2.1. Chemistry
2.1.1. Synthesis of 3,4-dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde (5)
In the present work, xanthone
of aminated xanthones with a 3,4-dioxygenated pattern of substitution. Recently, the multi-step
synthesis of compound has been reported [36] as consisting in: (i) a Friedel-Crafts acylation of
1,2,3-trimethoxybenzene (20) with the commercially available reagent 2-methoxybenzoyl chloride
21); (ii) an intramolecular nucleophilic aromatic substitution of (2-hydroxy-3,4-dimethoxyphenyl)
5 was used as a starting precursor for the synthesis of a set
5
(
(2-methoxyphenyl)methanone (22) and consecutive regioselective demethylation of the methoxy group
at position C-4 under conventional heating; (iii) a Duff formylation at position C-1 of 4-hydroxy-3-
methoxy-9H-xanthen-9-one (23); and (iv) a methylation of the hydroxyl group at position C-4 of
4-hydroxy-3-methoxy-9-oxo-9H-xanthene-1-carbaldehyde (24) (Scheme 1A). The difficulty in achieving
a regioselective demethylation at position C-4 necessary for Duff formylation at position C-1 demanded
the planning of an optimized synthetic route more appropriate for scaling-up. We hypothesized that
the implementation of an analog precursor of 20 with a suitable substituent at position C-1 might
provide a useful alternative for the synthesis of 5, avoiding the troublesome demethylation and Duff
formylation processes. Herein, the commercially available 1,2,3-trimethoxy-5-methylbenzene (25) was
selected for the Friedel-Crafts acylation with the acyl chloride 21 and the Lewis acid aluminum chloride
to furnish the intermediate (2-hydroxy-3,4-dimethoxy-6-methylphenyl)(2-methoxyphenyl)methanone
(26). Subsequent intramolecular nucleophilic aromatic substitution followed by elimination of methanol
under basic conditions and microwave (MW) irradiation provided the 3,4-dimethoxy-1-methyl-
9H-xanthen-9-one (27) in good yield quantity. This two-step methodology for the construction
of xanthone analogues of 27 has been previously described in the literature [32,41–43]. With the
application of MW methodology in the basic cyclization of 26 instead of the conventional heating
employed in the synthesis of 23, the reaction time was reduced from 48 h to 6 h. Furthermore,
xanthone 27 was obtained in higher reaction yield under MW irradiation (63% yield (27) vs. 38%
yield (23)). Subsequently, the Vohl-Ziegler bromination of 27, using dibenzoyl peroxide (DBP) and
N-bromosuccinimide (NBS) as a radical initiator and a brominating agent, respectively, afforded
the 1-(dibromomethyl)-3,4-dimethoxy-9H-xanthen-9-one (28) as the major product. A solvolytic
displacement of bromine atoms of the xanthone 28 was effectively attained using 1-butyl-3-
methylimidazolium tetrafluoroborate and water, under conventional heating, and furnished xanthone
5
in 64% yield quantity (Scheme 1B). Of note, the two-step procedure of Vohl-Ziegler bromination and
subsequent solvolytic displacement required lower reaction times (4.5 h (2 steps from 27 to ) vs. 54 h
(2 steps from 23 to ) and allowed the preparation of xanthone in higher reaction yields (51% yield
(2 steps from 27 to ) vs. 28% yield (2 steps from 23 to )), compared to the Duff formylation/methylation
5
5
5
5
5

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pathway. Overall, the replacement of starting precursor 20 by the methylated derivative 25 at position
C-1 of the aromatic ring was critical for the successful optimization of the preparation of xanthone 5.
Figure 2. Strategy of molecular hybridization used for the construction of the library of aminated
xanthones (
with the chemical moieties of known murine double minute 2 (MDM2)-p53 inhibitors (
molecules 1 (IC50 = 0.09 µM), 6 (IC50 = 2.6 µM), 7 (KI = 0.656 µM), and 8 (KI = 0.023 µM) (B).
D) by conjugation of the 3,4-dioxygenated substitution pattern of xanthones
4 and 5 (A)
C
), namely the

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Scheme 1.
) from 1,2,3-trimethoxybenzene (20). (
using the starting precursor 1,2,3-trimethoxy-5-methylbenzene (25). The compound numbering
(A
) Synthetic route for the preparation of 3,4-dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde
(5
B
) Optimized synthetic route for the preparation of the xanthone
5
used concerns the nuclear magnetic resonance (NMR) assignments. Note: [BmIm]BF4 = 1-butyl-
3-methylimidazolium tetrafluoroborate; DBP = dibenzoyl peroxide; HMTA = hexamethylenetetramine;
a
NBS = N-bromosuccinimide. Isolated yields.
2.1.2. Synthesis of a Library of Aminated Xanthones (30–40)
A library of aminated xanthones 30
obtained through a one-pot reductive amination of xanthone
19 (Scheme 2). In one-pot reductive aminations, the selection of the reducing agent is critical for
–
40 with a 3,4-dioxygenated pattern of substitution was
5
with the appropriate amine precursors
9
–
their efficiency, since a suitable agent must reduce the Schiff base intermediate selectively over the
carbonylated compounds. As an attempt to investigate the best reaction conditions, a solid-supported
reactant containing borohydride (MP-BH4) combined with methanol were initially employed, since
it was successfully used in the reductive amination of a xanthone derivative (data not shown).
When these conditions were applied to the reductive amination of
completely consumed and the alcohol derivative 1-(hydroxymethyl)-3,4-dimethoxy-9H-xanthen-9-one
29) was the major product formed. The replacement of the above-mentioned conditions by the milder
reducing agent sodium triacetoxyborohydride (STAB) and the solvent tetrahydrofuran allowed a total
consumption of the xanthone , favoring the one-pot amination/reduction pathway. Under the reaction
5, the starting precursor was not
(
5
conditions described in the Scheme 2, eleven aminated xanthones 30–40 were successfully synthesized
in moderate to good yields (11 examples, 39–73% yields).

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Scheme 2. Reductive amination of
5 with appropriate amine precursors 9–19 for the synthesis of
aminated xanthones 30
used concerns the NMR assignments. Isolated yields.
–
40 with a 3,4-dioxygenated substitution pattern. The compound numbering
a
The structure elucidation of the xanthones
5 and 27–40 was established on the basis of high-
resolution mass spectrometry (HRMS) (Figures S1–S15) and nuclear magnetic resonance (NMR)
techniques (Figures S16–S45). The 13C-NMR assignments were determined by bidimensional
heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC)
experiments (Supplementary Materials).

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2.2. Biological Activity Evaluation
2.2.1. Effect of Synthesized Xanthone Derivatives 30–40 on the MDM2-p53 Interaction Using A
Yeast-Based Screening Assay
Our group has previously developed a yeast-based screening assay to search for potential inhibitors
of the MDM2-p53 interaction [30 31 44 46]. In this yeast cell system, the expression of human wt
,
, –
p53 induces a growth inhibition proportional to the degree of its activity (Figure 3, p53, dimethyl
sulfoxide (DMSO)). On the other hand, this p53 inhibitory effect is reversed by yeast co-expression
of human MDM2 (Figure 3, MDM2-p53, DMSO). This established yeast screening assay was used
to study the potential inhibitory effect of 1-20
interaction. Figure 3 represents the maximal effect achieved with xanthone derivatives 30
The results showed that only the xanthone 37 caused a significant reversion of the inhibitory effect
of MDM2 on p53 (77.01 7.35% of reversion, n = 3), with an almost complete reestablishment of the
p53 growth inhibitory effect (63.55 4.26% vs. 69.22 2.23%; Figure 3, DMSO-treated p53-expressing
µ
M of xanthone derivatives 30
–
40 on the MDM2-p53
–
40 at 10 M.
µ
±
±
±
yeast vs. xanthone 37-treated MDM2-p53-expressing yeast). It should be noted that xanthone 37 did
not interfere with the growth of control yeast (transformed with empty vectors) or yeast expressing
human p53 alone (data not shown). As such, these results indicated that the xanthone 37 behaved as
a potential p53-activating agent through inhibition of interaction with MDM2, and it was, therefore,
selected for further investigation in human cancer cells.
Figure 3. Effect of xanthones 30
–40 on the MDM2-p53 interaction using a yeast-based assay. Effect of
10 M xanthone derivatives 30 40 and nutlin-3A on the growth of yeast co-expressing p53 and MDM2
µ
–
after 42 h treatment; results were plotted setting as 100% the growth of yeast transformed with the
empty vector (control yeast); nutlin-3A (inhibitor of the MDM2-p53 interaction) was used as positive
control. Data are mean
±
standard error of the mean (SEM) of three independent experiments; mean
values were statistically analyzed by one-way analysis of variance (ANOVA) with Tukey’s multiple
comparisons test, with significant differences from yeast co-expressing MDM2-p53 treated with DMSO.
Note: * p < 0.05; ** p < 0.01.
2.2.2. Evaluation of the Antitumor Activity of Xanthone 37 in Human Cancer Cell Lines
Using the sulforhodamine B (SRB) assay, it was confirmed that xanthone 37 inhibited the growth
of human HCT116 p53+/+ colon cancer cells, with an IC50 (concentration that causes 50% growth
inhibition) value of 8.67
±
0.59 µM (n = 4), and of the MDM2-overexpressing human HepG2 liver
carcinoma cells, with an IC50 value of 18.95 ± 0.39 µM (n = 4), after 48 h treatment.
To evaluate the dependency of the antitumor activity of xanthone 37 on the p53 pathway, we next
determined the impact of xanthone 37 on the colony forming ability of HCT116 p53+/+ cells and on the
respective p53-knockout (HCT116 p53−/−), using a colony formation assay (Figure 4A). The results
showed a significant reduction of the growth inhibitory activity of xanthone 37, at 3–5
p53+/+ cells, demonstrating a p53-dependent antitumor effect of xanthone 37. Accordingly, 10 and
20
M of xanthone 37 caused a G1-phase cell cycle arrest in HCT116 p53+/+ cells, but not in HCT116
µM in HCT116
µ

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p53−/− cells (Figure 4B) after 24 h treatment. It is of note that apoptosis analysis was also investigated
by Annexin-V assay; nevertheless, apoptotic events were not detected at 10 and 20 M of xanthone
µ
37, after 24 and 48 h treatment. In accordance with these results, we also observed that xanthone 37
upregulated the protein expression levels of MDM2, p53, and p21, in HCT116 p53+/+ cells for 24 h
treatment (Figure 4C).
Figure 4. Xanthone 37 inhibits the colony forming capability of HCT116 cells through induction of cell
cycle arrest and in a p53-dependent manner. (
and HCT116 p53−/− colon cancer cells after 11 days treatment with 3-10
plotted setting as 100% the colonies formed after treatment with dimethyl sulfoxide (DMSO). (
10 and 20
M of xanthone 37 on cell cycle progression of HCT116 p53+/+ and HCT116 p53−/− cells, after
24 h treatment. ( ) Effect of 10 and 20 M of xanthone 37 on the expression levels of MDM2, p53, and
A
) Evaluation of colony forming ability in HCT116 p53+/+
µM of xanthone 37; results were
B
) Effect of
µ
C
µ
p21 in HCT116 p53+/+ cells analyzed by Western Blot after 24 h treatment. Immunoblots represent one
of three independent experiments; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as
loading control. (
A
,
B
) Data are mean
±
SEM of three independent experiments; values significantly
different from HCT116 p53−/− cells using two-way ANOVA with Sidak’s multiple comparisons test (
or DMSO using Student’s t-test (B) are indicated: * p < 0.05; ** p < 0.01.
A)
Altogether, these results indicated that xanthone 37 had an in vitro p53-dependent antitumor
activity mediated by induction of cell cycle arrest.
2.3. In Slico Studies
It is common knowledge that the amino-terminal (residues 18–26) p53 α-helical peptide interacts
with a deep hydrophobic cleft within the amino-terminal domain of MDM2 [47]. The MDM2 binding
site consists of a large and a small pocket that interact with Phe19/Trp23 and Leu26 of p53, respectively
(Figure 5A) [48]. The crystal structure of the MDM2 complexed with the transactivation domain of p53
(Protein Data Bank (PDB) id 1YCR [49]) allowed the visual inspection of the interactions between the
Phe19, Trp23, and Leu26 residues of p53 and their respective pockets. Hydrogen-bonding interactions
are established between the indole group of Trp23 and carbonyl backbone of Leu54, and between the
NH backbone of Phe19 and the carbamoyl group of Gln72. Additional interactions are established
between p53 and Leu57, Gly58, Ile61, Met62, Val75, Val93, and His96 residues of MDM2 (Figure 5B).

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Figure 5.
(
A) Crystallographic p53 (for simplification, only Phe19, Trp23, and Leu26 residues are
represented as purple sticks) bound to the three pockets of MDM2 (solid surface). (
B) Binding mode
of crystallographic p53 (for simplification, only residues Phe19, Trp23, and Leu26, are represented as
purple sticks) in the hydrophobic cleft of MDM2 (transparent surface); MDM2 amino acid residues
involved in interactions with p53 are represented as green sticks. Structural data were obtained from
the X-ray crystal structure of MDM2-p53 complex (Protein Data Bank (PDB) id 1YCR). MDM2 is
represented as surface, where carbon, oxygen, nitrogen, and sulfur are represented in green, red, blue,
and yellow, respectively. Hydrogen-bonding interactions are depicted with a dashed blue line. Other
types of interactions are represented as a dashed red line.
The 1.6 Å X-ray crystal structure of the small-molecule inhibitor of MDM2-p53 interaction
1
in complex with MDM2 (PDB id 4HG7 [50]) was selected for further molecular docking studies of
the aminated xanthone derivatives 30 40 onto MDM2 protein, using the MDM2-p53 disruptors
–
1–4
as positive controls. In this work, AutoDock Vina [51] was the software chosen to predict docking
conformations, as it has been described as being the best software in predicting crystallographic
MDM2-p53 inhibitor poses (root-mean-square deviation (RMSD) < 1.0 Å) by redocking tests [52].
Docking scores for the most stable binding poses of xanthones 30
using the AutoDock Vina software [51]. The predicted docking scores for known MDM2-p53
inhibitors (positive controls ) ranged from 7.7 to 8.0 kcal
mol−1 (Table 1). The most stable
binding conformation of the synthesized xanthones 30 40 exhibited docking scores from 5.9 to
–40 on MDM2 were determined
1–
4
−
−
·
–
−
−7.4 kcal· mol−1, therefore being in the same range of binding affinity as the positive controls (Table 1).
Amongst the tested compounds, the xanthone 37 was identified as the most active compound
in the yeast-based screening assay. In addition, the predicted free energy values for the most stable
binding pose of xanthone 37 were more negative than the majority of the remaining ligands. Therefore,
the xanthone 37 was further analyzed in terms of docking poses and residues potentially involved in the
interaction with MDM2. Figure 6A shows the most stable conformation of xanthone 37 as suggested by
the docking protocol. The xanthone 37 was predicted to interact within the p53-binding site, mimicking
the key hydrophobic residues of p53 when bound to MDM2 (docking score of
Similarly to the binding mode of within the p53-binding site (Figure 6B), no hydrogen-bonding
interactions between the xanthone 37 and the MDM2 residues are predicted to be established. The
p-fluorobenzylamino group of xanthone 37 occupies the Trp23 pocket and makes -stacking interactions
with Phe91 residue, amide- interactions with the Leu57 and Gly58 residues, and CH- interactions
with the Leu54, Leu57, Gly58, Ile61, Val93, and Ile99 residues. Interestingly, the docking conformation
of p-fluorobenzylamino group and the p-chlorophenyl ring of are identical and superimposable
−
7.3 kcal
mol−1).
·
1
π
π
π
1
(Figure 6A,B). The methoxy groups of the xanthone scaffold are oriented to the Leu26 pocket and
make CH-CH interactions with the Leu54 residue. The adopted scaffold orientation of the xanthone 37
in the hydrophobic cleft where p53 is supposed to bind allows additional interactions to be formed,
strengthening the binding of xanthone 37 to MDM2. For example, apart from the Gly58 residue of
MDM2, the non-substituted aromatic ring of the xanthone scaffold is predicted to establish π-stacking

Molecules 2019, 24, 1975
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interactions with the Phe55 residue, and amide-
π
and CH-
π
interactions with the Gln59 residue.
The establishment of additional interactions within the MDM2 protein that are not observed in the
MDM2-p53 binding have been described for a considerable number of high-affinity p53-activating
agents [17]. The visual inspection of the xanthone 37 also suggests that an enhancement of its binding
affinity towards MDM2 protein can be achieved by molecular modifications in the xanthone scaffold of
37 in order to optimize its interaction with the amino acid residues surrounding the Phe19 pocket.
Table 1. Docking scores (kcal
·
mol−1) of xanthones 30
–
40 and positive controls
1–
4
onto MDM2 target.
Xanthones 30–40
Ligands
Free Energy of the Ligand: MDM2 (kcal·mol−1
)
30
31
32
33
34
35
36
37
38
39
40
−6.6
−6.2
−6.6
−5.9
−6.9
−7.2
−6.6
−7.3
−7.1
−7.3
−7.4
Inhibitors of MDM2-p53 Interaction
Ligands
Free Energy of the Ligand: MDM2 (kcal·mol−1
)
1
2
3
4
−7.7
−8.0
−7.9
−8.0
Figure 6.
(
A
) Predicted binding pose of the xanthone 37 (purple sticks) in the binding site of MDM2.
, purple sticks). MDM2
(B
) MDM2 (transparent surface) in complex with the crystallographic nutlin-3A (1
is represented as a transparent surface, where carbon, oxygen, nitrogen, and sulfur are represented
in green, red, blue, and yellow, respectively. Small molecule-MDM2 interactions are depicted with a
dashed red line. MDM2 residues involved in interactions with the ligands are represented as green
sticks and labeled.
3. Materials and Methods
3.1. Chemistry
All reagents and solvents were purchased from Sigma Aldrich (Sigma-Aldrich Co. Ltd., Gillinghan,
UK) and no further purification process was implemented. Solvents were evaporated using a rotary

Molecules 2019, 24, 1975
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evaporator under reduced pressure, Buchi Waterchath B-480. Microwave (MW) reactions were
performed using an Ethos MicroSYNTH 1600 Microwave Labstation from Milestone (Thermo Unicam,
Portugal). The internal reaction temperature was controlled by a fiber optic probe sensor. All reactions
were monitored by thin-layer chromatography (TLC) carried out on precoated plates with 0.2 mm of
thickness using Merck silica gel 60 (GF254) with appropriate mobile phases. Compounds 30–40 were
easily detectable at 254 nm or 365 nm and after revelation with a solution of ninhydrin in ethanol
3 mg·mL−1 (activated by heat).
Flash column chromatography using silica gel 60 (0.040–0.063 mm, Merck, Darmstadt, Germany),
flash cartridge chromatography (GraceResolv®, Grace Company, Deerfield, IL, USA), and Discovery®
DSC-SCX SPE cationic exchange cartridge (Grace Company, Deerfield, IL, USA) were used in the
purification of the synthesized compounds. Melting points (m.p.) were measured in a Köfler microscope
1
(Wagner and Munz, Munich, Germany) and are uncorrected. H- and 13C-nuclear magnetic resonance
(NMR) spectra were recorded at the University of Aveiro, Department of Chemistry in CDCl3 or
DMSO-d6 (Deutero GmbH, Ely, UK) at room temperature on a Bruker Avance 300 spectrometer
1
(300.13 MHz for H and 75.47 MHz for 13C, Bruker Biosciences Corporation, Billerica, MA, USA).
Chemical shifts are expressed in
δ (ppm) values relative to tetramethylsilane (TMS) as an internal
reference. Coupling constants are reported in hertz (Hz). 13C-NMR assignments were made by
bidimensional heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond
correlation (HMBC) NMR experiments (long-range C, H coupling constants were optimized to 7 Hz)
or by comparison with the assignments of similar molecules. High-resolution mass spectroscopy
(HRMS) spectra were measured on a Bruker FTMS APEX III mass spectrometer (Bruker Corporation,
Billerica, MA, USA) and recorded as electrospray ionization (ESI) mode in Centro de Apoio Cientifico e
Tecnolóxico á Investigación (CACTI, University of Vigo, Pontevedra, Spain). The following compounds
were synthesized and purified by the described procedures.
3.1.1. Synthesis of 3,4-dimethoxy-1-methyl-9H-xanthen-9-one (27)
Xanthone 27 (9.34 g, 63% yield) was synthesized from 1,2,3-trimethoxy-5-methylbenzene (25) and
characterized according to the previously described procedure [42].
3.1.2. Synthesis of 1-(dibromomethyl)-3,4-dimethoxy-9H-xanthen-9-one (28)
A mixture of 27 (2.52 g, 9.32 mmol), N-bromosuccinimide (3.32 g, 18.7 mmol), and dibenzoyl
peroxide (0.68 g, 2.8 mmol) in carbon tetrachloride (25 mL) was refluxed (85 ◦C) for 2 h. The reaction
was monitored using n-hexane/ethyl acetate in a proportion of 8:2. Once the reaction was completed,
the resulting orange suspension was cooled at 0 ◦C and stirred for 30 min in an ice bath. The solid was
filtered and washed with cold carbon tetrachloride. The filtrate was evaporated under reduced pressure
and further purified by flash column chromatography (SiO2, n-hexane/ethyl acetate in gradient) to
obtain the 28 as a white powder.
For 1-(dibromomethyl)-3,4-dimethoxy-9H-xanthen-9-one (28): White powder (3.14 g, 80% yield);
melting point (m.p.) 125–127 ◦C. 1H-NMR (CDCl3, 300.13 MHz):
δ = 8.91 (1H, s, H-1), 8.30 (1H, dd,
J = 8.0 and 1.6 Hz, H-8), 7.77 (1H, s, H-2), 7.73 (1H, ddd, J = 8.4, 7.0, and 1.6 Hz, H-6), 7.54 (1H, d,
J = 8.4 Hz, H-5), 7.39 (1H, ddd, J = 7.5, H-7), 4.11 (3H, s, 4-OCH3), 4.05 (3H, s, 3-OCH3) ppm; 13C-NMR
(CDCl3, 75.47 MHz):
δ = 177.8 (C-9), 156.5 (C-3), 155.0 (C-10a), 150.4 (C-4a), 139.5 (C-4), 137.6 (C-1),
135.0 (C-6), 126.9 (C-8), 124.3 (C-7), 122.0 (C-8a), 117.6 (C-5), 111.9 (C-2), 110.8 (C-9a), 61.9 (3-OCH3),
56.6 (4-OCH3), 39.2 (C-10) ppm. HRMS (ESI+): m/z [C16H12Br2O5 + H]+ calcd. for [C16H13Br2O5]:
426.91751; found: 426.91709.
3.1.3. Synthesis of 3,4-dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde (5)
Xanthone 28 (3.14 g, 7.34 mmol) was added to a solution of 1-butyl-3-methylimidazolium
tetrafluoroborate and water (6 mL, 5:1). The mixture was heated at 100 ◦C with stirring for 2.5 h. The
reaction was monitored using n-hexane/ethyl acetate in a proportion of 7:3. After completion of the

Molecules 2019, 24, 1975
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reaction, the resulting suspension was cooled at room temperature, diluted with water (10 mL), and
extracted with ethyl acetate (5 100 mL). The combined organic layers were dried over anhydrous
×
sodium sulfate, and after filtration of the solution, the solvent was evaporated under reduced pressure.
The crude product was then purified by flash column chromatography (SiO2, n-hexane/ethyl acetate in
gradient). A light yellow powder corresponding to 5 was obtained.
For 3,4-dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde (
5): Light yellow powder (1.33 g, 64%
yield); m.p. > 330 ◦C. 1H-NMR (CDCl3, 300.13 MHz): = 11.21 (1H, s, H-10), 8.31 (1H, dd, J = 8.0 and
δ
1.7 Hz, H-8), 7.77 (1H, ddd, J = 8.5, 7.0, and 1.7 Hz, H-6), 7.60 (1H, dd, J = 8.5 and 0.8 Hz, H-5), 7.56 (1H,
s, H-2), 7.43 (1H, ddd, J = 8.0, 7.0, and 0.8 Hz, H-7), 4.11 (3H, s, 4-OCH3), 4.07 (3H, s, 3-OCH3) ppm;
13C-NMR (CDCl3, 75.47 MHz):
δ
= 192.8 (C-10), 177.9 (C-9), 156.3 (C-3), 155.5 (C-10a), 150.9 (C-4a),
140.7 (C-4), 135.1 (C-6), 133.5 (C-1), 126.7 (C-8), 124.5 (C-7), 121.9 (C-8a), 117.8 (C-5), 116.1 (C-9a), 108.5
(C-2), 61.8 (4-OCH3), 56.6 (3-OCH3) ppm. HRMS (ESI+): m/z [C16H12O5 + H]+ calcd. for [C16H13O5]:
285.07575; found: 285.07598; [C16H12O5 + Na]− calcd. for [C16H12NaO5]: 307.05769; found 307.05750.
3.1.4. Synthesis of 1-(hydroxymethyl)-3,4-dimethoxy-9H-xanthen-9-one (29)
Xanthone 29 (52 mg, 52% yield) was synthesized from xanthone
5 and characterized according to
the previously described procedure [36].
3.1.5. General Procedure for the Synthesis of Xanthones (30–40)
Xanthone
amine precursors
4.0 equimolar quantity of sodium triacetoxyborohydride (119 mg, 0.563 mmol), the resulting mixture
was stirred at room temperature for 30 min. Subsequently, a 3.0 equimolar quantity of acetic acid (24
0.423 mmol) was added to the solution, The reaction was carried out at room temperature for 12 h
under N2 gas. For monitoring the synthesis of xanthones 30 40 by TLC, two chromatographic systems
were employed: (i) n-hexane/ethyl acetate (7:3) and methanol/triethylamine (10:0.1) for xanthones
30 36; (ii) n-hexane/ethyl acetate (7:3) and chloroform/acetone/triethylamine (5:5:0.1) for xanthones
37–40. Once completed of the reaction, three distinct work-up approaches were employed.
(a) For xanthones 30 32: the solvent was evaporated under reduced pressure and the resulting
crude product was basified with sodium hydroxide (10 mL, 5%, m/v) and extracted with chloroform
3 × 10 mL). The combined organic phases were then acidified and extracted with hydrochloric acid
5 M (3 30 mL). Afterwards, the aqueous phases were gathered and then basified with sodium
hydroxide (100 mL, 20%, m/v). The crude product was extracted successively with chloroform (3
5
(40 mg, 0.141 mmol) was dissolved in tetrahydrofuran (3 mL) and the appropriate
9–
19 (0.197 mmol) were added to the solution under N2 gas. After the addition of a
µL
–
–
–
(
×
×
100 mL). The organic layers were gathered and dried over anhydrous sodium sulfate. After filtration,
the solution was concentrated under reduced pressure, and the resulting crude product was purified
by flash cartridge chromatography (SiO2, chloroform/acetone/ammonium hydroxide, 90:10:1) to afford
the xanthones 30, 31, and 32 as pure compounds.
For 3,4-dimethoxy-1-(((2-morpholinoethyl)amino)methyl)-9H-xanthen-9-one (30): Green powder
(35 mg, 62% yield); m.p. 125–126 ◦C. 1H-NMR (CDCl3, 300.13 MHz):
δ = 8.25 (1H, dd, J = 8.0 and
1.6 Hz, H-8), 7.72 (1H, ddd, J = 8.1, 7.0, and 1.6 Hz, H-6), 7.57 (1H, dd, J = 8.4 and 0.8 Hz, H-5), 7.38 (1H,
ddd, J = 8.0, 7.1, and 0.9 Hz, H-7), 7.09 (1H, s, H-2), 4.34 (2H, s, H-10); 4.04 (3H, s, 4-OCH3), 4.01 (3H,
s, 3-OCH3), 3.66 (4H, t, J = 4.6 Hz, H-50), 2.90 (2H, t, J = 6.1 Hz, H-20), 2.60 (2H, t, J = 6.1 Hz, H-30),
2.47 (4H, t, J = 4.3 Hz, H-40) ppm; 13C-NMR (CDCl3, 75.47 MHz):
δ = 178.0 (C-9), 156.5 (C-3), 155.3
(C-10a), 152.1 (C-4a), 137.0 (C-1), 136.0 (C-4), 134.6 (C-6), 126.5 (C-8), 124.1 (C-7), 122.1 (C-8a), 117.7
(C-5), 114.6 (C-9a), 111.6 (C-2), 67.0 (C-50), 61.5 (4-OCH3), 57.4 (C-20), 56.4 (3-OCH3), 53.7 (C-10), 53.5
(C-40), 45.3 (C-30) ppm. HRMS (ESI+): m/z [C22H26N2O5 + H]+ calcd. for [C22H27N2O5]: 399.19145;
found: 399.19060.
For 1-(((3-(dimethylamino)propyl)(methyl)amino)methyl)-3,4-dimethoxy-9H-xanthen-9-one (31):
White powder (35 mg, 65% yield); m.p. 84–85 ◦C. 1H-NMR (CDCl3, 300.13 MHz):
δ
= 8.25 (1H, dd,
J = 8.0 and 1.6 Hz, H-8), 7.69 (1H, ddd, J = 8.5, 7.0, and 1.6 Hz, H-6), 7.54 (1H, dd, J= 7.4 and 0.7, H-5),

Molecules 2019, 24, 1975
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7.53 (1H, s, H-2), 7.35 (1H, ddd, J = 8.0, 7.1, and 1.0 Hz, H-7), 4.36 (2H, s, H-10); 4.05 (3H, s, 4-OCH3),
4.02 (3H, s, 3-OCH3), 2.65 (2H, t, J = 7.2 Hz, H-30), 2.44 (2H, t, J = 7.5 Hz, H-50), 2.39 (3H, s, H-20),
2.28 (6H, s, H-60), 1.82 (2H, m, J = 7.3 Hz, H-40) ppm; 13C-NMR (CDCl3, 75.47 MHz):
δ = 178.1 (C-9),
156.6 (C-3), 155.2 (C-10a), 151.9 (C-4a), 135.2 (C-1), 135.0 (C-4), 134.2 (C-6), 126.6 (C-8), 123.9 (C-7),
122.4 (C-8a), 117.6 (C-5), 114.5 (C-9a), 109.0 (C-2), 61.5 (4-OCH3), 60.1 (C-10), 57.8 (C-50), 56.4 (3-OCH3),
56.1 (C-30), 45.3 (C-60), 42.5 (C-20), 25.3 (C-40) ppm. HRMS (ESI+): m/z [C22H28N2O4 + H]+ calcd. for
[C22H29N2O4]: 385.21218; found: 385.21139.
For 1-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-3,4-dimethoxy-9H-xanthen-9-one (32): Yellow
powder (40 mg, 71% yield); m.p. 129–130 ◦C. 1H-NMR (CDCl3, 300.13 MHz):
δ = 8.25 (1H, dd, J = 8.0
and 1.6 Hz, H-8), 7.68 (1H, ddd, J = 8.5, 7.0, and 1.6 Hz, H-6), 7.53 (1H, dd, J = 8.4 and 0.7 Hz, H-5), 7.48
(1H, s, H-2), 7.35 (1H, ddd, J = 8.0, 7.1, and 1.0 Hz, H-7), 4.32 (2H, s, H-10), 4.03 (3H, s, 4-OCH3), 4.01
(3H, s, 3-OCH3), 3.63 (2H, t, J = 5.4 Hz), 2.58 (2H, t, J = 5.4 Hz) ppm; 13C-NMR (CDCl3, 75.47 MHz):
δ
= 178.1 (C-9), 156.5 (C-3), 155.2 (C-10a), 152.0 (C-4a), 138.8 (C-1), 134.8 (C-4), 134.2 (C-6), 126.6 (C-8),
123.9 (C-7), 122.5 (C-8a), 117.5 (C-5), 114.6 (C-9a), 108.2 (C-2), 61.5 (4-OCH3), 60.2 (C-10), 59.3 (C-50),
57.7 (C-40), 56.2 (3-OCH3), 53.5 (C-30), 53.2 (C-20) ppm. HRMS (ESI+): m/z [C22H26N2O5 + H]+ calcd.
for [C22H27N2O5]: 399.19145; found: 399.19059.
(b) For xanthones 33
product was extracted with chloroform (3
–
36: the solvent was evaporated under reduced pressure and the crude
10 mL). The organic phases were gathered, dried over
×
anhydrous sodium sulfate, and after filtration, the resulting solution was concentrated under reduced
pressure. Then, a solid phase extraction using a cation exchange cartridge Discovery® DSC-SCX was
applied to further purify the crude product. Initially, an activation of the cartridge with methanol
(100 mL) was carried out followed by loading the cartridge with the sample. Then, the elution was
performed with the following solvents/solutions: (i) dichloromethane/methanol (5:5), (ii) methanol, and
(iii) ammonium hydroxide/methanol (2%, v/v). The fractions obtained from the elution with ammonium
hydroxide/methanol (2%, v/v) were gathered and the solvent was evaporated under reduced pressure.
A flash cartridge chromatography with chloroform/acetone/ammonium hydroxide (90:10:1) was also
performed to obtain the xanthones 33, 34, 35, and 36 as pure compounds.
For 1-(((2-(diethylamino)ethyl)amino)methyl)-3,4-dimethoxy-9H-xanthen-9-one (33): Green
◦
1
powder (28 mg, 52% yield); m.p. 68–69 C. H-NMR (CDCl3, 300.13 MHz):
δ = 8.27 (1H, dd, J
= 8.0 and 1.4 Hz, H-8), 7.71 (1H, ddd, J = 8.5, 7.0, and 1.6 Hz, H-6), 7.56 (1H, dd, J = 8.4 and 0.7 Hz, H-5),
7.37 (1H, ddd, J = 8.0, 7.1, and 1.0 Hz, H-7), 7.14 (1H, s, H-2), 4.39 (2H, s, H-10); 4.05 (3H, s, 4-OCH3),
4.01 (3H, s, 3-OCH3), 2.87 (2H, t, J = 6.4 Hz, H-20), 2.68 (2H, t, J = 6.4 Hz, H-30), 2.55 (4H, m, J = 7.1
Hz, H-40), 1.01 (6H, t, J = 7.1 Hz, H-50) ppm; 13C-NMR (CDCl3, 75.47 MHz):
δ = 178.1 (C-9), 156.5
(C-3), 155.3 (C-10a), 152.1 (C-4a), 137.4 (C-1), 135.8 (C-4), 134.5 (C-6), 126.6 (C-8), 124.0 (C-7), 122.2
(C-8a), 117.7 (C-5), 114.5 (C-9a), 111.1 (C-2), 61.5 (4-OCH3), 56.4 (3-OCH3), 53.4 (C-30), 52.4 (C-10), 46.9
(C-40), 46.8 (C-20), 11.6 (C-50) ppm. HRMS (ESI+): m/z [C22H28N2O4 + H]+ calcd. for [C22H29N2O4]:
385.21218; found: 385.21150.
For 4-((3,4-dimethoxy-9-oxo-9H-xanthen-1-yl)methyl)piperazin-2-one (34): Orange powder
◦
1
(20 mg, 39% yield); m.p. 245–247 C. H-NMR (DMSO-d6, 300.13 MHz):
δ = 8.13 (1H, dd, J =
8.0 and 1.5 Hz, H-8), 7.82 (1H, ddd, J = 8.5, 7.0, and 1.6 Hz, H-6), 7.65 (1H, dd, J = 8.4 and 0.6, H-5),
7.45 (1H, ddd, J = 8.0, 7.1, and 1.0 Hz, H-7), 7.36 (1H, s, H-2), 4.24 (2H, s, H-10); 3.98 (3H, s, 4-OCH3),
3.90 (3H, s, 3-OCH3), 3.20 (2H, t, J = 5.3 Hz, H-50), 3.13 (2H, s, H-20), 2.66 (2H, t, J = 5.3 Hz, H-40) ppm;
13C-NMR (DMSO-d6, 75.47 MHz):
δ
= 176.7 (C-9), 168.0 (C-30), 156.2 (C-3), 154.6 (C-10a), 151.8 (C-4a),
136.9 (C-1), 135.0 (C-4), 134.7 (C-6), 126.1 (C-8), 124.3 (C-7), 121.8 (C-8a), 117.7 (C-5), 113.7 (C-9a), 109.2
(C-2), 60.9 (4-OCH3), 58.5 (C-10), 57.2 (C-50), 56.3 (3-OCH3), 48.7 (C-20), 30.7 (C-40) ppm. HRMS (ESI+):
m/z [C20H20N2O5 + H]+ calcd. for [C20H21N2O5]: 369.14450; found: 369.14369.
For 1-((5-amino-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-3,4-dimethoxy-9H-xanthen-9-one (35):
White powder (23 mg, 39% yield); m.p. 171–172 ◦C. 1H-NMR (CDCl3, 300.13 MHz):
δ = 8.26 (1H, dd,
J = 8.0 and 1.4 Hz, H-8), 7.69 (1H, ddd, J = 8.5, 7.0, and 1.6 Hz, H-6), 7.57 (1H, s, H-2), 7.54 (1H, dd, J =
8.4 and 0.7 Hz, H-5), 7.35 (1H, ddd, J = 8.0, 7.1, and 1.0 Hz, H-7), 6.99 (1H, t, J = 7.7 Hz, H-70), 6.57

Molecules 2019, 24, 1975
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(1H, d, J = 7.8 Hz, H-60), 6.53 (1H, d, J = 7.6 Hz, H-80), 4.48 (2H, s, H-10); 4.02 (3H, s, 4-OCH3), 3.97
(3H, s, 3-OCH3), 3.81 (2H, s, H-100), 2.93 (2H, t, J = 5.9 Hz, H-20), 2.66 (2H, t, J = 5.9 Hz, H-30) ppm;
13C-NMR (CDCl3, 75.47 MHz):
δ
= 178.2 (C-9), 156.7 (C-3), 155.2 (C-10a), 151.9 (C-4a), 144.1 (C-50),
139.3 (C-1), 136.3 (C-90), 134.8 (C-4), 134.2 (C-6), 126.6 (C-8), 126.4 (C-70), 123.8 (C-7), 122.5 (C-8a), 119.5
(C-40), 117.6 (C-5), 117.1 (C-80), 114.5 (C-9a), 112.7 (C-60), 108.0 (C-2), 61.5 (4-OCH3), 60.1 (C-10), 56.7
(C-100), 56.3 (3-OCH3), 51.0 (C-20), 24.9 (C-30) ppm. HRMS (ESI+): m/z [C25H24N2O4 + H]+ calcd. for
[C25H25N2O4]: 417.18088; found: 417.18018.
For 3,4-dimethoxy-1-(piperidin-1-ylmethyl)-9H-xanthen-9-one (36): Yellow powder (31 mg, 62%
yield); m.p. 108-109 ◦C. 1H-NMR (CDCl3, 300.13 MHz):
δ = 8.25 (1H, dd, J = 8.0 and 1.6 Hz, H-8), 7.66
(1H, ddd, J = 8.5, 8.0, and 1.6 Hz, H-6), 7.56 (1H, s, H-2), 7.52 (1H, dd, J = 8.3 and 0.7 Hz, H-5), 7.33 (1H,
ddd, J = 7.9, 7.1, and 0.9 Hz, H-7), 4.26 (2H, s, H-10); 4.03 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3), 2.57 (4H,
t, J = 4.6 Hz, H-20), 1.65 (4H, m, J = 5.5 Hz, H-30), 1,50 (2H, m, J = 5.5 Hz, H-40) ppm; 13C-NMR (CDCl3,
75.47 MHz) ppm:
δ = 177.5 (C-9), 156.1 (C-3), 154.7 (C-10a), 151.3 (C-4a), 138.9 (C-4), 134.2 (C-1), 133.8
(C-6), 126.0 (C-8), 123.3 (C-7), 121.9 (C-8a), 117.1 (C-5), 113.9 (C-9a), 107.8 (C-2), 60.8 (4-OCH3), 56.1
(3-OCH3), 55.7 (C-20), 54.3 (C-10), 25.7 (C-30), 23.8 (C-40) ppm. HRMS (ESI+): m/z [C21H23NO4 + H]+
calcd. for [C21H24NO4]: 354.16998; found: 354.16939.
(c) For xanthones 37
crude product was basified with sodium hydroxide (10 mL, 5%, m/v) and extracted with chloroform
(3 10 mL). The combined organic phases were then acidified and extracted with hydrochloric acid
5 M (3 30 mL). The resulting organic phase was dried over anhydrous sodium sulfate and, after
–40: the solvent was evaporated under reduced pressure and the resulting
×
×
filtration, the solution was concentrated under reduced pressure. Then, a solid phase extraction using
a cation exchange cartridge Discovery® DSC-SCX was applied to further purify the crude product.
Initially, an activation of the cartridge with methanol (100 mL) was carried out followed by loading the
cartridge with the sample. Then, the elution was performed with the following solvents/solutions:
(i) dichloromethane/methanol (5:5), (ii) methanol, and (iii) ammonium hydroxide/methanol (2%, v/v).
The fractions obtained from the elution with ammonium hydroxide/methanol (2%, v/v) were gathered
and the solvent was evaporated under reduced pressure to furnish the xanthones 37, 38, 39, and 40 as
pure compounds.
For 1-(((4-fluorobenzyl)amino)methyl)-3,4-dimethoxy-9H-xanthen-9-one (37): White powder,
(34 mg, 61% yield); m.p. 163–165 ◦C. 1H-NMR (CDCl3, 300.13 MHz):
δ
= 8.28 (1H, dd, J = 8.0 and
1.6 Hz, H-8), 7.71 (1H, ddd, J = 8.5, 7.0, and 1.6 Hz, H-6), 7.55 (1H, dd, J = 8.5 and 0.8 Hz, H-5), 7.40
(1H, ddd, J = 8.0, 7.0, and 0.8 Hz, H-7), 7.36 (2H, m, H-50), 7.01 (2H, m, H-40), 6.96 (1H, s, H-2), 4.30
(2H, s, H-10), 4.01 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3), 3.85 (2H, s, H-20) ppm; 13C-NMR (CDCl3,
75.47 MHz):
δ
= 178.1 (C-9), 160.3 (C-60), 156.3 (C-3), 155.3 (C-10a), 152.2 (C-4a), 138.6 (C-4), 135.9
(C-30), 135.7 (C-1), 134.5 (C-6), 129.8 (C-50), 126.7 (C-8), 124.0 (C-7), 122.2 (C-8a), 117.7 (C-5), 115.3 (C-40),
114.7 (C-9a), 110.9 (C-2), 61.5 (4-OCH3), 56.3 (3-OCH3), 53.3 (C-10), 52.5 (C-20) ppm. HRMS (ESI+): m/z
[C23H20FNO4 + H]+ calcd. for [C23H21FNO4]: 394.14491; found: 394.14410.
For 1-(((4-chlorobenzyl)amino)methyl)-3,4-dimethoxy-9H-xanthen-9-one (38): Light yellow
◦
1
powder (35 mg, 61% yield); m.p. 162–163 C. H-NMR (CDCl3, 300.13 MHz):
δ = 8.25 (1H, dd,
J = 8.0 and 1.6 Hz, H-8), 7.79 (1H, ddd, J = 8.5, 7.0, and 1.6 Hz, H-6), 7.66 (2H, m, H-50), 7.60 (1H, dd, J
= 8.5 and 0.9 Hz, H-5), 7.43 (1H, ddd, J = 8.0, 7.0, and 0.9 Hz, H-7), 7.39 (1H, s, H-2), 7.38 (2H, m, H-40),
4.53 (2H, s, H-20); 4.33 (2H, s, H-10), 4.07 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3) ppm; 13C-NMR (CDCl3,
75.47 MHz):
δ = 179.6 (C-9), 157.0 (C-3), 155.5 (C-10a), 152.0 (C-4a), 137.7 (C-4), 135.8 (C-1), 135.6 (C-6),
131.7 (C-50), 129.5 (C-40), 129.4 (C-30), 127.3 (C-60), 126.6 (C-8), 124.6 (C-7), 121.3 (C-8a), 117.9 (C-5),
115.5 (C-2), 114.8 (C-9a), 61.6 (4-OCH3), 57.1 (3-OCH3), 51.3 (C-10), 50.7 (C-20) ppm. HRMS (ESI+): m/z
[C23H20ClNO4 + H]+ calcd. for [C23H21ClNO4]: 410.11536; found: 410.11466.
For (R)-1-(((1-(4-chlorophenyl)ethyl)amino)methyl)-3,4-dimethoxy-9H-xanthen-9-one (39): White
powder (39 mg, 65% yield); m.p. 163–164 ◦C. 1H-NMR (CDCl3, 300.13 MHz):
δ = 8.26 (1H, dd, J =
8.0 and 1.6 Hz, H-8), 7.79 (1H, ddd, J = 8.5, 7.0, and 1.6 Hz, H-6), 7.70 (2H, m, H-60), 7.59 (1H, dd,
J = 8.5 and 0.8 Hz, H-5), 7.43 (1H, ddd, J = 8.0, 7.0, and 0.8 Hz, H-7), 7.42 (2H, m, H-50), 7.25 (1H,

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s, H-2), 4.54 (2H, s, H-10); 4.27 (1H, q, J = 6.8 Hz, H-10), 4.04 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3),
1.83 (3H, d, J = 6.8 Hz, H-30) ppm; 13C-NMR (CDCl3, 75.47 MHz):
= 179.6 (C-9), 156.8 (C-3), 155.4
δ
(C-10a), 151.9 (C-4a), 137.6 (C-4), 135.5 (C-6), 134.5 (C-1), 131.8 (C-70), 129.8 (C-60), 129.5 (C-50), 127.3
(C-40), 126.6 (C-8), 124.6 (C-7), 121.3 (C-8a), 117.9 (C-5), 115.6 (C-2), 114.9 (C-9a), 61.6 (4-OCH3), 58.9
(C-20), 57.0 (3-OCH3), 50.2 (C-10), 20.2 (C-30) ppm. HRMS (ESI+): m/z [C24H22ClNO4 + H]+ calcd. for
[C24H23ClNO4]: 424.13101; found: 424.13018.
For 1-(((4-bromobenzyl)amino)methyl)-3,4-dimethoxy-9H-xanthen-9-one (40): White powder
(47 mg, 73% yield); m.p. 161–162 ◦C. 1H-NMR (CDCl3, 300.13 MHz):
δ = 8.27 (1H, dd, J = 8.0 and
1.6 Hz, H-8), 7.73 (1H, ddd, J = 8.5, 7.0, and 1.6 Hz, H-6), 7.58 (1H, dd, J = 8.5 and 0.8 Hz, H-5), 7.48
(2H, m, H-50), 7.39 (2H, m, H-40), 7.38 (1H, ddd, J = 8.0, 7.0, and 0.8 Hz, H-7), 7.08 (1H, s, H-2), 4.31
(2H, s, H-10); 4.01 (3H, s, 4-OCH3), 4.01 (3H, s, 3-OCH3), 3.88 (2H, s, H-20) ppm; 13C-NMR (CDCl3,
75.47 MHz):
δ = 178.6 (C-9), 156.5 (C-3), 155.3 (C-10a), 152.2 (C-4a), 136.4 (C-4), 136.2 (C-1), 134.9 (C-6),
131.8 (C-50), 130.7 (C-40), 128.4 (C-30), 126.6 (C-8), 124.2 (C-7), 121.9 (C-8a), 121.8 (C-60), 117.7 (C-5),
114.7 (C-9a), 112.5 (C-2), 61.6 (4-OCH3), 56.7 (3-OCH3), 52.7 (C-10), 52.0 (C-20) ppm. HRMS (ESI+): m/z
[C23H20BrNO4 + H]+ calcd. for [C23H21BrNO4]: 454.06485; found: 454.06385.
3.2. Biological Evaluation
3.2.1. Yeast Strains and Growth Conditions
For the yeast assay, the previously obtained Saccharomyces cerevisiae cells co-expressing human
p53 or human MDM2, as well as yeast control (transformed with empty vectors) [30
,31], were used.
To induce expression of human proteins, yeast cells were grown in selective induction medium with 2%
(w/w) galactose and 2% (w/w) raffinose, in the presence of 1–20
at 30 C under continuous orbital shaking (200 rpm) for approximately 42 h, as described [30
µ
M of xanthones 30
–
40 or 0.1% DMSO,
◦
,
31].
Yeast cell growth was analyzed by counting the number of colony-forming units (CFU) after 2 days
incubation at 30 ◦C on Sabouraud Dextrose Agar plates from Liofilchem (Frilabo, Porto, Portugal).
Results were estimated considering 100% growth as the number of CFU obtained with untreated yeast
co-expressing p53 and MDM2.
3.2.2. Human Cancer Cell Lines and Culture Conditions
Human colon adenocarcinoma HCT116 cell lines expressing wt p53 (HCT116 p53+/+) and its
p53-null isogenic derivative (HCT116 p53−/−) were provided by B. Vogelstein (The Johns Hopkins
Kimmel Cancer Center, Baltimore, MD, USA). Human liver HepG2 carcinoma cell lines were
purchased from ATCC (Rockville, MD, USA). Cancer cells were cultured in RPMI-1640 medium
with ultraglutamine (Lonza, VWR, Carnaxide, Portugal), and supplemented with 10% fetal bovine
serum (FBS; Merck Millipore, VWR). Cells were maintained at 37 ◦C in a humidified atmosphere of
5% CO2.
3.2.3. Cell Proliferation Assay
Cell proliferation was determined using the SRB assay, as described [30
seeded in 96-well plates at 5.0
103 cells/well for HCT116 p53+/+ cells and 4.0
HepG2 and 24 h later treated with serial dilutions (3.13–50 M) of xanthone 37 or 0.25% DMSO for 48
,
31]. Briefly, cells were
×
×
103 cells/well for
µ
h. IC50 values were determined from the concentration-response curves.
3.2.4. Colony Formation Assay
HCT116 p53+/+ and HCT116 p53−/− cells were seeded in a 6-well plate at 1.0
and treated with 3, 4, 5, 7, and 10 µM of xanthone 37 or DMSO at the time of seeding. After 11 days
×
103 cells/well
incubation, colonies were fixed using 10% of methanol and 10% acetic acid for 10 min and stained with
0.5% crystal violet (Sigma-Aldrich) in 1:1 methanol/water for 15 min. Colonies with more than 20 cells
were counted.

Molecules 2019, 24, 1975
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3.2.5. Cell Cycle Analysis
HCT116 p53+/+ and HCT116 p53−/− cells were seeded in a 6-well plate at 1.5
24 h and treated with 10 and 20 µM of xanthone 37 or DMSO for 24 h. Cells were collected, fixed, and
×
105 cells/well for
stained with propidium iodide (Fluka, Sigma-Aldrich). Cell cycle was analyzed by flow cytometry, as
described [45].
3.2.6. Western Blot Analysis
HCT116 p53+/+ were seeded in a 6-well plate at 1.5
µM of xanthone 37 or DMSO for 24 h. Total protein extracts of cancer cells were obtained and analyzed
×
105 cells/well for 24 h and treated with 10 and
20
by Western blot, as described [45]. Membranes were probed with a mouse monoclonal anti-p53 (DO-1),
anti-MDM2 (D-12), or with a rabbit polyclonal anti-p21 (C-19), followed by an anti-mouse or anti-rabbit
horseradish-peroxidase (HRP)-conjugated secondary antibody. For loading control, membranes were
stripped and re-probed with mouse monoclonal anti-GAPDH (6C5). All antibodies were purchased
from Santa Cruz Biotechnology (Frilabo, Porto, Portugal). The signal was detected with enhanced
chemiluminescence (ECL) Amersham Kit from GE Healthcare (VWR) and with Molecular Imager®
ChemiDoc™ XRS+ System, using Image Lab™ software.
3.2.7. Statistical Analysis
Data’s statistical analysis was performed using the Prism 7 program (GraphPad Software Inc.,
California, USA). Differences between means were tested for significance using one-way ANOVA with
Tukey’s multiple comparisons test, two-way ANOVA with Sidak’s multiple comparisons test, and
Student’s t-test (* p < 0.05; ** p < 0.01).
3.3. In Silico Studies onto MDM2
The chemical structures of gambogic acid (
dihydro-2H,6H-pyrano[3,2-b]xanthen-6-one ( ), and the xanthones 30
ChemSketch (Advanced Chemistry Development, Inc. (ACD/Labs), Canada); the structure of known
2),
α
-mangostin (
3), 12-hydroxy-2,2-dimethyl-3,4-
4
–
40 were drawn using
MDM2-p53 inhibitor nutlin-3A (1) was obtained from PubChem [53]. The three-dimensional (3D)
structures of the xanthones and
1
were minimized using the ArgusLab version 4.0.1 software for
Windows by Hamiltonian (quantum mechanics) using the Parameterized Model number 3 (PM3)
semi-empirical method [54]. The 3D structure of MDM2 was extracted from Protein Data Bank (PDB
id 4HG7) [50]. Docking simulations involving MDM2 and the small molecules were undertaken in
AutoDock Vina (Scripps Research Institute, USA) [51]. AutoDock Vina considered the macromolecular
target conformation as a rigid unit, while the ligands were allowed to be flexible and adaptable
to the target. Vina explored the lowest binding affinity conformations and returned nine different
conformations for each ligand. AutoDock Vina was run using an exhaustiveness of 8 and a grid
box with the dimensions of 19.7 Å, 26.1 Å, and 14.1 Å, engulfing the binding cavity occupied by the
crystallographic nutlin-3A (PDB id 4HG7). Ligand conformations and interactions with the binding
cavity were visualized using PyMOL version 1.3 [55]. In order to validate the docking approach for
the macromolecular target structure used, nutlin-3A was docked onto the binding cavity of MDM2
using AutoDock Vina, and the obtained docking conformation was compared to crystallographic
nutlin-3A (PDB id 4HG7) using RMSD (not shown). Docking scores of xanthones and positive controls
were compared.
4. Conclusions
An alternative and efficient strategy for the preparation of carbaldehydic xanthones was
successfully developed. The use of a precursor with a methyl substituent at position C-1 revealed to be
crucial for the optimization of the synthesis of
5. The newly described approach required lower reaction
times and enabled the preparation of xanthone
5
in a higher global yield. In this work, a series of eleven

Molecules 2019, 24, 1975
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aminated xanthones (30
–
40) possessing a 3,4-dioxygenated pattern of substitution was efficiently
constructed in moderate to good yields. From this group of compounds, xanthone 37 was identified
for the first time as a putative p53-activating agent, using a yeast-based screening assay. Xanthone 37
was revealed to inhibit the growth of human HCT116 p53+/+ colon cancer cells, being that this effect is
associated with cell cycle arrest through activation of the p53 pathway. Nevertheless, further studies
are required to confirm the mechanism of action of 37, which may lead to the identification of a novel
xanthone derivative with promising antitumor activity.
These results demonstrated the potential usefulness of coupling amine-containing structural motifs
of known MDM2-p53 disruptors into the 3,4-dioxygenated xanthone scaffold as a starting point for the
design of novel and improved p53-activating agents with antitumor activity and drug-like properties.
Supplementary Materials: The following are available online. Figure S1–S15: HRMS spectra of the xanthone
derivatives 5 and 27–40. Figure S16–S45: 1H- and 13C-NMR spectra of the xanthone derivatives 5 and 27–40.
Author Contributions: A.L. performed the in silico studies and the synthesis of xanthones and wrote the
manuscript. A.L., A.S.G., and J.B.L. performed biological experiments and analyzed the data. P.B. contributed to
the experimental synthetic work. M.E.S. and L.S. conceived and designed the study, coordinated the experimental
work, and wrote the manuscript. A.P. coordinated the in silico studies. M.M.M.P., M.E.S., and L.S. provided
financial support for the study, analyzed the data, and wrote the manuscript.
Funding: The authors thank to national funds provided by FCT—Foundation for Science and Technology and
European Regional Development Fund (ERDF) and COMPETE under the Strategic Funding UID/Multi/
04423/2019, UID/QUI/50006/2019 (UCIBIO/REQUIMTE) the projects (3599-PPCDT) PTDC/DTP-FTO/1981/2014—
POCI-01-0145-FEDER-016581, POCI-01-0145-FEDER-028736, and PTDC/MAR-BIO/4694/2014 (POCI-01-0145-
FEDER-016790; 3599-PPCDT). FCT fellowships: PD/BD/114046/2015 (A.S. Gomes), SFRH/BD/128673/2017 (J. B.
Loureiro), and the Programa Operacional Capital Humano (POCH), specifically the BiotechHealth Programme
(PD/00016/2012).
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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Sample Availability: Samples of the compounds 5, 16–40 are available from the authors.
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