Synthesis of new glycosylated flavonoids with inhibitory activity on cell growth

Article abstract of DOI:10.3390/molecules23051093

Natural flavonoids and xanthone glycosides display several biological activities, with the glycoside moiety playing an important role in the mechanism of action of these metabolites. Herein, to give further insights into the inhibitory activity on cell gr

Full text of DOI:10.3390/molecules23051093

molecules
Article
Synthesis of New Glycosylated Flavonoids with
Inhibitory Activity on Cell Growth
Ana R. Neves 1,2,† ID , Marta Correia-da-Silva 1,2,†, Patrícia M. A. Silva 3 ID , Diana Ribeiro 3,
Emília Sousa 1,2, ID , Hassan Bousbaa 2,3 and Madalena Pinto 1,2
*
ID
1
Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences,
Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal;
anarcneves92@gmail.com (A.R.N.); m_correiadasilva@ff.up.pt (M.C.-d.-S.); madalena@ff.up.pt (M.P.)
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto,
Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos, S/N 4450-208 Matosinhos,
Portugal; hassan.bousbaa@iucs.cespu.pt
CESPU, Institute of Research and Advanced Training in Health Sciences and Technologies (IINFACTS),
Rua Central de Gandra, 1317, 4585-116 Gandra, Portugal; patricia_masilva@hotmail.com (P.M.A.S.);
dianaroberta@ua.pt (D.R.)
2
3
*
Correspondence: esousa@ff.up.pt; Tel.: +351-220428689
†
These authors contributed equally to this work.
Academic Editors: Tsukasa Iwashina and Thomas J. Schmidt
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 3 April 2018; Accepted: 2 May 2018; Published: 5 May 2018
Abstract: Natural flavonoids and xanthone glycosides display several biological activities, with the
glycoside moiety playing an important role in the mechanism of action of these metabolites. Herein,
to give further insights into the inhibitory activity on cell growth of these classes of compounds,
the synthesis of four flavonoids (5, 6, 9, and 10) and one xanthone (7) containing one or more
acetoglycoside moieties was carried out. Acetyl groups were introduced using acetic anhydride and
microwave irradiation. The introduction of one or two acetoglycoside moieties in the framework of
3,7-dihydroxyflavone (
Koenigs-Knorr reaction. The in vitro cell growth inhibitory activity of compounds
4
) was performed using two synthetic methods: the Michael reaction and the
, and 10 was
5, 6, 7, 9
investigated in six human tumor cell lines: A375-C5 (malignant melanoma IL-1 insensitive), MCF-7
(breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), U251 (glioblastoma astrocytoma),
U373 (glioblastoma astrocytoma), and U87MG (glioblastoma astrocytoma). The new flavonoid
3-hydroxy-7-(2,3,4,6-tetra-O-acetyl-β-glucopyranosyl) flavone (10) was the most potent compound in
all tumor cell lines tested, with GI50 values < 8
µM and a notable degree of selectivity for cancer cells.
Keywords: flavonoids; xanthones; growth inhibitory activity; acetylation; glycosylation
1. Introduction
Flavonoid and xanthone derivatives comprise two classes of oxygenated heterocycles associated
with a large number of biological and pharmacological activities [
occur in nature as glycosides, which have been shown to have several biological activities, namely,
antimicrobial [ ], antioxidant [ ], and antitumor [10 11]. Flavonoid glycosides are widely
1,2]. These compounds also
3–
5
6–
9
,
present in vegetables and fruits and can be found with different types of glycosides, while xanthone
glycosides are mainly found in the families Gentianaceae and Polygalaceae with a glucose moiety [12].
In particular, acetylated flavonoid and xanthone glycosides have been reported as bioactive compounds
with antioxidant [13,14] and antitumor activities [15–19]. The last step in flavonoid biosynthesis is, in
some plant species, terminated by acylation, which is known to increase the solubility and stability
of glycosylated flavonoids in lipophilic systems [20]. Thus, we were inspired to perform acetylation
Molecules 2018, 23, 1093; doi:10.3390/molecules23051093
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1093
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and glycosylation in natural flavonoids and xanthones. Rutin (
1
), diosmin (2), and mangiferin
(3
) were selected for acetylation due to their use in therapy and/or investigation in clinical trials,
and 3,4-dihydroxyflavone ( ) was selected for glycosylation to study the structure-activity relationship
4
(Figure 1). All these starting materials have previously been shown to display in vitro and in vivo
antitumor activity [11,21–24].
Figure 1. Structure of the selected precursors 1–4.
2. Results and Discussion
2.1. Synthesis
Rutin peracetate (
acetic anhydride under reflux for 4 h in solvent-free conditions (Scheme 1) [25]. Previously, compound
was obtained in the presence of pyridine with long reaction times [26]. The synthesis of compound
without using pyridine and with short reaction times is reported for the first time.
Several products that were difficult to separate were obtained when this acetylation method
was applied to diosmin ( ) and mangiferin ( ). Diosmin peracetate ( ) was successfully obtained in
only 35 min (62% yield) in the presence of NaF, under microwave (MW) irradiation (Scheme 1) [27].
Peracetylation of mangiferin ( ) was achieved in only 15 min (78% yield) under MW irradiation, but
5) was obtained from rutin (1) according to a classical acetylation method using
5
5
2
3
6
3
only when iodine was used as a catalyst (Scheme 1).
Previous methods described to synthesize compounds
solvent and catalyst, which was applied room temperature, leading to long reaction times [28
6
and
7
used highly toxic pyridine as
32].
and
–
6
Herein, the application of MW irradiation without pyridine allowed the synthesis of compounds
7 in short reaction times.
For glycosylation of flavone
selected as glucose donor and two reaction methods were applied: a Koenigs-Knorr
method [33 35] and a modified Michael method [36]. Both reactions were carried out at
4, 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (8) was
–
room temperature to decrease the number of side products [37]. Formation of glycosylated
products could not be observed using the Koenigs-Knorr approach, even with the addition
of 3 equivalents/OH of 2,3,4,6-tetra-O-acetyl-
modified Michael method, with K2CO3 in dry acetone, it was possible to obtain two
glycosylated products (Scheme 2), the 3,7-(2,3,4,6-tetra-O-acetyl- -glucopyranosyl) flavone ( ) and the
-glucopyranosyl) flavone (10), although with low yields (11% and
α
-D-glucopyranosyl bromide (
8
).
Using the
β
9
3-hydroxy-7-(2,3,4,6-tetra-O-acetyl-
β
2%, respectively).

Molecules 2018, 23, 1093
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Scheme 1. Synthesis of rutin peracetate (5), diosmin peracetate (6), and mangiferin peracetate (7).
Ac2O—Acetic anhydride; MW—Microwave.
Scheme 2.
Synthesis of 3,7-(2,3,4,6-tetra-O-acetyl-β-glucopyranosyl) flavone
(9)
and
3-hydroxy-7-(2,3,4,6-tetra-O-acetyl-β-glucopyranosyl) flavone (10).
2.2. Structure Elucidation
The structure of compounds
5
,
–7
was established comparing their 1H and 13C NMR data with the
39], while the structures of the compounds and 10 was elucidated
reported in the literature [29 30 38
,
,
9
for the first time, using infrared (IR), nuclear magnetic resonance (NMR), and high-resolution mass
spectrometry (HRMS) techniques (Figures S1–S4).
For instance, the IR spectrum of compound
9
showed a strong band at 1750 cm−1, which is
1
characteristic of the C=O ester stretching vibration. H and 13C NMR spectra of compound
9 indicated
the presence of a flavone moiety and of two sugar moieties. H and 13C NMR spectra of compound
1

Molecules 2018, 23, 1093
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10 indicated the presence of a flavone moiety and one acetylated sugar moiety. The assignments of
the carbon atoms directly bonded to proton atoms were achieved from heteronuclear single quantum
correlation (HSQC) experiments, and the chemical shifts of the carbon atoms not directly bonded to
proton atoms were deduced from heteronuclear multiple bond correlation (HMBC) correlations. For
instance, Figure 2 shows the correlations found for compound 10.
Figure 2. Main connectivities found in the HMBC spectrum of compound 10.
The position of the sugar moiety in compound 10 was evidenced by the correlation found in the
HMBC spectrum between the proton signals of H-1” (δH 5.16–5.36 ppm) and the carbon signal of C-7
(δC 160.9 ppm).
2.3. Growth Inhibition Activity in Human Tumor Cell Lines
Acetylated flavonosides
5, 6, 9, 10, and acetylated xanthonoside 7, as well as their precursors,
compounds , were evaluated for their in vitro growth inhibitory effect on six human tumor cell
1–4
lines: A375-C5 (IL-1 insensitive malignant melanoma), MCF-7 (breast adenocarcinoma), NCI-H460
(non-small-cell lung cancer), U251 (glioblastoma astrocytoma), U373 (glioblastoma astrocytoma), and
U87MG (glioblastoma astrocytoma). For the tested compounds, a dose–response curve was established,
and the results, summarized in Table 1, are expressed as the concentration that was able to cause 50%
cell growth inhibition (GI50).
Table 1. Cell growth inhibitory activity displayed by compounds 1–7, 9, and 10 and their calculated
iLogP values *.
GI50(µM)
Compounds
iLogP
A375-C5
MCF-7
NCI-H460
U251
U373
U87-MG
1
2
3
4
5
6
7
9
10
>150
>150
>150
>150
>150
>150
>150
>150
>150
ND
ND
ND
ND
ND
ND
ND
ND
ND
2.43
2.03
1.42
2.01
5.44
4.03
3.08
4.35
3.55
-
10.81 ± 1.42
9.65 ± 1.92
>150
58.06 ± 2.74
10.44 ± 0.72
7.34 ± 0.93
0.014 ± 0.002
12.55 ± 5.23
20.49 ± 2.14
>150
12.80 ± 1.83
14.32 ± 4.60
>150
9.89 ± 0.53
24.46 ± 4.08
ND
10.24 ± 1.01
20.17 ± 1.92
ND
18.87 ± 1.36
14.28 ± 1.62
ND
88.49 ± 0.72
99.90 ± 5.81
>150
>150
>150
>150
>150
32.86 ± 0.042
5.41 ± 0.83
0.011 ± 0.004
109.16 ± 1.19
100.37 ± 9.61
6.48 ± 0.67
0.010 ± 0.002
2.67 ± 0.49
0.009 ± 0.001
7.61 ± 0.55
0.009 ± 0.002
6.42 ± 2.01
Doxorubicin
0.009 ± 0.001
Doxorubicin was used as positive control. GI50—concentration for 50% of maximal inhibition of cell proliferation.
ND—not determined. * iLogP from SwissADME software using an in-house physics-based method implemented
from [40].
Concerning the precursors, flavone
the growth of IL-1 insensitive A375-C5, estrogen-dependent MCF-7, and NCI-H460 cell lines.
In contrast to compounds , and 10, diosmin peracetate ( ) did not show an inhibitory effect
on the growth of any of the six human tumor cell lines tested. Rutin peracetate ( ) exhibited a good
growth inhibitory activity in all the cell lines tested, mangiferin peracetate ( ) showed a moderate
4 was the only precursor that showed an inhibitory effect on
5,
7
6
5
7
growth inhibitory activity only in IL-1 insensitive A375-C5, estrogen-dependent MCF-7, and NCI-H460
cell lines, while compound 10 was the most potent compound in all human tumor cell lines tested,
with GI50 values < 8 µM.

Molecules 2018, 23, 1093
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The presence of the acetyl groups in compounds
growth inhibitory activity (comparing with compounds
a strategy used to increase the penetration of flavonoids through the cell membrane [41
comparing the peracetylated compounds ( ), some correlations can be found between lipophilicity
and the cell growth inhibitory effect, especially in melanoma cells; compound , with the highest
5
and
7
was found to be crucial for their cell
1
and
3, respectively). Acylation has been
,42]. When
5–7, 9
5
calculated logP value, was also associated with the highest inhibitory effect. It is noteworthy that
compound 10, the only investigated acetylated glycoside with a free hydroxyl, was the most active
derivative, highlighting the importance of both types of substituents. The position of the sugar moiety
in flavonoids seems to be critical for the inhibitory activity.
The selectivity index was calculated for compounds 4, 5, 7, 9, 10, and for Doxorubicin (Table 2).
Table 2. Selectivity index of the compounds 4, 5, 7, 9, 10, and control (Doxorubicin).
HPAEpiC
(GI50, µM)
Selectivity Index
Compounds
A375-C5
MCF-7
NCI-H460
U251
U373
U87-MG
4
5
7
9
10
16.03 ± 1.38
14.13 ± 5.06
110 ± 7.07
1.48
1.46
1.89
8.75
4.32
0.29
1.28
0.69
1.24
1.25
0.99
1.10
1.62
0.58
-
2.78
5.68
0.36
1.57
0.70
-
0.84
4.94
0.44
0.85
0.99
-
0.91
4.89
0.40
91.40 ± 0.85
31.71 ± 1.63
11.88
0.44
4.17
0.44
Doxorubicin 0.004 ± 0.09
As outlined in Table 2, the results clearly reflect a notable degree of selectivity of compound 10 in
all cell lines studied. Of note, human breast cancer cell line MCF-7 responded very well by exhibiting
a higher selectivity index than the other cancer cell lines. As to the other compounds, some had no
selectivity (4 and 7) while the others (5) or (9) exhibited some selectivity regarding the cancer lines in
which they were most active. Interestingly, all tested compounds exhibited a better degree of selectivity
than Doxorubicin.
3. Materials and Methods
3.1. General Information
Commercially available reagents were purchased from Sigma-Aldrich Co. (Sintra, Portugal) and
BioSolve (Dieuze, France). Dichloromethane was dried using the following procedure: pre-drying
with CaSO4 and reflux over calcium hydride and distillation onto 4 Å molecular sieves [43].
Extra-dry acetone was purchased from BioSolve (France). Reactions were controlled by thin layer
chromatography (TLC) using Merck silica gel 60 (GF254) plates. Compounds were visually detected
by absorbance at 254 and/or 365 nm and ferric chloride 10% in methanol (MeOH). Purification of
compounds by flash chromatography was carried out using Fluka silica gel 60 (0.04–0.063 mm).
MW reactions were performed using a MicroSYNTH 1600 from Milestone (ThermoUnicam, Lisboa,
Portugal) synthesizer in open reaction vessels. IR spectra were obtained in KBr microplate in a FTIR
spectrometer Nicolet is 10 from Thermo Scientific with Smart OMNI-Transmission accessory (Software
1
OMNIC 8.3, Thermo Scientific, Madison, WI, USA). H and 13C NMR spectra were performed in
University of Aveiro, Department of Chemistry and were taken in CDCl3 or DMSO-d6, at room
temperature, on Bruker Avance 300 instrument (300.13 MHz for 1H and 75.47 MHz for 13C, Bruker
Biosciences Corporation, Billerica, MA, USA). 13C NMR assignments were made by 2D HSQC and
HMBC experiments (long-range C, H coupling constants were optimized to 7 and 1 Hz). Chemical
shifts are expressed in ppm values relative to tetramethylsilane (TMS) as an internal reference.
Coupling constants are reported in hertz (Hz). HRMS mass spectra were measured on an APEX
III mass spectrometer, recorded as ESI (Electrospray) made in Centro de Apoio Científico e Tecnolóxico
á Investigation (CACTI, University of Vigo, Spain). Six human tumor cell lines were used to study

Molecules 2018, 23, 1093
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antitumor activity: MCF-7 ER (+) (breast adenocarcinoma), NCI-H460 (non-small-cell lung cancer),
and A375-C5 (melanoma), U251 (glioblastoma astrocytoma), U373 (glioblastoma astrocytoma) and
U87MG (glioblastoma astrocytoma).
3.2. Chemistry
Synthesis of rutin peracetate (
5
). Rutin (
1
, 0.3 g, 0.5 mmol) was added to acetic anhydride
◦
(12 mL) and the mixture was heated at 130 C, for 4 h. The solution obtained was poured into
ice and extracted with CH2Cl2. The organic layer was extracted with a saturated solution of
NaHCO3, dried with anhydrous Na2SO4, filtered, and then evaporated under reduced pressure.
The obtained oil was dissolved in ethyl acetate. A solid material was obtained by adding
petroleum ether 60–80 ◦C corresponding to 2-(3,4-di-O-acetylphenyl)-5,7-di-O-acetyl-3-[3,4,5-tri-O-
acetyl-
α
-L-rhamnopyranosyl-(1
→
6)-3,4,5-tri-O-acetyl-β-D-glucopyranosyloxy]-4H-chromen-4-one (5)
(0.43 g, 0.41 mmol, 73% yield); mp 119–120 ◦C (petroleum ether 60–80 ◦C).
Synthesis of diosmin peracetate ( ). Diosmin ( , 0.05 g, 0.08 mmol) and NaF (0.42 g, 10 mmol) were
6
2
mixed in acetic anhydride (4 mL) and the mixture was kept under MW irradiation (400 W) at 130 ◦C,
for 35 min. After cooling, the solution obtained was poured into ice and then extracted with CH2Cl2.
The organic layer was extracted with NaHCO3 followed by crystallization from MeOH/H2O provided
5,3-O-acetyl-2-(3-O-acetyl-4-methoxyphenyl)-7-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→6)-2,3,4-
tri-O-acetyl-β-D-glucopyranosyloxy] oxychromen-4-one (6) as a yellow solid (0.048 g, 0.051 mmol, 62%
yield); mp 135–138 ◦C.
Synthesis of mangiferin peracetate (7). Mangiferin (3, 0.2 g, 0.5 mmol) and iodine (0.009 g, 0.07 mmol)
were mixed in acetic anhydride (7 mL) and the reaction was kept under MW irradiation (400 W),
at 130 ◦C, for 15 min. After cooling, a saturated solution of sodium thiosulfate was added to convert
iodine (dark yellow) into iodide (yellow). The crude product was extracted with CH2Cl2 and the
organic layer was extracted with a saturated solution of NaHCO3 twice, dried with anhydrous Na2SO4,
and filtered. The solvent was evaporated under reduced pressure and the oil obtained was dissolved
◦
in ethyl acetate. A yellow solid was obtained with petroleum ether 60–80 C corresponding to
1,3,6,7-tetra-O-acetyl-2-C-(2,3,4,6-tetra-O-acetyl-
β-D-glucopyranosyl)-9H-xanthen-9-one (7) (0.294 g,
0.39 mmol, 78% yield); mp 143–147 ◦C (petroleum ether 60–80 ◦C).
Synthesis of 3,7-(2,3,4,6-tetra-O-acetyl- -glucopyranosyl) flavone ( ) and 3-hydroxy-7-(2,3,4,6-tetra-O-acetyl-
-glucopyranosyl) flavone (10). 3,7-Dihydroxyflavone ( , 0.100 g, 0.4 mmol) and K2CO3 (0.225 g,
1.57 mmol, 2eq/OH) were mixed in dry acetone (10 mL) and the mixture was kept under stirring for
15 min. 2,3,4,6-Tetra-O-acetyl- -D-glucopyranosyl bromide ( , 0.653 g, 1.57 mmol, 2eq/OH) was added
β
9
β
4
α
8
and the mixture was kept under stirring at room temperature, for 48 h. The suspension was filtered to
eliminate the K2CO3 and the filtrate was evaporated. The oil obtained was further purified through
flash column chromatography (100% CHCl3). The obtained fractions were gathered and crystallized
from MeOH to achieve 3,7-(2,3,4,6-tetra-O-acetyl-β-glucopyranosyl) flavone (9, 0.04 g, 0.045 mmol,
11.4% yield) and 3-hydroxy-7-(2,3,4,6-tetra-O-acetyl-
β-glucopyranosyl) flavone (10, 0.003 g, 0.004 mmol,
1.2% yield).
3,7-(2,3,4,6-Tetra-O-acetyl-
β
-glucopyranosyl) flavone (
9
). Mp 105–108 ◦C; IR (KBr)
υ
max: 2962, 2917, 2843,
1750, 1623, 1449, 1381, 12310, 1066, 1040 cm−1; 1H NMR (CDCl3, 300.13 MHz)
δ: 8.17 (1H, d, J = 8.8 Hz,
0
0
0
0
H-5), 8.02–7.98 (2H, m, H-2 ,6 ), 7.50–7.47 (3H, m, H-3 ,4 ,5 ), 7.10 (1H, d, J = 2.2 Hz, H-8), 7.05 (1H, dd,
J = 2.3 and 8.9 Hz, H-6), 5.71 (1H, d, J = 7.7 Hz, H-glucose), 5.34–5.04 (7H, m, H-glucose), 4.31–4.19
(2H, m, H-glucose), 3.98–3.91 (3H, m, H-glucose), 3.65–3.60 (1H, m, H-glucose), 2.09 (3H, s, COCH3),
2.08 (3H, s, COCH3), 2.06 (3H, s, COCH3), 2.05 (3H, s, COCH3), 2.04 (3H, s, COCH3), 2.01 (3H, s,
COCH3), 2.00 (3H, s, COCH3), 1.90 (3H, s, COCH3) ppm; 13C NMR (CDCl3, 75.47 MHz)
δ: 173.4 (C-4),
170.6 (COCH3), 170.5 (COCH3), 170.3 (COCH3), 170.2 (COCH3), 170.0 (COCH3), 169.7 (COCH3), 169.5
(COCH3), 169.4 (COCH3), 160.9 (C-7), 157.4 (C-2), 156.6 (C-9), 136.3 (C-3), 131.0 (C-10), 130.6 (C-40),

Molecules 2018, 23, 1093
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129.1 (C-30,50), 128.3 (C-20,60), 127.6 (C-5), 119.9 (C-10), 115.5 (C-6), 104.4 (C-8), 98.9 (C-glucose), 98.5
(C-glucose), 72.9 (C-glucose), 72.6 (C-glucose), 72.6 (C-glucose), 71.8 (C-glucose), 71.8 (C-glucose),
68.4 (C-glucose), 68.2 (C-glucose), 62.1 (C-glucose), 61.5 (C-glucose), 21.0 (COCH3), 20.8 (COCH3),
20.8 (COCH3), 20.7 (COCH3), 20.7 (COCH3) ppm; HRMS (ESI+) m/z calcd for C29H28O13 [M + H]
915.25535, found 915.25410.
3-Hydroxy-7-(2,3,4,6-tetra-O-acetyl-
2961, 2917, 2847, 1758, 1621, 1572, 1501, 1457, 1235, 1211, 1071 cm−1; 1H NMR (CDCl3, 300.13 MHz)
: 9.62 (3-OH), 8.24-8.18 (3H, m, H-5,20,60), 7.57–7.48 (3H, m, H-30,40,50), 7.13 (1H, d, J = 2.2 Hz, H-8),
β υmax: 3442,
-glucopyranosyl) flavone (10). Mp 222–223 ◦C; IR (KBr)
δ
7.06 (1H, dd, J = 2.3 and 8.9 Hz, H-6), 5.36–5.16 (4H, m, H-1”,2”,3”,4”), 4.33–4.20 (2H, m, H6”a,b),
4.03–3.97 (1H, m, H-5”), 2.09 (3H, s, COCH3), 2.08 (3H, s, COCH3), 2.07 (3H, s, COCH3), 2.06 (3H, s,
COCH3) ppm; 13C NMR (CDCl3, 75.47 MHz)
δ: 172.9 (C-4), 170.6 (COCH3), 170.3 (COCH3), 169.6
(COCH3), 169.4 (COCH3), 160.9 (C-7), 156.8 (C-9), 138.4 (C-3), 131.1 (C-10), 130.4 (C-40), 128.8 (C-30,50),
127.7 (C-20,60), 127.4 (C-5), 116.6 (C-10), 115.4 (C-6), 104.3 (C-8), 98.4 (C-1”), 72.6 (C-3”), 72.6 (C-5”),
71.1 (C-2”), 68.3 (C-4”), 62.1 (C-6”), 20.8 (COCH3), 20.8 (COCH3), 20.7 (COCH3), 20.7 (COCH3) ppm;
HRMS (ESI+) m/z calcd for C29H28O13 [M + H] 585.16027, found 585.15875.
3.3. Tumor Cell Growth Assay and Selectivity Index
The human tumor cell lines A375-C5 (melanoma), MCF-7 (breast adenocarcinoma), and NCI-H460
(non-small cell lung cancer) were grown in culture medium RPMI-1640 (Biochrom, Berlin, Germany),
U251 (glioblastoma astrocytoma), U373 (glioblastoma astrocytoma), and U87MG (glioblastoma
astrocytoma) were grown in culture medium DMEM (Biochrom), both supplemented with 5%
◦
heat-inactivated fetal bovine serum (FBS, Biochrom). All cell lines were maintained at 37 C in
a 5% CO2 humidified atmosphere (Hera Cell, Heraeus). Cell viability was routinely determined with
Trypan Blue (Sigma-Aldrich) exclusion assay and all experiments were performed with exponentially
growing cells, showing more than 95% viability.
The effect of compounds on the cell growth was evaluated according to the procedure adopted
by the National Cancer Institute (NCI) in the ‘In vitro Anticancer Drug Discovery Screen’, which
uses the protein-binding dye sulforhodamine B (SRB) to assess cell growth. Cells◦were plated in
106 cells/well) in complete culture medium and incubated at 37 C. Twenty-four
96-well plates (0.05
×
hours later, cells were treated with two-fold serial dilutions of the test compounds, ranging from
0 to 150 M, for 48 h. Control groups received the same amount of sterile Dimethyl Sulfoxide (DMSO,
µ
Sigma-Aldrich), used as compounds solvent, up to 0.25% concentration. Then, cells were fixed in situ
with 50% (m/v) trichloroacetic acid (Merck Millipore, Darmstadt, Germany), washed with distilled
water and stained with Sulforhodamine B (SRB; Sigma-Aldrich) for 30 min at room temperature.
The SRB-stained cells were washed 5 times with 1% (v/v) acetic acid (Merck Millipore) and left to dry
at room temperature. SRB complexes were solubilized, by adding 10 mM Tris buffer (Sigma-Aldrich)
for 30 min. Absorbance was measured at 515 nm in a microplate reader (Biotek Synergy 2,BioTek
Instruments, Inc., Winooski, VT, USA). A dose–response curve was obtained for each cell line with each
test compound, and the concentration that caused cell growth inhibition of 50% (GI50) was determined.
The degree of selectivity of the compounds was assessed by determining the selectivity index (SI)
as the ratio of GI50 in non-tumor Human Pulmonary Alveolar Epithelial Cells (HPAEpiC) over GI50 in
cancer cells. An SI value less than 2.0 indicates the general toxicity of the compound [44].
4. Conclusions
In this work, four acetylated flavonosides (5, 6, 9, and 10), and one xanthonoside (7) were
synthesized. As far as we know, compounds and 10 are described for the first time. Compound
9
5
was synthesized without pyridine in a shorter reaction time. Applying MW irradiation to the synthesis
of compounds 6 and 7 allowed to shorten the reaction times.

Molecules 2018, 23, 1093
8 of 10
The in vitro growth inhibitory activity of compounds
investigated. 3-Hydroxy-7-(2,3,4,6-tetra-O-acetyl-
5
–
7
and compounds
9
and 10 was
β
-glucopyranosyl) flavone (10) was the most
active compound in all the human tumor cell lines tested with notable selectivity for cancer cells.
The mechanism of action of this promising compound deserves to be further explored. The position of
the sugar moiety seems to influence the activity of these compounds.
These results are a starting point from which to investigate more stable chemical modifications in
order to optimize such molecules and develop detailed structure-activity relationship studies in future.
Supplementary Materials: The following are available online, Figure S1. 1H and 13C NMR of compound
9, Figure S2.
1H and 13C NMR of compound 10, Figure S3. HRMS for compound 9, Figure S4. HRMS for compound 10.
Author Contributions: A.R.N. performed the synthesis, purification and structure elucidation of compounds
5
,
6
, and
7
, and wrote the manuscript; M.C.-d.-S. performed the synthesis, purification and structure elucidation
of compounds
9
and 10. M.C.-d.-S., E.S., and M.P. designed the experimental work concerning the synthesis.
P.M. A.S. and D.R. performed the inhibition tumor cell growth assays and determined the selectivity index; H.B.
designed the experimental work concerning the antitumor screening. M.C.-d.-S., P.M. A.S., D.R., E.S., Hassan
Bousbaa, and Madalena Pinto revised the manuscript.
Funding: This work was supported through national funds provided by Foundation for Science and Technology
from the Minister of Science, Technology and Higher Education (FCT/MCTES-PIDDAC) and European Regional
Development Fund (ERDF) through the COMPETE—Programa Operacional Factores de Competitividade (POFC)
(POCI-01-0145-FEDER-016790), PPCDT—Promover a Produção Científica e Desenvolvimento Tecnológico e a
Constituição de Redes Temáticas (Project 3599), under the project PTDC/MAR-BIO/4694/2014 in the framework
of the programme PT2020 and INNOVMAR—Innovation and Sustainability in the Management and Exploitation
of Marine Resources, reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR in the framework of
North Portugal Regional Operational Programme (NORTE 2020).
Acknowledgments: Marta Correia-da-Silva thanks FCT for the postdoctoral fellowship SFRH/BPD/81878/2011
and Ana R. Neves and Patrícia M.A. Silva for the Ph.D. fellowships SFRH/BD/114856/2016 and
SFRH/BD/90744/2012, respectively.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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