Prenylated derivatives of baicalein and 3,7-dihydroxyflavone: Synthesis and study of their effects on tumor cell lines growth, cell cycle and apoptosis

Article abstract of DOI:10.1016/j.ejmech.2011.03.047

Fourteen baicalein and 3,7-dihydroxyflavone derivatives were synthesized and evaluated for their inhibitory activity against the in vitro growth of three human tumor cell lines. The synthetic approaches were based on the reaction with prenyl or geranyl br

Full text of DOI:10.1016/j.ejmech.2011.03.047

European Journal of Medicinal Chemistry 46 (2011) 2562e2574
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Prenylated derivatives of baicalein and 3,7-dihydroxyflavone: Synthesis and study
of their effects on tumor cell lines growth, cell cycle and apoptosis
,
,
b
,
a
,
,e
Marta Perro Neves a b, Honorina Cidade a , Madalena Pinto b, Artur M.S. Silva c, Luís Gales d
,
*
Ana Margarida Damas d e, Raquel T. Lima f g, M. Helena Vasconcelos f g, Maria de São José Nascimento a
,
,
,
,g
a Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP), Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
b Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha 164,
4050-047 Porto, Portugal
c Departamento de Química & QOPNA, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
d Unidade de Estrutura Molecular, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal
e Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Largo Prof. Abel Salazar 2, 4099-003 Porto, Portugal
f Grupo de Oncobiologia, IPATIMUP e Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal
g Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Fourteen baicalein and 3,7-dihydroxyflavone derivatives were synthesized and evaluated for their inhib-
itory activity against the in vitro growth of three human tumor cell lines. The synthetic approaches were
based on the reaction with prenyl or geranyl bromide in alkaline medium, followed by cyclization of the
respective monoprenylated derivative. Dihydropyranoflavonoids were also obtained by one-pot synthesis,
using Montmorillonite K10 clay as catalyst combined with microwave irradiation. In vitro screening of the
compounds for cell growth inhibitory activity revealed that the presence of one geranyl group was
associated with a remarkable increase in the inhibitory activity. Moreover, for the 3,7-dihydroxyflavone
derivatives a marked increase in growth inhibitory effect was also observed for compounds with furan and
pyran fused rings. The most active compounds were also studied regarding their effect on cell cycle profile
and induction of apoptosis. Overall the results point to the relevant role of the prenylation of flavone
scaffold in the growth inhibitory activity of cancer cells.
Received 23 December 2010
Received in revised form
15 March 2011
Accepted 22 March 2011
Available online 30 March 2011
Keywords:
Flavonoids
Prenylation
Antitumor
Cell cycle
Apoptosis
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
studies have shown that baicalein inhibited the growth of tumor
xenographs in mouse models [8]. In addition, the cytostatic effect of
Flavonoids have long been recognized for their significant
spectrum of pharmacological activities and have been extensively
studied on several tumor cell lines [1e4]. Considering their
mechanisms of action, their antitumor effect has been attributed to
the interference with several biological processes. In particular,
flavonoids have been described as interesting agents concerning
apoptosis induction [1,4]. In fact, baicalein (1) and 3,7-dihydroxy-
flavone (2) have been reported to have antitumor activity, by acting
as inducers of apoptosis in human tumor cell lines [5e8]. Many
studies have shown that baicalein can selectively cause cell death in
several tumor cells but not in normal peripheral blood cells. In vivo
baicalein in tumor cells was associated with cell cycle arrest at
different checkpoints, depending on the type of tumors [8]. Inter-
estingly, there is evidence that 3,7-dihydroxyflavone has pro-
apoptotic activity, related to interference with calpains and cas-
pases [7].
Although the oxygenation pattern may play an important role in
the biological activities of these molecules, the presence of the
diverse isoprenoid side chains seems to be also relevant [9e13]. In
fact, it has been demonstrated that isoprenylation of flavones
significantly increased their growth inhibitory effect in several
human tumor cell lines [4,14]. An additional ring moiety such as
pyran and furan also seemed to be favorable for the growth
inhibitory activity of these compounds [15,16]. In the course of our
research, which focused on the search of compounds with anti-
tumor activity, we have previously analysed the effect of several
prenylflavones, possessing prenyl side chain and/or dihydropyran
or dihydrofuran fused rings, on the in vitro growth of five human
* Corresponding author. Laboratório de Química Orgânica
e Farmacêutica,
Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do
Porto, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal. Tel.: þ351 222 078 916;
fax: þ351 222 003 977.
E-mail address: hcidade@ff.up.pt (H. Cidade).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.047

M.P. Neves et al. / European Journal of Medicinal Chemistry 46 (2011) 2562e2574
2563
tumor cell lines [17]. The results previously obtained showed that
all the flavones tested, in particular artelastin, exhibited a growth
inhibitory effect on cancer cells [4,17]. In fact, this prenylflavone has
already been integrated in the “In vitro Anticancer Drug Discovery
Screen” (NSC 710340) from the National Cancer Institute (NCI), in
which its capacity to inhibit the growth of a variety of human tumor
cell lines representing different tumor types was confirmed.
The use of eco-friendly approaches for the synthesis of a diversity
of bioactive small molecules has been increasing during recent
years. One of these methods is microwave-assisted organic synthesis
(MAOS) which offers considerable advantages due to the rapid
heating and substantial rate enhancements of a wide range of
organic reactions compared with conventional heating. Cleaner
reactions are also commonly achieved with this method, together
with improvements in yields and selectivity [18,19]. Montmoril-
lonite clays are generally employed as effective and environmentally
benign heterogeneous catalysts for several reactions. In fact, Mont-
morillonite clays may be considered very promising agents since
they are naturally abundant, inexpensive, nontoxic, chemically
versatile and recyclable. Therefore, these solid catalysts, particularly
when used in combination with microwave (MW) irradiation, seem
to be an interesting green alternative to conventional chemistry
practices [18,20].
8e16): eight (3e10) from baicalein (1) and six (11e16) from 3,7-
dihydroxyflavone (2). The ability of prenylflavonoids to inhibit the in
vitro growth of three human tumor cell lines, MCF-7 (breast
adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-
C5 (melanoma), was evaluated. Since several reports refer to the
ability of flavonoids to arrest cell growth at more than one stage of
the cell cycle [4], the effect of the synthesized prenylated flavonoids
on cell cycle progression was analysed. Considering that both
starting blocks (1 and 2) are recognized as apoptosis inducer agents
[7,8,21,22], further investigation was also carried out to demonstrate
the capability of the most active compounds to induce apoptosis.
2. Chemistry
Fourteen baicalein (1) and 3,7-dihydroxyflavone (2) derivatives
were synthesized according to the strategy illustrated in Schemes 1
and 2. The synthesis of flavonoids with prenyl and geranyl side
chains (3e8 and 11e14) was carried out by the reaction of the
correspondent starting material (1 or 2) with prenyl or geranyl
bromide, respectively, in the presence of anhydrous potassium
carbonate using two different methodologies, one based on
conventional thermal heating (classical synthesis) [23] and other by
MAOS [18]. The monoprenylated flavonoids 3 and 11 were then used
as precursors for the synthesis of dihydrofurano- (9, 15) and dihy-
dropyranoflavonoids (10 and 16). Dihydrofuranoflavonoids 9 and 15
were obtained by MW irradiation of the respective monoprenylated
product in the presence of N-methylpyrrolidone (NMP) [18].
On this basis, we used MAOS with or without Montmorillonite
K10 clay to synthesize prenylated derivatives of baicalein (1) and
3,7-dihydroxyflavone (2). Fourteen prenylated derivatives were
obtained, twelve of them being described for the first time (3e5 and
Scheme 1. Synthesis of prenylated derivatives of baicalein (1). The numbering used concerns the NMR assignments.

2564
M.P. Neves et al. / European Journal of Medicinal Chemistry 46 (2011) 2562e2574
Scheme 2. Synthesis of prenylated derivatives of 3,7-dihydroxyflavone (2). The numbering used concerns the NMR assignments.
Dihydropyranoflavonoid 10 was obtained by MW irradiation of
monoprenylated derivative 3 using Montmorillonite K10 clay
order to analyse alterations in the cell cycle profile and alterations
in the levels of apoptosis.
as
a heterogeneous catalyst [24]. Additionally, dihydropyran
derivatives 10 and 16 were also synthesized by one-pot synthesis
using Montmorillonite K10 clay to catalyze the direct condensation
of precursors 1 and 2 with prenyl bromide under MW irradiation
[18,25].
4. Results and discussion
4.1. Synthesis of prenylated derivatives 3e16
As shown in Schemes 1 and 2, the classical synthesis of baicalein
(1) derivatives resulted in three prenylated (3e5) and three
geranylated (6e8) compounds (Scheme 1, via a1 and b1,
3. Pharmacology
The effect of prenylflavonoids and their precursors on the in
vitro growth of three human tumor cell lines, MCF-7 (breast
adenocarcinoma), NCI-H460 (non-small cell lung cancer) and
A375-C5 (melanoma) was evaluated according to the procedure
adopted by the National Cancer Institute (NCI, USA) which uses the
protein-binding dye sulforhodamine B (SRB) to assess cell growth
[26,27]. Based on this procedure, a doseeresponse curve was
obtained for each cell line with each test compound and the
concentration that caused cell growth inhibition of 50% (GI50, cor-
responding to the concentration of compound that inhibited 50% of
net cell growth), was determined as described elsewhere [27].
To clarify the mechanism of action of the most active
compounds (GI50 < 12
mM), the NCI-H460 cell line was used for
treatment with the compounds and cells were further processed in
Fig. 1. Mechanistic pathway proposed for C-prenylation of baicalein (1).

M.P. Neves et al. / European Journal of Medicinal Chemistry 46 (2011) 2562e2574
2565
respectively), while the classical synthesis of 3,7-dihydroxyflavone
(2) derivatives produced two prenylated (11 and 12) and two ger-
anylated (13 and 14) compounds (Scheme 2, via a1 and b1,
respectively). The major products were the derivatives with one
prenyl or geranyl side chain on the oxygen at position 7, with
compounds possessing two or three prenyl or geranyl groups being
obtained as by-products. For baicalein (1) our results indicate that
the first hydroxyl group to be prenylated/geranylated is at C-7,
followed by that at C-6 and only then the C-prenylation/ger-
anylation occurs. The direct C-prenylation/geranylation occurs at
C-8, the most nucleophilic aromatic position (Fig. 1). In the case of
compound 2, the prenylation/geranylation occurs first at C-7, fol-
lowed by that of 3-position.
To improve the yields of the monoprenylated derivatives 3, 6, 11
and 13, baicalein (1) and 3,7-dihydroxyflavone (2) were submitted
to MW irradiation in the presence of prenyl or geranyl bromide in
alkaline medium (Scheme 1, via a2 and b2 and Scheme 2, via a2 and
b2, respectively). In this case, the results suggest that prenylation of
baicalein (1) and 3,7-dihydroxyflavone (2) occurs particularly at the
most acidic hydroxyl group of the flavone scaffold (7-OH), yielding
the monoprenylated derivatives (3, 6, 11 and 13) as major products
and the diprenylated derivatives (4, 7, 12 and 14) as by-products. In
fact, using this methodology the yields of the monoprenylated
compounds 3, 6, 11 and 13 were fairly increased and the yields of
diprenylated compounds 4, 7 and 14 were moderately reduced.
Additionally, the triprenylated by-products 5 and 8 were not
obtained using MW (Table 1) which also illustrated the selectivity
of MAOS in comparison with classical methods. Furthermore, the
reaction time was reduced from 8 h in the conventional heating to
2 h with MW. Compared to conventional heating these results
confirmed that for this family of compounds, MAOS improved
yields and selectivity in shorter reaction times.
Amongst the prenylated flavonoids, pyran and furan derivatives
represent an important group of biologically interesting natural
products [15,16]. For this reason, the monoprenylated derivatives
3 and 11 were used as building blocks for the synthesis of the
dihydrofurano- and dihydropyranoflavonoids 9, 10, 15 and 16. With
N-methylpyrrolidone (NMP) as solvent, flavone 3 produced dihy-
drofuranoflavone 9 in 48% yield (Scheme 1, via c) and flavone 11
produced dihydrofuranoflavone 15 in 36% yield (Scheme 2, via c).
When performed in the presence of Montmorillonite K10 clay in
chloroform, flavone 3 provided dihydropyranoflavone 10 in 34%
yield (Scheme 1, via d). However, the use of the latter methodology
with flavone 11 failed to give the desired dihydropyranoflavone 16
(Scheme 2, via d). Hence, a one-pot synthesis was performed using
Montmorillonite K10 clay to catalyze the direct condensation of
3,7-dihydroxyflavone (2) with prenyl bromide under MW irradia-
tion, resulting in dihydropyranoflavone 16 in 20% yield (Scheme 2,
via e). Using the one-pot synthesis, baicalein (1) gave rise to dihy-
dropyranoflavone 10 in 14% yield (Scheme 1, via e).
These results support our proposal that the dihydrofuran
derivatives 9 and 15 might have been formed through an aromatic
Claisen rearrangement followed by cyclization of the 1,1-dime-
thylallyl group on position 8 of the flavones scaffold (Fig. 2). On
the other hand, the formation of dihydropyran derivatives 10 and
16 can be explained by the proposed mechanism that goes via
the elimination of the prenyl group from the aromatic ring,
Fig. 2. Mechanistic pathway proposed for the synthesis of furan derivatives.

2566
M.P. Neves et al. / European Journal of Medicinal Chemistry 46 (2011) 2562e2574
Fig. 3. Mechanistic pathway proposed for the synthesis of pyran derivatives.
followed by prenylation of the flavone scaffold through an
aromatic electrophilic substitution at the most nucleophilic
aromatic position and subsequent cyclization of the C-prenyl
group (Fig. 3). However, further studies are required to confirm the
reaction mechanism.
a more planar conformation [angle of 8.2(5)ꢀ] in the crystal
structure [30], which was attributed to the double bond char-
acter of C2eC10.
The prenyl side chain remains approximately coplanar to the
benzopyran moiety with a maximum deviation of 0.46 Å for C400a.
Moreover, there are intramolecular hydrogen bonds between
C5-OH
C6-OH
C4=O
and
and
and intermolecular hydrogen bonds between
. In the crystal structure, the molecules are
>
>
>
4.2. Structure elucidation of prenylated derivatives 3e16
C4=O
>
packed into columns with their planes parallel to one another and
4.4 Å apart.
The structure elucidation of compounds 3e16 was established
on the basis of IR, HRMS and NMR techniques. The 1H and 13C NMR
data of prenylated flavonoids are reported in Tables 2e4. 13C NMR
assignments were determined by 2D heteronuclear single quantum
correlation (HSQC) and heteronuclear multiple bond correlation
(HMBC) experiments.
4.3. Effect on growth of human tumor cell lines, cell cycle profile
and apoptosis
The structure of compound 3 was also determined by X-ray
Prenylated (3e16) and non-prenylated flavonoids (1e2) were
evaluated for their capacity to inhibit the in vitro growth of three
human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460
(non-small cell lung cancer) and A375-C5 (melanoma). The results
are summarized in Table 5 and discussed below.
Although the number of compounds was limited, an
attempt was made to draw some considerations concerning
structureeactivity relationship (SAR). Regarding the baicalein
derivatives 3e10, compound 6 clearly demonstrated the best
crystallography.
A perspective view of the crystal structure,
obtained using ORTEP [28] and showing the atomic numbering,
is presented in Fig. 4. The benzopyran system defines a plane
(r.m.s. deviation of the fitted atoms of 0.0049 Å) that makes an
angle of 20.8(1)ꢀ with the phenyl ring. This is in good agree-
ment with potential energy calculations of the flavonoid skel-
eton that have shown a minimum energy configuration at an
angle of 22.8ꢀ [29]. However, the precursor baicalein (1) adopts
Table 2
1H NMR data of prenylated derivatives 3e10a of baicalein (1).
3
4
5
6
7
8
9
10
H-3
6.69 (s)
6.67 (s)
6.68 (s)
6.69 (s)
6.67 (s)
6.69 (s)
6.66 (s)
6.69 (s)
H-5
H-6
12.49 (OH, s)
5.44 (OH, s)
12.63 (OH, s)
e
12.68 (OH, s)
e
12.49 (OH, s)
5.41 (OH, s)
12.64 (OH, s)
e
12.68 (OH, s)
e
12.92 (OH, s)
5.37 (OH, s)
12.40 (OH, s)
5.37 (OH, s)
H-8
6.62 (s)
7.91e7.88 (m)
7.55e7.51 (m)
7.55e7.51 (m)
6.55 (s)
7.91e7.88 (m)
7.56e7.50 (m)
7.56e7.50 (m)
e
6.62 (s)
7.91e7.88 (m)
7.56e7.52 (m)
7.56e7.52 (m)
6.55 (s)
e
e
e
H-20,60
H-30,50
H-40
7.90e7.88 (m)
7.54e7.49 (m)
7.54e7.49 (m)
7.91e7.87 (m)
7.56e7.50 (m)
7.56e7.50 (m)
4.70 (d, J ¼ 6.6)
5.54 (brt, J ¼ 6.6)
1.78 (s)
2.18e2.12 (m)
2.18e2.12 (m)
5.11e5.05 (m)
1.66, 1.59 (2s)
4.62 (d, J ¼ 7.2)
5.60 (brt, J ¼ 7.2)
1.69 (s)
7.91e7.87 (m)
7.58e7.48 (m)
7.58e7.48 (m)
4.76 (d, J ¼ 7.2)
5.55 (brt, J ¼ 7.2)
1.72 (s)
2.08e2.00 (m)
2.08e2.00 (m)
5.09e5.01 (m)
1.67, 1.52 (2s)
4.65 (d, J ¼ 7.2)
5.55 (brt, J ¼ 7.2)
1.69 (s)
2.08e2.00 (m)
2.08e2.00 (m)
5.09e5.01 (m)
1.66, 1.60 (2s)
3.60 (d, J ¼ 6.5)
5.23 (brt, J ¼ 6.5)
1.82 (s)
2.08e2.00 (m)
2.08e2.00 (m)
5.09e5.01 (m)
1.60, 1.29 (2s)
7.88e7.85 (m)
7.57e7.53 (m)
7.57e7.53 (m)
4.63 (q, 6.7)
7.91e7.86 (m)
7.56e7.49 (m)
7.56e7.49 (m)
H-100
H-200
H-400
H-500
H-600
H-700
H-900
H-1000
H-2000
H-4000
H-5000
H-6000
H-7000
H-9000
H-10000
H-20000
H-40000
H-50000
H-60000
H-70000
H-90000
4.70 (d, J ¼ 6.8)
4.67 (d, J ¼ 6.6)
4.73 (d, J ¼ 7.2)
4.73 (d, J ¼ 6.6)
2.98 (t, J ¼ 6.8)
5.56 (brt, J ¼ 6.8)
5.54 (brt, J ¼ 6.6)
5.54 (brt, J ¼ 7.2)
5.54 (brt, J ¼ 6.6)
1.48 (d, J ¼ 6.7)
1.95 (t, J ¼ 6.8)
1.83, 1.79 (2s)
1.82, 1.79 (2s)
1.78, 1.72 (2s)
1.79 (s)
1.62, 1.25 (2s)
1.46 (s)
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
2.18e2.13 (m)
2.18e2.13 (m)
5.10e5.07 (m)
1.67, 1.61 (2s)
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
4.59 (d, J ¼ 7.3)
4.61 (d, J ¼ 7.3)
e
e
e
e
e
e
e
e
e
e
e
e
e
e
5.60 (brt, J ¼ 7.3)
5.60 (brt, J ¼ 7.3)
1.75, 1.69 (2s)
1.77, 1.72 (2s)
e
e
e
e
e
e
e
e
e
e
e
e
2.08e2.00 (m)
2.08e2.00 (m)
5.11e5.05 (m)
1.66, 1.59 (2s)
e
e
e
e
3.58 (d, J ¼ 6.2)
5.21 (brt, J ¼ 6.2)
e
1.82, 1.71 (2s)
e
e
e
e
e
e
e
e
e
Abbreviations: singlet (s), doublet (d), triplet (t), broad triplet (brt), quadruplet (q), multiplet (m).
Assignments of similar methylic protons of the prenyl or geranyl substituents can be interchangeable within the same column.
Assignments were confirmed by HSQC and HMBC experiments.
a
Values in parts per million (dH). Measured in CDCl3 at 300.13 MHz or 500.13 MHz. J values (Hz) are presented in parentheses.

M.P. Neves et al. / European Journal of Medicinal Chemistry 46 (2011) 2562e2574
2567
Table 3
13C NMR data of prenylated derivatives 3e10a of baicalein (1).
3
4
5
6
7
8
9
10
C-2
C-3
C-4
C-4a
C-5
C-6
C-7
C-8
C-8a
C-10
C-20,60
C-30,50
C-40
C-100
C-200
C-300
C-400
C-500
C-600
C-700
C-800
C-900
C-1000
C-2000
C-3000
C-4000
C-5000
C-6000
C-7000
C-8000
C-9000
C-10000
C-20000
C-30000
C-40000
C-50000
C-60000
C-70000
C-80000
C-90000
164.0
105.4
182.7
106.0
145.6
129.8
152.1
91.3
150.6
131.5
126.2
129.1
131.7
66.2
118.3
139.8
25.8, 18.3
e
163.8
105.6
182.7
106.2
153.5
131.6
158.7
91.5
153.2
131.4
126.2
129.1
131.7
66.0
118.8
138.7
25.8, 18.3
e
164.1
105.2
183.5
107.9
152.3
135.8
156.9
113.7
150.3
131.8
126.3
129.1
131.7
70.6
120.4
138.4
25.8, 18.0
e
164.0
105.4
182.7
106.0
145.6
129.8
152.1
91.4
150.6
131.5
126.2
129.1
131.7
66.3
118.1
142.8
16.8
39.5
26.2
123.5
132.0
25.7, 17.7
e
163.8
105.6
182.7
106.0
153.5
131.9
158.7
91.5
153.2
131.4
126.2
129.1
131.7
66.1
118.6
141.8
16.8
39.6
26.4
123.6
131.6
25.7, 17.7
69.1
120.0
142.0
16.4
39.5
26.2
124.0
131.5
25.7, 17.7
e
164.1
105.2
183.5
107.9
152.3
135.5
157.0
113.8
150.3
131.4
126.3
129.1
131.8
70.6
120.1
141.7
16.5
39.6
26.4
124.0
131.7
25.6, 17.7
69.5
120.1
141.7
16.5
163.6
105.3
182.6
105.6
146.1
129.1
151.9
113.9
147.0
131.7
126.1
129.2
131.8
91.5
14.1
44.4
25.9, 21.8
e
163.3
105.2
182.8
105.4
143.6
126.2
147.8
100.1
147.7
131.7
126.1
129.1
131.6
16.3
31.7
76.6
26.6
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
69.2
120.5
138.6
25.5, 17.9
e
69.5
120.4
138.5
25.8, 17.1
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
39.6
26.4
e
e
e
e
e
e
e
e
e
e
e
e
124.0
131.6
25.6, 16.5
22.7
122.6
135.6
16.5
39.6
26.4
124.0
135.8
25.6, 17.7
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
22.7
122.8
131.7
25.7, 18.0
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
Assignments of similar carbon atoms of the prenyl or geranyl substituents can be interchangeable within the same column.
Assignments were confirmed by HSQC and HMBC experiments.
a
Values in parts per million (dC). Measured in CDCl3 at 75.47 MHz or 125.77 MHz.
results (GI50 ¼ 4.1e5.3
m
M) in the three human tumor cell lines
growth inhibitory activity when compared with the corre-
sponding building block (2). Furthermore, the monoprenylated
derivative 11 exhibited a growth inhibitory activity similar to
3,7-dihydroxyflavone (2) toward the three human tumor cell
lines. Considering the effects of compounds 11 and 13, the
presence of a geranyl instead of a prenyl side chain was favorable
in this case. On the contrary, the existence of a second prenyl or
geranyl group on compounds 12 and 14 respectively, was asso-
studied. In fact, by comparing the structures 1 and 6 it can be
concluded that the presence of a geranyl group on position 7 of the
flavone scaffold was associated with a remarkable increase in the
growth inhibitory effect, suggesting the importance of this molec-
ular modification for the activity. Furthermore, the monopreny-
lated derivative 3 also made evident the positive influence of these
isoprenoid side chains regarding the growth inhibitory effect.
However, this positive influence was mainly observed on two of the
three human tumor cell lines studied (MCF-7 and NCI-H460).
Actually, the results obtained for the melanoma cell line (A375-C5)
were quite similar from those obtained for baicalein (1), which
might suggest the importance of the hydroxyl groups on positions 6
and 7 for the molecular mechanism of action for this tumor cell line.
On the other hand, the presence of more than one prenyl or geranyl
group on the baicalein (1) scaffold (compounds 4, 5, 7 and 8) was
associated with a complete loss of activity up to the concentrations
ciated with lower activity (GI50 > 50
mM) than with 3,7-dihy-
droxyflavone (2). These results prompted us to conclude that
prenylation at position C-3 had a negative influence on the
growth inhibitory activity. The results for the cyclic derivatives
15 and 16 revealed a pronounced growth inhibitory effect in the
three human tumor cell lines, with GI50 values ranging from 3.9
to 7.6 mM and 2.3e3.7 mM, respectively. In this case the cycliza-
tion of the isoprenoid side chain seemed to be favorable for the
studied effect.
tested (GI50 > 150
mM). Concerning the cyclic derivatives 9 and 10
The most active compounds (GI50 < 12 mM) were selected to
the results revealed that cyclization was not favorable for the
studied effect, since compounds 9 and 10 were as potent as bai-
calein (1).
Regarding the prenylated derivatives (11e16) of 3,7-
dihydroxyflavone (2), the presence of a geranyl side chain on
position 7 of compound 13 was related to an improvement in the
study their effect on cell cycle profile and apoptosis. To achieve this
purpose, the NCI-H460 cell line, the most sensitive to the tested
compounds, was used. Flow cytometry analysis of cell cycle
distribution and apoptosis were carried out, following treatment of
NCI-H460 cells for 48 h with the corresponding GI50 concentrations
of compounds 3, 6, 11, 13, 15 and 16.

2568
M.P. Neves et al. / European Journal of Medicinal Chemistry 46 (2011) 2562e2574
Table 4
1H NMR and 13C NMR data of prenylated derivatives 11e16a of 3,7-dihydroxyflavone (2).
11
12
13
14
15
16
H-3
e
e
e
e
e
e
H-5
H-6
H-8
H-20,60
H-30,50
H-40
H-100
H-200
H-400
H-500
H-600
H-700
H-900
H-1000
H-2000
H-4000
H-5000
H-6000
H-7000
H-9000
8.13 (d, J ¼ 8.7)
8.15 (d, J ¼ 8.9)
8.14 (d, J ¼ 8.9)
8.16 (d, J ¼ 8.9)
6.98 (dd, J ¼ 8.9, 2.3)
6.92 (d, J ¼ 2.3)
8.12e8.09 (m)
7.51e7.46 (m)
7.51e7.46 (m)
4.64 (d, J ¼ 7.0)
5.51 (brt, J ¼ 7.0)
1.77 (s)
2.15e2.12 (m)
2.15e2.12 (m)
5.10e5.07 (m)
1.67, 1.60 (2s)
4.64 (d, J ¼ 7.0)
5.37 (brt, J ¼ 7.0)
1.58 (s)
8.09 (d, J ¼ 8.6)
7.99 (d, J ¼ 8.9)
7.00 (dd, J ¼ 8.7, 2.0)
6.98 (dd, J ¼ 8.9, 2.3)
7.01 (dd, J ¼ 8.9, 2.2)
6.89 (d, J ¼ 8.6)
6.87 (d, J ¼ 8.9)
6.97 (d, J ¼ 2.0)
6.92 (d, J ¼ 2.3)
6.97 (d, J ¼ 2.2)
e
e
8.25e8.22 (m)
8.12e8.08 (m)
8.25e8.22 (m)
8.26e8.18 (m)
7.60e7.45 (m)
7.60e7.45 (m)
8.26e8.22 (m)
7.56e7.42 (m)
7.56e7.42 (m)
7.56e7.46 (m)
7.56e7.46 (m)
7.52e7.48 (m)
7.52e7.48 (m)
7.59e7.43 (m)
7.59e7.43 (m)
4.64 (d, J ¼ 6.7)
4.62 (d, J ¼ 6.7)
4.67 (d, J ¼ 6.5)
4.61 (q, J ¼ 6.6)
3.05 (t, J ¼ 6.8)
5.53 (brt, J ¼ 6.7)
5.52 (brt, J ¼ 6.7)
5.52 (brt, J ¼ 6.5)
1.47 (d, J ¼ 6.6)
1.94 (t, J ¼ 6.8)
1.84, 1.79 (2s)
1.83, 1.78 (2s)
1.79 (s)
1.67, 1.39 (2s)
1.41 (s)
e
e
e
e
e
e
e
e
e
e
e
e
2.17e2.10 (m)
2.17e2.10 (m)
5.10e5.02 (m)
1.67, 1.61 (2s)
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
4.59 (d, J ¼ 7.4)
5.38 (brt, J ¼ 7.4)
1.66, 1.60 (2s)
e
e
e
e
1.96e1.94 (m)
1.96e1.94 (m)
5.05e5.00 (m)
1.64, 1.56 (2s)
C-2
C-3
C-4
C-4a
C-5
C-6
C-7
C-8
C-8a
C-10
C-20,60
C-30,50
C-40
C-100
C-200
C-300
C-400
C-500
C-600
C-700
C-800
C-900
C-1000
C-2000
C-3000
C-4000
C-5000
C-6000
C-7000
C-8000
C-9000
144.1
138.1
172.8
114.5
126.7
115.4
163.6
100.5
157.3
131.2
127.5
128.6
129.9
65.5
118.4
139.5
25.8, 18.3
e
155.7
139.9
174.8
118.0
127.1
114.8
163.2
100.6
157.0
131.4
128.6
128.3
130.3
65.4
118.5
139.4
25.8, 18.3
e
144.2
138.1
172.8
114.5
126.7
115.4
163.6
100.6
157.3
131.2
127.5
128.6
129.9
65.6
118.2
142.5
16.8
39.5
27.1
123.6
132.0
26.2, 17.7
e
155.5
139.9
174.8
117.9
127.0
114.9
163.2
100.6
157.0
131.4
128.6
128.3
130.3
65.5
118.4
142.5
16.7
39.5
26.2
123.5
132.0
25.6, 17.7
68.7
119.5
142.4
16.4
143.7
137.8
173.0
115.4
127.0
115.3
163.6
118.4
153.2
131.5
127.3
128.6
129.8
90.4
14.2
44.3
25.9, 21.5
e
143.4
138.1
173.0
113.7
124.0
116.2
158.7
108.8
154.7
131.6
127.3
128.6
129.7
16.8
31.5
75.8
26.6
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
68.7
119.9
139.3
25.7, 17.9
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
39.5
26.2
123.9
131.6
25.6, 17.7
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
Abbreviations: singlet (s), doublet (d), triplet (t), broad triplet (brt), quadruplet (q), multiplet (m), double of doublets (dd).
Assignments for similar methylic protons and carbon atoms of the prenyl or geranyl substituents can be interchangeable within the same column. Assignments were
confirmed by HSQC and HMBC experiments.
a
Values in parts per million (dH and dC). Measured in CDCl3 at 300.13 MHz or 500.13 MHz and at 75.47 MHz or 125.77 MHz. J values (Hz) are presented in parentheses.
Regarding cell cycle analysis in the NCI-H460 cells, results
indicate that all the compounds studied, but particularly
compounds 3, 6 and 11, increased the percentage of cells in the G1
phase of the cell cycle (i.e. caused a G1 cell cycle arrest), with
a concomitant decrease in the percentage of cells in the S or/and
G2/M phases of the cell cycle (Fig. 5). The prenylated and gerany-
lated derivatives (3 and 6, respectively) of baicalein (1) caused the
highest increases in the % of cells in G1, which were greater than
10%.
which significantly increased the levels of apoptosis, increasing
from 8.4% (DMSO control) to 18.8% following 48 h treatment of the
cells with the GI50 concentration (4.1
(Table 6).
mM) of this compound
5. Conclusions
Ten acyclic prenylated flavonoids (3e8 and 11e14) were
synthesized using two different methodologies of prenylation:
a classic methodology, based on conventional heating and another,
using microwave-assisted organic synthesis (MAOS). Both strate-
gies afforded reasonable yields, with the monoprenylated
When analysing the capacity of the tested compounds (3, 6, 11,
13, 15 and 16) to induce apoptosis in the NCI-H460 cell line, results
indicated that the baicalein derivative (6) was the only compound

M.P. Neves et al. / European Journal of Medicinal Chemistry 46 (2011) 2562e2574
2569
Fig. 5. Cell cycle analysis of NCI-H460 cells treated with the most active derivatives
of baicalein (1) and 3,7-dihydroxyflavone (2). Cells were cultured with the GI50
concentration (as indicated in the graph) of the tested compounds (3, 6, 11, 13, 15
and 16) for 48 h. Untreated cells were used as control (blank). Appropriate DMSO
controls were also included (control 1 was the control for compound 11 and control
2 was the control for all the other comounds tested). Cell cycle analysis was possible
by flow cytometry following propidium iodide staining. Results represent the
mean ꢁ SEM of three independent experiments. * Represents p < 0.05 and **
represents p < 0.01 when comparing the treatment of each compound with the
blank.
Fig. 4. View of the crystal structure of compound
scheme. Displacement ellipsoids are drawn at the 50% probability level. Hydrogen
atoms are represented by circles of arbitrary size.
3 showing the atom-labeling
derivatives (3, 6, 11 and 13) being the major products. However,
MAOS afforded higher yields and selectivity in shorter reaction
times, when compared to conventional heating.
MW irradiation was also applied on the synthesis of dihy-
dropyran (10) and dihydrofuran (9, 15) derivatives from the
monoprenylated precursors 3 and 11, as well as on the one-pot
synthesis of dihydropyran derivatives (10 and 16).
The results here presented, allowed us to conclude that the
suitable prenylation of flavone scaffolds may result in interesting
derivatives with potent growth inhibitory activity, which deserve
further studies, namely regarding their molecular mechanism of
action and cellular toxicity, in order to investigate their potential as
antitumor agents.
The in vitro growth inhibitory effect of the synthesized
compounds was evaluated using three human tumor cell lines:
MCF-7, NCI-H460 and A375-C5. It is possible to conclude that the
monogeranylation of baicalein (1) and 3,7-dihydroxyflavone (2)
was associated with a remarkable improvement in the growth
inhibitory effect of the compounds. The growth inhibitory effect of
the pyran and furan derivatives of 3,7-dihydroxyflavone (2) in the
three human tumor cell lines is also noteworthy. Using the NCI-
H460 cells it was possible to observe that the effect of the
monogeranylated derivative (6) of baicalein (1) on cellular growth
could be attributed, at least in part, to induction of apoptosis. In
addition, by analysing the cell cycle profile of these cells following
treatment with the most potent compounds, it was possible to
observe an increase in the percentage of cells in G1 phase (cell cycle
arrest in G1), with the isoprenylated derivatives 3, 6 and 11 causing
significant effects.
6. Experimental
6.1. Synthesis
MW reactions were performed using a glassware setup for
atmosphericepressure reactions and a 100 mL Teflon reactor
(internal reaction temperature measurements with a fiber-optic
probe sensor) and were carried out using an Ethos MicroSYNTH
1600 Microwave Labstation from Milestone. The reactions were
monitored by thin-layer chromatography (TLC). Purifications of
compounds were carried out by flash chromatography using
Macherey-Nagel silica gel 60 (0.04e0.063 mm), GraceResolvÒ silica
gel cartridges (5 g/25 mL) and preparative thin-layer
Table 5
Effect of flavonoids (1e2) and prenylated flavonoids (3e16) on the growth of three
human tumor cell lines. Determination of the GI50 concentration of each compound.
Table 6
Effects of most active derivatives of baicalein (1) and 3,7-dihydroxyflavone (2) on
Compound
GI50
(
mM)
apoptosis of NCI-H460 cells.
MCF-7
NCI-H460
A375-C5
Compound
Apoptotic cells (% of total cells)
1
2
3
4
5
6
7
8
32.8 ꢁ 2.2
17.4 ꢁ 2.0
9.1 ꢁ 0.6
>150
26.7 ꢁ 2.9
15.4 ꢁ 1.2
6.8 ꢁ 0.8
>150
7.7 ꢁ 0.5
23.0 ꢁ 1.0
9.2 ꢁ 0.5
>150
Blank
Control 1 (DMSO)
Control 2 (DMSO)
9.5 ꢁ 1.89
11.2 ꢁ 4.88
8.4 ꢁ 1.18
9.1
>150
>150
>150
3 (6.8
6 (4.1
m
m
M)
M)
4.4 ꢁ 0.1
4.1 ꢁ 0.1
5.3 ꢁ 0.4
18.8 ꢁ 0.82*
11.9 ꢁ 2.54
12.0 ꢁ 3.12
9.5 ꢁ 1.99
13.1 ꢁ 2.29
>150
>150
>150
11 (12.6 mM)
>150
>150
>150
13 (4.9
15 (3.9
16 (2.7
m
m
m
M)
M)
M)
9
43.7 ꢁ 4.5
21.0 ꢁ 1.7
14.0 ꢁ 0.6
63.3 ꢁ 3.9
6.4 ꢁ 0.5
66.5 ꢁ 10.5
4.5 ꢁ 0.3
3.7 ꢁ 0.6
40.6 ꢁ 4.3
17.7 ꢁ 1.5
12.6 ꢁ 0.8
51.9 ꢁ 3.9
4.9 ꢁ 0.6
55.0 ꢁ 5.0
3.9 ꢁ 0.1
2.7 ꢁ 0.8
47.7 ꢁ 2.9
27.4 ꢁ 1.2
30.3 ꢁ 6.2
59.0 ꢁ 4.7
7.2 ꢁ 1.0
58.5 ꢁ 7.5
7.6 ꢁ 0.5
2.3 ꢁ 0.5
10
11
12
13
14
15
16
NCI-H460 cells were cultured with the GI50 concentration of the tested
compounds (3, 6, 11, 13, 15 and 16) for 48 h. Untreated cells were used as control
(blank). Appropriate DMSO treatments were also included (control 1 and control
2). Determination of apoptotic cells was possible following Annexin-V-FITC/PI
staining and analysis in a flow cytometer. Results represent the mean ꢁ SEM of
three independent experiments, except for compound 3 for which only two
independent experiments were performed.
Results are presented as the concentrations that were able to cause 50% cell growth
inhibition (GI50) after a continuous exposure of 48 h and represent means ꢁ SEM
from at least three independent experiments performed in duplicate.
* Represents p < 0.05 when comparing the treatment of each compound with the
blank.

2570
M.P. Neves et al. / European Journal of Medicinal Chemistry 46 (2011) 2562e2574
chromatography (TLC) using Macherey-Nagel silica gel 60 (GF254
)
5-hydroxy-8-(3-methylbut-2-enyl)-6,7-bis(3-methylbut-2-
enyloxy)-2-phenyl-4H-chromen-4-one (5). 1% (classic) as yellow
gum; IR (KBr) ymax: 3600e3300, 2963, 2919, 2852, 1654, 1610, 1585,
1443, 1376, 1355, 1289, 1094; 1H NMR data, see Table 2; 13C NMR
data, see Table 3; ESI-MS (þ) m/z (%): 476 (16), 475 (47, [M þ H] þ),
407 (17), 359 (3), 340 (21), 339 (100), 284 (11), 283 (67), 271 (5);
ESI-HRMS (þ) m/z: Anal. calc. for C30H35O5 (M þ H)þ: 475.2479;
found: 475.2485.
plates. Melting points were obtained in a Köfler microscope and are
uncorrected. IR spectra were measured on an ATI Mattson Genesis
series FTIR (software: WinFirst, v. 2.10) spectrophotometer in KBr
microplates (cmꢂ1). 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
d (ppm) values relative to
tetramethylsilane (TMS) as an internal reference; 13C NMR assign-
ments were made by 2D (HSQC and HMBC) NMR experiments
(long-range C, H coupling constants were optimized to 7 Hz). HRMS
mass spectra were recorded as FAB (Fast Atom Bombardment) or
ESI (electrospray ionization) mode on VG Autospec M. spectrom-
eter, on microTOF(focus) mass spectrometer or on APEXQe FT-ICR
MS (Bruker Daltonics), equipped with a 7T actively shielded magnet
at C.A.C.T.I.-University of Vigo, Spain. Prenyl bromide, geranyl
bromide, Montmorillonite K10 clay, baicalein and 3,7-dihydroxy-
flavone were purchased from Sigma Aldrich. The following mate-
rials were synthesized and purified by the described procedures.
6.4. Geranylation of baicalein (1) by conventional heating
A mixture of baicalein (1) (0.20 g, 0.74 mmol), geranyl bromide
(282 mL, 1.48 mmol) and anhydrous K2CO3 (0.21 g, 1.48 mmol) in
anhydrous acetone (92 mL) was refluxed at 65 ꢀC for 8 h. After
cooling, the solid was filtered and the solvent removed under
reduced pressure to afford the crude product. The brown-orange oil
obtained was dissolved in acetone and purified by flash chroma-
tography (SiO2, n-hexane: EtOAc). Three compounds were isolated:
7-(3,7-dimethylocta-2,6-dienyloxy)-5,6-dihydroxy-2-phenyl-4H-
chromen-4-one (6), 6,7-bis(3,7-dimethylocta-2,6-dienyloxy)-5-
hydroxy-2-phenyl-4H-chromen-4-one (7) and 8-(3,7-dimethy-
locta-2,6-dienyl)-6,7-bis(3,7-dimethylocta-2,6-dienyloxy)-5-
hydroxy-2-phenyl-4H-chromen-4-one (8).
6.2. Prenylation of baicalein (1) by conventional heating
A mixture of baicalein (1) (0.50 g, 1.85 mmol), prenyl bromide
(260 mL, 2.22 mmol) and anhydrous K2CO3 (0.51 g, 3.70 mmol) in
anhydrous acetone (70 mL) was refluxed at 65 ꢀC for 8 h. After
cooling, the solid was filtered and the solvent removed under
reduced pressure to afford the crude product. The brown-orange oil
obtained was dissolved in acetone and purified by flash chroma-
tography (SiO2; n-hexane: EtOAc). Three compounds were isolated:
5,6-dihydroxy-7-(3-methylbut-2-enyloxy)-2-phenyl-4H-chromen-
4-one (3), 5-hydroxy-6,7-bis(3-methylbut-2-enyloxy)-2-phenyl-
4H-chromen-4-one (4) and 5-hydroxy-8-(3-methylbut-2-enyl)-
6,7-bis(3-methylbut-2-enyloxy)-2-phenyl-4H-chromen-4-one (5).
6.5. Geranylation of baicalein (1) under MW irradiation
A mixture of baicalein (1) (0.20 g, 0.74 mmol), geranyl bromide
(452 mL, 2.28 mmol) and anhydrous K2CO3 (0.21 g, 1.48 mmol) in
anhydrous acetone (50 mL) was subjected to successive 20 min of
microwaveirradiationat200 Wofpotency. Totalirradiationtimewas
2 h and the final temperature was 61 ꢀC. After cooling, the solid was
filtered and the solvent removed under reduced pressure to afford
the crude product. The brown-orange oil obtained was dissolved in
acetone and purified by flash chromatography (SiO2, n-hexane:
EtOAc). Two compounds were isolated: 7-(3,7-dimethylocta-2,6-
dienyloxy)-5,6-dihydroxy-2-phenyl-4H-chromen-4-one (6) and 6,
7-bis(3,7-dimethylocta-2,6-dienyloxy)-5-hydroxy-2-phenyl-4H-chr
-omen-4-one (7).
6.3. Prenylation of baicalein (1) under MW irradiation
A mixture of baicalein (1) (0.37 g, 1.37 mmol), prenyl bromide
(210 mL, 1.82 mmol) and anhydrous K2CO3 (0.41 g, 2.74 mmol) in
anhydrous acetone (60 mL) was submitted to successive 30 min of
microwave irradiation at 200 W of potency. Total irradiation time
was 2 h and the final temperature was 60 ꢀC. After cooling, the solid
was filtered and the solvent removed under reduced pressure to
afford the crude product. The yellow-orange oil obtained was
dissolved in acetone and purified by flash chromatography (SiO2;
n-hexane: EtOAc). Two compounds were isolated: 5,6-dihydroxy-
7-(3-methylbut-2-enyloxy)-2-phenyl-4H-chromen-4-one (3) and
5-hydroxy-6,7-bis(3-methylbut-2-enyloxy)-2-phenyl-4H-chromen
-4-one (4).
5,6-dihydroxy-7-(3-methylbut-2-enyloxy)-2-phenyl-4H-
chromen-4-one (3). 47% (classic) and 77% (MW) as yellow crystals;
mp (n-hexane: EtOAc) 162e165 ꢀC; IR (KBr) ymax:3400e3100, 2961,
2917, 2851, 1659, 1607, 1576, 1492, 1446, 1354, 1291, 1184, 1107;
1H NMR data, see Table 2; 13C NMR data, see Table 3; ESI-MS (þ) m/z
(%): 361 (37), 340 (12), 339 (52, [M þ H]þ), 292 (9), 272 (15),
271 (100); ESI-HRMS (þ) m/z: Anal. calc. for C20H19O5 (M þ H)þ:
339.1227; found: 339.1235.
5-hydroxy-6,7-bis(3-methylbut-2-enyloxy)-2-phenyl-4H-
chromen-4-one (4). 22% (classic) and 6% (MW) as yellow crystals;
mp (n-hexane: EtOAc) 102e103 ꢀC; IR (KBr) ymax: 3500e3300,
2962, 2920, 2851, 1656, 1614, 1586, 1490, 1454, 1356, 1294, 1189,
1114; 1H NMR data, see Table 2; 13C NMR data, see Table 3; ESI-MS
(þ) m/z (%): 408 (17), 407 (67, [M þ H]þ), 360 (5), 340 (7), 339 (33),
272 (14), 271 (100); ESI-HRMS (þ) m/z: Anal. calc. for C25H27O5
(M þ H)þ: 407.1853; found: 407.1863.
7-(3,7-dimethylocta-2,6-dienyloxy)-5,6-dihydroxy-2-phenyl-
4H-chromen-4-one (6). 29% (classic) and 44% (MW) as yellow
solid; mp (n-hexane: EtOAc) 125e128 ꢀC; IR (KBr) ymax
:
3600e3100, 2957, 2913, 2849, 1655, 1604, 1572, 1454, 1353, 1348,
1296, 1181, 1098; 1H NMR data, see Table 2; 13C NMR data, see
Table 3; FAB-MS (þ) m/z (%): 408 (16), 407 (58, [M þ H]þ), 406 (18),
272 (17), 271 (100), 270 (97), 269 (14), 154 (8); FAB-HRMS (þ) m/z:
Anal. calc. for C25H27O5 (M þ H)þ: 407.1858; found: 407.1862.
6,7-bis(3,7-dimethylocta-2,6-dienyloxy)-5-hydroxy-2-
phenyl-4H-chromen-4-one (7). 27% (classic) and 27% (MW) as
yellow solid; mp (n-hexane: EtOAc) 76e78 ꢀC; IR (KBr) ymax
:
3600e3300, 2967, 2909, 2878, 1657, 1610, 1583, 1490, 1451, 1352,
1297, 1192, 1112; 1H NMR data, see Table 2; 13C NMR data, see
Table 3; FAB-MS (þ) m/z (%): 544 (6), 543 (15, [M þ H]þ), 407
(14), 406 (16), 272 (17), 271 (100), 270 (97), 269 (24); FAB-HRMS
(þ) m/z: Anal. calc. for C35H43O5 (M þ H)þ: 543.3110; found:
543.3105.
8-(3,7-dimethylocta-2,6-dienyl)-6,7-bis(3,7-dimethylocta-
2,6-dienyloxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (8). 2%
(classic) as yellow gum; IR (KBr) ymax: 3600e3200, 2959, 2921,
2852, 1652, 1612, 1447, 1378, 1358, 1284, 1161, 1101; 1H NMR data,
see Table 2; 13C NMR data, see Table 3; FAB-MS (þ) m/z (%): 680 (6),
679 (12, [M þ H]þ), 542 (13), 541 (6), 408 (26), 407 (100), 406 (69),
405 (47), 338 (9), 337 (31), 321 (13), 284 (20), 283 (81), 281 (19),
271 (7). FAB-HRMS (þ) m/z: Anal. calc. for C45H59O5 (M þ H)þ:
679.4363; found: 679.4358.

M.P. Neves et al. / European Journal of Medicinal Chemistry 46 (2011) 2562e2574
2571
6.6. Prenylation of 3,7-dihydroxyflavone (2) by conventional
heating
Total irradiation time was 2 h and the final temperature was 58 ꢀC.
After cooling, the solid was filtered and the solvent removed under
reduced pressure to afford the crude product. The yellow oil
obtained was dissolved in acetone and purified by flash chroma-
tography (SiO2, petroleum ether: Et2O) affording compound 13,
a mixture of compounds 13,14 and starting material (2). Compound
14 was obtained after purification by preparative TLC (SiO2,
petroleum ether: EtOAc, 7:3).
7-(3,7-dimethylocta-2,6-dienyloxy)-3-hydroxy-2-phenyl-4H-
chromen-4-one (13). 42% (classic) and 50% (MW) as yellow solid;
mp (CHCl3) 102e105 ꢀC; IR (KBr) ymax: 3600e3300, 2949, 2915,
2846, 1727, 1608, 1494, 1450, 1403, 1370, 1322, 1253, 1177, 1121,
1068, 1013; 1H NMR data, see Table 4; 13C NMR data, see Table 4;
ESI-MS (þ) m/z (%): 413 (34), 391 (19, [M þ H]þ), 301 (6), 255 (100),
391 (25); ESI-HRMS (þ) m/z: Anal. calc. for C25H27O4 (M þ H)þ:
391.1904; found: 391.1906.
A mixture of 3,7-dihydroxyflavone (2) (0.50 g,1.97 mmol), prenyl
bromide (686
mL, 5.90 mmol) and anhydrous K2CO3 (0.82 g,
5.90 mmol) in anhydrous acetone (100 mL) was refluxed at 65 ꢀC for
8 h. After cooling, the solid was filtered and the solvent removed
under reduced pressure to afford the crude product. The yellow oil
obtained was dissolved in acetone and purified by flash chroma-
tography (SiO2, n-hexane: EtOAc) affording a mixture of compounds
11,12 and starting material (2). Compounds 11 and 12 were obtained
after purification by preparative TLC (SiO2, n-hexane: EtOAc, 8:2 and
toluene: EtOAc, 95:5, respectively).
6.7. Prenylation of 3,7-dihydroxyflavone (2) under MW irradiation
A mixture of 3,7-dihydroxyflavone (2) (0.10 g, 0.39 mmol), prenyl
3,7-bis(3,7-dimethylocta-2,6-dienyloxy)-2-phenyl-4H-chro-
men-4-one (14). 10% (classic) and 4% (MW) as yellow gum; IR (KBr)
ymax: 2951, 2908, 2840, 1608, 1484, 1433, 1379, 1239, 1171, 1126,
1095; 1H NMR data, see Table 4; 13C NMR data, see Table 4; ESI-MS
(þ) m/z (%): 527 (100, [M þ H]þ), 391 (25), 279 (7), 233 (10), 201
(30); ESI-HRMS (þ) m/z: Anal. calc. for C35H43O4 (M þ H)þ:
527.3156; found: 527.3143.
bromide (184
mL, 1.59 mmol) and anhydrous K2CO3 (0.11 g,
0.79 mmol) in anhydrous acetone (70 mL) was submitted to
successive 20 min of microwave irradiation at 200 W of potency.
Total irradiation time was 2 h and the final temperature was 60 ꢀC.
After cooling, the solid was filtered and the solvent removed under
reduced pressure to afford the crude product. The yellow oil
obtained was dissolved in acetone and purified by flash chroma-
tography (SiO2, n-hexane: EtOAc) affording a mixture of compounds
11,12 and starting material (2). Compounds 11 and 12 were obtained
after purification by preparative TLC (SiO2, n-hexane: EtOAc, 8:2 and
toluene: EtOAc, 95:5, respectively).
3-hydroxy-7-(3-methylbut-2-enyloxy)-2-phenyl-4H-chro-
men-4-one (11). 35% (classic) and 44% (MW) as yellow solid; mp
(CHCl3) 166e169 ꢀC; IR (KBr) ymax: 3400e3100, 2966, 2925, 2867,
1603, 1563, 1498, 1447, 1407, 1376, 1254, 1181, 1123; 1H NMR data,
see Table 4; 13C NMR data, see Table 4; ESI-MS (þ) m/z (%): 345
(100), 323 (79, [M þ H]þ), 301 (10), 291 (25), 275 (20), 255 (14), 245
(11), 215 (4). ESI-HRMS (þ) m/z: Anal. calc. for C20H19O4 (M þ H)þ:
323.1278; found: 323.1277.
6.10. General procedure for Claisen rearrangement of
monoprenylated flavones 3 and 11 with NMP under MW irradiation
A solution of monoprenylated flavone 3 or 11 (0.10 g), in NMP
(15 mL), was transferred to a two-necked glassware apparatus,
provided with magnetic stirring bar, fiber-optic temperature control
and reflux condenser and heated for 3 ꢃ 15 min according to the
following microwave program: step 1: 5 min, ramp to 204 ꢀC, 800 W
maximum power; step 2: 10 min, hold at 204 ꢀC, 400 W maximum
power. After completion of the reaction, the mixture was poured
onto crushed ice, acidified (10% HCl) and extracted successively
with, Et2O (3 ꢃ100 mL), CH2Cl2 (3 ꢃ100 mL) and EtOAc (3 ꢃ100 mL).
The organic layers were washed with water (2 ꢃ 100 mL) and dried
with anhydrous sodium sulfate. The crude products were purified by
flash column chromatography cartridges (SiO2; CHCl3: Me2CO). One
compound was isolated from each building block (3 and 11):
5,6-dihydroxy-8,9,9-trimethyl-2-phenyl-8,9-dihydro-4H-furo[2,3-
h]chromen-4-one (9) and 3-hydroxy-8,9,9-trimethyl-2-phenyl-8,9-
dihydro-4H-furo[2,3-h]chromen-4-one (15), respectively.
3-hydroxy-7-(3-methylbut-2-enyloxy)-2-phenyl-4H-chro-
men-4-one (12). 2% (classic) and 18% (MW) as yellow gum; IR (KBr)
ymax: 2959, 2917, 2851, 1613, 1494, 1442, 1386, 1249, 1179, 1103;
1H NMR data, see Table 4; 13C NMR data, see Table 4; ESI-MS (þ) m/z
(%): 391 (100, [M þ H]þ), 361 (11), 345 (15), 323 (82), 255 (7); ESI-
HRMS (þ) m/z: Anal. calc. for C25H27O4 (M þ H)þ: 391.1904; found:
391.1904.
5,6-dihydroxy-8,9,9-trimethyl-2-phenyl-8,9-dihydro-4H-
furo[2,3-h]chromen-4-one (9). 48% as yellow crystals; mp (n-
hexane: EtOAc) 260e264 ꢀC; IR (KBr) ymax: 3400e3100, 2961, 2917,
2851, 1659, 1607, 1576, 1492, 1446, 1354, 1291, 1184, 1107; 1H NMR
data, see Table 2; 13C NMR data, see Table 3; ESI-MS (þ) m/z (%): 340
(17), 339 (100, [M þ H]þ), 183 (8); ESI-HRMS (þ) m/z: Anal. calc. for
C20H19O5 (M þ H)þ: 339.1227; found: 339.1224.
6.8. Geranylation of 3,7-dihydroxyflavone (2) by conventional
heating
A mixture of 3,7-dihydroxyflavone (2) (0.48 g, 1.97 mmol),
geranyl bromide (450 mL, 2.36 mmol) and anhydrous K2CO3 (0.55 g,
3.93 mmol) in anhydrous acetone (50 mL) was refluxed at 65 ꢀC for
8 h. After cooling, the solid was filtered and the solvent removed
under reduced pressure to afford the crude product. The yellow oil
obtained was dissolved in acetone and purified by flash chroma-
tography (SiO2, petroleum ether: Et2O) affording compound 13,
a mixture of compounds 13,14 and starting material (2). Compound
14 was obtained after purification by preparative TLC (SiO2,
petroleum ether: EtOAc, 7:3).
3-hydroxy-8,9,9-trimethyl-2-phenyl-8,9-dihydro-4H-furo
[2,3-h]chromen-4-one (15). 36% as yellow solid; mp (n-hexane:
EtOAc) 145e148 ꢀC; IR (KBr) ymax: 3400e3100, 2966, 2925, 2867,
1603, 1563, 1498, 1447, 1407, 1376, 1254, 1181, 1123; 1H NMR data,
see Table 4; 13C NMR data, see Table 4; ESI-MS (þ) m/z (%): 323
(100, [M þ H]þ), 199 (8). ESI-HRMS (þ) m/z: Anal. calc. for C20H19O4
(M þ H)þ: 323.1278; found: 323.1275.
6.9. Geranylation of 3,7-dihydroxyflavone (2) under MW
irradiation
6.11. General procedure for rearrangement of monoprenylated
flavones 3 and 11 with Montmorillonite K10 clay under MW
irradiation
A mixture of 3,7-dihydroxyflavone (2) (0.50 g, 1.97 mmol),
geranyl bromide (562
3.93 mmol) in anhydrous acetone (60 mL) was subjected to
successive 20 min of microwave irradiation at 200 W of potency.
m
L, 2.83 mmol) and anhydrous K2CO3 (0.56 g,
A slurry of the K10 clay (2.0 g, 20 equiv by weight) in CHCl3 (ca.
20 mL) was treated with the monoprenylated flavones 3 or 11
(0.10 g) in a 100 mL closed microwave reactor. The mixture under

2572
M.P. Neves et al. / European Journal of Medicinal Chemistry 46 (2011) 2562e2574
stirring was irradiated at 200 W for 3 ꢃ 15 min and final tempera-
tures range were 60e65 ꢀC. The reaction mixture was filtered under
vacuum, washed with CHCl3, Me2CO and MeOH and the filtrate
concentrated under vacuum. The recovered clay was reactivated by
washing with MeOH. The crude product was purified by flash
chromatography cartridges (SiO2; n-hexane: EtOAc). One compound
was isolated: 5,6-dihydroxy-8,8-dimethyl-2-phenyl-9,10-dihy-
dropyrano[2,3-f]chromen-4(8H)-one (10) from the building block 3.
5,6-dihydroxy-8,8-dimethyl-2-phenyl-9,10-dihydropyrano
[2,3-f]chromen-4(8H)-one (10). 34% as yellow crystals; mp
(n-hexane: EtOAc) 213e216 ꢀC; IR (KBr) ymax: 3525e3360, 2971,
2930, 2853, 1666, 1621, 1583, 1492, 1449, 1370, 1306, 1255, 1230,
1158, 1108; 1H NMR data, see Table 2; 13C NMR data, see Table 3;
ESI-MS (þ) m/z (%): 340 (16), 339 (100, [M þ H]þ), 337 (3), 183 (8);
ESI-HRMS (þ) m/z: Anal. calc. for C20H19O5 (M þ H)þ: 339.1227;
found: 339.1225.
6.14. Biological activity
6.14.1. Reagents and stock solutions of compounds
Fetal bovine serum (FBS), phosphate buffer saline (PBS), and
trypsin were purchased from Gibco Invitrogen Co. (Scotland, UK).
RPMI-1640 medium was purchased from Lonza (Basel, Switzerland).
Dimethylsulfoxide (DMSO), doxorubicin, ethylenediaminetetracetic
acid (EDTA), propidium iodide (PI), RNase, sulforhodamine B (SRB)
and trypan blue were purchased from SigmaChemical Co. (Saint
Louis, MO, USA). Trichloroacetic acid (TCA) and Tris were purchased
from Merck (Darmstadt, Germany). Stock solutions of compounds
1e16 were dissolved in DMSO and stored at ꢂ20 ꢀC. Appropriate
dilutions of the compounds were freshly prepared with culture
medium prior to the assays. Final concentrations of DMSO did not
interfere with cell growth (data not shown).
6.14.2. Cell culture
6.12. General procedure for the one-pot synthesis of
dihydropyranoflavones 15 and 16 with Montmorillonite K10 clay
under MW irradiation
Three human tumor cell lines were used: MCF-7 (breast
adenocarcinoma, ECACC, UK), NCI-H460 (non-small cell lung
cancer, a kind gift from NCI, Bethesda, USA) and A375-C5 (mela-
noma, ECACC, UK). The human tumor cell lines were grown as
monolayer and routinely maintained in RPMI-1640 medium, sup-
plemented with 5% heat-inactivated fetal bovine serum (FBS) at
37 ꢀC in a humidified atmosphere containing 5% CO2.
A slurry of K10 clay (6.0 g, 20 equiv by weight) in CHCl3 (ca.
20 mL) was treated with baicalein (1) and 3,7-dihydroxyflavone (2)
(0.30 g), followed by the addition of prenyl bromide (2 equiv) in
a 100 mL closed microwave reactor. The mixture under stirring was
irradiated at 200 W for 3 ꢃ 15 min and final temperature ranges
were 105e115 ꢀC. The reaction mixture was filtered under vacuum,
washed with CHCl3, Me2CO and MeOH and the filtrate concentrated
under vacuum. The recovered clay was reactivated by washing with
MeOH. The crude product was purified by flash chromatography
(SiO2; n-hexane: EtOAc). One compound was isolated from each
building block (1 and 2): 5,6-dihydroxy-8,8-dimethyl-2-phenyl-
9,10-dihydropyrano[2,3-f]chromen-4(8H)-one (10) and 3-hydroxy-
8,8-dimethyl-2-phenyl-9,10-dihydropyrano[2,3-f]chromen-4(8H)-
one (16), respectively.
3-hydroxy-8,8-dimethyl-2-phenyl-9,10-dihydropyrano[2,3-f]
chromen-4(8H)-one (16). 20% as yellow crystals; mp (n-hexane:
EtOAc) 217e221 ꢀC; IR (KBr) ymax: 3238, 2971, 2932, 2851, 1605,
1571, 1498, 1445, 1410, 1382, 1334, 1266, 1216, 1172, 1118; 1H NMR
data, see Table 4; 13C NMR data, see Table 4; ESI-MS (þ) m/z (%): 346
(19), 345 (100, [M þ Na]þ). ESI-HRMS (þ) m/z: Anal. calc. for
C20H18NaO4 (M þ Na)þ: 345.1097; found: 345.1095.
6.14.3. Cell growth inhibition assay
Cells were plated in 96-well plates at appropriate densities to
ensure exponential growth throughout the experimental period
(5.0 ꢃ 104 cells/mL for MCF-7 and NCI-H460 and 7.5 ꢃ 104 cells/mL
for A375-C5) before being allowed to attach overnight. Attached
cells were exposed for 48 h to five serial dilutions of each
compound (150; 75; 37.5; 18.75 and 9.37
7e9, 12 and 14 or 150; 50; 16.7; 5.56 and 1.9
1e3, 10 and 11 or 50; 16.7; 5.56; 1.9 and 0.62
m
M for compounds 4, 5,
M for compounds
M for compounds 6,
m
m
13, 15 and 16). Following this incubation period, adherent cells
were fixed in situ with trichloroacetic acid, before being washed
and stained with SRB [27,33]. The bound stain was solubilised and
absorbance was measured at 492 nm in a plate reader (Biotek
Instruments Inc. PowerWave XS, Winooski, USA). The effect of the
vehicle solvent (DMSO) on the growth of these cell lines was
evaluated in a preliminary experiment by exposing untreated
control cells to the maximum concentration of DMSO used in each
assay (0.25%).
6.13. X-ray crystallography
6.14.4. Cell cycle analysis
Suitable crystals were obtained by slow evaporation of a n-
hexane: EtOAc solution. They were monoclinic, space group P21/c,
cell volume V ¼ 1637.88(16) Å3, a ¼ 6.0010(3) Å, b ¼ 15.5301(9) Å,
NCI-H460 cells were plated in 6-well plates (5.0 ꢃ 104 cells/mL)
and incubated at 37 ꢀC for 24 h. Exponentially growing cells were
then incubated with complete medium (blank) or with the
compounds (3, 6, 11, 13, 15 or 16) at their respective GI50 concen-
trations (previously determined with the SRB assay, data from
Table 5). Appropriate controls were also tested. However, to avoid
having an enormous number of DMSO controls, we tested only two
control concentrations: one corresponding to the highest concen-
c ¼ 17.5785(10) Å and
b
¼ 91.215(5)ꢀ (uncertainties in parentheses).
There were four molecules per unit cell and the calculated density
was 1.372 g/cm3. Diffraction data was collected at 293 K with
a Gemini PX Ultra equipped with MoKa radiation (
A total of 3812 independent reflections were measured, of which
2545 were observed (I > 2 (I)). The structure was solved by direct
l
¼ 0.71073 Å).
s
tration used for the compounds that had GI50 below 10
2) and another one corresponding to the highest concentration
used for the compounds that had GI50 above 10 M (control 1).
Therefore, compound 11 which had a GI50 greater than 10 M had
an appropriate DMSO control (control 1). All other compounds
mM (control
methods using SHELXS-97 [31] with atomic positions and displace-
ment parameters refined with SHELXL-97 [32]. The non-hydrogen
atoms were refined anisotropically and the hydrogen atoms were
refined freely with isotropic displacement parameters. The refine-
ment converged to R(all data) ¼ 8.10% and wR2(all data) ¼ 14.28%.
Crystallographic data (excluding structure factors) forcompound
3 were deposited with the Cambridge Crystallographic Data Centre
as supplementary publication No CCDC 742276. Copies of the data
can be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK, (fax: þ44-(0)1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk).
m
m
which had GI50 smaller than 10
(control 2).
mM had a different DMSO control
Following 48 h treatment, cells were pelleted and fixed in 70%
ethanol at 4 ꢀC for at least 12 h and subsequently ressuspended in
PBS containing 0.1 mg/mL RNase A and 40 mg/mL propidium iodide.
Cellular DNA content (for cell cycle distribution analysis) was
measured by flow cytometry using an Epics XL-MCL Coulter flow

M.P. Neves et al. / European Journal of Medicinal Chemistry 46 (2011) 2562e2574
2573
microtubules and interferes with DNA replication in MCF-7 human breast
cancer cells, Life Sci. 77 (2005) 293e311.
cytometer (Brea, CA, USA) by plotting at least 20 000 events per
sample, as previously described [34e36]. The percentage of cells in
the G1, S and G2/M phases of the cell cycle was determined using
the FlowJo 7.2 software (Tree Star, Inc., Ashland, OR, USA) after cell
debris exclusion.
[5] F.V. So, N. Guthrie, A.F. Chambers, K.K. Carroll, Inhibition of proliferation of
estrogen receptor-positive MCF-7 human breast cancer cells by flavonoids in
the presence and absence of excess estrogen, Cancer Lett. 112 (1997) 127e133.
[6] L.S. Po, Z.-y. Chen, D.S.C. Tsang, L.K. Leung, Baicalein and genistein display
differential actions on estrogen receptor (ER) transactivation and apoptosis in
MCF-7 cells, Cancer Lett. 187 (2002) 33e40.
[7] A. Monasterio, M.C. Urdaci, I.V. Pinchuk, N. López-Moratalla, J.J. Martínez-Irujo,
Flavonoids induce apoptosis in human leukemia U937 cells through caspase-
and caspase-calpainedependent pathways, Nutr. Cancer 50 (2004) 90e100.
[8] M. Li-Weber, New therapeutic aspects of flavones: the anticancer properties
of Scutellaria and its main active constituents Wogonin, Baicalein and Baicalin,
Cancer Treat. Rev. 35 (2009) 57e68.
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noids: pharmacology and biotechnology, Curr. Med. Chem. 12 (2005) 713e739.
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oxyprenylatedsecondaryplantmetabolites, Phytochemistry68(2007)939e953.
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enzymes as a tool for flavonoid prenylation, Trends Pharmacol. Sci. 26 (2005)
606e608.
[13] H. Cidade, M. Neves, A. Kijjoa, Natural prenylated flavones: chemistry and
biological activities e an overview. in: G. Brahmachari (Ed.), Natural Products:
Chemistry, Biochemistry and Pharmacology. Narosa Publishing House PVT.
Ltd, New Delhi, 2009, pp. 463e519.
6.14.5. Analysis of cellular apoptosis
NCI-H460 cells were plated in 6-well plates (5.0 ꢃ 104 cells/mL)
and incubated at 37 ꢀC for 24 h. Exponentially growing cells were
then incubated for 48 h with complete medium (blank) or with the
compounds (3, 6, 11, 13, 15 or 16) at their respective GI50 concen-
trations (previously determined with the SRB assay, data from
Table 5). Appropriate controls were also tested: compound 11 had
a GI50 greater than 10
1; all the other compounds had GI50 concentrations smaller than
10 M e therefore their DMSO control was control 2.
Cells were then harvested and the Human Annexin-V-FITC/PI
mM e therefore its DMSO control was control
m
apoptosis kit (Bender MedSystems, Vienna, Austria) was used
according to the manufacturer’s instructions to detect apoptotic
cells. Flow cytometry was carried out using an Epics XL-MCL
Coulter flow cytometer (Brea, CA, USA) plotting at least 20 000
events per sample. Data obtained from the flow cytometer was
analysed using the FlowJo 7.2 software (Tree Star, Inc., Ashland, OR,
USA), as previously described [36].
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noids on cell proliferation and caspase activation in a human colonic cell line
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(2005) 400e424.
6.14.6. Statistical analysis
[16] H. Sakagami, Y. Jiang, K. Kusama, T. Atsumi, T. Ueha, M. Toguchi, I. Iwakura,
K. Satoh, T. Fukai, T. Nomura, Induction of apoptosis by flavones, flavonols (3-
hydroxyflavones) and isoprenoid-substituted flavonoids in human oral tumor
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Artelastocarpin and carpelastofuran, two new flavones, and cytotoxicities of
prenyl flavonoids from Artocarpus elasticus against three cancer cell lines,
Planta Med. 67 (2001) 867e870.
[18] R.A.P. Castanheiro, M.M.M. Pinto, S.M.M. Cravo, D.C.G.A. Pinto, A.M.S. Silva,
A. Kijjoa, Improved methodologies for synthesis of prenylated xanthones by
microwave irradiation and combination of heterogeneous catalysis (K10 clay)
with microwave irradiation, Tetrahedron 65 (2009) 3848e3857.
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Angew. Chem. Int. Ed. 43 (2004) 6250e6284.
All experimental data are presented as means ꢁ SEM from at
least three independent experiments (most performed in dupli-
cate). Statistical analysis was carried out using an unpaired
student’s t-test. All analysis were performed by comparing treat-
ment with compounds with blank cells (cells treated with medium
only). * Indicates p < 0.05 and **p < 0.01.
Acknowledgments
The authors thank Fundação para a Ciência e a Tecnologia (FCT)
(I&D 4040/2007 and 62/1992), POCTI, POCI, FCT/COMPETE/FEDER
PTDC/SAU-FCF/100930/2008, PTDC/EQU-FTT/81496/2006, U. Porto,
and Santander Totta for financial support and for the PhD grants of
Marta Perro Neves and Raquel T. Lima (SFRH/BD/21770/2005, and
SFRH/BD/21759/2005, respectively). The authors thank Sara Cravo
for technical support in microwave equipment, Kanthima Choosang
for help in the biological assays and Jonathan Flor for participation
in some synthetic steps. The authors are also indebted to the
National Cancer Institute, Bethesda, MD, USA, for the generous gift
of the NCI-H460 cell line. IPATIMUP is an Associate Laboratory of
the Portuguese Ministry of Science, Technology and Higher
Education and is partially supported by FCT, the Portuguese Foun-
dation for Science and Technology.
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