Activity of novel quinoxaline-derived chalcones on in vitro glioma cell proliferation

Article abstract of DOI:10.1016/j.ejmech.2011.12.023

Gliomas are the most common and devastating tumors of the central nervous system (CNS). Many pieces of evidence point out the relevance of natural compounds for cancer therapy and prevention, including chalcones. In the present study, eight synthetic quin

Full text of DOI:10.1016/j.ejmech.2011.12.023

European Journal of Medicinal Chemistry 48 (2012) 255e264
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Activity of novel quinoxaline-derived chalcones on in vitro glioma cell
proliferation
,
,
Tânia R. Mielcke a b, Alessandra Mascarello c, Eduardo Filippi-Chiela d, Rafael F. Zanin b f, Guido Lenz d,
Paulo César Leal c, Louise D. Chirardia c, Rosendo A. Yunes c, Ricardo J. Nunes c, Ana M.O. Battastini e,
Fernanda B. Morrone b f, Maria M. Campos a
,
,f,g,
*
a Postgraduate Program in Medicine and Health Sciences, PUCRS, Porto Alegre, RS, Brazil
b School of Pharmacy, PUCRS, Porto Alegre, RS, Brazil
c Department of Chemistry, UFSC, Florianópolis, SC, Brazil
d Biophysics Department, UFRGS, Porto Alegre, RS, Brazil
e Department of Biochemistry, UFRGS, Porto Alegre, RS, Brazil
f Institute of Toxicology and Pharmacology, PUCRS, Porto Alegre, RS, Brazil
g School of Dentistry, PUCRS, Porto Alegre, RS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2011
Received in revised form
12 December 2011
Accepted 14 December 2011
Available online 22 December 2011
Gliomas are the most common and devastating tumors of the central nervous system (CNS). Many pieces
of evidence point out the relevance of natural compounds for cancer therapy and prevention, including
chalcones. In the present study, eight synthetic quinoxaline-derived chalcones, structurally based on the
selective PI3Kg inhibitor AS605240, were evaluated for anti-proliferative activity and viability inhibition
using glioma cell lines from human and rat origin (U-138 MG and C6, respectively), at different time-
periods of incubation and concentrations. The results revealed that four chalcones (compounds 1, 6, 7
and 8), which present methoxy groups at A-ring, displayed higher efficacies and potencies, being able to
inhibit either cell proliferation or viability, in a time- and concentration-dependent manner, with an
efficacy that was greater than that seen for the positive control compound AS605240. Flow cytometry
analysis demonstrated that incubation of C6 cells with compound 6 led to G1 phase arrest, likely indi-
cating an interference with apoptosis. Furthermore, compound 6 was able to visibly inhibit AKT acti-
vation, allied to the stimulation of ERK MAP-kinase. The chalcones tested herein, especially those
displaying a methoxy substituent, might well represent promising molecules for the adjuvant treatment
of glioma progression.
Keywords:
Synthesis
Quinoxaline
Chalcones
Anti-cancer activity
Anti-proliferative
Glioma cells
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Treatment options for patients with GBM are very limited, or in
most situations they are ineffective. Despite the increasing
Gliomas comprise several types of primary brain tumors,
accounting for approximately 50% of all neoplasms of the central
nervous system (CNS) [1e3]. Glioblastoma multiforme (GBM) is the
most common, aggressive and deadly malignant glioma in adults
[4e6]. This kind of brain tumor is characterized by marked cell
proliferation and invasiveness with a rapid progression, presenting
a high grade of recurrence [2,7e9]. Therefore, the prognosis for the
patients with these tumors is poor. The mean survival is around one
year [8,10,11] and few patients survive beyond five years [12].
advances in radiotherapy, chemotherapy, and surgical techniques,
the survival rate for these patients remains low [13]. Therefore,
there is an urgent need for novel and effective therapies for treating
these tumors. In this regard, molecules based on natural products
represent very interesting therapeutic alternatives.
Extensive research over the past decades has identified
numerous dietary and botanical natural compounds with clear
anti-cancer effects. They might also present synergistic beneficial
effects when used in combination with known chemotherapeutic
drugs [14,15]. Previous studies have shown that the simple chem-
ical structure and the uncomplicated process of synthesis make
plant-derived polyphenols an attractive scaffold for the develop-
ment of new compounds [16,17].
* Corresponding author. School of Dentistry, Pontifícia Universidade Católica do
Rio Grande do Sul, Avenida Ipiranga, 6681, Partenon, 90619-900, Porto Alegre,
Brazil. Tel.: þ55 51 3320 4367; fax: þ55 51 3320 3868.
Chalcones are a group of natural precursors of flavonoid and
isoflavonoids synthesis in high plants [18e21], and they are cancer
E-mail addresses: camposmartha@yahoo.com.br, maria.campos@pucrs.br
(M.M. Campos).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.023

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T.R. Mielcke et al. / European Journal of Medicinal Chemistry 48 (2012) 255e264
preventive components found in human diet rich in fruits and
vegetables [20,22]. Concerning the chemical structural level, these
compounds are open-chained molecules, in which the two
aromatic rings are joined by a three-carbon
a,b-unsaturated
carbonyl system (1,3-diphenyl-2-propen-1-one) [16,23,24]. Several
studies have shown that chalcones display a wide variety of bio-
logical and pharmacological proprieties that include anti-
proliferative and anti-cancer activities [25e27]. Clinical trials
have shown that these compounds reach reasonable plasma
concentrations, they are not associated to marked toxicity [28] and
flavonoids and their derivatives are able to cross the brain blood
barrier [29,30].
A large number of chemical structures have been tested, and it
was reported that hydroxy-derivatives of chalcones display marked
anti-proliferative effects on cancer cells. These groups are likely
necessary for the inhibitory effects of glutathione S-transferase,
what is associated to cancer cell sensitization for chemotherapy
agents [15,31]. Other chemical modifications of chalcones have
been investigated, such as hydrogenation and bromination across
the carbonecarbon double bond, but they failed in potentiate the
anti-tumor effects of chalcones [16]. Recently, it was demonstrated
that quinoxaline derivatives display a broad spectrum of biological
activities. Of relevance, some of these compounds have been
described as potential candidates for the treatment of cancer and
disorders associated with angiogenesis [32].
Fig. 1. PI3Kg inhibitor AS605240 (a) and chalcones proposed in this research (b).
revealed that all structures were geometrically pure and configured
E (JH
¼ w16.0 Hz).
-
Hb
a
3. Biological results and discussion
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is
implicated in a variety of cellular processes including cell growth,
proliferation, and survival, which is frequently deregulated in
cancer, including glioma [33e35]. The alteration of this pathway
through mutation of its coding genes increases the activation status
of the signaling and can thus lead to cellular transformation [33]
and cancer development [36]. Given the frequency of deregulated
PI3K signaling, the modulation of this pathway might have thera-
peutic potential in glioma and other cancer types. AS605240
As a first approach, we performed a screening using both U-138
MG and C6 glioma cell lines, in order to assess the inhibitory effects
of the chalcones. This was carried out following 48 h of incubation,
at concentrations between 0.1 and 10
produced a significant decrease of C6 rat glioma cell line viability, at
g/mL (Table 1). The estimated IC50 values (accompanied by
the confidence interval) for the rat cells ranged from 2.66
(2.09e3.38) g/ml to 9.19 (8.93e9.48) g/ml. Concerning the U-138
mg/mL. All tested chalcones
5
m
m
m
[5-(quinoxalin-6-ylmethylene)thiazolidine-2,4-dione] is
a
low-
MG glioma cells, with exception of the compound 5, all other
compounds produced a significant inhibition of cell viability at
toxicity and selective inhibitor of the PI3K
g
isoform, which is able
to suppress inflammation in different experimental models [37],
and further studies are currently testing its effects on tumor cells
[38], considering the involvement of this signaling pathway in
cancer development. Then, in the present study, we have examined
the in vitro effects of eight novel synthetic quinoxaline-derived
5 mg/mL. However, for the human cell line, it was not possible to
calculate the IC50 values, as the calculated percentages of inhibition
did not exceed 50% (Table 1). As demonstrated in the present study
for the tested chalcones, Zamin et al. (2009) also showed that rat C6
and human U-138 glioma cell lines displayed a different sensitivity
to the treatment with the compounds resveratrol and quercetin,
two well-studied natural polyphenols [29].
From this experimental set, it was possible to observe that four
of tested chalcones (compounds 1, 6, 7 and 8) displayed higher
efficacies and potencies. This is especially notable if we compare
with the low inhibitory rates obtained for reference chemotherapy
drugs, such as cisplatin and doxorubicin [44]. Of note, these four
chalcones present a similar chemical structure, having methoxy
groups in the ring A. In fact, previous studies demonstrated that the
activity of chalcones is largely dependent on the presence and
position of the substituted groups added in both rings A and B. It
has been reported that dimethoxylation and trimethoxylation are
highly beneficial for the anticancer activity [17,45e47]. In our
studies, chalcone 6, dimethoxylated in positions 2 and 5 at A-ring,
displays the best activity.
chalcones, structurally based on the selective PI3K
g inhibitor
AS605240 (Fig.1), on viability, proliferation, cell cycle, and signaling
pathways, by using human U-138 MG and rat C6 glioma cell
lineages.
ERK1 and ERK2 are members from the family of MAP-kinases
(mitogen-activated protein kinases), and are activated by various
growth factors, inducing the transition from the quiescent state
into the cell cycle. ERK signaling pathways are also involved in cell
proliferation, differentiation, actin cytoskeleton reorganization,
and cell migration. Moreover, ERKs are also involved in the stress
response and cell death [39e42]. Therefore, this is another relevant
signaling pathway to be investigated in cancer research.
2. Chemistry
The quinoxaline-6-carbaldehyde (12) was synthesized from 3,4-
diaminobenzoic acid (9) as previously described [43] and presented
in Fig. 2, with yield of 80%. Eight chalcones (1e8) were prepared by
aldolic condensation between quinoxaline-6-carbaldehyde (12)
and corresponding acetophenones, in methanol and KOH 50% w/v,
under magnetic agitation and room temperature. The novel chal-
cones were obtained with yields between 41% and 93%, and the
structures were confirmed by chemical identification data: 1H
NMR, 13C NMR, IR and elementary analysis. 1H NMR spectra
As a next step, we assayed the compounds 1, 6, 7 and 8 in
concentrations ranging from 0.1 to 10 mg/mL, at different periods of
incubation (24, 48 and 72 h), by using a hemocytometer. Cell
counting assay revealed marked inhibitory effects on the prolifer-
ation of either human or rat cell lines (Table 2). For this assay, the
estimated IC50 values ranged from 2.29 (2.03e2.58)
(2.55e2.80) g/mL, indicating a high potency for these compounds
(Table 2). Of high interest, the chemotherapy drug doxorubicin,
incubated during 48 h, at 2- g/mL concentration, produced an
mg/ml to 2.67
m
m

T.R. Mielcke et al. / European Journal of Medicinal Chemistry 48 (2012) 255e264
257
Fig. 2. Synthesis of compounds. (i) glyoxal, acetic acid, CH3CH2OH, reflux; (ii) LiAlH4, THF; (iii) CCP, dichloromethane; (iv) corresponding acetophenones, CH3OH, KOH 50% w/v,
magnetic stirring, 24 h, r.t.
inhibition of 27 ꢀ 3% and 31 ꢀ 4%, in C6 and U-138 MG, cells,
respectively (results not shown).
The concentration-dependent effects of compounds 1, 6, 7 and
8, at both the MTT and the cell counting assays are depicted in the
Figs. 3e6. From these figures, it is possible to observe that
compounds 1, 6, 7 and 8 presented anti-tumor-like effects in
of 100 nM, 10 mM and 30 mM [37], during 48 h, and subsequently
evaluated in the MTT and cell counting assays. The results show
that AS605240 failed to significantly affect C6 and U-138 MG cell
viability and proliferation at the concentration of 100 nM, whereas
this compound visibly reduced these parameters at 10 and 30
(Fig. 7). The percentages of inhibition for the concentration of
30
mM
concentrations as low as 0.1e0.5
mg/mL. Furthermore, it is feasible
m
M were 35 ꢀ 2% and 73 ꢀ 4% for C6; and 10 ꢀ 3% and 10 ꢀ 0.2%
to observe that maximal inhibitions for these four chalcones were
observed between 48 h and 72 h of treatment, whereas the
compounds 1, 6, 7 and 8 failed to significantly alter cell viability and
proliferation when incubated for 24 h. This assembly of results
revealed a concentration-related profile of inhibition for the tested
chalcones on either the cell viability or proliferation. In addition,
the effects of these four chalcones were found to be time-
dependent, being maximal between 48 and 72 h following
in vitro treatment.
for U-138 MG, considering the viability and the proliferation,
correspondingly (Fig. 8). It is worth mentioning that the concen-
tration of 5 mg/ml of the compound 6 (which corresponds to 16 mM)
displayed inhibitions of 50 ꢀ 7% for C6 viability and 85 ꢀ 8% for C6
proliferation; and 36 ꢀ 1% for U-138 MG viability and 87 ꢀ 4% for
proliferation of this cell line. This series of results suggest a greater
efficacy to compound 6, in comparison to the reference compound
AS605240, especially concerning the cell lineage U-138 MG.
Previous literature data demonstrated that natural and
synthetic chalcones are able to induce cell cycle arrest and
apoptosis in different cancer cell lines [17,26]. To examine the
possible mechanisms responsible for the inhibitory effects dis-
played by the quinoxaline-derived chalcones in our study, we have
used flow cytometry analysis. For this purpose, we have selected
the compound 6, which presented the more favorable profile in our
experiments. The results demonstrate that incubation of compound
6 caused an accumulation of cells in sub-G1/G1 phases in C6 cells at
6 and 12 h (results not shown). When assessed after 24 h of incu-
AS605240 is a well-characterized selective PI3K
g inhibitor,
which blocks human recombinant PI3K in an ATP-competitive
g
manner, displaying marked anti-inflammatory effects in several
animal disease models [37,48]. The chalcones derived from qui-
noxaline-6-carbaldehyde analyzed in this work are structurally
based on this selective PI3Kg inhibitor. Therefore, some experi-
ments were conducted with the aim of comparing the effects of
quinoxaline-derived chalcones to those displayed by AS605240.
The glioma cells were treated with AS605240 at the concentrations
bation, compound 6 (2.5
mg/ml) visibly increased the cell pop-
ulation of the sub-G1/G1 phases from 57.9 to 66.4, when compared
with the control group (Fig. 8). It is feasible to suggest that part of
Table 1
Effects of quinoxaline-derived chalcones on the viability of rat and human glioma
cell lines, according to assessment by MTT assay.
Glioma cell line
Table 2
Effects of quinoxaline-derived chalcones 1, 6, 7 and 8 on the proliferation of rat and
human glioma cell lines, as assessed by cell counting in a hemocytometer.
Rat C6
Human Uꢁ138 MG
Chalcones
Imax (%)a
IC50
(m
g/ml)b
Imax (%)a
IC50 (m
g/ml)b
Glioma cell line
1
2
3
4
5
6
7
8
53 ꢀ 3
55 ꢀ 5
56 ꢀ 6
60 ꢀ 6
26 ꢀ 6
50 ꢀ 7
51 ꢀ 7
52 ꢀ 7
4.42 (4.03e4.84)
8.39 (7.66e9.20)
9.19 (8.93e9.48)
4.63 (3.40e6.30)
e
34 ꢀ 3
15 ꢀ 5
16 ꢀ 5
21 ꢀ 7
9 ꢀ 2
e
e
e
e
e
e
e
e
Rat C6
Human U-138 MG
Chalcones
Imax (%)a
IC50
(m
g/ml)b
Imax (%)a
IC50 (m
g/ml)b
1
6
7
8
90 ꢀ 4
85 ꢀ 8
82 ꢀ 6
67 ꢀ 7
2.19 (1.85e2.59)
1.35 (1.10e1.65)
1.00 (0.21e4.65)
1.53 (1.21e1.95)
84 ꢀ 2
87 ꢀ 4
84 ꢀ 3
86 ꢀ 3
2.67 (2.55e2.80)
2.64 (2.46e2.84)
2.29 (2.03e2.58)
2.48 (2.17e2.79)
2.66 (2.09e3.38)
4.16 (3.51e4.93)
4.60 (4.07e5.20)
36 ꢀ 1
33 ꢀ 3
42 ꢀ 2
a
a
The maximal percentages of inhibition were calculated at the concentration of
g/ml.
The maximal percentages of inhibition were calculated at the concentration of
g/ml.
The compounds were tested at concentrations ranging between 1 and 10 mg/ml.
5
m
5
m
b
b
The compounds were tested at concentrations ranging between 1 and 10
m
g/ml.

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T.R. Mielcke et al. / European Journal of Medicinal Chemistry 48 (2012) 255e264
Fig. 3. Effects of compound 1 on glioma cell viability and proliferation. The viability of rat glioma cells C6 (A) and human glioma cells U-138 MG (B) were assessed by MTT assay. Cell
viability is provided as the percentage of control group viability. Proliferation of C6 (C) and U-138 MG (D) cells was assessed by cell counting in a hemocytometer. Each column
represents the mean of 3 independent experiments performed in triplicate and the lines indicate the standard error means. Data were analyzed by oneway ANOVA, followed by
multiple comparisons post-hoc test (Tukey test). *Significantly different from the control group *(P < 0.05); ** (P < 0.01).
the decrease in glioma cell viability caused by 6 is likely associated
with cell cycle arrest at the G1 phase.
It is obviously necessary to investigate the ability of these
chalcones to cross the bloodebrain barrier, as cerebral sites need to
be reached. Of interest, previous studies have shown that flavo-
noids and their derivatives were able to cross this barrier [29,30].
Further in vitro and in vivo studies should be performed for verify
this matter. Thus, the synthetic quinoxaline-derived compounds 1,
6, 7 and 8 might well represent promising molecules for the
adjuvant treatment of glioma progression.
It has been demonstrated that alterations of the PI3K/Akt acti-
vation might lead to cellular transformation [33] and cancer
development [46]. In tumor cells, PI3K plays an important role in
tumor initiation, growth and proliferation. Inhibition of PI3K may
thus have the potential to inhibit formation of secondary-site
metastases [49e52]. To examine the possible modulation of
PI3Kg, we performed flow cytometry experiments with compound
6 and the selective PI3K
g
inhibitor AS605240, as the positive
4. Conclusions
control drug. As expected, both the compound 6 and AS605240
were able to reduce AKT activation, according to assessment at 15
and 30 min. Of note, differently from AS605240, the compound 6
led to increased activation of ERK 1/2 MAP-kinase, indicating
additional mechanisms of action for this compound (Fig. 9).
Nevertheless, we can infer that inhibition of PI3K signaling may be
related with the reduction of cell proliferation observed in our
previous results and can be one of the mechanisms involved in the
anti-tumor actions of chalcone 6. Furthermore, the ability of
compound 6 in positively modulating ERK 1/2 activation might
possibly accounts for cell death, likely by inducing apoptosis. In fact,
previous studies suggest that apoptosis might be induced by the
disruption of MAP-kinase signal transduction. The duration and
intensity of ERK activation appear to be important in determining
the cell fate (growth, survival or apoptosis), indicating that ERK
may have a dual role in the regulation of cell survival and death
[53,54]. According to Werlen and col. (2003), depending on the
affinity of the ligand, the ERK pathway is activated before p38 and
JNK, resulting in cell apoptosis [55]. In support of this, our results
showed that compound 6 led to marked ERK 1/2 activation, which
was accompanied by increased granulosity of the cells (data not
shown).
The present results show for the first time the activity of
synthetic quinoxaline-derived chalcones in two different glioma
cell lines. These results clearly suggest that compounds 1, 6, 7 and 8
might represent promising molecules for the treatment of glioma
progression, although the in vivo efficacy of these chalcones
remains to be confirmed in future studies. Nevertheless, these
chalcones might represent potential useful alternatives for treating
gliomas, even when used alone or in combination with currently
available chemotherapy agents.
5. Materials and methods
5.1. Preparation of the compounds
All reagents used were obtained commercially (SigmaeAldrich),
except the quinoxaline-6-carbaldehyde, which was obtained as
previously described [43] by condensation between 3,4-
diaminobenzoic acid (9) and glyoxal at reflux with ethanol and
acetic acid, generating the quinoxalinecarboxylic acid (10) with
yield of 25%. Compound 10 was then reduced to its primary alcohol
(11) with LiAlH4 in THF, which was after oxidized to aldehyde (12)

T.R. Mielcke et al. / European Journal of Medicinal Chemistry 48 (2012) 255e264
259
Fig. 4. Effects of compound 6 on glioma cell viability and proliferation. The viability of rat glioma cells C6 (A) and human glioma cells U-138 MG (B) were assessed by MTT assay. Cell
viability is provided as the percentage of control group viability. Proliferation of C6 (C) and U-138 MG (D) cells was assessed by cell counting in a hemocytometer. Each column
represents the mean of 3 independent experiments performed in triplicate and the lines indicate the standard error means. Data were analyzed by oneway ANOVA, followed by
multiple comparisons post-hoc test (Tukey test). *Significantly different from the control group *(P < 0.05); ** (P < 0.01).
with pyridineechlorochromate in dichloromethane (Fig. 2), with
yield of 80%. The novel chalcones (1e8)were prepared by magnetic
stirring with acetophenone (1 mmol), methanol (30 ml), KOH 50%
w/v (5 ml) and quinoxaline-6-carbaldehyde (12) (1 mmol), at room
temperature for 24 h. Distilled water and chloridric acid 10% were
added in the reaction for total precipitation of the compounds. The
compounds were then obtained by vacuum filtration and later
recrystallized in dichloromethane/hexane. The purified chalcones
were obtained with yields between 41% and 93%.
H20, H60), 8.16 (d, 1H, J ¼ 8.0 Hz, H4), 8.33 (s, 1H, H1), 8.88 (dd, 2H,
J ¼ 8.0/1.0 Hz, H6, H7). 13C NMR (CDCl3)
d
55.34 (OCH3), 114.15 (C30,
C50), 124.76 (C ), 128.82 (C10), 130.20 (C3), 130.75 (C1), 131.17 (C20,
a
C60, C4), 137.24 (C2), 141.71 (C
b, C4a), 144.02 (C8a), 146.28 (C6),
146.53 (C7), 164.04 (C40), 187.27 (C]O). IR nmax/cmꢁ1 1654 (C]O),
1599 (C]C), 1259, 1017 (CeO), 3447 (CeN), 1504, 1416, 1367, 1223,
1181, 817, 610 (Ar) (KBr). Anal. Calcd for C18H14N2O2: C 74.47, H 4.86,
N 9.65. Found: C 74.65, H 4.94, N 10.73. Yield: 68%.
5.2.2. (2E)-1-(1,3-benzodioxol-5-yl)-3-(quinoxalin-6-yl)prop-2-en-
1-one (2)
5.2. Physicoechemical data of the compounds
Yellow solid, m.p. 225e226 ꢂC; 1H NMR (DMSO-d6)
d 6.16 (s,
2H, eOCH2O-), 7.09 (d, 1H, J ¼ 8.0 Hz, H50), 7.70 (s, 1H, H20), 7.91 (d,
The structures were identified using melting points (m.p.),
infrared spectroscopy (IR), 1H and 13C nuclear magnetic resonance
spectroscopy (NMR) and elementary analysis. Melting points were
determined with a Microquimica MGAPF-301 apparatus and are
uncorrected. IR spectra were recorded with an Abb Bomen FTLA
2000 spectrometer on KBr disks. Elementary analyses were ob-
tained with a CHNS EA 1110. Percentages of C and H were in
agreement with the product formula (within 0.4% of theoretical
values to C). NMR (1H and 13C NMR) were recorded on Varian
Oxford AS-400 (400 MHz), using tetramethylsilane as internal
standard. 1H NMR spectra revealed that all the structures were
1H, J ¼ 8.0 Hz, H60), 7.92 (d,1H, J ¼ 16.0 Hz, H
), 8.12 (d,1H, J ¼ 8.0 Hz,
a
H3), 8.14 (d, 1H, J ¼ 16.0 Hz, H ), 8.43 (d, 1H, J ¼ 8.0 Hz, H4), 8.53 (s,
b
1H, H1), 8.95 (d, 2H, J ¼ 8.0 Hz, H6, H7).13C NMR (DMSO-d6)
d 102.85
(eOCH2Oe), 108.71 (C20), 108.93 (C50), 125.25 (C ), 126.16 (C60),
a
129.89 (C3),130.26 (C1),131.26 (C4),132.74 (C10),137.34 (C2),142.60
(Cb
),143.13 (C4a),143.84 (C8a),146.95 (C6),147.16 (C7),148.78 (C30),
152.50 (C40), 187.56 (C]O). IR nmax/cmꢁ1 1651 (C]O), 1595 (C]C),
1255, 1034 (CeO), 3440 (CeN), 3048, 2909, 1496, 1444, 1367, 1314,
1114, 969, 925, 793, 651 (Ar) (KBr). Anal. Calcd for C18H12N2O3: C
71.05, H 3.97, N 9.21. Found: C 71.76, H 4.04, N 10.94. Yield: 88%.
geometrically pure and configured E (JH
¼ w16.0 Hz).
a-Hb
5.2.3. (2E)-1-(4-bromophenyl)-3-(quinoxalin-6-yl)prop-2-en-1-
one (3)
5.2.1. (2E)-1-(4-methoxyphenyl)-3-(quinoxalin-6-yl)prop-2-en-1-
Cream solid, m.p. 131e132 ꢂC; 1H NMR (CDCl3)
d
7.69 (dd,
one (1)
2H, J ¼ 8.0/1.0 Hz, H30, H50), 7.70 (d, 1H, J ¼ 16.0 Hz, H
a), 7.95 (d,
Cream solid, m.p. 160e161 ꢂC; 1H NMR (CDCl3)
d 3.92 (s, 3H,
2H, J ¼ 8.0 Hz, H20, H60), 8.02 (d, 1H, J ¼ 16.0 Hz, H
b), 8.08 (m,
OCH3), 7.02 (d, 2H, J ¼ 8.0 Hz, H30, H50), 7.76 (d, 1H, J ¼ 16.0 Hz, H
a),
1H, H3), 8.17 (d, 1H, J ¼ 8.0 Hz, H4), 8.34 (s, 1H, H1), 8.89
8.00 (d, 1H, J ¼ 16.0 Hz, H ), 8.09 (m, 1H, H3), 8.10 (d, 2H, J ¼ 8.0 Hz,
b

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T.R. Mielcke et al. / European Journal of Medicinal Chemistry 48 (2012) 255e264
Fig. 5. Effects of compound 7 on glioma cell viability and proliferation. The viability of rat glioma cells C6 (A) and human glioma cells U-138 MG (B) were assessed by MTT assay. Cell
viability is provided as the percentage of control group viability. Proliferation of C6 (C) and U-138 MG (D) cells was assessed by cell counting in a hemocytometer. Each column
represents the mean of 3 independent experiments performed in triplicate and the lines indicate the standard error means. Data were analyzed by oneway ANOVA, followed by
multiple comparisons post-hoc test (Tukey test). *Significantly different from the control group *(P < 0.05); ** (P < 0.01).
(dd, 2H, J ¼ 8.0/1.0 Hz, H6, H7). 13C NMR (CDCl3)
d
124.35 (C
a
),
(C8a), 145.08 (C10, C ), 146.23 (C6), 147.08 (C7), 153.96 (C40), 188.72
b
128.82 (C3), 129.46 (C40), 130.27 (C20, C60), 130.73 (C30, C50), 131.17
(C]O). IR nmax/cmꢁ1 1661 (C]O), 1588 (C]C), 1281, 1030 (CeO),
3418 (CeN), 1516, 1348, 847 (NO2), 3107, 1315, 1215, 1105, 985, 826,
755, 706, 515 (Ar) (KBr). Anal. Calcd for C17H17N3O3: C 66.88, H 3.63, N
13.76. Found: C 65.63, H 3.36, N 13.08. Yield: 77%.
(C1), 132.03 (C10), 132.18 (C4), 137.14 (C2), 143.15 (C
b, C4a), 144.14
(C8a), 146.46 (C6), 146.62 (C7), 188.31 (C]O). IR nmax/cmꢁ1 1656
(C]O), 1589 (C]C), 1279, 1026 (CeO), 3447 (CeN), 1070 (C-Br),
3000, 1493, 1393, 1370, 1218, 1179, 1000, 866, 820, 516 (Ar) (KBr).
Anal. Calcd for C17H11BrN2O: C 60.20, H 3.27, N 8.26. Found: C 59.79,
H 3.24, N 8.91. Yield: 72%.
5.2.6. (2E)-1-(2,5-dimethoxyphenyl)-3-(quinoxalin-6-yl)prop-2-
en-1-one (6)
Yellow solid, m.p. 142e144 ꢂC; 1H NMR (CDCl3)
d 3.83 (s, 3H,
5.2.4. (2E)-1-phenyl-3-(quinoxalin-6-yl)-2-propen-1-one (4)
OCH3), 3.91 (s, 3H, OCH3), 6.98 (d, 1H, J ¼ 8.0 Hz, H30), 7.08 (d, 1H,
Cream solid, m.p. 158e159 ꢂC; 1H NMR (CDCl3)
d
7.60 (t, 2H,
J ¼ 8.0 Hz, H40), 7.27 (s, 1H, H60), 7.68 (d, 1H, J ¼ 16.0 Hz, H
a), 7.86 (d,
J ¼ 8.0 Hz, H30, H50), 7.69 (t, 1H, J ¼ 8.0 Hz, H40), 8.03 (d, 1H,
1H, J ¼ 16.0 Hz, H ), 8.03 (dd, 1H, J ¼ 8.0/1.0 Hz, H3), 8.13 (d, 1H,
b
J ¼ 16.0 Hz, H
a
), 8.14 (d, 1H, J ¼ 8.0 Hz, H10), 8.16 (d, 1H, J ¼ 16.0 Hz,
J ¼ 8.0 Hz, H4), 8.28 (s, 1H, H1), 8.87 (dd, 2H, J ¼ 8.0/1.0 Hz, H6, H7).
13C NMR (CDCl3) 56.11 (OCH3), 56.72 (OCH3), 113.60 (C30), 114.69
(C60),120.19 (C40), 128.92 (C ), 129.57 (C3),129.36 (C10), 130.32 (C1),
130.42 (C4), 137.32 (C2), 141.09 (C , C4a), 143.40 (C8a), 145.64 (C6),
Hb
), 8.24 (d, 2H, J ¼ 8.0 Hz, H20, H60), 8.40 (dd, 1H, J ¼ 8.0/1.0 Hz,
d
H90), 8.48 (s, 1H, H2), 8.95 (dd, 2H, J ¼ 8.0/1.0 Hz, H5, H6). 13C NMR
a
(CDCl3)
d
124.77 (C
a
), 128.81 (C30, C50), 128.97 (C20, C60, C10), 130.25
b
(C2), 131.01 (C9), 133.28 (C40), 137.05 (C10), 138.22 (C1), 142.57
145.93 (C7), 153.15 (C50), 153.93 (C20), 191.83 (C]O). IR nmax/cmꢁ1
1659 (C]O), 1588 (C]C), 1276, 1027 (CeO), 3444 (CeN), 3042,
2929, 2832, 1497, 1456, 1407, 1367, 1323, 1222, 1159, 977, 861, 818,
713 (Ar) (KBr). Anal. Calcd for C19H16N2O3: C 71.24, H 5.03, N 8.74.
Found: C 70.56, H 4.41, N 8.60. Yield: 56%.
(Cb, C8), 143.34 (C3), 146.37 (C6), 146.56 (C5), 189.12 (C]O). IR
nmax/cmꢁ1 1656 (C]O), 1601 (C]C), 1279, 1018 (CeO), 3421 (CeN),
3053, 1497, 1440, 1367, 1308, 1216, 984, 864, 828, 770, 692, 659 (Ar)
(KBr). Anal. Calcd for C17H12N2O: C 78.44, H 4.65, N 10.76. Found: C
81.01, H 4.69, N 13.65. Yield: 41%.
5.2.7. (2E)-1-(3,4-dimethoxyphenyl)-3-(quinoxalin-6-yl)prop-2-
5.2.5. (2E)-1-(4-nitrophenyl)-3-(quinoxalin-6-yl)prop-2-en-1-one (5)
en-1-one (7)
Ocher solid, m.p. 235e236 ꢂC; 1H NMR (CDCl3)
d
7.67 (d, 1H,
Yellow solid, m.p. 143e144 ꢂC; 1H NMR (CDCl3)
d 4.00 (s, 6H,
J ¼ 16.0 Hz, H
a
), 8.02 (s,1H, H1), 8.06 (d,1H, J ¼ 8.0Hz, H3), 8.17 (d,1H,
OCH3), 6.97 (d, 1H, J ¼ 8.0 Hz, H50), 7.67 (s, 1H, H20), 7.74 (dd, 1H,
J ¼ 8.0 Hz, H4), 8.19 (d, 2H, J ¼ 8.0 Hz, H20, H60), 8.36 (d, 2H, J ¼ 8.0 Hz,
J ¼ 8.0/1.0 Hz, H60), 7.77 (d, 1H, J ¼ 16.0 Hz, H
a), 8.01 (d, 1H,
H30, H50), 8.37 (d,1H, J ¼ 16.0 Hz, H
b
), 8.88 (d, 2H, J ¼ 8.0 Hz, H6, H7).
J ¼ 16.0 Hz, H ), 8.09 (dd, 1H, J ¼ 8.0/1.0 Hz, H3), 8.16 (d, 1H,
b
13C NMR (CDCl3)
d
123.80 (C
a
), 124.24 (C30, C50), 128.54 (C3), 129.75
J ¼ 8.0 Hz, H4), 8.34 (s, 1H, H1), 8.88 (dd, 2H, J ¼ 8.0/1.0 Hz, H6,
(C20, C60), 130.71 (C1), 131.37 (C4), 136.20 (C2), 140.32 (C4a), 143.32
H76). 13C NMR (CDCl3) 56.11 (OCH3), 56.17 (OCH3), 110.03 (C50),
d

T.R. Mielcke et al. / European Journal of Medicinal Chemistry 48 (2012) 255e264
261
Fig. 6. Effects of compound 8 on glioma cell viability and proliferation. The viability of rat glioma cells C6 (A) and human glioma cells U-138 MG (B) were assessed by MTT assay. Cell
viability is provided as the percentage of control group viability. Proliferation of C6 (C) and U-138 MG (D) cells was assessed by cell counting in a hemocytometer. Each column
represents the mean of 3 independent experiments performed in triplicate and the lines indicate the standard error means. Data were analyzed by oneway ANOVA, followed by
multiple comparisons post-hoc test (Tukey test). *Significantly different from the control group *(P < 0.05); ** (P < 0.01).
110.75 (C20), 123.24 (C60), 124.19 (C
a
), 128.64 (C3), 130.18 (C10, C1,
), 143.18 (C4a), 143.90 (C8a), 145.52 (C6),
Eagle’s medium (DMEM), supplemented with 15% and 5% fetal
bovine serum (FBS), for U-138 MG and C6 lines, respectively.
Culture cells were maintained at 37 ꢂC, a minimum relative
humidity of 95%, at atmosphere of 5% CO2, and were allowed to
reach confluence.
C4), 136.88 (C2), 142.10 (C
b
145.80 (C7), 149.40 (C30), 153.60 (C40), 187.24 (C]O). IR nmax/cmꢁ1
1654 (C]O),1581 (C]C),1281, 1022 (CeO), 3487 (CeN),1518,1453,
1419, 1358, 1316, 1161, 984, 918, 776, 718, 636 (Ar) (KBr). Anal. Calcd
for C19H16N2O3: C 71.24, H 5.03, N 8.74. Found: C 70.98, H 4.33, N
8.11. Yield: 51%.
5.3.2. Cell viability assay
Glioma cell lines were seeded at 1 ꢃ 103 cells/well in DMEM/5%
FBS for C6 line, or DMEM/15% FBS for U-138 MG in 96-well plates.
They were then exposed to increasing concentrations (0.1, 0.5, 1, 5
5.2.8. (2E)-1-(2,4-dimethoxyphenyl)-3-(quinoxalin-6-yl)prop-2-
en-1-one (8)
Cream solid, m.p. 119e120 ꢂC; 1H NMR (CDCl3)
d
3.90 (s, 3H,
and 10 mg/ml) of the eight quinoxaline-derived chlacones, for 24 h,
OCH3), 3.96 (s, 3H, OCH3), 6.53 (s, 1H, H30), 6.60 (dd, 1H, J ¼ 8.0/
48 h or 72 h. Parallel control experiments were carried out with the
addition of 5% and 15% FBS (cell viability control) or DMSO (vehicle
control, 0.01%), in the absence of chalcones. The glioma cell lines
1.0 Hz, H50), 7.77 (d, 1H, J ¼ 16.0 Hz, H
a
), 7.84 (d, 1H, J ¼ 8.0 Hz, H60),
7.88 (d, 1H, J ¼ 16.0 Hz, H ), 8.04 (dd, 1H, J ¼ 8.0/1.0 Hz, H3), 8.12 (d,
b
1H, J ¼ 8.0 Hz, H4), 8.28 (s, 1H, H1), 8.86 (dd, 2H, J ¼ 8.0/1.0 Hz, H6,
H7). 13C NMR (CDCl3) 55.62 (OCH3), 55.85 (OCH3), 98.62 (C30),
105.45 (C10), 109.77 (C50), 128.84 (C
), 129.77 (C3), 129.85 (C1),
130.01 (C4), 133.23 (C60), 137.46 (C2), 139.84 (C
), 143.21 (C4a),
were also treated with the PI3K
g
inhibitor, AS605240, at the
M, for 48 h.
d
concentrations of 100 nM, 10 M and 30 m
m
a
Cell viability was evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-
diphenyl tetrazolium bromide (MTT) assay [56]. This assay
measures the activity of cellular dehidrogenases (mainly from
mitochondria) and, indirectly, the cell viability, even of the spon-
taneously detached cells in the culture medium. The method
is based on the reduction of a tetrazolium bromide salt (MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]). It
provides a quantitative measure of the number of metabolically
viable cells. The results were expressed as the percentage of cell
viability in relation to the control.
b
143.75 (C8a), 145.28 (C6), 145.65 (C7), 160.69 (C20), 164.63 (C40),
189.71 (C]O). IR nmax/cmꢁ1 1651 (C]O), 1609 (C]C), 1274, 1026
(CeO), 3447 (CeN), 2967, 1500, 1473, 1422, 1367, 1323, 1214, 1128,
974, 865, 821, 632 (Ar) (KBr). Anal. Calcd for C19H16N2O3: C 71.24, H
5.03, N 8.74. Found: C 70.80, H 4.77, N 8.29. Yield: 93%.
5.3. Pharmacology
5.3.1. General cell culture procedures
U-138 MG human and C6 rat GBM cell lines were obtained from
the American Type Culture Collection (ATCC, Rockville, Maryland,
USA). The cells were grown in culture flasks in Dulbecco’s Modified
5.3.3. Cell counting
U-138 MG and C6 cells were seeded at 1 ꢃ104 and 5 ꢃ 103 cells/
well, respectively, in appropriate medium. Both lineages were

262
T.R. Mielcke et al. / European Journal of Medicinal Chemistry 48 (2012) 255e264
Fig. 7. Effects of AS605240 on glioma cell viability and proliferation. The viability of rat glioma cells C6 (A) and human glioma cells U-138 MG (B) were assessed by MTT assay. Cell
viability is provided as the percentage of control group viability. Proliferation of C6 (C) and U-138 MG (D) cells was assessed by cell counting in a hemocytometer. Each column
represents the mean of 3 independent experiments performed in triplicate and the lines indicate the standard error means. Data were analyzed by oneway ANOVA, followed by
multiple comparisons post-hoc test (Tukey test). *Significantly different from the control group *(P < 0.05); ** (P < 0.01).
seeded in 24-well plates and allowed to grow for 24 h. The medium
was changed prior to treatment with the different chalcones (0.1,
were washed with calcium- and magnesium-free phosphate-buff-
ered saline (CMF), and 200 l of 0.5% trypsin/EDTA solution (Gibco,
m
0.5, 1, 5 and 10
m
g/ml) and also with AS605240 (100 nM, 10
m
M and
BRL) was added to detach the cells. After that, the cells were
counted in a Neubauer chamber.
30 M). After 48 h of treatment the medium was removed, the cells
m
5.3.4. Cell cycle analysis
The effects of compound 6 on C6 cell cycle phase distribution
was assessed using flow cytometry. The cells (3 ꢃ 105 cells per well)
were cultured in triplicate in 6-well plates. After 6, 12 or 24 h of
incubation, the cells were treated with vehicle (0.1% DMSO) or
compound 6 (2.5
mg/ml). Cells were harvested, fixed with 70%
ethanol and stained with PSSI solution (Triton, RNAse, Propidium
Iodide and PBS). Data acquisition and analysis were performed on
flow cytometer (Guava EasyCyte 8HT Flow Cytometry System,
Millipore Corporation, MA, USA), and data from 10,000 cells were
collected for each data file. Cell cycle analysis was performed with
CellQuest software (Becton Dickinson, Canada, Inc.).
5.3.5. PI3K and ERK 1/2 MAP-kinase analysis
C6 rat glioma cells (2 ꢃ 105 cells per well) were plated in 6-well
plates, with 5% FBS DMEM medium and allowed to grow for 24 h.
The cells remained incubated for more 24 h with DMEM 0.5%,
to stop the cell cycle. Cells were treated with the compound 6
(5
mg/ml), AS605240 (30 mM), or vehicle (0.1% DMSO), in the
presence of FBS 5% (used to induce protein activation). After 15 min
and 30 min of incubation, the cells were detached with 0.5%
trypsin/EDTA solution (Gibco, BRL), and then incubated with
Phosflow Fix Buffer (BD) for 10 min, and permeabilizated with
Phosflow Perm Buffer III (BD). The antibodies for AKT and ERK 1/2
were incubated during 30 min. Data acquisition and analysis was
performed on flow cytometer (Facscanto) and FlowJo 7.6.3software.
Fig. 8. Effect of compound 6 treatment on cell cycle distribution. C6 cells were treated
for 6, 12 and 24 h with dimethylsulfoxide (control) or chalcone 6 (2.5 g/mL), collected,
m
fixed, stained with PSSI solution and subjected to flow cytometry cell cycle analysis.
Values are the relative number of cells in the sub-G1/G1, S and G2/M phases of cell
cycle. Each column represents the mean of 6 independent experiments performed in
triplicate and the lines indicate the standard error means. Data were analyzed by
oneway ANOVA, followed by Bonferroni’s post-hoc test. *Significantly different from
the control group *(P < 0.05); ** (P < 0.01).

T.R. Mielcke et al. / European Journal of Medicinal Chemistry 48 (2012) 255e264
263
Fig. 9. Effects of compound 6 (5
mg/ml, red line) and AS605240 (30
mM, black line) on AKT (A and B) and ERK 1/2 MAP-kinase (C and D) activation, following addition of cell culture
medium supplemented with 5% FBS. Cells were treated with compound 6 (5
m
g/ml) or AS605240 (30 M) during 15 (A and C) and 30 min (B and D). The antibodies for AKT and ERK
m
1/2 were incubated during 30 min and the cells were subjected to flow cytometry cell cycle analysis. The gray line represents the negative control, and the dotted line indicates the
positive control with 5% FBS plus DMSO 0.01%. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
5.3.6. Statistical analysis
T.R.M. is a post-graduate student in Medicine and Health Sciences
supported by the Institution (PROBOLSAS Program/PUCRS). The
authors thank Mr. Juliano Soares by his excellent technical
assistance.
The percentage of inhibition is presented as mean ꢀ standard
error mean. The estimated IC50 values are provided as the
geometric means accompanied by the 95% confidence limit. Data
were analyzed by oneway analysis of variance (ANOVA), followed
by Tukey’s or Bonferroni’s tests, depending on the experimental
protocol, using the software GraphPad PrismÒ 4.02. P values less
than 0.05 (P < 0.05) were considered as indicative of significance.
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