Synthesis of dihydropyrimidin-2-one/thione library and cytotoxic activity against the human U138-MG and Rat C6 glioma cell lines

Article abstract of DOI:10.1590/S0103-50532011000700025

Two series of 4-aryl-3,4-dihydropyrimidin-2(1H)-(thio)ones including monastrol (1a), have been synthesized by an environment-friendly methodology based on the combined use of citric acid or oxalic acid and TEOF (triethylorthoformate). The library was eval

Full text of DOI:10.1590/S0103-50532011000700025

J. Braz. Chem. Soc., Vol. 22, No. 7, 1379-1388, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
0
103 - 5053 $6.00+0.00
Short Report
S
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity against
the Human U138-MG and Rat C6 Glioma Cell Lines
a
b
b
Rômulo F. S. Canto, Andressa Bernardi, Ana Maria O. Battastini,
,
c
,a
Dennis Russowsky* and Vera Lucia Eifler-Lima*
a
Laboratório de Síntese Orgânica Medicinal/LaSOM, Programa de Pós Graduação em
Ciências Farmacêuticas, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752,
0610-00 Porto Alegre-RS, Brazil
9
b
Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul,
Rua Ramiro Barcelos, 2660, 90035-003 Porto Alegre-RS, Brazil
c
Instituto de Química, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500,
1501-970 Porto Alegre-RS, Brazil
9
Duas séries de 4-aril-3,4-diidropirimidin-2(1H)-(tio)onas incluindo monastrol (1a), foram
sintetizadas por uma metodologia não agressiva ao meio ambiente baseada no uso combinado de
ácido cítrico ou ácido oxálico na presença de TEOF (ortoformato de trietila). A atividade como
inibidores da proliferação celular da quimioteca de compostos foi avaliada em duas linhagens de
gliomas (U138-MG-humana e C6-rato). Os compostos derivados da tiouréia 1f e 1d mostraram
atividade citotóxica maior do que a do monastrol. O composto derivado da uréia 2d apresentou a
maior atividade citotóxica dentre todos os compostos analisados.
Two series of 4-aryl-3,4-dihydropyrimidin-2(1H)-(thio)ones including monastrol (1a), have
been synthesized by an environment-friendly methodology based on the combined use of citric
acid or oxalic acid and TEOF (triethylorthoformate). The library was evaluated as inhibitor of cell
proliferation on two glioma cell lines (human-U138-MG and Rat-C6). The compounds derived
from thiourea 1f and 1d were more cytotoxic than monastrol. The compound derived from urea
2
d showed the highest cytotoxic activity among the analyzed compounds.
Keywords: dihydropyrimidin-2(1H)-ones, Biginelli reaction, triethylorthoformate, TEOF,
monastrol, cancer, glioma
Introduction
activity relationship) studies concerning this interaction
have been performed.
2
The 4-aryl-3,4-dihydropyrimidin-2(1H)-ones (DHPMs)
are a class of compounds that has a huge interest in the
medicinal chemistry community in recent years.
Moreover, this class of heterocycles revealed other
3
pharmacological activities such as anti-inflammatory,
calcium channel modulators, antifungal and antibacterial,
4
5
1
Using high throughput screening (HTS) Mayer et al.
melanin concentrating hormone receptor (MCH1-R)
6
have evaluated a library of 16,330 small molecules that
vary in functional groups and charge. These findings
have lead to discover a small molecule which they named
monastrol, whose acts by inhibiting the motility of the
mitotic kinesin Eg5, a motor protein required for spindle
bipolarity. This revealed the potential of this compound as
antitumor prototype and since that, some SAR (structure
antagonists, chemical modulators of heat shock protein 70
7
8
(Hsp70), hepatitis B replicationinhibitors, andinhibitors of
9
the fatty acid transporters. This set of potentialities linked to
the possibility of chemical modulation in all positions of the
dihydropirimidinone/thione rings make DHPMs a privileged
structure, justifying the great interest in their synthesis.
The original three component reaction of DHPMs
consisted of a simple one-pot condensation of benzaldehyde
4
b, ethyl acetoacetate 5 and urea 6b (Schemes 1 and 2),
*e-mail: dennis@iq.ufrgs.br, veraeifler@ufrgs.br
catalyzed by few drops of hydrochloric acid under refluxing

1
380
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity
J. Braz. Chem. Soc.
1
0
ethanol. However, these original Biginelli conditions
suffer of poor yields despite the long reaction times and
harsh conditions, and cannot also be applied when acid
sensitive reactants are required. It can be found in the
literature several reports of catalytic methods to improve
the yields and scope of this reaction. The majority use
Multimodal combinations of target agents with radiation
2
4
and chemotherapy may enhance treatment efficacy, but
despite these treatments, gliomas recur early due to their
2
5
high proliferation, infiltrative and invasive behaviours.
2
6
Recently, Muller et al. have observed that monastrol
(1a), enastron (3a) and dimethylenastron (3b) exhibited
antiproliferative activity against human glioblastoma cells
(Figure 1). Their investigations revealed that these mitotic
kinesin Eg5 inhibitors did not show cytotoxic effect on
resting cells, in contrast with the antimicrotubular agent
paclitaxel. This is an important result because it shows that
these compounds should present less neurotoxic activity
than the classical tubulin inhibitors.
1
1
12
Brønsted or Lewis acid catalysis, with methods based
on metal salts with non-nucleophilic anions having
pronounced catalytic activity.
The most effective ones involve reagents which have
dehydrating properties in conjunction with protic or
1
3
Lewis acidic behavior, for instance: ethyl polyphosphate,
1
4
15
TMSCl, propane phosphonic acid anhydride and
1
6
TMSCl/NaI. However, some of these methods with
expensive catalysts have been reported with unsatisfactory
yields. Besides, modern techniques employing microwave
The aim of the current study was to develop two series
(oxo-serie and thio-serie) of DHPMs analogues employing
an economical and environment-friendly methodology, in
a rapid and efficient approach, to evaluate their cytotoxic
effects against the human glioma cell line U138-MG
and the rat glioma cell line C6. This work, to the best of
our knowledge, is the first report on the study which the
monocyclic analogues of monastrol with different pattern of
substitution on aromatic ring are evaluated against glioma
cells that justify our wish to report our results.
17
18
19
irradiation, solid-phase organic synthesis, ionic liquids,
20
and solvent-free organic reactions with different acids have
recently been reported to promote the DHPM ring formation.
In this context, we have reported the synthesis of a
small library of DHPM, including monastrol (1a), using a
Lewis acid as catalyst and this series was evaluated against
seven human cancer cell lines. The results showed that the
oxo-monastrol analogue (2a) just shows cytostatic activity,
while monastrol (1a) was cytotoxic against the seven cancer
cell lines. We have also found that the 3,4-methylenodioxy
derivative (piperastrol, 1c) was approximately 30 times
more potent than monastrol (1a) against five of the seven
tested cancer cell lines, and it was also more potent than the
positive control doxorrubicine against three of the tested
cell lines (Figure 1).
21
Results and Discussion
Chemistry
In connection with our early investigations regarding the
antiproliferative activities of the DHPMs, we developed an
alternative method to prepare this heterocyclic compounds
in absence of metal salts as catalysts, due to the possible
risk of trace contamination for biological assays. Taking
in account that the cyclocondensations of an aldehyde (4),
ethyl acetoacetate (5) and urea (6b) in the acid catalyzed
Biginelli reaction produce 2 equivalents of water per
These results have encouraged us to carry out new
studies in order to investigate the in vitro antiproliferative
activity of a DHPM library against the glioma cell lines.
Gliomas are the main primary central nervous system
(
CNS) tumors in humans, accounting for almost 80% of
2
2
27
brain malignances. Glioblastoma multiforme (GBM),
classified with a grade 4, represents the most aggressive
of these tumors. Despite considerable progress in research
regarding the molecular aspects of malignant gliomas, the
equivalent of DHPM formed, we envisaged that the use
of a dehydrating agent could accelerate the reaction. The
literature reports the use of dehydrating agents, however
28
in the presence of metals, such as FeCl . The orthoesters
3
23
prognosis of these tumors continues to be dismal.
have been described as mild, non-toxic, non pollutant,
Figure 1. Chemical structures of some bioactive dihydropyrimidinones.

Vol. 22, No. 7, 2011
Canto et al.
1381
Scheme 1. Biginelli reaction catalyzed by Brønsted acid/TEOF system.
2
9
inexpensive, and effective dehydrating agents, therefore,
we considered the use of the triethylorthoformate (TEOF)
as an adjuvant reagent in the dehydration steps during the
DHPM synthesis.
absence of TEOF (74%, cf. entry 1) to confirm the influence
of TEOF. Although the use of citric acid/TEOF system at
room temperature and 48 h does not has been effective to
afford 1b, the reactions which were performed in presence
o
First, we investigate the reaction between benzaldehyde
of 1 or 2 equiv. of TEOF at 100 C during 2 h lead to the
(
4b), ethyl acetoacetate (5), and thiourea (6a) in the
production of the desired product in 81% yield and 92%
yield, respectively (entries 10 and 11, respectively). Similar
result was observed for the oxalic acid/TEOF system (93%,
entry 12). The effect of TEOF seems to be more evident
with weak Bronsted acids as citric acid or oxalic acids
related to the strong mineral acid HCl.
presence of citric acid and oxalic acid to compare their
reactivity with the classical method employing HCl as
catalyst (Scheme 1).
In our hands, the use of 10 mol% of HCl as catalyst
o
at 100 C during 2 h, afforded the DHPM 1b in 74%
yield (Table 1, entry 1). To modulate the efficacy of
citric acid, the reactions at three different temperatures
were investigated. None product was produced at
room temperature, even in 48 h (Entry 2), while the
yield gives rise with the increase of temperature, from
The comparison of these results with those obtained in
absence of TEOF (entries 4 and 5) disclose the rule that the
dehydrating agent plays in the efficiency of the process. We
believe that TEOF is important for the loosing of water in
3
1
the intermediate formation at weak acidic pH conditions.
o
o
5
0 C and 100 C, respectively during 2 h (entries 3
The increase of the yields with 2 equiv. of TEOF can
indicate their association with the loosing of a second equiv.
and 4, respectively). The use of oxalic acid in the same
conditions, afforded the product 1b in 65% yield (entry 5).
27
of water in the last step. While this work was running, the
32
33
use of pure citric acid and oxalic acid as promoters of the
Biginelli reaction was published. However, in our hands,
the reported results were not reproducible. In any way, we
demonstrate the importance of TEOF as dehydrating agent.
We extend the best condition highlighted above to the
reactions of ethyl acetoacetate (5) with a series of aromatic
aldehydes 4a-g and thiourea (6a) or urea (6b) to afford the
respective DHPMs 1a-g and 2a-g (Scheme 2). The results
are presented in Table 2.
The H NMR, C NMR and IR spectroscopic data
of compounds 1a-g and 2a-g were compatible with the
proposed structures as well as the melting points were in
accordance with those reported in the literature.
Table 1. Optimization of reaction conditions for synthesis of 1b
a
o
Entry
Acid
HCl
TEOF
time / h
T / ( C) Yield / (%)
1
2
3
4
5
6
7
8
9
-
-
2
48
12
2
100
r.t
74
-
citric ac.
citric ac.
citric ac.
oxalic ac.
-
-
50
16
34
65
-
-
100
100
100
100
100
r.t.
-
2
1
2
2
2
1
2
2
6
-
6
-
1
13
HCl
2
84
-
citric ac.
citric ac.
citric ac.
oxalic ac.
48
2
1
1
1
0
1
2
100
100
100
81
92
93
2
According to the Table 2, all aromatic aldehydes 4a-g
readily undergo reaction, with both urea (6b) and thiourea
2
aNumber of equiv. of TEOF.
(
6a) giving good-to-high yields of the corresponding
A second set of investigations was dedicated to evaluate
the influence of TEOF in this reaction. Reactions performed
in presence of 1 or 2 equiv. of TEOF for 6 h and in absence
of Bronsted acids did not afford none product, evidencing
the necessity of employment of Bronsted acid to activate the
DHPMs 1/2a-g with times varying from 1 to 2 h.
30
dehydrating form of TEOF (entries 6 and 7, respectively).
On the other hand, the reaction carried out in presence of
HCl/TEOF during 2 h, afforded the desired DHPM 1b in
84% yield (entry 8). We can compare this result with that in
Scheme 2. Synthesis of dihydropyrimidinones 1a-g and 2a-g.

1
382
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity
J. Braz. Chem. Soc.
Table 2. Synthesis of dihydropyrimidinones via Scheme 2
Biology
Yield (%) / time (h)
Entry
Ar
X
Product
To investigate the antiproliferative potential of
monastrol (1a), oxo-monastrol (2a), sulfur (1b-g), and oxo
2b-g) analogues, initially we used the cell counting assay
Citric acid Oxalic acid
1
2
3
4
5
6
7
8
9
3-HO-C H4
S
S
1a
1b
1c
1d
1e
1f
88 / 1.0
92 / 2.0
84 / 2.0
97 / 2.0
80 / 2.0
82 / 1.5
77 / 2.0
87 / 1.0
93 / 1.0
81 / 1.0
92 / 1.5
86 / 1.0
75 / 1.5
74 / 2.0
69 / 2.0
93 / 2.0
73 / 1.0
82 / 2.0
78 / 2.0
68 / 2.0
75 / 2.0
77 / 1.0
67 / 1.0
84 / 2.0
89 / 1.5
68 / 1.0
66 / 1.0
78 / 2.0
6
(
6
C H5
in glioma cell lines. Thus, the glioma cells were treated with
-1
3,4(-OCH O-)-C H
S
several concentrations of monastrol (8.5-685 mmol L ) for
2
6
3
2
4, 48 or 72 h.
3-O N-C H
4
S
2
6
The time-course experiments revealed a significant
4-NC-C H4
S
6
decrease in the cell number of C6 and U138-MG treated
with monastrol, which shows also concentration-dependent
effect (data not shown). Thus, the cell cultures were treated
4-(Me) N-C H
4
S
2
6
2-F-C H4
S
1g
2a
2b
2c
2d
2e
2f
6
3-HO-C H4
O
O
O
O
O
O
O
-1
6
with all compounds at 150 μmol L for 48 h and to compare
6
C H5
the effect with monastrol.
1
1
1
1
1
0
1
2
3
4
3,4(-OCH O-)-C H
The results of the inhibitory effect of all compounds
against the growth of both C6 and U138-MG glioma cell
proliferation are presented in Figure 2. Figure 2 shows
that all tested compounds were active in the described
panels, including monastrol (1a), although their activities
on U138-MG cells have been more pronounced. For both
2
6
3
3-O N-C H
4
2
6
4-NC-C H4
6
4-(Me) N-C H
4
2
6
2-F-C H4
2g
6
Figure 2. Effect of analogues of monastrol on cell proliferation (A and B) and on cell viability (C and D) of glioma cell lines. Semi-confluent cultures of
-
1
C6 and U138-MG glioma cells were treated with 150 mmol L of oxo or sulfur analogues of monastrol for 48 h. The cell number and the cell viability
were represented in relation to the respective controls. The values are represented as means ± S.D. of three independent experiments made in duplicate.
#
Data are analyzed by ANOVA followed by post-hoc comparisons (Tukey test). Significantly different from the control and DMSO groups (P < 0.01);
*
Significantly different from the monastrol (1a) group (P < 0.05); **Significantly different from the monastrol (1a) group (P < 0.01); ***Significantly
&
different from the monastrol (1a) group (P < 0.001); significantly different between the two groups (P < 0.01).

Vol. 22, No. 7, 2011
Canto et al.
1383
cell lines cultures, the compounds 1d, 1f and 2d displayed
high inhibition of the cell proliferation, being the compound
two fold higher activity against both the C6 and U138-MG
glioma cells compared to its analogue monastrol (1a). In
the oxo-series, other substitutions such as meta-OH, para-
N(CH ) or para-CN, ortho-F, 3,4-(-OCH O-) or even no
2d most effective (Figures 2A and 2B). Monastrol (1a)
presents an effect in a range of 35-44% of inhibition for
both glioma cell lines, while compound 1e showed higher
antiproliferative effect against the human U138-MG cells
than the C6 glioma cells (34.3% and 64.9%, respectively).
The cytotoxic effect of monastrol 1a and its analogues
were confirmed by MTT assay where, in agreement to cell
counting results, the same compounds exhibited higher
cytotoxicity in comparison to monastrol (Figure 2C and 2D).
Even if the structure-activity relationship can not be
formulate because the target is unknown and the chemical
variations were performed only in the aromatic ring of the
DHPMs, some considerations can be made. The library
was designed to cover a wide range of functional groups,
leading to different electronic and conformational effects
3
2
2
substitution at the benzene nucleus results in compounds
with similar or lower effect than 1a. Besides, the oxo-
analogue 2d is more active than its thio counterpart 1d.
The Figure 3 below, shows representative pictures
-1
of U138-MG glioma cells treated with 150 μmol L of
monastrol (1a) and its analogues 1d, 1e, 1f and 2d for 48 h.
Note the decrease of total number of cells with monastrol
treatment when compared to DMSO (negative control
cultures). As observed in Figure 3, when the cells were
treated with compounds 1d, 1e, 1f and 2d, this reduction is
more evident, confirming the results with cell counting assay.
Among the fourteen DHPMs synthesized, the three
more active against both glioma cells lines were the
compounds 1d and 1f from thio-series and the compound
2d from the oxo-series (Figure 4).
21
in the compounds. As disclosed in previous studies, the
introduction of electron donor methylenedioxy group (1c)
led to a highest activity against several cancer cell lines as
the template monastrol and doxorubicin. In contrast with
the present work, the most effective were the compounds
1
d and 2d, containing electron withdrawing groups at
3-position of aromatic ring. This leads us to think, in a first
moment, that these DHPMs act by a different mechanism
of action in the glioma cell lines, supported by the fact of
these compounds show different activities.
For the thio-series, the removal of the 3-hydroxy
group of monastrol (1a) to generate 1b does not affect the
activity what indicates that this functional group is not
essential for the cytotoxic activity on the gliomas. On the
other hand, the introduction of substituents at meta- and
para-positions improves this cytotoxicity. In comparison
with monastrol, the replacement of meta-OH by a meta-
NO group (compound 1d) results in a marked increase
2
of antiproliferative effect in U138-MG glioma cells. This
effect is also observed when the meta-OH group of 1a is
substituted either by a para-N(CH ) group in compound
Figure 3. Representative pictures of U138-MG cell cultures treated
with monastrol and analogues. After 48 h of treatment with DMSO
3
2
-1
(
non-treated cultures), 150 mmol L of monastrol (1a) or analogues of
1f or by a para-CN group in compound 1e.
monastrol, the cells were visualized using a Nikon inverted microscope.
Panels correspond to contrast phase photomicrographs. The pictures are
representative of three different experiments.
The compound 5-ethoxycarbonyl-6-methyl-4-(3-
nitrophenyl)-3,4-dihydropyrimidin-2(1H)-one (2d) has
Figure 4. The active DHPMs 1d, 1e, 1f and 2d against the proliferation of glioma cell lines.

1
384
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity
J. Braz. Chem. Soc.
Conclusions
General procedure for the synthesis of compounds 1a-g
and 2a-g under citric acid/TEOF system and oxalic acid/
TEOF system
The synthetic studies showed that triethylorthoformate
(TEOF), associated with citric acid or oxalic acid, acts as an
efficient promoter system of the Biginelli reaction yielding
dihydropyrimidinones in good to high yields. The use of
TEOF as dehydrating agent enhances drastically the yields
obtained when weak acids are employed. This method
offers a simple, inexpensive, versatile and environmental-
friendly free of metals approach to synthesize a library
of 3,4-dihydropyrimidin-2(1H)-(thio)ones, addressed to
biological assays.
A mixture of ethyl acetoacetate (5, 2.5 mmol,
325 mg), aromatic aldehyde (4a-g, 2.5 mmol), thiourea
(6a, 5.0 mmol, 360 mg) or urea (6b, 5.0 mmol, 300 mg),
citric acid (0.25 mmol, 48 mg, 10 mol%) or oxalic acid
(0.25 mmol, 22.51 mg, 10 mol%) and triethylorthoformate
(5.0 mmol, 741 mg) were placed in a 50 mL round bottom
flask and heated under stirring in a pre-heated oil batch
o
(100 C) for the time indicated in the Table 1. The reaction
The results of the biological assays on U138-MG glioma
cell line and C6 glioma cell line showed that monastrol
presents cytotoxicity against both cell lines. In addition,
four other analogues (1f, 1d, 1e and 2d) presented higher
cytotoxic effect on the same cell lines, where compound
was stopped by addition of 5 mL of water and the crude
mixture was cooled in an ice-batch under vigorous stirrer.
The solid formed was filtered, washed with small portions
of cold ethanol and then, dried under vacuum to afford the
desired product with good purity grade.
2
d was the most effective (two fold higher than monastrol).
The overall profile of 2d makes it suitable candidate to
The NMR and IR data of compounds 1a-g and 2a-g are
presented in the items bellow.
extend the pharmacological investigations. The anti-tumor
effects of the dihydropyrimidinones/thiones analogues of
monastrol on U138-MG and C6 gliomas cells have not
been reported previously. Other biological and medicinal
studies are currently underway in our research group
aiming to identify the cytotoxic mechanism of action
found here.
5-Ethoxycarbonyl-6-methyl-4-(3-hydroxyphenyl)-3,4-
35
dihydropyrimidin-2-(1H)-thione (1a)
1
mp 184-187 ºC. H NMR (250 MHz, DMSO-d )
6
d 1.14 (t, 3H, J 7.1 Hz), 2.30 (s, 3H), 4.04 (q, 2H,
J 7.1 Hz), 5.11 (d, 1H, J 3.7 Hz), 6.65-6.69 (m, 3H),
7.10-7.18 (m, 1H), 9.46 (s, 1H, OH), 9.62 (br s, 1H,
1
3
NH), 10.31 (br s, 1H, NH). C NMR (62.5 MHz,
Experimental
DMSO-d ) d 14.1, 17.2, 53.9, 59.6, 100.8, 113.2,
6
1
14.6, 117.0, 129.5, 144.8, 144.9, 157.5, 165.2, 174.2.
-
1
Chemical analysis
IR (neat) nmax/cm : 3299, 3180, 2984, 1663, 1573, 1474,
445, 1370, 1282, 1188, 1153, 1113, 1024, 788, 752,
1
All chemicals are research grade and were used as
700.
1
obtained. Nuclear magnetic resonance spectra ( HNMR
1
3
and C NMR) were recorded in an Varian INOVA-300
spectrometer or Brucker Avance DPX-250 NMR
spectrometer with standard pulse sequences operating
5-Ethoxycarbonyl-6-methyl-4-phenyl-3,4-dihydro-
pyrimidin-2-(1H)-thione (1b)
mp 204-207 ºC. H NMR (300 MHz, DMSO-d ) d 1.09
(t, 3H, J 7.0 Hz), 2.29 (s, 3H), 4.00 (q, 2H, J 7.0 Hz), 5.17
(d, 1H, J 3.7 Hz), 7.23-7.37 (m, 5H), 9.65 (d, 1H, J 1.8 Hz,
NH), 10.34 (br s, 1H, NH). C NMR (75 MHz, DMSO-d )
d 14.1, 17.2, 54.1, 59.9, 100.7, 126.4, 127.7, 128.6, 143.5,
145.1, 165.1, 174.2. IR (neat) nmax/cm : 3324, 3170, 2980,
1666, 1572, 1464, 1370, 1326, 1283, 1192, 1175, 1116,
1028, 1002, 758, 722, 691, 651.
34
1
6
1
at 300 MHz or 250 MHz for H NMR and 75 MHz or
1
3
6
2.5 MHz for C NMR, respectively, using DMSO-d6 as
13
solvent. Chemical shifts are reported as d values (ppm)
relative to TMS (0.0 ppm). The NMR multiplicities br s,
s, d, t, q, and m stand for broad singlet, singlet, doublet,
triplet, quartet and multiplet, respectively. FT-IR spectra
were recorded in a Perkin Elmer Spectrometer BXII
using an ATR probe. TLC analyses were performed on
Merck´s silica plates 60 F254. Melting-points (mp) were
determined on a System Kofler type WME apparatus
6
-1
5-Ethoxycarbonyl-6-methyl-4-(3,4-methylenedioxyphenyl)-
3,4-dihydropyrimidin-2-(1H)-thione (1c)
mp 156-159 ºC. H NMR (250 MHz, DMSO-d ) d 1.11
(t, 3H, J 7.0 Hz), 2.28 (s, 3H), 3.99 (q, 2H, J 7.0 Hz), 5.08
(d, 1H, J 3.8 Hz), 5.99 (s, 2H), 6.64-6.72 (m, 1H), 6.87 (m,
36
1
and are uncorrected. The term room temperature means
6
o
2
0-30 C. All products were identified through their
spectroscopic data and the melting-points which were
confirmed by comparison with those reported in the
literature.
1
3
2H), 9.61 (br s, 1H, NH), 10.33 (br s, 1H, NH). C NMR
(62.5 MHz, DMSO-d ) d 14.1, 17.2, 53.6, 59.6, 100.6,
6

Vol. 22, No. 7, 2011
Canto et al.
1385
1
1
1
1
01.0, 106.7, 108.1, 119.6, 137.4, 145.0, 146.7, 147.3,
5-Ethoxycarbonyl-6-methyl-4-(3-hidroxyphenyl)-3,4-
-1
5
65.0, 173.9. IR (neat) nmax/cm : 3312, 3176, 2981, 1662,
574, 1484, 1445, 1371, 1336, 1266, 1235, 1189, 1110,
038, 938, 917, 815, 748, 657.
dihydropyrimidin-2-(1H)-one (2a)
1
mp 163-166 ºC. H NMR (300 MHz, DMSO-d ) d 1.11
6
(t, 3H, J 7.0 Hz), 2.22 (s, 3H), 3.98 (q, 2H, J 7.0 Hz), 5.04
(
s, 1H), 6.64-7.09 (m, 4H), 7.64 (s, 1H, NH), 9.15 (s, 1H,
13
5
-Ethoxycarbonyl-6-methyl-4-(3-nitrophenyl)-3,4-dihydro-
NH). C NMR (75 MHz, DMSO-d ) d 14.2, 17.6, 52.9,
6
38
pyrimidin-2-(1H)-thione (1d)
59.2, 100.1, 113.2, 114.9, 116.7, 129.4, 147.0, 148.5, 152.2,
1
-1
mp 206-209 ºC. H NMR (250 MHz, DMSO-d )
157.3, 165.9. IR (neat) nmax/cm : 3513, 3341, 3237, 3116,
1723, 1675, 1633, 1599, 1452, 1296, 1218, 1089, 1026,
872, 775, 701.
6
d 1.13 (t, 3H, J 7.1 Hz), 2.34 (s, 3H), 4.05 (q, 2H,
J 7.1 Hz), 5.36 (d, 1H, J 3.6 Hz), 7.70-7.72 (m, 2H), 8.10-
8
1
.11 (m, 1H), 8.17-8.20 (m, 1H), 9.81 (br s, 1H, NH),
1
3
0.55 (br s, 1H, NH). C NMR (62.5 MHz, DMSO-d )
5-Ethoxycarbonyl-6-methyl-4-phenyl-3,4-dihydro-
6
34
d 14.0, 17.3, 53.5, 59.8, 99.8, 121.2, 122.8,130.5, 133.0,
pyrimidin-2-(1H)-one (2b)
-
1
1
1
3
1
45.5, 146.0, 147.8, 164.9, 174.5. IR (neat) nmax/cm :
175, 2990, 1707, 1659, 1593, 1529, 1472, 1343, 1278,
184, 1100, 893, 727, 688.
mp 210-212 ºC. H NMR (300 MHz, DMSO-d ) d 1.09
6
(t, 3H, J 7.1 Hz), 2.25 (s, 3H), 3.98 (q, 2H, J 7.1 Hz), 5.15
(d, 1H, J 2.9 Hz), 7.22-7.32 (m, 5H), 7.74 (br s, 1H, NH),
13
9
.20 (br s, 1H, NH). C NMR (75 MHz, DMSO-d ) d 12.2,
6
5
-Ethoxycarbonyl-6-methyl-4-(4-cyanophenyl)-3,4-
16.0, 52.1, 57.3, 97.4, 124.4, 125.3, 126.4, 143.1, 145.2,
150.2, 163.3. IR (neat) nmax/cm : 3237, 3113, 2975, 1722,
1697, 1643, 1454, 1419, 1313, 1290, 1217, 1086, 879,
3
9
-1
dihydropyrimidin-2-(1H)-thione (1e)
o
1
mp 130-133 C; H NMR (300 MHz, DMSO-d ) d 1.09
6
(
(
t, 3H, J 7.0 Hz), 2.30 (s, 3H), 4.00 (q, 2H, J 7.0 Hz), 5.24
d, 1H, J 3.5 Hz), 7.40 (d, 2H, J 8.3 Hz), 7.83 (d, 2H,
772, 756, 697, 660.
J 8.3 Hz), 9.74 (br s, 1H, NH), 10.47 (br s, 1H, NH).
5-Ethoxycarbonyl-6-methyl-4-(3,4-methylenedioxyphenyl)-
13
37
C NMR (75 MHz, DMSO-d ) d 14.0, 17.2, 53.8, 59.8,
3,4-dihydropyrimidin-2-(1H)-one (2c)
6
1
9
1
1
1
9.8, 127.5, 129.9, 132.7, 133.2, 138.8, 145.9, 148.5, 164.9,
mp 188-190 ºC. H NMR (300 MHz, DMSO-d ) d 1.11
6
-1
74.5. IR (neat) nmax/cm : 3299, 3274, 2984, 2239, 1651,
557, 1456, 1370, 1320, 1282, 1198, 1167, 1108, 1032,
003, 842, 758, 611.
(t, 3H, J 7.0 Hz), 2.25 (s, 3H), 3.99 (q, 2H, J 7.0 Hz), 5.08
(d, 1H, J 2.6 Hz), 5.98 (s, 2H), 6.69-6.75 (m, 2H), 6.84-
6.86 (m, 1H), 7.71 (br s, 1H, NH), 9.20 (br s, 1H, NH).
13
C NMR (75 MHz, DMSO-d ) d 14.1, 17.8, 53.7, 59.2,
6
5
-Ethoxycarbonyl-6-methyl-4-(4-N,N-dimethyl-
99.3, 101.0, 106.7, 108.0, 119.4, 138.9, 146.4, 147.3, 148.3,
152.1, 165.4. IR (neat) nmax/cm : 3354, 3221, 3104, 2965,
1688, 1637, 1488, 1446, 1373, 1295, 1242, 1223, 1167,
37
-1
aminophenyl)-3,4-dihydropyrimidin-2-(1H)-thione (1f)
1
mp 206-208 ºC. H NMR (300 MHz, DMSO-d )
6
d 1.11 (t, 3H, J 7.0 Hz), 2.28 (s, 3H), 2.85 (s, 6H), 3.97 (q,
1090, 1039, 928, 810, 794, 674.
2H, J 7.0 Hz), 5.04 (d, 1H, J 3.2 Hz), 6.66 (d, 2H, J 8.5
Hz), 7.01 (d, 2H, J 8.5 Hz), 9.55 (br s, 1H, NH), 10.24 (br
5-Ethoxycarbonyl-6-methyl-4-(3-nitrophenyl)-3,4-
1
3
38
s, 1H, NH). C NMR (75 MHz, DMSO-d ) d 14.1, 17.1,
dihydropyrimidin-2-(1H)-one (2d)
6
1
4
1
1
8
0.1, 53.5, 59.5, 101.3, 112.2, 127.1, 131.2, 144.3, 150.0,
mp 225-227 ºC. H NMR (250 MHz, DMSO-d ) d 1.08
6
-1
65.3, 173.8. IR (neat) nmax/cm : 3322, 3169, 2982, 1666,
576, 1523, 1462, 1364, 1327, 1284, 1182, 1116, 1023,
04, 755, 650.
(t, 3H, J 6.9 Hz), 2.26 (s, 3H), 3.98 (q, 2H, J 6.9 Hz), 5.29
(s, 1H), 7.64-8.10 (m, 4H), 8.90 (s, 1H, NH), 9.37 (s, 1H,
13
NH); C NMR (62.5 MHz, DMSO-d ) d 14.0, 17.9, 53.6,
6
5
9.4, 98.4, 121.2, 122.3, 130.0, 133.0, 147.0, 147.8, 149.3,
-1
5
-Ethoxycarbonyl-6-methyl-4-(2-fluorphenyl)-3,4-
151.9, 165.0. IR (neat) nmax/cm : 3326, 3090, 2963, 1706,
1686, 1626, 1523, 1456, 1345.1, 1310, 1266, 1221, 1086,
900, 816, 794, 739, 685.
41
dihydropyrimidin-2-(1H)-thione (1g)
1
mp 140-143 ºC. H NMR (300 MHz, DMSO-d ) d 1.03
6
(t, 3H, J 7.0 Hz), 2.30 (s, 3H), 3.93 (q, 2H, J 7.0 Hz), 5.45
(
d, 1H, J 3.1 Hz), 7.13-7.34 (m, 4H), 9.59 (s, 1H, NH),
5-Ethoxycarbonyl-6-methyl-4-(4-cyanophenyl)-3,4-
13
12f
1
1
1
3
1
0.37 (s, 1H, NH); C NMR (75 MHz, DMSO-d ) d 14.5,
dihydropyrimidin-2-(1H)-one (2e)
6
o
1
7.8, 49.4, 60.2, 100.0, 116.1 , 116.4, 125.3, 129.9, 130.5,
mp 130-133 C; H NMR (300 MHz, DMSO-d ) d 1.07
6
-
1
31.27, 146.1, 165.5, 174.7. IR (neat) nmax/cm : 3184,
007, 1716, 1653, 1583, 1479, 1380, 1318, 1264, 1185,
102, 846, 760, 744, 646.
(t, 3H, J 7.1 Hz), 2.25 (s, 3H), 3.97 (q, 2H, J 7.1 Hz), 5.21
(s, 1H), 7.42 (d, 2H, J 8.1 Hz), 7.80 (d, 2H, J 8.1 Hz),
13
7.88 (s, 1H, NH), 9.33 (s, 1H, NH); C NMR (75 MHz,

1
386
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity
J. Braz. Chem. Soc.
3
DMSO-d ) d 14.5, 18.3, 54.3, 59.8, 98.7, 110.5, 119.2,
Glioma cells were plated in a 96-well plate at 10 per
6
1
27.8, 133.0, 149.8, 150.5, 152.3, 165.6. IR (neat)
well and, after reaching semi-confluence, the cultures were
treated with monastrol 1a for 24, 48 or 72 h or its analogues
1b-g, 2b-g for 48 h.After the end of treatment, each culture
medium containing the drug was removed and the cells
were washed twice with 100 mL of PBS. After removing
the PBS, 90 mL of culture medium and 10 mL of MTT were
added to each of the wells. The cells were incubated for 3 h
and the solution was then removed from the precipitate. A
total of 100 mL of DMSO were added to the wells and the
level of absorbance was read by an ELISA plate reader
at 490 nm. This absorbance was linearly proportional to
the number of live cells with active mitochondria. The
cell viability was calculated using the equation 1 bellow,
-1
nmax/cm : 3299, 2980, 2230, 1700, 1652, 1543, 1385, 1363,
251, 1200, 1075, 1019, 935, 825, 756.
1
5
-Ethoxycarbonyl-6-methyl-4-(4-N,N-dimethyl-
40
aminophenyl)-3,4-dihydropyrimi- din-2-(1H)-one (2f)
1
mp 257-259 ºC. H NMR (300 MHz, DMSO-d ) d 1.05
6
(
t, 3H, J 7.0 Hz), 2.23 (s, 3H), 2.84 (s, 6H), 4.00 (q, 2H,
J 7.0 Hz), 5.03 (d, 1H, J 3.1 Hz), 6.64 (d, 2H, J 8.5 Hz),
.04 (d, 2H, J 8.5 Hz), 7.59 (br s, 1H, NH), 9.09 (br s, 1H,
7
1
3
NH). C NMR (75 MHz, DMSO-d ) d 14.2, 17.7, 40.2,
6
5
1
1
7
3.3, 59.1, 99.9, 112.2, 126.9, 132.7, 147.6, 149.8, 152.3,
-1
65.5. IR (neat) nmax/cm : 3241, 3115, 2973, 1720, 1700,
647, 1525.9, 1456, 1365, 1290, 1219, 1088, 1047, 880,
84, 659.
whereAbs is the absorbance of cells treated with different
s
formulations and Abscontrol is the absorbance of negative
control cells (incubated with cell culture medium only,
equation 1).
5
-Ethoxycarbonyl-6-methyl-4-(2-fluorphenyl)-3,4-
12
dihydropyrimidin-2-(1H)-one (2g)
1
mp 235-237 ºC. H NMR (300 MHz, DMSO-d ) d 1.20
Cell viability (%) = (Abs /Abscontrol)100
(1)
6
s
(
(
t, 3H, J 7.1 Hz), 2.44 (s, 3H), 4.07 (q, 2H, J 7.1 Hz), 5.62
s, 1H), 7.28-7.48 (m, 4H), 7.87 (s, 1H, NH), 9.43 (s, 1H,
Cell counting
1
3
NH); C NMR (75 MHz, DMSO-d ) d 13.8, 17.5, 54.5,
6
6
1
1
1
1.1, 115.2, 122.4, 124.0, 127.8, 129.3, 135.8, 154.7, 157.2,
The human glioma cells (U138-MG) were seeded at
1 × 10 cells per well in DMEM/15% FBS in 24-well
plates, and allowed to grown. After reaching semi-
confluence, the glioma cells were treated with monastrol
-1
4
58.3, 160.3. IR (neat) nmax/cm : 3229, 3158, 2973, 1747,
699, 1616, 1539, 1436, 1385, 1319, 1229, 1199, 1144,
075, 933, 755.
(
1a) for 24, 48 or 72 h. The same procedure was repeated
Cytotoxicity assays
for the compounds 1b-g, 2b-g for 48 h. At the end of
the treatment, the medium was removed. The cells
were washed with phosphate buffered saline (PBS) and
200 μL of 0.25% trypsin/EDTA solution was added to
detach the cells, which were counted immediately in a
hemocytometer. The procedure was the same for the rat
Maintenance of cell lines
The human glioblastoma cell line U138-MG and the
rat glioma cell line C6 were obtained from American
Type Culture Collection (ATCC) (Rockville, Maryland,
USA). The cells were grown and maintained in Dulbecco’s
modified Eagle’s medium (DMEM) containing antibiotics
penicillin/streptomycin 0.5 U mL , and supplemented
with 5% (C6) or 15% (U138-MG) (v/v) fetal bovine
serum (FBS). Cells were kept at a temperature of 37 C, a
3
glioma cells (C6) except that they were seeded at 5 × 10
cells per well in DMEM/5% FBS.
-
1
Statistical analysis
o
minimum relative humidity of 95%, and an atmosphere of
Data are expressed as mean ± S.D. and analyzed for
statistical significance by one-way analysis of variance
(ANOVA) followed by post-hoc for multiple comparisons
5
% CO in air. All the experiments throughout this study
2
were conducted in serum supplemented DMEM.
(Tukey test) using a GraphPad Prism Software. Differences
Assessment of glioma cell viability
between mean values were considered significant when
P < 0.05.
The method MTT provides a quantitative measure of the
number cells with metabolically active mitochondria and
it is based on the mitochondrial reduction of a tetrazolium
bromide salt, MTT ([3-(4,5-dimethylthiazol-2-yl)-
Supplementary Information
2
,5-diphenyltetrazolium bromide]), to a chromophore,
Supplementary data are available free of charge at
http://jbcs.sbq.org.br as PDF file.
formazan product, whose absorbance can be determined
by spectrophotometric measurement.

Vol. 22, No. 7, 2011
Canto et al.
1387
Acknowledgments
P. S.; D’Oca, M. G. M.; Russowsky D.; Quim. Nova 2005,
2
8, 1010; Dondoni, A.; Massi, A.; Minghini, E.; Sabbatini, S.;
The authors are thankful to CNPq/MCT (VLEL-
Universal No. 477657/2008-7, PQ10/2008, 566201/2008
postgraduate, INCT-if (Instituto Nacional de Ciência e
Tecnologia para Inovação Farmacêutica) and PRONEX/
FAPERGS/CNPq and DR-Universal No. 484615/2007-6),
for support the program on the antitumoral agents.
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Sudalai, A.; Tetrahedron Lett. 2003, 44, 3305.
1
3. Kappe, C. O.; Falsone, S. F.; Synlett 1998, 718.
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9. Look, G. C.; Murphy, M. M.; Campbell, D. A.; Gallop, M. A.;
Tetrahedron Lett. 1995, 36, 2937.
40. Attaby, F. A.; Ramla, M. M.; Harukuni, T.; Phosphorus, Sulfur
Silicon Relat. Elem. 2008, 183, 2956.
0. DeWolfe, R. H.; Jensen, J. L.; J. Am. Chem. Soc. 1963, 85,
41. Gholap, A. R.; Venkatesan, K.; Daniel, T.; Lahoti, R. J.;
Srinivasan, K. V.; Green Chem. 2004, 6, 147.
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2. Ramu, E.; Kotra, V.; Bansal, N.; Varala, R.; Adapa, S. R.;
Rasāyan J. Chem. 2008, 1, 188.
Submitted: November 3, 2010
Published online: March 15, 2011

J. Braz. Chem. Soc., Vol. 22, No. 7, S1-S15, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
0
103 - 5053 $6.00+0.00
Supplementary Information
SI
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity against
the Human U138-MG and Rat C6 Glioma Cell Lines
a
b
b
Rômulo F. S. Canto, Andressa Bernardi, Ana Maria O. Battastini,
,
c
,a
Dennis Russowsky* and Vera Lucia Eifler-Lima*
a
Laboratório de Síntese Orgânica Medicinal/LaSOM, Programa de Pós Graduação em
Ciências Farmacêuticas, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752,
0610-00 Porto Alegre-RS, Brazil
9
b
Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul,
Rua Ramiro Barcelos, 2660, 90035-003 Porto Alegre-RS, Brazil
c
Instituto de Química, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500,
1501-970 Porto Alegre-RS, Brazil
9
1
Figure S1. H NMR (250 MHz, DMSO-d ) of compound 1a (monastrol).
6
*e-mail: dennis@iq.ufrgs.br, veraeifler@ufrgs.br

S2
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity
J. Braz. Chem. Soc.
13
Figure S2. C NMR (62.5 MHz, DMSO-d ) of compound 1a (monastrol).
6
-
1
Figure S3. IR (ATR, cm ) of compound 1a (monastrol).

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1
Figure S4. H NMR (300 MHz, DMSO-d ) of compound 1b.
6
13
Figure S5. C NMR (75 MHz, DMSO-d ) of compound 1b.
6

S4
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity
J. Braz. Chem. Soc.
-
1
Figure S6. IR (ATR, cm ) of compound 1b.
1
Figure S7. H NMR (250 MHz, DMSO-d ) of compound 1c.
6

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13
Figure S8. C NMR (62.5 MHz, DMSO-d ) of compound 1c.
6
-
1
Figure S9. IR (ATR, cm ) of compound 1c.

S6
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity
J. Braz. Chem. Soc.
1
Figure S10. H NMR (250 MHz, DMSO-d ) of compound 1d.
6
13
Figure S11. C NMR (62.5 MHz, DMSO-d ) of compound 1d.
6

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-
1
Figure S12. IR (ATR, cm ) of compound 1d.
Figure S13. HRMS of compound 1d.

S8
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity
J. Braz. Chem. Soc.
1
Figure S14. H NMR (300 MHz, DMSO-d ) of compound 1e.
6
13
Figure S15. C NMR (75 MHz, DMSO-d ) of compound 1e.
6

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-
1
Figure S16. IR (ATR, cm ) of compound 1e.
1
Figure S17. H NMR (300 MHz, DMSO-d ) of compound 1f.
6

S10
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity
J. Braz. Chem. Soc.
13
Figure S18. C NMR (75 MHz, DMSO-d ) of compound 1f.
6
-
1
Figure S19. IR (ATR, cm ) of compound 1f.

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Figure S20. HRMS of compound 1f.
1
Figure S21. H NMR (300 MHz, DMSO-d ) of compound 1g.
6

S12
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity
J. Braz. Chem. Soc.
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Figure S22. C NMR (75 MHz, DMSO-d ) of compound 1g.
6
-
1
Figure S23. IR (ATR, cm ) of compound 1g.

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Figure S24. HRMS of compound 1g.
1
Figure S25. H NMR (250 MHz, DMSO-d ) of compound 2d.
6

S14
Synthesis of Dihydropyrimidin-2-one/thione Library and Cytotoxic Activity
J. Braz. Chem. Soc.
13
Figure S26. C NMR (62.5 MHz, DMSO-d ) of compound 2d.
6
-
1
Figure S27. IR (ATR, cm ) of compound 2d.

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Figure S28. HRMS of compound 2d.