D. L. da Silva et al. / Bioorg. Med. Chem. 20 (2012) 2645–2650
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while NCI-H460 cells were sensitive to three adducts (BA2, BA24
and BA33). Only 786-0, NCI-H460 and PC-3 cells were sensitive
to monastrol (BA2), a known anticancer agent, at a concentration
1330, 1278, 1217, 1184, 1148, 1119, 859, 821, 789, 753, 672. 1H
NMR (DMSO-d6): d 1.14 (t, 3H, J = 7.0 Hz, CH3), 2.28 (s, 3H, CH3),
3.71 (s, 6H, 2 Â OCH3), 4.04 (q, 2H, J = 7.0 Hz, CH2), 5.11 (d, 1H,
JH-NH = 3.2, CH), 6.46 (s, 2H, CHAR), 8.42 (s, 1H, OH), 9.58 (s, 1H,
NH), 10.29 (s, 1H, NH); 13C NMR (DMSO-d6): d 14.1 (CH3), 17.1
(CH3), 53.9 (CH), 56.0 (2 Â OCH3), 59.6 (CH2), 100.9 (C), 103.9
(2 Â CHAR), 133.7 (CAR), 135.3 (CAR), 144.7 (C), 147.9 (2 Â CHAR),
165.3 (COO), 174,3 (C(S)). Anal. calcd. for C16H20N2SO5: C, 54.53;
H, 5.72; N, 7.95. Found: C, 54.16; H, 5.63; N, 7.49.
lower than 10 lg/mL. Interestingly, the adduct BA33 was 12-fold
more potent than monastrol against PC-3 cells while BA24 and
BA33 were threefold more potent on NCI-H460 cells (Table 3).
Among the tested compounds, BA24 exhibited a large spectrum
of action affecting all the cell lines at concentrations lower than
10 lg/mL (Table 3). The Biginelli adducts in this study were more
active against cancer cells than those previously reported by
Russowsky et al.21 Overall, the potency of Biginelli adducts was
dependent on the histological origin of cancer cells.
4.2. Biological activities
4.2.1. Scavenging of reactive nitrogen species
The ability of Biginelli adducts BA2 and BA9-BA33 to scavenge
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, a reactive nitrogen
species (RNS), was determined according to Gülcin,25 with modifi-
cations. The screening of potential candidates was done by incu-
3. Conclusion
The potential to scavenge reactive nitrogen/oxygen species
(RNS and ROS) and the ability to inhibit the growth of cancer cells
were investigated for 26 Biginelli adducts. Among them, BA14,
BA15, BA22 and BA23 were found to be promising RNS and ROS
scavengers, being indeed more potent than the known antioxidant
agent resveratrol. The uncontrolled production of RNS/ROS leads to
cellular damages that may result in a series of diseases.24 Thus,
such adducts may be used as lead compounds for the design of
effective antioxidant agents. Sixteen monastrol-analogs had their
antiproliferative activity against cancer cells evaluated for the first
time. The capacity to inhibit cancer cells growth of the Biginelli ad-
ducts was dependent on the histological origin of cells. The adduct
BA24 was the only exception, being active against all cell lines at
bating each compound-test (160
lM) in an ethanolic medium
containing 100 M DPPH. The systems were maintained under
l
stirring and absence of light for 30 min and the absorbance re-
corded at 517 nm. Those compounds with potential scavenging
activity were then tested in the range of 0–160 lM to determine
the concentration necessary to scavenge DPPH radicals by 50%
(SC50). The best compounds were subjected to kinetic studies at
the respective SC50 values. Results presented are from three inde-
pendent experiments, each done in triplicate.
4.2.2. Scavenging of reactive oxygen species
The capacity of Biginelli adducts to scavenge superoxide anions
concentrations lower than 10 lg/mL. For each cancer cell studied,
(ÁO2À) was evaluated in 50 mM phosphate buffer (pH 7.8) contain-
at least two adducts were more potent than monastrol (BA2).
Notably, BA20 and BA33 were as potent and the reference drug
doxorubicin against NCI-ADR/RES and PC-3, respectively. These re-
sults disclose some monastrol analogs as lead compounds for
obtaining new anticancer agents.
ing 13 mM
EDTA, 2 M riboflavin and compound-test at 0–200
mixtures were incubated for 10 min at 25 °C in the presence of
L-metionine, 75
lM nitroblue tetrazolium, 100
lM
l
lM. Reaction
Á
À
fluorescent light to induce O2 formation. Controls consisted of
reaction mixtures kept at 25 °C for 10 min in absence of light.
À
The percentage of ÁO2 scavenged by each compound-test was
4. Experimental
4.1. Chemistry
determined through spectrophotometric analysis at 575 nm. Re-
sults presented are from three independent experiments, each
done in triplicate.
4.1.1. General procedures
4.2.3. Antiproliferative assay
All chemicals were obtained from commercially available
sources and used without further purification. Anhydrous condi-
tions were not required for the reaction. Melting points (uncor-
rected) were determined on Mettler FP 80 HT apparatus.
Elemental analyses were performed on CHN Perkin-Elmer 2400
apparatus. Infrared spectra were recorded on Perkin Elmer, Spec-
trum One spectrophotometer (ATR). 1H and 13C NMR spectra were
recorded on a Bruker AVANCE DPX-200 spectrometry at 200 MHz
and 50 MHz respectively, in DMSO-d6.
Human tumor cell lines U251 (glioma), NCI-ADR/RES (multiple
drugs-resistant ovarian), 786-0 (renal), NCI-H460 (lung, non-small
cells), PC-3 (prostate), OVCAR-03 (ovarian) and HT-29 (colon) were
kindly provided by Frederick Cancer Research & Development Cen-
ter—National Cancer Institute—Frederick, MA, USA. Stock cultures
were grown in RPMI 1640 (GIBCO BRL, Life Technologies) supple-
mented with 5% of fetal bovine serum and penicillin (final concen-
tration of 1 mg/mL) and streptomycin (final concentration of
200 U/mL).26–28 Cells in 96-well plates (100
posed to Biginelli adducts (0.25–250 g/mL) for 48 h at 37 °C and
lL cells/well) were ex-
l
4.1.2. Synthesis of DHPMs
5% of CO2. Afterward cells were fixed with 50% trichloroacetic acid,
submitted to sulforhodamine B assay for cell proliferation quanti-
tation at 540 nm.23 The concentration of compound that inhibits
cell growth by 50% (GI50) was determined through non-linear
regression analysis using software ORIGIN 7.5 (OriginLab Corpora-
tion). Doxorubicin was used as a reference drug. Results presented
are from two independent experiments, each done in triplicate.
Aldehydes (3 mmol), ethyl acetoacetate (4.5 mmol) and
(thio)urea (4.5 mmol) were dissolved in 3 mL of ethanol containing
p-sulfonic acid calix[4]arene (0.5 mol %). The mixture was heated
under reflux and stirred for 8 h.20 All DHPMs were characterized
by NMR (1H and 13C), infrared, melting point and elemental analy-
sis. Characterization data for compounds BA9, BA11-BA14, BA16-
BA21, BA24-BA28, BA30 and BA32 were recently reported by da
Silva et al.20 Data for compounds BA2, BA10, BA15, BA22, BA29,
BA31 and BA33 are listed as Supplementary data.
Acknowledgments
Authors are thankful to the financial support provided by Fun-
dação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG),
Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível
4.1.2.1. Ethyl 4-(4-hydroxy-3,5-dimethoxyphenyl)-6-methyl-
2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (BA23).
Yield: 80%. Yellow solid. Mp 189–191 °C. IR (ATR, cmÀ1): 3473,
3326, 3193, 2945, 2840, 1663, 1619, 1516, 1454, 1432, 1371,