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K. E. Machado et al. / Bioorg. Med. Chem. 19 (2011) 6285–6291
4.1.7. 4-[2-(1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N’-
[(1E)-(4-hydroxyphenyl)methylene]benzenesulfonyl hydrazone
(14)
N, 10.21; O, 17.49; S, 5.84. Found: C, 56.88; H, 3.10; Cl, 6.22; N,
10.25; O, 17.42; S, 5.91. Unpublished compound.
Sulfonyl-hydrazide 11 (400 mg, 1.00 mmol) was added to a
4.2. Cell culture and viability assay (MTT assay)31
solution of p-hydroxybenzaldehyde (100 lL, 1.00 mmol) and 2
drops of hydrochloride acid concentrate in 20 ml of ethanol. The
reaction was carried out as described for compound 13. Yield:
Murine B16F10 melanoma cells (ATCC, Manassas, VA) main-
tained in Dulbecco’s modified Eagle’s medium were cultured in
DMEM (GIBCO, São Paulo, SP, Brazil) supplemented with 10% fetal
calf serum, 100 U/ml penicillin, 100 lg/ml streptomycin and
10 mM HEPES, pH 7.4 at 37 °C in a 5% CO2 humidified atmosphere
77%. Mp: 163.6–165.8 °C. IR (KBr): 3186(
N(C@O)2], 1596 ( Ph–OH), 1353 and 1164 (
(Ar.) cmÀ1 1H NMR (200 MHz, DMSO-d6) d: 2.31–2.51 (t, 2H,
m
NH), 1701 and 1652
[m
m
m
–SO2–), 780
.
J = 8.2 Hz, –CH–Ph), 5.13 (t, 2H, J = 8.2 Hz, –CH2–N–C@O), 6.42–
6.61 (m, 4H, Ar), 7.46–7.53 (m, 6H, ArH.), 7.91–8.03 (m, 6H, ArH,
–N@CH– and –OH), 11.55 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) d 33.38 (1C, NCH2CH2), 44.51 (1C, NCH2CH2); 121.83
(2C, Ar), 124.57 (2C, Ar), 125.71 (2C, Ar), 127.05 (2C, Ar), 127.64
(1C, Ar), 127.97 (1C, Ar), 128.48 (1C, Ar), 129.38 (2C, Ar), 131.22
(2C, Ar), 134.35 (1C, Ar), 137.23 (2C, Ar), 139.33 (2C, Ar), 144.32,
(1C, Ar), 147.56 (1C, C@N), 159.44 (1C, OH–C Ar), 163.22 (2C,
C@O). Anal. Calcd for C27H21N3O5S: C, 64.92; H, 4.24; N, 8.41; S,
6.42. Found: C, 64.93; H, 4.29; N, 8.49; S, 6.31. Unpublished
compound.
in plastic culture flasks. Test compounds were added to cells in a
maximum volume of 20 ll. For compounds soluble in DMSO, the
same volume of the solvent was added to control wells. Treatments
with the imides at indicated concentrations were carried out until
12, 24, 48 and 72 h. Cell viability was assessed by using MTT (3-
(4,5-dimethiazol-zyl)-2-5-diphenyltetrazolium bromide, Sigma
Chemical Co., St. Louis, MO, USA) assay.
4.3. Cell cycle analysis
To assess cell cycle arrest, a PI/RNase solution kit (Immunostep,
Salamanca, Spain) was used. Cells (1 Â 106 cells/well) were incu-
bated with vehicle or cyclic imide 4 at 77.75 1.3 lM. After 24 h
4.1.8. 4-[2-(1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N’-
[(1E)-(4-nitrophenyl)methylene]benzenesulfonyl hydrazone
(15)
Sulfonyl-hydrazide 11 (400 mg, 1.00 mmol) was added to a solu-
tion of p-nitrobenzaldehyde (151 lg, 1.00 mmol) and 2 drops of
of incubation, cells were harvested and cell cycle analysis was as-
sessed according to the kit protocol. Briefly, cells were washed
with PBS buffer, fixed with 70% ethanol, washed with PBS buffer
supplemented with 2% bovine albumin and stained with 500 ll
PI/RNase solution. Analysis was performed by flow cytometry
(FACSCantoII™, Becton Dickinson Immunocytometry Systems).
The data were analyzed by WinMID software.
hydrochloride acid concentrate in 20 ml of ethanol. The reaction
was carried out as described for compound 13. Yield: 67%. Mp:
193.7–195.0 °C. IR (KBr): 3189(
N(C@O)2], 1517 and 1341 ( –NO2), 1341 and 1162 (
(Ar.) cmÀ1 1H NMR (200 MHz, DMSO-d6) d: 2.98–3.02 (t, 2H,
m
NH), 1697 and 1654
[m
m
m
SO2), 776
4.4. Analysis of the apoptotic effects with ethidium bromide
and acridine orange42
.
J = 8.0 Hz, –CH2–Ph), 4.21–4.26 (t, 2H, J = 8.0 Hz, –CH2–N–C@O–),
7.20–7.22 (d, 2H, J = 10.0 Hz, ArH.), 7.49–7.53 (m, 2H, ArH), 7.76–
7,86 (m, 4H, ArH), 7.99 (s, 1H, –N@C–), 8.18–8.21 (d, 2H,
J = 10.0 Hz,ArH), 8.33 (m, 4H, ArH.), 11.93 (s, 1H, NH). 13C NMR
(50 MHz, DMSO-d6) d: 33.55 (1C, NCH2CH2), 41.91 (1C, NCH2CH2);
121.93 (2C, Ar), 122.04 (2C, Ar), 124.27 (2C, Ar), 126.07 (2C, Ar),
127.43 (2C, Ar), 127.51 (1C, Ar), 127.96 (1C, Ar), 128.37 (2C, Ar),
129.97 (1C, Ar), 131.42 (2C, Ar), 134.69 (1C, Ar), 137.04 (2C, Ar),
140.00 (1C, Ar), 145.02 (1C, C@N), 148.10 (1C, NO2–C Ar), 163.57
(2C, C@O). Anal. Calcd for C27H20N4O6S: C, 61.36; H, 3.81; N,
10.60; S, 6.07. Found: C, 61.49; H, 3.85; N, 10.65; S, 6.22. Unpub-
lished compound.
For determination of apoptotic death with ethidium bromide
and acridine orange, 1 Â 106 cells/well were incubated with cyclic
imide 4 at 77.75 1.3 lM. After 24 h, the coverslips covering the
bottom of the plate were removed, washed with PBS and covered
with a solution of ethidium bromide and acridine orange (1:1) at
1% concentration. Cells were analyzed by a fluorescence micro-
scope (Olympus BX41).
4.5. Analysis of the apoptotic effects with annexin43
For determination of apoptotic death, an Annexin V-FITC Apop-
tosis Detection kit was used according to the manufacturer’s
instructions. 1 Â 106 cells/well were incubated with cyclic imide
4.1.9. 4-[(6-Chloro-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-
yl)methyl]-N’-[(1E)-(4-nitrophenyl)methylene]benzenesulfonyl
hydrazone (16)
4 at 77.75 1.3 lM. After 12 h of incubation, cells were harvested,
washed with PBS buffer, annexin buffer (1:10) and double-stained
with Annexin V-FITC solution. After incubation, 300 l of annexin
l
Sulfonyl-hydrazide 12 (400 mg, 0.96 mmol) was added to a
buffer was added and fluorescence was analyzed by flow cytome-
try. Analyses were performed by flow cytometry (FACSCanto™,
Becton Dickinson Immunocytometry Systems). The data were ana-
lyzed by WinMID software.
solution of p-nitrobenzaldehyde (145 lg, 0.96 mmol) and 2 drops
of hydrochloride acid concentrate in 20 ml of ethanol. The reaction
was carried out as described for compound 13. Yield: 60%. Mp:
204.4–206.8 °C. IR (KBr): 3435
(
m
NH), 1696 and 1657
[m
N(C@O)2], 1341 and 1166 ( SO2), 848 (
m
m
ArH) cmÀ1 1H NMR
.
Acknowledgments
(200 MHz, DMSO-d6) d 4.19 (s, 2H, CH2), 7.55 (s, 1H, N@CH),
7.60–7.53 (m, 3H, ArH), 7.83–7.73 (m, 4H, ArH), 7.97 (s, 1H,
ArH); 8.43–8.12 (m, 5H, ArH); 11.09 (s, 1H, NH). 13C NMR
(50 MHz, DMSO-d6) d 33.76 (1C, CH2); 121.74 (2C, Ar), 123.03
(2C, Ar), 124.56 (1C, Ar), 126.43 (1C, Ar), 127.71 (1C, Ar), 128.27
(2C, Ar), 128.70 (2C, Ar), 128.95 (2C, Ar), 129.15 (1C Ar), 130.69
(1C, Ar), 131.53 (1C, Ar), 132.23 (1C, Ar), 138.23 (1C Ar), 140.02
(1C Ar), 140.37 (1C, N–CH2–Ar), 145.07 (1C, –Cl–C Ar); 146.50
(1C, C@N); 148.39 (1C, NO2–C Ar); 163.15 (1C, C@O); 163.43 (1C,
C@O). Anal. Calcd for C26H17ClN4O6S: C, 56.89; H, 3.12; Cl, 6.46;
This research was supported by grants and scholarship from
CNPq (Conselho Nacional de Desenvolvimento Científico e Tec-
nológico), and CAPES (Coordenação de Pessoal de Nível Superior).
References and notes
1. Alonso, S. R.; Otiz, P.; Pollán, M.; Pérez-Gomez, B.; Sánchez, L.; Acuña, M. J.;
Pajares, R.; Martínez-Tello, F. J.; Hortelano, C. M.; Piris, M. A.; Rodríguez-
Peralto, J. L. Am. J. Pathol. 2004, 164, 193.