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M. Szumilak et al. / European Journal of Medicinal Chemistry 45 (2010) 5744e5751
3.38e3.47 (m, 4H, C-1xH2, C-1xxH2), 7.69 (dd, J ¼ 8.6, 1.5 Hz, 2H,
C-8H, C-80H), 7.97 (bs, 2H, C-4NH2, C-40NH2), 8.14 (d, J ¼ 8.6 Hz, 2H,
C-7H, C-70H), 8.19 (s, 2H, C-5H, C-50H), 9.07 (bs, 2H, C-4NH2, C-
(DMSO) dC: 25.88, 27.72, 35.96, 67.83, 69.78, 122.91, 126.50, 127.92,
133.50, 135.89, 144.03, 163.16, 165.78 ppm. Anal. Calcd for
C26H26N4O10: C, 56.31; H, 4.69; N, 10.10; Found: C, 56.28; H, 4.39; N,
10.18.
40NH2), 9.28 (t, J ¼ 5.9 Hz, 2H, 2 CONH) ppm. 13C NMR (DMSO) dC
:
21.52, 26.89, 37.29, 41.64, 55.43, 115.91, 120.85, 127.46, 128.34,
133.27, 138.34, 143.06, 147.27, 167.10 ppm. Anal. Calcd for
C27H33N8O4$H2O: C, 60.77; H, 6.61; N, 23.62; Found: C, 60.92; H,
6.40; N, 23.65.
6.1.4.6. N,N0-Di(3-nitrophthaloyl)-3,30-diamino-N-methyldipropyl-
amine (8c). Yield: 52%, m.p. 148.4e150.1 ꢁC (EtOH); 1H NMR
(DMSO) dH: 1.76e1.89 (m, 4H, C-2xH2, C-2xxH2), 2.16 (s, 3H, CH3),
2.41 (t, J ¼ 7.0 Hz, 4H, C-3xH2, C-3xxH2), 3.66e3.73 (m, 4H, C-1xH2,
C-1xxH2), 8.0e8.1 (m, 2H, C-4H, C-40H), 8.13 (dd, J ¼ 7.5, 0.9 Hz, 2H,
C-3H, C-30H), 8.22 (dd, J ¼ 8.0, 0.9 Hz, 2H, C-5H, C-50H) ppm. 13C
NMR (DMSO) dC: 22.68, 35.41, 52.65, 123.02, 126.62, 128.02, 134.57,
135.98, 144.07, 163.26, 165.90 ppm. Anal. Calcd for C23H21N5O8: C,
55.75; H, 4.27; N, 14.13; Found: C, 55.58; H, 4.32; N, 13.98.
6.1.4. General procedure for the synthesis of 7aec and 8aec
To a solution of phthalic anhydride (10 mmol) or 3-nitrophthalic
anhydride (10 mmol) in acetic acid (15 ml) appropriate polyamines
(5 mmol) were added. The reaction was refluxed for 7 h, poured
into ice bathing water. The precipitate was filtered off and crys-
tallized from appropriate solvent.
6.2. Bioassays
6.1.4.1. N,N0-Diphthaloyl-1,4-bis(3-aminopropyl)piperazine
(7a). Yield: 86.9%, m.p. 178.3e179.9 ꢁC (EtOH); 1H NMR (CDCl3) dH
:
6.2.1. Cell line and cell culture conditions
1.74e1.86 (m, 4H, C-2xH2, C-2xxH2), 2.03e2.64 (cluster, 8H pipera-
zine CH2, 4H, C-3xH2, C-3xxH2), 3.72 (t, J ¼ 7.0 Hz, 4H, C-1xH2,
C-1xxH2), 7.64e7.74 (m, 4H, C-4H, C-40H, C-5H, C-50H), 7.76e7.90
(m, 4H, C-3H, C-30H, C-6H, C-60H) ppm. 13C NMR (CDCl3) dC: 25.41,
36.89, 53.15, 56.21, 123.20, 132.38, 133.84, 168.44 ppm. Anal. Calcd
for C26H28N4O4: C, 67.75; H, 6.08; N, 12.17; Found: C, 67.45; H, 6.18;
N, 12.08.
A375, a human melanoma cell line with high metastatic potential
was maintained in RPMI 1640 medium (Lonza, Switzerland), sup-
plemented with 10% fetal bovine serum (FBS), penicillin (10 U/ml)
and streptomycin (50 mg/ml) in standard conditions. For experi-
ments, culture medium was substituted with fresh medium con-
taining 0.5% FBS. In all experiments, cells in logarithmic phase of
growth were used.
6.1.4.2. N,N0-Diphthaloyl-4,9-dioxa-1,12-dodecanediamine
(7b). Yield: 94.8%, m.p. 117.0e118.8 ꢁC (EtOH); 1H NMR (CDCl3) dH
:
6.2.2. Cell proliferation analysis
1.46e1.64 (m, 4H, C-5xH2, C-6xH2), 1.89e1.98 (m, 4H, C-2xH2, C-
9xH2), 3.32 (t, J ¼ 6.0 Hz, 4H, C-1xH2, C-10xH2), 3.45 (t, J ¼ 6.1 Hz, 4H,
C-3xH2, C-8xH2), 3.79 (t, J ¼ 6.7 Hz, 4H, C-4xH2, C-7xH2), 7.68e7.73
(m, 4H, C-4H, C-40H, C-5H, C-50H), 7.81e7.87 (m, 4H, C-3H, C-30H,
C-6H, C-60H) ppm. 13C NMR (CDCl3) dC: 26.55, 28.94, 35.97, 68.55,
70.92, 123.22, 132.30, 133.89, 168.42 ppm. Anal. Calcd for
C26H28N2O6: C, 67.17; H, 6.03; N, 6.03; Found: C, 67.18; H, 5.96; N,
6.09.
To determined IC50 value (concentration of tested compounds
causing 50% inhibition of cell growth) colorimetric MTT assay was
used. A375 cells were seeded in 24-well plates and 6 h later
adherent cells were exposed to tested compounds at different
concentrations. After 44 h, the MTT reagent (thiazolyl blue tetra-
zolium bromide; SigmaeAldrich; 0.84 mg/ml in PBS) was added to
each well for additional 3 h. After removal of the medium, dimethyl
sulfoxide was added to each well to dissolve the blue formazan
crystals and the absorbance was determined at 540 nm. Data show
the mean of at least three independent experiments ꢀ SD. IC50
values were calculated by concentrationeresponse curve fitting
using a Microsoft Excel-based analytic method.
6.1.4.3. N,N0-Diphthaloyl-3,30-diamino-N-methyldipropylamine
(7c). Yield: 50.1%, m.p. 105.8e106.8 ꢁC (EtOH); 1H NMR (CDCl3) dH
:
1.73e1.88 (m, 4H, C-2xH2, C-2xxH2), 2.17 (s, 3H, CH3), 2.39 (t,
J ¼ 7.0 Hz, 4H, C-3xH2, C-3xxH2), 3.69e3.76 (m, 4H, C-1xH2, C-1xxH2),
7.65e7.74 (m, 4H, C-4H, C-40H, C-5H, C-50H), 7.77e7.87 (m, 4H,
C-3H, C-30H, C-6H, C-60H) ppm. 13C NMR (CDCl3) dC: 26.54, 36.59,
41.79, 55.43, 123.22, 132.27, 133.87, 168.39 ppm. Anal. Calcd for
C23H23N3O4: C, 68.41; H, 5.70; N, 10.40; Found: C, 68.11; H, 5.32; N,
10.39.
6.2.3. Cell cycle analysis
Flow cytometric analysis measuring cellular DNA content was
performed to evaluate the distribution of the cells through the cell
cycle phases. Melanoma cells A375 were incubated with tested
compounds at indicated concentrations for 24 h. Untreated and
treated cells were harvested, washed with PBS, fixed in ice-cold 70%
ethanol and stained with propidium iodide/RNAse solution (BD
Pharmingen, San Diego, CA, USA). The cell cycle profiles were
obtained by flow cytometry (BectoneDickinson FACSCalibur, San
Diego, CA, USA). Up to 10,000 cells per sample were analyzed.
ModFit LT 3.0 software was used to calculate the percentages of
cells in each cell cycle phase.
6.1.4.4. N,N0-Di(3-nitrophthaloyl)-1,4-bis(3-aminopropyl)piperazine
(8a). Yield: 86.6%, m.p. 203.0e204.6 ꢁC (DMF/H2O); 1H NMR
(DMSO) dH: 1.60e1.71 (m, 4H, C-2xH2, C-2xxH2), 1.91e2.18 (cluster,
8H piperazine CH2, 4H, C-3xH2, C-3xxH2), 3.60 (t, J ¼ 6.6 Hz, 4H,
C-1xH2, C-1xxH2), 7.98e8.08 (m, 2H, C-4H, C-40H), 8.15 (dd, J ¼ 7.5,
0.9 Hz, 2H, C-3H, C-30H), 8.25 (dd, J ¼ 8.0, 0.9 Hz, 2H, C-5H,
C-50H) ppm. 13C NMR (DMSO) dC: 25.73, 36.72, 52.54, 55.83, 122.89,
126.54, 127.87, 133.56, 135.82, 144.21, 163.12, 165.81 ppm. Anal.
Calcd for C26H26N6O8: C, 56.69; H, 4.72; N,15.25; Found: C, 56.58; H,
4.51; N, 15.35.
6.2.4. Cell death analysis by acridine orange/ethidium bromide
double staining
6.1.4.5. N,N0-Di(3-nitrophthaloyl)-4,9-dioxa-1,12-dodecanediamine
(8b). Yield: 75.17%, m.p. 104.6e106.0 ꢁC (EtOH); 1H NMR (DMSO)
dH: 1.20e1.35 (m, 4H, C-5xH2, C-6xH2), 1.66e1.90 (m, 4H, C-2xH2,
C-9xH2), 3.20 (t, J ¼ 6.7 Hz, 4H, C-1xH2, C-10xH2), 3.36 (t, J ¼ 6.8 Hz,
4H, C-3xH2, C-8xH2), 3.64 (t, J ¼ 6.8 Hz, 4H, C-4xH2, C-7xH2),
8.0e8.08 (m, 2H, C-4H, C-40H), 8.15 (dd, J ¼ 7.5, 0.9 Hz, 2H, C-3H,
C-30H), 8.26 (dd, J ¼ 8.0, 0.9 Hz, 2H, C-5H, C-50H) ppm. 13C NMR
The apoptotic and necrotic cells were monitored by double
staining with acridine orange and ethidium bromide using a fluo-
rescence microscope (Olympus BX 41, Japan). A375 cells were
cultured for 44 h with or without tested compounds at indicated
concentrations. To the combined cell populations (adherent and
floating) in 100
ml PBS, 20 ml of staining solution (1:1) mixture of