1004 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Antonini et al.
1
was stirred at room temperature for 1 h and then partitioned between
CHCl3 (30 mL) and an excess of 1 M aqueous Na2CO3 (2 × 30
mL). The organic layer was worked up to give a residue that was
purified by flash chromatography on a silica gel column eluted with
CHCl3/MeOH (30:1, v/v) to obtain 13c as a solid used as such for
nonane (10c). Yield 30%. mp 200–201 °C. H NMR (CDCl3) δ:
0.95 (t, 12H, 4 × CH3), 2.00 (m, 4H, 2 × CH2), 2.38 (s, 3H, 5-CH3),
2.43–2.60 (m, 8H, 4 × CH2), 2.65 (t, 4H, 2 × CH2), 2.95 (t, 4H,
2 × CH2), 3.53 (q, 4H, 2 × CH2), 4.44 (t, 4H, 2 × CH2), 6.79 (d,
2H, ar), 7.38 (d, 2H, ar), 8.24 (d, 2H, ar), 8.76 (d, 2H, ar), 9.22 (t,
2H, 2 × NH, ex), 9.61 (s, 2H, ar). Anal. Calcd (C45H55N11O2) C,
H, N.
1
the next step. Yield 45%. mp 135–137 °C. H NMR (CDCl3) δ:
0.92 (t, 6H, 2 × CH3), 2.44 (s, 3H, CH3), 2.56 (q, 4H, 2 × CH2),
3.01 (t, 2H, CH2), 4.57 (t, 2H, CH2), 7.28–7.40 (m, 2H, ar), 7.53
(d, 1H, ar), 7.81 (d, 1H, ar), 7.91–8.02 (m, 2H, ar), 8.08 (d, 1H,
ar), 8.82 (d, 1H, ar), 9.55 (s, 1H, ar).
Biophysical and Biological Evaluation. 1. Fluorescence Bind-
ing Studies. The fluorometric assays have been described previ-
ously.17 The C50 values for ethidium displacement from CT-DNA
and synthetic [poly(dA-dT)]2 (AT) and [poly(dG-dC)]2 (GC)
oligonucleotides were determined using aqueous buffer (10 mM
Na2HPO4, 10 mM NaH2PO4, and 1 mM EDTA at pH 7.0)
containing 1.26 µM ethidium bromide and 1 µM CT-DNA, AT,
and GC, respectively.17,18
All measurements were made in 10 mm quartz cuvettes at 20
°C using a Perkin-Elmer LS5 instrument (excitation at 546 nm and
emission at 595 nm) following serial addition of aliquots of a stock
drug solution [∼5 mM in dimethylsulfoxide (DMSO)]. The C50
values are defined as the drug concentrations that reduce the
fluorescence of the DNA-bound ethidium by 50% and are calculated
as the mean from three determinations.
The intermediate derivatives 13a, 13b, and 14a-14c were
prepared in a similar manner from 11a, 11d, and 11c.
1,9-Bis{2-[2-(dimethylamino)ethyl]-6-oxo-2,6-dihydroindazo-
lo[4,3-gh]isoquinolin-5-yl}-5-methyl-1,5,9-triazanonane (9a). Ex-
ample of the General Procedure for the Preparation of 9a-9c
and 10a-10c. A solution of bis(3-aminopropyl)ethylamine (0.035
mL, 0.19 mmol) and 13a (0.18 g, 0.39 mmol) in 2-ethoxyethanol
(5 mL) and triethylamine (0.5 mL) were heated at 120 °C for 5 h
under stirring. The resulting mixture was partitioned between CHCl3
(30 mL) and an excess of 1 M aqueous Na2CO3 (2 × 30 mL). The
organic layer was worked up to give a residue that was purified by
flash chromatography on a silica gel column eluted first with CHCl3/
MeOH (1:1 v/v) and then with CHCl3/MeOH (1:1 v/v) containing
32% aqueous NH3 (50 mL for 1 L of eluent) to obtain 9a. Yield
29%. mp 85–86 °C. 1H NMR (CDCl3) δ: 1.99 (m, 4H, 2 × CH2),
2.32 (s, 12H, 4 × CH3), 2.37 (s, 3H, 5-CH3), 2.63 (t, 4H, 2 ×
CH2), 2.88 (t, 4H, 2 × CH2), 3.52 (m, 4H, 2 × CH2), 4.48 (t, 4H,
2 × CH2), 6.77 (d, 2H, ar), 7.34 (d, 2H, ar), 8.23 (d, 2H, ar), 8.72
(d, 2H, ar), 9.20 (t, 2H, 2 × NH, ex), 9.60 (s, 2H, ar). Anal. Calcd
(C41H47N11O2) C, H, N.
The target compounds 9b, 9c, and 10a-10c were prepared in
the same experimental conditions from 13b, 13c, and 14a-14c,
respectively. All of the target compounds were converted to water-
soluble dimaleate salts by usual methods.6
Data for 1,9-Bis{2-[3-(dimethylamino)propyl]-6-oxo-2,6-di-
hydroindazolo[4,3-gh]isoquinolin-5-yl}-5-methyl-1,5,9-triaza-
nonane (9b). Yield 20%. Dimaleate mp 115–116 °C. 1H NMR
(DMSO-d6) δ: 1.98–2.21 (m, 4H, 2 × CH2), 2.42 (s, 12H, 4 ×
CH3), 2.51 (s, 3H, 5-CH3), 2.59 (t, 4H, 2 × CH2), 2.78 (t, 4H, 2 ×
CH2), 3.44–3.63 (m, 8H, 4 × CH2), 4.45 (t, 4H, 2 × CH2), 6.77
(d, 2H, ar), 7.37 (d, 2H, ar), 8.23 (d, 2H, ar), 8.75 (d, 2H, ar), 9.22
(t, 2H, 2 × NH, ex), 9.60 (s, 2H, ar). Anal. Calcd (C43H51N11O2)
C, H, N.
Data for 1,9-Bis{2-[2-(diethylamino)ethyl]-6-oxo-2,6-dihy-
droindazolo[4,3-gh]isoquinolin-5-yl}-5-methyl-1,5,9-triaza-
nonane (9c). Yield 30%. Dimaleate mp 109–110 °C. 1H NMR
(CDCl3) δ: 0.95 (t, 12H, 4 × CH3), 1.97 (m, 4H, 2 × CH2), 2.35
(s, 3H, 5-CH3), 2.47–2.58 (m, 8H, 4 × CH2), 2.61 (t, 4H, 2 ×
CH2), 2.85 (t, 4H, 2 × CH2), 3.53 (q, 4H, 2 × CH2), 4.45 (t, 4H,
2 × CH2), 6.77 (d, 2H, ar), 7.37 (d, 2H, ar), 8.23 (d, 2H, ar), 8.75
(d, 2H, ar), 9.22 (t, 2H, 2 × NH, ex), 9.60 (s, 2H, ar). Anal. Calcd
(C45H55N11O2) C, H, N.
Data for 1,9-Bis{2-[2-(dimethylamino)ethyl]-6-oxo-2,6-dihy-
droindazolo[3,4-fg]isoquinolin-5-yl}-5-methyl-1,5,9-triaza-
nonane (10a). Yield 15%. Dimaleate mp 79–80 °C. 1H NMR
(CDCl3) δ: 1.98 (m, 4H, 2 × CH2), 2.25 (s, 12H, 4 × CH3), 2.35
(s, 3H, 5-CH3), 2.62 (t, 4H, 2 × CH2), 2.85 (t, 4H, 2 × CH2), 3.50
(m, 4H, 2 × CH2), 4.50 (t, 4H, 2 × CH2), 6.75 (d, 2H, ar), 7.32 (d,
2H, ar), 8.20 (d, 2H, ar), 8.74 (d, 2H, ar), 9.18 (t, 2H, 2 × NH,
ex), 9.55 (s, 2H, ar). Anal. Calcd (C41H47N11O2) C, H, N.
2. In Vitro Cytotoxicity. The human colon adenocarcinoma cell
line (HT29) was used for cytotoxicity testing in vitro using the
SRB assay.20 Cells were maintained as stocks in Dulbecco’s
modified eagle medium (DMEM) (Gibco) supplemented with 10%
fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco). Cell
cultures were passaged twice weekly using trypsin-EDTA to detach
the cells from their culture flasks. The rapidly growing cells were
harvested, counted, and incubated under the appropriate concentra-
tions (7 × 105 cells/well) in 96-well microtiter plates. After
incubation for 24 h, target and reference compounds dissolved in
culture medium were applied to the culture wells in quadruplicate
and incubated for 72 h at 37 °C in a 5% CO2 atmosphere and 95%
relative humidity. At the same time, a plate was tested to value the
cell population before the drug addition (Tz). The culture fixed with
cold trichloroacetic acid was stained by 0.4% SRB (Sigma-Aldrich,
Milan, Italy) dissolved in 1% acetic acid. Bound stain was
subsequently solubilized with 10 mM Trizma (Sigma-Aldrich,
Milan, Italy), and the absorbance was read on the microplate reader
Dynatech model MR 700 at a wavelength of 515 nm. The cytotoxic
activity was evaluated by measuring the drug concentration resulting
in a 50% reduction in the net protein increase (as measured by
SRB staining) in control cells during the drug incubation (GI50),
the drug concentration resulting in total growth inhibition (TGI),
and the drug concentration resulting in a 50% reduction in the
measured protein at the end of the drug treatment as compared to
that at the beginning (LC50). The percentage of growth inhibition
was calculated as [(Ti - Tz)/(C - Tz)] × 100 for concentrations
for which Ti g Tz and as [(Ti - Tz)/Tz] × 100 for concentrations
for which Ti < Tz, where Tz is the absorbance at time zero, C is
the absorbance in the presence of the vehicle, and Ti is the
absorbance in the presence of the drug at different concentrations.
GI50, TGI, and LC50 were obtained by interpolation on a graph of
the percentage of growth versus log(M). Each quoted value
represents the mean of triplicate experiments.
3. Apoptotic Assays. Apoptosis of HT-29 cells treated with the
vehicle or the LC50 concentration of target compounds was
evaluated by annexin V binding and biparametric PI/annexin V
cytofluorimetric analysis.21 To detect early stages of apoptosis, the
expression of annexin V, a Ca2+-dependent phospholipid-binding
protein with high affinity for phosphatidylserine was employed.
Moreover, simultaneous staining of cells with FITC-annexin V
and PI, allows for the discrimination of intact cells (annexin V-/
PI-), early apoptotic cells (annexin V+/PI-), and late apoptotic or
necrotic cells (annexin V+/PI+). Apoptotic cells become annexin
V+ after nuclear condensation has started but before the cells
becomes permeable to PI. Briefly, 2 × 106 HT-29 cells treated with
the LC50 of selected compounds for 6, 12, and 24 h were
resuspended in 0.2 mL of binding buffer [10 mM N-2-hydroxy-
ethylpiperazine-N′-2-ethanesulfonic acid (HEPES)/NaOH at pH 7.4,
Data for 1,9-Bis{2-[3-(dimethylamino)propyl]-6-oxo-2,6-di-
hydroindazolo[3,4-fg]isoquinolin-5-yl}-5-methyl-1,5,9-triaza-
1
nonane (10b). Yield 20%. Dimaleate mp 152–153 °C. H NMR
(CDCl3) δ: 1.70–1.98 (m, 4H, 2 × CH2), 2.22 (s, 12H, 4 × CH3),
2.29 (s, 3H, 5-CH3), 2.40 (t, 4H, 2 × CH2), 2.58 (t, 4H, 2 × CH2),
3.35–3.56 (m, 8H, 4 × CH2), 4.50 (t, 4H, 2 × CH2), 6.78 (d, 2H,
ar), 7.39 (d, 2H, ar), 8.22 (d, 2H, ar), 8.75 (d, 2H, ar), 9.22 (t, 2H,
2 × NH, ex), 9.60 (s, 2H, ar). Anal. Calcd (C43H51N11O2) C, H, N.
Data for 1,9-Bis{2-[2-(diethylamino)ethyl]-6-oxo-2,6-dihy-
droindazolo[3,4-fg]isoquinolin-5-yl}-5-methyl-1,5,9-triaza-