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Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 14 4505
Table 1. Cytotoxicity Activity and DNA Binding Affinity of
DIP Derivatives 4a-d, along with Carboplatin and
Chelerythrine for Comparison
DNA binding constants,
IC50 (µM)a on
cell line A2780
K (M-1) × 104
compounds
on salmon testes DNA
carboplatin
5.22 ( 0.14
4.63 ( 0.52
1.56 ( 0.18
1.53 ( 0.09
11.7 ( 1.20
0.087 ( 0.01
NA
chelerythrine
46.6 ( 4.2
2.56 ( 0.08
2.89 ( 0.1
2.02 ( 0.06
12.9 ( 0.45
4a
4b
4c
4d
a IC50 is the concentration of drug necessary to kill 50% of the
cells.
Figure 2. ORTEP representation of structure 4b. Selected
bond lengths showing delocalization of charge [Å]: N1-C31
1.340(3), N2-C31 1.333(3), N2-C33 1.472(3), N1-C32 1.475-
(3), N2-C40 1.457(3), N1-C11 1.389(3).
Like most of the BCPAs, the cytotoxicity of the DIP
framework is likely to come from the intercalation of
the aromatic platform between the DNA base pairs. To
evaluate the DNA binding affinity of the DIP frame-
work, ITC experiments were undertaken on a DNA
solution.22 Binding constants in the region of 104-105
M-1 were obtained (Table 1), which are comparable to
other phenanthridinium-based DNA intercalating agents.
Further studies concerning the mode of action of the
DIPs are ongoing.
In summary, it has been shown that the fusion of a
dihydro-imidazo moiety onto a phenanthridinium frame-
work leads to important stability advantages. The
general cytotoxicity associated with the phenanthri-
dinium moiety is maintained in the resulting DIP
framework and even dramatically improved in the case
of molecule 4d. The DIP derivatives have also interest-
ing DNA binding properties. The simplicity and gener-
ality of the annelation reaction14 could be used to
strengthen the most labile BCPAs and should provide
new horizons to some clinically abandoned phenanthri-
dinium antitumor agents. Additionally, it could facilitate
the development of new druglike heterocycles.
shaken. In the case of the reference molecule, a purple
product instantaneously precipitates from the D2O layer
and shifts toward the CDCl3 layer where 5-methyl-
1
dihydro-phenanthridine was characterized by H and
13C NMR spectroscopy. No evidence of reduction was
observed from compound 4b. A similar experiment was
also undertaken with the biological reducing agent
NADH leading to reduction of the reference within an
1 h, but leaving 4b unaffected, even after 24 h exposure
time. Note that NADH is the coenzyme involved in the
bioreduction of BCPAs.17
The susceptibility of the control 5-methyl-phenan-
thridinium bromide and DIP 4b to undertake addition
of hydroxide in aqueous medium was investigated by
the method of Albert and Serjeant using UV spectro-
photometry measurements.20 It was found that the
reference 5-methyl-phenanthridinium bromide becomes
affected around pH 8. The pseudo-base formation seems
to be accompanied by an irreversible oxidation process,
preventing the determination of an accurate pK(ROH),
i.e: pH value at which 50% of the starting material is
transformed into its pseudobase. Nevertheless, the
apparent sensitivity of this reference to alkaline pH is
in accordance with the pK(ROH) of BCPAs sanguinarine,
chelerythrine, and nitidine (Figure 1), reported to be
5.75, 6.67, and 9.76, respectively.12,21 On the other hand,
due to the introduction of an amidinium moiety, raising
the electron density around the positive center, the DIP
4b shows a much higher resistance to pseudobase
formation, with a calculated pK(ROH) of 11.4.
Crystallographic analysis of 4b (Figure 2) shows the
bonds N2-C31 and C31-N1 to be equidistant. This
confirms the charge delocalization over the two nitrogen
atoms, which is responsible for the higher stability of
the DIP framework.
The demonstrated high stability of the DIP frame-
work is expected to disfavor the cell metabolism pro-
cesses that are involved in the case of BCPAs (Figure
1). In vitro cytotoxicity studies were undertaken with
human ovarian tumor cell line A2780. A growth inhibi-
tion assay with 24 h drug exposure and a 3 day recovery
period reveals that DIP derivatives 4a-c have cytotox-
icity within the same order of magnitude as both
chelerythrine and the clinically used antitumor agent
carboplatin. However, compound 4d shows much higher
cytotoxicity, with an IC50 value in the nanomolar range
(Table 1).
Acknowledgment. This work was supported by the
EPSRC and Cancer Research UK. We thank Prof A.
Cooper for the DNA binding studies at the UK Center
for Microcalorimetry funded by the BBSRC, and Dr.
Jesus M. de la Fuente for fruitful discussions.
Supporting Information Available: More details for the
reduction resistance experiment, pKa(ROH) measurement, in
vitro cytotoxicity assay, and DNA binding studies; synthetic
procedures; analytical data. The cif file for compound 4b is
also available. This material is available free of charge via the
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