Synthesis and cytotoxic activity of benzo[c][1,7] and [1,8]phenanthrolines analogues of nitidine and fagaronine

Article abstract of DOI:10.1016/j.bmc.2004.04.038

Fagaronine and nitidine are natural benzo[c] phenanthridinium alkaloids, which display antileukemic activity. Both act as topoisomerase I and topoisomerase II inhibitors. The objective of the present study was to prepare noncharged isosters of these compounds, with replacement of the aromatic A ring by a pyridine ring, present in other topoisomerase I inhibitors. Various 7,8- and 8,9-dimethoxy and metylenedioxy benzo[c][1,7] and [1,8]phenanthrolines were readily synthesized by benzyne-mediated cyclization of the corresponding substituted N-(2-halobenzyl)-5-quinolinamines or 5-isoquinolinamines. In both series, compounds bearing oxygenated substituents at positions 8 and 9 exhibited cytotoxic properties towards L1210 murine leukemia cells, which may result from their capacities to intercalate into DNA. Topoisomerase I inhibition was observed for all active compounds.

Full text of DOI:10.1016/j.bmc.2004.04.038

Bioorganic & Medicinal Chemistry 12 (2004) 3943–3953
Synthesis and cytotoxic activity of benzo[c][1,7] and
[1,8]phenanthrolines analogues of nitidine and fagaronine
Soizic Prado,a Sylvie Michel,a Francois Tillequin,a,* Michel Koch,a Bruno Pfeiffer,b
ß
c
d
e
e
ꢀ c
ꢀ
ꢀ
ꢀ
Alain Pierre, Stephane Leonce, Pierre Colson, Brigitte Baldeyrou, Amelie Lansiaux
and Christian Baillye
aLaboratoire de Pharmacognosie de l’Universitꢀe Renꢀe Descartes, UMR/CNRS No. 8638,
Facultꢀe des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l’Observatoire, 75006 Paris, France
bLes Laboratoires Servier, 1 rue Carle Hebert, 92415 Courbevoie Cedex, France
cInstitut de Recherches Servier, Division Recherche Cancꢀerologie, 125 Chemin de Ronde, 78290 Croissy sur Seine, France
dBiospectroscopy and Physical Chemistry Unit, Department of Chemistry and Natural and Synthetic Drugs Research Center,
University of Liꢁege, Sart-Tilman, 4000 Liꢁege, Belgium
eINSERM U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, 59045 Lille Cedex, France
Received 13 January 2004; accepted 27 April 2004
Available online 2 June 2004
Abstract—Fagaronine and nitidine are natural benzo[c] phenanthridinium alkaloids, which display antileukemic activity. Both act
as topoisomerase I and topoisomerase II inhibitors. The objective of the present study was to prepare noncharged isosters of these
compounds, with replacement of the aromatic A ring by a pyridine ring, present in other topoisomerase I inhibitors. Various 7,8-
and 8,9-dimethoxy and metylenedioxy benzo[c][1,7] and [1,8]phenanthrolines were readily synthesized by benzyne-mediated cycli-
zation of the corresponding substituted N-(2-halobenzyl)-5-quinolinamines or 5-isoquinolinamines. In both series, compounds
bearing oxygenated substituents at positions 8 and 9 exhibited cytotoxic properties towards L1210 murine leukemia cells, which may
result from their capacities to intercalate into DNA. Topoisomerase I inhibition was observed for all active compounds.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
ptothecin, are topoisomerase I poisons, which were
launched in 1996 and 1994, respectively.5;6 Anthracy-
clines, exemplified by daunorubicin and doxorubicin,
are typical examples of topoisomerase II inhibitors
currently used in cancer chemotherapy.7;8
DNA topoisomerases are ubiquitous enzymes that cat-
alyze the breaking and rejoining of DNA strands. They
are involved in the regulation of the topological states of
DNA, resolving the topological problems that arise
during several essential nuclear processes, including
transcription, recombination, and replication in the
course of cell division.1;2 Two major types of topo-
isomerases occur in eukaryotic cells: type I and type II,
depending on their mechanism of action, which implies
either a single or a double-stranded DNA transient
breakage. Both enzymes represent important targets for
the development of new antitumor drugs.3 Indeed, the
biological activity of several anticancer agents appears
to be correlated with their ability to stabilize enzyme–
DNA cleavable complexes.4 Topotecan and irinotecan,
derived from the structure of the natural alkaloid cam-
It is interesting to note that most of the anticancer
agents, which interfere with topoisomerases were ini-
tially developed from a natural product, isolated either
from a higher plant or from a microorganism. In this
context, benzo[c]phenanthridinium alkaloids, illustrated
by fagaronine (1) and nitidine (2), both isolated from
trees belonging to the genus Zanthoxylum (Rutaceae),
appear as interesting models for the development of new
antitumor drugs.9–11 Indeed, these compounds have
OH
OCH3
O
H3CO
H3CO
H3CO
H3CO
O
N+
N+
* Corresponding author. Tel.: +33-1-53-73-98-10; fax: +33-1-40-46-96-
58; e-mail: francois.tillequin@univ-paris5.fr
1
2
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.038

3944
S. Prado et al. / Bioorg. Med. Chem. 12 (2004) 3943–3953
been shown to be cytotoxic and to act as topoisomerase
I inhibitors at low concentrations and topoisomerase II
inhibitors at higher concentrations.12–14 Preclinical in
vivo experiments conducted by the NCI. revealed
the antileukemic activity of fagaronine in rodents.15
Nevertheless, these trials were rapidly discontinued, due
to several points, which appeared as severe drawbacks
for future development, including a quaternary structure
limiting the intracellular penetration of the drug and a
narrow spectrum of action, restricted to leukemias. The
imminium charge of these alkaloids had been first
speculated as necessary for biological activity, in con-
nection with its reactivity towards nucleophilic attack.
Later on, Bisagni and Janin demonstrated that non-
charged analogues of nitidine could act as topoisomer-
ase poisons and exhibited potent cytotoxic activity.16
Based on this finding, the group of LaVoie recently
developed promising structural analogues in the
benzo[i]phenanthridine series bearing four oxygenated
substituents at positions 2, 3, 8 and 9, in the form of
methoxy or methylenedioxy groups in reference to the
natural models.17–19 Interestingly, in the related 11H-
indeno[1,2-c] isoquinoline-5,11-dione and 5H-dibenzo
[c,h][1,6] naphthyridine-6-one series, several compounds
bearing only two ortho-oxygenated substituents also
revealed potent cytotoxic and topoisomerase I inhibiting
activities, some of them being effective in regressing tu-
mor growth in vivo.20–22 In this context, the replacement
of the aromatic A ring of nonquaternary benzo[c]phen-
anthridines by a pyridine ring, present in some other
topoisomerase I inhibitors such as intoplicine,23;24 ap-
peared to us as an interesting pharmacomodulation in
this series of natural alkaloids analogues. We describe
here the synthesis and biological activities of several 7,8-
and 8,9-dimethoxy and methylenedioxy benzo[c][1,7]
and [1,8]phenanthrolines.
2. Results and discussion
Scheme 1 outlines the synthetic procedures used for the
preparation of benzo[c][1,7]phenanthrolines 3 and 4,
and benzo[c][1,8]phenanthrolines 5–7. The key step of
this approach was a benzyne-mediated cyclization of a
R1
R2
Br
R1
R2
X
Br
H
R3
20 R = R = OCH , R = H
O
1
2
3
3
14 R = R = OCH , X = Br
1
2
3
21 R , R = OCH O, R = H
1
2
2
3
15 R , R = OCH O, X = Cl
1
2
2
25 R = H, R = R = OCH
1
2
3
3
Y
Y
z
z
i)
ii)
NH2
NH2
12 Y = N, Z = CH
13 Y = CH, Z = N
12 Y = N, Z = CH
13 Y = CH, Z = N
1
8
Y
8a
4a
Y
z
7
2
z
Br
3'
X
2'
R1
R2
4'
R1
R2
6
3
5
4
NH
N
1'
5'
7'
6'
R3
16
R = R = OCH , X = Br, Y = N, Z = CH
1 2 3
22 R , R = OCH O, R = H, Y = N, Z = CH
1
2
2
3
17 R , R = OCH O, X = Cl, Y = N, Z = CH
1
2
2
23 R , R = OCH O, R = H, Y = CH, Z = N
1
2
2
3
18 R = R = OCH , X = Br, Y = CH, Z = N
1
2
3
24 R = H, R = R = OCH , Y = CH, Z = N
1
2
3
3
19 R , R = OCH O, X = Cl, Y = CH, Z = N
1
2
2
iv)
iii)
1
12
Y
12 a Y
11
z
2
z
X
10 10b
10a
R1
R2
9
8
iv)
R1
R2
3
4a
4b
4
NH
N
6a
7
5
6
R3
3 R = R = OCH , R = H, Y = N, Z = CH
1
2
3
3
8 R = R = OCH , X = Br, Y = N, Z = CH
1
2
3
4 R = R = OCH O, R = H, Y = N, Z = CH
1
2
2
3
9 R , R = OCH O, X = Cl, Y = N, Z = CH
1
2
2
5 R = R = OCH , R = H, Y = CH, Z = N
1
2
3
3
10 R = R = OCH , X = Br, Y = CH, Z = N
1
2
3
6 R , R = OCH O, R = H, Y = CH, Z = N
1
2
2
3
11 R , R = OCH O, X = Cl, Y = CH, Z= N
1
2
2
7 R = H, R = R = OCH , Y = CH, Z = N
1
2
3
3
Scheme 1. Synthetic approach to the preparation of benzo [c][1,7] and [1,8] phenanthrolines derivates. (i) neutral, alumina, toluene reflux , (ii) toluene
reflux; (iii) NaBH4, MeOH, 0 °C; (iv) (1) LDA, THF, )78 °C; (2) air oxidation.

S. Prado et al. / Bioorg. Med. Chem. 12 (2004) 3943–3953
3945
conveniently substituted N-(2-halobenzyl)-5-quinolin-
amine or 5-isoquinolinamine. Lithium diisopropylamide
was used for this purpose, since this reagent had previ-
ously given satisfactory results for the cyclization of
various anils into benzo[c]phenanthridines25 and het-
erocyclic structural analogues, such as pyranophenan-
thridines,26 bearing methoxy and methylenedioxy
groups.
The perturbation of the cell cycle induced by com-
pounds 3–6 was studied in the same cell line by flow
cytometry. Interestingly, the cell cycle specificity ob-
served depended on the nature of the oxygenated sub-
stituents on the benzo[c]phenanthroline basic core.
Indeed, compounds 3 and 5, both bearing two methoxy
groups at positions 8 and 9, induced an accumulation in
the G2 + M phases of the cell cycle, whereas compounds
4 and 6, with a methylenedioxy substituent at the same
positions induced an accumulation in the G1 phase,
suggesting therefore the implications of at least two
distinct mechanisms of action.
The required N-(2-halobenzyl)-5-quinolinamines and
isoquinolinamines 8–11, with oxygenated substituents at
positions 4 and 5 on the benzyl moiety, were prepared
by condensation of 5-aminoquinoline (12) and 5-am-
inoisoquinoline (13) either with 6-bromoveratraldehyde
(14) or with 6-chloropiperonal (15), which afforded the
corresponding Schiff bases 16–19 in 82–95% yield. Those
imines were subsequently reduced with sodium boro-
hydride to the 5-quinolinamines and 5-isoquinolinam-
ines 8–11 in almost quantitative yield. Alternatively,
compounds 8 and 10 could be prepared in a one step
procedure27 by alkylation of 5-aminoquinoline (12) and
5-aminoisoquinoline (13) with 2-bromo-4,5-dimeth-
oxybenzyl bromide (20).28 Similarly, the use of 2-bromo-
4,5-methylenedioxybenzyl bromide (21)29 gave access to
N-(2-bromo-4,5-methylenedioxybenzyl)-5-quinolinamine
(22) and N-(2-bromo-4,5-methylenedioxybenzyl)-5-iso-
quinolinamine (23). This second approach gave lower
overall yields than the first one, but was more versa-
tile, since it also permitted the preparation of N-
(2-bromo-5,6-dimethoxybenzyl)-5-isoquinolinamine (24)
by condensation of 5-aminoisoquinoline (13) with 2-
bromo-5,6-dimethoxybenzyl bromide (25).30
In an effort to elucidate their molecular mechanism of
action, at least partially, compounds 3–6 were tested as
potential inhibitors of topoisomerases I and II using
conventional DNA relaxation assays.32 No poisoning of
human topoisomerase II was detected. Double stranded
DNA cleavage, reflecting the stabilization of topoiso-
merase II–DNA covalent complexes, was observed with
the reference drug etoposide but not with 3–6, even
when tested at concentrations up to 50 lM (data not
shown). In contrast, drug-induced stabilization of
topoisomerase I–DNA complexes was evidenced. The
stimulation of topoisomerase I–mediated DNA single
strand breaks was clearly shown with the reference drug
camptothecin and to a lower but noticeable extent with
3–6 (Fig. 1a). The most efficient compound is the
dimethoxy derivative 5, which stimulated DNA cleavage
by the enzyme in a concentration-dependent manner
(Fig. 1b). The amount of nicked DNA species increased
gradually with the increasing drug concentration but the
poisoning effect remains weaker than that detected with
camptothecin, by about 35% (Fig. 1c). In concentration-
dependent experiments with camptothecin (data not
shown), topoisomerase I inhibition starts to appear at
about 1 lM whereas it requires a concentration 3–5
times higher to get a similar extent of DNA cleavage by
topoisomerase I with compound 5, which is the most
potent of the four analogues tested. Moreover, even at
high concentrations, the level of topoisomerase I inhi-
bition seen with 5 never reaches that observed with
camptothecin. The two dimethoxy derivatives 5 and 3
are both slightly more potent than the corresponding
methylenedioxy analogs 4 and 6 at stabilizing topoiso-
merase I–DNA complexes.
Cyclization of N-(2-halobenzyl)-5-quinolinamines 8 and
9 or 22, and N-(2-halobenzyl)-5-isoquinolinamines 10,
11 or 23, and 24 using LDA in THF at )78 °C25 was
followed by spontaneous air-oxidation of the unstable
dihydrophenanthroline intermediates during the work-
up,31 and gave the desired benzo[c][1,7]phenanthrolines
3 and 4, and benzo[c][1,8]phenanthrolines 5–7, respec-
tively.
The study of the cytotoxic properties of the benzo[c]-
phenanthrolines 3–7 was carried out in vitro on the
L1210 murine leukemia cell line. The results (IC50) are
reported in Table 1. In both [1,7] and [1,8] series, the
compounds bearing oxygenated substituents at positions
8 and 9 were found to be cytotoxic, with IC50 values
within the same range of magnitude as fagaronine (1). In
contrast, 7,8-dimethoxybenzo[c][1,8]phenanthroline (7)
was about 10-fold less potent.
DNA sequencing experiments were carried out to
investigate further the inhibition of topoisomerase I by
these compounds. In this case, the DNA substrate was a
117-bp restriction fragment labelled at the 30-end with
32P and subjected to cleavage by topoisomerase I in the
absence or presence of the alkaloids at 10 or 50 lM. The
cleavage products were resolved on polyacrylamide
sequencing gels. A typical gel is presented in Figure 2. A
few well resolved cleavage sites were identified with
camptothecin, especially at the TÅG positions 26, 48 and
81. These three main sites were also detected in the
presence of the benzo[c]phenanthrolines 3–6 but cleav-
age intensities are considerably weaker. Here again, we
found that 5 was the most efficient topoisomerase I
poisons among the four compounds tested. The poi-
soning activity of this compound is clearly detected but
Table 1. Inhibition of L1210 cell proliferation and cell cycle pertur-
bation induced by compounds 3–7 in comparison with fagaronine (1)
Compound Cytotoxicity IC50 (lM)
Cell cycle perturbationa
1
3
4
5
6
7
6.4
7.1
8.8
6.6
4.4
43.1
35% G2 + M (25 lM)
44% G2 + M (25 lM)
61% G1 (50 lM)
76% G2 + M (25 lM)
61% G1 (10 lM)
Not tested
a Percentage of cells blocked in a specific phase of the cell cycle at the
indicated concentration.

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S. Prado et al. / Bioorg. Med. Chem. 12 (2004) 3943–3953
Figure 2. Cleavage of the 117-mer DNA fragment by topoisomerase I
in the presence of the benzo[c]phenanthrolines 3–6. The 30-end labeled
fragment (DNA) was incubated in the absence (lane TopoI) or pres-
ence of the alkaloids at 10 or 50 lM. Camptothecin (CPT) was used at
20 lM. Topoisomerase I cleavage reactions were analyzed on a 8%
denaturing polyacrylamide gel. Numbers at the side of the gels show
the nucleotide positions, determined with reference to the guanine
track labeled G. The nucleotide positions and sequences of the main
cleavage sites are indicated.
Figure 1. Topoisomerase I inhibition. (a) Effect of benzo[c]phen-
anthrolines 3–6 on the relaxation of plasmid DNA by topoisomerase I.
Gel (b) shows the concentration-dependent effect of 5 tested at 0.1–
50 lM, compared to CPT at 20 lM. Native supercoiled pLAZ3 DNA
(0.3 lg) (lane DNA) was incubated with 4 units topoisomerase I in the
absence (lane TopoI) or presence of drug at the indicated concentra-
tion (lM). Reactions were stopped with sodium dodecylsulfate and
treatment with proteinase K. DNA samples were separated by elec-
trophoresis on 1% agarose gels containing ethidium (1 lg/mL) and
then photographed under UV light. Nck, nicked; Rel, relaxed; Sc,
supercoiled DNA. (c) Comparison of the extent of topoisomerase
I-mediated DNA cleavage measured with 3–6 or CPT. The histogram
shows the formation of nicked DNA (form II, %) for each compound.
Band intensities from three gels such as the one shown in panel
(a) were compiled for the quantitative analysis.
sidered as a primary target for the benzo[c]phenanthr-
olines tested here.
In parallel, we studied the interaction of the alkaloids
with DNA by different spectroscopic methods. Binding
to DNA was first visualized from absorption and fluo-
rescence measurements (Fig. 3). Interaction of 5 with
DNA causes bathochromic and hypochromic shifts and
the titration shows a well resolved isosbestic point at
290 nm suggesting a homogeneous interaction (Fig. 3a).
Similar titrations were observed by absorption spec-
troscopy with the other compounds although in these
cases the spectral shifts were less pronounced (data not
shown). The interaction with DNA was also evidenced
by means of circular dichroism and fluorescence. A
weak positive CD signal appeared at 320 nm upon
interaction of 5 with DNA (spectra not shown) and in
the fluorescence titration (excitation set at 300 nm),
addition of DNA induces a pronounced quenching of
the intrinsic fluorescence of 5 in the 350–450 nm region
(Fig. 3b).
relatively discrete compared to the camptothecin. In
other words, there is no doubt that this compound be-
haves as a conventional topoisomerase I poison in the
molecular assays but its limited potency suggests that
topoisomerase I is perhaps not its major target. This
consideration prompted us to evaluate the cytotoxicity
of 5 using a pair of murine leukemia cell lines, sensitive
(P388) or resistant (P388CPT5) to camptothecin.33;34
With this cell system, which we have previously em-
ployed with other toposiomerase I poisons,35 we mea-
sured IC50 values of 3.1 and 2.4 lM for compound 5 on
the P388 and P388CPT5 cell lines, respectively. The
benzo[c][1,8]phenanthroline derivative is thus more or
less equally toxic to the parental cells and the CPT-
resistant cells expressing a mutated top1 gene. For this
reason, we believe that topoisomerase I cannot be con-
To determine the mode of binding of the compounds to
DNA, we used electric linear dichroism (ELD), which is
useful to probe the orientation of small molecules bound
to nucleic acids.36 A representative data set obtained

S. Prado et al. / Bioorg. Med. Chem. 12 (2004) 3943–3953
3947
Figure 3. DNA titrations of 5 measured by (a) absorption and (b) fluorescence spectroscopy. To 1 mLof drug solution at (a) 20 lM or (b) 2.5 lM
were added aliquots of a concentrated calf thymus DNA solution. The phosphate–DNA/drug ratio increased from 0 to 20 (top to bottom curves at
(a) 270 nm or (b) 390 nm). For the fluorescence measurement, the excitation was set at 300 nm. Spectra were recorded in 1 mM sodium cacodylate
buffer, pH 7.0.
-0.0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
-0.0
-0.1
-0.2
-0.3
-0.4
-0.5
(a)
(b)
250
300
350
400
0
2
4
6
8
10 12 14
wavelength (nm)
E (kV/cm)
-0.0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.0
-0.1
-0.2
-0.3
-0.4
-0.5
(c)
(d)
0
10
20
DNA
5
3
6
4
P/D
Figure 4. ELD data for the binding to DNA. Dependence of the reduced dichroism DA/A on (a) the wavelength, (b) the electric field strength and (c)
the DNA–drug ratio (P=D) for ð Þ compound 5 bound to calf thymus DNA and ðsÞ DNA alone. Conditions: (a) 13.6 kV/cm, P=D ¼20 (200 lM calf
ꢀ
thymus DNA, 10 lM drug), (b) 360 nm, P=D ¼20 for the DNA–drug complexes and 260 nm for the DNA alone (c) 13.6 kV/cm, 360 nm, in 1 mM
sodium cacodylate buffer, pH 7.0. The histograms in panel (d) compare the reduced dichroism values (DA/A) measured with DNA at 260 nm and the
different drug–DNA complexes at 360 nm.
with 5 is presented in Figure 4a–c. The ELD spectrum of
5 bound to DNA exhibits negative reduced dichroism
values (DA/A) in the 300–400 nm drug absorption band
(where DNA does not absorb) (Fig. 4a) and the mag-
nitude of the DA/A signal is dependent on both the
electric field strength (Fig. 4b) and the DNA/drug ratio
(Fig. 4c). The intensity of the DA/A signals measured at
360 nm with the different drug–DNA complexes is at
least equal to that measured with DNA alone at 260 nm
(Fig. 4d). Such strong negative signals are typical of
DNA intercalating agents.37 The more negative DA/A
values recorded with 5 are often obtained with potent
intercalating agents and likely reflect a drug-induced
stiffening of the DNA macromolecule, which favours its
orientation in the electric field. Altogether, the spectro-
scopic data are fully consistent with an intercalative
binding mode, as expected for such planar molecules.
Intercalation of these benzo[c]phenanthrolines into
DNA occurs independently of the target DNA se-
quence. DNase I footprinting experiments revealed no
preferential binding sites (no footprints). These com-
pounds interact equally well with AT and GC sequences.
This is also observed by ELD. Binding of compound 5
to the polynucleotides poly(dAT)2 and poly(dGC)2
produced negative reduced dichroism signals at 360 nm
of intensities comparable to those obtained with the
polynucleotides alone at 260 nm (Fig. 5), indicating that

3948
S. Prado et al. / Bioorg. Med. Chem. 12 (2004) 3943–3953
poly(dGC)2
poly(dAT)2
-0.0
-0.1
-0.2
-0.3
-0.0
-0.1
-0.2
-0.3
-0.4
0
2
4
6
8
10 12 14
0
2
4
6
8
10 12 14
E (kV/cm)
E (kV/cm)
Figure 5. Variation of the reduced dichroism (DA/A) for compound 5 bound to poly(dAT)2 or poly(dGC)2, as a function of the electric field strength.
DA/A was measured at 360 nm and at a DNA/drug ratio of 20 in 1 mM sodium cacodylate buffer, pH 7.0 symbols ðdÞ and ðsÞ refer to the drug–
polynucleotide complex and the polynucleotide alone, respectively.
the compound can intercalate between both A–T and
G–C base pairs.
Lancaster Synthesis Ltd (Morecambe, Lancashire,
England).
4.2. General procedure for the synthesis of imines 16–19
3. Conclusion
An solution of 5-aminoquinoline (12) or 5-aminoiso-
quinoline (13) (101 mg, 0.7 mmol) and 6-bromoveratr-
aldehyde (14) (172 mg, 0.7 mmol) or 6-chloropiperonal
(15) (129 mg, 0.7 mmol) in 1,2-dichlorobenzene (30 mL)
was refluxed in a Dean–Stark apparatus for 24 h. The
solvent was evaporated under reduced pressure. Crys-
tallization of the residue from toluene afforded the
imines 16–19 as pale yellow prisms.
We report an efficient strategy for the synthesis of novel
benzo[c][1,7] and [1,8]phenanthrolines. In both series,
compounds bearing oxygenated substituents at positions
8 and 9 exhibit interesting cytotoxic activities, which
may result from their capacities to intercalate into
DNA. Topoisomerase I inhibition was observed but
apparently it does not constitute a primary event
responsible for the cytotoxic action. Further mechanistic
studies are needed to identify their molecular targets.
4.3. N-(Quinolin-5-yl)-2-bromo-4,5-dimethoxyphenyl-
methanimine (16)
4. Experimental
4.1. General
Yield 82%, mp 207 °C (from toluene), UV k (MeOH)
238, 285 nm; IR (KBr) m 2910, 1595, 1514, 1440, 1262,
1024 cmÀ1; 1H NMR (400 MHz, CDCl3) d 3.90 (6H, s, 2
OCH3), 7.10 (1H, s, H-30), 7.15 (1H, dd, J ¼ 7, 2, H-6),
7.42 (1H, dd, J ¼ 8:5, 4, H-3), 7.57 (1H, dd, J ¼ 8, 2, H-
8), 7.71 (1H, dd, J ¼ 8, 7, H-7), 7.90 (1H, s, H-60), 8.66
(1H, dd, J ¼ 8:5, 2, H-4), 8.88 (1H, s, H-70), 8.92 (1H,
dd, J ¼ 4, 2, H-2); 13C NMR (75 MHz, CDCl3) d 56.0
(OCH3), 56.2 (OCH3), 110.2 (C-6), 113.5 (C-60), 115.2
(C-30), 118.5 (C-20), 124.2 (C-3), 127.1 (C-8), 128.7 (C-
4a), 129.6 (C-4), 130.8 (C-10), 132.5 (C-7), 148.5 (C-8a),
148.9 (2C, C-40, C-50), 150.8 (C-2), 152.6 (C-5), 159.8 (C-
70); DCI–MS m=z 371, 373 [MH]þ. Anal.
(C18H15BrN2O2): C, H, Br, N.
Melting points were determined on a hot stage Reichert
microscope and are uncorrected.Mass spectra were re-
corded with a Nermag R-10-10C spectrometer using
desorption–chemical ionization (DCI–MS; reagent gas:
NH3). UV spectra (kmax in nm) were recorded in spec-
troscopic grade MeOH on a Beckman Model 34 spec-
trophotometer. IR spectra (mmax in cmÀ1) were obtained
from potassium bromide pellets or sodium chloride films
on a Perkin–Elmer 257 instrument. 1H NMR (d [ppm], J
[Hz]) spectra were run at 400 MHz and 13C NMR
spectra at 75 MHz, using Bruker AVANCE-400 and
AC-300 spectrometers, respectively. When necessary,
the structures of the novel compounds were insured and
the signals unambiguously assigned by 2D NMR tech-
niques: 1H–1H COSY, 1H–1H NOESY, 13C–1H HMQC,
and 13C–1H HMBC. These experiments were performed
using standard Bruker microprograms. Flash column
chromatographies were conducted using silica gel 60
Merck (35–70 lM) with an overpressure of 300 mbars.
Microanalyses were in agreement with calculated values
0.4%. 5-Aminoquinoline (12) and 5-aminoisoquinoline
(13) were purchased from Aldrich Chemical Company
(Steinheim, Germany). 6-Bromoveratraldehyde (14)
and 6-chloropiperonal (15) were purchased from
4.4. N-(Quinolin-5-yl)-2-chloro-4,5-methylenedioxyphen-
ylmethanimine (17)
Yield 85%, mp 266 °C (from toluene), UV k (MeOH)
287 nm; IR (KBr) m 2923, 1585, 1570, 1504, 1482, 1254,
1120, 1050, 922, 837 cmÀ1; 1H NMR (400 MHz, CDCl3/
CD3OD 9:1) d 6.25 (2H, s, OCH2O), 7.30 (1H, s, H-30),
7.40 (1H, dd, J ¼ 7, 1, H-6), 7.60 (1H, dd, J ¼ 8:5, 4, H-
3), 7.80 (1H, dd, J ¼ 8, 7, H-7), 7.90 (1H, s, H-60), 7.95
(1H, dd, J ¼ 8, 1, H-8), 8.77 (1H, dd, J ¼ 8:5, 2, H-4),
8.95 (1H, s, H-70), 9.00 (1H, dd, J ¼ 4, 2, H-2); 13C
NMR (75 MHz, CDCl3/CD3OD 9:1) d 102.3 (OCH2O),

S. Prado et al. / Bioorg. Med. Chem. 12 (2004) 3943–3953
3949
106.9 (C-6), 109.8 (C-60), 113.5 (C-30), 120.7 (C-3), 124.3
(C-20), 124.5 (C-3), 126.8 (C-8), 126.9 (C-4a), 129.9 (C-
4), 130.5 (C-10), 133.2 (C-7), 147.3 (C-8a), 147.7 (C-50),
148.7 (C-40), 150.2 (C-2), 151.2 (C-5), 158.2 (C-70); DCI–
MS m=z 311, 313 [MH]þ. Anal. (C17H11ClN2O2): C, H,
Cl, N.
1474, 901 cmÀ1; 1H NMR (400 MHz, CDCl3) d 3.75 (3H,
s, OCH3), 3.90 (3H, s, OCH3), 4.52 (2H, d, J ¼ 5, CH2-
70), 4.70 (1H, t, J ¼ 5, D2O exch., NH), 6.66 (1H, dd,
J ¼ 7, 2, H-6), 6.95 (1H, s, H-60), 7.08 (1H, s, H-30), 7.37
(1H, dd, J ¼ 8:5, 4, H-3), 7.57 (2H, m, H-7, H-8), 8.25
(1H, dd, J ¼ 8:5, 1, H-4), 8.90 (1H, dd, J ¼ 4, 1, H-2);
13C NMR (75 MHz, CDCl3) d 48.6 (C-70), 56.0 (OCH3),
56.2 (OCH3), 105.5 (C-6), 112.4 (C-60), 113.5 (C-20),
115.7 (C-30), 118.5 (C-4a), 119.0 (C-3), 119.4 (C-8), 128.7
(C-4), 129.3 (C-10), 130.3 (C-7), 143.1 (C-5), 148.6 (C-
8a), 148.9 (C-50), 149.1 (C-40), 149.9 (C-2); DCI–MS m=z
373, 375 [MH]þ. Anal. (C18H17BrN2O2): C, H, Br, N.
4.5. N-(Isoquinolin-5-yl)-2-bromo-4,5-dimethoxyphenyl-
methanimine (18)
Yield 90%, mp 168–169 °C (from toluene), UV k
(MeOH) 285, 340 nm; IR (NaCl) m 2923, 1482, 1254,
1120, 837 cmÀ1; 1H NMR (400 MHz, CDCl3) d 3.85 (3H,
s, OCH3), 3.90 (3H, s, OCH3), 7.09 (1H, s, H-30), 7.30
(1H, dd, J ¼ 7, 1, H-6), 7.62 (1H, dd, J ¼ 8; 7, H-7),
7.87 (1H, dd, J ¼ 8, 1, H-8), 7.92 (1H, s, H-60), 8.12 (1H,
d, J ¼ 6, H-4), 8.58 (1H, d, J ¼ 6, H-3), 8.83 (1H, s, H-
70), 9.28 (1H, s, H-1); 13C NMR (75 MHz, CDCl3) d 56.1
(OCH3), 56.3 (OCH3), 108.3 (C-60), 113.1 (C-30), 114.7
(C-6), 115.0 (C-4), 116.5 (C-20), 123.1 (C-8), 124.9 (C-10),
125.5 (C-7), 126.9 (C-8a), 129.6 (C-4a), 141.0 (C-3),
146.0 (C-50), 146.8 (C-40), 150.1 (C-1), 150.6 (C-5), 157.9
(C-70); DCI–MS m=z 371, 373 [MH]þ. Anal.
(C18H15BrN2O2): C, H, Br, N.
4.9. N-(2-Chloro-4,5-methylenedioxybenzyl)-5-quinolin-
amine (9)
Yield 97%, mp 259 °C (from toluene), UV k (MeOH)
237, 346 nm; IR (NaCl) m 3400, 2955, 2925, 1597, 1486,
1266, 1052, 797 cmÀ1 1H NMR (400 MHz, CDCl3/
;
CD3OD 9:1) d 4.51 (2H, d, J ¼ 5, CH2-70), 4.80 (1H, t,
J ¼ 5, D2O exch., NH), 5.98 (2H, s, OCH2O), 6.60 (1H,
dd, J ¼ 6, 2, H-6), 6.91 (1H, s, H-30, H-60), 7.35 (1H, dd,
J ¼ 8, 4, H-3), 7.58 (2H, m, H-7, H-8), 8.22 (1H, dd,
J ¼ 8, 2, H-4), 8.89 (1H, dd, J ¼ 4, 2, H-2); 13C NMR
(75 MHz, CDCl3/CD3OD 9:1) d 48.7 (C-70), 101.5
(OCH2O), 105.5 (C-6), 113.2 (C-60), 113.4 (C-30), 118.5
(C-4a), 120.6 (C-3), 124.3 (C-20), 127.8 (C-8), 129.9 (C-
4), 130.8 (C-10), 133.2 (C-7), 142.8 (C-5), 147.5 (C-8a),
147.7 (C-50), 147.8 (C-40), 150.1 (C-2); DCI–MS m=z 313,
315 [MH]þ. Anal. (C17H13ClN2O2): C, H, Cl, N.
4.6. N-(Isoquinolin-5-yl)-2-chloro-4,5-methylenedioxy-
phenylmethanimine (19)
Yield 95%, mp 167 °C (from toluene), UV k (MeOH)
244, 274, 340 nm; IR (NaCl) m 2910, 1585, 1477, 1383,
1
1248, 1040 cmÀ1; H NMR (400 MHz, CDCl3) d 5.93
(2H, s, OCH2O), 7.09 (1H, s, H-30), 7.27 (1H, dd, J ¼ 7,
1, H-6), 7.61 (1H, dd, J ¼ 8, 7, H-7), 7.86 (1H, dd,
J ¼ 8, 1, H-8), 7.88 (1H, s, H-60), 8.11 (1H, d, J ¼ 6, H-
4), 8.57 (1H, d, J ¼ 6, H-3), 8.91 (1H, s, H-70), 9.27 (1H,
s, H-1); 13C NMR (75 MHz, CDCl3) d 101.8 (OCH2O),
108.6 (C-60), 110.3 (C-30), 113.4 (C-20), 116.9 (2C, C-4,
C-6), 125.2 (C-10), 126.2 (C-8a), 128.2 (C-8), 128.9 (C-7),
129.4 (C-4a), 141.9 (C-3), 142.3 (2C, C-40, C-50), 147.2
(C-1), 147.6 (C-5), 153.1 (C-70); DCI–MS m=z 311, 313
[MH]þ. Anal. (C17H11ClN2O2): C, H, Cl, N.
4.10. N-(2-Bromo-4,5-dimethoxybenzyl)-5-isoquinolin-
amine (10)
Yield 92%, mp 245 °C (from toluene), UV k (MeOH)
245, 285, 345 nm; IR (KBr) m 3410, 2925, 2853, 1583,
1503, 1385, 1263, 1030 cmÀ1
;
1H NMR (400 MHz,
CDCl3) d 3.75 (3H, s, OCH3), 3.90 (3H, s, OCH3), 4.50
(2H, d, J ¼ 5, CH2-70), 4.85 (1H, t, J ¼ 5, D2O exch.,
NH), 6.74 (1H, d, J ¼ 7, H-6), 6.95 (1H, s, H-60), 7.07
(1H, s, H-30), 7.35 (1H, d, J ¼ 8, H-8), 7.45 (1H, dd,
J ¼ 8, 7, H-7), 7.61 (1H, d, J ¼ 6, H-4), 8.48 (1H, d,
J ¼ 6, H-3), 9.15 (1H, s, H-1); 13C NMR (75 MHz,
CDCl3) d 48.5 (C-70), 56.1 (OCH3), 56.3 (OCH3), 108.3
(C-6), 113.2 (C-60), 114.7 (C-4), 115.0 (C-30), 116.5 (C-
20), 125.5 (C-8), 126.1 (C-4a), 128.0 (C-7), 129.3 (2C, C-
10, C-8a), 142.0 (C-5), 142.1 (C-3), 148.7 (2C, C-40, C-50),
153.0 (C-1); DCI–MS m=z 373, 375 [MH]þ. Anal.
(C18H17BrN2O2): C, H, Br, N.
4.7. General procedure for the reduction of imines 16–19
Sodium borohydride (1 g, 26 mmol) was added over
30 min to a solution of the imine (1.6 mmol) in MeOH
(100 mL) at 0 °C. The reaction mixture was stirred at
0 °C for 2 h. The solvent was evaporated under reduced
pressure. Water (150 mL) and CH2Cl2 (150 mL) were
added to the residue. The organic layer was dried over
anhydrous Na2SO4, filtered and evaporated under re-
duced pressure. Crystallization from toluene gave the N-
benzylquinolinamines 8 and 9, and the N-benzyliso-
quinolinamines 10 and 11 as pale yellow crystals.
4.11. N-(2-Chloro-4,5-methylenedioxybenzyl)-5-isoquino-
linamine (11)
Yield 90%, mp 161 °C (from toluene), UV k (MeOH)
239, 300, 333 nm; IR (NaCl) m 3326, 2955, 1586, 1476,
4.8. N-(2-Bromo-4,5-dimethoxybenzyl)-5-quinolinamine
(8)
1368, 1248, 1039, 789 cmÀ1 1H NMR (400 MHz,
;
CDCl3) d 4.50 (2H, d, J ¼ 5, CH2-70), 4.88 (1H, t, J ¼ 5,
D2O exch., NH), 5.95 (2H, s, OCH2O), 6.71 (1H, d,
J ¼ 7, H-6), 6.89 (1H, s, H-60), 6.90 (1H, s, H-30), 7.32
(1H, d, J ¼ 8, H-8), 7.45 (1H, dd, J ¼ 8, 7, H-7), 7.62
Yield 96%, mp 214 °C (from toluene), UV k (MeOH)
256, 288, 328 nm; IR (KBr) m 3400, 2964, 2836, 1588,

3950
S. Prado et al. / Bioorg. Med. Chem. 12 (2004) 3943–3953
(1H, d, J ¼ 6, H-4), 8.53 (1H, d, J ¼ 6, H-3), 9.25 (1H,
s, H-1); 13C NMR (75 MHz, CDCl3) d 45.8 (C-70), 101.8
(OCH2O), 108.2 (C-6), 108.4 (C-60), 108.7 (C-4), 111.2
(C-30), 116.7 (C-8), 125.0 (C-20), 125.9 (C-4a), 128.0 (C-
7), 128.8 (C-10), 129.3 (C-8a), 141.8 (C-5), 142.1 (C-3),
147.1 (C-50), 147.5 (C-40), 152.9 (C-1); DCI–MS m=z 313,
315 [MH]þ. Anal. (C17H13ClN2O2): C, H, Cl, N.
(75 MHz, CDCl3) d 45.9 (C-70), 101.8 (OCH2O), 108.4
(C-6), 108.3 (C-20), 108.8 (C-60), 110.9 (C-4), 111.3 (C-
30), 113.4 (C-4a), 116.8 (C-8), 127.8 (C-8a), 128.1 (C-7),
128.8 (C-10), 141.7 (C-5), 142.0 (C-3), 145.7 (2C, C-40, C-
50), 152.9 (C-1); DCI–MS m=z 357, 359 [MH]þ. Anal.
(C17H13BrN2O2): C, H, Br, N.
4.15. N-(2-Bromo-5,6-dimethoxybenzyl)-5-isoquinolin-
amine (24)
4.12. General procedure for the one step synthesis of N-
benzylquinolinamines 8 and 22, and N-benzylisoquinolin-
amines 10, 23 and 24
Yield 34%, amorphous solid, UV k (MeOH) 330 nm; IR
(KBr) m 3254, 3065, 2883, 1575, 1335, 789 cmÀ1 1H
;
A mixture of 5-aminoquinoline (12) or 5-aminoiso-
quinoline (13) (0.69 g, 4.85 mmol) and neutral alumina
(9.65 g) in anhydrous toluene (140 mL) was stirred for
30 min at room temperature under argon. The respective
2-bromo-dialkoxybenzyl bromide (2-bromo-4,5-di-
methoxybenzyl bromide (20),27 2-bromo-4,5-methylene-
NMR (400 MHz, CDCl3) d 3.82 (3H, s, OCH3), 3.88
(3H, s, OCH3), 4.61 (2H, d, J ¼ 5, CH2-70), 4.85 (1H, t,
J ¼ 5, D2O exch., NH), 6.81 (1H, d, J ¼ 9, H-40), 7.10
(1H, d, J ¼ 8, H-6), 7.30 (1H, d, J ¼ 9, H-30), 7.35 (1H,
d, J ¼ 8, H-8), 7.49 (1H, t, J ¼ 8, H-7), 7.57 (1H, d,
J ¼ 6, H-4), 8.45 (1H, d, J ¼ 6, H-3), 9.17 (1H, s, H-1);
13C NMR (75 MHz, CDCl3) d 42.9 (C-70), 55.8 (OCH3),
61.6 (OCH3), 108.4 (C-6), 113.1 (C-4), 113.4 (C-40),
115.3 (C-20), 116.3 (C-8), 126.2 (C-4a), 128.0 (C-30),
128.1 (C-7), 129.3 (C-8a), 131.7 (C-10), 141.9 (C-3), 142.2
(C-5), 148.8 (C-50), 152.3 (C-60), 152.7 (C-1); DCI–MS
m=z 373, 375 [MH]þ. Anal. (C18H17BrN2O2): C, H, Br,
N.
dioxybenzyl
bromide
(21),28
or
2-bromo-5,6-
dimethoxybenzyl bromide (25)29) (4.85 mmol) was ad-
ded, and the suspension was refluxed under argon for
48 h. After addition of water (50 mL), the mixture was
stirred for 30 min and the alumina was filtered off. The
filtrate was extracted with CH2Cl2 (3 ꢁ 75 mL). The or-
ganic layers were dried over anhydrous MgSO4, filtered
and evaporated under reduced pressure. Column chro-
matography of the residue over silica gel (eluent:
CH2Cl2/MeOH 99:1) gave N-benzylquinolinamines 8
and 22, and N-benzylisoquinolinamines 10, 23 and 24 in
15–35% yield.
4.16. General procedure for the synthesis of benzo[c]-
phenanthroline derivatives 3–7
A 2 M lithium diisopropylamide solution in THF (2 mL,
4.02 mmol) was added at )78 °C under argon to a
solution of the respective N-benzylquinolinamine or N-
benzylisoquinolinamine (0.67 mmol) in anhydrous THF
(16 mL). The reaction mixture was stirred at )78 °C for
4 h and allowed to warm to room temperature within
16 h. The solution was evaporated to dryness in vacuo
and the residue was dissolved in CH2Cl2 (50 mL) and
washed with H2O (50 mL). The organic layer was dried
over anhydrous MgSO4, filtered and evaporated under
reduced pressure. Column chromatography of the resi-
due over silica gel (eluent: CH2Cl2/MeOH 90:10) affor-
ded the benzo[c]phenanthrolines 3–7.
4.13. N-(2-Bromo-4,5-methylenedioxybenzyl)-5-quinolin-
amine (22)
Yield 21%, mp 209 °C (from toluene), UV k (MeOH)
229, 330 nm; IR (KBr) m 3407, 2952, 1576, 1443, 1273,
1074, 798 cmÀ1; 1H NMR (400 MHz, CDCl3) d 4.51 (2H,
d, J ¼ 5, CH2-70), 4.80 (1H, t, J ¼ 5, D2O exch., NH),
5.93 (2H, s, OCH2O), 6.60 (1H, dd, J ¼ 7, 2, H-6), 6.90
(1H, s, H-60), 7.05 (1H, s, H-30), 7.32 (1H, dd, J ¼ 8:5, 4,
H-3), 7.57 (2H, m, H-7, H-8), 8.22 (1H, dd, J ¼ 8:5, 1,
H-4), 8.90 (1H, dd, J ¼ 4, 1, H-2); 13C NMR (75 MHz,
CDCl3) d 48.7 (C-70), 101.8 (OCH2O), 105.5 (C-6), 113.0
(C-60), 113.7 (C-30), 114.3 (C-20), 118.5 (C-4a), 119.2 (C-
3), 119.5 (C-8), 128.6 (C-4), 130.3 (C-7), 130.7 (C-10),
142.8 (C-5), 147.7 (C-50), 147.8 (C-40), 149.3 (C-8a),
150.1 (C-2); DCI–MS m=z 357, 359 [MH]þ. Anal.
(C17H13BrN2O2): C, H, Br, N.
4.17. 8,9-Dimethoxybenzo[c][1,7]phenanthroline (3)
Yield 63%, amorphous solid, UV k nm (MeOH) (log e)
212 (4.09), 275.5 (4.50), 360 (3.13); IR (KBr) m 2970,
1
2960, 1521, 1496, 1397, 12861170, 1152, 819 cmÀ1; H
NMR (400 MHz, CDCl3) d 4.13 (3H, s, OCH3), 4.20
(3H, s, OCH3), 7.39 (1H, s, H-7), 7.62 (1H, dd, J ¼ 8, 4,
H-3), 7.89 (1H, s, H-10), 8.22 (1H, d, J ¼ 9, H-12), 8.63
(1H, d, J ¼ 9, H-11), 9.04 (1H, dd, J ¼ 4, 2, H-2), 9.28
(1H, s, H-6), 9.60 (1H, dd, J ¼ 8, 2, H-4); 13C NMR
(75 MHz, CDCl3) d 56.2 (2C, 2 OCH3), 101.7 (C-10),
107.2 (C-7), 120.7 (C-10b), 121.8 (C-3), 122.9 (C-6a),
123.5 (C-11), 127.5 (C-4a), 128.4 (C-12), 128.5 (C-10a),
132.9 (C-4), 140.3 (C-4b), 148.4 (C-12a), 150.3 (C-8),
150.6 (2C, C-2, C-6), 153.3 (C-9); DCI–MS m=z 291
[MH]þ. Anal. (C18H14N2O2): C, H, N.
4.14. N-(2-Bromo-4,5-methylenedioxybenzyl)-5-isoquino-
linamine (23)
Yield 17%, mp 202 °C (from toluene), UV k (MeOH)
222, 274, 333 nm; IR (KBr) m 3326, 1585, 1103,
1
931 cmÀ1; H NMR (400 MHz, CDCl3) d 4.47 (2H, d,
J ¼ 5, CH2-70), 5.00 (1H, t, J ¼ 5, D2O exch., NH), 5.93
(2H, s, OCH2O), 6.68 (1H, d, J ¼ 7, H-6), 6.89 (1H, s,
H-60), 7.05 (1H, s, H-30), 7.32 (1H, d, J ¼ 8, H-8), 7.42
(1H, dd, J ¼ 8, 7, H-7), 7.62 (1H, d, J ¼ 6, H-4), 8.55
(1H, d, J ¼ 6, H-3), 9.25 (1H, s, H-1); 13C NMR

S. Prado et al. / Bioorg. Med. Chem. 12 (2004) 3943–3953
3951
4.18. 8,9-Methylenedioxybenzo[c][1,7]phenanthroline (4)
(C-9), 121.4 (C-11), 122.9 (C-6a), 123.6 (C-10b), 126.1
(C-12), 127.2 (C-12a), 128.7 (C-10a), 135.9 (C-4a), 138.7
(C-4b), 145.1 (C-3), 147.7 (C-6), 150.4 (2C, C-7, C-8),
151.3 (C-1); DCI–MS m=z 291 [MH]þ. Anal.
(C18H14N2O2): C, H, N.
Yield 51%, amorphous solid, UV k nm (MeOH) (log e)
211 (3.15), 275 (3.58), 343 (2.40), 360 (2.29); IR (KBr) m
2950, 1471, 1446, 1262, 1034, 829 cmÀ1
;
1H NMR
(400 MHz, CDCl3) d 6.30 (2H, s, OCH2O), 7.33 (1H, s,
H-7), 7.62 (1H, dd, J ¼ 8, 4, H-3), 8.15 (1H, d, J ¼ 9, H-
12), 8.43 (1H, s, H-10), 8.95 (1H, d, J ¼ 9, H-11), 9.03
(1H, dd, J ¼ 4, 1, H-2), 9.40 (1H, s, H-6), 9.54 (1H, dd,
J ¼ 8, 1, H-4); 13C NMR (75 MHz, CDCl3) d 101.5 (C-
10), 102.0 (OCH2O), 105.1 (C-7), 121.3 (C-10b), 121.8
(C-3), 123.6 (C-6a), 123.7 (C-11), 127.5 (C-4a), 128.6
(C-12), 130.6 (C-10a), 133.0 (C-4), 140.6 (C-4b), 148.5
(C-12a), 150.7 (2C, C-2, C-6), 152.1 (2C, C-8, C-9);
DCI–MS m=z 275 [MH]þ. Anal. (C17H10N2O2): C, H, N.
4.22. Cytotoxicity
Murine leukemia L1210 cells from the American Type
Culture Collection (Rockville Pike, MD) were grown in
RPMI medium 1640 supplemented with 10% foetal calf
serum, 2 mM -glutamine, 100 U/mLpenicillin, 100 lg/
L
mLstreptomycin and 10 mM HEPES buffer (pH 7.4).
The cytotoxicity was measured by microculture tetra-
zolium assay essentially as described.38 Cells were ex-
posed for 48 h to nine graded concentrations in triplicate
of the test drug. Results are expressed as IC50 (mean,
n ¼3), which is defined as the drug concentration
inhibiting the absorbance by 50% with respect to that of
untreated cells.
4.19. 8,9-Dimethoxybenzo[c][1,8]phenanthroline (5)
Yield 56%, amorphous solid, UV k nm (MeOH) (log e)
258 (2.75), 341 (2.45); IR (KBr) m 2963, 2925, 1616,
1
1520, 1488, 1422, 1277, 1030, 850, 802 cmÀ1; H NMR
(400 MHz, CDCl3) d 4.09 (3H, s, OCH3), 4.13 (3H, s,
OCH3), 7.38 (1H, s, H-7), 7.82 (1H, s, H-10), 7.98 (1H,
d, J ¼ 9, H-12), 8.42 (1H, d, J ¼ 9, H-11), 8.81 (1H, d,
J ¼ 6, H-3), 8.96 (1H, d, J ¼ 6, H-4), 9.27 (1H, s, H-6),
9.53 (1H, s, H-1); 13C NMR (75 MHz, CDCl3) d 56.2
(2C, 2 OCH3), 101.6 (C-10), 107.1 (C-7), 117.4 (C-4),
121.3 (C-11), 123.2 (C-10b), 123.4 (C-6a), 125.3 (C-12),
127.6 (C-12a), 128.1 (C-10a), 136.0 (C-4a), 139.0 (C-4b),
144.8 (C-3), 150.5 (C-6), 150.9 (C-1), 153.2 (2C, C-8, C-
9); DCI–MS m=z 291 [MH]þ. Anal. (C18H14N2O2): C, H,
N.
4.23. Cell cycle analysis
For the cell cycle analysis, L1210 cells (5 ꢁ 105 cells/mL)
were incubated for 21 h with various concentrations of
drugs. Cells were then fixed by 70% ethanol (v/v), wa-
shed, and incubated in PBS containing 100 lg/mL
RNAse and 50 lg/mLpropidium iodide for 30 min at
20 °C. For each sample, 10,000 cells were analyzed on an
XLMCL flow cytometer (Beckman Coulter, France).
Results are expressed as % of cells arrested in the given
phases of the cell cycle.
4.20. 8,9-Methylenedioxybenzo[c][1,8]phenanthroline (6)
4.24. Topoisomerase Iinhibition
Yield 35%, amorphous solid, UV k nm (MeOH) (log e)
218 (2.98), 279 (3.15), 306 (2.83); IR (KBr) m 2918, 1628,
Recombinant topoisomerase I protein was produced
and purified from baculovirus infected Sf9 cells.39 Su-
percoiled pKMp27 DNA (0.4 lg) was incubated with
4 units topoisomerase I at 37 °C for 1 h in relaxation
buffer (50 mM Tris pH 7.8, 50 mM KCl, 10 mM MgCl2,
1 mM dithiothreitol, 1 mM EDTA) in the presence of
varying concentrations of the drug under study. Reac-
tions were terminated by adding SDS to 0.25% and
proteinase K to 250 lg/mL. DNA samples were then
added to the electrophoresis dye mixture (3 lL ) and
electrophoresed in an ethidium-containing 1% agarose
gel at room temperature for 2 h at 120 V. Gels were,
washed and photographed under UV light.32
1
1525, 1460, 1255, 1029, 855 cmÀ1; H NMR (400 MHz,
CDCl3) d 6.30 (2H, s, OCH2O), 7.76 (1H, s, H-7), 8.21
(1H, d, J ¼ 9, H-12), 8.44 (1H, s, H-10), 8.76 (1H, d,
J ¼ 6, H-3), 8.83 (1H, d, J ¼ 9, H-11), 8.95 (1H, d,
J ¼ 6, H-4), 9.40 (1H, s, H-6), 9.42 (1H, s, H-1); 13C
NMR (75 MHz, CDCl3) d 101.7 (C-10), 103.6 (OCH2O),
106.1 (C-7), 117.9 (C-4), 123.9 (C-11), 124.9 (2C, C-6a,
C-10b), 125.8 (C-12a), 126.6 (C-12), 128.8 (C-10a), 136.0
(C-4a), 139.6 (C-4b), 146.0 (C-3), 150.0 (C-8), 152.3 (C-
6), 152.5 (C-1), 153.0 (C-9); DCI–MS m=z 275 [MH]þ.
Anal. (C17H10N2O2): C, H, N.
4.21. 7,8-Dimethoxybenzo[c][1,8]phenanthroline (7)
4.25. Purification of the DNA restriction fragment and
radiolabeling
Yield 28%, amorphous solid, UV k nm (MeOH) (log e)
270 (3.46), 307 (2.83); IR (KBr) m 2964, 2930, 1577,
1459, 1279, 1085, 1017, 820 cmÀ1; H NMR (400 MHz,
The 117-bp DNA fragment was prepared by 30-[32P]-end
labeling of the Eco RI-Pvu II double digest of the pBS
plasmid (Stratagene) using a-[32P]-dATP (Amersham,
3000 Ci/mmol) and AMV reverse transcriptase (Roche).
The labeled digestion products were separated on a 6%
polyacrylamide gel under nondenaturing conditions in
TBE buffer (89 mM Tris–borate pH 8.3, 1 mM EDTA).
1
CDCl3)d 4.10 (3H, s, OCH3), 4.12 (3H, s, OCH3), 7.76
(1H, d, J ¼ 9, H-9), 8.06 (1H, d, J ¼ 9, H-12), 8.44 (1H,
d, J ¼ 9, H-10), 8.56 (1H, d, J ¼ 9, H-11), 8.83 (1H, d,
J ¼ 6, H-3), 9.05 (1H, d, J ¼ 6, H-4), 9.28 (1H, s, H-1),
9.82 (1H, s, H-6); 13C NMR (75 MHz, CDCl3) d 56.7
(OCH3), 61.9 (OCH3), 117.3 (C-4), 118.7 (C-10), 118.8

3952
S. Prado et al. / Bioorg. Med. Chem. 12 (2004) 3943–3953
After autoradiography, the requisite band of DNA was
excised, crushed and soaked in water overnight at 37 °C.
This suspension was filtered through a Millipore
0.22 lM filter and the DNA was precipitated with eth-
anol. Following washing with 70% ethanol and vacuum
drying of the precipitate, the labeled DNA was re-sus-
pended in 10 mM Tris adjusted to pH 7.0 containing
10 mM NaCl.
centrations ranging from 0.1–500 lM bp in 1 mM so-
dium cacodylate buffer, pH 7.0.
4.29. Electric linear dichroism
Calf thymus DNA (Pharmacia) was deproteinized with
sodium dodecyl sulfate (protein content <0.2%) and
extensively dialyzed against 1 mM sodium cacodylate
buffered solution pH 7.0. ELD measurements were per-
formed with a computerized optical measurement sys-
tem using the procedures previously outlined.40 All
experiments were conducted with a 10 mm pathlength
Kerr cell having 1.5 mm electrode separation. The
samples were oriented under an electric field strength
varying from 1 to 14 kV/cm. The drug under test was
present at 10 lM concentration together with the DNA
at 200 lM concentration unless otherwise stated. This
electro-optical method has proved most useful to
determine the orientation of the drugs bound to DNA.
It has the additional advantage that it senses only the
orientation of the polymer-bound ligand: free ligand is
isotropic and does not contribute to the signal.36
4.26. Sequencing of topoisomerase I-mediated DNA
cleavage sites
0
Each reaction mixture contained 2 lLof 3 -end [32P]
labeled DNA (ꢂ1 lM), 5 lLof water, 2 lLof 10X
topoisomerase I buffer, 10 lLof drug solution at the
desired concentration (50 lM final concentration). After
10 min incubation to ensure equilibration, the reaction
was initiated by addition of 2 lL(20 units) topoiso-
merase I. Samples were incubated for 45 min at 37 °C
prior to adding SDS to 0.25% and proteinase K to
250 lg/mLto dissociate the drug–DNA–topoisomerase I
cleavable complexes. The DNA was precipitated with
ethanol and then resuspended in 5 lLof formamide–
TBE loading buffer, denatured at 90 °C for 4 min then
chilled in ice for 4 min prior to loading on to the
sequencing gel. DNA cleavage products were resolved
by polyacrylamide gel electrophoresis under denaturing
conditions (0.3 mm thick, 8% acrylamide containing 8 M
urea). After electrophoresis (about 2.5 h at 60 Watts,
1600 V in TBE buffer), gels were soaked in 10% acetic
acid for 10 min, transferred to Whatman 3 MM paper,
and dried under vacuum at 80 °C. A Molecular
Dynamics 425E PhosphorImager was used to collect
data from the storage screens exposed to dried gels
overnight at room temperature. Each resolved band was
assigned to a particular bond within the DNA fragment
by comparison of its position relative to sequencing
standards generated by treatment of the DNA with
dimethylsulfate followed by piperidine-induced cleavage
at the modified guanine residues.
Acknowledgements
This work was done under research grants from the
ꢀ
Chancllerie des Universites de Paris (Bourse Aguirre-
Basualdo to S.P.) and from the Ligue Nationale Contre
le Cancer and the Institut de Recherches sur le Cancer
ꢀ ꢀ
de Lille (to C.B.). Support by the ‘Actions integrees
Franco-Belge, Programme Tournesol’ is also acknowl-
edged.
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